Interaction of connexin43 and protein kinase C-delta during FGF2 signaling

<p>Abstract</p> <p>Background</p> <p>We have recently demonstrated that modulation of the gap junction protein, connexin43, can affect the response of osteoblasts to fibroblast growth factor 2 in a protein kinase C-delta-dependent manner. Others have shown that the C-terminal tail of connexin43 serves as a docking platform for signaling complexes. It is unknown whether protein kinase C-delta can physically interact with connexin43.</p> <p>Results</p> <p>In the present study, we investigate by immunofluorescent co-detection and biochemical examination the interaction between Cx43 and protein kinase C-delta. We establish that protein kinase C-delta physically interacts with connexin43 during fibroblast growth factor 2 signaling, and that protein kinase C delta preferentially co-precipitates phosphorylated connexin43. Further, we show by pull down assay that protein kinase C-delta associates with the C-terminal tail of connexin43.</p> <p>Conclusions</p> <p>Connexin43 can serve as a direct docking platform for the recruitment of protein kinase C-delta in order to affect fibroblast growth factor 2 signaling in osteoblasts. These data expand the list of signal molecules that assemble on the connexin43 C-terminal tail and provide a critical context to understand how gap junctions modify signal transduction cascades in order to impact cell function.</p

Implication de l'endothéline-1 et de la communication jonctionnelle dans les processus de différenciation et prolifération (modèles trophoblastique villeux et ostéoblastique humains)

La communication intercellulaire par jonction gap (CIJG) permet la diffusion de cytoplasme à cytoplasme de petites molécules (ions, seconds messagers, etc...) via des canaux intercellulaires composés de connexines (Cx). Ces dernières années, il a été montré que la CIJG était associée à des pathologies variées, et modulait différentes fonctions incluant l'homéostasie tissulaire, la différenciation cellulaire et le contrôle de la croissance. À partir de deux modèles cellulaires, le trophoblaste villeux et la lignée ostéoblastique hFOB1.19, nous avons mis en évidence l'implication de la CIJG dans des processus de prolifération/différenciation cellulaires. Par ailleurs, nous avons montré qu'un facteur pléiotropique, l'endothéline-1 (ET1), inhibe la différenciation trophoblastique et ostéoblastique. Cette action s'accompagne de l'inhibition de la CIJG qui se traduit par une diminution de l'expression de la Cx43 et du couplage jonctionnel. De plus, l'ET1 induit une mobilisation du Ca2+ cytosolique, et dans le trophoblaste villeux, une activation des canaux SOCC et NSCC a été démontrée. En outre, le niveau d'expression de la Cx43 influence la réponse à l'ET1 dans le modèle ostéoblastique, suggérant des interactions entre la Cx43 et les molécules de la différenciation, lors de l'action de l'ET1.Gap junctions are clusters of intercellular channels made of proteins called connexins (Cx). They permit cell-to-cell communication by allowing passage of low-molecular weight metabolites (ions, second mesengers, etc). These last years, it was shown that gap junctional intercellular communication (GJIC) is associated with various pathologies and modulates different processes including tissue homeostasis, cell differentiation, and growth control. By means of two cell types, the villous trophoblast and osteoblastic cell line hFOB1.19, we have demonstrated that GJIC is involved in processes of proliferation and differentiation. Moreover, we have shown that a pleiotropic factor, endothelin-1 (ET1), inhibits the trophoblast and osteoblast differentiation. This effect was associated with a decrease of Cx43 expression and cell coupling. In addition, ET1 induced Ca2+ increase in the two cell types, and in the villous trophoblast, the activation of NSCC and SOCC channels was demonstrated for the first time. Interestingly, the level of Cx43 expression modulates the ET1 response in the osteoblastic model, suggesting interactions between Cx43 and differentiating mediators activated by ET1.POITIERS-BU Sciences (861942102) / SudocSudocFranceF

Effective knockdown of ADAMTS5 in SW982 synovial fibroblasts cells transduced with ADAMTS5 targeting shRNA lentiviral particles.

<p>SW982 cells were untransduced (unt), or transduced with non-targeting shRNA (Ctl) or with two distinct ADAMTS5-targeting shRNA (denoted #3 or #5). Post-transduction the cells were treated in the presence or absence of IL-1β (1ng/ml, 16h) to induce ADAMTS5 expression. Subsequently, a western blot of whole cell extracts from these cells were run and probed with anti-ADAMTS5 or anti-GAPDH antibodies as indicated. GAPDH abundance was used as a loading control reference. A representative blot is shown.</p

Cx43 is robustly expressed in synovial fibroblasts and hMSCs.

<p>(A-B), Sections of murine knee joint were labeled with anti-Cx43 antibodies (brown). Cx43 is detected in the synovial fibroblasts (arrows) in the synovial lining (SL), as well as in the articular chondrocytes in the articular cartilage (AC). (C), A sections of murine knee joint were labeled with non-immune IgG as a negative control. (D), Quantitative real time RT-PCR was performed on RNA isolated from hMSCs and synovial fibroblasts in culture using primer pairs for the genes encoding Cx37, Cx40, Cx43, Cx45 and Cx46. Histograms indicate means ± standard deviation. (E), A western blot of whole cell extracts from hMSCs or synovial fibroblasts probed with anti-Cx43 antibodies reveals expression of Cx43 in both cell types. The expression of Cx43 in hMSCs is composed of a larger percentage of the higher molecular weight phosphorylated Cx43 than in SW982 cells, which express mostly the non-phosphorylated form. The images are from non-contiguous lanes of the same blot and exposure. GAPDH abundance was used as a loading control reference. (F) Immunofluorescnece microscopy reveals that hSERT+ SW982 cells (red) can take up serotonin/5-HT (green) and communicate it with hMSCs, indicating functional gap junctional communication between the two cell types. A representative image is shown.</p

Co-culture of hMSCs transduced with ADAMTS5-targeting shRNA effectively reduces ADAMTS5 expression in flow cytometry sorted RFP-labeled SW982.

<p>(A) Following co-culture of non-targeting control shRNA or ADAMTS5-targeting shRNA transduced hMSCs with RFP labeled SW982 cells, the cells were sorted by flow cytometry. The gating threshold (RFP+ sorted) was set based on the analysis of co-cultured hMSCs with unlabeled SW982 cells (no SW982-RFP cells). RFP-positive SW982 cells that were co-cultured with hMSCs previously transduced with non-targeting control-shRNA or ADAMTS5-targeting shRNA were sorted directly into TriPure reagent for RNA isolation. (B) The histogram shows the relative gene expression for ADAMTS5 in the RFP-positive cell population isolated after co-culture with hMSC-shRNA-control or hMSC-shRNA-ADAMTS5 cells as determined by quantitative real time RT-PCR. The result is from a representative experiment.</p