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 tissue accumulation in skin and visceral organs including the heart. To study the mechanisms of myocardial fibrosis in TSK mice, we established several strains of TSK mice myocardial fibroblasts in culture and examined the regulation of collagen gene expression in these cells. These strains displayed increased collagen gene expression in comparison with myocardial fibroblasts established from normal mice. On an average, the TSK myocardial fibroblast cultures showed a 4-fold increase in collagen synthesis and 4.4- and 3.6-fold increases, respectively, in alpha 1(I) and alpha 1(III) collagen mRNA steady state levels. The increased alpha 1(I) and alpha 1(III) collagen mRNA levels were mainly due to increased transcription rates (3.4- and 3.8-fold higher, respectively) of the respective genes. Furthermore, we showed that the up-regulation of alpha 1(I) procollagen gene transcription in TSK mice myocardial 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 fibroblasts showed lower DNA binding activity to oligonucleotides spanning the mapped regulatory sequence as well as to a consensus AP-1 sequence, but not to a consensus SP-1 sequence, and supershift experiments with an AP-1 antibody confirmed the interaction of these oligonucleotides with AP-1 protein. These observations indicate that a strong negative regulatory sequence contained within -0.675 to -0.804 kilobase of the alpha 1(I) procollagen promoter binds AP-1 transcription factor and mediates inhibition of gene transcription in normal murine myocardial fibroblasts. The TSK mice myocardial fibroblasts lack this inhibitory control, due to lower available amounts and/or decreased binding activity to this inhibitory sequence, and hence display increased alpha 1(I) procollagen gene expression

Type X collagen biosynthesis and expression in avian tibial dyschondroplasia

AbstractObjective: Tibial dyschondroplasia (TD) is an abnormality of growth plate cartilage characterized by the presence of non-vascularized, non-mineralized tissue. The objective of this study was to examine structural and functional alterations of the growth plate-specific type X collagen in RTD cartilage.Design: Collagen biosynthesis was examined in organ cultures and in cultured chondrocytes from normal growth plate and TD cartilage. Thermal stability of type X collagen extracted from normal and TD cartilage organ cultures to protease digestions by trypsin plus chymotrypsin bacterial collagenase was determined. The expression of collagen genes was examined in cultured normal and TD chondrocytes.Results: Synthesis of total collagen and of type X collagen was greater than threefold higher in organ cultures from the TD lesion compared with normal growth plate. The increase in type X collagen synthesis in the lesion was compensated by a reduction in the relative proportions of types II and XI collagens. The thermal denaturation and collagenase cleavage properties of purified types II and X collagens from TD cartilage were normal. The expression of type X collagen gene was threefold higher in cultured TD chondrocytes compared to chondrocytes from normal growth plate. Normal growth plate chondrocytes in primary cultures synthesized predominantly type X collagen (80% of total collagen). In contrast, TD chondrocytes synthesized mainly types I and II collagens and type X collagen represented only 22% of total collagen. TD cells initiated the synthesis of type I collagen within 5 days of primary culture, whereas normal chondrocytes did not synthesize this collagen during the same culture period. Although type X collagen synthesis was reduced in TD chondrocytes, the mRNA levels for type X collagen were substantially higher than in normal chondrocytes.Conclusion: Accumulation of type X collagen in TD cartilage results from its increased biosynthesis which is due largely to increased expression of the gene for this collagen, although, the chondrocyte culture studies suggest the possibility of postranscriptional defect in type X collagen synthesis or processing in TD lesion. Moreover, the TD chondrocytes in contrast with normal chondrocytes display evidence of prompt loss of their specific phenotype during short-term primary cultures