| Background:The skin epidermis and its appendages provide a protective barrier that is impermeable to harmful microbes and also prevents dehydration. To perform their functions while being confronted with the physicochemical traumas of the environment, these tissues undergo continual rejuvenation through homeostasis, and, in addition, they must be primed to undergo wound repair in response to injury.The skin s elixir for maintaining tissue homeostasis, regenerating hair, and repairing the epidermis after injury is its stem cells, which reside in the adult hair follicle, sebaceous gland, and epidermis. Stem cells have the remarkable capacity to both self-perpetuate and also give rise to the differentiating cells that constitute one or more tissues. In recent years, scientists have begun to uncover the properties of skin stem cells and unravel the mysteries underlying their remarkable capacity to perform these feats.Epidemal stem cells surface have high level of integrinβ1. So Integrinβ1 was thought to be a marker of epidermal stem cell. A great deal of research show that integrinβ1 plays an important role in epidermal cell proliferation and differentiation. Epidermal stem cell reside in epidermis basal layer and hair follicles bulge,where integrinβ1 are concentrated.The connection between epidermal stem cell and basal layer extracellular matrix is rely on integrinβ1,which maintaining stem cell property. Researchers found down regulation integrinβ1 in epidermal stem cell would result in terminal differentiation.So accurate regulation of integrinβ1 sustains skin integrality and Homeostasis.Calcium is the second messenger,and calcium ion play an important role in maintaining epidermis Homeostasis.Within normal,intact epidmis,it is to be present calcium ion gradien.At epidermis basal layer ,calcium ion concentration is very low, about 0.5mmol/L.However,calcium ion concentration at granular layer of epidermis is about 1.4mmol/L. A great quantity of previous studies confirm skin calcium ion concentration influence cell proliferation and differentiationPiero C et.al by using human osteogenic sarcoma cell line (MG-63) demonstrated that two promoters control the transcription of the humanβ1 integrin gene, suggesting a probable complicated model forβ1 integrin expression. Absence of TATA and CCAAT box, high G + C content, presence of multiple copies of GC-boxes, and heterogeneity in transcription start sites are features different from promoter regions of housekeeping genes. As a general rule the different expression is based on the different susceptibility of transcriptionally active regions to specific transcription factors in response to various stimuli. The two promoters drive the expression of the uniqueβ1-gene resulting in the synthesis of at least two mRNAs that diverge only in the complete 5'-untranslated regions but share the same coding sequence as it is indicated by the isolation of two cDNAs with this structure.However the mechanism of integrinβ1 transcriptional control is unknown as yet.HaCaT(Human adult skin keratinocytos ,HaCaT) cell line originates from normal human epidermal cells, which have the capacity to proliferate infinitely. Researcher has showed that HaCaT were transplanted into subcutaneous skin of the nude mice, Which could differentiate into epithelial tissues, with expression of special markers for differentiation. So HaCaT cell line can provide an ideal model for the researchers to investigate regulation mechanism of human epidermal cell proliferation and differentiation.Objective:HaCaT cells and luciferase reporter gene system containing integrinβ1 promoter were enrolled in our study to determine integrinβ1 promoter s key activity region . To move forward a single step to investigate signal transduction mechanism of regulating epidermal stem cell proliferation and differentiation.Methods:1. Using previous constructed integrinβ1 promoter total ,distal, proximal and series of shortened vectors to transfects HaCaT cell so that inspection and measurement its relative luciferase activity and determine integrinβ1 promoter s key activity region .meanwhile,studying calcium on influence of integrinβ1 promoter and expression.2. Carrying out electrophoretic mobility shift assay and combining with a proteomics method to definite integrinβ1 promoter s key activity region and specific transcription factors.Results:1. In epidermis cell,integrinβ1 promoter s key activity region lies between -352bp and -212bp in distal promoter region.2. Electrophoretic mobility shift assay shows that there are specific transcription factors bind to key regulation region.Combine proteomics methods with transcription factors prediction system indicate Sp1 and MAZ to cooperate to regulate integrinβ1 promoter activity.Conclusion:1. The distal promoter region of integrinβ1 plays an important role in regulating of integrinβ1 transcription in epidermis cell.2. Using previous constructed integrinβ1 promoter total ,distal, proximal and series of shortened vectors to transfect HaCaT cell and measure its relative luciferase activity ,found that integrinβ1 promoter s key activity region lies between -352bp and -212bp in distal promoter region.3. Calcium regulates integrinβ1 promoter activity and expression,would which influence epidermis cell migration via integrinβ1.
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