Role Of P42/p44 MAPK Signal Transduction Pathway In Expression Of HIF-1α And VEGF In Human Retinal Pigment Epithelium Cells Under Hypoxia And Serum

Background Neovascularization is associated with various ocular disorders, often causing severe loss of vision and eventually blindness. Among these disorders, diabetic retinopathy (DR) and age-related macular degeneration (AMD) are the most prevalent in the World. VEGF is up-regulated by hypoxia and is a major stimulatory factor for retinal and choroidal neovascularization. Retinal microvascular endothelial cells, pericytes and retinal pigment epithelium cells all can produce VEGF. In particular, RPE cells have the most potential to express VEGF.Under hypoxia, HIF-1 can be expressed and transactivates the downstream genes such as VEGF, EPO and glycolytic enzyme as an adaption to environment. Neovascularization induced by VEGF in the eye is significant for some related eye diseases such as AMD and DR.Hypoxia can activate several signal pathways. Among them, mitogen-actived protein kinases (MAPKs) are ubiquitous enzymes involved in signal transduction. Their activity is essential in numerous cellular functions, including proliferation and programmed cell death. Extracellular signal-regulated kinases (ERKs) 1 and 2 are the most well-characterizedMAPKs. They could take part in the proliferation and differentiation of cells when stimulated by hypoxia, cytokine, growth factor, hormone and mitogen receptor, etc. It is shown that hypoxia can induce the expression of HIF-1 and lead to the transcriptional expression of downstream gene VEGF and the p42/p44 MAPK signal transduction pathway could play a role. The role of p42/p44 MAPK is to transactivate HIF-1 but have no effect on the expression of HIF-1.Purpose (1) To investigate the phosphorylation of p42/p44 MAPK in RPE cells stimulated by hypoxia and serum; (2) To evaluate the effect of p42/p44 MAPK signal transduction pathway on transactivation and expression of HIF-1α; (3) To investigate the effect of p42/p44 MAPK signal transduction pathway on VEGF secreting in RPE cells; (4) To investigate the synergistic effect of hypoxia and serum.Methods (1) 200μM C0Cl₂ was added to cell culture medium, which mimics hypoxia. Three groups were designed respectively for hypoxia, 10%FCS and combined group. The passage human RPE cells pretreated by PD98059, a specific upstream inhibitor of p42/p44 MAPK, were exposed to CoCl₂ for 0, 10, 30, 60, 120min and 1, 4, 8, 24h differently, and the RPE cells untreated by PD98059 were control. The expression of phospho-p42/p44 MAPK protein was detected by Western blot and immunohistochemistry staining. (2) In the three groups, cells of the PD98059 group and control were cultured for 1, 4, 8 and 24h under hypoxia, 10%FCS and combined conditions. Western blot, RT-PC and immunofluorescence staining were used to detect the expression of mRNA and protein of HIF-1α . (3) With enzyme-linked immunosorbent assay (ELISA), the expression of VEGF protein secreted by RPE cells in culture medium was measured and the expression of VEGF protein in cells was measured by immunofluorescence staining at 1, 3, 6, 12 and 24h under hypoxia, 10%FCS and combined conditions. (4) With the conditioned medium of cultured RPE cells treated by PD98059 for 24h, the effect of the secreted VEGF in supernatant of PD98059 group and control group under hypoxia on the proliferation of cultured human umbilical vein endothelial cells (HUVEC)was examined by 3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The HUNVC was cultured for 1,2, 3, and 4d.Results (1) Western blot showed that phospho-p42/p44 MAPK protein was expressed at 1, 4, 8 and 24h under hypoxia, 10%FCS and combined conditions. The level of phospho-p42/p44 MAPK of hRPE in the group treated with PD98059 was decreased and only the bands of phospho-p42 MAPK were visible. Immunohistochemistry staining proved that PD98059 could decrease the expression of phospho-p42/p44 MAPK of RPE cells in the three groups (P <0.01). (2) Western blot showed that hypoxia could induce the expression of HIF-la protein. However, 10%FCS couldn't induce the sustained expression of HIF-la. The band of control group was a diffused band compared with that of the PD98059 group which was a band near to 103kD but there was no significant difference in the level of HIF-la expression. Immunohistochemistry staining indicated that HIF-1α was mainly expressed in the nucleus of RPE cells and the expression was increased by time under hypoxia and combination conditions. However, 10%FCS couldn't induce obvious expression of HIF-la. But there was no significant difference between the PD98059 group and control group. Expression of HIF-1α mRNA of RPE cells under hypoxia, 10%FCS and combined conditions was measured by semi-quantify RT-PCR. There was no significant difference between PD98059 group and control group. Hypoxia, 10% FCS and combined conditions all can induce the expression of HIF-la mRNA. At 4h, the level was highest and the level was decreased after 8h. (3) ELISA proved that the amount of VEGF in hRPE supernatant was significantly increased in time-dependent manner (P<0.01), and it was down-regulated by PD98059 under hypoxia, 10%FCS and combined conditions, respectively (P<0.01). Immunofluorescence staining showed that the expression of VEGF protein in RPE cells was downregulated in the PD98059 group compared with control group under hypoxia, 10%FCS and combined conditions. (4) MTT showed that A value in the PD98059 group was lower than that in the control at all...