Abstract
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.
Highlights
Increased Collagen Biosynthesis and Increased Expressionof Type I and Type111 Procollagen Genes in Tight Skin(TSK) Mouse Fibroblasts*
Secondary culturesof 5 normal and 5 TSK mice dermal fibroblasts were incubated in media containing 10%fetal calf serum and 50 rg/ml ascorbic acid and after labeling with [14C]prolinefor 72 h the amount of [‘4C]hydroxyproline synthesized was determined
The results showed that TSKmice dermaflibroblasts produced significantly greater amounts of [‘4C]hydroxyproline thantheir normal counterparts
Summary
Mice Skin-In preliminary experiments, we found that the method of obtaining cultures of dermal fibroblasts from skin explants anchored at the bottom of aculturedish usually employed inour laboratories [18]did not lead to any observable outgrowth of fibroblasts. In attempts to improve the establishment of fibroblast cell lines from normal and TSKmice skin, we utilized an enzymatic digestion method to obtain isolated fibroblasts before seeding into culture dishes. The suspended cells were transferred into culture plastic tubes and resuspended in MEM containing 10% FCS, and thestock protease inhibitor mixture and aliquots of cell suspensions were used to determine DNA content employing a diamine method [19]. Northern blots of control and TSK poly(A+)RNA hybridized to procollagen cDNA probes were exposed for variable lengths of time for optimal visualization and densitometrically scanned on aJoyce-Loebl densitometer. The dot blots were washed as described above, air dried, and exposed to x-ray film with intensifying screen at -70 "C for 4 days. The dots were densitometrically scanned on a Joyce-Loebl densitometer
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