Pentosan polysulfate regulates hepcidin 1-facilitated formation and function of osteoclast derived from canine bone marrow

Suranji Wijekoon; Takafumi Sunaga; Yanlin Wang; Carol Mwale; Sangho Kim; Masahiro Okumura

doi:10.1371/journal.pone.0265596

. 2022 Mar 17;17(3):e0265596. doi: 10.1371/journal.pone.0265596

Pentosan polysulfate regulates hepcidin 1-facilitated formation and function of osteoclast derived from canine bone marrow

Suranji Wijekoon ^1,^*,^#, Takafumi Sunaga ^1,^‡, Yanlin Wang ^1,^‡, Carol Mwale ^1,^‡, Sangho Kim ^1,^‡, Masahiro Okumura ^1,^#

Editor: Dominique Heymann²

PMCID: PMC8929557 PMID: 35299233

Abstract

Hepcidin which is the crucial regulator of iron homeostasis, produced in the liver in response to anemia, hypoxia, or inflammation. Recent studies have suggested that hepcidin and iron metabolism are involved in osteoporosis by inhibiting osteoblast function and promoting osteoclastogenesis. Pentosan polysulfate (PPS) is a heparin analogue and promising novel therapeutic for osteoarthritis (OA). This study was undertaken to determine whether PPS inhibits hepcidin-facilitated osteoclast (OC) differentiation and iron overload. Canine (n = 3) bone marrow mononuclear cells were differentiated to OC by macrophage colony-stimulating factor and receptor-activator of nuclear factor kappaB ligand with the treatment of hepcidin1 (200, 400, 800, 1200 nmol/L) and PPS (1, 5, 10, 20, 40 μg/mL). Differentiation and function of OC were accessed using tartrate-resistant acid phosphate staining and bone resorption assay while monitoring ferroportin1 (FPN1) and iron concentration by immunocytochemistry. Gene expression of OC for cathepsin K (CTK), matrix metallopeptidase-9, nuclear factor of activated-T-cells cytoplasmic 1 and FPN1 was examined. Hepcidin1 showed significant enhancement of OC number at 800 nmol/L (p<0.01). PPS impeded hepcidin-facilitated OC at 1, 5 and 10 μg/mL and reduction of resorption pits at 5 and 10 μg/mL (p< 0.01). All OC specific genes were downregulated with PPS, specifically in significant manner with CTK at higher concentrations. However, heparin induced FPN1 internalization and degradation was inhibited at higher concentrations of PPS while restoring iron-releasing capability of OC. We demonstrate for the first time that PPS is a novel-inhibitor of hepcidin-facilitated OC formation/function which might be beneficial for treatment of OA and osteoporosis.

Introduction

Hepcidin is a recently discovered cysteine-rich cationic, small endogenous peptide hormone synthesized in hepatocytes and involved in the regulation of iron homeostasis [1, 2]. Secreted hepcidin inhibits iron transport by binding to the iron export channel ferroportin which is located in the basolateral plasma membrane of gut enterocytes and the plasma membrane of reticuloendothelial cells (macrophages), while breaking down the ferroportin in to lysosomes [3]. Further, hepcidin inhibits the release of iron into the circulation by regulating its associated receptor ferroportin 1 (FPN 1) [1].

Hepcidin expression is exaggerated in chronic inflammatory conditions due to cancer, infectious autoimmune disorders, hypoxia, and anemia [4–9]. Interleukin 6 (IL-6) plays a key role in inflammatory induction of hepcidin and ultimately causes hypoferremia due to ferroportin degradation and iron sequestration in tissue macrophages [10]. The IL-6 blockers have been shown as highly effective on the anemia of chronic disease, commonly observed in rheumatoid arthritis (RA). Several studies have focused on the role of hepcidin in RA and found that the circulating serum hepcidin level is reported to increase in the RA patients with anemia of chronic disease (ACD) [11–13]. With understanding of all those factors hepcidin is recognized as a major inducer of ACD in patients with RA.

Some recent reports have indicated that iron metabolism can affect bone metabolism. Elevated ferritin level/iron overload is a risk factor for progressive bone loss in healthy postmenopausal women and middle-aged men [14]. However, the direct effects of hepcidin on bone metabolism are still unknown although it shows potential of pharmacological target. Bone metabolism in the body involves osteoblastic bone formation as well as osteoclastic bone resorption. Previous studies showed that hepcidin could increase intracellular iron and calcium levels and promote mineralization in osteoblasts [15–17] and osteoclasts (OC) [1].

Pentosan polysulfate (PPS) is a semi-synthetic sulfated polysaccharide drug manufactured from European beech wood hemicellulose by sulfate esterification. Average molecular weight of PPS is around 5700 Da [18]. From the results of previous in vitro and in vivo studies, the spectrum of pharmacological activities exhibited by PPS would qualify it as disease-modifying osteoarthritis drugs [19] due to its ability of preserving the integrity of the articular cartilage and bone whilst improving the characteristic of the synovial joint fluid [20–25]. Further we have previously identified that the inhibitor effect of PPS on formation and function of bone marrow derived-OC which was differentiated with the presence of receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) [26].

Bone homeostasis between bone resorption by OC and bone formation by chondrocytes/osteoblasts is tightly orchestrated to preserve skeletal health and integrity throughout life [27]. Osteoclasts originate from the monocyte/macrophage hematopoietic lineage, whereas osteoblasts originate from multipotent mesenchymal stem cells. Osteoclastogenesis is a tightly regulated process by diverse cytokines, steroids, and lipids [28, 29]. Among them, M-CSF and RANKL predominantly involve differentiation, maturation, and function of this giant cell [30, 31]. However, excessive activation of the immune mediators at the inflammatory conditions can enhance the OC production and eventually the severe bone erosion can be occurred [32]. Therapeutic interventions targeting osteoclastogenesis might enable it to restore bone mass in arthritic patients.

This study was undergone to find out whether PPS is able to control the formation and function of hepcidin 1 treated OC and thereby intracellular iron concentration. To the best of our knowledge, the present study is the first attempt to identify the effect of PPS on hepcidin facilitated differentiation and intracellular iron concentration in bone marrow derived OC. We hypothesized that the PPS, which carry different effects by improving the symptoms of osteoarthritis and RA, would be more likely to have an inhibitory effect on hepcidin.

Material and methods

Preparation of sample collection site

Proximal femur of healthy beagle dogs (n = 3) was used to collect the 5 mL of bone marrow samples in to 10 mL syringe containing 1 mL Dulbecco’s modified eagle’s medium (DMEM, Life technologies, New York, USA) and 1000 U/mL of heparin (Nipro, Osaka, Japan). The use of all samples from healthy experimental Beagle dogs (mean age: 12.9 months; range: 12–14 months) was in accordance with Hokkaido University Institutional Animal Care and Use Committee guidelines (approval number: 21–0022). Briefly, dogs were put under general anesthesia induced with propofol (Intervet, Tokyo, Japan) at 6 mg/kg intravenously and maintained on isoflurane (Intervet) and oxygen. Meloxicam (Boehringer-Ingelheim Animal Health, Tokyo, Japan) at 0.2 mg/kg subcutaneously was administered for pain management. The bone marrow aspiration site was aseptically prepared by clipping the hair around the proposed site of collection and scrubbed with 70% ethanol and then povidone iodine.

Osteoclastic differentiation from canine bone marrow

Separation of bone marrow mononuclear cell (BMMs) fraction was done and preceded as described previously [33, 34]. Briefly, BMMs were obtained by density gradient centrifugation over lymphoprep (Axis-sheild PoC AS, Oslo, Norway) to remove red blood cells. Isolated BMMs cell fraction (5 × 10⁶ cells/mL) was incubated with DMEM containing penicillin/streptomycin (100 units/mL, Wako pure chemical, Tokyo, Japan) and 10% heat-inactivated fetal bovine serum (FBS, Nichirei Bioscience INC., Tokyo, Japan) for 24 h to separate the non-adherent from adherent cells. Non-adherent cells were collected as a source of immature OC precursors, suspended in DMEM, counted, seeded on 48-wells plates (Corning, New York, USA) at 2 × 10⁵ cells/well, and cultured in DMEM with the presence of 20 ng/ml recombinant human M-CSF (Invitrogen, Maryland, USA) for 3 days. After 3 days, adherent cells were used as OC precursors after washing out the non-adherent cells, including lymphocytes and further cultured in the presence of 25 ng/mL M-CSF, 50 ng/mL recombinant human RANKL (Sigma-Aldrich, St Louis, Missouri, USA) and 0, 200, 400, 800 nmol/L hepcidin 1 to generate osteoclast-like multinucleated giant cells. The selected concentrations of hepcidin are within the previously proved non-cytotoxic range for mouse macrophages [1]. The cells were treated with 1, 5, 10, 20 μg/mL concentration of PPS (Cartrophen Vet-Biopharm-100 mg/ ml, New South Wales, Australia) for 1-week. The selected concentrations of PPS are within the previously proved non-cytotoxic range for bone marrow derived cells [35]. Triplicate cultures for each concentration of PPS were maintained by changing the media in every 48 h ensuring their constancy of concentrations.

Tartrate-resistant acid phosphate (TRAP) staining

Cultured BMMs with M-CSF and RANKL in the presence of 0, 200, 400, 800 nmol/L hepcidin and 1, 5, 10, 20 μg/mL PPS were subjected to TRAP stain (Cosmo Bio Co., LTD, Tokyo, Japan) after 7 days. Cells were washed with 1% phosphate buffered saline (PBS) and fixed with 10% formalin neutral buffer solution for 5 min at room temperature. After washing with 500 μL deionized water 3 times, cells were stained for TRAP according to the manufacturer’s instructions. Cells containing ≥3 nuclei were considered as OC and counted.

Resorption pit formation assay

Non-adherent cells, collected from BMMs fraction of 3 dogs were cultured at 2 × 10⁵ cells/well density on calcium phosphate (CaP-coated) bone resorption assay plate 48 (PG Research, Tokyo, Japan). The cells were maintained in DMEM with the presence of 20 ng/ml recombinant human M-CSF (Invitrogen, Maryland, USA) for 3 days in triplicate cultures. After 3 days, adherent cells were used as OC precursors after washing out the non-adherent cells and further cultured in the presence of 25 ng/mL M-CSF, 50 ng/mL recombinant human RANKL, hepcidin (0, 200, 400, 800 nmol/L) and PPS (5 μg/mL). After 7 days, the CaP-coated plate was treated with 5% sodium hypochlorite (Sigma-Aldrich, St Louis, Missouri, USA) for 5 min according to the manufacturer’s instructions. The resorption pit area was analyzed and counted by Image-J software (Image J software version 1.43, National Institute of Health).

