TGF-β1 regulates pannexin1 channels and evokes apoptotic response in osteoblasts

Objective

Extracellular ATP is suggested to be involved in cell-cell interactions and TGF-β1 stimulates ATP release through pannexin1 channels. However, the role of TGF-β1 in regulating of pannexin1 channels and cell apoptosis remains unclear. In the present study, the aim was to clarify the role of TGF-β1 in relation to pannexin1 channels and cell apoptosis in osteoblasts.

Design

The detection of pannexin1 expression induced by TGF-β1 was achieved using immunofluorescence labeling and western blot analysis. The activity of pannexin1 channels was detected by the dye uptake assay. This study employed the MEK inhibitor U0126 to block ERK signaling in order to investigate the signaling pathway which is involved in the effect of TGF-β1 on pannexin1. In order to determine the expression of cleaved caspase-3 in osteoblasts, immunofluorescence labeling was employed. Flow cytometry was performed to detect the rate of apoptotic cells.

Results

Initially, the data of this study showed that TGF-β1 increase the expression of pannexin1 in both primary osteoblasts and the MC3T3 cell line. This study also confirmed that TGF-β1 triggers osteoblast ethidium bromide (EtBr) dye uptake by pannexin1 channels. The inhibition of ERK signaling pathway eliminated TGF-β1’s ability to promote pannexin1. Pannexin 1 is up-regulated in osteoblasts that undergo apoptosis due to high concentration of TGF-β1.

Conclusions

According to these findings, high concentration of TGF-β1 up-regulates the expression of pannexin1 and the activation of pannexin1 channels. The ERK signaling pathway mediates this regulation, which induces the apoptosis of osteoblasts.