Abstract
The excess accumulation of type I collagen within tissues leads to organ dysfunction and occurs as a result of an imbalance between synthesis and degradation. This chapter outlines several methods to assess the in vitro production of type I collagen that are employed in our laboratory. We describe Western immunoblotting of intact α1(I) collagen using antibodies directed to α1(I) collagen amino and carboxyl propeptides. The measurement of α1(I) collagen mRNA levels using real-time polymerase chain reaction is then outlined. Finally, methods to determine the transcriptional regulation of α1(I) collagen using a nuclear run-on assay are described.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Prockop, D. J., Kivirikko, K. I., Tuderman, L., and Guzman, N. A. (1979) The biosynthesis of collagen and its disorders (second of two parts). N. Engl. J. Med. 301(2), 77–85.
Prockop, D. J., Kivirikko, K. I., Tuderman, L., and Guzman, N. A. (1979) The biosynthesis of collagen and its disorders (first of two parts). N. Engl. J. Med. 301(1), 13–23.
Vuorio, E. and de Crombrugghe, B. (1990) The family of collagen genes. Annu. Rev. Biochem. 59, 837–872.
Raghow, R., Lurie, S., Seyer, J. M., and Kang, A. H. (1985) Profiles of steady state levels of messenger RNAs coding for type I procollagen, elastin, and fibronectin in hamster lungs undergoing bleomycin-induced interstitial pulmonary fibrosis. J. Clin. Invest. 76(5), 1733–1739.
Penttinen, R. P., Kobayashi, S., and Bornstein, P. (1988) Transforming growth factor beta increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability. Proc. Natl. Acad. Sci. USA 85(4), 1105–1108.
Stefanovic, B., Hellerbrand, C., Holcik, M., Briendl, M., Aliebhaber, S., and Brenner, D. A. (1997) Posttranscriptional regulation of collagen alpha1(I) mRNA in hepatic stellate cells. Mol. Cell Biol. 17(9), 5201–5209.
Doege, K. J. and Fessler, J. H. (1986) Folding of carboxyl domain and assembly of procollagen I. J. Biol. Chem. 261(19), 8924–8935.
Wilson, R., Lees, J. F., and Bulleid, N. J. (1998) Protein disulfide isomerase acts as a molecular chaperone during the assembly of procollagen. J. Biol. Chem. 273(16), 9637–9643.
Rocnik, E. F., van d, V., Cao, H., Hegele, R. A., and Pickering, J. G. (2002) Functional linkage between the endoplasmic reticulum protein Hsp47 and procollagen expression in human vascular smooth muscle cells. J. Biol. Chem. 277(41), 38,571–38,578.
Kivirikko, K. I., Myllyla, R., and Pihlajaniemi, T. (1989) Protein hydroxylation: prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit. FASEB J. 3(5), 1609–1617.
Berg, R. A., Steinmann, B., Rennard, S. I., and Crystal, R. G. (1983) Ascorbate deficiency results in decreased collagen production: under-hydroxylation of proline leads to increased intracellular degradation. Arch Biochem. Biophys. 226(2), 681–686.
Rowe, L. B. and Schwarz, R. I. (1983) Role of procollagen mRNA levels in controlling the rate of procollagen synthesis. Mol. Cell Biol. 3(2), 241–249.
Schwarz, R. I. (1985) Procollagen secretion meets the minimum requirements for the rate-controlling step in the ascorbate induction of procollagen synthesis. J. Biol. Chem. 260(5), 3045–3049.
Fitzgerald, J., Lamande, S. R., and Bateman, J. F. (1999) Proteasomal degradation of unassembled mutant type I collagen pro-alpha1(I) chains. J. Biol. Chem. 274(39), 27,392–27,398.
Rishikof, D. C., Ricupero, D. A., Poliks, C. F., and Goldstein, R. H. (1999) Amino acid availability regulates type I procollagen accumulation in human lung fibroblasts. J. Cell Biochem. 75(1), 130–137.
Bienkowski, R. S. (1984) Collagen degradation in human lung fibroblasts: extent of degradation, role of lysosomal proteases, and evaluation of an alternate hypothesis. J. Cell Physiol. 121(1), 152–158.
Rennard, S. I., Stier, L. E., and Crystal, R. G. (1982) Intracellular degradation of newly synthesized collagen. J. Invest. Dermatol. 79(Suppl 1), 77s–82s.
Berg, R. A., Schwartz, M. L., and Crystal, R. G. (1980) Regulation of the production of secretory proteins: intracellular degradation of newly synthesized “defective” collagen. Proc. Natl. Acad. Sci. USA 77(8), 4746–4750.
Barile, F. A., Guzowski, D. E., Ripley, C., Siddiqi, Z. A., and Bienkowski, R. S. (1990) Ammonium chloride inhibits basal degradation of newly synthesized collagen in human fetal lung fibroblasts. Arch Biochem. Biophys. 276(1), 125–131.
Berg, R. A., Schwartz, M. L., Rome, L. H., and Crystal, R. G. (1984) Lysosomal function in the degradation of defective collagen in cultured lung fibroblasts. Biochemistry 23(10), 2134–2138.
Fisher, L. W., Stubbs, J. T., III, and Young, M. F. (1995) Antisera and cDNA probes to human and certain animal model bone matrix noncollagenous proteins. Acta. Orthop. Scand. 266(Suppl), 61–65.
Fisher, L. W., Lindner, W., Young, M. F., and Termine, J. D. (1989) Synthetic peptide antisera: their production and use in the cloning of matrix proteins. Connect Tissue Res. 21(1–4), 43–48.
Ririe, K. M., Rasmussen, R. P., and Wittwer, C. T. (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245(2), 154–160.
Barsh, G. S., Roush, C. L., and Gelinas, R. E. (1984) DNA and chromatin structure of the human alpha 1 (I) collagen gene. J. Biol. Chem. 259(23), 14,906–14,913.
Pfarr, D. S., Rieser, L. A., Woychik, R. P., Rottman, F. M., Rosenberg, M., and Reff, M. E. (1986) Differential effects of polyadenylation regions on gene expression in mammalian cells. DNA 5(2), 115–122.
Maatta, A., Ekholm, E., and Penttinen, R. P. Effect of the 3′-untranslated region on the expression levels and mRNA stability of alpha 1(I) collagen gene. Biochim. Biophys. Acta 1260(3), 294–300.
Fine, A., Matsui, R., Zhan, X., Poliks, C. F., Smith, B. D., and Goldstein, R. H. (1992) Discordant regulation of human type I collagen genes by prostaglandin E2. Biochim. Biophys. Acta 1135(1), 67–72.
Rossert, J., Terraz, C., and Dupont, S. (2000) Regulation of type I collagen genes expression. Nephrol. Dial Transplant. 15(Suppl 6), 66–68.
Acknowledgments
This work was supported by the National Institutes of Health grants K08-HL04232 and R01-HL66547 and the VA Research Enhancement Award Program.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
Rishikof, D.C., Kuang, PP., Subramanian, M., Goldstein, R.H. (2005). Methods for Measuring Type I Collagen Synthesis In Vitro. In: Varga, J., Brenner, D.A., Phan, S.H. (eds) Fibrosis Research. Methods in Molecular Medicine, vol 117. Humana Press. https://doi.org/10.1385/1-59259-940-0:129
Download citation
DOI: https://doi.org/10.1385/1-59259-940-0:129
Publisher Name: Humana Press
Print ISBN: 978-1-58829-479-1
Online ISBN: 978-1-59259-940-0
eBook Packages: Springer Protocols