Jimenez S A, Williams C J, Myers J C, Bashey R I
J Biol Chem. 1986 Jan 15;261(2):657-62.
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.
紧皮（TSK）小鼠是一种突变品系，表现出结缔组织异常，其特征是皮肤、皮下组织以及某些内部器官（如心脏）中胶原蛋白过度积累。先前已证明，来自患病小鼠的皮肤器官培养物中胶原蛋白生物合成增加，但其负责机制尚未明确。为了研究导致这种蛋白质产生增加的分子改变，建立了正常和TSK小鼠真皮成纤维细胞系，并对胶原蛋白生物合成以及I型和III型前胶原基因的表达进行了研究。将5只正常小鼠和5只TSK小鼠的真皮成纤维细胞传代培养，在含有10%胎牛血清和50微克/毫升抗坏血酸的培养基中培养，用[14C]脯氨酸标记72小时后，测定合成的[14C]羟脯氨酸量。结果显示，TSK小鼠真皮成纤维细胞产生的[14C]羟脯氨酸量明显高于正常对照（118±28.3×10（-2）对53.7±21.9×10（-2）dpm/微克DNA；p<0.004）。随后，通过将多聚腺苷酸化RNA与分别作为α1（I）、α2（I）和α1（III）前胶原链转录本特异性探针的人cDNA克隆α12、Hf 32和RJ 5进行Northern印迹杂交，分析正常和TSK小鼠成纤维细胞中三种前胶原基因的表达。发现TSK小鼠成纤维细胞中所有三种胶原蛋白转录本水平持续升高（高达5倍），而β-肌动蛋白mRNA水平未改变。结果表明，TSK小鼠真皮成纤维细胞在培养中产生过量胶原蛋白，同时I型和III型前胶原基因的表达显著增加。
