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J. Biol. Chem., Vol. 261, Issue 2, 657-662, Jan, 1986

Increased collagen biosynthesis and increased expression of type I and type III procollagen genes in tight skin (TSK) mouse fibroblasts

SA Jimenez, CJ Williams, JC Myers and RI Bashey

The Tight Skin (TSK) mouse is a mutant strain that displays connective tissue abnormalities characterized by excessive accumulation of collagen in skin, subcutaneous tissues, and some internal organs such as the heart. Increased collagen biosynthesis by skin organ cultures from affected mice has been previously demonstrated, but the mechanisms responsible have not been identified. In order to examine the molecular alterations responsible for the increased production of this protein, normal and TSK mouse dermal fibroblast cell lines were established, and studies of collagen biosynthesis and expression of Types I and III procollagen genes were performed. Secondary cultures of 5 normal and 5 TSK mice dermal fibroblasts were incubated in media containing 10% fetal calf serum and 50 micrograms/ml ascorbic acid and after labeling with [14C]proline for 72 h the amount of [14C]hydroxyproline synthesized was determined. The results showed that TSK mice dermal fibroblasts produced significantly greater amounts of [14C]hydroxyproline than their normal counterparts (118 +/- 28.3 X 10(- 2) versus 53.7 +/- 21.9 X 10(-2) dpm/micrograms of DNA; p less than 0.004). Subsequently, the expression of three procollagen genes in normal and TSK mice fibroblasts was analyzed by Northern blot hybridization of polyadenylated RNA to the human cDNA clones alpha 12, Hf 32, and RJ 5 which are specific probes for transcripts of alpha 1(I), alpha 2(I), and alpha 1(III) procollagen chains, respectively. It was found that TSK mice fibroblasts consistently displayed increased levels (up to 5-fold) of all three collagen transcripts while beta- actin mRNA levels were unchanged. The results demonstrate that TSK mice dermal fibroblasts produce excessive amounts of collagen in culture concomitant with a dramatic increase in the expression of Types I and III procollagen genes.
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