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Synthesis of sulphated proteoglycans by primary cultures of rat hepatocytes-modulation by matrix substratum

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Abstract

Primary cultures of rat hepatocytes maintained on different matrix proteins such as collagen (Co IV) fibronectin (Fn), Laminin (Ln) or different tissue biomatrices were metabolically labelled with 35[S]-SO4 and the synthesis of sulphated proteoglycans was studied. The incorporation of the label into total glycosaminoglycan (GAG) was significantly higher in cells maintained on Co IV compared to those maintained on Fn or Ln. Similarly the incorporation of label was maximum in those cells maintained on the aortic biomatrix compared to liver or mammary gland biomatrix. About 80–95% of the GAG synthesised and secreted by cells maintained on individual matrix proteins and liver biomatrix was heparan sulphate (HS). But in the case of cells maintained on collagen IV aortic or mammary biomatrix in addition to HS, significant amount of chondroitin sulphate (CS) was also found. Nearly 50% of the total 35[S]-GAG was associated with the cell layer after 24 h in culture in the case of cells maintained on individual matrix protein while those maintained on tissue biomatrix, retained about 70% of the 35[S]-labelled proteoglycans (PG) with the cell layer. Analysis of the cell surface 35[S]-labelled proteoglycans isolated from cells maintained on different biomatrix showed that it is a hybrid proteoglycan consisting of CS and HS. While the PG isolated from cells maintained on liver biomatrix consists of HS and CS in the ratio of 3:2 that from cells maintained on aorta or mammary gland matrix was about 2:3 indicating an alteration in the nature of the cell surface PGs produced by cells maintained on different tissue biomatrix. These results indicate that depending on the nature of the matrix substratum with which the cells are in contact, the nature and quantity of sulphated proteoglycans produced by hepatocytes vary.

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References

  1. Yamada KM: Cell surface interactions with extracellular materials. Ann Rev Biochem 52: 761–799, 1983

    Google Scholar 

  2. Kleinman HK, Klebe RJ, Martin GR: Role of collagenous matrices in the adhesion and growth of cells. J Cell Biol 88: 473–485, 1981

    Google Scholar 

  3. Sudhakaran PR, Stamatoglou SC, Hughes RC: Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes. Exp Cell Res 167: 505–516, 1986

    Google Scholar 

  4. Bissell DM, Arenson DM, Maher JJ, Roll FJ: Support of cultured hepatocytes on a laminin rich gel. J Clin Invest 79: 801–811, 1987

    Google Scholar 

  5. Li, NL, Aggeler J, Farson DA, Hatier L, Hassell J and Bissell MJ: Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells. Proc Natl Acad Sci USA 84: 136–140, 1987

    Google Scholar 

  6. Schmidhauser C, Bissell MJ, Myers CA and Casperson GF: Extracellular Matrix and hormones transcriptionally regulate bovine β-casein 5′ sequences in stably transfected mouse mammary cells. Proc Natl Acad Sci USA 87: 9118–9122, 1990

    Google Scholar 

  7. Hughes RC and Stamatoglou SC: Adhesive interactions and the metabolic activity of hepatocytes. J Cell Sci 81: 273–291, 1987

    Google Scholar 

  8. Hedin U, Bottger BA, Forsberg E, Johansson S and Thyberg J: Diverse effects of fibronectin and laminin in phenotypic properties of cultured arterial smooth muscle cells. J Cell Biol 107: 307–319, 1988

    Google Scholar 

  9. Streuli CH and Bissell MJ: Expression of extracellular matrix components is regulated by substratum. J Cell Biol 110: 1405–1415, 1990

    Google Scholar 

  10. Laurie GW, Leblond CP, Martin GR: Light microscopic immunelocalization of type IV collagen, laminin, heparan sulphate proteoglycan, and fibronectin in the basement membranes of a variety of rat organs. Am J Anat 167: 71–82, 1983

    Google Scholar 

  11. Hook M, Kjellen L, Johansson S, Robinson J: Cell surface glycosaminoglycans. Ann Rev Biochem 53: 847–869, 1984

    Google Scholar 

  12. Pratt RM, Larsen MA, Johnston MC: Migration of cranial neural crest in a cell-free hyluronate-rich matrix. Dev Biol 44: 298–305, 1975

    Google Scholar 

  13. Turley EA, Roth S: Spontaneous glycosylation of glycosaminoglycan substrates by adherent fibroblasts. Cell 17: 109–115, 1979

    Google Scholar 

  14. Toole BP, Biswas C, Gross J: Hyaluronate and invasiveness of the rabbit V2 carcinomas. Proc Natl Acad Sci USA 76: 6299–6303, 1979

    Google Scholar 

  15. Banerjee SD, Cohn RH, Bernfield MR: Basal lamina of embryonic salivary epithelia. Production by epithelium and role in maintaining lobular morphology. J Cell Biol 73: 445–463, 1977

    Google Scholar 

  16. Markwald RR, Fitzharris TP, Smith WNA: Structural analysis of endocardial cytodifferentiation. Dev Biol 42: 160–180, 1975

    Google Scholar 

  17. Morris E, Hopwood JJ, Dorfman A: Biosynthesis of glycosaminoglycans in the developing retina. Dev Biol 58: 313–327, 1977

    Google Scholar 

  18. Kart LD, Mainglia CA, Sartorelli A: Influence of collagen substrata on glycosaminoglycan production by B 16 melanoma cells. Proc Nail Acad Sci USA 80: 3738–3742, 1983

