Connexin 43 Modulates Osteogenic Differentiation of Bone Marrow Stromal Cells Through GSK-3beta/Beta-Catenin Signaling Pathways

Abstract

Background/Aims: Bone marrow stromal cells (BMSCs) are multipotent precursors that give rise to osteoblasts, and contribute directly to bone formation. Connexin 43 (Cx43) is the most ubiquitous gap junction protein expressed in bone cell types, and plays crucial roles in regulating intercellular signal transmission for bone development, differentiation and pathology. However, the precise role and mechanism of Cx43 in BMSCs are less known. Here, we investigate the function of Cx43 in osteogenic differentiation of BMSCs in vitro. Methods: BMSCs were isolated by whole bone marrow adherent culture. Knock down of Cx43 was performed by using lentiviral transduction of Cx43 shRNA. BMSCs were induced to differentiate by culturing in a-MEM, 10% FBS, 50 µM ascorbic acid, 10 mM beta-glycerophosphate, and 100 nM dexamethasone. Alkaline phosphatase (ALP) activity and alizarin red S staining were used to evaluate osteogenic differentiation in calcium nodules. Target mRNAs and proteins were analyzed by using real-time quantitative PCR (qPCR) and western blotting. Results: Cx43 expression markedly increased during osteogenic differentiation. Osteogenic differentiation was suppressed following lentiviral-mediated knockdown of Cx43 expression, as judged by decreased levels of Runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), osteocalcin (Bglap), Osterix (Osx), alkaline phosphatase (ALP) activity and the number of calcium nodules in response to osteogenic differentiation stimuli. Knock down of Cx43 reduced the level of phosphorylation of GSK-3beta at Ser9 (p-GSK-3beta), resulting in decreased beta-catenin expression and activation. Furthermore, treatment of Cx43-knockdown cells with lithium chloride (LiCl), a GSK-3beta inhibitor, reduced osteogenic differentiation and decreased GSK-3beta levels, as well as partially rescued levels of both total and activated beta-catenin. Conclusion: These findings indicate that Cx43 positively modulates osteogenic differentiation of BMSCs by up-regulating GSK-3beta/beta-catenin signaling pathways, suggesting a potential role for Cx43 in determining bone mass and bone mineral density by modulating osteogenesis.

Simplet-dependent regulation of β-catenin signaling influences skeletal patterning downstream of Cx43

“…This model of Cx43 and β-catenin cooperating to inhibit joint formation is also supported in other model systems. In primary rat BMSCs, Cx43 increases with osteoblast differentiation, and KD suppresses both differentiation and β-catenin expression/activation (Lin et al, 2018). Further, Cx43 has been shown to promote βcatenin signaling in developing rabbit (Liu et al, 2016) and mouse (Moorer et al, 2017) skeletons, although the mechanism(s) of this regulation may not be transcriptional.…”

Imperatorin promotes osteogenesis and suppresses osteoclast by activating AKT/GSK3 β/β‐catenin pathways

“…Although they all have certain curative effects, they also have different limitations including thromboembolism and oesophageal irritation. This leads to the activation of β-catenin and the accumulation of β-catenin in the nucleus [37][38][39][40] However, our results suggest that although imperatorin can inhibit the differentiation of osteoclasts and the expression of NFATC1 and TRAP at high concentration (75 µmol/L), it can promote the osteoclast differentiation and expression of these genes at low concentration (25 µmol/L). Therefore, we intended to find a potential drug for its treatment from natural compounds.…”

“…[33][34][35] Whereas, Fzd receptor complex, leading to the recruitment, phosphorylation and inactivation of GSK3β. This leads to the activation of β-catenin and the accumulation of β-catenin in the nucleus [37][38][39][40] However, our results suggest that although imperatorin can inhibit the differentiation of osteoclasts and the expression of NFATC1 and TRAP at high concentration (75 µmol/L), it can promote the osteoclast differentiation and expression of these genes at low concentration (25 µmol/L). Osteoclast differentiation is a process involving cell proliferation, commitment, fusion and activation.…”

“…44 The inactivation (phosphorylation) of GSK3β is the intermediate product after the activation of Wnt/β-catenin pathway. 37,38 Further, GSK3β plays an important role in many pathways, including, growth factors, hedgehogs, G-protein coupling ligands, cytokines and Wnt. 45,46 It is also a key downstream factor of AKT and plays an essential role in osteoclast differentiation.…”

Small G protein signaling modulator 3 (SGSM3) knockdown attenuates apoptosis and cardiogenic differentiation in rat mesenchymal stem cells exposed to hypoxia

“…In fact, changes in Cx43 expression and distribution were shown in myocardium diseases such as hypertrophic cardiomyopathy, heart failure and ischemia [49]. In addition, Cx43 is important for maintaining late-passage MSCs during adipogenesis and regulates the osteogenic differentiation of bone marrow-derived MSCs [50,51]. These results imply that Cx43 may play a role in the cardiac differentiation of MSCs.…”