Connexin 43 Modulates β‐Catenin–Dependent Transcription and Secretory Responses to Oscillatory Fluid Flow in Osteocytes

TL;DR

This study shows that Connexin 43 in osteocytes influences β-catenin signaling and gene responses to mechanical forces, linking cell membrane activity to gene regulation.

Contribution

The study reveals a novel physical interaction between Cx43 and β-catenin, showing how Cx43 modulates transcriptional responses to mechanical loading in osteocytes.

Findings

Cx43 knockout cells showed reduced active β-catenin but increased β-catenin/TCF transcriptional activity.

Cx43 deficiency altered flow-responsive gene expression and secretome profiles, including increased OPG and reduced PGE₂.

Cx43 physically interacts with β-catenin, linking mechanosensing to nuclear gene regulation in osteocytes.

Abstract

Osteocytes detect and transduce mechanical cues into biochemical signals that regulate bone remodeling. Connexin 43 (Cx43) is the predominant gap junction protein in osteocytes, but its role in β‐catenin signaling during mechanical loading remains unclear. Wild‐type (WT) and Cx43 knockout (KO) OCY454 osteocytes were subjected to oscillatory fluid flow (10 dynes/cm² 1 Hz) using the Flexcell Streamer system. β‐catenin activation was assessed by Western blot and TOPflash reporter assays. Gene expression and secreted factors were quantified by qPCR and ELISA. Co‐immunoprecipitation revealed a Cx43–β‐catenin interaction in WT cells. KO cells exhibited reduced active β‐catenin protein but paradoxically elevated β‐catenin/TCF transcriptional activity. Cx43 deficiency altered flow‐responsive expression of Rankl and Col1a1, reduced baseline Ptgs2 and Gja1, and shifted the secretome toward increased OPG and reduced PGE₂ after flow. Cx43 regulates β‐catenin signaling through physical interaction and modulation of transcriptional output, linking membrane‐level mechanosensing to nuclear gene regulation. These findings extend prior models of Cx43 as a mechanosensory scaffold by demonstrating its role in coordinating β‐catenin activation and transcriptional responses to mechanical loading in osteocytes.

Figures

Loss of connexin 43 (Cx43) impairs gap junction communication in osteocytes. (A) Western blot analysis confirmed reduced Cx43 expression in knockout (KO) osteocytes compared to wild type (WT). (B) Functional impairment of gap junctions was validated using a parachute dye transfer assay, showing reduced dye spread in KO cells. Quantification was performed across n = 8 experiments. All western blot quantification was normalized to total protein measured from the stain‐free blot.

Cx43 regulates β‐catenin signaling through direct interaction. (A) Co‐immunoprecipitation (Co‐IP) revealed Cx43 association with β‐catenin in WT osteocytes. (B) Western blot analysis showed altered phosphorylation states of β‐catenin, with decreased non‐phospho (active) β‐catenin in KO cells. Data represent n = 3 independent assays.

Increased β‐catenin/TCF transcriptional activity in Cx43‐deficient osteocytes. TOPflash luciferase reporter assays demonstrated significantly elevated transcriptional activity in KO cells compared to WT under static and flow conditions. Cells were exposed to oscillatory fluid flow (2 h, 1 Hz, 10 dynes/cm²), and cells were harvested 4 h after initiating flow.

Fluid flow modulates osteocyte gene expression in a Cx43‐dependent manner. Cells were subjected to oscillatory fluid flow (15 min, 1 Hz, 10 dynes/cm²), and RNA was harvested 4 h after initiating fluid flow. Quantitative PCR analysis revealed significant genotype and fluid flow interactions in RANKL and col1a1 expression, suggesting these genes' response to loading is dependent on Cx43. Ptgs2 and Gja1 expression were decreased in KO cells. Gene expression was normalized to Gapdh. Results represent n = 5–6 biological replicates, analyzed by two‐way ANOVA (p < 0.05).

