The involvement of the ERK-MAPK pathway in TGF-β1–mediated connexin43-gap junction formation in chondrocytes

ABSTRACT

Purposes: Gap junction intercellular communication (GJIC) exhibits a key role in maintaining the homeostasis of articular cartilage. Connexin43 (Cx43) protein is predominant in the structures that form gap junctions. We aim to determine the potential underlying mechanisms of TGF-β1 (Transforming growth factor-β1)—regulated cell communication in chondrocytes.

Materials and methods: After exposure of chondrocytes to recombinant TGF-β1, quantitative real-time PCR was used to detect expression levels of Cx43 mRNA. Western blot analysis was used to check Cx43 and mitogen-activated protein kinase (MAPK) family components. Immunofluorescence staining was performed to confirm ERK-MAPK pathway activation and Cx43 protein distribution. MAPK inhibitors (ERK inhibitor U0126, JNK inhibitor SP 600125 and P38 inhibitor SP 203580) were applied to verify the specificity effects of ERK-MAPK pathway. GJIC between chondrocytes were evaluated using Scrape loading/dye transfer (SLDT) assay.

Results: It was first found that TGF-β1modulatedthe Cx43protein expressions and its sub-cellular distribution. TGF-β1 promoted gap junction intercellular communication (GJIC) formations in chondrocytes, especially in a higher cell intensity. ERK-MAPK signaling pathway was activated in TGF-β1-mediated gap junctions among chondrocytes. Furthermore, the inhibitor of ERK attenuated the increases of Cx43 expressions and functional gap junction formations induced by TGF-β1, while cross-talk between ERK-MAPK and Smad signal pathways exists shown in the process.

Conclusions: This study provides evidence to show the importance of the ERK-MAPK pathway in TGF-β1—mediated Cx43 expression and functional gap junction formation.

KEYWORDS:

• GJIC
• TGF-β1
• MAPK
• connexin43
• chondrocyte
• ERK

Acknowledgments

This study was supported by the National Nature Science Foundation of China (81600840, 81771047) to Jing Xie. We also acknowledged Dr. Chenghui Li in the Analytical and Testing Centre of Sichuan University for her excellent assistance in CLSM imaging.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [81600840,81771047].