Regulation of heat-shock-protein 47(Hsp47) and procollagen by TGF-? in avian tendon cells

by 1965- Pan, Hongjie

The rupture of chicken gastrocnemius tendon poses a serious problem for the poultry industry. The cause of tendon rupture is unclear, virus infections, extreme environmental conditions or inappropriate growth factor expression have been considered to play a role in pathogenesis. Histological examination in most cases reveals fibrosis and rupture of collagen fibers. I investigated effects of heat-shock, growth factors and mechanical stress on procollagen and heat shock protein 47 (Hsp47) expression in chicken tendon fibroblasts. Hsp47 expression and synthesis rapidly increased in response to heat shock, and its response was reversible after removal of heat shock. Type I procollagen expression transiently increased and then decreased with heat shock. Both Hsp47 and procollagen expression was enhanced by human TGF-?1 treatment. Mechanical stress increased Hsp47 expression and protein production, but had no effect on type I procollagen expression. Because transforming growth factor-? (TGF-?) is a major regulator of collagen I have investigated the effects of TGF-? on collagen expression. As a part of my effort I generated the chicken TGF-?4 cDNA and expressed the corresponding protein and partially characterized it. Chicken TGF-?4 has 82% amino acid sequence identity to human TGF-?1. I expressed this protein and characterize it in vitro. I failed to recover the active TGF-?4 after purification and refolding when the chicken mature TGF-?4 protein was expressed in E. coli using a pET-28 vector. I generated the 5’ end of TGF-?4 cDNA using the modified 5’RACE. cDNA was produced from mRNA purified from the embryonic chicken tendon fibroblasts using the thermal stable reverse transcriptase and random hexamers at 70°C. Both the first and nested PCR was performed using GC-rich PCR kit. The alignment of obtained sequences showed that the 5’ end contained 271 more oligonucleotides with 70% of GC-bases (Genbank accession No: AF395834) than the original partial sequence recovered from the chicken TGF-?4 (Genbank accession No: M31160). I in-frame cloned cDNA expressing the TGF-?4 precursor into pcDNA3.1/V5-His-TOPO plasmid and expressed it in CHO-K1 cell line. The recombinant protein TGF-?4 was purified with Probond Resin under native conditions, and then activated by strong acidification. Protein bioassay showed that recombinant chicken TGF-?4 shares the TGF-? superfamily-related activities. Recombinant chicken TGF-?4 increased Hsp47 expression at both the protein and mRNA levels. I also found that human TGF-?1, chicken TGF-?4, and mechanical stress increased Hsp47 protein synthesis through activation and translocation of HSF1 into the nucleus as heat stress does. Therefore I conclude that chicken TGF-?4 coregulates type I procollagen and Hsp47 protein production in chicken tendon cells as human TGF-?1 does in mammalian species.