Cx43 regulates osteocyte secretome composition following mechanical stimulation. (A) Cells were subjected to oscillatory fluid flow (15 min, 1 Hz, 10 dynes/cm²), and conditioned media were collected 4 h after initiating flow. Secreted OPG, PGE₂, and the RANKL/OPG ratio were quantified by ELISA. (B) Cells were exposed to oscillatory fluid flow (2 h, 1 Hz, 10 dynes/cm²), and intracellular proteins were measured 4 h after initiating flow. Intracellular OPG, RANKL, sclerostin, and the RANKL/OPG ratio were quantified by ELISA. Data represent n = 12 experiments, though secreted factors were not detected in every sample. Statistical analysis was performed using two‐way ANOVA with Tukey's post hoc testing (p < 0.05).

Full text

Introduction

1

Bone is a dynamic tissue that continually adapts its structure in response to mechanical demands. Osteocytes, the most abundant and long‐lived bone cells, are central to this process, acting as mechanosensors that detect and transduce physical cues into biochemical signals that regulate bone formation and resorption. Mechanical forces such as weight‐bearing activity generate interstitial fluid flow within the lacunocanalicular network, producing shear stresses on osteocytes that activate anabolic signaling pathways, including WNT/β‐catenin [1, 2, 3]. Understanding how these load‐induced signals are initiated and integrated at the cellular level is critical for identifying strategies to preserve skeletal integrity and prevent fragility.

Osteocytes reside within the mineralized matrix and extend dendritic processes through canaliculi to detect mechanical stimuli [4]. Fluid shear stress deforms the osteocyte cytoskeleton, prompting the release of paracrine factors such as prostaglandin E₂ and sclerostin, and transmitting signals via gap junctions to osteoblasts and osteoclasts [5]. This multicellular communication network orchestrates localized bone remodeling in response to mechanical loading.

Connexin 43 (Cx43) is the predominant gap junction protein in osteocytes and osteoblasts, facilitating both intercellular communication and hemichannel signaling. Recent reviews, including Ma L et al. 2024 [6], have synthesized evidence that Cx43 not only mediates direct cell–cell communication but also serves as a scaffold for signaling complexes, influencing mechanotransduction and bone homeostasis. Mechanistic studies have shown that Cx43 interacts directly with β‐catenin to regulate gene expression in response to mechanical loading [7], and that osteoblast‐ and osteocyte‐specific deletion of Cx43 alters skeletal responsiveness to load [8]. These findings suggest that Cx43 may influence β‐catenin–dependent transcription through both structural and signaling roles. We hypothesized that β‐catenin binds to Cx43, sequestering it and thereby modulating nuclear translocation and activation of anabolic signaling in response to mechanical loading.

Materials and Methods

2

Cell Culture and Differentiation

2.1

OCY454 cells were maintained in α‐MEM supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin‐streptomycin (Gibco) at 33°C under permissive conditions. To induce differentiation, cells were cultured at 37°C for 7 days prior to experimentation, as previously described [9].

Generation and Validation of Connexin 43 Knockout

2.2

Connexin 43 (Cx43) knockout OCY454 cells were generated using CRISPR/Cas9 targeting the Gja1 gene with the single‐guide RNA sequence 5′‐AACCCTACCCCCCCA‐3′, designed to introduce a frameshift mutation near the N‐terminus of Cx43. The guide RNA vector, Cas9, and puromycin resistance cassette were cotransfected into OCY454 cells, and puromycin‐resistant single‐cell clones were isolated and expanded as previously described in Hoppock et al [9]. Clones were screened using the Surveyor mismatch endonuclease assay, and Sanger sequencing of the targeted Gja1 locus confirmed the presence of nonsense mutations generated by frameshift. Loss of Cx43 protein expression in the validated clone used for this study was further verified by Western blotting (anti‐Cx43, Sigma‐Aldrich). Gap junction–mediated intercellular communication was assessed using the parachute dye‐transfer assay performed exactly as previously described in Hoppock et al [9]. Briefly, donor OCY454 cells were loaded with calcein‐AM, washed, trypsinized, and gently added onto confluent unlabeled recipient cells. After co‐culture, cells were collected, and calcein transfer was quantified by flow cytometry.

