TRPV4 calcium influx controls sclerostin protein loss independent of purinergic calcium oscillations

Abstract

Skeletal remodeling is driven in part by the osteocyte’s ability to respond to its mechanical environment by regulating the abundance of sclerostin, a negative regulator of bone mass. We have recently shown that the osteocyte responds to fluid shear stress via the microtubule network-dependent activation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species and subsequent opening of TRPV4 cation channels, leading to calcium influx, activation of CaMKII, and rapid sclerostin protein downregulation. In addition to the initial calcium influx, purinergic receptor signaling and calcium oscillations occur in response to mechanical load and prior to rapid sclerostin protein loss. However, the independent contributions of TRPV4-mediated calcium influx and purinergic calcium oscillations to the rapid sclerostin protein downregulation remain unclear. Here, we showed that NOX2 and TRPV4-dependent calcium influx is required for calcium oscillations, and that TRPV4 activation is both necessary and sufficient for sclerostin degradation. In contrast, calcium oscillations are neither necessary nor sufficient to acutely decrease sclerostin protein abundance. However, blocking oscillations with apyrase prevented fluid shear stress induced changes in osterix (Sp7), osteoprotegerin (Tnfrsf11b), and sclerostin (Sost) gene expression. In total, these data provide key mechanistic insights into the way bone cells translate mechanical cues to target a key effector of bone formation, sclerostin.

1. Introduction

The skeleton dynamically responds to mechanical stimuli to direct bone formation and resorption. Bone embedded osteocytes sense and respond to mechanically induced fluid shear stress (FSS) in the lacunar canalicular network to regulate effectors of bone remodeling. Mechanistically, osteocytes exhibit a rapid increase in intracellular calcium (Ca²⁺) followed by prolonged cytosolic Ca²⁺ oscillations in response to mechanical stimulation [1–5]. While these distinct Ca²⁺ responses are well characterized both in vitro and in vivo, the mechanisms of how these Ca²⁺ responses integrate into signal pathways that regulate effectors of bone formation and remodeling, such as RANKL, PGE2, and sclerostin, and the precise roles of Ca²⁺ influx versus Ca²⁺oscillation have remained ambiguous [6–8].

Sclerostin regulation is of particular interest. Sclerostin is an osteocyte-derived glycoprotein that inhibits the canonical Wnt/β-catenin signaling pathway and its deletion dramatically increases bone mass [9]. Mechanical stimulation reduces sclerostin abundance in osteocytes and derepresses bone formation [10–12]. Understanding the hierarchical effects of these pathways and their biological consequences on bone cells is critical to developing therapeutic applications in skeletal fragility.

We recently described a pathway by which osteocytes sense and respond to mechanical load and rapidly decrease sclerostin protein abundance [13]. In this pathway, FSS initiates Ca²⁺ influx through TRPV4 channels in the osteocyte plasma membrane as a result of mechano-activation of NADPH-oxidase 2 (NOX2) and the production of reactive oxygen species (ROS) [13]. TRPV4-dependent Ca²⁺ influx activates Ca²⁺/calmodulin-dependent kinase II (CaMKII) and decreases sclerostin protein within 5 min of FSS. Similar to our work, others have shown that TRPV4 is among the mechanically activated plasma membrane channels contributing to Ca²⁺ influx in osteocytes and their progenitors [14–16]. However, we did not systematically test the independent contributions of the TRPV4-dependent initial Ca²⁺ influx and the subsequent prolonged cytosolic Ca²⁺ oscillations to rapid loss of sclerostin protein. Disentangling these two modes of Ca²⁺ signaling may provide useful insights into how osteocytes respond to mechanical stimulation.

The Ca²⁺oscillations observed in mechanically stimulated osteocytes [1–5] are a result of ATP release and signaling through both P2X and P2Y type purinergic receptors [17–21]. P2X receptors are ATP-gated cation channels that allow for Ca²⁺influx, and P2Y receptors are G protein-coupled receptors that lead to Ca²⁺release from intracellular, IP₃-sensitive stores [22]. P2X7 receptors have been strongly linked to osteocyte mechanical responses, and mice lacking P2X7R show a decreased response to mechanical load [23,24]. Further, the magnitude of ATP release and frequency of Ca²⁺ waves following mechanical load is proportional to the strain perceived by the osteocyte [1,3,17]. While ATP-purinergic receptor dependent Ca²⁺ oscillations are a consequence of mechanical load, no data link purinergic receptor signaling directly to sclerostin protein abundance, and little is known about how the initial influx of Ca²⁺ regulates subsequent Ca²⁺ oscillations.

