Molecular and Cellular Biochemistry

, Volume 361, Issue 1–2, pp 235–241 | Cite as

Thrombin-dependent modulation of β1-integrin-mediated signaling up-regulates prolidase and HIF-1α through p-FAK in colorectal cancer cells

  • Ewa Karna
  • Lukasz Szoka
  • Jerzy Palka


Products of prolidase [E.C.] activity, proline or hydroxyproline, contribute to up-regulation of hypoxia-inducible factor-1α (HIF-1α). Prolidase activity is regulated by β1-integrin signaling. We studied the effects of echistatin (a well-known disintegrin) and thrombin (a serine protease capable of activation of integrin α2β1 receptor) on prolidase activity and expressions of prolidase, α2β1-integrin receptor, focal adhesion kinase (FAK), MAP-kinases (ERK1 and ERK2), and nuclear HIF-1α in human colon adenocarcinoma (DLD-1) cells. It has been found that treatment of the cells with thrombin contributes to decrease in the expression of prolidase and simultaneously increase in its phosphorylation, resulting in maintenance of the enzyme activity. The phenomenon was accompanied by thrombin-dependent recovery of depressed autophosphorylation of FAK (pY397) under the effect of FAK inhibitor (1,2,4,5-benzenetetramine tetrahydrochloride). Although integrin α2β1 receptor expression was not affected by thrombin, the signaling induced by thrombin up-regulated nuclear HIF-1α expression. It was accompanied by increase in the expression of MAP kinases, ERK1 and ERK2. It suggests that integrin-dependent signaling through p-FAK is up-regulated in DLD-1 cells and it may represent potential target for anti-cancer therapy.