Preparation of cell extracts and analysis

Total RNA and protein were extracted using TRIZol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Total RNA was quantified by spectrophotometry at 260 nm using Biowave DNA-WPA, 7123 V1.8.0 (Biochrom, Cambridge, UK) and stored at -20 °C until use. RNA with a 260/280 nm ratio in the range 1.8–2.0 was considered high quality and total of 500 ng RNA was reverse transcribed (RT) into cDNA using ReverTra Ace qPCR RT Master Mix (Toyobo Co, Osaka, Japan) and amplified by PCR using TaKaRa Ex taq (TaKaRa Bio, Tokyo, Japan) according to manufacturer’s recommended protocol. This technique was employed to amplify mRNAs specific for cathepsin k (CTK), matrix metallopeptidase-9 (MMP9), nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and ferroportin 1 (FPN1). The PCR conditions were an initial denaturation of 94 °C for 1 min followed by 35 cycles of 94 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s and then a finishing step of 72 °C for 1 min. Gel electrophoresis was performed to detect mRNA bands. Quantitative real-time PCR (qPCR) was performed with KAPA SYBR FAST qPCR kit (KAPA biosystems, Woburn, MA, USA) to determine the relative mRNA expression by the two step method. The qPCR conditions were an initial denaturation of 95 °C for 20 s followed by 40 cycles of 95 °C for 3 s and 60 °C for 20 s then a pre-melt condition of 60 °C for 90 s followed by a final melt step. The standard curve method was used to determine the relative mRNA quantification. All PCR reactions were validated by the presence of a single peak in the melt curve analysis and single band on gel electrophoresis. The amount of 2 μL of cDNA template was added to each 10 μL of premixture with specific primers. The following primer sets were used: cathepsin k, 5’- ACCCATATGTGGGACAGGAT-3’ (forward) and 5’-TGGAAAGAGGTCAGGCTTGC-3’ (reverse); MMP9, 5’-GGCAAATTCCAGACCTTTGA-3’ (forward) and 5’-TACACGCGAGTGAAGGTGAG-3’(reverse); NFATc1, 5’-CACAGGCAAGACTGTCTCCA-3′ (forward) and 5’-TCCTCCCAATGTCTGTCTCC-3′ (reverse); FPN1, 5’-CAGTCTATGGGCTGGTGGTG-3′ (forward) and 5’-TCTGGATCGTGATGGCAGTG-3′ (reverse); GAPGH, 5’-CTGA ACGGGAAGCTCACTGG-3′ (forward) and 5’-CGATG CCTGCTTCACTACCT-3′ (reverse). All reactions were normalized to the housekeeping gene b-Actin.glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Immunocytochemical detection of ferroportin 1 (FPN 1)

Osteoclast precursors resulting from canine bone marrow cells (2 × 10⁵ cells) were cultured in 8-wells culture slide (Iwaki, Tokyo, Japan) in 200 μL of DMEM, 10% FBS with OC differentiation factors (25 ng/mL M-CSF, 50 ng/mL recombinant human RANKL). Cells were treated separately and combined with hepcidin (0, 200, 400, 800, 1200 nmol/L) and PPS (5, 10, 20, 40 μg/mL) for 20 hours. The cells were fixed with 4% paraformaldehyde for 20 min. After being blocked in 1% bovine serum albumin (BSA) for 45 minutes at room temperature, the cells were then incubated with primary antibodies (rabbit anti-ferroportin 1, 1:50, Lifespan Biosciences, Seattle, Washington, USA) in a humid chamber at 4 °C overnight. After being washed three times with PBS, the cells were incubated with a secondary antibody (goat anti-rabbit, 1:500, Alexa Fluor 488, Abcam, Cambridge, UK) away from light at room temperature for 45 minutes. After washing three times with PBS, the cells were stained with DAPI. The OC were observed using laser scanning confocal microscope (Zeiss, Illinois, USA).

Immunofluorescent analysis of intracellular iron concentration

Fluorescence of the Phen Green FL is quenched upon binding iron ion, so the change of emission intensity of the indicator is correlated with iron concentration. The weaker the fluorescence intensity is an indicator of the higher the concentration of intracellular iron [1]. Osteoclast precursors cells were seeded in an 8-wells culture slide of 2×10⁵ cells/well. After cells adhered to the coverslips, the medium was replaced with fresh medium containing 25 ng/mL M-CSF, 50 ng/mL RANKL and 800, nmol/L hepcidin 1 and 20 μg/mL PPS and allowed to incubate for 20 h. After treatment, the cells were washed twice with PBS and incubated with Phen Green FL (Carlsbad, California, USA) away from light at 34°C in a humidified atmosphere containing 5% CO2 for 30min. Unbound fluorescent indicator was removed by washing with PBS two times. A confocal laser scanning microscope was used to record the signal intensity from Phen Green FL, with excitation at 488 and emission at 521 nm.

Data analysis

Data were analyzed using Statistical Package for the Social Sciences v.16 (SPSS inc., Chicago, IL, USA). Statistical significance of quantitative qPCR data, number of OC and resorption pits were determined by analysis of variance (ANOVA), comparing the mean values of the treatments. Where significant differences were observed, multiple comparison of group means was performed using Post Hoc Bonferroni. The results were considered significant at a 95% confidence level (p < .05). All quantitative results are presented as mean ± SE.

Results

Hepcidin 1 facilitate M-CSF and RANKL-induced osteoclastogenesis

Effect of hepcidin 1 on M-SCF and RANKL treated BMMs were evaluated. Dose-dependent stimulatory effect of hepcidin 1 was observed up to the 800 nmol/L concentration by counting the TRAP positive multinucleated cells (≥3 nuclei) seeded in 48 wells plates (Fig 1A). However, TRAP-stained cell number reduced at the 1200 nmol/L concentration. Significant enhancement of OC number was detected at the 800 nmol/L concentration (p<0.05) (Fig 1B).

Fig 1 — (A) The cells were treated with various concentrations of hepcidin followed by M-CSF (20 ng/mL) and RANKL (50 ng/mL) for 7 days. The cells were stained for TRAP stain and TRAP-positive cells (≥3 nuclei) were counted. Scale bar- 200 μm. (B) Bar graphs show the number of OC cells/well. Data are representative of three independent experiments and expressed as means ± SE. Means with *are significantly different from 0 μg/mL of hepcidin (*p < 0.05, **p < 0.01).

PPS inhibits osteoclastogenesis

Effects of different concentrations of PPS were evaluated over the BMMs treated M-CSF and RANKL. Dose dependent inhibition of TRAP-stained multinucleated cells (≥3 nuclei) (Fig 2A) were detected significantly at all the concentrations of PPS from 1 (p<0.001), 5, 10, 20 μg/mL (p<0.005) (Fig 2B).

Fig 2 — (A) The cells were treated with various concentrations of PPS followed by M-CSF (20 ng/mL) and RANKL (50 ng/mL) for 7 days. The cells were stained for TRAP stain and TRAP-positive cells (≥3 nuclei) were counted. Scale bar- 100 μm. (B) Bar graphs show the number of OC cells/well. Data are representative of three independent experiments and expressed as means ± SE. Means with *are significantly different from 0 μg/mL of PPS (*p < 0.05, **p < 0.01).

PPS inhibits hepcidin-facilitated OC formation and function in dose dependent manner

The effect of different concentrations of PPS (1, 5, and 10 μg/mL) on OC differentiation from BMMs stimulated with M-CSF, RANKL and hepcidin 800 nmol/L was evaluated. The number of TRAP-positive multinucleated cells (≥3 nuclei) generated in 48 well plate was reduced (Fig 3A) with the administration of PPS at the concentration of 1, 5 and 10 μg/mL in significant manner (p < 0.05) compared to the control samples those were not treated with PPS (Fig 3B). The effect of PPS on hepcidin-facilitated OC function was assessed via counting the bone resorption pits formed by OC generated from 3 dogs. Cells were plated on CaP-coated plates and stimulated with M-CSF, RANKL and hepcidin 800 nmol/L in the presence and absence of PPS. Cells stimulated with M-CSF, RANKL and hepcidin formed a number of resorption pits suggesting that the bone resorption activity of RANKL-treated cells made them into functionally active state resembling OC. The concentrations of 5 and 10 μg/mL PPS significantly reduced the formation of resorption pits (Fig 4A) in number and in overall area compared with treatment with M-CSF, RANKL and hepcidin alone (Fig 4B). Gene expression of Cathepsin K, MMP-9, NFATc1 and FPN1 was investigated (Fig 4C). Quantitative qPCR data showed that PPS at 5–10 μg/mL significantly downregulates the gene expression (p<0.05) level of CTK while inhibiting expression level of MMP-9 and NFATc1 in concentration-dependent manner (Fig 4D). However, the expression of FPN1 was upregulated toward the higher concentrations of PPS.

Fig 3 — The cells were treated with various concentrations of PPS followed by 800 nmol/L hepcidin 1, M-CSF (20 ng/mL) and RANKL (50 ng/mL) for 7 days. (A) The cells were stained for TRAP stain and TRAP-positive cells (≥3 nuclei) were counted. Scale bar- 200 μm. (B) Bar graphs show the number of OC cells/well. Data are representative of three independent experiments and expressed as means ± SE. Means with *are significantly different from 0 μg/mL of PPS (*p < 0.05, **p < 0.01).

Fig 4 — Canine BMMs, cultured with M-CSF (20 ng/mL), RANKL (50 ng/mL) and 800 nmol/L hepcidin 1 for 7 days with or without indicated doses of PPS. (A) The cells were washed and the resorption pits were counted. (B) The numbers of pits were analyzed with Image-J software. Scale bar- 200 μm. Column indicates means ± SE of three experiments performed in triplicate. (C) and (D) Gene expression of CTK, MMP9, NFATc1 and FPN1 was investigated. Quantitative qPCR showed that PPS downregulates the gene expression of MMP9, NFATc1 and CTK (CTK at 5 and 10 μg/mL of PPS; p<0.05) while upregulating FPN1 expression in concentration manner. Data expressed as mean ± SE for each PPS concentration after normalizing for the expression of the GAPDH. Means with *are significantly different from 0 μg/mL of NaPPS (*p < 0.05, **p < 0.01).