In this study, we systematically examined the roles of the primary TRPV4-dependent Ca²⁺ influx and the subsequent Ca²⁺ oscillations in osteocyte control of CaMKII signaling and sclerostin downregulation. Understanding the respective contributions of each of these Ca²⁺ signal pathways to the osteocyte mechano-response could have therapeutic implications. For example, if oscillations prove to be a feedback loop repressing the mechano-response, then limiting them could enhance anabolic responses to load. Conversely, if Ca²⁺ oscillations reinforce mechanical load signals then mimicking or sustaining them could be a viable approach to increasing mechanical load responses of bone.

2. Methods

2.1. Chemicals and reagents

GSK1016790A, GSK2193874, and apyrase were purchased from Sigma. Fluo-4 AM ester and ATP disodium trihydrate were purchased from ThermoFisher. PPADS (LX-550-347-M010) was purchased from Enzo LifeSciences. gp91-dsTAT was from AnaSpec (AS-63818). Antibodies were purchased as follows: sclerostin (R&D Systems, AF1589), phospho-CaMKII Thr286 (Cell Signaling Technology, 12716S), and total CaMKII (Cell Signaling Technology, 3362).

2.2. Cell culture and treatments

Osteocyte-like Ocy454 cells (provided by Dr. Divieti-Pajevic, Boston University) were cultured on type I rat tail collagen (BD Biosciences) coated dishes in α-MEM supplemented with 10% FBS at 33 °C [25]. Prior to experiments, Ocy454 cells were seeded at 125,000 cells/cm² into a tissue culture treated vessel and maintained at 37 °C and 5% CO₂ for a minimum of 24 h. We observe detectable sclerostin at this time-point even though it is further enriched by time in culture (Supplemental Fig. 1A). The specificity of the antibody (Sost+/+ versus Sost−/− long bone extracts) have also been validated (Supplemental Fig. 1B). To activate Ca²⁺ influx through TRPV4 in the absence of FSS, cells were treated with the TRPV4 agonist GSK1016790A (15 μM, 30 min). To inhibit TRPV4-dependent Ca²⁺ influx, Ocy454 cells were treated with the TRPV4 antagonist GSK2193874 (15 μM, 30 min) prior to FSS. To inhibit NOX2-dependent production of ROS, cells were treated with gp91-dsTAT (10 μM, 30 min). To activate Ca²⁺ oscillations in the absence of FSS, cells were treated with ATP (1 μm, 5 min). To inhibit FSS-induced Ca²⁺ oscillations, cells were treated with the nonselective P2 purinergic receptor antagonist PPADS (3 μm, 20 min) or apyrase (10 U/mL, 20 min) prior to FSS.

2.3. Fluid flow

Cells in tissue culture treated 96-well plates were exposed to 4 dynes/cm² pulsatile fluid flow for 1 to 5 min, as indicated, using a custom FSS device [26]. Fluid flow was performed in HEPES-buffered Ringer solution containing 140 mM NaCl, 4 mM KCl, 1 mM MgSO₄, 5 mM NaHCO₃, 10 mM glucose, 1.8 mM CaCl₂ and 10 mM HEPES (pH 7.3).

2.4. Calcium imaging

Prior to the experiment, the cells were seeded into optically clear 96-well plates (Corning) and incubated overnight at 37 °C. Cells were then loaded with Fluo-4 AM ester (5 μM, 30 min) in 37 °C Ringer solution, washed, and allowed to rest for 15 min, as described [27]. Time-lapse fluorescence intensity measurements were collected using ImageJ Time Series Analyzer plugin and data analyzed using Microsoft Excel software. A “primary influx” was defined as an increase in Fluo-4 fluorescence intensity of at least 70% from baseline following FSS, while “% cells oscillating” was defined as a Fluo-4 fluorescence increase of at least 20% over baseline and occurring after the termination of flow or subsequent to the initial mechanically induced Ca²⁺ peak. The “# of oscillations per oscillating cell” represents the number of oscillations per cell in cells that exhibited at least one oscillation. Results represent a minimum of three independent experiments performed on separate days with new cultures (n ≥ 149 cells/treatment group). All conditions were run with controls on each experimental day.

