POSITIVE REGULATION OF CORNEAL TYPE-V COLLAGEN MESSENGER-RNA - ANALYSIS BY CHICKEN-HUMAN HETEROKARYON FORMATION

Our previous studies have suggested that type V collagen is at least o ne factor responsible for the characteristically small, uniform diamet er of striated collagen fibrils of the corneal stroma. These fibrils, which are heterotypic combinations of collagen types I and V, contain four- to fivefold more type V collagen than those of tendon and sclera . The latter are much larger and more heterodisperse, This high conten t of type V collagen in cornea is reflected by an equally elevated con tent of alpha 1(V) chain mRNA in corneal fibroblasts. Thus, the increa sed production of the molecule in cornea appears to be regulated at th e level of transcription and/or mRNA stability. One possible explanati on for this is that corneal fibroblasts contain positive regulatory fa ctors that specifically upregulate transcription of the type V collage n genes and/or increase their, mRNA stability. To test this possibilit y, we have produced transient heterokaryons by fusing chicken corneal fibroblasts with two human noncorneal cell lines selected as containin g little if any alpha 1(V) mRNA. If the chicken corneal cells contain positive regulators that can act across species, these regulators shou ld result in increased levels of the human alpha 1(V) transcript. The results were evaluated by reverse transcript-polymerase chain reaction employing a primer pair selected for its ability specifically to ampl ify part of the human alpha 1(Tr) mRNA. In fusions between chicken cor neal fibroblasts and the human cell lines, after a lag of 10-14 h the heterokaryon-containing cultures showed de novo appearance or upregula tion of human alpha 1(V) chain mRNA compared with that of the parental cell lines. Cultures of the mixed cell types that had not been fused showed no such upregulation, so the effect was not mediated by diffusi ble substances acting between the cells. Chicken tendon fibroblasts, a low producer of type V collagen, when tested in the same assay, evoke d no detectible increase in the human transcript, Thus, corneal cells do contain positive regulators for alpha 1(V) chain mRNA, and this eff ect is at least somewhat cell specific. (C) 1996 Academic Press,Inc.