The Cell Biology of Fibroblast Cyclooxygenase

  • Amiram Raz
  • Angela Wyche
  • Diana Fagan
  • Philip Needleman
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 259)


We have prepared polyclonal antisera against sheep seminal vesicles cy-clooxygenase (COX) which cross-reacted with human COX. We employed this antisera in studies with human dermal fibroblast cultures to immunoprecipi-tate selectively the COX enzyme. Labeling of the cells with [35S]-methionine, solubilization of cellular COX followed by its immunoprecipitation, SDS-PAGE electrophoresis and fluorography enabled us to determine directly the synthetic rate of COX protein and its modulation by the monokine inter-leukin-1 (IL-1).

The immunoprecipitated [35S]-labeled COX, as judged from SDS-PAGE electrophoresis, has a molecular size of approximately 73,000 daltons, simi-lar to that of native sheep COX and [3H]-acetyl COX. IL-1 stimulation of enhanced COX synthesis was time and dose dependent; as little as 0.03 units/ml of IL-1 produced significant stimulation of [35S]-labeled COX synthesis. Maximum stimulation was 3–10-fold after preincubation of the cells with IL-1 for 12–16 hours.

IL-1 treatment of cells in serum-free media yielded parallel dose response curves for stimulation of PGE2 formation, cellular solubilzed COX activity and synthesis of newly formed COX, suggesting that this IL-1 effect is mediated solely via induction of new COX protein synthesis. In contrast, IL-1 effect on cells incubated in the presence of fetal calf serum is more complex. Serum synergistically augments the IL-1 effect on PGE2 synthesis in intact cells but concurrently blunts IL-1 induction of COX synthesis, thus suggesting that a factor (or factors) in serum may stimulate PGE2 production by activating cellular phospholipase(s).