Oscillatory Fluid Flow

2.3

OCY454 cells were sub‐cultured onto glass slides and maintained for 24 h until reaching 80%–90% confluence, a density at which stable cell–cell contacts and functional gap junctions are established prior to mechanical stimulation. Fluid flow was applied using the Osci‐Flow controller (Flexcell International). Two loading protocols were applied: (1) 2 h of flow at 1 Hz and 10 dynes/cm², followed by 2 h of post‐flow incubation; and (2) 15 min of flow under the same parameters, followed by 3 h 45 min of post‐flow incubation. Static control cells were maintained in identical chambers without flow. The applied shear stress of 10 dynes/cm² falls within the estimated physiological range experienced by osteocytes in vivo (∼8–30 dynes/cm²) during moderate to high mechanical loading in the lacunocanalicular network [10, 11], providing a model that closely approximates native mechanical cues. All treatments were performed in α‐MEM supplemented with 10% FBS, with fresh media applied during post‐flow incubation.

TOPflash Luciferase Reporter Assay

2.4

To assess Wnt/β‐catenin signaling, cells were transfected with the TOPflash luciferase reporter construct (Addgene) using Lipofectamine 3000 (Thermo Fisher Scientific). A Renilla luciferase vector was co‐transfected to normalize transfection efficiency. Luciferase activity was measured using the Dual‐Luciferase Reporter Assay System (Promega) 24 h post‐transfection following static or 2 h flow conditions. Values were normalized to Renilla and expressed as fold change relative to static controls [12].

Co‐Immunoprecipitation and Western Blot Analysis

2.5

Cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific). For co‐immunoprecipitation, 500 µg of lysate was incubated overnight at 4°C with anti‐β‐catenin antibodies—either total β‐catenin (CST #9562S) or active, non‐phosphorylated β‐catenin (CST #8814S)—followed by capture with Protein A/G magnetic beads (Thermo Fisher Scientific). Eluted proteins were resolved by SDS‐PAGE and transferred to PVDF membranes. Blots were probed with an antibody against Cx43 (Sigma‐Aldrich) and visualized using enhanced chemiluminescence. Band intensities were quantified using a ChemiDoc system (Bio‐Rad) and normalized to total protein from the stain‐free gel image. In parallel, western blots were performed to quantify active β‐catenin, total β‐catenin, and the active/total β‐catenin ratio for all expression analyses.

Quantitative PCR (qPCR)

2.6

Total RNA was extracted using the RNeasy Mini Kit (Qiagen) and reverse‐transcribed using the iScript cDNA Synthesis Kit (Bio‐Rad). Gene expression of Col1a1, Gja1, Ptgs2, Tnfsf11 (RANKL), Dmp1, Tnfrsf11b (OPG), Sost, and Wnt1 was quantified using SsoAdvanced Universal SYBR Green Supermix (Bio‐Rad, Hercules, CA, USA) on a CFX96 Touch Real‐Time PCR Detection System (Bio‐Rad). Gapdh served as the reference gene. All primers were Bio‐Rad PrimePCR SYBR Green Assays (Bio‐Rad) pre‐validated according to MIQE guidelines, intron‐spanning when possible, and optimized for amplification efficiency. The following assays were used: Col1a1 (qMmuCED0045486, Chr17:94,449,161–94,450,082), Gja1 (qMmuCED0045489, Chr10:56,975,432–56,976,351), Ptgs2 (qMmuCED0045488, Chr1:150,109,001–150,109,920), Tnfsf11 (qMmuCED0045493, Chr14:43,741,210–43,742,129), Dmp1 (qMmuCED0045485, Chr5:104,223,876–104,224,795), Tnfrsf11b (qMmuCED0045494, Chr15:51,395,221–51,396,140), Sost (qMmuCED0045492, Chr11:101,961,432–101,962,351), Wnt1 (qMmuCED0045495, Chr15:98,456,321–98,457,240), and Gapdh (qMmuCED0027497, Chr6:125,107,123–125,108,042).

ELISA

2.7

Conditioned media and cell lysates were collected following treatments. OPG, RANKL, SOST, and PGE_2_ levels were measured using ELISA kits (R&D Systems) per the manufacturer's instructions. Absorbance was read at 450 nm with background correction. Concentrations were interpolated from standard curves generated with known standards.

