Abstract
Gap junction (GJ) has been indicated to have an intimate correlation with adhesion junction. However, the direct interaction between them partially remains elusive. In the current study, we aimed to elucidate the role of N-cadherin, one of the core components in adhesion junction, in mediating connexin 43, one of the functional constituents in gap junction, via transforming growth factor-β1(TGF-β1) induction in osteoblasts. We first elucidated the expressions of N-cadherin induced by TGF-β1 and also confirmed the upregulation of Cx43, and the enhancement of functional gap junctional intercellular communication (GJIC) triggered by TGF-β1 in both primary osteoblasts and MC3T3 cell line. Colocalization analysis and Co-IP experimentation showed that N-cadherin interacts with Cx43 at the site of cell–cell contact. Knockdown of N-cadherin by siRNA interference decreased the Cx43 expression and abolished the promoting effect of TGF-β1 on Cx43. Functional GJICs in living primary osteoblasts and MC3T3 cell line were also reduced. TGF-β1-induced increase in N-cadherin and Cx43 was via Smad3 activation, whereas knockdown of Smad3 signaling by using siRNA decreased the expressions of both N-cadherin and Cx43. Overall, these data indicate the direct interactions between N-cadherin and Cx43, and reveal the intervention of adhesion junction in functional gap junction in living osteoblasts.
Highlights
Cell–cell communication, a characteristic feature of virtually all multicellular organisms, mediates a series of responses to the internal or external environment necessary for cell survival
We aimed to explore the effect of N-cadherin on transforming growth factor-β1 (TGF-β1)-mediated cell–cell communication in N-cadherin colocalizes with connexin 43 (Cx43) to form a protein complex in osteoblast lineage and its mechanism
As for primary osteoblasts, the Pearson’ correlation coefficients of Cx43 and N-cadherin colocalization are 0.68 in the TGF-β1 increases the expression of N-cadherin in osteoblast lineage control group and 0.79 in the TGF-β1 group, both belong to the “strong” level according to Vadim’s research.[19]
Summary
Coefficients of Cx43 and N-cadherin colocalization are 0.68 in the TGF-β1 increases the expression of N-cadherin in osteoblast lineage control group and 0.79 in the TGF-β1 group, both belong to the “strong” level according to Vadim’s research.[19]. The expression of N- Immunoprecipitations with N-cadherin antibody followed by cadherin was approximately 4-fold and 2-fold higher induced by 10 ng·mL−1 TGF-β1 in primary osteoblast and MC3T3-E1 cells, respectively (Fig. 1c, e). Fluorescent signal of N-cadherin was almost up to 1.50-fold and 1.25-fold to the control groups in primary mediation of Cx43, primary osteoblast and MC3T3-E1 cells were treated with TGF-β1 (10 ng·mL−1) in the presence or absence of 100 nmol·L−1 N-cadherin siRNA. To. The expression of Cx43, one of the ubiquitous channel forming investigate whether N-cadherin is essential for TGF-β1-induced proteins, in osteoblasts isolated from 2-day-old mice was tested by GJIC activity, we performed scrape loading/dye transfer (SL/DT). Consistent with the western blotting, treatment with TGF-β1 increased GJIC activity, with the transmission β1 significantly upregulated Cx43 gene expression in primary speed of LY dye increased, whereas knock-down of N-cadherin osteoblasts. TGF-β1 (10 ng.mL-1) Normal control TGF-β1/(ng.mL-1) Normal control a Colocalization of N-cadherin and Cx43
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