Bradykinin potentially stimulates cell proliferation in rabbit corneal endothelial cells through the ZO-1/ZONAB pathway

Bradykinin (BK) has been demonstrated to induce proliferation in several types of cell in ex vivo corneas. However, the mechanisms underlying the action of BK on corneal endothelial cells (CECs) remain largely unknown. The present study aimed to investigate the effect of BK on rabbit corneal endothelial cell (RCEC) proliferation, and assess the involvement of the zonula occludens-1(ZO-1)/ZO-1associated nucleic acid binding protein (ZONAB) pathway. Cell proliferation and cell cycle distribution was analyzed following treatment with BK (0.01, 0.1,1.0 or 10.0 µM) for the indicated time intervals (24, 48, 72 and 96 h), or following BK treatment combined with transfection of ZONAB-small interfering (si)RNA for 72 h. In addition, the expression of tight junction ZO-1, nuclear ZONAB, proliferating cell nuclear antigen(PCNA) and cyclin D1 were evaluated using western blotting or immunofluorescence. BK treatment was demonstrated to induce time- and concentration-dependent cell proliferation and cell cycle progression, along with the upregulation of tight junction ZO-1 and nuclear ZONAB, as well as PCNA and cyclin D1 protein expression. Furthermore, knockdown with ZONAB-siRNA inhibited cell proliferation, induced cell cycle arrest and downregulated PCNA and cyclin D1 protein expression. ZONAB knockdown therefore successfully reversed the increase in proliferation induced by BK treatment. Taken together, these results suggested that BK stimulated RCEC proliferation, potentially via the ZO-1/ZONAB pathway. The signaling paradigm disclosed in the present study potentially serves as an important therapeutic target for cornea regeneration and transplantation.