Dermal Fibroblast Ureteral Obstruction Rabbit Kidney Phate Buffer Saline Eicosanoid Production 
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  1. 1.
    K. Nishikawa, A.R. Morrison, P Needleman, Exaggerated prostaglandin biosynthesis and its influence on renal resistance in the isolated hydronephrotic rabbit kidney, J. Clin. Invest. 59:1143–1150 (1977).PubMedCrossRefGoogle Scholar
  2. 2.
    A.R. Morrison, K. Nishikawa, P. Needleman, Unmasking of thromboxane A2 synthesis by ureteral obstruction in the rabbit kidney, Nature 267:259–260 (1977).PubMedCrossRefGoogle Scholar
  3. 3.
    R. Zipser, S. Meyers, P. Needleman, Exaggerated prostaglandin and thromboxane synthesis in the rabbit with renal vein constriction, Circ. Res. 47:231–237 (1980).PubMedCrossRefGoogle Scholar
  4. 4.
    E.A. Lianos, G.A. Giuseppe, M.J. Dunn, Glomerular prostaglandin and thromboxane synthesis in rat nephrotoxic serum nephritis, J. Clin. Invest. 72:1439–1448 (1983).PubMedCrossRefGoogle Scholar
  5. 5.
    R.D. Zipser, J.B. Patterson, H.W. Kao, C.J. Hauser, R. Locke, Hypersensitive prostaglandin and thromboxane response to hormones in rabbit colitis, Am. J. Phvsiol. 249:G457-G463 (1985).Google Scholar
  6. 6.
    M.K. McGuire, J.E. Meats, N.M. Ebsworth, L. Harvey, G. Murphy, G.G. Russell, J.J. Reynolds, Properties of rheumatoid and normal synovial tissue in vitro and cells derived from them. Production of prostaglandins and collagenase in response to factors derived from cultured blood mononuclear cells and from synovium, Rheumatol. Int. 2:113–120 (1982).PubMedCrossRefGoogle Scholar
  7. 7.
    A.S. Evers, S. Murphree, J.E. Saffitz, B.A. Jakschik, P. Needleman, Effects of endogenously produced leukotrienes, thromboxane, and prostaglandins on coronary vascular resistance in rabbit myocardial infarction, J. Clin. Invest. 75:992–999 (1985).PubMedCrossRefGoogle Scholar
  8. 8.
    J.G. Clark, K.M. Kostal, B.A. Marino, Bleomycin-induced pulmonary fibrosis in hamsters, J. Clin. Invest. 72:2082–2091 (1983).PubMedCrossRefGoogle Scholar
  9. 9.
    A.R. Morrison, K. Nishikawa, P. Needleman, Thromboxane A2 synthesis by ureteral obstruction in the rabbit kidney, Nature 267:259–260 (1978).CrossRefGoogle Scholar
  10. 10.
    R.B. Nagle, R.E. Bulger, R.E. Cutler, H.R. Jervis, E.P. Benditt, Unilateral obstructive nephropathy in the rabbit: I. Early morphologic, physiologic, and histochemical changes, Lab. Invest. 28:456–467 (1973).PubMedGoogle Scholar
  11. 11.
    T. Okegawa, P.E. Jonas, K. DeSchryver, A. Kawasaki, P. Needleman, Metabolic and cellular alterations underlying the exaggerated renal prostaglandin and thromboxane synthesis in ureter obstruction in rabbits. Inflammatory response involving fibroblasts and mononuclear cells, J. Clin. Invest. 71:81–90 (1983).PubMedCrossRefGoogle Scholar
  12. 12.
    C.J. Mathia, M.J. Welch, D. Schwartz, S.M. Spaethe, P. Needleman. In-111 labeled cells to differentiate the sequential blood cell invasion of the injured rabbit kidney in vivo, submitted (1987).Google Scholar
  13. 13.
    D.F. Reingold, S. Waters, S. Holmberg, P. Needleman, Differential biosynthesis of prostaglandins by hydronephrotic rabbit and cat kidneys, J. Pharmacol. Exp. Ther. 216:510–515 (1981).PubMedGoogle Scholar
  14. 14.
    J.B. Lefkowith, T. Okegawa, K. DeSchryver-Kecksemeti, P. Needleman, Macrophage — dependent arachidonate metabolite in hydronephrosis, Kidney Int. 26:10–17 (1984).PubMedCrossRefGoogle Scholar
  15. 15.
    N. Feuerstein, M. Foegh, P.W. Ramwell, Recently reported stimulation of TxB2 and 6-keto PGF synthesis by rat peritoneal macrophages incubated with E. coli 055:BS lipopolysaccharide, Br. J. Pharmacol. 72: 389–391 (1981).PubMedCrossRefGoogle Scholar
  16. 16.
    P.V. Halushka, J.A. Cook, W.C. Wise, Thromboxane Ao synthesis and prostacyclin production by lipopolysaccharide-stimulated peritoneal macrophages, J. Reticuloendothe. Soc. 30:445–450 (1981).Google Scholar
  17. 17.
