The UV responsive elements in the human mimecan promoter: Afunctional characterization

[摘要] Purpose: A major environmental stress encountered by humans is solarUV light, which can cause a spectrum of eye diseases, such asphotokeratitis, cataract, pterygia, and ocular neoplasms. Mammaliandefense mechanisms in response to adverse effects of UV light result ininduction of a number of genes. Studies on the transcriptionalregulation of genes that are expressed in the eye will increaseunderstanding of both the physiological functions of these genes in themammalian UV response, and the molecular bases for abnormalitiesassociated with the above diseases. Mimecan is an extracellular matrixproteoglycan that is abundantly expressed in the cornea. The purpose ofthis study was to determine and characterize the UV responsiveregulatory elements of the human mimecan promoter.Methods: Transcriptional activity of the promoter was evaluated,before and after UV irradiation, using transient transfection of humanmimecan promoter/luciferase reporter constructs into corneal keratocytesand non-corneal cells. Site directed mutagenesis and correspondingfunctional assays were used to determine the contribution of UVresponsive regions to human mimecan transcription. Co-transfectionexperiments were used to investigate the role of transcription factorsthat bind these elements in the promoter and mediate the UV response.mRNA expression was analyzed by reverse transcription-polymerase chainreaction (RT-PCR).Results: The shortest promoter construct that was strongly activatedfollowing UV irradiation contained three initiator elements, an E-boxelement that is conserved between species, and the entire first intronof the human mimecan gene. Deletion of the intronic p53 binding sitefrom this construct considerably diminished transcription and the UVresponse of the promoter. Surprisingly, deletion of the E-box sequencefrom this construct completely abolished both transcription and UVresponse of the promoter. These results demonstrated that the E-box isessential to transcription of the human mimecan gene and also isrequired for activation by p53. The role of the E-box, and the E-boxbinding protein, USF-1, in transcription and UV responses of the humanmimecan promoter were confirmed by co-transfection experiments usingdominant negative transcription factor, A-USF. In addition to thesepositive regulators, we demonstrate that the region between nucleotides-1314 and -1907 contains a transcriptional repressor site that is activein a time dependent manner following UV irradiation. Finally, we showthat UV irradiation results in changes in mimecan mRNA levels in bovinecorneal keratocytes in a time-dependent manner.Conclusions: The human mimecan promoter contains several UVresponsive regulatory elements that are conserved between human andbovine species and include the intronic p53 DNA binding site, the E-boxin the proximal promoter, and the region between nucleotides -1314 and-1907. The E-box plays an important role in transcription and UVresponse of the human mimecan promoter. UV irradiation modulatesexpression of mimecan mRNA in bovine corneal keratocytes and non-cornealcells.

[学科分类] 生物化学/生物物理