Molecular mechanism of the regulation of type VII collagen gene expression : Elucidation of pathogenesis and development therapy for dystophic epidermolysis bullosa

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Type VII collagen is the predominat component of the anchoring fibrils, attachment structures stabilizing the association of the cutaneous basement membrane to the underlying dermis. Alterations in the type VII collagen protein structure or luck of its expression due to mutations in the corresponding gene COL7A1 are the hallmark of dystrophic epidermolysis bullosa, a mechano-bullous skin disease characterized by extreme fragility of the skin and leading to development of sub-lamina densa blisters.1) It has been previously demonstrated that various cytokines (TGF-β, TNF-α, IL-1 β) upregulate the expression of COL7A1 in dermal fibroblasts. To determine the effect of various cytokines on COL7A1 expression in keratinocytes, we performed northern blot hybridization with COL7A1 cDNA probes. The results showed the down-regulation of COL7A1 expression by TNF-α, or IL-1 β in keratinocytes, wherase TGF-β up-regulates COL7A1 expression as shown in previous paper. These results indicate that effec … More t of TNF-α or IL-1 β on COL7A1 expression is cell-type specific. 2) To gain insight into the molecular mechanism underlying the up-regulation of COL7A1 by two cytokines (TNF-α, IL-1 β) on COL7A1 transcription in dermal fibroblast, we performed transient cell transfection with a series of 5'-deletion COL7A1 promoter/luciferase reporter gene constructs, and electrophoresis mobility shift assays with an oligonucleotide spanning the response element of each cytokines. The results showed that TNF-α effect was mediated by RelA (p65) homodimer binding the TNF-α response element in the COL7A1 promoter. Furthermore, an additive effect was observed when TGF-β and INF-α were added simultaneously to the dermal fibroblasts. IL-1 β up-regulates the COL7A1 expression mediated by NF-κB and AP-1 binding the NF-κB binding site (as an enhancer) and AP-1 binding site (as a suppresor) in the COL7A1 promoter region, respectively. 3) DEB is an inherited skin disorder caused by COL7A1 mutations. Over 100 mutations within exons or exon-intron borders of COL7A1 have been publishied. However, no mutation has been detected in COL7A1 promoter region. To examine the COL7A1 mutation in promoter region, we rescreened COL7A1 of DEB patients whose mutatons have not been detected using PCR amplification, followed by heteroduplex analysis. The results of direct sequencing of all 118 exons and their flanking intronic sequences showed that no mutation was detected in COL7A1 promoter region. However, one novel pathogenic mutation 7687-6 C→T was identified in intron 102 of COL7A1. Less