PPS inhibits the hepcidin-induced FPN1 internalization and degradation

Canine bone marrow derived OC were treated with rabbit anti-ferroportin 1 after 20 hours treatment of hepcidin (200, 400, 800 and 1200 nmol/L) to visualize the expression of FPN1 protein. Immunofluorescence showed the expression of FPN1 protein in OC treated MCSF and RANKL (Fig 5A). The intensity of fluorescence was reduced towards the 800 and 1200 nmol/L concentration of hepcidin (Fig 5B). Separate groups of OC were teared with different concentrations of PPS for overnight after treatment of 20 hours of 800 nmol/L hepcidin. However, heparin induced FPN1 internalization and degradation was inhibited with higher concentrations of PPS (5, 10, 20 and 40 μg/mL). Higher the fluorescence intensity was visualized toward the 40 μg/mL PPS (Fig 5C and 5D).

Fig 5 — (A) Immunofluorescence analysis of ferroportin 1 (FPN1) protein. Canine bone marrow derived OC were treated with rabbit anti-ferroportin 1 after 20 hours treatment of hepcidin (200, 400 and 800 nmol/L) to visualize the expression of FPN1 protein. The figure shows the strong detection of localization of FPN1 at the membrane of hepcidin untreated OC (control). (B) The green fluorescence intensity significantly weakened toward the higher concentration of hepcidin (800 nmol/L). Scale bar- 50 μm. (C) PPS inhibits the hepcidin-induced FPN1 internalization and degradation. OC were treated with rabbit anti-ferroportin 1 after 20 hs treatment of PPS (5, 10, 20 and 40 μg/mL) and hepcidin 1 (800 nmol/L) to visualize the expression of FPN1 protein. The figure shows the strong inhibition of localization of FPN1 at the membrane of hepcidin treated OC (control). (D) Heparin induced FPN1 internalization and degradation was inhibited with higher concentrations of PPS (5, 10, 20 and 40 μg/mL). Higher the fluorescence intensity was visualized toward the 40 μg/mL PPS. Scale bars- 50 and 100 μm. (E) Confocal microscopy analysis of iron concentration in OC. Cell medium was replaced with fresh medium containing 25 ng/mL M-CSF, 50 ng/mL RANKL and 800, nmol/L hepcidin 1 and 20 μg/mL PPS and allowed to incubate for 20 h. Fluorescence of the Phen Green FL is quenched upon binding iron and emission intensity was weakened when the iron concentration is high. E, shows high fluorescence intensity in untreated cells implying low intracellular iron. (F) Fluorescence images showed that intracellular iron concentration was increased (weakened fluorescence intensity) with hepcidin 800 nmol/L compared to the untreated cells. (G) fluorescence images have proven the inhibitory effect of PPS on hepcidin-induced iron accumulation by visualizing greater intensity of fluorescence at 20 μg/mL.

PPS repressed the hepcidin-induced intracellular iron accumulation

Fluorescence of the Phen Green FL is quenched upon binding iron and emission intensity was weakened when the iron concentration is high. Fluorescence images showed that intracellular iron concentration was increased (weakened fluorescence intensity) with hepcidin 800 nmol/L compared to the untreated cells (Fig 5E and 5F). However, fluorescence images have proven the inhibitory effect of PPS on hepcidin-induced iron accumulation by visualizing greater intensity of fluorescence (Fig 5G).

Discussion

Hepcidin-centered therapeutic studies are a fascinating field of research to regulate the iron homeostasis, anemia of inflammation and bone metabolism. Numerous therapeutic modalities which are identified targeting the property of cytokines-based hepcidin repression seem effective over the patients of OA and RA [36, 37]. The present study demonstrates for the first time that PPS is a novel inhibitor of hepcidin-facilitated OC formation and function from bone marrow-derived stem cells. With the multiple facets of action of PPS, this novel finding would be upgraded the uses of DMOARs and specially, PPS for the betterment of OA and RA associated anemia and iron imbalance.

Similar to the finding of mouse macrophage-generated OC culture [1], our findings demonstrate that hepcidin facilitate MSCF and RANKL induced osteoclastogenesis from canine macrophages in dose dependent manner up to 800 nmol/L concentration. The fact that understands in this study is concentration over the 800 nmol/L was no able to further facilitate canine OC formation which is controversy with previous mouse study of Zhao and the team [1], could be due to species variation affecting the degree of hepcidin effect on bone metabolism. Hepcidin exerts its synergistic effect of upregulating the target genes of OC such as, CTK, MMP9 and NFATc1 which are needed in bone resorption activity of OC while inhibiting FPN1 which is well known iron exporter from inside to outside the cell. By confirming our previous research findings PPS deters osteoclastogenesis in a dose dependent manner at the range of concentration 1–20 μg/mL. The outcome of studies suggests that the inhibitory action of PPS over OC differentiation and function could be applied in treatment of pathological bone disorders such as osteoporosis or inflammatory arthritis where OC plays a vital role.

Current study data distinguished the novel capability of PPS by allowing it to interact with hepcidin at the process of canine bone-derived OC differentiation. Former research data demonstrated that the ability of hepcidin on enhancing differentiation and intracellular iron in OC with the use of mouse monocyte/macrophage cell line (RAW264.7) that can differentiate into multinucleated cells with an osteoclastic phenotype under the induction of RANKL [1, 38]. The results of previous study suggested that PPS at concentrations of 1 and 5 μg/mL suppressed M-CSF and RANKL-induced bone resorption activity and formation of actin-rings in matured OC (S1 Fig). The resorption lacunae, pit formation and actin ring formation are essential for OC bone resorption and is the most obvious character of mature OC during osteoclastogenesis [26, 39, 40]. With consisting of the former data on effect of PPS, our current findings further imply that hepcidin-facilitated bone resorption of OC can be condensed with the presence of 5, 10 μg/mL concentrated PPS in significant manner.

In our study, PPS at higher concentrations significantly suppressed the cathepsin K genomic expression in hepcidin-treated OC while reducing the MMP9 and NFATc1 expression in dose dependent manner. Once the Mitogen-activated protein kinase signaling cascade is activated, NFATc1 is activated as a master transcription factor for OC differentiation [30, 41]. Further, NFATc1 plays a dynamic role in upregulating expressions of genes required for OC maturation, such as cathepsin K and MMP-9 which are requisite for the bone resorption processes mediated by mature OC [42]. We further speculated that the existence of FPN1 at the membrane premature OC cells, and the expression of FPN1 was markedly downregulated by hepcidin in a concentration dependent manner, markedly at the 800 nmol/L. Intriguingly, PPS is able to inhibit the hepcidin-induced internalization and degradation of FPN1 molecule and that was visualized in gene expression level and by immunocytochemistry. With the increment of hepcidin, it binds to FPN1 molecules and provokes their internalization and degradation, and iron release is decreased progressively [3]. With that inhibition of degradation of FPN1 molecule in OC by PPS, facilitate the iron releasing into the outside of OC cells.

Some of the limitations of our study are the not analyzing the protein level FPN expression. We believe that current presentation of data would be a good platform to initiate protein level work together with advanced protein analysis for each factors, focused on this study other than the current finding of specific gene expression level for OC to understanding how the PPS affect those cells in transcriptional level. To the author’s knowledge, no similar studies have previously been reported to determine a priori what the crucial pathways to address. Current study took place to explain the fundamentals of the PPS effect in cell culture level using changes of differentiation and functional changes associated with osteoclasts. Further, undifferentiated bone marrow mononuclear cells (BMMs or osteoclast precursor cells) do not show reduced FPN levels after hepcidin treatment could be the true factor hence BMM cells might have comparatively less hepcidin-induced FPN1 internalization and degradation compared to mature osteoclasts, which needs additional step forward to examine the variability of cellular response to hepcidin and understand the variability of metabolism.

Previous studies have indicated that OC development comprises high iron requirements [43]. With that phenomenal statement, it further supplemented the inhibitory effect of PPS on osteoclastogenesis and their functional capability even at the presence of hepcidin. Outcome of this study confirms that PPS regulates hepcidin 1-facilitated formation and function of OC derived from canine bone marrow thereby inhibiting iron accumulation in the cells. However, further investigations would be required to clarify the mechanism of action of PPS on hepcidin inhibition. This in vitro study will pave the other clinical aspect of PPS to explore its additional therapeutic application against hepcidin compound which plays a vital role in the anemia of inflammation observed in many RA patients.

Supporting information

S1 Fig. Osteoclasts actin ring formation (Rhodamin Phalloidin) was monitored after treatment of PPS with different concentartions.

Osteoclasts derived from canine bone marrow were treated with various concentrations (0, 5, 10, 20 μg/mL) of PPS followed by M-CSF (20 ng/mL) and RANKL (50 ng/mL) for 7 days stained with phalloidin, which detects filamentous actin. An actin ring is a characteristic actin structure that is essential for bone resorption by osteoclasts. Scale bar- 100 μm. PPS showed inhibitory effect of formation of number of acting ring and the differention of osteoclast in a concentartion dependent manner.

(TIFF)

Click here for additional data file.^{(8.9MB, tiff)}

S1 Table. Primer sequences used to polymerize the osteoclast specific genes.

(DOCX)

Click here for additional data file.^{(15.7KB, docx)}

Data Availability

Data Availability: All relevant data are within the paper and its Supporting information files.

