TY - JOUR

T1 - Mycophenolic acid inhibits the autocrine PDGF-B synthesis and PDGF-BB-induced mRNA expression of Egr-1 in rat mesangial cells

AU - Sabuda-Widemann, Danuta

AU - Grabensee, Bernd

AU - Schwandt, Christina

AU - Blume, Cornelia

PY - 2009/1

Y1 - 2009/1

N2 - Background. Uncontrolled mesangial cell (MC) proliferation within the context of glomerular disease contributes to the development of glomerulosclerosis. Mesangial autocrine growth factor stimulation has been described as a pathogenic factor. We investigated the effects of mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil (MMF), on proliferation factors of cultured rat MCs. MPA was tested on the expression of platelet-derived growth factor-B (PDGF-B) and its receptor β (PDGFR-β), the immediate early gene (IEG) c-fos and the early growth response gene-1 (Egr-1), and AP-1 activation. Methods. Growth-arrested rat MCs were stimulated with 10% fetal calf serum (FCS) or 10-25 ng/ml platelet-derived growth factor-BB (PDGF-BB) in the presence or absence of MPA (0.019-10 μM) with or without guanosine (100 μM). MC proliferation was quantified by 5-bromo-2′-deoxyuridine (BrdU) incorporation and direct cell counting. Cytotoxicity of MPA was evaluated using the MTT and LDH tests. Protein expression of PDGF-B and its receptor PDGFR-β was quantified by western blot analysis. The effect of MPA on gene expression of PDGF-B, Egr-1 and c-fos was determined by the reverse transcriptase-polymerase chain reaction (RT-PCR). AP-1 activation was analysed by an electrophoretic mobility shift assay (EMSA). Results. Exposure of MCs to MPA caused a concentration-dependent inhibition of FCS-induced cell proliferation (cell number increase) with an IC50 of 0.44 ± 0.03 μM and DNA synthesis with an IC50 of 0.52 ± 0.02 μM without cell cytotoxicity in the therapeutic range. MPA decreased the PDGF-B protein expression and mRNA self-induction of PDGF-B but did not alter the protein expression of PDGFR-β. MPA strongly inhibited the PDGF-BB-induced mRNA expression of Egr-1 decreasing to 7.6 ± 2.5% after 30 min (P ≤ 0.001) and to 4.7 ± 3.1% after 1 h (P ≤ 0.05), both being compared to the maximal expression induced by PDGF-BB. PDGF-BB-induced c-fos expression under MPA was unchanged after 30 min and decreased to 57 ± 26% after 1 h (n.s.). MPA treatment did not affect PDGF-BB-induced AP-1 activity determined after 1 h and 2 h. The inhibitory MPA effect on PDGF-BB-induced PDGF-B expression was not significantly restored by guanosine (56 ± 18% versus 32 ± 17% after 2 h, n.s.), and MPA inhibition of PDGF-BB-induced Egr-1 expression was not reversed by exogenous guanosine. Conclusions. Treatment of cultured MCs with MPA inhibits MC proliferation correlating with a downregulation of the PDGF-B gene and protein expression and a suppression of Egr-1 mRNA expression. Since exogenous guanosine was not able to reverse the inhibitory MPA effect on PDGF-B and Egr-1 expression, we conclude that the antiproliferative effect of MPA on MCs may not solely depend on dGTP depletion but on a specific interference with the autocrine PDGF-B synthesis and Egr-1 expression of MCs.

AB - Background. Uncontrolled mesangial cell (MC) proliferation within the context of glomerular disease contributes to the development of glomerulosclerosis. Mesangial autocrine growth factor stimulation has been described as a pathogenic factor. We investigated the effects of mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil (MMF), on proliferation factors of cultured rat MCs. MPA was tested on the expression of platelet-derived growth factor-B (PDGF-B) and its receptor β (PDGFR-β), the immediate early gene (IEG) c-fos and the early growth response gene-1 (Egr-1), and AP-1 activation. Methods. Growth-arrested rat MCs were stimulated with 10% fetal calf serum (FCS) or 10-25 ng/ml platelet-derived growth factor-BB (PDGF-BB) in the presence or absence of MPA (0.019-10 μM) with or without guanosine (100 μM). MC proliferation was quantified by 5-bromo-2′-deoxyuridine (BrdU) incorporation and direct cell counting. Cytotoxicity of MPA was evaluated using the MTT and LDH tests. Protein expression of PDGF-B and its receptor PDGFR-β was quantified by western blot analysis. The effect of MPA on gene expression of PDGF-B, Egr-1 and c-fos was determined by the reverse transcriptase-polymerase chain reaction (RT-PCR). AP-1 activation was analysed by an electrophoretic mobility shift assay (EMSA). Results. Exposure of MCs to MPA caused a concentration-dependent inhibition of FCS-induced cell proliferation (cell number increase) with an IC50 of 0.44 ± 0.03 μM and DNA synthesis with an IC50 of 0.52 ± 0.02 μM without cell cytotoxicity in the therapeutic range. MPA decreased the PDGF-B protein expression and mRNA self-induction of PDGF-B but did not alter the protein expression of PDGFR-β. MPA strongly inhibited the PDGF-BB-induced mRNA expression of Egr-1 decreasing to 7.6 ± 2.5% after 30 min (P ≤ 0.001) and to 4.7 ± 3.1% after 1 h (P ≤ 0.05), both being compared to the maximal expression induced by PDGF-BB. PDGF-BB-induced c-fos expression under MPA was unchanged after 30 min and decreased to 57 ± 26% after 1 h (n.s.). MPA treatment did not affect PDGF-BB-induced AP-1 activity determined after 1 h and 2 h. The inhibitory MPA effect on PDGF-BB-induced PDGF-B expression was not significantly restored by guanosine (56 ± 18% versus 32 ± 17% after 2 h, n.s.), and MPA inhibition of PDGF-BB-induced Egr-1 expression was not reversed by exogenous guanosine. Conclusions. Treatment of cultured MCs with MPA inhibits MC proliferation correlating with a downregulation of the PDGF-B gene and protein expression and a suppression of Egr-1 mRNA expression. Since exogenous guanosine was not able to reverse the inhibitory MPA effect on PDGF-B and Egr-1 expression, we conclude that the antiproliferative effect of MPA on MCs may not solely depend on dGTP depletion but on a specific interference with the autocrine PDGF-B synthesis and Egr-1 expression of MCs.

KW - AP-1

KW - Egr-1

KW - Mesangial cell proliferation

KW - MPA

KW - PDGF

UR - http://www.scopus.com/inward/record.url?scp=58049215479&partnerID=8YFLogxK

U2 - 10.1093/ndt/gfn462

DO - 10.1093/ndt/gfn462

M3 - Article

C2 - 18723570

AN - SCOPUS:58049215479

VL - 24

SP - 52

EP - 61

JO - Nephrology Dialysis Transplantation

JF - Nephrology Dialysis Transplantation

SN - 0931-0509

IS - 1

ER -