Inflammation Research

, Volume 67, Issue 2, pp 191–201 | Cite as

Transforming growth factor-β1 and phosphatases modulate COX-2 protein expression and TAU phosphorylation in cultured immortalized podocytes

  • Maya S. Abdallah
  • Christopher R. J. Kennedy
  • Joseph S. Stephan
  • Pamela Abou Khalil
  • Mohammad Mroueh
  • Assaad A. Eid
  • Wissam H. Faour
Original Research Paper


Objective and design

The aim of this study is to elucidate TGF-β1 signaling pathways involved in COX-2 protein induction and modulation of TAU protein phosphorylation in cultured podocytes.

Materials, treatment and methods

In vitro cultured immortalized podocytes were stimulated with TGF-β1 in presence and absence of pharmacologic inhibitors for various signaling pathways and phosphatases. Then, COX-2 protein expression, as well as P38MAPK, AKT and TAU phosphorylation levels were evaluated by western blot analysis.


TGF-β1 induction of COX-2 protein levels was completely blocked by pharmacologic inhibitors of phosphatases, P38 MAPK, or NF-қB pathways. Time course experiments showed that TGF-β1 activated p38 MAPK after 5 min of stimulation. Interestingly, podocyte co-incubated with TGF-β1, high glucose and/or PGE2 showed strong increase in p38 MAPK and AKT phosphorylation as well as COX- 2 protein expression levels. Levels of phosphorylated AKT were further reduced and levels of phosphorylated p38 were increased when PGE2 was added to the culture media. Interestingly, selective phosphatases inhibitors completely abrogated PGE2-induced P38 MAPK and TAU phosphorylation. Also, inhibition of phosphatases reversed TGF-β1–induced COX-2 protein expression either alone or when incubated with high glucose or PGE2.


These data suggest TGF-β1 mediates its effect in podocyte through novel signaling mechanisms including phosphatases and TAU protein phosphorylation.


Cyclooxygenase Diabetic nephropathy Podocytes TGF-β1 TAU 



Dr Wissam H. Faour is a recipient of a grant from the Lebanese National Council for Scientific Research and an Assistant Professor of Pharmacology at the School of Medicine at the Lebanese American University. This project has been funded with support from the National Council for Scientific Research in Lebanon, Grant number: 01-08-2015.

Compliance with ethical standards

Conflict of interest

No competing financial interests exist.