2.5. Western blotting

Western blotting was performed using whole cell extracts isolated from cells in culture following FSS, as described [28,29]. For some samples, extracts were split and run on parallel blots for sclerostin/GAPDH and pCaMKII/tCaMKII. Membranes were blocked in 5% non-fat dry milk, 3% bovine serum albumin in 0.1% PBS-T (unless otherwise stated) and probed with the indicated primary antibodies overnight at 4 °C. Bands were detected by enhanced chemiluminescence detection as described [30]. Blots were normalized using GAPDH (for sclerostin) or total CaMKII (for pCaMKII). Quantification of band intensities was performed using ImageLab (BioRad) software. Specificity of the Sclerostin antibody was verified (Supplemental Fig. 1).

2.6. RNA isolation and quantitative reverse transcription and real time PCR

RNA was isolated from cultured OCY454 cells, 1 μg of RNA was reverse transcribed, and ~250 ng of cDNA subjected to quantitative real time PCR as described [31]. Data were normalized to three housekeeping genes (Rpl13, Hprt, and Gapdh) using geNorm v3.5 software (Ghent University Hospital Ghent, Belgium), as described [28]. PCR primers used were: Sost, GGA ATG ATG CCA CAG AGG TCA T and CCC GGT TCA TGG TCT GGT T; Sp7, CCC TAT GGC TCG TGG TAC AAG and CAT GTC CCA CCA AGG AGT AGG T; Tnfrsf11b, GCG TGC AGC GGC ATC T and AGG CTC TCC ATC AAG GCA AGA; Tnfsf11, ACC AGC ATC AAA ATC CCA AGT T and TCA GAA TTG CCC GAC CAG TT; Rpl13, CGA AAC AAG TCC ACG GAG TCA and GAG CTT GGA GCG GTA CTC CTT; Gapdh, CGT GTT CCT ACC CCC AAT GT and TGT CAT CAT ACT TGG CAG GTT TCT; and Hprt, AGC AGT ACA GCC CCA AAA TGG and AAC AAA GTC TGG CCT GTA TCC AA.

2.7. Statistical analysis

Experiments were repeated a minimum of 3 times with triplicate samples, unless indicated otherwise. All data were analyzed using GraphPad Prism 8, as appropriate. For normally distributed data, samples were compared by a t-test, one-way or two-way ANOVA for unpaired samples. For nonparametric data, a two-tailed Mann-Whitney test or Kruskal-Wallis test was performed. A p-value of < 0.05 was used as a threshold for statistical significance.

3. Results

3.1. Activation of NOX2 and TRPV4 is required for FSS-induced intracellular Ca²⁺ oscillations in Ocy454 cells

We have previously shown that FSS rapidly (within 5 min) reduces sclerostin protein abundance in Ocy454 cells as a result of TRPV4-dependent Ca²⁺ influx and the activation of CaMKII (Fig. 1A) [13]. However, we did not examine the interrelationship between TRPV4-dependent Ca²⁺ influx and Ca²⁺ oscillations, nor did we distinguish their independent contributions to the rapid loss of sclerostin protein. In fact, both Ca²⁺ influx and Ca²⁺ oscillations occur in this 5 min timeframe of sclerostin protein loss and could contribute to its regulation. To assess the interrelationship between Ca²⁺ influx and Ca²⁺ oscillations, we exposed Ocy454 cells to FSS and performed live cell Ca²⁺ imaging while inhibiting either TRPV4-dependent Ca²⁺ influx or purinergic signaling. During the 1 min of FSS stimulation, approximately 63% of the cells exhibited a robust Ca²⁺ influx (Fig. 1B–D). Additionally, approximately 20% of the Ocy454 cells continued to exhibit transient intracellular Ca²⁺ oscillations post FSS with oscillating cells producing up to eight Ca²⁺ oscillations. Ca²⁺ oscillations occurred most commonly in cells that exhibited an initial Ca²⁺ influx (Fig. 1D) and were only observed in a small fraction (< 5%) of Ocy454 cells that did not exhibit a primary Ca²⁺ influx above threshold. Consistent with these findings, blocking the initial FSS-induced Ca²⁺ influx with the TRPV4 antagonist GSK2193874 reduced the percentage of cells undergoing subsequent Ca²⁺ oscillations, as well as the number of Ca²⁺ oscillations that occurred per oscillating cell (Fig. 1E). These data show that the initial TRPV4-mediated Ca²⁺ influx is required for Ca²⁺ oscillations. Inhibiting purinergic receptors with PPADS decreased Ca²⁺ oscillations without affecting the number of cells experiencing initial Ca²⁺ influx or magnitude of influx, suggesting these receptors do not contribute to FSS induced primary Ca²⁺ influx (Fig. 1E, Supplemental Fig. 2).