FAK HIF-1α Integrins Prolidase Thrombin Colon adenocarcinoma 


  1. 1.
    Hanks SK, Calalb MB, Harper MC, Patel SK (1992) Focal adhesion protein-tyrosine kinase phosphotylated in response to cell attachment to fibronectin. Proc Natl Acad Sci USA 89:8487–8491PubMedCrossRefGoogle Scholar
  2. 2.
    Juliano RL, Varner JA (1993) Adhesion molecules in cancer, the role of integrins. Curr Opin Cell Biol 5:812–818PubMedCrossRefGoogle Scholar
  3. 3.
    Meredith JE Jr, Winitz S, Artur Lewis J, Hess S, Xiang-Dong R, Renshaw MW, Shwartz MA (1996) The regulation of growth and intracellular signaling by integrins. Endocr Rev 17:207–220PubMedGoogle Scholar
  4. 4.
    Adams JC (2001) Cell-matrix contact structures. Cell Mol Life Sci 58:371–392PubMedCrossRefGoogle Scholar
  5. 5.
    Bosco M, Chan C, Matsuura N, Tokada Y, Zetter BR, Hemler ME (1991) In vitro and in vivo consequences of VLA-2 expression on rhabdomyosarcoma cells. Science 25:1600–1602Google Scholar
  6. 6.
    Schaller MD, Otey CA, Hildebrand JD, Parsons JT (1995) Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains. J Cell Biol 130:1181–1187PubMedCrossRefGoogle Scholar
  7. 7.
    Parsons JT (2003) Focal adhesion kinase: the first ten years. J Cell Sci 116:1409–1416PubMedCrossRefGoogle Scholar
  8. 8.
    Palka J, Phang J (1997) Prolidase activity in fibroblasts is regulated by interaction of extracellular matrix with cell surface integrin receptor. J Cell Biochem 67:166–175PubMedCrossRefGoogle Scholar
  9. 9.
    Myara I, Charpentier C, Lemonnier A (1984) Prolidase and prolidase deficiency. Life Sci 34:1985–1998PubMedCrossRefGoogle Scholar
  10. 10.
    Yaron A, Naider F (1993) Proline–dependent structural and biological properties of peptides and proteins. Crit Rev Biochem Mol Biol 28:31–81PubMedCrossRefGoogle Scholar
  11. 11.
    Emmerson KS, Phang JM (1993) Hydrolysis of proline dipeptides completely fulfills the proline requirement in a proline-auxotropic Chinese hamster ovary cell line. J Nutr 123:909–914PubMedGoogle Scholar
  12. 12.
    Jackson SH, Dennis AW, Greenberg M (1975) Iminopeptiduria: a genetic defect in recycling of collagen; a method for determining prolidase in erythrocytes. Can Med Assoc J113:759–763Google Scholar
  13. 13.
    Surazynski A, Donald SP, Cooper SK, Whiteside MA, Salnikow K, Liu Y, Phang JM (2008) Extracellular matrix and HIF-1 signaling: The role of prolidase. Int J Cancer 122:1435–1440PubMedCrossRefGoogle Scholar
  14. 14.
    Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, Kriegsheim Av, Hebestreit HF, Mukherji M, Schofield CJ, Maxwell PH, Pugh CW, Ratcliffe PJ (2001) Targeting of HIF-alpha to the von Hippel Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292:468–472PubMedCrossRefGoogle Scholar
  15. 15.
    Juliano R (1996) Cooperation between soluble factors and integrin-mediated cell anchorage in the control of cell growth and differentiation. Bioessays 18:911–917PubMedCrossRefGoogle Scholar
  16. 16.
    Seger R, Krebs EG (1995) The MAPK signaling cascade. FASEB J 9:726–735PubMedGoogle Scholar
  17. 17.
    Labat-Robert J, Robert L (2000) Interaction between cells and extracellular matrix: signaling by integrins and the elastin-laminin receptor. Prog Mol Subcell Biol 25:57–70PubMedCrossRefGoogle Scholar
  18. 18.
    Calvete JJ, Wang Y, Mann K, Schafer W, Niewiarowski S, Stewart GJ (1992) The disulfide bridge pattern of snake venom disintegrins, flavoridin and echistatin. FEBS Lett 309:316–320PubMedCrossRefGoogle Scholar
  19. 19.
    Jung SM, Moroi M (2000) Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1). J Biol Chem 275:8016–8026PubMedCrossRefGoogle Scholar
  20. 20.
    Myara I, Charpentier C, Lemonnier A (1982) Optimal conditions for prolidase assay by proline colorimetric determination: application to imidodipeptiduria. Clin Chim Acta 125:193–205PubMedCrossRefGoogle Scholar
  21. 21.
    Lowry OH, Rosenbregh NI, Far AL, Randall IR (1951) Protein measurement with the Folin reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  22. 22.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  23. 23.
    Carmichael J, Degraff W, Gazdar A, Minna J, Mitchell J (1987) Evaluation of a tetrazolinum-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 47:936–942PubMedGoogle Scholar
  24. 24.
    Surażyński A, Palka J, Wolczynski S (2001) Phosphorylation of prolidase increases the enzyme activity. Mol Cell Biochem 220:95–101PubMedCrossRefGoogle Scholar
  25. 25.
    Arata J, Tada J, Yamada T, Oono T, Yasutomi H, Oka E (1991) Angiopathic pathogenesis of clinical manifestations in prolidase deficiency. Arch Dermatol 127:124–125PubMedCrossRefGoogle Scholar
