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

ABSTRACT: 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.

SUBMITTER: He L

PROVIDER: S-EPMC5979832 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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

He Lixian L Zhou Zhou Z Shao Yi Y Yang Zhen Z Zhou Shuangshuang S Zou Xuexiang X Zhou Ying Y Tan Gang G

International journal of molecular medicine 20180322 1

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 w ...[more]

PMID: 29568941

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Project description:Corneal endothelial cells are rich in mitochondria, a potential source of reactive oxygen species (ROS). ROS have been implicated in endothelial cell loss during aging or in endothelial dystrophies. In this study we examined the anti-oxidative role of mitochondrial superoxide dismutase (SOD2) in corneal endothelial cells.SOD2 expression was examined by RT-PCR and western blot analysis in fresh rabbit corneal endothelium (RCE) and cell cultures. SOD2 activity, total reactive oxygen species (ROS), mitochondrial ROS, mitochondrial membrane potential (MMP), and apoptotic levels were examined in untreated, SOD2 siRNA and viral vector shRNA treated RCE cells. Scrambled siRNA and shRNA sequence targeting non-mammalian genes were used as controls.SOD2 is expressed in both fresh and cultured rabbit corneal endothelium. SOD2 expression was reduced by ~80%-90% in cultured RCE using either siRNA or shRNA approaches. SOD2 activity was decreased by ~70%-80% for both approaches. Total cell ROS was significantly increased in shSOD2 lentivirus treated cells (9%±6%) relative to control transduction (0.4%±0.1%). MitoSOX™ staining for mitochondrial ROS in siSOD2 treated RCE cells was dramatically increased. Two minutes of UV irradiation increased total ROS levels by 15%, whereas in shSOD2 treated cells UV induced ROS was increased 29%±5% (p<0.05). MMP was reduced in shSOD2 viral treated cells by 66%±3%, significantly greater than in control transduced cells (15%±8%, p<0.05). Apoptosis increased by 1.5 fold in shSOD2 virus treated samples compared with scrambled virus and untreated cells.SOD2 is expressed in both fresh and cultured rabbit corneal endothelium. siRNA and shRNA approaches are able to efficiently knockdown SOD2 expression and reduce enzyme activity in RCE cells. Decreased SOD2 activity causes elevated ROS production, mitochondrial membrane potential loss and early cell apoptosis. These results indicate that SOD2 is a significant anti-oxidative enzyme in RCE cells.

Dataset Information

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

Publications

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

Similar Datasets

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