Fig. 1.

Fluid shear stress activates TRPV4-dependent Ca²⁺ influx which drives subsequent Ca²⁺ oscillations. (A) Mechano-transduction pathway in osteocytes that regulates sclerostin abundance. Fluid shear stress (FSS) acts on the microtubule (MT) network to activate NADPH Oxidase 2 (NOX2), which produces reactive oxygen species (ROS). ROS activates TRPV4 channels and extracellular Ca²⁺ influx into the osteocyte, the activation of CaMKII, and a rapid down regulation of sclerostin. (B–C) Ca²⁺ imaging (Fluo-4) of Ocy454 cell exposed to 4 dynes/cm² FSS for 60s and imaged for up to 6 min. Pseudo colored images (B) and corresponding Ca²⁺ trace (C) indicating Fluo-4 fluorescence change over time are shown from a representative experiment. The time stamp of the image is shown in the upper left corner and the corresponding point in the trace for each representative image is shown in the upper right corner. After establishing a baseline ①, an initial Ca²⁺ influx ② is induced by the application of FSS. After FSS has ended, several subsequent Ca²⁺ oscillations are observed (③ through ⑧ in B and C). (D) Graph represents the percentage of cells exhibiting Ca²⁺ influx alone, those displaying both a Ca²⁺ influx and Ca²⁺ oscillation(s), those displaying only Ca²⁺ oscillation(s), or cells that did not have a Ca²⁺ response. (E) Quantitation of the Ca²⁺ responses of Ocy454 cells treated with the TRPV4 antagonist GSK2193874 (15 μM) or the purinergic receptor blocker PPADS (3 μM) and subjected to 4 dynes/cm² fluid shear stress. (F) Quantitation of the Ca²⁺ responses of Ocy454 cells treated with the NOX2 inhibitor gp91-dsTAT (10 μM) subjected to 4 dynes/cm² fluid shear stress. Graphs depict means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control. n ≥ 149 cells from 3 independent experiments.

The mechanically activated Ca²⁺ influx is probably not exclusively through TRPV4. In our prior work, when we inhibit TRPV4 pharmacologically or genetically we still observed a residual FSS-induced Ca²⁺ influx [13]. In bone, other Ca²⁺ channels have been implicated in the mechanically induced Ca²⁺ influx, including T-type voltage gated calcium channels, polycystin, and Piezo channels [32–38]. Accordingly, to more broadly disrupt mechano-signaling, we targeted NOX2. NOX2-generated ROS is both necessary and sufficient for the mechanically activated Ca²⁺ influx and sclerostin downregulation in Ocy454 cells exposed to FSS [13]. Consistent with our prior work, treatment of Ocy454 cells with gp91-dsTAT, a NOX2 inhibitor, reduced the percent of cells with a primary influx subsequent to FSS, as well as the peak amplitude of the Ca²⁺ response (Fig. 1F). Furthermore, inhibition of NOX2 activity with gp91-dsTAT reduced the percentage of cells (22% vs < 1%) undergoing Ca²⁺ oscillations and the number of Ca²⁺ oscillations per cell. Together, these data establish that mechano-activated Ca²⁺ influx is required for oscillations, while disrupting Ca²⁺ oscillations had no impact on the initial Ca²⁺ influx.

