Nitric Oxide Contributes to Rapid Sclerostin Protein Loss Following Mechanical Load

Abstract

In response to mechanical loading of bone, osteocytes produce nitric oxide (NO•) and decrease sclerostin protein expression, leading to an increase in bone mass. However, it is unclear whether NO• production and sclerostin protein loss are mechanistically linked, and, if so, the nature of their hierarchical relationship within an established mechano-transduction pathway. Prior work showed that following fluid-shear stress (FSS), osteocytes produce NOX2-derived reactive oxygen species, inducing calcium (Ca²⁺) influx. Increased intracellular Ca²⁺ results in calcium-calmodulin dependent protein kinase II (CaMKII) activation, which regulates the lysosomal degradation of sclerostin protein. Here, we extend our discoveries, identifying NO• as a regulator of sclerostin degradation downstream of mechano-activated CaMKII.

Pharmacological inhibition of nitric oxide synthase (NOS) activity in Ocy454 osteocyte-like cells prevented FSS-induced sclerostin protein loss. Conversely, short-term treatment with a NO• donor in Ocy454 cells or isolated murine long bones was sufficient to induce the rapid decrease in sclerostin protein abundance, independent of changes in Sost gene expression. Ocy454 cells express all three NOS genes, and transfection with siRNAs targeting eNOS/Nos3 was sufficient to prevent FSS-induced loss of sclerostin protein, while siRNAs targeting iNOS/Nos2 mildly blunted the loss of sclerostin but did not reach statistical significance. Similarly, siRNAs targeting both eNOS/Nos3 and iNOS/Nos2 prevented FSS-induced NO• production. Together, these data show iNOS/Nos2 and eNOS/Nos3 are the primary producers of FSS-dependent NO•, and that NO• is necessary and sufficient for sclerostin protein control.

Further, selective inhibition of elements within this sclerostin-controlling mechano-transduction pathway indicated that NO• production occurs downstream of CaMKII activation. Targeting Camk2d and Camk2g with siRNA in Ocy454 cells prevented NO• production following FSS, indicating that CaMKII is needed for NO• production. However, NO• donation (1min) resulted in a significant increase in CaMKII activation, suggesting that NO• may have the ability to tune CaMKII response. Together, these data support that CaMKII is necessary for, and may be modulated by NO•, and that the interaction of these two signals is involved in the control of sclerostin protein abundance, consistent with a role in bone anabolic responses.

1. Introduction

Bone is an adaptable, mechano-responsive tissue, able to accommodate changes in load by removing, repairing, and accumulating bone according to mechanical strain¹. This remodeling occurs through the coordination of bone resorption by osteoclasts and the deposition of new bone matrix by osteoblasts². Control of these two processes is moderated by osteocytes, which receive mechanical input from experienced mechanical strain and covert it to the chemical signals that coordinate bone anabolism and catabolism³ in a process termed ‘mechano-transduction’. One of the primary effectors of osteocyte mechano-transduction is a secreted glycoprotein, sclerostin (gene name Sost). Sclerostin is a WNT/β-catenin antagonist⁴ that suppresses osteoblastogenesis and matrix deposition⁵. In response to mechanical loading, sclerostin protein abundance is reduced via transcriptional⁶ and post-translational^7–9 control, de-inhibiting Wnt/β-catenin signaling and unleashing osteoblast differentiation and new bone formation.

Nitric oxide (NO•) is a free radical signaling molecule that is produced by osteocytes in response to mechanical loading cues¹⁰, which has a long-studied role in bone physiology¹¹. NO• is produced by three synthases, neuronal (nNOS/Nos1), inducible (iNOS/Nos2), and endothelial (eNOS/Nos3), which are widely expressed throughout a variety of tissues, including bone^12,13. Genetic deletion of two of the nitric oxide synthase genes (Nos2 and Nos3) in bone cells reveals a role consistent with bone anabolism. Nos3 knockout mice have low bone mass and decreased osteoblast activity^14,15. Basally, the skeletal phenotype of adult Nos2 knockout mice is no different than control mice. However, reloading of Nos2^−/− mice after tail suspension exposes a less robust response to reloading relative to control, with decreased bone mass and osteoblast activity¹⁶. Additionally, there are defects in the mineralization capacity of primary osteoblasts isolated from these Nos2 deleted mice after unloading¹⁶. In contrast to Nos2 and Nos3, Nos1 knockout mice have increased bone mass, suggesting a distinct role in bone physiology¹⁷.

