A Study In Molecular Mechanism Of The ECM Secreted By Senescence Fibroblasts Induced By UVB Promoting The Proliferation Of HaCAT Cells

Background UVB,an inherent component of sunlight,crosses the epidermis,reaching the upper dermis composed mainly of fibroblast and ECM.Higher levels of senescent cells were previously found in fibroblasts from skin biopsies from old donors compared with younger counterparts.Moreover,the experiments ex vivo have shown repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers UVB stress-induced premature senescence(UVB-SIPS).Fibroblasts are well known for their function in maintaining the homeostasis of ECM in tissues,which could be disturbed by repeated UV exposure.less was known about how alternations of ECM in the photoaging skin influence the processes of epithelial cells,such as survival,proliferation, cell cycle progression and signal transduction pathways.All these processes is implied to be the essential for skin cancer development, involving clonal expansion of initiated cells giving rise to pre-malignant and then to malignant lesionsSeveral researches have cast a light on the relationship of senescence and tumorigenesis,which suggested senescent fibroblasts induced by a variety of stresses promote preneoplastic and neoplastic epithelial cell growth,but not normal epithelial cell,by secretion of dissoluble factors, deposition of ECM and direct cell-cell interaction.Among these, extracellular matrix(ECM)deposited by senescent fibroblasts accounts for at least 40%of growth stimulation.Senescent HSFs induced by successive UVB exposure has also share a similar phenotype with other types of senescent fibroblast,in which the increase of certain extracellular matrix(ECM)components,such as collagens,fibronectin and osteonectin, could be found.Whereas,whether ECM deposited by HSFs in UVB-SIPS exhibit a similar effect on growth stimulation remains unknown.HaCaT cell is one of human preneoplastic epidermal keratinocytes, having acquired only some mutations that predispose to malignancy.Thus, it is common model cell for the research on the mechanisms of tumorigenesis.The research in the effect of ECM on HaCaT can offer a better comprehension for such progression.Meantime,two well-established models allow us to investigate how the ECM secreted by persenescet HSFs and HSFs in UVB-SIPS influence the functions of HaCaT.One is the induction of premature senescence fibroblasts by repeated UVB exposure;another is preparation of extracellular matrix of fibroblasts.Previous observations have suggested that ECM secreted by senescent fibroblasts can preferentially promote HaCaT(preneoplastic keratinocyte) cells proliferation and progression.Although,the mechanisms responsible for these effects have yet to be understood,the ERK1/2 and PI3-k/AKT signaling pathway have been implicated to play critical roles in the regulation of survival and proliferation in response to cell-ECM interaction in many type of cells.We therefore hypothesized that HaCaT adhesion to ECM secreted by human skin fibroblasts(HSFs)treated might activate PI3-k/AKT and members of the MAPK family such as extracellular signal-regulated kinase ERK1/2 in human HaCaT cells,and then sought to characterize such signals.In further studies,we sought to characterize upstream signaling elements that might be involved in ERK1/2 and AKT activation in response to cell-ECM attachment.In particular,we focused on the cell's focal adhesion kinase(FAK),a 125kD tyrosine kinase,appears to play a central role in integrin-mediated signal transduction,which normally localizes with surface integrin receptors, and is activated by cell binding to ECM,and PI3-K-AKT and RAS-RAF-ERK1/2 pathway are the well-known downstream effectors of FAK.Given all aforementioned information,we hypothesized that ECM secreted by HSFs promotes cell proliferation via the activation of ERK and PI3K/AKT pathways in HaCaT cells,we try to seek the evidence to support this hypothesis.The modulation of these signaling pathways may lead to better prevention or clinical management of UV-induced skin aging and skin cancer.Methods 1)presenescent human skin fibroblasts(HSF)were exposed to repeated subcytotoxic UVB at 15mJ/cm~2 until the HSF developed senescent response which confirmed by determining the expression of SA-βgal and DNA