  1. 1.
    Ritz E, Rychlik I, Locatelli F, Halimi S. End-stage renal failure in type 2 diabetes: A medical catastrophe of worldwide dimensions. Am J Kidney Dis. 1999;34:795–808.CrossRefPubMedGoogle Scholar
  2. 2.
    Schrijvers BF, De Vriese AS, Flyvbjerg A. From hyperglycemia to diabetic kidney disease: the role of metabolic, hemodynamic, intracellular factors and growth factors/cytokines. Endocr Rev. 2004;25:971–1010.CrossRefPubMedGoogle Scholar
  3. 3.
    Wharram BL, Goyal M, Wiggins JE, Sanden SK, Hussain S, Filipiak WE, et al. Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene. J Am Soc Nephrol. 2005;16:2941–52.CrossRefPubMedGoogle Scholar
  4. 4.
    Shankland SJ. The podocyte’s response to injury: role in proteinuria and glomerulosclerosis. Kidney Int. 2006;69:2131–47.CrossRefPubMedGoogle Scholar
  5. 5.
    Ziyadeh FN, Hoffman BB, Han DC, Iglesias-De La Cruz MC, Hong SW, Isono M, et al. Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice. Proc Natl Acad Sci USA. 2000;97:8015–20.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Han DC, Hoffman BB, Hong SW, Guo J, Ziyadeh FN. Therapy with antisense TGF-beta1 oligodeoxynucleotides reduces kidney weight and matrix mRNAs in diabetic mice. Am J Physiol Renal Physiol. 2000;278:F628-34.CrossRefPubMedGoogle Scholar
  7. 7.
    Chen S, Jim B, Ziyadeh FN. Diabetic nephropathy and transforming growth factor-beta: transforming our view of glomerulosclerosis and fibrosis build-up. Semin Nephrol 2003; 23:532–43.CrossRefPubMedGoogle Scholar
  8. 8.
    Ziyadeh FN, Sharma K, Ericksen M, Wolf G. Stimulation of collagen gene expression and protein synthesis in murine mesangial cells by high glucose is mediated by autocrine activation of transforming growth factor-beta. J Clin Invest 1994; 93:536–42.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Mason RM, Wahab NA. Extracellular matrix metabolism in diabetic nephropathy. J Am Soc Nephrol. 2003;14:1358–73.CrossRefPubMedGoogle Scholar
  10. 10.
    Tsilibary EC. Microvascular basement membranes in diabetes mellitus. J Pathol 2003; 200:537–46.CrossRefPubMedGoogle Scholar
  11. 11.
    Dessapt C, Baradez MO, Hayward A, Dei Cas A, Thomas SM, Viberti G, et al. Mechanical forces and TGFbeta1 reduce podocyte adhesion through alpha3beta1 integrin downregulation. Nephrol Dial Transpl. 2009;24:2645–55.CrossRefGoogle Scholar
  12. 12.
    Wu DT, Bitzer M, Ju W, Mundel P, Bottinger EP. TGF-beta concentration specifies differential signaling profiles of growth arrest/differentiation and apoptosis in podocytes. J Am Soc Nephrol. 2005;16:3211–21.CrossRefPubMedGoogle Scholar
  13. 13.
    Schiffer M, Bitzer M, Roberts IS, Kopp JB, ten Dijke P, Mundel P, et al. Apoptosis in podocytes induced by TGF-beta and Smad7. J Clin Invest 2001;108:807–16.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Xavier S, Niranjan T, Krick S, Zhang T, Ju W, Shaw AS, et al. TbetaRI independently activates Smad- and CD2AP-dependent pathways in podocytes. J Am Soc Nephrol. 2009;20:2127–37.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kidger AM, Keyse SM. The regulation of oncogenic Ras/ERK signalling by dual-specificity mitogen activated protein kinase phosphatases (MKPs). Semin Cell Dev Biol 2016; 50:125–32.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Worby CA, Dixon JE. Pten Annu Rev Biochem 2014; 83:641–69.CrossRefPubMedGoogle Scholar
  17. 17.
    Lin J, Shi Y, Peng H, Shen X, Thomas S, Wang Y, et al. Loss of PTEN promotes podocyte cytoskeletal rearrangement, aggravating diabetic nephropathy. J Pathol. 2015;236:30–40.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Santamaria B, Marquez E, Lay A, Carew RM, Gonzalez-Rodriguez A, Welsh GI, et al. IRS2 and PTEN are key molecules in controlling insulin sensitivity in podocytes. Biochim Biophys Acta. 2015;1853:3224–34.CrossRefPubMedGoogle Scholar
  19. 19.
    Sun J, Li ZP, Zhang RQ, Zhang HM. Repression of miR-217 protects against high glucose-induced podocyte injury and insulin resistance by restoring PTEN-mediated autophagy pathway. Biochem Biophys Res Commun. 2017;483:318–24.CrossRefPubMedGoogle Scholar
  20. 20.
    Jung KY, Chen K, Kretzler M, Wu C. TGF-beta1 regulates the PINCH-1-integrin-linked kinase-alpha-parvin complex in glomerular cells. J Am Soc Nephrol. 2007;18:66–73.CrossRefPubMedGoogle Scholar
  21. 21.
    Massague J. TGF-beta signal transduction. Annu Rev Biochem. 1998;67:753–91.CrossRefPubMedGoogle Scholar
  22. 22.
    Massague J, Gomis RR. The logic of TGFbeta signaling. FEBS Lett. 2006;580:2811–20.CrossRefPubMedGoogle Scholar
  23. 23.
    Yu L, Border WA, Huang Y, Noble NA. TGF-beta isoforms in renal fibrogenesis. Kidney Int 2003; 64:844–56.CrossRefPubMedGoogle Scholar
  24. 24.
    Smith WL, DeWitt DL, Garavito RM. Cyclooxygenases: structural, cellular, and molecular biology. Annu Rev Biochem 2000; 69:145–82.CrossRefPubMedGoogle Scholar
  25. 25.