The molecular basis for NO•’s influence on bone-resident cells is not entirely clear, although its effects on osteoblast proliferation or differentiation¹⁸, cell survival effects in osteocytes¹⁹, and osteoblast glycolysis²⁰ have been reported. Previously, we described a signal transduction pathway regulating the post-translational control of sclerostin^7–9. Among the findings of this paper was evidence that NO• also may influence sclerostin protein abundance. Here, we investigate the role that NO• plays within this pathway in osteocytes and tie NO• to the post-translational control of a fundamentally important effector of bone formation, sclerostin.

2. Methods and Materials

2.1. Cell culture

Ocy454 osteocyte-like cell line (provided by P. Divieti-Pajevic, Boston University) were cultured as previously reported^7,9,21. Briefly, Ocy454 cells were cultured in αMEM (Corning) with 10% FBS (Benchmark, GenClone) and 1% penicillin/streptomycin (Corning Cellgro). For cell expansion, cells were plated on rat tail collagen I (BD Biosciences) coated plasticware and maintained at 33°C and 5% CO₂ with media changes every 2 to 3 days. For experiments, cells were maintained at 37°C and 5% CO₂.

2.2. Cell Treatment with Nitric Oxide Donor

The NO• donor, diethylamine NONOate sodium salt hydrate (Sigma-Aldrich), was prepared as 64mM stock and diluted in water just prior to use and administered at a final concentration of 10μM.

2.3. Fluid Shear Stress

Assay was performed as previously reported²². Briefly, cells were plated in Stripwell (Costar) 96-well-size vessels at 35,000 or 40,000 cells per well the day before flow. Processively by strip, media was replaced with 37°C, pH 7.4 mouse Ringer’s solution (140 mM NaCl, 4 mM KCl, 1 mM MgSO₄, 10 mM Hepes, 10 mM glucose, 5 mM NaHCO₃, 1.8 mM CaCl₂). Strips were allowed to acclimate on a benchtop warming mat (38°C) for 20 minutes. Cells were stimulated at a flow rate of 38ml/min (flow rate modelled at 7 dynes/cm², 5min), unless otherwise indicated, in Ringer’s solution for 5 minutes by peristaltic pump (Harvard) via custom-printed tips²³. After completion of fluid shear stress (FSS) exposure, cells were allowed to incubate on benchtop warming mat from 0-10min, as indicated. Cells were lysed in 1X RIPA buffer supplemented with 2% SDS, 1% HALT protease and phosphatase inhibitor (Thermo) and 1% EDTA (0.5M) or 2x Laemmli buffer with 5% BME, 1% HALT (Thermo Fisher) and 1% EDTA (0.5M).

2.4. Griess Assay

Following exposure of cells to FSS, Ringer’s solution from wells and tubing was collected. Additional Ringer’s solution was added to no-flow samples collected from wells to volume-match FSS samples. Colorimetric Griess assay (Thermo Fisher) was performed using 280μl each sample/standard. Standards were produced and protocol followed per manufacturer’s instructions. Plates were read at 548nm on μQuant (Bio-Tek Instruments) plate reader. Fluorometric Griess assay (Invitrogen) was performed using 20μl each sample/standard. Plates were read at 350/450nm on Tristar 3 (Berthold) plate reader.

