Hypoxia induces myofibroblast formation and stimulates production of collagen Ⅰ in myofibroblasts through ERK1/2 pathway / 中国病理生理杂志

ABSTRACT

AIM: To investigate the effect of hypoxia on the myofibroblast transdifferentiation from fibroblasts,and associated signaling of hypoxia on the production of collagen Ⅰ in cultured rat renal cortical myofibroblasts.METHODS: The study is composed of two relevant parts.In the first part,a normal rat renal interstitial fibroblast cell line NRK-49F was treated with hypoxia(1% O2) or normoxia(21% O2) for 6 h,12 h and 24 h.The expression of hypoxia inducible factor-1?(HIF-1?) was examined by Western blotting in order to make sure the hypoxic condition is reliable.The myofibroblast transformation from fibroblasts induced by hypoxia was assayed by detecting the protein levels of ?-smooth muscle actin(?-SMA).In the second part,the object was done on the primary cultured rat renal cortical myofibroblasts.Myofibroblasts were subjected to hypoxic or normoxic conditions for variety of times.The levels of HIF-1? in cell lysates and collagen I protein in supernatant culture medium and the activation of extracellular signal-regulated kinase(ERK)1/2 MAPK pathway were analyzed by Western blotting.RT-PCR was carried out to measure the levels of collagen I mRNA at different time points(2 h,4 h and 6 h).The distribution of HIF-1? in myofibroblasts was demonstrated by immunocytochemistry.The changes of collagen I production were detected after PD98059,a specific inhibitor of ERK1/2 activation pretreatment and during the hypoxia incubation.The activity of gelatinase matrix metalloproteinase-2(MMP-2) and MMP-9 in the supernatant medium from the cultured cells were assayed by gelatin zymography.RESULTS: Significant increased levels of HIF-1? protein appeared in cell lysates under hypoxia for 6 h.Furthermore,HIF-1? was translocated into nuclei of myofibroblasts after 6 h exposure of myofibroblasts to hypoxia.The levels of ?-SMA protein increased in NRK-49F under hypoxia for 12 h(187%?32%,P