INCREASED ALPHA-1(I) PROCOLLAGEN GENE-EXPRESSION IN TIGHT-SKIN (TSK) MICE MYOCARDIAL FIBROBLASTS IS DUE TO A REDUCED INTERACTION OF A NEGATIVE REGULATORY SEQUENCE WITH AP-1 TRANSCRIPTION FACTOR

The TSK mouse, a model of fibrosis, displays exaggerated connective ti ssue accumulation in skin and visceral organs including the heart, To study the mechanisms of myocardial fibrosis in TSK mice, we establishe d several strains of TSK mice myocardial fibroblasts in culture and ex amined the regulation of collagen gene expression in these cells, Thes e strains displayed increased collagen gene expression in comparison w ith myocardial fibroblasts established from normal mice. On an average , the TSK myocardial fibroblast cultures showed a 4-fold increase in c ollagen synthesis and 4.4- and 3.6-fold increases, respectively, in al pha 1(I) and alpha 1(III) collagen mRNA steady state levels, The incre ased alpha 1(I) and alpha 1(III) collagen mRNA levels were mainly due to increased transcription rates (3.4- and 3.8-fold higher, respective ly) of the respective genes, Furthermore, we showed that the up-regula tion of alpha 1(I) procollagen gene transcription in TSK mice myocardi al fibroblasts was due to the lack of the strong inhibitory influence of a regulatory sequence contained in the promoter region encompassing nucleotides -675 to -804, Nuclear extracts from TSK mice myocardial f ibroblasts showed lower DNA binding activity to oligonucleotides spann ing the mapped regulatory sequence as well as to a consensus AP-1 sequ ence, but not to a consensus SP-l sequence, and supershift experiments with an AP-1 antibody confirmed the interaction of these oligonucleot ides with AP-1 protein. These observations indicate that a strong nega tive regulatory sequence contained within -0.675 to -0.804 kilobase of the alpha 1(I) procollagen promoter binds AP-1 transcription factor a nd mediates inhibition of gene transcription in normal murine myocardi al fibroblasts, The TSK mice myocardial fibroblasts lack this inhibito ry control, due to lower available amounts and/or decreased binding ac tivity to this inhibitory sequence, and hence display increased alpha 1(I) procollagen gene expression.