synthesis,presenescent and senescent HSF were shifted to dishes and incubated in serum-free medium for 3 days to generate lawns,respectively,the ECM were prepared by removing the cells wth EDTA.2)HaCaT cell were plated onto ECM prepared with EGF-free KBM medium.The rate of apoptosis and cell-cycle progression in HaCaT cells was detected with Flow Cytometry method after 36 hours.The proliferation of HaCaT was measured with MTT assay at 96h following seeding.3)HaCaT cells were plated onto the ECM prepared with EGF-free medium.The level of phosphorylation of FAK,ERK1/2 and AKT was measured by Western blot method in a time-dependent manner.4)HaCaT cells were plated onto them with EGF-free medium.In some works Cytochalasin D,U0126 and wortmaninn were used to block FAK, ERK1/2 and PI3-K/AKT,respectively.The rate of HaCaT cells apoptosis and cell-cycle progression was detected with Flow Cytometry method after 36 hours.The proliferation of HaCaT was measured with MTT assay. The level of phosphorylation of FAK and AKT was measured by Western blot method in a time-dependent manner.Results 1)We show that repeated exposure of skin HDFs to subcytotoxic doses of UVB lead to the long-term appearance of several biomarkers of senescence such as senescence associated_-galactosidase activity and overexpression of several genes known to be overexpressed in senescent HDFs,And prepare the the extracellular matrices(ECM) secreted by UVB-induced senescent(UVB-SCIP)fibroblasts and by presenescent fibroblasts.2)ECM secreted from HSFs in UVB-SCIP had 13.15%and 29.27% (P＜0.05)more stimulatory effect than ECM secreted from presenescent HSFs and non-ECM,respectively.HaCaT cultured in non-ECM,ECM from presenescent HSFs and HSFs in UVB-SCIP undergone average 4.01%,5.47%and 7.12%apoptosis,respectively.Compared with non-ECM groups,the groups cultured in ECM secreted from HSFs in UVB-SCIP and presenescent HSFs had a 23.68%and 8.55%increase of cell proportion in S phase(p＜0.05)3)The study showed that attachment to ECM prepared(non-ECM controls,ECM deposited by HSFs in UVB-SCIP and ECM by presenescent HSFs)induced both FAK and ERK1/2 phosphorylation in a time-dependent manner,both FAK and ERK1/2 phosphorylation begin at 30min,and reach the peak at the time of 120 min,the most intense phosphorylation effect of FAK and ERK was found on ECM deposited by HSFs in UVB-SCIP;phosphorylated Akt was detected in HaCaT cells under a condition of either attachment or suspension,and there was slight difference in the phosphorylated level among non-ECM groups, presenescent ECM groups and UVB-SCIP ECM groups.4)treatment with U0126 completely prevented ERK1/2 phosphorylation in all of three groups,the blockade of ERK1/2 completely inhibit the effect of proliferation induced by ECM.The pretreatment with wortmannin dramatically enhanced the proportion of apoptotic cells among all of three groups by 15.8%,23.4%and 21.8%,respectively.The pretreatment with 2μM Cytochalasin D in HaCaT inhibit phosphorylation of FAK and AKT.At the same time,the rate of cells apoptosis increased significantly.Conclusion 1)The studies show that repeated stresses with subcytotoxic doses of UVB(15mJ/cm~2)induce stress-induced premature senescence (SIPS)of skin human diploid fibroblasts(HDFs).This model could be used to test whether HDFs in UVB-induced premature senescence are able to promote epithelial cell growth and tumorigenesis in photoaging skin. 2)ECM from HSFs in UVB-SCIP can not support more survival than that from presenescent HSFs,but the former could accelerate cell-cycle procession of HaCaT and promote the proliferation of HaCaT cells.3)ECM deposited by HSFs in UVB-SCIP could induce the most rapid and intense phosphorylation of ERK1/2 and FAK.For AKT,attachment to non-ECM and ECM deposited by HSFs in UVB-SCIP or by presenescent HSFs induce a slight but not significant phosphorylation of AKT.4)ERK1/2 signaling promote proliferation via accelerating cell-cycle progression,but not decreasing the proportion of apoptosis induced by EGF-withdrawn and suspension.ECM produced by HSF in UVB-SCIP can not induce significantly the phosphorylation of AKT,but PI3-K/AKT still worked as a protector of HaCaT cells from apoptosis in our system, the inhibition of which would overwhelm the proliferation effect induced by ECM deposited by HSFs in UVB-SCIP.