Statistical Analysis

2.8

Analyses were performed in GraphPad Prism 9. Data are presented as mean ± standard deviation (SD). Comparisons between two groups were conducted using unpaired two‐tailed Student's t‐tests. For experiments involving multiple factors (e.g., genotype × flow condition), two‐way ANOVA with Tukey's post hoc test was applied. Replicate numbers (n) and statistical tests are detailed in figure legends. Statistical significance was defined as p < 0.05.

Use of AI Tools

2.9

During manuscript preparation, Microsoft Copilot (an AI assistant powered by large language models) was used to assist with reference verification, citation formatting, and editorial refinement of select sentences for clarity and conciseness. All scientific content, interpretations, and conclusions were developed and verified by the authors. Copilot was not involved in data analysis, experimental design, or authorship decisions.

Results

3

Cx43 Deficiency Impairs Gap Junction Function and Disrupts Mechanotransduction

3.1

Western blot analysis confirmed reduced Cx43 expression in KO osteocytes compared to WT controls (Figure 1A). Total protein loading was verified using stain‐free imaging prior to ECL exposure. Functional impairment of gap junction communication was validated using a parachute dye transfer assay, which showed significantly reduced dye spread in KO cells (p < 0.05, Figure 1B).

Loss of connexin 43 (Cx43) impairs gap junction communication in osteocytes. (A) Western blot analysis confirmed reduced Cx43 expression in knockout (KO) osteocytes compared to wild type (WT). (B) Functional impairment of gap junctions was validated using a parachute dye transfer assay, showing reduced dye spread in KO cells. Quantification was performed across n = 8 experiments. All western blot quantification was normalized to total protein measured from the stain‐free blot.

Cx43 Regulates β‐Catenin Signaling Through Direct Interaction and Phosphorylation Control

3.2

Co‐immunoprecipitation revealed an interaction between Cx43 and active, non‐phosphorylated β‐catenin in WT osteocytes (Figure 2A). Inactive, phosphorylated β‐catenin did not associate to Cx43. Western blot analysis showed no significant difference in active β‐catenin levels between WT and KO cells, although KO cells exhibited a trend toward higher total β‐catenin (p = 0.067) (Figure 2B). As a result, the active/total β‐catenin ratio was significantly higher in WT cells, indicating that a greater proportion of β‐catenin was maintained in the active, non‐phosphorylated state in the presence of Cx43. These data support a model in which Cx43 facilitates β‐catenin activation through direct interaction and post‐translational regulation.

Cx43 regulates β‐catenin signaling through direct interaction. (A) Co‐immunoprecipitation (Co‐IP) revealed Cx43 association with β‐catenin in WT osteocytes. (B) Western blot analysis showed altered phosphorylation states of β‐catenin, with decreased non‐phospho (active) β‐catenin in KO cells. Data represent n = 3 independent assays.

Cx43 Deficiency Enhances β‐Catenin/TCF Transcriptional Activity

3.3

TOPflash luciferase reporter assays demonstrated significantly elevated β‐catenin/TCF transcriptional activity in KO cells compared to WT in response to fluid flow (Figure 3). This increase occurred despite reduced levels of active β‐catenin protein, suggesting that loss of Cx43 may improve nuclear access or alter co‐factor engagement for β‐catenin.

Increased β‐catenin/TCF transcriptional activity in Cx43‐deficient osteocytes. TOPflash luciferase reporter assays demonstrated significantly elevated transcriptional activity in KO cells compared to WT under static and flow conditions. Cells were exposed to oscillatory fluid flow (2 h, 1 Hz, 10 dynes/cm²), and cells were harvested 4 h after initiating flow.

