Transcriptional control of the mouse Col7a1 gene in keratinocytes: basal and transforming growth factor-beta regulated expression.
Anchoring fibrils at the cutaneous basement membrane zone of the stratified squamous epithelia are essential to maintaining skin integrity, as absence of these structures leads to the chronic blistering disease, dystrophic epidermolysis bullosa. Type VII collagen, the major component of anchoring fibrils, is synthesized primarily by basal keratinocytes and to a lesser degree by dermal fibroblasts. To elucidate the transcriptional control elements of the type VII collagen gene (Col7a1), 3 kb of 5' flanking sequence of the mouse gene was cloned, sequenced, and fused to the chloramphenicol acetyltransferase reporter gene. Promoter deletion analyses revealed that 560 bp of Col7a1 5' flanking sequence was sufficient and necessary for basal level of transcription in cultured murine keratinocytes. Mutagenesis of DNA sequences with similarity to consensus binding sites for transcription factors, including Sp1/Sp3, AP2, AP1, and Smads, within the p-560Col7a1 promoter/chloramphenicol acetyltransferase construct, coupled with DNA binding assays, revealed the importance of these sites for basal Col7a1 expression. The effect of transforming growth factor beta, an activator of Col7a1 expression in keratinocytes and dermal fibroblasts, was examined using the same Col7a1 promoter/chloramphenicol acetyltransferase constructs. These analyses demonstrated that transforming growth factor beta1 stimulation of Col7a1 transcription is dependent on a putative interaction between Smads and AP1. Interestingly, the Smad-like binding site was essential for both basal and transforming growth factor beta1 stimulated Col7a1 transcription. Collectively, these findings attest to the complex regulation of Col7a1 transcription in epidermal keratinocytes.
Naso
Michael
M
Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Uitto
Jouni
J
Klement
John F
JF
D024506:5' Flanking Region; D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D024143:Collagen Type VII / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D017353:Gene Deletion; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D005816:Genetic Complementation Test; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D024363:Transcription Initiation Site; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D024143:Collagen Type VII; D016212:Transforming Growth Factor beta