    Google Scholar 

  19. Angellow JC, Hauscka SD: Hyaluronate-cell interaction. Effects of exogenous hyaluronate on muscle fibroblast cell surface composition. Exp Cell Res 125: 389–400, 1980

    Google Scholar 

  20. Prinz R, Klein U, Sudhakaran PR, Sinn W, Ullrich R, von Figura K: Metabolism of sulphated glycosaminoglycans in rat hepatocytes: Synthesis of heparan sulphate and distribution into extracellular and intracellular pools. Biochem Biophys Acta 630: 402–413, 1980

    Google Scholar 

  21. Engvall E, Ruoslahti E: Binding of soluble forms of fibroblast surface protein, fibronectin to collagen. Int J Cancer 20: 1–5, 1977

    Google Scholar 

  22. Seglen OP: Preparation of isolated rat liver cells. Methods Cell Biol 13: 29–83, 1976

    Google Scholar 

  23. Sudhakaran PR, Sinn W, von Figura K: Regulation of heparan sulphate metabolism by adenosine 3′5′-cyclic monophosphate in hepatocytes in culture, Biochem J 192: 395–402, 1980

    Google Scholar 

  24. Rojkind M, Gatmaitan Z, Mackensen S, Giambone M, Ponce P, Reid LM: Connective tissue biomatrix: Its isolation and utilization for long term culture of normal rat hepatocytes. J Cell Biol 87: 255–263, 1980

    Google Scholar 

  25. Sudhakaran PR, Prinz R, Filipovic I, von Figura K, Buddecke E: Homologous low density lipoprotein does not affect proteoglycan metabolism of cultured skin fibroblasts and arterial smooth muscle cells. Hoppe-Seyler's Z Physiol Chem 361: 129–134, 1980

    Google Scholar 

  26. Saito H, Yamagata J, Suzuki S: Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J Biol Chem 243: 1536–1542, 1968

    Google Scholar 

  27. Shiveley JE, Conrad HF: Formation of anhydro sugars in the chemical depolymerisation of heparin. Biochemistry 15: 3932–3942, 1976

    Google Scholar 

  28. Lowry OH, Rosehrough NJ, Farr AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275, 1951

    CAS  PubMed  Google Scholar 

  29. David G, Bernfield MR: Collagen reduces glycosaminoglycan degradation by cultured mammary epithelial cells: Possible mechanism for basal lamina formation. Proc Natl Acad Sci USA 76: 786–790, 1979

    Google Scholar 

  30. Gallagher JT, Gasiunas N, Scher SL: Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels. Biochem J 190: 243–254, 1980

    Google Scholar 

  31. Pratt RM, Yamada KM, Olden K, Ohanian SH, Hascall VC: Tunicamycin-induced alterations in the synthesis of sulfated proteoglycans and cell surface morphology in the chick embryo fibroblast. Exp Cell Res 118: 245–252, 1979

    Google Scholar 

  32. Bissell DM, Stamatoglou SC, Nermut MV, Hughes RC: Interaction of rat hepatocytes with type IV collagen, fibronectin and laminin matrices. Distinct matrix controlled modes of attachment and spreading. Eur J Cell Biol 40: 72–78, 1986

    Google Scholar 

  33. Rojkind M, Valadez G: In D. Evered and J. Whelan (eds). ‘Fibrosis.’ Pitman, London, 1985, p. 208

    Google Scholar 

  34. Ruoslahti E: Structure and Biology of Proteoglycans. Ann Rev Cell Biol 4: 229–255, 1988

    Google Scholar 

  35. Bernfield MK, Kokenyesi M, Kato MT, Hinkes J, Spring RL, Gallo, Iose EJ: Ann Rev Cell Biol 8: 365–393, 1992

    Google Scholar 

  36. Bernfeld M, Sanderson RD: Syndecan, a developmentally regulated cell surface proteoglycan that binds extra cellular matrix and growth factors. Philos-Trans-R-Soc Lond [Biol] 327: 171–186, 1990

    Google Scholar 

  37. Saunders S, Jalkanen PI, O'Farrell S, Bernfield M: Molecular cloning of syndecan, an integral membrane proteoglycan. J Cell Biol 108: 1547–1556, 1989

    Google Scholar 

  38. Liebersbach BF and Sanderson RD: Expression of syndecan-1 inhibits cell invasion into type I Collagen. J Biol Chem 269: 20013–20019, 1994

    Google Scholar 

  39. Woods A and Couchman JR: Syndecan 4 is a selectively enriched and widespread focal adhesion component. Mol Biol Cell 5: 183–192, 1994

    Google Scholar 

  40. Gressner AM, Pazen H, Greiling H: The biosynthesis of glycosaminoglycans in normal rat liver and in response to experimental hepatic injury. Hoppe-Seyler's Z Physiol Chem 358: 825–833, 1977

    Google Scholar 

  41. Unnikrishnan VS, Sudhakaran PR: Metabolism of glycosaminoglycans in experimental liver fibrosis, Indian J Biochem Biophys 22: 304–308, 1985

    Google Scholar 

  42. Stamatoglou SC, Hughes RC: Cell adhesion molecules in liver and pattern formation FASEB J 8: 420–427, 1994

    Google Scholar 

  43. Martinez-Hemandez A, Amenta PS: The extracellular matrix in hepatic regeneration. FASEB J 9: 1401–1410, 1995

    Google Scholar 

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Santhosh, A., Mathew, S. & Sudhakaran, P.R. Synthesis of sulphated proteoglycans by primary cultures of rat hepatocytes-modulation by matrix substratum. Mol Cell Biochem 165, 1–7 (1996). https://doi.org/10.1007/BF00229739

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