Cx43 Modulates Osteocyte Gene Expression in Response to Mechanical Loading

3.4

Following short‐duration flow (15 min of oscillatory fluid flow and 3 h and 45 min of post‐flow incubation), qPCR analysis revealed distinct genotype‐ and flow‐dependent patterns across the genes examined (Figure 4). For Ptgs2 and Gja1, there was a significant main effect of genotype, with KO cells showing reduced Gja1 expression under static conditions, while Ptgs2 levels did not differ significantly between groups. Col1a1 exhibited a significant genotype × flow interaction, driven by higher Col1a1 expression in KO cells compared to WT under static conditions. RANKL also showed a significant interaction, with KO cells displaying increased RANKL expression under flow relative to their own static condition. Together, these findings indicate that Cx43 deletion alters baseline expression of several mechanically responsive genes and modifies the pattern of flow‐induced regulation.

Fluid flow modulates osteocyte gene expression in a Cx43‐dependent manner. Cells were subjected to oscillatory fluid flow (15 min, 1 Hz, 10 dynes/cm²), and RNA was harvested 4 h after initiating fluid flow. Quantitative PCR analysis revealed significant genotype and fluid flow interactions in RANKL and col1a1 expression, suggesting these genes' response to loading is dependent on Cx43. Ptgs2 and Gja1 expression were decreased in KO cells. Gene expression was normalized to Gapdh. Results represent n = 5–6 biological replicates, analyzed by two‐way ANOVA (p < 0.05).

Cx43 Regulates Osteocyte Secretome Composition Following Mechanical Loading

3.5

ELISA quantification of secreted and intracellular proteins revealed genotype‐dependent differences in osteocyte signaling output (Figure 5). For secreted factors collected after short‐duration flow (Figure 5A), OPG showed a significant genotype × flow interaction: KO cells expressed higher OPG than WT under both static and flow conditions, and flow reduced OPG only in KO cells. PGE₂ exhibited a significant main effect of genotype, with KO cells producing lower levels than WT across conditions. The RANKL/OPG ratio showed a significant main effect of genotype, with KO static values significantly lower than WT static.

Cx43 regulates osteocyte secretome composition following mechanical stimulation. (A) Cells were subjected to oscillatory fluid flow (15 min, 1 Hz, 10 dynes/cm²), and conditioned media were collected 4 h after initiating flow. Secreted OPG, PGE₂, and the RANKL/OPG ratio were quantified by ELISA. (B) Cells were exposed to oscillatory fluid flow (2 h, 1 Hz, 10 dynes/cm²), and intracellular proteins were measured 4 h after initiating flow. Intracellular OPG, RANKL, sclerostin, and the RANKL/OPG ratio were quantified by ELISA. Data represent n = 12 experiments, though secreted factors were not detected in every sample. Statistical analysis was performed using two‐way ANOVA with Tukey's post hoc testing (p < 0.05).

For intracellular proteins measured after longer‐duration flow (Figure 5B), OPG and sclerostin showed significant main effects of genotype, with KO cells expressing higher levels than WT under both static and flow conditions. RANKL displayed a significant genotype × flow interaction, driven by higher RANKL in KO cells under flow. The intracellular RANKL/OPG ratio showed no significant effects, indicating that genotype‐dependent differences in intracellular protein abundance did not translate into ratio‐level changes. Together, these findings indicate that Cx43 deletion alters both the baseline abundance and flow‐related modulation of key osteocyte‐derived signaling molecules.

Discussion

4

This study demonstrates that Connexin 43 (Cx43) plays a multifaceted role in osteocyte mechanotransduction, regulating gap junction communication, β‐catenin signaling, and transcriptional and secretory responses to fluid flow. Loss of Cx43 impaired intercellular dye transfer and reduced expression of Cx43 protein, confirming functional knockout (Figure 1). These structural deficits were accompanied by altered β‐catenin dynamics: although active β‐catenin levels were reduced in KO cells (Figure 2B), transcriptional activity measured by TOPflash was paradoxically elevated (Figure 3), consistent with reports that Cx43 can modulate transcription factor activity independent of its channel function [7].