To confirm the biological consequence of these distinct Ca²⁺ events on signal transduction and loss of sclerostin protein, we performed western blots on whole cell extracts of Ocy454 cells exposed to acute FSS and then harvested 5 min later. Application of FSS or mimicking the Ca²⁺ influx via direct activation of Ca²⁺ influx with the TRPV4 agonist GSK1016790 was sufficient to rapidly decrease sclerostin protein and activate CaMKII in Ocy454 cells (Fig. 2A). Conversely, the TRPV4 antagonist, GSK2193874, which blocks both Ca²⁺ influx and oscillations, prevented FSS-induced CaMKII phosphorylation and loss of sclerostin (Fig. 2B). Likewise, blocking both Ca²⁺ influx and oscillations with NOX2 inhibition with gp91-dsTAT prevented the mechanically induced activation of CaMKII and decrease in sclerostin protein following FSS (Fig. 2C). These data confirm Ca²⁺ influx is necessary for the rapid loss of sclerostin protein following FSS. At first glance, this data establishes that activation of TRPV4-dependent Ca²⁺ influx is also sufficient to decrease sclerostin protein abundance. However, the kinetics of TRPV4 agonist action on intracellular Ca²⁺ are distinct compared to those seen with FSS, as in agonist treated cells intracellular Ca²⁺ is sustained longer than is seen with FSS and oscillations cannot be resolved. Thus, it remained unclear if Ca²⁺ oscillations are also part of the pathway regulating the loss of sclerostin protein. Alternatively, it may be the integral of Ca²⁺ in the cytosol that controls sclerostin abundance.

Fig. 2.

TRPV4 and NOX2 activation are required for Sclerostin downregulation. (A) Ocy454 were treated with vehicle (control) and exposed to 4 dynes/cm² FSS (Flow) or treated the TRPV4 agonist GSK1016790 (15 μM) to mimic the Ca²⁺ influx observed with FSS. Five minutes following FSS or treatment with GSK1016790, whole-cell lysates were prepared, then immunoblotted for sclerostin, pCaMKII, total (t)CaMKII, and GAPDH. Sclerostin/GAPDH and pCaMKII/tCaMKII ratios are shown. (B–C) Ocy454 were treated with vehicle (control) or (B) the TRPV4 antagonist GSK2193874 (15 μM) or (C) the NOX2 inhibitor gp91-dsTAT (10 μM) and subjected to static culture conditions (No Flow) or 4 dynes/cm² FSS (Flow). Whole-cell lysates were prepared and immunoblotted as above. Representative blots are shown. Graphs depict mean ± SD from n ≥ 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 *p < 0.05 compared to the no flow, control treated samples.

3.2. ATP-driven oscillations are neither necessary nor sufficient for CaMKII activation and Sclerostin downregulation

Having established that NOX2 ROS-dependent TRPV4 Ca²⁺ influx is required for Ca²⁺ oscillations and reductions in sclerostin protein abundance, we examined if oscillations were necessary or sufficient to drive the downstream signaling that converges on sclerostin. Low concentrations of ATP (1 μM) are sufficient to induce Ca²⁺ oscillations in Ocy454 cells that closely approximate those observed post-FSS (Fig. 3A). Despite mimicking the Ca²⁺ oscillations observed after FSS, ATP failed to activate CaMKII or downregulate sclerostin protein in Ocy454 cells (Fig. 3B). These data strongly suggest that ATP is insufficient to mimic mechanical load responses, at least with respect to loss of sclerostin.

Fig. 3.

ATP-induced Ca²⁺ oscillations are insufficient to induce rapid loss of sclerostin protein. (A) A representative Ca²⁺ trace of an Ocy454 cell treated with ATP (1 μM) indicating Fluo-4 fluorescence (F) change over time. (B) Western blots of whole cell lysates of Ocy454 cells treated with or without ATP (1 μM, 5 min) probed for sclerostin, pCaMKII, total (t)CaMKII, and GAPDH. Representative blots are shown. Graphs depict mean ± SD from 3 independent experiments. No significant differences in sclerostin or pCaMKII were detected between control and ATP treated samples.

Next, we examined if ATP-dependent Ca²⁺ oscillations are required for FSS-induced loss of sclerostin protein. To accomplish this, Ocy454 cells were cultured in the presence of apyrase to degrade extracellular ATP during FSS. In cells exposed to fluid shear in the presence of apyrase, Ca²⁺ influx was unaffected, whereas the percentage of cells undergoing Ca²⁺ oscillations were reduced 10-fold (Fig. 4A). However, the FSS-induced rapid downregulation of sclerostin and phosphorylation of CaMKII still occurred at similar magnitudes as in control cells (Fig. 4B). These data confirm that Ca²⁺-influx, but not Ca²⁺ oscillations, are required for the rapid loss of sclerostin in FSS-stimulated Ocy454 cells.

