Growth-dependent modulation of type I collagen production and mRNA levels in cultured human skin fibroblasts

Abstract

Five human skin fibroblast lines were studied for type I collagen production and type I procollagen mRNA levels through the different growth phases. The cells were plated at low density and followed for 11 days at daily intervals through the stages of rapid growth and visual confluency until the cultures reached stationary growth phase. Each day one culture flask was labeled with [3H]proline for 24 h, and analyzed for production of radiolabeled type I collagen into culture medium. The cell layers were counted and subjected to isolation of cytoplasmic RNA and determination of type I procollagen mRNA levels. The results revealed an approx. 2-fold increase in procollagen production and mRNA levels when the cells reached visual confluency. Thereafter the synthesis rates and mRNA levels remained relatively constant, although a decreasing tendency of both parameters was observed upon further culturing. The results confirm that determination of cell density is important when cell cultures are used for measurement of collagen synthesis or mRNA levels. For determination of pro alpha 2(I) collagen mRNA an 1193 bp cDNA clone was constructed using RNA extracted from human fetal calvaria. Sequencing of the clone revealed some nucleotide and amino acid differences between the previously published sequences. This suggests the presence of more individual variation in procollagen coding sequences than expected.