Funding Statement

The research was funded by Japan Racing Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Zhao G, Di D, Wang B, Huang X, Xu Y. Effects of mouse hepcidin 1 treatment on osteoclast differentiation and intracellular iron concentration. Inflammation. 2015; 38:718–727. doi: 10.1007/s10753-014-9982-2 [DOI] [PubMed] [Google Scholar]
2.Krause A, Neitz S, Magert HJ, Schulz A, Forssmann WG, Schulz-Knappe P et al. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett. 2000;480:147–150. doi: 10.1016/s0014-5793(00)01920-7 [DOI] [PubMed] [Google Scholar]
3.Drakesmith H, Nemeth E, Ganz T. Iron out Ferroportin. Cell Metab. 2015;22:777–787. doi: 10.1016/j.cmet.2015.09.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Ganz T. Molecular pathogenesis of anemia of chronic disease. Pediatr Blood Cancer. 2006;46:554–557. doi: 10.1002/pbc.20656 [DOI] [PubMed] [Google Scholar]
5.Viatte L, Vaulont S. Hepcidin, the iron watcher. Biochimie.2016;91:1223–1228. doi: 10.1016/j.biochi.2009.06.012 [DOI] [PubMed] [Google Scholar]
6.Ashby DR, Gale DP, Busbridge M, Murphy KG, Duncan ND, Cairns TD, et al. Plasma hepcidin levels are elevated but responsive to erythropoietin therapy in renal disease. Kidney Int. 2009;75:976–981. doi: 10.1038/ki.2009.21 [DOI] [PubMed] [Google Scholar]
7.Macdougall IC, Malyszko J, Hider RC, Bansal SS. Current status of the measurement of blood hepcidin levels in chronic kidney disease. Clin. J. Am. Soc. Nephrol. 2010;5:1681–1689. doi: 10.2215/CJN.05990809 [DOI] [PubMed] [Google Scholar]
8.Weiss G. Anemia of chronic disorders: new diagnostic tools and new treatment strategies. Semin. Hematol. 2015;52:313–320. doi: 10.1053/j.seminhematol.2015.07.004 [DOI] [PubMed] [Google Scholar]
9.Wang CY, Babitt JL. Hepcidin regulation in the anemia of inflammation. Curr. Opin. Hematol. 2016;23:189–197. doi: 10.1097/MOH.0000000000000236 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Ludwiczek S, Aigner E, Theurl I, Weiss G. Cytokine-mediated regulation of iron transport in human monocytic cells. Blood. 2003;101:4148–4154. doi: 10.1182/blood-2002-08-2459 [DOI] [PubMed] [Google Scholar]
11.Demirag MD, Haznedaroglu S, Sancak B, Konca C, Gulbahar O, Ozturk M.A, et al. Circulating hepcidin in the crossroads of anemia and inflammation associated with rheumatoid arthritis. Intern. Med. 2009;48, 421–426. doi: 10.2169/internalmedicine.48.1578 [DOI] [PubMed] [Google Scholar]
12.van Santen S, van Dongen-Lases EC, de Vegt F, Laarakkers CM, van Riel PL, van Ede AE, et al. Hepcidin and hemoglobin content parameters in the diagnosis of iron deficiency in rheumatoid arthritis patients with anemia. Arthritis Rheum. 2011;63: 3672–3680. doi: 10.1002/art.30623 [DOI] [PubMed] [Google Scholar]
13.Sellam J, Kotti S, Fellahi S, Bastard JP, Meyer M, Liote F, et al. Serum hepcidin level is not an independent surrogate biomarker of disease activity or of radiographic progression in rheumatoid arthritis: results from the ESPOIR cohort. Ann. Rheum. Dis. 2013;72: 312–314. doi: 10.1136/annrheumdis-2012-202119 [DOI] [PubMed] [Google Scholar]
14.Kim BJ, Ahn SH, Bee SJ, Kim EH, Lee SH, Kim HK. et al. Iron overload accelerates bone loss in healthy postmenopausal women and middle-aged men: a 3-year retrospective longitudinal study. J. Bone Miner. Res. 2012;27:2279–2290. doi: 10.1002/jbmr.1692 [DOI] [PubMed] [Google Scholar]
15.Zhang P, Xu YJ, Zhao DY, Ma Y, Xiao L, Feng YS, et al. Increased intracellular iron and mineralization of cultured hFOB 1.19 cells following hepcidin activation through ferroportin-1. Saudi Medical Journal. 2010;31:1303–1308. [PubMed] [Google Scholar]
16.Xu Y, Li G, Du B, Zhang P, Xiao L, Sirois P, et al. Hepcidin increases intracellular Ca2+ of osteoblast hFOB1.19 through L-type Ca2+ channels. Regulatory Peptides. 2011;172:58–61. doi: 10.1016/j.regpep.2011.08.009 [DOI] [PubMed] [Google Scholar]
17.Li GF, Xu YJ, He YF, Du BC, Zhang P, et al. Effect of hepcidin on intracellular calcium in human osteoblasts. Molecular and Cel. Biochem. 2012;366:169. doi: 10.1007/s11010-012-1294-y [DOI] [PubMed] [Google Scholar]
18.Ghosh P, Edelman J, March L, Smith M. Effects of Pentosan Polysulfate in osteoarthritis of the knee: a randomized, double-blind, placebo-controlled pilot study. Curr. Ther. Res. 2005;66:552–571. doi: 10.1016/j.curtheres.2005.12.012 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Kumagai K, Shirabe S, Miyata N, Murata M, Yamauchi A, Kataoka Y, et al. Sodium pentosan polysulfate resulted in cartilage improvement in knee osteoarthritis—an open clinical trial. Clin. Pharmacol. 2010;10:7. doi: 10.1186/1472-6904-10-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Burkhardt D, Ghosh P. Laboratory evaluation of antiarthritic agents as potential chondroprotective agents. Semin. Arthritis Rheum. 1987;17:3–34. [PubMed] [Google Scholar]
21.Edelman J, March L, Ghosh PA. Double-blind placebo-controlled clinical study of a pleotropic osteoarthritis drug (pentosan polysulphate Cartrophen v) in 105 patients with osteoarthritis of the knee and hip joints. Osteoarthr Cartil. 1994;2:35. [Google Scholar]
22.Ghosh P, Smith M, Wells C. Second line agents in osteoarthritis. In: Dixon JS, Furst DE, editors. Second Line Agents in the Treatment of Rheumatic Diseases. New York: Marcel Dekker Inc. 1992;363–427. [Google Scholar]
23.Ghosh P. The pathobiology of osteoarthritis and the rationale for the use of pentosan polysulfate for its treatment. Semin. Arthritis Rheum. 1999;28:211–67. doi: 10.1016/s0049-0172(99)80021-3 [DOI] [PubMed] [Google Scholar]
24.Bwalya EC, Kim S, Fang J, Wijekoon HMS, Hosoya K, Okumura M. Pentosan polysulfate inhibits IL-1β-induced iNOS, c-Jun and HIF-1α upregulation in canine articular chondrocytes. PLoS One. 2017;12(5):e0177144. doi: 10.1371/journal.pone.0177144 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Wijekoon HMS, Kim S, Bwalya EC, Fang J, Aoshima K, Hosoya K, et al. Anti-arthritis effect of pentosane polysulafe in the rats with collagen induced arthritis. Res. Vet. Sci. 2019;122:179–185. doi: 10.1016/j.rvsc.2018.11.028 [DOI] [PubMed] [Google Scholar]
26.Wijekoon HMS, Bwalya EC, Kim S, Fang J, Hosoya K, Okumura M. Inhibitory effects of pentosan polysulfate on formation and function of osteoclasts derived from canine bone marrow. Journal of BMC Veterinary Research. 2018;14:152. doi: 10.1186/s12917-018-1466-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Corral DA, Amling M, Priemel M, Loyer F, Fuchs S, Ducy P, et al. Dissociation between bone resorption and bone formation in osteopenic transgenic mice. Proc. Natl. Acad. Sci. USA. 1998;95:13835–13840. doi: 10.1073/pnas.95.23.13835 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Teitelbaum SL, Ross FP. Genetic regulation of osteoclast development and function. Nat. Rev. Genet. 2003;4:638–649. doi: 10.1038/nrg1122 [DOI] [PubMed] [Google Scholar]
29.Wijekoon S, Bwalya EC, Fang J, Kim S, Hosoya K, Okumura M. Chronological differential effects of pro-inflammatory cytokines on RANKL-induced osteoclast differentiation of canine bone marrow-derived macrophages. J. Vet. Med. Sci. 2017;79(12):2030–2035. doi: 10.1292/jvms.17-0393 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423:337–342. doi: 10.1038/nature01658 [DOI] [PubMed] [Google Scholar]
31.Kim J, Kim E, Lee B, Min J, Song D, Lim J, et al. The effects of Lycii Radicis cortex on RANKL-induced osteoclast differentiation and activation in RAW 264.7 cells. Int. J. Mol. Med. 2016;37:649–658. doi: 10.3892/ijmm.2016.2477 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Gao Y, Grassi F, Ryan MR, Terauchi M, Page K, Yang X. et al. IFN-γ stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation. J. Clin. Invest. 2007;117:122–132. doi: 10.1172/JCI30074 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Li F, Chung H, Reddy SV, Lu G, Kurihara N, Zhao AZ, et al. Annexin II stimulates RANKL expression through MAPK. J. Bone Miner Res. 2005;20:1161–1167. doi: 10.1359/JBMR.050207 [DOI] [PubMed] [Google Scholar]
34.MacDonald BR, Takahashi N, McManus LM, Holahan J, Mundy GR, Roodman GD. Formation of multinucleated cells that respond to osteotropic hormones in long term human bone marrow cultures. Endocrinology. 1987;120:2326–33. doi: 10.1210/endo-120-6-2326 [DOI] [PubMed] [Google Scholar]
35.Ghosh P, Wu J, Shimmon S, Zannettino ACW, Gronthos S, Itescu S. Pentosan polysulfate promotes proliferation and chondrogenic differentiation of adult human bone marrow-derived mesenchymal precursor cells. Arthritis Res. Ther. 2010;12:28. doi: 10.1186/ar2935 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Hashizume M, Uchiyama Y, Horai N, Tomosugi N, Mihara M. Tocilizumab, a humanized anti-interleukin-6 receptor antibody, improved anemia in monkey arthritis by suppressing IL-6-induced hepcidin production. Rheumatol Int. 2010;30:917–923. doi: 10.1007/s00296-009-1075-4 [DOI] [PubMed] [Google Scholar]
37.Doyle MK, Rahman MU, Frederick B, Birbara CA, de vries D, Toedter G, et al. Effects of subcutaneous and intravenous golimumab on inflammatory biomarkers in patients with rheumatoid arthritis: results of a phase 1, randomized, open-label trial. Rheumatol. 2010;52,1214–1219. [DOI] [PubMed] [Google Scholar]
38.Das SK, Ren R, Hashimoto T, Kanazawa K. Fucoxanthin induces apoptosis in osteoclast-like cells differentiated from RAW264.7 cells. J. Agri. Food Chem. 2010;58:6090–6095. doi: 10.1021/jf100303k [DOI] [PubMed] [Google Scholar]
39.Hsu H, Lacey DL, Dunstan CR, Solovyev I, Colombero A, Timms E, et al. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc. Natl. Acad. Sci. 1999;96:3540–3545. doi: 10.1073/pnas.96.7.3540 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Jun AY, Kim HJ, Park KK, Son KH, Lee DH, Woo MH, et al. Extract of Magnoliae Flos inhibits ovariectomy-induced osteoporosis by blocking osteoclastogenesis and reducing osteoclast-mediated bone resorption. Fitoterapia. 2012;83:1523–1531. doi: 10.1016/j.fitote.2012.08.020 [DOI] [PubMed] [Google Scholar]
41.Ikeda F, Nishimura R, Matsubara T, Tanaka S, Inoue J, Reddy SV, et al. Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation. J. Clin. Invest. 2004;114:475–84. doi: 10.1172/JCI19657 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Altman R. Measurement of structural modification in osteoarthritis. Osteoarthr Cartil. 2004;12:69–76. doi: 10.1016/j.joca.2003.09.011 [DOI] [PubMed] [Google Scholar]
43.Roodman GD. Osteoclasts pump iron. Cell Metab. 2009;9:405–406. doi: 10.1016/j.cmet.2009.04.005 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0265596.r001

