CONTROL OF COLLAGENASE IN HUMAN FIBROBLAST CULTURES

胶原酶在启动间质性胶原降解中起关键作用。 胶原蛋白在各种形态发生的重要事件。 我们将使用 培养的皮肤成纤维细胞，以检查胶原酶刺激的作用， 蛋白质，包括20 kDa表皮细胞因子和血小板衍生的生长 因子，对这些靶细胞表达胶原酶的影响。 我们将 评估对这些肽激动剂的反应是否是发育性的 通过检查（i）由酶反应的胶原酶表达来调节 活性，免疫反应酶蛋白的合成和 可杂交的mRNA和（ii）靶成纤维细胞的促有丝分裂反应 对细胞因子的反应。 我们将使用培养的成纤维细胞从不同阶段的 发展，包括从遗传“衰老综合征”，以解决是否 这两种反应反映了不同的机制途径。 这些 研究细胞因子对肿瘤细胞增殖的作用机制， 胶原酶表达将通过定义基因产物 胶原酶本身，可以以任何方式影响其自身的合成， 其他激动剂对正常细胞和各种类型细胞的作用 大疱性表皮病，并通过检查在核酸的影响， 例如类维生素A和糖皮质激素的抑制剂水平 胶原酶合成。 由于有证据表明，在细胞中存在胶原酶的结构突变形式， 隐性营养不良性大疱性表皮病（RDEB），我们将采用培养 从这种疾病的成纤维细胞作为胶原酶mRNA的来源，克隆 puerapy突变型胶原酶基因 作为分离和 RDEB cDNa克隆的初始表征，我们将采用 正常人皮肤胶原酶pCol 185.2的全长cDNA克隆。 我们 目标是集中精力定义突变位点， 结构改变的蛋白质，这样我们就可以更好地理解 监管机制，提供更好的诊断和遗传咨询， 并设计出合理的治疗方法， 遗传性皮肤病

Collagenase is critical in initiating the degradation of interstitial collagens in a variety of morphogenetically important events. We shall use cultured skin fibroblasts to examine the effects of collagenase-stimulatory proteins, including a 20 kDa epidermal cytokine and platelet-derived growth factor, on the expression of collagenase by these target cells. We shall assess whether the response to these peptide agonists is developmentally modulated by examining (i) collagenase expression as reflected by enzyme activity, synthesis of immunoreactive enzyme protein and alterations in hybridizable mRNA and (ii) the mitogenic response of the target fibroblasts to the cytokines. We shall use cultured fibroblasts from various stages of development including from the genetic "aging syndromes" to address whether these two responses are reflective of separate mechanistic pathways. These investigations of the mechanisms for the effects of cytokines on collagenase expression will be complemented by defining if the gene product itself, collagenase, can in any way affect its own synthesis, by probing the effects of other agonists on normal cells and cells from various types of epidermolysis bullosa, and by examining the effects at a nucleic acid level of agents such as retinoids and glucocorticoids that inhibit collagenase synthesis. Since there is evidence for a structurally mutant form of collagenase in recessive dystrophic epidermolysis bullosa (RDEB), we shall employ cultured fibroblasts from this disease as a source for collagenase mRNA to clone the putatively mutant collagenase gene. As a probe for the isolation and initial characterization of the RDEB cDNa clones we shall employ a full-length cDNA clone for normal human skin collagenase pCol 185.2. Our goal is to focus intensely on defining the site(s) of mutation leading to the structurally altered protein, so that we can better understand regulatory mechanisms, provide improved diagnosis and genetic counseling, and devise rational therapeutic modalities in this devastating genodermatosis.