To clarify, the “active” β‐catenin antibody used in this study recognizes β‐catenin that is not phosphorylated at Ser33/Ser37/Thr41, the N‐terminal residues targeted by the destruction complex for ubiquitination and degradation. Conversely, the phospho‐β‐catenin antibody detects phosphorylation at these same residues, which marks β‐catenin for turnover rather than transcriptional activation. Activating phosphorylation at Ser552 (AKT) or Ser675 (PKA), which enhances β‐catenin transcriptional activity, occurs at distinct regulatory sites not examined here. This observation is in line with the model proposed over a decade ago by Plotkin and Laird [13] and expanded in Ma L et al. 2024 [6], which describes the C‐terminal domain of Cx43 as a scaffold for signaling proteins and a target for phosphorylation events that regulate these interactions. Our data provide direct experimental support for that model in osteocytes, showing that Cx43 physically associates with β‐catenin and that its absence alters β‐catenin activation status, thereby linking the structural scaffold role described in these reviews to a specific transcriptional effector pathway. Although imaging‐based co‐localization under flow would provide additional spatial resolution, this was not technically feasible because the Flexcell Streamer system places cells within a sealed flow chamber that cannot be accessed or imaged during loading. Nonetheless, our co‐immunoprecipitation experiments confirm a physical interaction between Cx43 and β‐catenin, and the flow‐dependent transcriptional responses observed in WT and KO cells support the functional relevance of this interaction during mechanical stimulation. Future studies using imaging‐compatible flow platforms will be required to define the spatial dynamics of Cx43–β‐catenin interactions in real time. This finding aligns with prior observations of Cx43‐dependent regulation of WNT/β‐catenin signaling under mechanical loading [7, 14] and specifically reflects regulation at the destruction‐complex phosphorylation sites rather than the AKT‐ or PKA‐dependent activating sites described in other contexts. Additional work has shown that phosphorylation of specific serine residues in the C‐terminal tail can alter Cx43's binding affinity for β‐catenin and other transcriptional regulators [15] and our findings indicate that this regulation occurs upstream of the N‐terminal β‐catenin phosphorylation events that control its stability.

Consistent with this, under static conditions we observed a significantly higher active/total β‐catenin ratio in WT cells, accompanied by a trend (p = 0.067) toward higher total β‐catenin in KO cells and no significant difference in active β‐catenin levels between genotypes. These findings align with prior reports that Cx43 knockdown can alter β‐catenin abundance in osteocytes [7]. Together, the static data indicate that Cx43 promotes a greater proportion of β‐catenin in the active pool, whereas its absence permits accumulation of total β‐catenin with a lower fraction in the active state, supporting a role for Cx43 in coordinating both β‐catenin abundance and activation status.

Although active β‐catenin levels were reduced in KO cells, several lines of evidence help explain the enhanced transcriptional response to flow. Cx43's C‐terminal tail functions as a scaffold for β‐catenin and components of the destruction complex, and loss of this interaction can increase β‐catenin turnover while simultaneously reducing cytoplasmic sequestration, thereby enhancing nuclear availability [16, 17]. In addition, Cx43 has been shown to regulate transcription factor complexes independently of β‐catenin abundance, including effects on TCF/LEF activity and co‐factor [18, 19]. Mechanical loading can also activate β‐catenin–dependent transcription through pathways that do not require stabilization of the Ser33/Ser37/Thr41‐unphosphorylated form, such as PI3K–Akt, PKA, and GSK3β‐dependent signaling [20, 21, 22]. Together, these mechanisms provide a coherent explanation for how β‐catenin transcriptional output can increase in Cx43‐deficient osteocytes despite reduced levels of “active” β‐catenin protein.

In our earlier review [23], we proposed a model to explain why Cx43 deficiency enhances the skeletal response to mechanical load. This model posited that Cx43 binds β‐catenin under basal conditions, sequestering it in the cytoplasm. Mechanical loading increases β‐catenin levels, and in the absence of Cx43, more β‐catenin would be available for nuclear translocation, thereby amplifying the activation of anabolic genes in Cx43‐deficient cells. The present findings partially support this framework: consistent with the model, Cx43 knockout cells exhibit greater transcriptional activation in response to oscillatory fluid flow. However, in contrast to the predicted increase in β‐catenin protein, we observed a reduction in active β‐catenin levels in the knockout. This discrepancy suggests that Cx43 may influence β‐catenin stability or activation state, such that its absence accelerates β‐catenin turnover while still permitting enhanced transcriptional output—possibly through altered co‐factor recruitment, promoter accessibility, or compensation by parallel mechanosensitive pathways. Thus, while the transcriptional phenotype aligns with our prior model, the protein‐level data refine it, indicating additional regulatory layers that integrate Cx43 with β‐catenin signaling during mechanotransduction.

