Marchant J K, Hahn R A, Linsenmayer T F, Birk D E
Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
J Cell Biol. 1996 Dec;135(5):1415-26. doi: 10.1083/jcb.135.5.1415.
A number of factors have been implicated in the regulation of tissue-specific collagen fibril diameter. Previous data suggest that assembly of heterotypic fibrils composed of two different fibrillar collagens represents a general mechanism regulating fibril diameter. Specifically, we hypothesize that type V collagen is required for the assembly of the small diameter fibrils observed in the cornea. To test this, we used a dominant-negative retroviral strategy to decrease the levels of type V collagen secreted by chicken corneal fibroblasts. The chicken alpha 1(V) collagen gene was cloned, and retroviral vectors that expressed a polycistronic mRNA encoding a truncated alpha 1(V) minigene and the reporter gene LacZ were constructed. The efficiency of viral infection was 30-40%, as determined by assaying beta-galactosidase activity. To assess the expression from the recombinant provirus, Northern analysis was performed and indicated that infected fibroblasts expressed high steady-state levels of retroviral mRNA. Infected cells synthesized the truncated alpha 1(V) protein, and this was detectable only intracellularly, in a distribution that colocalized with lysosomes. To assess endogenous alpha 1(V) protein levels, infected cell cultures were assayed, and these consistently demonstrated reductions relative to control virus-infected or uninfected cultures. Analyses of corneal fibril morphology demonstrated that the reduction in type V collagen resulted in the assembly of large-diameter fibrils with a broad size distribution, characteristics similar to fibrils produced in connective tissues with low type V concentrations. Immunoelectron microscopy demonstrated the amino-terminal domain of type V collagen was associated with the small-diameter fibrils, but not the large fibrils. These data indicate that type V collagen levels regulate corneal fibril diameter and that the reduction of type V collagen is sufficient to alter fibril assembly so that abnormally large-diameter fibrils are deposited into the matrix.
许多因素与组织特异性胶原纤维直径的调节有关。先前的数据表明，由两种不同的纤维状胶原组成的异型纤维的组装是调节纤维直径的一般机制。具体而言，我们假设V型胶原是角膜中观察到的小直径纤维组装所必需的。为了验证这一点，我们使用了显性负性逆转录病毒策略来降低鸡角膜成纤维细胞分泌的V型胶原水平。克隆了鸡α1(V)胶原基因，并构建了表达编码截短α1(V)小基因和报告基因LacZ的多顺反子mRNA的逆转录病毒载体。通过检测β-半乳糖苷酶活性确定病毒感染效率为30%-40%。为了评估重组前病毒的表达，进行了Northern分析，结果表明感染的成纤维细胞表达高水平的逆转录病毒mRNA稳态。感染的细胞合成了截短的α1(V)蛋白，并且仅在细胞内可检测到，其分布与溶酶体共定位。为了评估内源性α1(V)蛋白水平，对感染的细胞培养物进行了检测，结果始终显示相对于对照病毒感染或未感染的培养物有所降低。角膜纤维形态分析表明，V型胶原水平的降低导致了具有广泛尺寸分布的大直径纤维的组装，其特征类似于在V型胶原浓度低的结缔组织中产生的纤维。免疫电子显微镜显示V型胶原的氨基末端结构域与小直径纤维相关，但与大纤维无关。这些数据表明V型胶原水平调节角膜纤维直径，并且V型胶原的减少足以改变纤维组装，从而使异常大直径的纤维沉积到基质中。