    Faour WH, He Y, He QW, de Ladurantaye M, Quintero M, Mancini A, et al. Prostaglandin E(2) regulates the level and stability of cyclooxygenase-2 mRNA through activation of p38 mitogen-activated protein kinase in interleukin-1 beta-treated human synovial fibroblasts. J Biol Chem. 2001;276:31720–31.CrossRefPubMedGoogle Scholar
  26. 26.
    Tomasoni S, Noris M, Zappella S, Gotti E, Casiraghi F, Bonazzola S, et al. Upregulation of renal and systemic cyclooxygenase-2 in patients with active lupus nephritis. JASN. 1998;9:1202–12.PubMedGoogle Scholar
  27. 27.
    Weichert W, Paliege A, Provoost AP, Bachmann S. Upregulation of juxtaglomerular NOS1 and COX-2 precedes glomerulosclerosis in fawn-hooded hypertensive rats. Am J Physiol Renal Physiol. 2001;280:F706–14.CrossRefPubMedGoogle Scholar
  28. 28.
    Takano T, Cybulsky AV. Complement C5b-9-mediated arachidonic acid metabolism in glomerular epithelial cells : role of cyclooxygenase-1 and -2. Am J Pathol. 2000;156:2091–101.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Wang JL, Cheng HF, Zhang MZ, McKanna JA, Harris RC. Selective increase of cyclooxygenase-2 expression in a model of renal ablation. Am J Physiol. 1998;275:F613–22.PubMedGoogle Scholar
  30. 30.
    Fujihara CK, Antunes GR, Mattar AL, Andreoli N, Malheiros DM, Noronha IL, et al. Cyclooxygenase-2 (COX-2) inhibition limits abnormal COX-2 expression and progressive injury in the remnant kidney. Kidney Int. 2003;64:2172–81.CrossRefPubMedGoogle Scholar
  31. 31.
    Cheng H, Wang S, Jo Y-I, Hao C-M, Zhang M, Fan X, et al. Overexpression of cyclooxygenase-2 predisposes to podocyte injury. J Am Soc Nephrol. 2007;18:551–9.CrossRefPubMedGoogle Scholar
  32. 32.
    Martineau LC, McVeigh LI, Jasmin BJ, Kennedy CR. p38 MAP kinase mediates mechanically induced COX-2 and PG EP4 receptor expression in podocytes: implications for the actin cytoskeleton. Am J Physiol Renal Physiol. 2004;286:F693–701.CrossRefPubMedGoogle Scholar
  33. 33.
    Faour WH, Gomi K, Kennedy CR. PGE(2) induces COX-2 expression in podocytes via the EP(4) receptor through a PKA-independent mechanism. Cell Signal. 2008;20:2156–64.CrossRefPubMedGoogle Scholar
  34. 34.
    Faour WH, Thibodeau JF, Kennedy CR. Mechanical stretch and prostaglandin E2 modulate critical signaling pathways in mouse podocytes. Cell Signal. 2010;22:1222–30.CrossRefPubMedGoogle Scholar
  35. 35.
    Cheng H, Fan X, Moeckel GW, Harris RC. Podocyte COX-2 exacerbates diabetic nephropathy by increasing podocyte (pro)renin receptor expression. JASN. 2011;22:1240–51.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Wang L, Chang J-H, Paik S-Y, Tang Y, Eisner W, Spurney RF. Calcineurin (CN) activation promotes apoptosis of glomerular podocytes both in vitro and in vivo. Mol Endocrinol. 2011;25:1376–86.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG, et al. Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest. 1997;99:342–8.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Ziyadeh FN, Wolf G. Pathogenesis of the podocytopathy and proteinuria in diabetic glomerulopathy. Curr Diabetes Rev. 2008;4:39–45.CrossRefPubMedGoogle Scholar
  39. 39.
    Stitt-Cavanagh EM, Faour WH, Takami K, Carter A, Vanderhyden B, Guan Y, et al. A maladaptive role for EP4 receptors in podocytes. JASN. 2010;21:1678–90.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Lasa M, Mahtani KR, Finch A, Brewer G, Saklatvala J, Clark AR. Regulation of cyclooxygenase 2 mRNA stability by the mitogen-activated protein kinase p38 signaling cascade. Mol Cell Biol. 2000;20:4265–74.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Jovanovic DV, Di Battista JA, Martel-Pelletier J, Jolicoeur FC, He Y, Zhang M, et al. IL-17 stimulates the production and expression of proinflammatory cytokines, IL-ß and TNF-α, by human macrophages. J Immunol. 1998;160:3513–21.PubMedGoogle Scholar
  42. 42.
    Faour WH, Mancini A, He QW, Di Battista JA. T-cell-derived interleukin-17 regulates the level and stability of cyclooxygenase-2 (COX-2) mRNA through restricted activation of the p38 mitogen-activated protein kinase cascade: role of distal sequences in the 3′-untranslated region of COX-2 mRNA. J Biol Chem. 2003;278:26897–907.CrossRefPubMedGoogle Scholar
  43. 43.
    Eid AA, Ford BM, Block K, Kasinath BS, Gorin Y, Ghosh-Choudhury G, et al. AMP-activated protein kinase (AMPK) negatively regulates nox4-dependent activation of p53 and epithelial cell apoptosis in diabetes. J Biol Chem. 2010;285:37503–12.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Institut Européen des MembranesUniversité de MontpellierMontpellierFrance
  2. 2.Division of Nephrology, Department of Medicine, Kidney Research CentreThe Ottawa HospitalOttawaCanada
  3. 3.Gilbert and Rose-Marie Chagoury School of MedicineLebanese American UniversityByblosLebanon
  4. 4.School of PharmacyLebanese American UniversityByblosLebanon
  5. 5.School of MedicineAmerican University of BeirutBeirutLebanon

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