Decision Letter 0

Dominique Heymann

1 Dec 2021

PONE-D-21-31424Pentosan polysulfate regulates hepcidin 1-facilitated formation and function of osteoclast derived from canine bone marrowPLOS ONE

Dear Dr. Wijekoon,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact.

Please submit your revised manuscript by 20th January 2022. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Dominique Heymann, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for submitting the above manuscript to PLOS ONE. During our internal evaluation of the manuscript, we found significant text overlap between your submission and the following previously published works, some of which you are an author.

https://www.frontiersin.org/articles/10.3389/fphar.2016.00160/full

https://bmcvetres.biomedcentral.com/articles/10.1186/s12917-018-1466-4

We would like to make you aware that copying extracts from previous publications, especially outside the methods section, word-for-word is unacceptable. In addition, the reproduction of text from published reports has implications for the copyright that may apply to the publications.

Please revise the manuscript to rephrase the duplicated text, cite your sources, and provide details as to how the current manuscript advances on previous work. Please note that further consideration is dependent on the submission of a manuscript that addresses these concerns about the overlap in text with published work.

We will carefully review your manuscript upon resubmission, so please ensure that your revision is thorough

3. Thank you for stating the following financial disclosure:

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

At this time, please address the following queries:

a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.

b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

c) If any authors received a salary from any of your funders, please state which authors and which funders.

d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf

4. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

"Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors demonstrate, in vitro, that pentosan polysulfate impairs hepcidin 1-induced osteoclast formation and function. Data is nicely presented and organized.

Flaws that should be addressed by the authors include:

- the animal model choice is not clear. What is the relevance of the canine model for that study? Could not the authors have used PBMC for such purpose? Or rodent bone marrow cells?

- no statistical analysis was reported, yet the authors used asterisks in graphs. Would that indicate statistical significance diferences? Which tests were applied? Gene expression show small error bars, but no asterisks. Please indicate methods used.

- manuscript present several grammar and typos errors. Please revise.

Reviewer #2: Hepcidin, a peptide hormone released mainly by liver hepatocytes, acts as a key regulator of systematic iron homeostasis. This function is achieved by hepcidin binding to ferroportin (FPN), the iron exporter that high expressed in macrophages and intestinal cells. FPN is internalized and degraded upon binding to the hepcidin, leading to a decrease in the export of intracellular iron from macrophages and intestinal cells. Hepcidin treatment thus increases intracellular iron levels and promotes proliferation and osteoclast differentiation of RAW264.7 cells (PMID: 25059214; PMID: 34108442). In the current manuscript, Wijekoon et al., found that pentosane polysulfate (PPS), a heparin analogue, could inhibit osteoclast differentiation during hepcidin treatment. Further studies showed reduced mRNA levels in Cathepsin K, MMP9, and NFATC1, but an increase in FPN1. In contrast, immunofluorescence staining showed decreased FPN1 levels and increased iron levels in osteoclasts after hepcidin treatment; and this hepcidin's effects could be reversed by PPS treatment. These results lead to the conclusion that PPS might be beneficial for treatment of OA and osteoporosis via its inhibitory effect on hepcidin. This study is of interest to the field. However, several concerns remain to be resolved, which are listed below.

1. Hepcidin has been reported to promote the proliferation and differentiation of osteoclast (OC) precursor cells (PMID: 25059214; PMID: 34108442). In addition to inhibiting OC differentiation, does PPS treatment affect the proliferation of OC precursor cells?

2. Some experiments lack necessary control groups. PPS treatment could inhibit OC differentiation without hepcidin treatment (in Fig 2). What is the underlying mechanism? It is better to include PPS treatments at different doses (e.g., 1, 5, 10 μg/mL) without hepcidin in Fig 3 and Fig 4, so that the effect of PPS on OC differentiation and gene expression with or without hepcidin can be compared.

3. In Fig 4D, are there any significant differences in the transcription levels of MMP9, NFATc1 and FPN1? It is better to use Q-PCR here, which is more accurate than that of the reverse transcription PCR. In addition, hepcidin is known to regulate the stability of FPN1 protein. How is the transcription level of FPN1 increased after PPS treatment? Does PPS regulate FPN1 expression via a hepcidin-independent manner?

4. The published paper and the current manuscript have shown that hepcidin promotes the differentiation of OC precursor cells. In Fig 5, it is better to examine the FPN and iron levels in OC precursor cells. In Fig 5, what are the small size cells surrounding the OCs? If they are the undifferentiated bone marrow mononuclear cell (BMMs), why they did not show reduced FPN levels after hepcidin treatments? The FPN and iron levels were also not changed in these cells after PPS treatments (Fig 5D-G).

5. Western blot is a more accurate method to detect the expression level of FPN protein (or MMP9, NFATc1 and so on).

6. If PPS regulates OC differentiation via changing FPN or iron levels, it would be of interest to add iron mimic such as FAC to see whether it can diminish the effects by PPS.

7. The OCs numbers in Fig1 and Fig3 do not appear to have significant changes. It would be more convincing to show the entire well instead of the enlarged field of the view.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2022 Mar 17;17(3):e0265596. doi: 10.1371/journal.pone.0265596.r002

Author response to Decision Letter 0

19 Dec 2021

Dear Editor-in-Chief and Reviewers,

Subject: Submission of revised paper

Thank you for your email dated 1st December 2021 enclosing reviewer’s comments. We have carefully reviewed the comments and have revised the manuscript accordingly. Our responses are given in a point-by-point manner below.

Thank you for your consideration of this manuscript. We hope the revised version is now suitable for publication and look forward to hearing from you in due time regarding our submission and to respond to any further questions and comments you may have. Thank you for your consideration of this manuscript.

suranjisk@gmail.com

Sincerely,

Dr. Suranji Wijekoon (PhD., Mphill., BVSc., MSLCVS)

(Corresponding Author)

Response to reviewer 1:

Thank you for your review of our paper. We have answered each of your points below.

[In this manuscript, the authors demonstrate, in vitro, that pentosan polysulfate impairs hepcidin 1-induced osteoclast formation and function. Data is nicely presented and organized]

Thank you for the valuable comment

[The animal model choice is not clear. What is the relevance of the canine model for that study? Could not the authors have used PBMC for such purpose? Or rodent bone marrow cells]

Thank you very much for the comment.

Spontaneous inflammatory arthropathies might be a good model for human juvenile rheumatoid arthritis are available for further clinical perspectives on use of PPS. Enough amount for reliably repeatable use of bone marrow could be obtained from dogs for implication to therapeutic use of PPS to arthritis. It is known that the monocyte/macrophage lineage gives rise to osteoclast. Cells in the bone marrow are mainly precusors that upon maturation migrate out of the bone marrow and enter the circulation, hence it contains high amount of precursor cells.

[No statistical analysis was reported, yet the authors used asterisks in graphs. Would that indicate statistical significance diferences? Which tests were applied? Gene expression show small error bars, but no asterisks. Please indicate methods used]

Thank you for the valuable comment.

Data were analyzed using Statistical Package for the Social Sciences v.16 (SPSS inc., Chicago, IL, USA). Statistical significant of quantitative qPCR data, number of OC and resorption pits were determined by analysis of variance (ANOVA), comparing the mean values of the treatments. Where significant different observed, multiple comparison of group means was performed using Post Hoc Bonferroni. The results were considered significant at a 95% confidence level (p < .05). All quantitative results are presented as mean ± SE. Fig 4D indicates the gene expression levels of Quantitative qPCR data showed that PPS at 5-10 μg/mL significantly downregulate the gene expression (p<0.05) level of CTK while inhibiting expression level of MMP-9 and NFATc1 in concentration-dependent manner (figure 4D). However, the expression of FPN1 was upregulated toward the higher concentrations of PPS.

[manuscript present several grammar and typos errors. Please revise]

Thank you for the valuable comment. All the grammar and typo errors were addressed.

Response to reviewer 2:

[This study is of interest to the field. However, several concerns remain to be resolved, which are listed below.]

Thank you for the valuable comment

1. [Hepcidin has been reported to promote the proliferation and differentiation of osteoclast (OC) precursor cells (PMID: 25059214; PMID: 34108442). In addition to inhibiting OC differentiation, does PPS treatment affect the proliferation of OC precursor cells?]

Thank you very much for your question. We will briefly give the background information of effect of PPS.