Fig. 4.

ATP-dependent Ca²⁺ oscillations are not required for the FSS induced rapid loss of sclerostin protein. (A) Ocy454 were subjected to 4 dynes/cm² FSS in the presence or absence of apyrase (10 U/mL) to remove extracellular ATP. The percentage of cells exhibiting an initial Ca²⁺ influx and percentage of cells showing subsequent oscillations were quantified. Graphs depict means ± SEM from n ≥ 149 cells from 3 independent experiments. ***p < 0.001 compared to control. (B) Ocy454 were treated with vehicle (control) or apyrase (10 U/mL) and subjected to static culture conditions (No Flow) or 4 dynes/cm² FSS (Flow). Five minutes following FSS, whole-cell lysates were prepared and immunoblotted for sclerostin, pCaMKII, total (t)CaMKII, and GAPDH. Representative blots are shown. Sclerostin/GAPDH and pCaMKII/tCaMKII ratios are shown. Graphs depict mean ± SD from n = 3 independent experiments. *p < 0.05 compared to the corresponding no flow control.

3.3. ATP-driven oscillations are required for fluid shear stress induced changes in osteoprotegerin (Tnfrsf11b), osterix (Sp7), and sclerostin (Sost) gene expression

To assess if Ca²⁺ oscillations contributed to osteocyte function following acute mechanical stimulation, Ocy454 cells were exposed to FSS for 5 min in the presence or absence of apyrase (Fig. 5). RNA was isolated 6 h after FSS and subjected to qRT-PCR. FSS increased the gene expression of osterix (Sp7), osteoprotegerin (Tnfrsf11b), and sclerostin (Sost). Apyrase blocked this FSS-induced increase in gene expression. The expression of RANKL (Tnfsf11) was slightly increased by FSS but did not reach statistical significance. Apyrase reduced Tnfsf11 gene expression relative to the control-treated FSS group. These data demonstrate that ATP dependent Ca²⁺ oscillations contribute to long term changes in gene expression following a short bout of FSS.

Fig. 5.

FSS-induced ATP-dependent Ca²⁺ oscillations regulate osteocyte gene expression. Ocy454 were treated with vehicle (control) or apyrase (10 U/ml) and subjected to static culture conditions (No Flow) or 4 dynes/cm² FSS (Flow) for 5 min. Four to six hours post flow, RNA was isolated, reverse transcribed and gene expression was quantified by real time quantitative PCR. Average raw CT values are available in Supplemental Table I. Graphs depict mean ± SEM from n = 6 independent samples. *p < 0.05, **p < 0.01 compared to the corresponding no flow control. n.s., not significant.

4. Discussion

While our prior work showed a requirement for mechanically induced Ca²⁺ for the rapid loss of sclerostin protein, we did not discriminate the effects of Ca²⁺ influx versus Ca²⁺ oscillation. In the present study, we temporally resolved the Ca²⁺ signaling responses, influx and oscillations, and tied them to a measurable changes in osteocyte function. Our data confirm that the initial FSS-induced Ca²⁺ influx, activation of CaMKII, and loss of sclerostin protein occurred via NOX2 ROS and the opening of TRPV4 channels. We also showed the hierarchical relationship between Ca²⁺ influx and Ca²⁺ oscillations, in that the subsequent Ca²⁺ oscillations that occur following fluid shear stresses are also NOX2 and TRPV4 dependent. Conversely, consistent with published work [39,40], we confirmed that inhibition of purinergic receptors or their signaling was sufficient to reduce FSS-induced oscillations with no effect on primary influx. Our data demonstrate that, with respect to the rapid loss of sclerostin protein, it is the initial Ca²⁺ influx that is wholly responsible for the rapid loss of sclerostin protein. Sclerostin protein is rapidly lost following FSS when oscillations are inhibited by the purinergic receptor inhibitor PPADs, but not when NOX2 or TRPV4 were inhibited. In addition, induction of oscillations with ATP alone was insufficient to cause the rapid loss of sclerostin protein. The rapidity of this loss of sclerostin protein would strongly suggest post-translational control, presumably by degradation.