Similar β‐catenin regulatory phenomena have been reported in other mechanosensitive cell types, including endothelial cells exposed to shear stress [24]. and chondrocytes under compressive loading [25]. suggesting that Cx43‐mediated modulation of transcription factor activity may be a conserved feature of mechanically active tissues. Gene expression analysis revealed that Cx43 deficiency altered baseline expression of several mechanically responsive genes, including reduced Gja1 and modestly lower Ptgs2 levels under static conditions. Col1a1 and Rankl exhibited significant genotype × flow interactions: KO cells showed higher Col1a1 expression than WT under static conditions, and Rankl expression increased in KO cells under flow relative to their own static condition. These patterns indicate that Cx43 deletion modifies both baseline transcription and the way osteocytes integrate mechanical cues [26, 27]. The altered secretome profile we observed—including consistently higher OPG and lower PGE₂ production in KO cells across conditions, as well as genotype‐dependent differences in the secreted RANKL/OPG ratio—parallels reports that osteocyte‐derived factors are sensitive to both mechanical and metabolic cues [28, 29]. Intracellular OPG and sclerostin were also elevated in KO cells, and RANKL showed a genotype × flow interaction, indicating that Cx43 deletion influences both the baseline abundance and flow‐related modulation of key osteocyte signaling molecules. These changes highlight that Cx43 shapes the balance of pro‐ and anti‐resorptive signals, particularly under static conditions, with potential consequences for downstream remodeling pathways. Notably, altered OPG/RANKL ratios have been implicated in postmenopausal osteoporosis [30]. and diabetic bone disease [31]. underscoring the potential clinical relevance of our findings.

Importantly, the disconnect between β‐catenin protein levels and transcriptional activity in KO cells highlights a non‐canonical regulatory mechanism, potentially involving altered promoter accessibility or co‐factor dynamics [32]. It is also important to note that the phosphorylation sites examined in this study (Ser33/Ser37/Thr41) regulate β‑catenin degradation, whereas activating phosphorylation at Ser552 or Ser675 enhances transcriptional activity through distinct pathways. Thus, our findings reflect changes in β‑catenin stability rather than AKT‑ or PKA‑mediated activation. Cx43 expression declines with age, and osteocyte mechanosensitivity is impaired in aged bone [28]. Evidence from Zhao D et al. 2020 [32] shows that Cx43 channel function modulates skeletal responses to unloading. This work complements recent mechanistic findings by Zeng Y et al. 2022 [33], who identified Piezo1‐mediated Ca²⁺ influx as an upstream activator of Cx43 hemichannels via PI3K–Akt signaling. Together, these studies suggest that Cx43's regulation of β‐catenin may be integrated with other mechanosensitive pathways. Targeting Cx43–β‐catenin interactions, or their upstream modulators, could represent a novel therapeutic strategy for restoring mechanosensitivity in aging and disease.

In summary, this study identifies Connexin 43 as a key integrator of osteocyte mechanotransduction, linking gap junction communication to β‐catenin–dependent transcriptional responses under oscillatory fluid flow. Loss of Cx43 disrupted canonical and non‐canonical β‐catenin signaling, altered the expression of mechanically responsive genes, and reshaped the osteocyte secretome, with potential consequences for bone remodeling. These findings position Cx43 not only as a structural conduit for intercellular communication but also as a regulatory scaffold that modulates transcription factor activity in response to mechanical cues. Future work should define the specific post‐translational modifications and binding partners that govern Cx43–β‐catenin interactions, determine how these mechanisms are altered in aging and disease, and test whether restoring Cx43 function or targeting its upstream mechanosensitive pathways can rescue osteocyte mechanosensitivity and improve skeletal outcomes in vivo.