We have conducted several studies to understand the PPS activity on OC differentiation, proliferation, and function. All findings were already published in reputed journals. In certain studies, PPS at concentration of 5 μg/mL exerted an inhibitory effect on canine osteoclastogenesis through suppression of key transcription factors such as NFATc1, c-Fos while visualizing co-localization patterns. This information may partially support the suggestion that PPS may exert its inhibitory effect on OC by direct interaction with transcription factors, subsequently deterring the target genes like Cathepsin K and MMP-9 which are needed in bone resorption activity of OC. To further study the effects of PPS on osteoclastogenesis, we examined whether PPS affected RANKL-induced OC function by bone resorption assays and actin formation (Functional structure of OC). The results suggested that PPS at concentrations of 1 and 5 μg/mL suppressed RANKL-induced bone resorption activity and formation of actin-rings of matured OC. The stimulation of M-CSF and RANKL make mature OC result in resorption lacunae, pit formation and actin ring formation which is a prerequisite for OC bone resorption and is the most obvious character of mature OC during osteoclastogenesis. The outcome of this study suggests that the inhibitory action of PPS over OC differentiation and function could be applied in treatment of pathological bone disorders where OC play central role. Intracellular colocalization and interaction of PPS with c-Jun transcriptional factor were observed in this study by immunofluorescence assay emphasizing that the site of action of drug of interest. Binding of c-Fos to the NFATc1 promoter is important for its activation. Suppression of NFATc1 by PPS is the consequence of the downregulation of c-Fos, with the subsequent down-regulation of AP-1 activity and attenuation of OC–specific gene expression required for efficient OC differentiation and bone resorption. Some other finding related to efficacy of PPS over the osteoclast functional unit (actin ring) is added into supplementary files. With these all finding, we concluded that PPS could inhibit the proliferative ability of OC by affecting target genes and functional units.

2. [Some experiments lack necessary control groups. PPS treatment could inhibit OC differentiation without hepcidin treatment (in Fig 2). What is the underlying mechanism? It is better to include PPS treatments at different doses (e.g., 1, 5, 10 μg/mL) without hepcidin in Fig 3 and Fig 4, so that the effect of PPS on OC differentiation and gene expression with or without hepcidin can be compared.]

Thank you very much for the comments and valuable suggestions. However, we have already demonstrated the effect of PPS on OC formation and functions. And those were already published. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930774/. Briefly, we are explaining the underlying mechanism of OC inhibition by PPS. Intracellular colocalization and interaction of PPS with c-Jun transcriptional factor were observed in our previous study by immunofluorescence assay emphasizing that the site of action of drug of interest. Binding of c-Fos to the NFATc1 promoter is important for its activation. Suppression of NFATc1 by PPS is the consequence of the down-regulation of c-Fos, with the subsequent down-regulation of activator protein 1 transcription factor (AP-1) activity and attenuation of OC–specific gene expression required for efficient OC differentiation and bone resorption. Further extension of the study up to detailed work by evaluating specific binding affinity of PPS with specific protein at nuclear, sub nuclear domain or nuclear speckles in OC would be much awarded the PPS as therapeutic perspective.

To avoid the repetition of those findings, we displayed the effect of PPS in different doses (e.g., 1, 5, 10 μg/mL) without hepcidin in fig 2. We have sited previous experiments in this regard to avoid the confusion of readers. And compared the current results with previous findings comparing all most all the aspects. But thank you again for pointing this out.

Thank you for the valuable comments and agreed with your statement.

In figure legend that was mentioned as RT PCR, but it was correctly motioned in material and method. That was corrected and rephased. Thank you. All quantitative results are presented as mean ± SE. Fig 4D indicates the gene expression levels of Quantitative qPCR data showed that PPS at 5-10 μg/mL significantly downregulate the gene expression (p<0.05) level of CTK while inhibiting expression level of MMP-9 and NFATc1 in concentration-dependent manner (figure 4D). However, the expression of FPN1 was upregulated toward the higher concentrations of PPS.

4. [The published paper and the current manuscript have shown that hepcidin promotes the differentiation of OC precursor cells. In Fig 5, it is better to examine the FPN and iron levels in OC precursor cells. In Fig 5, what are the small size cells surrounding the OCs? If they are the undifferentiated bone marrow mononuclear cell (BMMs), why they did not show reduced FPN levels after hepcidin treatments? The FPN and iron levels were also not changed in these cells after PPS treatments (Fig 5D-G).]

Thank you for the comment and suggestions for advancement of future experiments.

5.[Western blot is a more accurate method to detect the expression level of FPN protein (or MMP9, NFATc1 and so on).]

Thank you for the suggestion and agreed with it. We definitely include those additional tests to our future experiments to continue this study to further clarify the mode of action of PPS with regards to the iron metabolism in high demanding cells.

6.[If PPS regulates OC differentiation via changing FPN or iron levels, it would be of interest to add iron mimic such as FAC to see whether it can diminish the effects by PPS.]

Thank you for the comment.

It very true and we were initially planned to add iron mimic to see how PPS works on it. However, we have already identified and confirmed the efficacy of PPS over the osteoclast genesis and function of OC. Here we more focused on the hepcidin facilitated OC formation and how PPS react over it. This current data very clearly emphasized the ability of PPS on regulating the hepcidin induced OC formation and function. With those fundamentals we will move further to understand the detailed molecular aspects of PPS effect on iron metabolism.

7. [The OCs numbers in Fig1 and Fig3 do not appear to have significant changes. It would be more convincing to show the entire well instead of the enlarged field of the view.]

Thank you for the comment and agreed well with it.

We have changed Fig 1 and Fig 3 according to the suggestion. Most profusely Fig 3 was changed adding entire well.

Thank you very much for very constructive and supportive feedback to improve the current layout and to advance the research into the next level in future studies.

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file.^{(56KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0265596.r003

Decision Letter 1

Dominique Heymann

21 Jan 2022

PONE-D-21-31424R1

Pentosan polysulfate regulates hepcidin 1-facilitated formation and function of osteoclast derived from canine bone marrow

PLOS ONE

Dear Dr. Wikekoon,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we have decided that your manuscript does not meet our criteria for publication and must therefore be rejected.

Specifically:

Several key concerns are not fully addressed. As specifiied by the reviewer 2, the revised manuscript only shows ferriportin (FPN)(a receptor of hepcidin) expression at transcriptional level, but not at protein level (Fig 4). The undifferentiated bone marrow mononuclear cells (BMMs or osteoclast precursor cells) do not show reduced FPN levels after hepcidin treatment (Fig 5).

More important and that is a big issue, ame images appear duplicated and used in different figures. The “PPS 10 μg/mL” group in Fig 2 is similar to “Hepcidin 800 nmol/L + PPS 1 μg/mL” group in Fig 3, and the “PPS 20 μg/mL” group in Fig 2 is similar to “Hepcidin 800 nmol/L + PPS 5 μg/mL” group in Fig 3. It is then extremely complicated to determine which are the true data.

I am sorry that we cannot be more positive on this occasion, but hope that you appreciate the reasons for this decision.Yours sincerely,

Dominique Heymann, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have provided answers to all questions and made the changes in the manuscript accordingly.

Reviewer #2: The revised manuscript has addressed some, but not all of the concerns raised previously. Several key concerns are not fully addressed/ For examples, the revised manuscript only showed ferriportin (FPN)(a receptor of hepcidin) expression at transcriptional level, but not protein level (Fig 4). The undifferentiated bone marrow mononuclear cells (BMMs or osteoclast precursor cells) did not shown reduced FPN levels after hepcidin treatment (Fig 5). Additionally, same images appeared to be used in different figures. The “PPS 10 μg/mL” group in Fig 2 appeared to be same as the “Hepcidin 800 nmol/L + PPS 1 μg/mL” group in Fig 3, and the “PPS 20 μg/mL” group in Fig 2 appeared to be same data as the “Hepcidin 800 nmol/L + PPS 5 μg/mL” group in Fig 3. These issues may result in an incorrect quantification and conclusion.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

- - - - -

For journal use only: PONEDEC3

PLoS One. 2022 Mar 17;17(3):e0265596. doi: 10.1371/journal.pone.0265596.r004

Author response to Decision Letter 1

17 Feb 2022

H.M. Suranji Wijekoon

Graduate School of Veterinary Medicine

Department of Veterinary Clinical Sciences

Laboratory of Veterinary Surgery

Kita 18, Nishi 9, Kita-Ku, (060-0818), Sapporo

Hokkaido, Japan

31.01.2021

Dear Editor-in-Chief,

Subject: Submission of revised paper

Thank you for your email dated 1st December 2021 enclosing reviewer’s comment and 26th dated information requested after considering appeal. We have carefully reviewed the comments and have revised the manuscript accordingly. Our responses are given in a point-by-point manner below for the concerns raised by the reviewers and Academic Editor and details on the revisions carried out on the manuscript since its original submission.

Data Availability:

All relevant data are within the paper and its supporting information files.

Enclosed is a manuscript to be considered for publication in PLOSONE. The research reported in this manuscript has been ethically clearance with obtaining approval from Graduate School of Veterinary Medicine, Hokkaido University (approval No 21-0022) and no financial or personal conflicts of interest.

We confirm that this work is original and has not been published elsewhere nor is it currently under consideration for publication elsewhere.

suranjisk@gmail.com

Sincerely,

Dr. Suranji Wijekoon (PhD., Mphill., BVSc., MSLCVS)

(Corresponding Author)

Response to reviewer 1:

Thank you for your review of our paper. We have answered each of your points below.

[In this manuscript, the authors demonstrate, in vitro, that pentosan polysulfate impairs hepcidin 1-induced osteoclast formation and function. Data is nicely presented and organized]

Thank you for the valuable comment

[The animal model choice is not clear. What is the relevance of the canine model for that study? Could not the authors have used PBMC for such purpose? Or rodent bone marrow cells]

Thank you very much for the comment.

Thank you for the valuable comment.

[manuscript present several grammar and typos errors. Please revise]

Thank you for the valuable comment. All the grammar and typo errors were addressed.

Response to reviewer 2:

[This study is of interest to the field. However, several concerns remain to be resolved, which are listed below.]

Thank you for the valuable comment. We believe that we have addressed almost all the concerns with very detailed explanation, rewrite the certain part of manuscript agreeing all most all the valuable comments and further clarified the few concerns by elucidating future study expansion based on current findings.

Thank you very much for your question. We will briefly give the background information of effect of PPS.

Thank you for the valuable comments and agreed with your statement.

Thank you for the comment and suggestions for advancement of future experiments.

