REDUCTION OF TYPE-V COLLAGEN USING A DOMINANT-NEGATIVE STRATEGY ALTERS THE REGULATION OF FIBRILLOGENESIS AND RESULTS IN THE LOSS OF CORNEAL-SPECIFIC FIBRIL MORPHOLOGY

A number of factors have been implicated in the regulation of tissue-s pecific collagen fibril diameter. Previous data suggest that assembly of heterotypic fibrils composed of two different fibrillar collagens r epresents 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 us ed a dominant-negative retroviral strategy to decrease the levels of t ype V collagen secreted by chicken corneal fibroblasts. The chicken al pha 1(V) collagen gene was cloned, and retroviral vectors that express ed a polycistronic mRNA encoding a truncated alpha 1(V) minigene and t he reporter gene LacZ were constructed. The efficiency of viral infect ion was 30-40%, as determined by assaying beta-galactosidase activity. To assess the expression from the recombinant provirus, Northern anal ysis was performed and indicated that infected fibroblasts expressed h igh steady-state levels of retroviral mRNA. Infected cells synthesized the truncated alpha 1(V) protein, and this was detectable only intrac ellularly, in a distribution that colocalized with lysosomes. To asses s endogenous alpha 1(V) protein levels, infected cell cultures were as sayed, and these consistently demonstrated reductions relative to cont rol 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 distribut ion, characteristics similar to fibrils produced in connective tissues with low type V concentrations. Immunoelectron microscopy demonstrate d the aminoterminal 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 assemb ly so that abnormally large-diameter fibrils are deposited into the ma trix.