Original Article

Journal of Investigative Dermatology (1982) 79, 398–402; doi:10.1111/1523-1747.ep12530243

Colchicine-Induced Modulation of Collagenase in Human Skin Fibroblast Cultures. I. Stimulation of Enzyme Synthesis in Normal Cells

Eugene A Bauer and Karl-Johan Valle

Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, U.S.A.

Received 4 January 1982; Accepted 16 April 1982.

Top

Abstract

Microtubule-active agents affect the secretion of a variety of proteins, including collagenase. To gain insight into the mechanisms involved in this process, we examined the effects of colchicine on the synthesis, secretion, and activity of human skin collagenase. When added to monolayer cultures of human skin fibroblasts, 10-6 M colchicine produced a mean 3-fold increase in trypsin-activatable collagenase in the culture medium. Stimulation was not observed with lumicolchicine, The enhanced accumulation of collagenase was dose-dependent with 10-9, 10-8, 10-7, and 10-6 M colchicine giving collagenase activities/mg protein that were 100 plusminus 6%, 165 plusminus 20%, 186 plusminus 34%, and 297 plusminus 62% of control, respectively. Although the effect on collagenase was seen under conditions independent of cellular growth (i.e., in serum-free medium), maximum stimulation occurred in subconfluent cultures. The colchicine-induced increase in activity was paralleled by an increase in immunoreactive enzyme protein, suggesting stimulation of enzyme synthesis. The catalytic efficiency of the enzyme (activity per unit immunoreactive protein) was unchanged, however, indicating that a structurally normal enzyme was being synthesized. To examine the process in more detail, the biosynthesis of 3H-labeled collagenase was quantitated in these cultures by specific immunoprecipitation. Although 10-6 M colchicine produced no increase in total protein synthesis, an increased rate of collagenase synthesis was seen after only 1.5 hr. These data suggest that colchicine has a specific effect on the synthesis of collagenase and may be a useful probe for studying its regulation.