2.5. Ex Vivo Samples

Camk2d^flox/flox/Camk2g^flox/flox mice were backcrossed for ten generations into the C57BL/6 background and generously provided by the Oslon group²⁴. Mice were then crossed with osteocalcin-cre mice (RRID:IMSR_JAX:019509), which are also in the C57BL/6 background. Mice were maintained with ad libitum access to water and standard rodent chow under Animal Care and Use Committee-approved protocols at the University of Maryland Baltimore, protocol# 00000134. Five male and six female mice between 6-23 weeks of age were utilized and no sexual differences were observed in the data. Briefly, as described²⁵, following euthanasia, femur or ulna/radius were isolated from surrounding soft tissues, epiphyses were removed and marrow was flushed. Bones were maintained in 37°C complete αMEM during collection, before transfer to 37°C mouse Ringer’s solution for treatment. After treatment, bones were transferred to Laemmli lysis buffer. Bones were homogenized with 1mm metal beads in a Precellys Evolution (Bertin Technologies) homogenizer.

2.6. Western Blot

Western blots were performed as previously reported⁸. Briefly, cell lysates were sonicated and electrophoresed on 10% or 4-20% SDS-Page gels (Bio-Rad) and transferred to PVDF film (Millipore). Total protein stain imaging was taken on Odyssey CLx (LI-COR) following five minutes Revert 700 Total Protein Stain (LI-COR) incubation. Membranes intended for ECL development were blocked in phosphate buffered saline with 0.1% Tween-20 and 5% non-fat dry milk. Membranes intended for goat-derived ECL antibodies were blocked in 3% bovine serum albumen as well. Membranes intended for LI-COR development were blocked in Intercept (LI-COR). ECL membranes were developed with Pierce ECL Western Blotting Substrate (Thermo Scientific) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). Blots were analyzed using ImageLab software (Bio-Rad). Loading controls were probed sequentially on the same membrane as its experimental target for all probes except p-CaMKII. For p-CamKII, two membranes were produced and processed at the same time, keeping all conditions and handling as identical as possible until p-CaMKII or panCaMKII probing. Antibody sources, catalog numbers, and dilutions are presented in Supplementary Table I.

2.7. RT-qPCR

RT-qPCR was performed as previously reported²⁶. Briefly, cells were lysed in TRIzol (Sigma). RNA was isolated using Direct-zol RNA Miniprep kit (Zymo) per manufacturer’s instructions and measured for quantity and purity using μQuant plate reader. RNA was converted to cDNA using High-Capacity RNA-to-cDNA kit (Thermo Fisher) on a MyCycler thermocycler (Bio-Rad). Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher) was used to prepare samples, which were run on Applied Biosystems 7300 sequence detection system. Relative gene expression for each sample was simultaneously normalized to three housekeeping genes, Gapdh, Hprt, and Rpl13. PCR primer sequences are presented in Supplementary Table II.

2.8. Transient Transfection

Transfections of Ocy454 cells were performed using JetPrime transfection kit (Polyplus) per manufacturer’s recommendation. Transfection target, concentrations, and manufacturer’s reference numbers presented in Supplementary Table 3.

2.9. Data Analysis

Analysis and graphing was performed using GraphPad 10 (Prism). Multigroup comparisons performed via two-way ANOVA with Holm-Sidak’s post hoc correction. Two-group comparisons performed via unpaired t-test. Graphs show mean with ± standard deviation. Asterisks represent p values: *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.

3. Results

3.1. Nitric Oxide is Necessary for Sclerostin Protein Control

We recently described a mechanical transduction pathway that controls lysosomal degradation of sclerostin protein following mechanical stimulation, thereby coordinating later bone anabolism^7–9. We demonstrated that mechanical stimulation of osteocytes in vivo or in vitro caused NOX2-dependent generation of reactive oxygen species, resulting in the activation of TRPV4 Ca²⁺ channels and an increase in intracellular calcium. This increase in calcium stimulates activation of CaMKII, leading to the minute-scale degradation of sclerostin by the lysosome.

NO• is a signaling compound capable of supporting multiple protein modifications and is rapidly produced by osteocytes following mechanical stimulation¹⁰. NO• production and its interaction with CaMKII is commonly studied in cardiac and neuronal systems, but less is known about the relationship of NOS and CaMKII in bone.

To determine the potential role of NO• in the control of sclerostin protein abundance, Ocy454 osteocyte-like cells were transfected with myc-tagged Sost plasmid (mycSost) and treated with vehicle or the non-specific NOS inhibitor LNAME prior to mechanical stimulation. Following exposure to FSS via pulsatile flow, vehicle treated cells exhibited an ~40% reduction in sclerostin protein abundance (Fig. 1A). In contrast, treatment with LNAME fully blocked the effects of FSS on sclerostin protein abundance. These data indicate that NO• production is required for the loss of sclerostin protein following mechanical stimulation.