However, in this study we basically focused on how the PPS act on Hepcidin treated OC at the differentiating and matured stages where they are actively involved in bone resorption in osteoporosis, osteoarthritis, and rheumatoid arthritis. Small cells surrounding the OC are undifferentiated mononuclear cells which remained in the culture slides even after the 7 days. If we compare those mononuclear cells to mature OC, we can’t see very clear FPN changes. But if we carefully see those small cells in the Fig 5.CD, fluorescence intensities are slightly increased combined with PPS treatment. Interesting point in this juncture is selectivity of treatment efficacy of PPS which profusely shows the therapeutic application against OC among other cells. Those mature OC occupy a large area with several nuclei and cells are massive to visualize the changes compared to tiny cells. However, as you suggested it`s a very valuable point to look for the FPN and iron changes quantitatively within OC precursor cells. On the other hand undifferentiated bone marrow mononuclear cells (BMMs or osteoclast precursor cells) do not show reduced FPN levels after hepcidin treatment could be the true factor hence BMM cells might have comparatively less hepcidin-induced FPN1 internalization and degradation compared to mature osteoclasts. We also don’t know. We believe that it could be due to different level of metabolic capacity in the cells. And those undifferentiated cells need different time frame to react on those compounds. Osteoclasts are highly active cell types which carries higher grade metabolic activity rate compared to other cells. Those all novel findings will be addressed again with future progressive study and will apply protein work on top of that.

5.[Western blot is a more accurate method to detect the expression level of FPN protein (or MMP9, NFATc1 and so on).]

6.[If PPS regulates OC differentiation via changing FPN or iron levels, it would be of interest to add iron mimic such as FAC to see whether it can diminish the effects by PPS.]

Thank you for the comment.

7. [The OCs numbers in Fig1 and Fig3 do not appear to have significant changes. It would be more convincing to show the entire well instead of the enlarged field of the view.]

Thank you for the comment and agreed well with it.

We have changed the figure.

Thank you very much for very constructive and supportive feedback to improve the current layout and to advance the research into the next level in future studies.

Appeal

Dear Editor,

Receive the decision. Thank you very much.

However I just want to clarify certain points regarding the facts you have considered to reject the following manuscript. Please kindly consider the explanation. Thank you for understanding.

After reading the clarification we believe you will reconsider the decision OR you will just keep this in a side; however I respect the final decision whatever you will take.

Please ask for further clarification if needed. Thank you for considering our paper.

PONE-D-21-31424R1

Pentosan polysulfate regulates hepcidin 1-facilitated formation and function of osteoclast derived from canine bone marrow

PLOS ONE

Reviewer #1: All comments have been addressed

Reviewer #2: Raised few concerns. We have carefully reviewed the comments again and we identified one mistake in figure, happened in second submission when we responding the first revision. And other concern of Reviewer 2 was addressed, and we have responded in submission of revision. However, reviewer# 2 has mentioned that we haven’t addressed two points but accepted intelligible fashion of writing and written in standard English.

Our responses are given in a point-by-point manner below for the two concerns raised by reviewer after we submitted the revision addressing totally 12 major issues in well manner.

Specifically:

1- As specifiied by the reviewer 2, the revised manuscript only shows ferriportin (FPN)(a receptor of hepcidin) expression at transcriptional level, but not at protein level (Fig 4). The undifferentiated bone marrow mononuclear cells (BMMs or osteoclast precursor cells) do not show reduced FPN levels after hepcidin treatment (Fig 5).

Clarification;

We have addressed all the key concerns of Reviewer 2 and however we respect this statement. In that response we have mentioned very clearly that ferriportin (FPN)(a receptor of hepcidin) expression in protein level was not analyzed in this study; however we are planning to add that technique together in the future work. Because the current study took place to explain the fundamentals of the PPS effect in cell culture level using changes of differentiation and functional changes associated with osteoclasts. Specific gene expression level for Osteoclasts were examined to understanding how the PPS affect those cells in transcriptional level. We have understood that the importance of protein level study but that will be included in our next step of the research for deeper understanding of the molecular aspects. As this is a very initial finding of the field of veterinary aspect additional steps will be taken progressively. We believe that current presentation of data would be a good platform to initiate protein level work as we clearly mentioned and submitted on previous submission of revision 1 `Response to reviewer` document.

Further, undifferentiated bone marrow mononuclear cells (BMMs or osteoclast precursor cells) do not show reduced FPN levels after hepcidin treatment could be the true factor hence BMM cells might have comparatively less hepcidin-induced FPN1 internalization and degradation compared to mature osteoclasts. We also don’t know. We believe that it could be due to different level of metabolic capacity in the cells. And those undifferentiated cells need different time frame to react on those compounds. Osteoclasts are highly active cell types which carries higher grade metabolic activity rate compared to other cells. Those all novel findings will be addressed again with future progressive study and will apply protein work on top of that. We have given such a very clear explanation to Reviewer 2 and we wondered how it ended with the same comment. All other concerns of reviewer 2 were well addressed according to his statements.

2- More important and that is a big issue, ame images appear duplicated and used in different figures. The “PPS 10 μg/mL” group in Fig 2 is similar to “Hepcidin 800 nmol/L + PPS 1 μg/mL” group in Fig 3, and the “PPS 20 μg/mL” group in Fig 2 is similar to “Hepcidin 800 nmol/L + PPS 5 μg/mL” group in Fig 3. It is then extremely complicated to determine which is the true data.

Clarification;

If you carefully reobserve our first submission before revision you will understand that the image issue is a mistake that happened while preparing images again for revision 1. Because that duplication was not there at the first submission and we are not in the level of making such a way unless there is a mistake. Hereby I again request to RECONSIDER that matter and hope you will understand the issue. Kindly recheck the first submission docs which you used to send to reviewer for their comments. Images were duplicated while revision.

Thank you very much for very constructive and supportive feedback to improve the current layout and to advance the research into the next level in future studies.

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file.^{(64KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0265596.r005

Decision Letter 2

Dominique Heymann

7 Mar 2022

Pentosan polysulfate regulates hepcidin 1-facilitated formation and function of osteoclast derived from canine bone marrow

PONE-D-21-31424R2

Dear Dr. Dr Wijekoon,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Dominique Heymann, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have provided answers to all questions and made the changes in the manuscript accordingly.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

PLoS One. doi: 10.1371/journal.pone.0265596.r006

Acceptance letter

Dominique Heymann

9 Mar 2022

PONE-D-21-31424R2

Pentosan polysulfate regulates hepcidin 1-facilitated formation and function of osteoclast derived from canine bone marrow

Dear Dr. Wijekoon:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Pr. Dominique Heymann

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Osteoclasts actin ring formation (Rhodamin Phalloidin) was monitored after treatment of PPS with different concentartions.

(TIFF)

Click here for additional data file.^{(8.9MB, tiff)}

S1 Table. Primer sequences used to polymerize the osteoclast specific genes.

(DOCX)

Click here for additional data file.^{(15.7KB, docx)}

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file.^{(56KB, doc)}

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file.^{(64KB, doc)}

Data Availability Statement

Data Availability: All relevant data are within the paper and its Supporting information files.

[pone.0265596.ref001] 1.Zhao G, Di D, Wang B, Huang X, Xu Y. Effects of mouse hepcidin 1 treatment on osteoclast differentiation and intracellular iron concentration. Inflammation. 2015; 38:718–727. doi: 10.1007/s10753-014-9982-2 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref002] 2.Krause A, Neitz S, Magert HJ, Schulz A, Forssmann WG, Schulz-Knappe P et al. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett. 2000;480:147–150. doi: 10.1016/s0014-5793(00)01920-7 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref003] 3.Drakesmith H, Nemeth E, Ganz T. Iron out Ferroportin. Cell Metab. 2015;22:777–787. doi: 10.1016/j.cmet.2015.09.006 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265596.ref004] 4.Ganz T. Molecular pathogenesis of anemia of chronic disease. Pediatr Blood Cancer. 2006;46:554–557. doi: 10.1002/pbc.20656 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref005] 5.Viatte L, Vaulont S. Hepcidin, the iron watcher. Biochimie.2016;91:1223–1228. doi: 10.1016/j.biochi.2009.06.012 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref006] 6.Ashby DR, Gale DP, Busbridge M, Murphy KG, Duncan ND, Cairns TD, et al. Plasma hepcidin levels are elevated but responsive to erythropoietin therapy in renal disease. Kidney Int. 2009;75:976–981. doi: 10.1038/ki.2009.21 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref007] 7.Macdougall IC, Malyszko J, Hider RC, Bansal SS. Current status of the measurement of blood hepcidin levels in chronic kidney disease. Clin. J. Am. Soc. Nephrol. 2010;5:1681–1689. doi: 10.2215/CJN.05990809 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref008] 8.Weiss G. Anemia of chronic disorders: new diagnostic tools and new treatment strategies. Semin. Hematol. 2015;52:313–320. doi: 10.1053/j.seminhematol.2015.07.004 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref009] 9.Wang CY, Babitt JL. Hepcidin regulation in the anemia of inflammation. Curr. Opin. Hematol. 2016;23:189–197. doi: 10.1097/MOH.0000000000000236 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265596.ref010] 10.Ludwiczek S, Aigner E, Theurl I, Weiss G. Cytokine-mediated regulation of iron transport in human monocytic cells. Blood. 2003;101:4148–4154. doi: 10.1182/blood-2002-08-2459 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref011] 11.Demirag MD, Haznedaroglu S, Sancak B, Konca C, Gulbahar O, Ozturk M.A, et al. Circulating hepcidin in the crossroads of anemia and inflammation associated with rheumatoid arthritis. Intern. Med. 2009;48, 421–426. doi: 10.2169/internalmedicine.48.1578 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref012] 12.van Santen S, van Dongen-Lases EC, de Vegt F, Laarakkers CM, van Riel PL, van Ede AE, et al. Hepcidin and hemoglobin content parameters in the diagnosis of iron deficiency in rheumatoid arthritis patients with anemia. Arthritis Rheum. 2011;63: 3672–3680. doi: 10.1002/art.30623 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref013] 13.Sellam J, Kotti S, Fellahi S, Bastard JP, Meyer M, Liote F, et al. Serum hepcidin level is not an independent surrogate biomarker of disease activity or of radiographic progression in rheumatoid arthritis: results from the ESPOIR cohort. Ann. Rheum. Dis. 2013;72: 312–314. doi: 10.1136/annrheumdis-2012-202119 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref014] 14.Kim BJ, Ahn SH, Bee SJ, Kim EH, Lee SH, Kim HK. et al. Iron overload accelerates bone loss in healthy postmenopausal women and middle-aged men: a 3-year retrospective longitudinal study. J. Bone Miner. Res. 2012;27:2279–2290. doi: 10.1002/jbmr.1692 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref015] 15.Zhang P, Xu YJ, Zhao DY, Ma Y, Xiao L, Feng YS, et al. Increased intracellular iron and mineralization of cultured hFOB 1.19 cells following hepcidin activation through ferroportin-1. Saudi Medical Journal. 2010;31:1303–1308. [PubMed] [Google Scholar]