Top

References

  1. Valle, K-J, Bauer, EA: Biosynthesis of collagenase by human skin fibroblasts in monolayer culture. J Biol Chem 1979 254: 10115–10122,  | PubMed | ChemPort |
  2. Gross, J: Aspects of the animal collagenases, Biochemistry of Collagen 1976 Edited by GN Ramachandran, AH Reddi. New York, Plenum, pp 275–317,
  3. Bauer, EA, Uitto, J: Collagen in cutaneous diseases. Int J Dermatol 1979 18: 251–270,  | PubMed | ChemPort |
  4. Bauer, EA, Gedde-Dahl, T, Eisen, AZ: The role of human skin collagenase in epidermolysis bullosa. J Invest Dermatol 1977 68: 119–124,  | Article | PubMed | ChemPort |
  5. Yamanishi, Y, Dabbous, MK, Hashimoto, K: Effect of collagenolytic activity in basal cell epithelioma of the skin on reconstituted collagen and physical properties and kinetics of the crude enzymes. Cancer Res 1972 32: 2551–2560,  | PubMed | ChemPort |
  6. Bauer, EA, Gordon, JM, Reddick, ME, Eisen, AZ: Quantitation and immunocytochemical localization of human skin collagenase in basal cell carcinoma. J Invest Dermatol 1977 69: 363–367,  | Article | PubMed | ISI | ChemPort |
  7. Eisen, AZ: Human skin collagenase: relationship to the pathogenesis of epidermolysis bullosa dystrophica. J Invest Dermatol 1969 52: 449–453,  | PubMed | ChemPort |
  8. Lazarus, GS: Collagenase and connective tissue metabolism in epidermolysis bullosa. J Invest Dermatol 1972 58: 242–248,  | Article | PubMed | ISI | ChemPort |
  9. Bauer, EA, Eisen, AZ: Recessive dystrophic epidermolysis bullosa. Evidence for increased collagenase as a genetic characteristic in cell culture. J Exp Med 1978 148: 1378–1387,  | Article | PubMed | ISI | ChemPort |
  10. Valle, K-J, Bauer, EA: Enhanced biosynthesis of human skin collagenase in fibroblast cultures from recessive dystrophic epidermolysis bullosa. J Clin Invest 1980 66: 176–187,  | PubMed | ChemPort |
  11. Dresden, MH, Heilman, SA, Schmidt, JD: Collagenolytic enzymes in human neoplasms. Cancer Res 1972 32: 993–996,  | PubMed | ChemPort |
  12. Bauer, EA, Uitto, J, Walters, RC, Eisen, AZ: Enhanced collagenase production by fibroblasts derived from human basal cell carcinomas. Cancer Res 1979 39: 4594–4599,  | PubMed | ChemPort |
  13. Ehrlich, HP, Bornstein, P: Microtubules in transcellular movement of procollagen. Nature [New Biol] 1972 238: 257–260,
  14. Dehm, P, Prockop, DJ: Time lag in the secretion of collagen by matrix-free tendon cells and inhibition of the secretory process by colchicine and vinblastine. Biochim Biophys Acta 1972 264: 375–382,  | PubMed |
  15. Diegelmann, RF, Peterkofsky, B: Inhibition of collagen secretion from bone and cultured fibroblasts by microtubular disruptive drugs. Proc Natl Acad Sci USA 1972 69: 892–896,  | Article | PubMed | ChemPort |
  16. Ehrlich, HP, Ross, R, Bornstein, P: Effects of antimicrotubular agents on the secretion of collagen. A biochemical and morphological study. J Cell Biol 1974 62: 390–405,  | Article | PubMed |
  17. Lohmander, S, Moskalewski, S, Madsen, K, Thyberg, J, Friberg, U: Influence of colchicine on the synthesis and secretion of proteoglycans and collagen by fetal guinea pig chondrocytes. Exp Cell Res 1976 99: 333–345,  | Article | PubMed |
  18. Chou, I-N, Zeiger, J, Solomon, JA, Black, PH: Stimulation of plasminogen activator expression and induction of DNA synthesis by microtubule-disruptive drugs. Biochem Biophys Res Commun 1981 101: 1266–1273,  | Article | PubMed |
  19. Harris, ED, Krane, SM: Effects of colchicine on collagenase in cultures of rheumatoid synovium. Arthritis Rheum 1971 14: 669–684,  | PubMed | ChemPort |
  20. Gordon, S, Werb, Z: Secretion of macrophage neutral proteinase is enhanced by colchicine. Proc Natl Acad Sci USA 1976 73: 872–876,  | Article | PubMed |
  21. Bauer, EA: Cell culture density as a modulator of collagenase expression in normal human fibroblast cultures. Exp Cell Res 1977 107: 269–276,  | Article | PubMed | ChemPort |
  22. Busiek, DF, Bauer, EA: Environmental pH modulation of collagenase in normal human fibroblast cultures. Biochim Biophys Acta 1979 585: 389–397,  | PubMed | ChemPort |
  23. Bauer, EA, Stricklin, GP, Jeffrey, JJ, Eisen, AZ: Collagenase production by human skin fibroblasts. Biochem Biophys Res Commun 1975 64: 232–240,  | Article | PubMed | ChemPort |
  24. Nagai, Y, Lapiere, CM, Gross, J: Tadpole collagenase: preparation and purification. Biochemistry 1966 5: 3123–3130,  | Article | PubMed | ISI | ChemPort |
  25. Bauer, EA, Eisen, AZ, Jeffrey, JJ: Radioimmunoassay of human collagenase. Specificity of the assay and quantitative determination of in vivo and in vitro human skin collagenase. J Biol Chem 1972 247: 6679–6685,  | PubMed | ChemPort |
  26. Stricklin, GP, Bauer, EA, Jeffrey, JJ, Eisen, AZ: Human skin collagenase: isolation of precursor and active forms from both fibroblast and organ cultures. Biochemistry 1977 16: 1607–1615,  | Article | PubMed | ISI | ChemPort |
  27. Stricklin, GP, Eisen, AZ, Bauer, EA, Jeffrey, JJ: Human skin collagenase: chemical properties of precursor and active forms. Biochemistry 1978 17: 2331–2337,  | Article | PubMed | ISI | ChemPort |
  28. King, J, Laemmli, UK: Polypeptides of the tail fibres of bacteriophage T4. J Mol Biol 1971 62: 465–477,  | Article | PubMed | ISI | ChemPort |
  29. Lowry, O, Rosebrough, N, Farr, A, Randall, R: Protein measurement with the Folin phenol reagent. J Biol Chem 1951 193: 265–275,  | PubMed | ISI | ChemPort |
  30. Burton, K: A study of the conditions and mechanisms of diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 1956 62: 315–323,  | PubMed | ISI | ChemPort |
  31. Ostlund, RE, Pfleger, B, Schonfeld, G: Role of microbubules in low density lipoprotein processing by cultured cells. J Clin Invest 1979 63: 75–84,  | PubMed | ISI | ChemPort |
  32. Snyder, JA, McIntosh, JR: Biochemistry and physiology of microtubules. Annu Rev Biochem 1976 45: 699–720,  | Article | PubMed | ChemPort |

Extra navigation

.
ADVERTISEMENT