Positive Regulation of Corneal Type V Collagen mRNA: Analysis by Chicken–Human Heterokaryon Formation

Abstract

Our previous studies have suggested that type V collagen is at least one factor responsible for the characteristically small, uniform diameter 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 content of type V collagen in cornea is reflected by an equally elevated content of α1(V) chain mRNA in corneal fibroblasts. Thus, the increased production of the molecule in cornea appears to be regulated at the level of transcription and/or mRNA stability. One possible explanation for this is that corneal fibroblasts contain positive regulatory factors that specifically upregulate transcription of the type V collagen genes and/or increase their mRNA stability. To test this possibility, we have produced transient heterokaryons by fusing chicken corneal fibroblasts with two human noncorneal cell lines selected as containing little if any α1(V) mRNA. If the chicken corneal cells contain positive regulators that can act across species, these regulators should result in increased levels of the human α1(V) transcript. The results were evaluated by reverse transcript–polymerase chain reaction employing a primer pair selected for its ability specifically to amplify part of the human α1(V) mRNA. In fusions between chicken corneal fibroblasts and the human cell lines, after a lag of 10–14 h the heterokaryon-containing cultures showedde novoappearance or upregulation of human α1(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 diffusible substances acting between the cells. Chicken tendon fibroblasts, a low producer of type V collagen, when tested in the same assay, evoked no detectible increase in the human transcript. Thus, corneal cells do contain positive regulators for α1(V) chain mRNA, and this effect is at least somewhat cell specific.