[pone.0265596.ref016] 16.Xu Y, Li G, Du B, Zhang P, Xiao L, Sirois P, et al. Hepcidin increases intracellular Ca2+ of osteoblast hFOB1.19 through L-type Ca2+ channels. Regulatory Peptides. 2011;172:58–61. doi: 10.1016/j.regpep.2011.08.009 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref017] 17.Li GF, Xu YJ, He YF, Du BC, Zhang P, et al. Effect of hepcidin on intracellular calcium in human osteoblasts. Molecular and Cel. Biochem. 2012;366:169. doi: 10.1007/s11010-012-1294-y [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref018] 18.Ghosh P, Edelman J, March L, Smith M. Effects of Pentosan Polysulfate in osteoarthritis of the knee: a randomized, double-blind, placebo-controlled pilot study. Curr. Ther. Res. 2005;66:552–571. doi: 10.1016/j.curtheres.2005.12.012 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265596.ref019] 19.Kumagai K, Shirabe S, Miyata N, Murata M, Yamauchi A, Kataoka Y, et al. Sodium pentosan polysulfate resulted in cartilage improvement in knee osteoarthritis—an open clinical trial. Clin. Pharmacol. 2010;10:7. doi: 10.1186/1472-6904-10-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265596.ref020] 20.Burkhardt D, Ghosh P. Laboratory evaluation of antiarthritic agents as potential chondroprotective agents. Semin. Arthritis Rheum. 1987;17:3–34. [PubMed] [Google Scholar]

[pone.0265596.ref021] 21.Edelman J, March L, Ghosh PA. Double-blind placebo-controlled clinical study of a pleotropic osteoarthritis drug (pentosan polysulphate Cartrophen v) in 105 patients with osteoarthritis of the knee and hip joints. Osteoarthr Cartil. 1994;2:35. [Google Scholar]

[pone.0265596.ref022] 22.Ghosh P, Smith M, Wells C. Second line agents in osteoarthritis. In: Dixon JS, Furst DE, editors. Second Line Agents in the Treatment of Rheumatic Diseases. New York: Marcel Dekker Inc. 1992;363–427. [Google Scholar]

[pone.0265596.ref023] 23.Ghosh P. The pathobiology of osteoarthritis and the rationale for the use of pentosan polysulfate for its treatment. Semin. Arthritis Rheum. 1999;28:211–67. doi: 10.1016/s0049-0172(99)80021-3 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref024] 24.Bwalya EC, Kim S, Fang J, Wijekoon HMS, Hosoya K, Okumura M. Pentosan polysulfate inhibits IL-1β-induced iNOS, c-Jun and HIF-1α upregulation in canine articular chondrocytes. PLoS One. 2017;12(5):e0177144. doi: 10.1371/journal.pone.0177144 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265596.ref025] 25.Wijekoon HMS, Kim S, Bwalya EC, Fang J, Aoshima K, Hosoya K, et al. Anti-arthritis effect of pentosane polysulafe in the rats with collagen induced arthritis. Res. Vet. Sci. 2019;122:179–185. doi: 10.1016/j.rvsc.2018.11.028 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref026] 26.Wijekoon HMS, Bwalya EC, Kim S, Fang J, Hosoya K, Okumura M. Inhibitory effects of pentosan polysulfate on formation and function of osteoclasts derived from canine bone marrow. Journal of BMC Veterinary Research. 2018;14:152. doi: 10.1186/s12917-018-1466-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265596.ref027] 27.Corral DA, Amling M, Priemel M, Loyer F, Fuchs S, Ducy P, et al. Dissociation between bone resorption and bone formation in osteopenic transgenic mice. Proc. Natl. Acad. Sci. USA. 1998;95:13835–13840. doi: 10.1073/pnas.95.23.13835 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265596.ref028] 28.Teitelbaum SL, Ross FP. Genetic regulation of osteoclast development and function. Nat. Rev. Genet. 2003;4:638–649. doi: 10.1038/nrg1122 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref029] 29.Wijekoon S, Bwalya EC, Fang J, Kim S, Hosoya K, Okumura M. Chronological differential effects of pro-inflammatory cytokines on RANKL-induced osteoclast differentiation of canine bone marrow-derived macrophages. J. Vet. Med. Sci. 2017;79(12):2030–2035. doi: 10.1292/jvms.17-0393 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265596.ref030] 30.Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423:337–342. doi: 10.1038/nature01658 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref031] 31.Kim J, Kim E, Lee B, Min J, Song D, Lim J, et al. The effects of Lycii Radicis cortex on RANKL-induced osteoclast differentiation and activation in RAW 264.7 cells. Int. J. Mol. Med. 2016;37:649–658. doi: 10.3892/ijmm.2016.2477 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265596.ref032] 32.Gao Y, Grassi F, Ryan MR, Terauchi M, Page K, Yang X. et al. IFN-γ stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation. J. Clin. Invest. 2007;117:122–132. doi: 10.1172/JCI30074 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265596.ref033] 33.Li F, Chung H, Reddy SV, Lu G, Kurihara N, Zhao AZ, et al. Annexin II stimulates RANKL expression through MAPK. J. Bone Miner Res. 2005;20:1161–1167. doi: 10.1359/JBMR.050207 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref034] 34.MacDonald BR, Takahashi N, McManus LM, Holahan J, Mundy GR, Roodman GD. Formation of multinucleated cells that respond to osteotropic hormones in long term human bone marrow cultures. Endocrinology. 1987;120:2326–33. doi: 10.1210/endo-120-6-2326 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref035] 35.Ghosh P, Wu J, Shimmon S, Zannettino ACW, Gronthos S, Itescu S. Pentosan polysulfate promotes proliferation and chondrogenic differentiation of adult human bone marrow-derived mesenchymal precursor cells. Arthritis Res. Ther. 2010;12:28. doi: 10.1186/ar2935 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265596.ref036] 36.Hashizume M, Uchiyama Y, Horai N, Tomosugi N, Mihara M. Tocilizumab, a humanized anti-interleukin-6 receptor antibody, improved anemia in monkey arthritis by suppressing IL-6-induced hepcidin production. Rheumatol Int. 2010;30:917–923. doi: 10.1007/s00296-009-1075-4 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref037] 37.Doyle MK, Rahman MU, Frederick B, Birbara CA, de vries D, Toedter G, et al. Effects of subcutaneous and intravenous golimumab on inflammatory biomarkers in patients with rheumatoid arthritis: results of a phase 1, randomized, open-label trial. Rheumatol. 2010;52,1214–1219. [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref038] 38.Das SK, Ren R, Hashimoto T, Kanazawa K. Fucoxanthin induces apoptosis in osteoclast-like cells differentiated from RAW264.7 cells. J. Agri. Food Chem. 2010;58:6090–6095. doi: 10.1021/jf100303k [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref039] 39.Hsu H, Lacey DL, Dunstan CR, Solovyev I, Colombero A, Timms E, et al. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc. Natl. Acad. Sci. 1999;96:3540–3545. doi: 10.1073/pnas.96.7.3540 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265596.ref040] 40.Jun AY, Kim HJ, Park KK, Son KH, Lee DH, Woo MH, et al. Extract of Magnoliae Flos inhibits ovariectomy-induced osteoporosis by blocking osteoclastogenesis and reducing osteoclast-mediated bone resorption. Fitoterapia. 2012;83:1523–1531. doi: 10.1016/j.fitote.2012.08.020 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref041] 41.Ikeda F, Nishimura R, Matsubara T, Tanaka S, Inoue J, Reddy SV, et al. Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation. J. Clin. Invest. 2004;114:475–84. doi: 10.1172/JCI19657 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0265596.ref042] 42.Altman R. Measurement of structural modification in osteoarthritis. Osteoarthr Cartil. 2004;12:69–76. doi: 10.1016/j.joca.2003.09.011 [DOI] [PubMed] [Google Scholar]

[pone.0265596.ref043] 43.Roodman GD. Osteoclasts pump iron. Cell Metab. 2009;9:405–406. doi: 10.1016/j.cmet.2009.04.005 [DOI] [PubMed] [Google Scholar]

PERMALINK

Pentosan polysulfate regulates hepcidin 1-facilitated formation and function of osteoclast derived from canine bone marrow

Suranji Wijekoon

Takafumi Sunaga

Yanlin Wang

Carol Mwale

Sangho Kim

Masahiro Okumura

Roles

Abstract

Introduction

Material and methods

Preparation of sample collection site

Osteoclastic differentiation from canine bone marrow

Tartrate-resistant acid phosphate (TRAP) staining

Resorption pit formation assay

Preparation of cell extracts and analysis

Immunocytochemical detection of ferroportin 1 (FPN 1)

Immunofluorescent analysis of intracellular iron concentration

Data analysis

Results

Hepcidin 1 facilitate M-CSF and RANKL-induced osteoclastogenesis

Fig 1. Shows effect of hepcidin 1 on canine OC differentiation.

PPS inhibits osteoclastogenesis

Fig 2. Shows inhibitory effect of PPS on canine OC differentiation.

PPS inhibits hepcidin-facilitated OC formation and function in dose dependent manner

Fig 3. Inhibitory effect of PPS on OC treated with hepcidin 1.

Fig 4. PPS inhibits bone resorption and OC specific genes.

PPS inhibits the hepcidin-induced FPN1 internalization and degradation

Fig 5. Immunofluorescence images.

PPS repressed the hepcidin-induced intracellular iron accumulation

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Dominique Heymann

Roles

Author response to Decision Letter 0

Decision Letter 1

Dominique Heymann

Roles

Author response to Decision Letter 1

Decision Letter 2

Dominique Heymann

Roles

Acceptance letter

Dominique Heymann

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases