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. Author manuscript; available in PMC: 2017 Jun 1.
Published in final edited form as: Osteoarthritis Cartilage. 2016 Jan 8;24(6):1036–1046. doi: 10.1016/j.joca.2015.12.017

Reduced Response of Human Meniscal Cells to Osteogenic Protein 1 during Osteoarthritis and Pro-inflammatory Stimulation

Kadie S Vanderman 1, Richard F Loeser 2, Susan Chubinskaya 3, Andrea Anderson 4, Cristin M Ferguson 1
PMCID: PMC4875791  NIHMSID: NIHMS758240  PMID: 26778533

Abstract

Objective

Many cell types lose responsiveness to anabolic factors during inflammation and disease. Osteogenic Protein 1 (OP1/BMP7) was evaluated for the ability to enhance extracellular matrix synthesis in healthy and OA meniscus cells. Mechanisms of cell response to OP1 were explored.

Design

Meniscus and cartilage tissues from healthy tissue donors and osteoarthritis patients undergoing total knee arthroplasties were acquired. Primary cell cultures were stimulated with OP1 and/or inflammatory factors (IL1α, IL1β, or fibronectin fragments (FnF)) and cellular responses were analyzed by RT-qPCR and immunoblots. Frozen section immunohistochemistry was conducted to assess OP1 and receptor proteins in normal and OA meniscus.

Results

OP1 treatment of normal meniscus cells resulted in significant, dose-dependent increases in ACAN (aggrecan) and COL2A1, and decreased MMP13 gene transcription, while only ACAN was upregulated (p<0.01) at the highest dose of OP1 in OA meniscus cells. OP1 induced significantly more ACAN gene transcription in normal meniscus than normal articular cartilage (p=0.05), and no differences between normal and OA cartilage were detected. Receptor expression and kinetics of canonical signaling activation were similar between normal and OA specimens. Normal meniscus cells treated with inflammatory factors were refractory to OP1 stimulation. Smad1 phosphorylation at an inhibitory site was induced (p=0.01 for both normal and OA meniscus) by inflammatory cytokine treatment.

Conclusions

The meniscus demonstrates resistance to OP1 stimulation in OA and in the presence of inflammatory mediators. MAPK-mediated Smad1 linker phosphorylation is a possible mediator of the loss of anabolic extracellular matrix production in the inflammatory cytokine affected meniscus.

Keywords: meniscus, osteoarthritis, OP1, BMP, extracellular matrix, cytokine

Introduction

The meniscus is a crescent-shaped fibrocartilaginous tissue that provides important biomechanical and biological functions in the knee such as supporting joint stability, reducing joint contact stress upon the articular cartilage, and contributing biologically to joint nutrition, lubrication, and proprioception [1]. The meniscus is frequently injured and not easily repaired, particularly in the inner region which lacks vascularization and retains more cartilage-like properties [2]. Approximately 35% of the general population between 19–50 years of age has meniscus tears identified by MRI, with incidence increasing with age [3], and up to 91% in symptomatic osteoarthritis (OA) patients [4]. Following meniscus injury, OA is often induced where changes to the articular cartilage structure become apparent within several years [5]. Approximately 50% of patients with tears treated by meniscectomy have radiographic OA within 16 years [6]. This suggests that the meniscus is an important target in studies of OA.

OA is a process of lost tissue homeostasis and abnormal remodeling, in which catabolic breakdown exceeds matrix synthesis in a process driven by inflammatory mediators within the affected joint [7, 8]. Consistent with other OA affected tissues, injured menisci produce inflammatory factors and matrix metalloproteases which enhance meniscus degeneration and OA progression [9–12]. Although it has not been studied in the meniscus, it is likely that inflammatory cytokines released from the injured meniscus also inhibit anabolic factors associated with matrix synthesis. Retention of meniscus extracellular matrix following meniscus injury and catabolic factor release is likely to slow or prevent OA progression [9–12].

Osteogenic Protein 1 (OP1/BMP7), a Bone Morphogenetic Protein (BMP) and Transforming Growth Factor β (TGFβ) family member, is a candidate for stimulating meniscus matrix synthesis. OP1 stimulates matrix synthesis in cells of related tissues such as articular cartilage, ligament, and bone. Treatment of articular cartilage with OP1 both in vitro and in vivo is chondroprotective and enhances anabolic matrix synthesis and proteoglycan production without inducing abnormal anabolism, such as osteophyte formation [13]. OP1 is also anti-catabolic in articular cartilage, as treatment prevents matrix metalloprotease stimulation by modulating the effect of inflammatory and catabolic mediators such as Interleukin 1β (IL1β) and fragments of the matrix protein fibronectin (FnF) [13]. Research has shown that the animal meniscus responds to OP1 with enhanced proteoglycan and collagen production [14–16]. However, no studies have investigated OP1 effects on the human meniscus using tissue from normal and OA joints. Information about the responsiveness to OP1 is needed if it is to be considered as a potential therapeutic intervention.

OP1 signaling occurs via a common signaling pathway characteristic of BMP family members. Following binding to a receptor complex, specific type I receptors (ALK-2, -3, or -6) transduce the signal by phosphorylating the C-terminus SVS motif at Ser463/Ser465 of intracellular targets Smads 1, 5, and 8 [17, 18]. A conformation change of Smad1/5/8 opens a binding site for the Smad4 cofactor. The Smad1/5/8 and Smad4 complex then translocate to the nucleus to interact with transcription factors on promoter elements of target genes. [19].

The objective of this study was to evaluate the ability of OP1 to stimulate meniscus matrix synthesis in cells derived from normal and OA meniscus tissue. Identification of a decreased response to OP1 in cells from OA joints led us to explore potential mechanisms for resistance to OP1 in OA meniscus cells.

Methods

Cell Culture

Normal human menisci and femoral articular cartilage (28–82 years, mean= 67) were obtained from organ donors through the Gift of Hope Organ and Tissue Donor Network (Itasca, IL), or from the National Disease Research Interchange (NDRI, Philadelphia, PA). Normal tissue had a score of 0–2 within the International Cartilage Research Society Cartilage Morphology Score (ICRS) or a meniscus modified ICRS which ranged from 0–4 [9]. OA meniscus and/or cartilage (54–74 years, mean=65) were obtained as discarded tissue from total knee arthroplasties performed at Wake Forest Baptist Medical Center (Winston-Salem, NC). Human donor tissue research was approved by the IRBs at Rush University and Wake Forest University School of Medicine. All comparisons between meniscus and articular cartilage were from the same knee and therefore were donor-matched samples (n=14 total matched donors). In experiments using inner vs outer meniscus regions, the inner 1/3 of the meniscus was excised and processed separately from the outer region. Tissue was digested and cells were harvested as described previously [9]. Primary meniscus cells and articular chondrocytes were grown as high density monolayer cultures with 10% serum in DMEM media (Gibco) until confluent, changed to mini-ITS or serum free media upon reaching confluence. Cells were treated with OP1 (25–500 ng/mL for initial dose response and 100 ng/mL used for all subsequent experiments, Stryker Biotech). In signaling experiments, OP-1 stimulation was compared to IL1α, IL1β (10 ng/mL, R&D Systems), TGFβ1, IGF1 (100 ng/mL, R&D Systems), GDF5 (100 ng/mL, Prospec), or FnF (1 μM), a recombinant fragment of fibronectin protein containing domains 7–10 of full length fibronectin [20] for 24 hours or as indicated. These factors were chosen for comparison to OP1 because IL1α, IL1β, and FnF are known to stimulate MAPK signaling, TGFβ stimulates Smad2 signaling, IGF1 stimulates AKT activation, and GDF5 stimulates Smad1/5/8 signaling. All treatment doses were selected to be within biologically relevant ranges of those found in synovial fluid or joint tissue except IL1α and IL1β, for which doses used were selected based on previous studies examining their role in mediating catabolic signaling [13, 21].

Quantitative Real-time PCR

Total RNA was extracted using TRIzol (Invitrogen) and concentrations were determined by a nanodrop 1000 spectrophotometer (Thermo Scientific). Total RNA was used to synthesize cDNA (Retroscript RT kit) and RT-qPCR amplification was performed on a 7900HT Fast Real-Time PCR System with Taqman Mastermix and manufacturer recommended primer-probe sets (Applied Biosystems). All data were normalized to the endogenous control gene TATA box-binding protein (TBP) measured in parallel samples and calculated using the 2-ΔΔct method. In preliminary studies we have found TBP to be more reliable than GAPDH or other “housekeeping” genes as a constitutively expressed control in meniscus cells. Graphs showing Normalized Expression reflect this calculation. Graphs showing Relative Expression present results normalized to TBP and then expressed as fold differences relative to unstimulated control cells (set to 0) for each donor in each experiment. Unstimulated normal cells from each donor sample served as the control for OP-1 stimulated normal cells from the same donor and unstimulated OA cells from each OA patient served as the control for the OP-1 stimulated OA cells from the same patient. Endogenous OP1 and BMP receptor expression levels were determined using RT-qPCR arrays for which RNA was purified using the RNEasy Mini kit (Qiagen) and used for the RT2 Profiler™ PCR Array Human TGFβ / BMP Signaling Pathway (SABiosciences) for the Applied Biosystems 7900HT thermocycler according to the manufacturer’s protocol. For these experiments, the geometric average from a panel of endogenous control genes including B2M HPRPT1, RPL13A, GAPDH, ACTB was used to normalize raw cts of OP1 and BMP receptors using the 2-ΔΔct method and graphed as normalized data.

Immunoblot Analysis

Cell signaling studies were performed as previously described [9], with cultures lysed at the indicated time points and lysates used to analyze cell signaling proteins by immunoblot. Cell-conditioned media was collected 24 hours post stimulation for MMP analyses. Phospho-Smad1/5/8 (Ser463/Ser465)/(Ser426/Ser428), phospho-Smad1linker region (Ser206), total-Smad1, phospho-Smad2 (Ser465/Ser467), total-Smad 2, phospho-p38 (Thr180/Tyr182), total-p38, phospho–ERK-1/2 (Thr202/Tyr204), total-ERK-1/2, phospho-Akt (Ser473), total-Akt, and secondary anti-mouse and anti-rabbit antibodies were all obtained from Cell Signaling Technology. The phospho-JNK (Tyr183/Tyr185) and JNK-2 antibodies were obtained from Invitrogen. The β-actin, MMP2, and MMP13 antibodies were from Abcam. MMP1 (Abnova) and MMP3 (Millipore) antibodies were also used. Densitometry was performed using ImageJ 1.48v analysis software. MMP-1,-3, and -13 were normalized to MMP2 since its levels in conditioned media were not changed by the treatments tested. Phospho-Smad1linker region was normalized to total-Smad1. Phospho-Smad2 was normalized to total-Smad2. Because the phospho-Smad1/5/8 antibody recognizes a phosphorylation site in all 3 of the Smads and therefore cannot distinguish Smad1 from Smad5 or Smad8, β-actin was used to normalize phospho-Smad1/5/8 densitometry.

Immunohistochemistry

Human meniscus tissue excised from the body of the lateral meniscus (n=3 normal and n=3 OA) was snap frozen in OCT (Fisher Scientific) and sectioned at 5 μm. Immunostaining was automated (Leica Bond-max™, Leica Microsystems Inc., Bannockburn, IL) following manual optimization (Vector labs) protocols. Sections were washed with PBS between each of the following steps: fixed for 10 minutes in acetone, treated with Bloxall, Dako Serum Free Protein Block, followed by primary antibody diluted in Dake antibody Diluent. Antibodies consisted of anti-mature OP1 (Stryker Biotech), ALK2 (LSBio), ALK3 (Gene Tex), ALK6 (Bioorybt), and secondary anti-rabbit (Vector labs). RTU Elite was then added. The DAB chromagen was added and neutralized within 2 minutes. Slides were counterstained with Gill’s hematoxylin and mounted with MM24. Slides were scanned with an Olympus VS110, version 2.7, with VS-ASW FL software. The objectives are true Olympus 40X and images shown at 10x. OlyVIA is the viewing software for the VSI image format.

Statistical Analysis

Results are shown as dot plots which reflect individual data points, and the mean ± CI from at least 3 independent donors. Data were analyzed with SAS version 9.4 (SAS Institute, Inc. Cary, NC). Experimental control-normalized data were log or square root transformed to correct for skewness if necessary. In order to account for within-donor correlations, all regression analyses were conducted using mixed modelling techniques: dose response experiments used a repeated measures framework; experiments of donor-matched tissues used linear mixed models; and longitudinal regression models were used to evaluate time courses. T-tests were used to compare inflammatory cytokine stimulation to inflammatory cytokine with OP1 co-treatment. Post hoc tests were conducted with Dunnett’s adjustment for multiple comparisons to evaluate differences from the control treatment within normal and OA groups or Tukey-Kramer adjustment for multiple comparisons to further define differences between normal and OA groups.

Results

Comparison of Normal and OA Meniscus Cell Response to OP1

We stimulated normal and OA meniscus cells with increasing doses of OP1 and analyzed the effects on extracellular matrix and matrix metalloprotease (MMP) gene transcription. Following OP1 treatment in normal cells, we observed dose-dependent increases in transcript levels of ACAN (aggrecan) which were significantly greater than the untreated controls starting at the 100ng/ml dose. However, compared to normal cells, ACAN expression was upregulated to a lesser degree in OA meniscus cells following OP1 treatment, with significant difference between untreated OA control cells only detected at the highest (500ng/ml) dose of OP1 (Figure 1). OP-1 significantly increased COL2A1 (collagen type II) mRNA levels in cells isolated from normal meniscus at the 100, 300, and 500 ng/mL dose as compared to untreated control cells (0 ng/mL) from normal meniscus. No differences were detected in OA meniscus cells treated with OP1 when compared to the untreated OA control cells. Gene expression of MMP13 was significantly downregulated during OP1 stimulation in normal cells at each OP-1 dose compared to unstimulated normal cells, but not in OA cells compared to unstimulated OA cells. A modest increase in MMP1 occurred in normal cells at the 100 ng/ mL dose of OP1 (fold change of 0.302, p=0.0278) and in OA cells at the 300 ng/mL dose (fold change of 0.811, p=0.0321), but significance was not found at other doses (data not shown). Transcript levels of COL1A1, COL3A1, and MMP3 were not significantly changed in normal or OA cells in response to OP1 at any dose (data not shown). From this experiment, we chose to stimulate cells in all subsequent experiments with OP1 at 100 ng/mL, a biologically relevant dose [21, 22].

Figure 1. OP1 dose effect on gene expression in normal and OA meniscus cells.

Figure 1

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Meniscal cells from normal (n=7 independent donors) and OA (n=5 independent donors) tissues were treated with 0–500 ng/mL of OP1 for 24 hours. All p values are compared to unstimulated controls from the same donor (the 0 ng/mL OP1 dose for the respective normal or OA cells) unless otherwise indicated by brackets to point out the direct comparisons. P values reported on graph or +p=0.03, *p<0.01, **p<0.001 by mixed model with repeated measures and post hoc Dunnett’s adjustments or Turkey Kramer adjustments. ACAN= aggrecan, COL2A1= collagen type II, MMP13= matrix metalloprotease 13

The OP1 response in cells from donor-matched meniscus and articular cartilage from both normal and OA knee joints were compared. Data for each gene were normalized to TBP and then presented as the expression relative to unstimulated cells for each experiment (i.e. OP1 stimulated normal meniscus cells relative to unstimulated normal meniscus cells). OP1 stimulated significantly greater ACAN expression in normal meniscus cells than either OA meniscus cells or articular chondrocytes from either normal or OA joints (Figure 2A). COL2A1 was also increased by OP1 in normal meniscus to a significantly greater extent than in cells from OA meniscus. Differences in MMP13 expression in meniscus and cartilage were not detected. No differences in gene expression were identified between normal and OA chondrocytes. The inner region of the meniscus is often lost to degeneration during OA [2]. We analyzed the response of cells from healthy meniscus tissue by region to determine if the loss of inner region tissue was responsible for reduced response to OP1 in OA meniscus. The mRNA levels of ACAN and COL2A1 upon OP1 stimulation were comparable between the healthy meniscus regions, and no significant differences were detected (Figure 2B). MMP13 was more significantly down-regulated in the inner meniscus cells compared to other regions and articular chondrocytes.

Figure 2. Effect of OP1 stimulation on meniscus cells versus articular chondrocytes.

Figure 2

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A) OP1 (100 ng/mL) 24 hour stimulation of matrix gene expression in meniscal cells and chondrocytes from normal, matched donors, and corresponding effect in tissue-matched OA donors (n=5 each). B) OP1 (100 ng/mL) for 24 hours effect on normal meniscus cells by region vs. cells from whole meniscus and chondrocytes (n=4 matched tissue donors). Data for each gene were normalized to the endogenous control gene, TBP, and presented as the expression relative to the expression of unstimulated normal or OA cells, respectively. Significantly different groups indicated by brackets to point out the direct comparisons. P values reported on graph by linear mixed models and post hoc Turkey Kramer adjustments. M= meniscus, C= cartilage, IM= inner meniscus, OM= outer meniscus

Endogenous Expression of OP1 and BMP Receptors in Meniscus

We evaluated endogenous levels of OP1 and type I BMP receptors in normal and OA meniscus cells by mRNA and immunohistochemistry (IHC). No significant differences were found between normal and OA meniscus mRNA levels of BMP7 (OP1) and the type I BMP receptors ACVR1 (Activin receptor-like kinase-2 or ALK2), BMPR1A (ALK3), and BMPR1B (ALK6)(Figure 3 A, B). IHC staining showed an increased amount of OP1 in normal tissue over OA tissue (Figure 3C). BMP type I receptor protein staining was apparent in both normal and OA menisci.

Figure 3. Endogenous levels of OP1 and receptors in meniscus.

Figure 3

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A) BMP7 (OP1) RNA expression from cultured cells of n=3 independent normal and OA donors. B) BMP Type I receptor RNA levels ACVR1 (ALK2), BMPR1A (ALK3), and BMPR1B (ALK6) in cultured cells from n=3 independent normal and OA donors. C) OP1 and type I receptor protein localization in meniscus tissue by frozen section immunohistochemistry. Data from A and B were normalized to the geometric average from a panel of endogenous control genes. IHC images representative of n=3 independent donors. Scale bar 100 μM. ALK= Activin receptor-like kinase, No 1°= no primary antibody control

Normal and OA meniscus cell response to OP1 during inflammatory factor treatment

OP1 has been shown to reduce MMP production by modulating the effect of inflammatory and catabolic mediators in articular cartilage [13]. Normal and OA meniscus cells, with or without OP1, were treated with FnF, IL1α, or IL1β. Immunoblots of conditioned media were probed for MMP-1, -3, or -13 and normalized to MMP2, for which levels remained stable. Densitometry of immunoblots revealed that normal cells secreted significantly more MMP1 and MMP3 when stimulated with inflammatory cytokines (Figure 4 A, B, C). MMP13 was not detected by immunoblot of normal cells. Likewise, OA cells secreted significantly more MMP1 and MMP13 when stimulated with inflammatory cytokines (Figure 4 D, E, F, G). Across both normal and OA meniscus, OP1 (100 ng/mL) addition did not alleviate MMP secretion basally or with inflammatory factor addition. We also tested the ability of OP1 to modulate gene transcription in parallel experiments. OP1 co-stimulation with any of the inflammatory factors was not able to influence gene expression for either normal or OA donors (Figure 5). In general, IL1α, IL1β, and FnF increased MMP -1, -3, and -13 gene expression and decreased anabolic matrix gene transcription, but OP1 was not able to restore gene expression in the presence of any of the tested inflammatory factors.

Figure 4. MMP release from meniscus cells following inflammatory factor treatment and OP1 co-treatment.

Figure 4

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Meniscus cells treated for 24 hours with fibronectin fragments (FnF, 1uM), inflammatory cytokines (10ng/mL), or OP1 (100 ng/mL). A) Immunoblot of normal human meniscus cells and B, C) densitometry of MMP1 and MMP3 from 3 normal human menisci. D) Immunoblot of OA human meniscus cells and E, F, G) densitometry of MMP1, MMP3, and MMP13 from 3 OA human menisci. MMP-1,-3, and -13 were normalized to MMP2 since its levels in conditioned media were not changed by cell treatment. P values by linear mixed models and post hoc Dunnett’s adjustments reported on graphs vs. unstimulated controls for densitometry from n=3 independent normal and OA donors, each.

Figure 5. Gene expression of matrix proteins and MMPs in meniscus cells following inflammatory factor treatment and OP1 cotreatment.

Figure 5

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Meniscus cells treated for 24 hours with fibronectin fragments (FnF, 1uM), inflammatory cytokines (10ng/mL), or OP1 (100 ng/mL). Data for each gene were normalized to the endogenous control gene, TBP. P values are reported on graphs or are *p<0.01, **p<0.001 vs. unstimulated controls by linear mixed models and post hoc Dunnett’s adjustments. No differences were detected between n=3 independent normal and n=3 OA meniscus donors.

OP1 Effects on Normal and OA Meniscus Cell Signaling

Phosphorylation of the c-terminus of the Smad1/5/8 transcription factors (p-Smad1/5/8) is the result of canonical BMP signaling activation. Whether or not OP1 activates non-canonical pathways such as MAPKs or AKT in the meniscus has not been reported. Thus, we examined the kinetics of OP1-induced phosphorylation of these proteins in normal and OA meniscus. We found that OP1 only activated canonical, Smad1/5/8 phosphorylation (P-Smad1/5/8) in both normal and OA meniscus and not non-canonical signaling, as compared to TGFβ, IL1β, GDF5, or IGF1 (Figure 6A normal donor, Figure 6B OA donor). Smad1/5/8 phosphorylation significantly increased over time in response to OP1 treatment in normal and OA donors (p<0.001), beginning at 30 minutes and peaking at 1.5 hours, but no differences in kinetics were found between normal and OA donors (Figure 6C). As expected, IL1β and IGF1 failed to induce significant, canonical Smad1/5/8 phosphorylation. IL1β was the only stimulus which induced phosphorylation of the MAPKs (p38, ERK, JNK), while IGF1 was the only stimulus which induced AKT phosphorylation in both normal and OA meniscus cells. GDF5 was a less potent stimulator of canonical Smad1/5/8 phosphorylation than OP1. TGFβ induced both Smad1/5/8 phosphorylation and Smad2 phosphorylation in normal and OA meniscus.

Figure 6. Effect of OP1 on kinetics of canonical Smad1/5/8 signaling and non-canonical signaling.

Figure 6

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OP1 (100 ng/mL) treatment over time in both (A) normal meniscus cells and (B) OA meniscus cells as compared to positive controls for each signaling pathway. Positive controls consisted of TGFβ activation of Smad2, IL1β activation of MAPKs (p38, ERK, JNK), and IGF1 activation of AKT. GDF5 was also evaluated for activation of Smad1/5/8. (C) Densitometry of P-Smad 1/5/8 from n=4 independent normal donors and n=4 independent OA donors reveals a difference over time (p<0.001 by longitudinal mixed models) in response to OP1 stimulation, but no difference between disease state. P-Smad 1/5/8= canonical Smad1/5/8 phosphorylation. Non-canonical pathways evaluated include P-Smad2=phosphorylated Smad2, P-p38= phosphorylated p38, P-ERK= phosphorylated extracellular signal-regulated kinases, P-JNK= phosphorylated c-Jun N-terminal kinases, and P-AKT= phosphorylated AKT/protein kinase B

Smad1 Linker Region Phosphorylation in meniscal cells

The ability of Smad1 to regulate transcription can be inhibited by phosphorylation of the interdomain linker region by inflammatory factor-activated MAPK [16, 23–25]. We compared the intensity of canonical (P-Smad1/5/8) and non-canonical linker region (P-Smad1, S206) Smad1 phosphorylation between normal and OA meniscus cells, and also evaluated TGFβ-associated Smad2 C-terminus phosphorylation (P-Smad2). In normal cells, significant increases in P-Smad1/5/8 were detected in response to OP1 at 90 minutes and to TGFβ at 30 minutes. OA meniscus cells had a trend of higher basal P-Smad1/5/8 than normal cells although no significant differences were detected (Figure 7B). Phosphorylation at Serine 206 in the Smad1 linker region was significantly increased in response to IL1β in both normal and OA samples versus unstimulated controls (Figure 7C). TGFβ significantly increased phosphorylated Smad2 above control levels in both normal and OA meniscus cells (Figure 7D). As expected, IL1β did not alter P-Smad1/5/8 or P-Smad2. No differences were detected between intensity of phosphorylation of the evaluated proteins between normal and OA tissues.

Figure 7. Direct comparison of normal and OA meniscus cell activation of canonical and non-canonical Smad phosphorylation.

Figure 7

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OP1 (100 ng/mL), ILIβ (10 ng/mL), or TGFβ (100 ng/mL) stimulation at select time points. A) Immunoblot representative of n=3 independent experiments. B) Densitometry of canonical Smad 1/5/8 phosphorylation (p-Smad1/5/8) from n=3 independent normal and OA donors. C) Densitometry of non-canonical linker region Smad1 (p-Smad1 (S206)), an inhibitory phosphorylation for Smad1 transcription factor activity, from n=3 independent normal and OA donors. D) Densitometry of phosphorylated Smad 2 (p-Smad2) from n=3 independent normal and OA donors. Phospho-Smad1linker region was normalized to Total-Smad1. Phospho-Smad2 was normalized to Total-Smad2. Because the phospho-Smad1/5/8 antibody recognizes a phosphorylation site in all 3 of the Smads and therefore cannot distinguish Smad1 from Smad5 or Smad8, β-actin was used to normalize phospho-Smad1/5/8 densitometry. P values by linear mixed models and post hoc Dunnett’s adjustments reported on graphs vs. unstimulated controls for densitometry from n=3 normal and OA donors, each.

Discussion

These results identify OP1 as an anabolic agent in normal human meniscus cells which does not promote catabolic processes. Importantly, the reduced ability of OP1 to stimulate extracellular matrix gene expression and to reduce MMP expression in meniscus cells obtained from end-stage OA knee joints suggests that factors associated with OA inhibit the response to OP1. We found evidence that these could include IL1α, IL1β, and FnF which have been found to be present in torn meniscus and/or OA joint fluid [26, 27]. We also confirmed previous work [13] that unlike the meniscus, OA chondrocytes do not appear to become unresponsive to OP1 suggesting differences in the regulation of OP1 signaling between these two joint tissues.

Previous work has shown that OP1 stimulates proteoglycan and collagen production in pig, rat, and sheep meniscus [14–16]. We further explored this using human tissue and found that ACAN and COL2A1 were upregulated in normal meniscus cells and MMP13 was downregulated. Our study also evaluated the effect of OP1 on meniscus cells derived from human OA joints, in which the stimulatory effects of OP1 on extracellular matrix gene expression was lost in all evaluations except for ACAN induction using a high dose (500 ng/mL) of OP1. However, OP1 did not induce COL1A1, COL3A1, or MMPs in normal or OA meniscus cells, which supports that OP1 induces neither fibrotic nor catabolic processes in the meniscus. Lack of induction of these genes is also consistent with published reports in articular cartilage [13, 28].

Unlike meniscus, OP1 has been extensively studied in articular cartilage. Surprisingly, we found that the normal meniscus expressed higher levels of ACAN in response to OP1 than matched articular cartilage from the same donor, while OA meniscal cells but not OA chondrocytes lost responsiveness to OP1. ACAN and COL2A1 are OP1 responsive genes in articular cartilage [28, 29] and this held true in the meniscus. We did not find a differential response to OP1 stimulated ACAN or COL2A1 expression by separated meniscus regions, ruling out the possibility that the inner region, which is higher in aggrecan and collagen type II expression and often lost to degeneration during OA [2, 30], was responsible for OP1 response. This suggests that anabolic insensitivity of meniscus cells to OP1 in OA is related to disease state and not due to loss of the inner meniscus region in OA.

We unexpectedly identified that normal articular chondrocytes were less responsive to OP1 than normal meniscal cells as measured by ACAN levels. Our comparative experiments used meniscus cells and chondrocytes from the same donor knees. Thus the pre-existing knee joint environment was shared, and cells were processed and treated in parallel, nearly eliminating the possibility of differential cell stress or treatment. It is also unlikely that articular chondrocytes have high endogenous levels of the Smad1 linker region phosphorylation. Several papers, from which we based our cell isolation, cell treatment, and immunoblot techniques, showed that IL1β or tert-butyl hydroperoxide to induce oxidative stress were required to detect phosphorylation in the linker region of Smad1 [21, 31]. Articular cartilage expresses 5 times as many proteoglycans as the meniscus on a protein level [32]. Our data of endogenous expression suggests that articular chondrocytes express approximately 40 times more ACAN than the meniscus cells on a gene level. This suggests that OP1 could have less effect on articular chondrocytes due to the already elevated expression of ACAN and due to complex regulation of this gene in articular cartilage [33]. As a more fibro-cartilagenous tissue, the healthy meniscus may have more ability to respond to OP1 and gain a more cartilage-like ECM composition.

Type I BMP receptors assist in signal transduction across the cell membrane and confer specificity of intracellular BMP signals [34, 35]. We evaluated expression of endogenous OP1 and the BMP type I receptors (ALK -2, -3, and -6) in normal and OA tissues to assess if OA impacts receptors available for signal transduction. OP1 and BMP receptor protein staining was apparent in both normal and OA meniscus. We did not identify differences in mRNA levels of OP1 or receptors between normal and OA meniscus. OP1 has been shown to bind strongly to ALK2 or weakly to ALK6 [36]. Because we observed similar expression of these receptors in normal and OA meniscus, we chose to look further downstream in the BMP signaling pathway for a possible explanation for anabolic resistance to OP1 in OA meniscal cells.

The signaling events incited by inflammatory factors released from injured and OA menisci, including IL1α, IL1β, and FnF [26, 27] culminate in meniscus tissue breakdown and OA progression [9–12]. It has been less studied, but inflammatory factors also inhibit intrinsic meniscal repair [37], and likely set up the joint for subsequent damage. Our objective in the present study was to model the inflammation which occurs following meniscus injury and OA. Hence we used a high-dose (10 ng/mL) of IL1α and IL1β to induce inflammation, as has been previously published [13, 21]. Using this model in articular chondrocytes, OP1 was not able to compensate for IL1β induced downregulation in aggrecan expression [21], consistent with our findings. However, OP1 was able to decrease MMP1 and MMP13 promoter activity in human chondrocytes following 25 ng/mL IL1β [38], while we saw no OP1-induced change in MMP gene and protein expression in our experiments. Hence our choice of 10 ng/mL was likely the most informative dose for this first study of OP1 effects in the meniscus during inflammation and OA. We also used an OA-physiological dose of fibronectin fragment (FnF) (1 μM) [27]. In articular chondrocytes, OP1 was able to block, as well as restore, PG loss induced by FnF [39], while we failed to identify any restoration of anabolic gene transcription or reduction of MMP gene and protein expression. Our study indicates inhibition of OP1 occurs during inflammation and also at a physiological concentration FnF in the meniscus. In other studies, the meniscus has been found to be more sensitive to catabolic factors than articular cartilage [40]. Inflammatory factors also inhibit intrinsic meniscal repair [37], and likely set up the joint for subsequent damage. Although there is variation in methodology, this further suggests differences in meniscus and cartilage metabolism, and a potentially greater sensitivity of the meniscus to inflammation-inhibited repair processes.

BMP signal transduction from the cell surface to the nucleus occurs via the Smad transcription factors, which are central mediators of this pathway [41]. Ligand-activated BMP receptors phosphorylate the Smad1/5/8 transcription factors at the canonical, activating c-terminus. This results in co-factor recruitment, nuclear accumulation, association with other transcription cofactors, and gene transcription [42]. Alternatively, non-canonical BMP signaling can activate the MAPKs, and AKT pathways [42, 43]. We identified similar kinetics of canonical, activated Smad1/5/8 (p-Smad1/5/8) between normal and OA donors. OP1 did not activate non-canonical pathways in the meniscus, including the p38, ERK, and JNK MAPKs, Smad2, or AKT.

Convergence of inflammatory cytokine activated MAPK and BMP signaling results in BMP signaling constraint at the level of Smad1. This occurs via non-canonical, inhibitory phosphorylation of Smad1 at the interdomain linker region by MAPKs. Smad1 linker region phosphorylation at serines of PXSP motifs by MAPKs has been shown to limit nuclear accumulation, reduce transcription activity, and induce Smad1 degradation in developmental models [16, 23–25]. This potential mechanism for OP1 resistance had not been studied in the meniscus, but many of the inflammatory cytokines associated with the torn or degenerative menisci, including IL1 and FnF, activate the MAPKs [44], and phosphorylated p38 MAPK has been identified histologically from torn menisci [45]. In articular cartilage, IL1β has been shown to increase Smad1 linker region phosphorylation through p38 MAPK [21, 31]. In our study, Smad1 linker region phosphorylation, as indicated by the antibody to detect phosphorylation at serine 206, was enhanced during IL1β stimulation in normal and OA meniscus cells. In conjunction with our experiments showing that cells treated with inflammatory cytokines were also refractory to OP1 stimulation, this suggests that Smad1 linker region phosphorylation may be a mechanism contributing to OP1 insensitivity following inflammatory events.

TGFβ signaling in articular chondrocytes has been shown to be altered during aging and OA, with a switch in prominence from Smad2/3 to the BMP-related Smad1/5/8 resulting in MMP13 expression and loss of tissue homeostasis [46]. In articular cartilage, TGFβ stimulated phosphorylation of Smad1/5/8 is thought to recapitulate chondrocyte terminal differentiation and induces cartilage degeneration, synovial fibrosis, and osteophyte formation [47]. In contrast to chondrocytes, TGFβ induced significant increases in phosphorylation of Smad1/5/8 in cells isolated from both normal and OA menisci, as well as significant increases in phosphorylation of Smad2 in both normal and OA meniscal cells. We noted comparable levels of TGFβ stimulated phosphorylation of Smad1/5/8 and Smad2 when comparing normal and OA human menisci. These findings further exemplify differences in chondrocytes and meniscal cells and raise the question of whether the meniscus is protected from altered TGFβ signaling during OA. Further study of the meniscus in comparison to articular cartilage may indicate signaling differences which can be targeted to assist knee joint homeostasis.

Despite the strengths of using a human model of disease, our study carries limitations specific to human tissue and cell-culture research. The use of human subjects and associated variability may have resulted in failure to statistically identify differences in Smad phosphorylation between normal and OA tissues. We attempted transfection experiments in normal human meniscus cells using Smad1 [48] and Smad1 linker region mutants [19] to determine if the inhibitory site phosphorylation at serines of PXSP motifs in the Smad1 linker region was responsible for the reduced response to OP1, but did not observe consistent effects. The inability to accomplish these experiments reliably may exemplify the importance of Smad1 to normal meniscus cell phenotype and stress regulation, but they also highlight the limitations of cell culture studies using primary cells. The meniscus contains a mixed cell population reflective of vascularization and different embryonic origins of inner and outer meniscal cell regions [2]. No cell lines are available which are representative of meniscal cells. Further evaluation of the functional consequence of OP1 signaling and Smad1 linker region phosphorylation in the OA meniscus pertinent to anabolic insensitivity may require in vivo approaches. These were beyond the scope of the current study.

In summary, these findings support the presence of significant anabolic resistance to OP1 in OA menisci. In our experimental model, treatment with inflammatory mediators promotes the inhibition to OP1 and also results in Smad1 interdomain linker phosphorylation at a site known to inhibit Smad1 transcriptional activity [16, 23–25]. This is the most likely explanation for OP1 insensitivity in OA meniscus and cytokine treated normal meniscus. Our data also exemplify key differences between meniscus and articular chondrocyte responses to OP1 as well as TGFβ. Normal meniscus cells were more responsive to OP1 than normal chondrocytes but inflammatory mediators induced OP1 insensitivity in normal meniscal cells, similar to OA meniscus cells. Unlike reports in chondrocytes [46, 47], there was no change in prominence of Smad1/5/8 and Smad2 activation following TGFβ stimulation in normal and OA meniscus. The BMP pathway in general is known to have variant outcomes following a common intracellular signaling system due to species and cell type differences [49]. Our work, conducted in human adult primary meniscus cells exemplifies cell type specific differences. This is of particular interest during injury and OA, and highlights the importance of considering tissue specific and whole joint effects of potential therapeutics.

Supplementary Material

supplement. Supplementary Figure 1: Endogenous protein levels of OP1 and receptors in meniscus.

OP1 and type I receptor protein localization in meniscus tissue by frozen section immunohistochemistry. Whole frozen tissue sections at 10× magnification show region of imaging for normal, OA, and No 1° (no primary antibody control) meniscus by blue boxes. The final images shown in Figure 3 at 100× magnification are superimposed along the top of the 10× whole tissue section, left side = outer meniscus, right side = inner meniscus image. IHC images representative of n=3 independent donors. Scale bar 100 μM. ALK= Activin receptor-like kinase.

Acknowledgments

We would like to thank the Wake Forest School of Medicine Orthopaedic Surgery Joint Service and Surgical Pathology for their assistance in human tissue acquisition. We would like to thank the National Disease and Research Interchange (NDRI) and Dr. Arkady Margulis at Rush University Medical Center for procuring meniscus from the Gift of Hope Organ and Tissue Donor Network. We also would like to acknowledge the donor’s family and the members of Dr. Chubinskaya’s laboratory (Mrs. Arnavaz Hakimiyan and Dr. Lev Rappoport) for assistance with human tissue and OP1 immunostaining.

Role of the funding source

This study was funded by the Goldberg Arthritis Research Grant from the Orthopaedic Research and Education Foundation (Ferguson) and the Tyner and Daniels Research Endowment Fund (Wake Forest Dept. of Orthopaedic Surgery). Research reported in this publication was supported by the National Institute of Arthritis And Musculoskeletal And Skin Diseases of the National Institutes of Health under Awards Number NIH/NIAMS K08AR059172 (Ferguson) and NIH/NIAMS R37 AR049003 (Loeser). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Additional support was received from the Rush University Ciba-Geigy Endowed Chair (Chubinskaya). We acknowledge use of meniscus and articular cartilage provided by the National Disease Research Interchange (NDRI), with support from NIH grant 2 U421 OD011158. The study sponsors had no role in the study design; in the acquisition, analysis, or interpretation of data; in drafting the manuscript; or in the decision to submit the manuscript to OAC.

Footnotes

Author Contributions

Vanderman: Conception and design, analysis and interpretation of the data, drafting of the article, critical revision of the article for important intellectual content, final approval of the article, obtaining of funding, collection and assembly of data.

Loeser: Conception and design, analysis and interpretation of the data, critical revision of the article for important intellectual content, final approval of the article, obtaining of funding.

Chubinskaya: Critical revision of the article for important intellectual content, final approval of the article, collection and assembly of data. Suppled human tissue, provided initial morphological joint assessment, provided OP1 and anti-mature OP1 antibody and assistance with OP1 immunohistochemistry.

Anderson: Analysis and interpretation of the data, drafting of the article, critical revision of the article for important intellectual content, final approval of the article.

Ferguson: Conception and design, analysis and interpretation of the data, critical revision of the article for important intellectual content, final approval of the article, obtaining of funding.

Competing Interests

The authors have no competing interests to report. Funding sources disclosed above.

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Contributor Information

Kadie S. Vanderman, Email: kvanderm@wakehealth.edu.

Richard F. Loeser, Email: richard_loeser@med.unc.edu.

Susan Chubinskaya, Email: susanna_chubinskaya@rush.edu.

Andrea Anderson, Email: amanders@wakehealth.edu.

Cristin M. Ferguson, Email: ferguson@wakehealth.edu.

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Associated Data

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

Supplementary Materials

supplement. Supplementary Figure 1: Endogenous protein levels of OP1 and receptors in meniscus.

OP1 and type I receptor protein localization in meniscus tissue by frozen section immunohistochemistry. Whole frozen tissue sections at 10× magnification show region of imaging for normal, OA, and No 1° (no primary antibody control) meniscus by blue boxes. The final images shown in Figure 3 at 100× magnification are superimposed along the top of the 10× whole tissue section, left side = outer meniscus, right side = inner meniscus image. IHC images representative of n=3 independent donors. Scale bar 100 μM. ALK= Activin receptor-like kinase.

NIHMS758240-supplement.tif (2.4MB, tif)

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