Study Of HIF1a Effect And Its Signal Pathway In UV-Induced Skin Cancer

Study of HIF1 a effect and its Signal Pathway in UV-InducedSkin CancerUltraviolet (UV) radiation from the sun is a significant risk to human skin.Solar UV radiation causes cumulative damage in the form of photoagingand skin cancer. Previous studies have implicated specific intracellularsignals mediating the transformation response, including epidermalgrowth factor receptor (EGFR), AP1, NFκB, PI3 kinase, and MAP kinase.Activation of these receptors and signal elements contribute importantlyto changes in expression of various genes involved in skin photoagingand skin cancer. Hypoxia inducible factor alpha (HIF1α), one of themembers of the HIF transcription complex, has recently been identifiedas a contributor to altered patterns of gene expression in various types ofcancer, and as such is considered to be a valid therapeutic target forcancer treatment. HIF1αexpression is modulated by theEGFR/PI3K/AKT pathway in human prostate cancer cells. However, thequestion whether this pathway is also involved in UV-induced expressionof HIF1αremains to be answered. In addition to HIF, two othertranscription factors, DEC1 and DEC2, recently known to interact withHIF, have been shown to be involved in cell proliferation and survival.Recent findings show that UV-induced HIF1αexpression requires DEC1,and that over-expression of DEC1 provides cells with an unusual survival mechanism under hypoxic conditions. The interactions of DEC1 andHIF1α, and their respective roles in the development of cellulartransformation, in response to UV irradiation are unknown. Thehypothesis to be tested in this project is that EGFR and Racl/PI3K/AKTmediate UV-induced HIF1αexpression and its target genes, and DEC1and HIF1αare reciprocally regulated in response to UV irradiation.The specific aims of this proposed project are:Specific Aim 1-To define the role of UVB in inducing expression ofHIF1αand its taget gen VEGF and TFR HaCaT cells were treated withvarious dose of UVB (10,20 and 30mJ/cm~2),then cultured for 6h,12h and24h,collected and detected the expression of HIF1α/VEGF/TFR.Specific Aim 2- To define the role of EGFR in UV-inducedexpression of HIF1α/VEGF/TFR. This specific aim is designed to testthe hypothesis that EGFR mediates the expression of HIF1αand its targetgenes. (a) EGFR inhibitors will be applied to determine whether theexpression of HIF1αwill be inhibited. (b) Forced expression of EGFR(e.g. over-expression of EGFR, knockout of EGFR) will also be utilizedto determine whether modulation of EGFR would affect the expression ofHIF1αand its target genes.Specific Aim 3- To determine the role of PI3K/AKT pathway inUV-induced expression of HIF1αand its target genes. This specificaim is to test the hypothesis that PI3 kinase/AKT pathway mediates UV-induced expression of HIF1αand its target genes. PI3 kinaseinhibitors will be applied to determine whether the expression of HIF1αand its targets genes will be inhibited.Specific Aim 4- To investigate the role of DEC1 in UV-inducedexpression of HIF1α/VEGF/TFR in response to UV irradiation. (a)Forced expression of DEC1 (e.g. DEC1 knockout, over-expression ofDEC1) will be used to determine whether UV-induced HIF1αand itstarget genes expression is DEC1-dependent.Specific Aim 5- To determine the role of HIF1αin UV-inducedexpression of its target genes. Forced expression of HIF1α(e.g.HIF1αRNAi ) will be used to determine whether UV-induced itstarget genes expression is HIF1α-dependent.RESULTResultl- To determine whether UVB could induce HIF-1αexpression,HaCaT cells were treated with various dose of UVB (10,20 and30mJ/cm~2)for 12h.. The total cellular protein extracts were prepared forimmunoblot assays of HIF-1αprotein levels, levels of HIF-1αprotein wereinduced by UVB in a dose-dependent manner. The maximum expressionof HIF-1αwas induced by 30mJ/cm~2 UVB. To determine the kinetics ofHIF-1αexpression induced by UVB, cells were treated with 30mj/cm~2UVB for various times as indicated. The maximum UVB-induction of HIF-1αexpression was at 12h after the treatment, the level of VEGF andTFR protein in HaCaT cells after exposure of UVB was analyzedTreatment of cells with 30mJ/cm~2 UVB significantly increased VEGFand TFR levels in a time-dependent manner, when compared withuntreated cells(P＜0.05). We also assayed for their expression at mRNAlevel. HaCaT cells were treated with various dose of UVB (10,20 and30mJ/cm~2)for 4h.and cells were treated with 30mJ/cm~2 UVB for varioustimes as indicated(1,2,4,8,16h), and cellular RNA extracts were preparedfor analysis of HIF-1α/VEGF/TFR mRNA levels.We foundHIF-1αmRNA expression were unaffected by UVB treatment(P＞0.05).VEGF mRNA expression in a dose-dependent manner (P＜0.05)and reacha peak at 4h in HaCaT cells when compared with untreated cells. TFRmRNA expression in a dose-dependent manner (P＜0.05)and reach a peakat 8h in HaCaT cells when compared with untreated cellsResult2- UVB can induces activation of EGFR and enhance theexpression HIF1αin HaCaT cells. HaCaT cells (80% confluence) wereirradiated with UVB(30mJ/cm~2) and subsequently cultured for0.25h,0.5h, 1h,2h,4h,8h. Flow Cytometry detect total receptor andtyrosine-phosphorylated EGFR, respectively. EGF-R phosphorylationwas detectable within 15 minutes after UV. After a peak at 30 minutes,EGF-R phosphorylation decreased to near basal levels between 2 and 8hours after UV exposure. UV exposure did not change the level of total EGF-R in HaCaT. To investigate whether expression of HIF1αfollowing UVB irradiation depend on EGFR, we examined HIF1αexpression in MEF cells (forced expression of egfr e.g. egfr knock out,over-expression of egfr). MEF cells were treated with UVB (30mj/cm~2)for 12h. The result show that egfr(-/-) MEF were not capable ofinducing HIF1αexpression following UVB irradiation andegfr(+/+)MEF cells were strongly enhanced HIF1αexpression after UVBirradiation compared to the egfr(+/-)MEF cells. This analysisconfirmed that activation of EGFR was essential for HIF1αexpressionfollowing UVB irradiation. Our result also show that EGFR plays a vitalrole in the induction of VEGF and TFR expression. The VEGFconcentration was determined by ELISA in the supernatant of The MEFcells after UVB irradiation, the egfr (-/-) MEF were not capable ofincreasing VEGF expression following UVB irradiation(p＞0.05), andegfr(+/+)MEF were strongly enhanced VEGF expression(p＜0.05) afterUVB irradiation compared to control. The EGFR inhibitor PD153505significantly decreased the TFR levels induced by UVB Thusdemonstrating that UVB-induced HIF1α/VEGF/TFR expression need arequirement for signal transduction via the EGFR.Result3- To investigate whether PI3K were involved in the expression ofHIF1α/VEGF/TFR expression, HaCaT cells were cultured in DMEMsupplemented with 10% FBS for 24h, followed by the addition of PI3K inhibitors, LY294002 and wortmannin, 1h prior to the treatment of UVB.Cells were then treated with UVB(30 mJ/cm~2) for 12h.. LY294002 andwortmannin significantly decreased the HIF1α/VEGF/TFR protein levelsinduced by UVB, These results suggest that PI3K signaling was requiredfor induction of HIF-1α/VEGF/TFR induced by UVB. UVB-inducedVEGF expression via PI3K signaling pathway. LY294002 andwortmannin were able to inhibit UVB-induced HIF-1αexpression in adose-dependent manner.Result4- we examined HIF1α/VEGF/TFR expression in HaCaTcells.(forced expression of DEC1 e.g. DEC1 knock out, over-expressionof DEC1). HaCaT(dec1+/-,dec1+/+,dec1-/-) cells (80%confluence)were starved by replacing the medium with 0.1%FBS DMEMand culturing for 24h. The cells were irradiated with UVB(30mJ/cm~2) andsubsequently cultured for 12h.Immunoprecipitation of lysates withanti-HIF1αwas followed by Western immunoblotting, cells were thenirradiated by UVB(30mJ/cm~2)or shammed at the time points(6,12,24h)The VEGF and TFR expression was determined after UVBirradiation(30mJ/cm~2) at the time points (6,12,24h)., The result showthat. The DEC1(-/-) HaCaT cells were not capable of increasing HIF1α/VEGF/TFR expression following UVB irradiation(p＞0.05). In contrast,DEC1(+/+) HaCaT were strongly enhanced VEGF/HIF1α/TFRexpression after UVB irradiation. This analysis showed that UV induced HIF1αand VEGF expression in DEC1-dependent manner. Cellswere treated with 30mj/cm~2 UVB for various times asindicated(1,2,4,8,16h), and cellular RNA extracts were prepared foranalysis of VEGF/TFR mRNA levels. DEC1(+/+) HaCaT were stronglyenhanced VEGF/TFR mRNA levels after UVB irradiation(P＜0.05).DEC1(+/+) HaCaT VEGF mRNA expression reach a peak at 16h inHaCaT cells when compared with untreated cells.Result4- we examined VEGF/TFR expression in HaCaTcells.(forced expression of HIF1αe.g. HIF1αRNAi). HaCaT andHaCaT(HIF1αRNAi) (80% confluence)were starved by replacing themedium with 0.1%FBS DMEM and culturing for 24h. The cells wereirradiated with UVB(30mJ/cm~2) and subsequently cultured for 24h. TheVEGF and TFR expression were detected. HaCaT (HIF1αRNAi)werestrongly attenuate VEGF/TFR levels after UVB irradiation(P＜0.05)when compared with HaCaT cells.ConclusionIn conclusion, this work demonstrates that UVB is able to induceHIF-1αa/ VEGF/TFR expression via the EGFR/PI3K/AKT/DEC1signaling pathway. HIF1αalso modulate its target gens expression.