Fig 1. Nitric Oxide is Necessary for Sclerostin Protein Control.

(A) Fold change in relative sclerostin protein abundance in mycSost-transfected Ocy454 cells. Pretreatment with vehicle or L-NAME (1mM, 30min). No-flow control or fluid shear stress (FSS) 38ml/min (7 dynes/cm², 5min), samples collected 10min after FSS. Representative blots for sclerostin and α-tubulin. (B) Relative gene expression of nitric oxide synthase (NOS) isoforms in Ocy454 cells. (C) Relative abundance of individual NOS isoform protein in Ocy454 cells transfected with scramble (Sc) or specified isoform siRNA. Number represents fold change in protein abundance of knockdown relative to control, normalized to β-actin. (D) Fold change in relative sclerostin expression in Ocy454 cells with mycSost and specified siRNA transfections. No-flow control or FSS, collected 5m after FSS. Representative blots for sclerostin and α-tubulin. (E) Fold change in relative nitrite production in Ocy454 cells with mycSost and specified siRNA transfections. No-flow control or FSS, collected immediately after FSS.

Osteocytes have been shown to express eNOS^27,28, but the relative abundance of nNOS (gene: Nos1), iNOS (Nos2), or eNOS (Nos3) isoforms in Ocy454 cells has not been reported. Consistent with primary osteocytes, Nos3 was found to have higher expression than Nos1 or Nos2, as indicated via RT-qPCR (Fig. 1B).

Next, Ocy454 cells were transfected with scramble or isoform-specific siRNA before exposure to FSS. Western blots of Ocy454 cell homogenates confirmed reduced expression of respective NOS isoform protein after isoform-specific siRNA transfection (Fig. 1C). Following mechanical stimulation, scramble siRNA transfected cells exhibited the expected fluid shear stress-dependent decrease in sclerostin protein abundance (Fig. 1D). Knockdown of eNOS inhibited the loss of sclerostin protein. siRNA knockdown of iNOS appears to blunt the loss of sclerostin after FSS. This partial inhibition could be due to the lower efficacy of the iNOS knockdown relative to eNOS knockdown (Fig 1C), or potentially due to lesser participation of iNOS-derived NO• in anabolic sclerostin control. In contrast, knockdown of nNOS had no effect on sclerostin protein loss after FSS (Fig. 1D), despite robust knockdown of nNOS protein (Fig. 1C). Neither plasmid nor siRNA transfection significantly altered cell viability (Fig. S1).

As nNOS knockdown did not prevent the loss of sclerostin after FSS (Fig. 1D) and because global knockout of nNOS is mice results in increased bone mass, we chose to focus on the role of eNOS- and iNOS-derived NO•. eNOS or iNOS knockdown cells were exposed to FSS, with samples collected at a timepoint suitable for nitrite analysis as a proxy for NO• production. After exposure to FSS, scramble siRNA transfected cells demonstrated a significant increase in nitrite concentration, while eNOS and iNOS siRNA transfected cells did not (Fig. 1E). In total, these data support that NO• production in response to fluid shear stress occurs via eNOS and iNOS, and the resultant NO• is required for loss of sclerostin protein abundance.

3.2. NO• Production is Downstream of CaMKII

To determine whether NO• production falls within this previously reported mechanical response pathway that regulates sclerostin degradation (Fig. 2A), various components of this pathway were inhibited prior to mechanical stimulation. Following pre-treatment with apocynin (NOX inhibitor), or GSK219 (TRPV4 channel inhibitor), nitrite production was not increased by mechanical stimulation, as compared to vehicle treated samples (Fig. 2B). Apocynin is not fully specific to NOX2 inhibition, but inhibition of TRPV4 channels, which allow Ca²⁺ influx after NOX2 activation²⁹, also prevented significant NO• production. FSS of vehicle treated cells or LNAME (non-specific NOS inhibitor) served as positive and negative controls, respectively. These data suggest NO• production in response to fluid shear stress occurs downstream of TRPV4-dependent Ca²⁺ influx.

