Mechanical load enhances procollagen processing in dermal fibroblasts by regulating levels of procollagen C-proteinase

Mechanical forces are emerging as key regulators of cell function. We hypot hesize that mechanical load may influence dermal fibroblast activity. We as sessed the direct effects of mechanical load on human dermal fibroblast pro collagen synthesis and processing in vitro. Cells were loaded in a biaxial loading system (Flexercell 3000), Hydroxyproline levels were measured in th e medium and cell layer as an estimate of procollagen synthesis and process ing to insoluble collagen, Mechanical load (in the presence of serum or TGF -beta) enhanced procollagen synthesis by 45 +/- 3% (P < 0.001), and 38 +/- 4% (P < 0.001), respectively, over unloaded growth factor controls after 48 h, Insoluble collagen deposition was enhanced in the same cultures by 115 +/- 8% (P < 0.01) and 72% +/- 9% (P < 0.01), respectively. This effect was inhibited using L-arginine suggesting that procollagen C-proteinase, the en zyme which directly cleaves the C-terminal propeptide of procollagen to for m insoluble collagen, is required for the fiber formation observed. Procoll agen mRNA levels in loaded samples increased by more than two-fold in both serum and TGF-beta-treated cultures at 48 h. Procollagen C-proteinase mRNA levels were also enhanced by a similar magnitude, although the increase was observed at 24 h, Procollagen C-proteinase protein levels were also increa sed at this time. Protein and mRNA levels of the procollagen C-proteinase e nhancer protein, which binds the C-terminal propeptide of procollagen to en hance the rate of peptide cleavage, were unaffected by mechanical load. Thi s study demonstrates that mechanical load promotes procollagen synthesis in dermal fibroblasts by enhancing gene expression and posttranslational proc essing of procollagen. (C) 1999 Academic Press.