Importantly, FSS-stimulated Ca^2+` oscillations may serve roles in osteocyte function beyond the regulation of sclerostin protein abundance. We observed that a short bout of FSS increases the gene expression of Sp7, Tnfrsf11b, and Sost hours after exposure. Further, the FSS-induced upregulation of these genes requires ATP-dependent signaling as apyrase treatment – which blocks Ca²⁺ oscillations – attenuates the effects of FSS on expression of these genes. These data show that Ca²⁺ influx is responsible for the rapid loss of sclerostin protein, while Ca²⁺ oscillations contribute to gene expression changes.

On its face, the upregulation of Sost gene expression after fluid flow is paradoxical. We expected the gene expression of Sost would diminish after flow. These data would suggest that, with respect to sclerostin, Ca²⁺ oscillations serve as a feedback loop, in which the increase in gene expression is “refilling” sclerostin protein levels following the rapid loss of the protein. If this were the case, then attenuating Ca²⁺ oscillations in mechano-activated osteocytes may enhance the bone anabolic response. This could have obvious therapeutic applications to skeletal fragility, although more data is needed to confirm these findings. It is also possible that a longer flow time would result in genome level reorganization that would lead not only to decreased sclerostin protein levels, but also downregulation of the Sost mRNA. Indeed, in contrast to what we observed with short term fluid flow, long term mechanical stimulation typically decreases Sost mRNA in osteocytes [42–44].

The lack of an effect of Ca²⁺ oscillations on sclerostin protein may not be too surprising given the small percentage of Ocy454 cells (< 25% of cells demonstrate even a single oscillation) that oscillate after FSS. It is unlikely this small percentage of osteocytes would be sufficient to drive the observed ≥50% loss of sclerostin protein across the population of cells.

Notably, these cells rarely demonstrated Ca²⁺ oscillations in the absence of an initial Ca²⁺ influx. Even this small percentage (~5%) of cells typically had an initial Ca²⁺ influx, albeit below our criteria of a 70% increase in baseline Fluo-4 fluorescence. This result was somewhat surprising, as we expected a paracrine action of ATP on neighboring cells that may not have exhibited an initial Ca²⁺ influx. Elevated intracellular Ca²⁺ opens pannexin channels and connexin43 hemi-channels, which have been implicated in ATP release from osteocytes, and ATP is sufficient to activate purinergic calcium signaling [21,45,46]. While we expected this ATP to activate adjacent cells, we observed instead that the ATP effect was mostly autocrine in that it reinforced signaling in a cell that had already mounted a mechano-response. Perhaps this is a unique property of Ocy454 cells, as others have observed paracrine activation of cells following mechanically induced ATP release [47,48]. Alternatively, this may be a caveat of the system we used in which all cells experienced FSS and thus had the potential to release ATP. This result may be far different in bone in which only a subset of cells may experience a strain and yet signal to distal effector cells.

5. Conclusion

Here, we showed that it is the primary Ca²⁺ influx via TRPV4 that is responsible for the activation of CaMKII and the rapid, acute downregulation of sclerostin protein in response to FSS in Ocy454 cells. Blocking TRPV4 or its upstream activator, NOX2, prevents the FSS-induced Ca²⁺ influx and subsequent activation of pCaMKII and decrease in sclerostin. This initial influx via TRPV4 also seems to be required for the FSS-induced oscillations that occur in osteocytes. These Ca²⁺ oscillations are neither necessary nor sufficient for the downregulation of sclerostin protein. However, FSS induced oscillations appear to be important for gene level regulation of osteocyte genes, such as sclerostin (Sost), osterix (Sp7), and osteoprotegerin (Tnfrsf11b). Understanding the way bone cells perceive and respond to their environmental cues is important for understanding and altering bone homeostasis with obvious therapeutic applications in skeletal fragility.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bone.2020.115356.

Abbreviations:

Ca²⁺

calcium

TRPV4

transient receptor potential cation channel subfamily V member 4

NOX2

NADPH Oxidase 2

ROS

reactive oxygen species

CaMKII

calcium/calmodulin-dependent kinase II

RANKL

receptor activator of NFκB

OPG

osteoprotegerin

PGE2

prostaglandin E2

FSS

fluid shear stress

ATP

adenosine triphosphate