    S.L. Humes, R.J. Bonney, L. Peius, M.E. Dahlgren, S.J. Sadowski, F.A. Kuehl, P. Davis, Macrophage synthesize and release prostaglandins in response to inflammatory stimuli, Nature (Lond.) 269:149–151.Google Scholar
  18. 18.
    I. Gery and B.H. Waksman, Potentiation of the T-lymphocyte response to mitogens. II. The cellular source of potentiating mediator(s), J. Exp. Med. 136:143–155 (1971).CrossRefGoogle Scholar
  19. 19.
    J.H. Korn, P.V. Halushka, E.C. LeRoy, Mononuclear cell modulation of connective tissue function: Suppression of fibroblast growth by stimulation of endogenous prostaglandin production, J. Clin. Invest. 65:543–554 (1980).PubMedCrossRefGoogle Scholar
  20. 20.
    S.M. D’Souza, D.J. Englis, A. Clark, R.G. Russell, Stimulation of production of prostaglandin E in gingival cells exposed to products of human blood mononuclear cells, Biochem. J. 198:391–396 (1981).PubMedGoogle Scholar
  21. 21.
    P.E. Jonas, K.M. Leahy, K. DeSchryver-Kecksemeti, P. Needleman, Cellular interactions and exaggerated arachidonic acid metabolism in rabbit renal injury, J. Leukocyte Biol. 35:55–64 (1984).PubMedGoogle Scholar
  22. 22.
    P.E. Jonas and P. Needleman, Mechanism of enhanced fibroblast arachidonic acid metabolism by mononuclear cell factor, J. Clin. Invest. 74: 2249–2253 (1984).CrossRefGoogle Scholar
  23. 23.
    P. Hawley-Nelson, J.E. Sullivan, M. Kung, H. Hennings, S.H. Yuspa, Optimized conditions for the growth of human epidermal cells in culture, J. Invest. Dermatol. 75:176–179 (1980).PubMedCrossRefGoogle Scholar
  24. 24.
    C.R. Albrightson, N.L. Baenziger, P. Needleman, Exaggerated human vascular cell prostaglandin biosynthesis mediated by monocytes: role of monokines and interleukin 1, J. Immunol. 135:1872–1877.Google Scholar
  25. 25.
    F.J. Van Der Ondera, M. Buytonhek, D.K. Nugteren, D.A. Van Dorp. Purification and characterization of prostaglandin endoperoxide synthetase from sheep vesicular glands, Biochem. Biophys. Acta 487:315–331 (1977).CrossRefGoogle Scholar
  26. 26.
    A.T. Meukh, G.F. Sudina, N.B. Golub, S.D. Vanfolomeer, Purification of prostaglandin H synthase and a fluorometric assay for its activity, Analytical Biochem. 150:91–96 (1985).CrossRefGoogle Scholar
  27. 27.
    C.L. Tai and H.H. Tai, A radioimmunoassay for prostaglandin endoperoxide synthetase, Prostagl. Leuket. Medicine 14:243–254 (1984).Google Scholar
  28. 28.
    G.J. Roth, Preparation of [acetyl- H] aspirin and use in quantitating PGH synthase, Methods in Enzvmology 86:392–400 (1984).CrossRefGoogle Scholar
  29. 29.
    M.S. McHardy, S. Schlesinger, J. Lindstrom, J.P. Merlie, The effects of inhibiting oligosaccharide trimming by 1-dexoxynojirimycin on the nicotinic acetylcholine receptor, J. Biol. Chem. 261:14825–14832 (1986).Google Scholar
  30. 30.
    U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227:680–685 (1970).PubMedCrossRefGoogle Scholar
  31. 31.
    R.J. Zucali, C.A. Dinarello, D.J. Obion, M.A. Gross, L. Anderson, R.S. Weiner, Interleukin-1 stimulates fibroblasts to produce granulocytes-macrophage colony stimulating activity and prostaglandin E2, J. Clin. Invest. 77:1857–1863 (1986).PubMedCrossRefGoogle Scholar
  32. 32.
    J.F. Balaudine, B. Rochemontiex, K. Williamson, P. Seckinger, A. Cruchand, J.M. Dayer, Prostaglandin E2 and collagenase production by fibroblasts and synovial cells is regulated by urine derived human interleukin-1 and inhibitor(s), J. Clin. Invest. 78:1120–1124 (1986).CrossRefGoogle Scholar
  33. 33.
    J. Chang, S.C. Gilman, A.-J. Lewis, Interleukin 1 activates phospholipase A2 in rabbit chondrocytes: A possible signal for IL 1 action, J. Immunol. 136:1283–1287 (1986).PubMedGoogle Scholar
  34. 34.
    R.W. Godrey, W.T. Johnson, S.T. Hoffstein, Biochem. Biophys. Res. Commun. 142:235–241 (1987).CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1989

Authors and Affiliations

  • Amiram Raz
    • 1
  • Angela Wyche
    • 1
  • Diana Fagan
    • 1
  • Philip Needleman
    • 1
  1. 1.Department of PharmacologyWashington University School of MedicineSt. LouisUSA

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