  26. 26.
    Slack-Davis JK, Martin KH, Tilghman RW, Iwanicki M, Ung EJ, Autry C, Luzzio MJ, Cooper B, Kath JC, Roberts WG, Parsons JT (2007) Cellular characterization of a novel focal adhesion kinase inhibitor. J Biol Chem 282:14845–14852PubMedCrossRefGoogle Scholar
  27. 27.
    Golubovskaya VM, Nyberg C, Zheng M, Kweh F, Magis A, Ostrov D, Cance WG (2008) A small molecule inhibitor, 1, 2, 4, 5-benzenetetraamine tetrahydrochloride, targeting the Y397 site of focal adhesion kinase decreases tumor growth. J Med Chem 51:7405–7416PubMedCrossRefGoogle Scholar
  28. 28.
    Bornstein P, Sage H (1989) Regulation of collagen gene expression. Prog Nucleic Acid Res Mol Biol 37:67–106PubMedCrossRefGoogle Scholar
  29. 29.
    Carey DJ (1991) Control of growth and differentiation of vascular cells by extracellular matrix. Ann Rev Physiol; 53:161–177CrossRefGoogle Scholar
  30. 30.
    Ruoslahti E (1992) Control of cell motility and tumor invasion by extracellular matrix interaction. Br J Cancer 66:239–242PubMedCrossRefGoogle Scholar
  31. 31.
    Mizejewski GJ (1999) Role of integrins in cancer: survey of expression patterns. Proc Soc Exp Biol Med 222:124–138PubMedCrossRefGoogle Scholar
  32. 32.
    Andrews RK, Berndt MC (2000) Snake venom modulators of platelet adhesion receptors and their ligands. Toxicon 38:775–791PubMedCrossRefGoogle Scholar
  33. 33.
    Mercer B, Markland F, Minkin C (1998) Contortrostatin, a homodimeric snake venom disintegrin, is a potent inhibitor of osteoclast attachment. J Bone Miner Res 13:409–414PubMedCrossRefGoogle Scholar
  34. 34.
    Coelho AL, De Freitas MS, Oliveira-Carvalho AL, Moura-Neto V, Zingali RB, Barja-Fidalgo C (1999) Effects of jarastatin, a novel snake venom disintegrin, on neutrophil migration and actin cytoskeleton dynamics. Exp Cell Res 251:379–387PubMedCrossRefGoogle Scholar
  35. 35.
    Tsai TJ, Sheu JR, Chen YM, Yen CJ, Chen CF, Huang TF (1995) Disintegrin modulates rat glomerular mesangial cell behavior. Nephron 70:83–90PubMedCrossRefGoogle Scholar
  36. 36.
    Yagami-Hiromasa T, Sato T, Kurisaki T, Kamijo K, Nabeshima Y, Fujisawa-Sehara A (1995) A metalloprotease- disintegrin participating in myoblast fusion. Nature 377:652–656PubMedCrossRefGoogle Scholar
  37. 37.
    Fenton JW II (1988) Regulation of thrombin generation and functions. Semin Thromb Hemost 14:234–240PubMedCrossRefGoogle Scholar
  38. 38.
    Lim ST, Mikolon D, Stupack DG, Schlaepfer DD (2008) FERM control of FAK function: implications for cancer therapy. Cell Cycle 7:2306–2314PubMedGoogle Scholar
  39. 39.
    Stupack DG, Teitz T, Potter MD, Mikolon D, Houghton PJ, Kidd VJ, Lahti JM, Cheresh DA (2006) Potentiation of neuroblastoma metastasis by loss of caspase-8. Nature 439:95–99PubMedCrossRefGoogle Scholar
  40. 40.
    White DE, Muller WJ (2007) Multifaceted roles of integrins in breast cancer metastasis. J Mammary Gland Biol Neoplasia 12:135–142PubMedCrossRefGoogle Scholar
  41. 41.
    Kuphal S, Bauer R, Bosserhoff AK (2005) Integrin signaling in malignant melanoma. Cancer Metastasis Rev 24:195–222PubMedCrossRefGoogle Scholar
  42. 42.
    Palka JA, Phang JM (1994) Prolidase activity is regulated by cell surface extracellular matrix interaction in normal fibroblast and MCF-7 cells. Proc Am Assoc Cancer Res 35:531Google Scholar
  43. 43.
    Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, Koos RD, Semenza GL (1996) Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 16:4604–4613PubMedGoogle Scholar
  44. 44.
    Jiang BH, Rue E, Wang GL, Roe R, Semenza GL (1996) Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J Biol Chem 271:17771–17778PubMedCrossRefGoogle Scholar
  45. 45.
    Gleadle JM, Ratcliffe PJ (1997) Induction of hypoxia-inducible factor-1, erythropoietin, vascular endothelial growth factor, and glucose transporter-1 by hypoxia: evidence against a regulatory role for Src kinase. Blood 89:503–509PubMedGoogle Scholar
  46. 46.
    Iyer NV, Kotch LE, Agani F, Leung SW, Laughner E, Wenger RH, Gassmann M, Gearhart JD, Lawker AM, Yu AY, Semenza GL (1998) Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev 12:149–162PubMedCrossRefGoogle Scholar
  47. 47.
    Li QQ, Sun YP, Ruan CP, Xu XY, Ge JH, He J, Xu ZD, Wang Q, Gao WC (2011) Cellular prion protein promotes glucose uptake through the Fyn-HIF-2α-Glut1 pathway to support colorectal cancer cell survival. Cancer Sci 102:400–406PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  1. 1.Department of Medicinal ChemistryMedical University in BialystokBialystokPoland

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