Fig 2. NO• Production is Downstream of CaMKII.

(A) Representation of ‘Mechano-Pathway’ in osteocytes. (B) Fold change in nitrite production in Ocy454 cells pretreated with vehicle, LNAME (1mM, 30min), Apocynin (500μM, 1hr), or GSK219 (15μM, 10min). No-flow control or FSS at 35ml/min (4 dynes/cm², 5min), collected immediately after FSS. (C) Fold change in nitrite production in Ocy454 cells with scrambled (Scr) or CaMKIIδ/γ (CaMδγ) siRNA transfection. No-flow control or FSS, collected immediately after FSS. (D) Fold change of relative CaMKII activation (phosphorylation at T286) in Ocy454 cells with scrambled (Scr) or i/eNOS (ieNOS) siRNA transfection. No-flow control or FSS, collected within 1m of FSS. Representative blots for phospho-CaMKII and panCaMKII.

Next, we examined the calcium signaling integrator CaMKII as it relates to NO• production. CaMKII activation is required for the loss of sclerostin protein following multiple bone anabolic signals, including fluid shear stress^7–9 and parathyroid hormone⁹. Of the four CaMKII isoforms, CaMKIIδ and CaMKIIγ are primarily expressed in osteocytes³⁰. These isoforms function redundantly²⁴, so Ocy454 cells were co-transfected with CaMKIIδ- (Camk2d) and CaMKIIγ- (Camk2g) targeted siRNA prior to mechanical stimulation. Scramble transfected samples demonstrated a significant increase in nitrite production following FSS, but co-knockdown of CaMKIIδ/γ in siRNA-transfected cells inhibited the production of NO• (Fig. 2C); further indicating that NO• production falls within this pathway, subsequent to CaMKII. As siRNA transfection against both eNOS and iNOS prevented nitrite production following FSS (Fig. 1E), we co-transfected Ocy454 cells with siRNA against eNOS and iNOS to maximally suppress NO• production. Co-transfection with eNOS and iNOS siRNA (ieNOS) prior to FSS did not blunt the activation of CaMKII, as measured via phosphorylation of site T286 (Fig. 2D), further supporting that NO• production occurs downstream of CaMKII activation.

3.3. Nitric Oxide Donation Alters Protein Abundance Within Minutes

We have demonstrated above the physiological role that NO• plays in sclerostin-mediated anabolic signaling following FSS, but wanted to further investigate the ability of NO• to effect elements of this signaling pathway independent of mechanical stimulation. Thus, we exposed cells to NONOate, a pharmacological NO• donor. Activation of CaMKII, as determined by phosphorylation at site T286, was increased following one minute of treatment with NONOate, compared to samples collected immediately after treatment (Fig. 3A). Sclerostin protein abundance was also significantly decreased after 5min of NONOate treatment (Fig. 3B). This effect was also observed in ex vivo bone samples. Isolated and flushed control murine long bones exhibited a ~47% reduction in sclerostin protein abundance following 15min of NONOate treatment (Fig. 3C). In contrast, the response to NONOate was fully abrogated in CaMKIIδ/γ double conditional knockout (dCKO) murine long bones, although a lower abundance of sclerostin is found in CaMKIIδ/γ dCKO long bones in the untreated state. These data suggest that while CaMKII is required for NO• production, NO• may also modulate CaMKII to direct sclerostin protein loss in response to FSS.

Fig 3. Nitric Oxide Donation Alters Protein Abundance Within Minutes.

(A) Fold change in relative CaMKII activation (p-T286) in mycSost transfected Ocy454 cells with NONOate (10μM), sample collection immediately (0) or 1 minute (1) after treatment. Representative blots for phospho-CaMKII and panCaMKII. (B) Fold change of relative sclerostin protein expression in mycSost transfected Ocy454 cells following treatment with vehicle or NONOate (10μM, 5min). Representative blots for sclerostin and α-tubulin. C) Fold change of relative sclerostin protein expression following vehicle or NONOate (NONO) (10μM, 15min) treatment in ex vivo CaMKIIδ/γ double conditional knockout (CaMKII) or littermate control (Ctrl) long bone homogenate. Representative blots for sclerostin and total protein. Bones were flushed of marrow and flash frozen before homogenization. (D) Fold change in relative Sost gene expression in Ocy454 cells following vehicle or NONOate (10μM) treatment. Samples collected at indicated time points. (E) Representation of NO’s role within ‘Mechano-Response’ pathway.

As short-term, minute-scale changes to sclerostin protein abundance were observed, changes to Sost gene expression were also quantified. Two, four, or 24 hours after NONOate treatment, no significant changes in Sost gene expression were detectable via RT-qPCR, relative to vehicle treated controls (Fig. 3D). These data suggest that protein expression changes following NO• exposure occur post-translationally, rather than at the level of gene expression, consistent with our prior observations^7–9.

Previously published data⁹, in conjunction with the data presented here, suggest that NO• production does indeed fall within this mechanical response pathway, and that NO• has the ability to support the modulation of calcium influx (Fig. S2) and CaMKII activation to regulate sclerostin degradation and, ultimately, bone formation (Fig. 3E).

4. Discussion

Here, we demonstrated that NO• production participates in a mechano-transduction pathway controlling sclerostin protein abundance. Hierarchically, our data support that mechanical stimulation leads to calcium influx and activation of CaMKII^7,8 prior to the production of NO• by osteocytes, and that NO• is required for the minute-scale loss of sclerostin protein following fluid shear stress⁹. Exogenous NO•, via NONOate, was sufficient to rapidly decrease sclerostin protein. While we have shown that activated CaMKII is necessary for NO• production, NO• also appears to feedback to tune CaMKII activation. Regulation of CaMKII activity by NO• has been described in other cell systems^31–34, and can occur via nitrosylation at Cys280/289 or by increased auto-phosphorylation at Thr286³⁵. NO•’s ability to suppress TRPV4-mediated calcium influx via negative feedback has been demonstrated in other tissues^36,37, consistent with our findings that NO• appears to tune calcium influx following mechanical stimulation (Fig. S2).

We demonstrated that Ocy454 osteocyte-like cells express all three NOS isoforms, and that eNOS is the most relevant to sclerostin protein control, although iNOS also may contribute to NO• production and sclerostin protein loss. Our siRNA transfection knockdown of NOS2/iNOS was less effective than NOS1/nNOS or NOS3/eNOS knockdown, which could contribute to the downward trending, but not significant, loss of sclerostin expression with iNOS knockdown in Figure 1D. These findings are in concordance with previous in vivo studies, where eNOS isoform-specific knockout mice were deficient in bone volume and deposition^14,15, while adult iNOS knockout mice have no significant basal bone phenotype, but are resistant to bone deposition after re-loading¹⁶. Our in vitro data indicate that nNOS does not appear to be involved in the loss of sclerostin protein following FSS, which is distinct from the roles of iNOS and eNOS and consistent with in vivo data demonstrating that nNOS deletion leads to high bone mass in mice¹⁷.

These data reveal an important mechanism through which NO• can influence bone formation, beyond control of osteoblast mineralization. While these data were generated primarily in vitro in Ocy454 osteocyte-like cells, they align with many other observations both in vitro and in vivo. As sclerostin is a β-catenin pathway inhibitor that suppresses osteoblast differentiation, our findings agree with previous research, which has demonstrated that NO• donation supports both increased β-catenin signaling³⁸ and osteoblast differentiation²⁰. Additionally, Ocy454 cells have been shown to express many mature murine osteocyte markers and be broadly representative of primary osteocytes^21,39,40, but like any cell line, they are not identical to in situ primary cells.

These findings are significant because there are many clinically available treatments for osteoporosis, but very few that target osteoblast function⁴¹. In vitro and ex vivo NO• donation is sufficient to stimulate decreased sclerostin protein abundance, which supports osteoblastogenesis and matrix deposition. NO• donation may yet be a promising area of investigation into the prevention or adjuvant treatment of osteoporosis¹¹.