Mycophenolic acid inhibits the autocrine PDGF-B synthesis and PDGF-BB-induced mRNA expression of Egr-1 in rat mesangial cells.
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 beta (PDGFR-beta), 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 microM) with or without guanosine (100 microM). 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-beta 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 microM and DNA synthesis with an IC50 of 0.52 +/- 0.02 microM 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-beta. MPA strongly inhibited the PDGF-BB-induced mRNA expression of Egr-1 decreasing to 7.6 +/- 2.5% after 30 min (P <or= 0.001) and to 4.7 +/- 3.1% after 1 h (P <or= 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.
Sabuda-Widemann
Danuta
D
Department for Nephrology, Heinrich-Heine Universität, Düsseldorf, Germany.
Grabensee
Bernd
B
Schwandt
Christina
C
Blume
Cornelia
C
D000818:Animals; D001483:Base Sequence; D000077214:Becaplermin; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D051766:Early Growth Response Protein 1 / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D017781:Genes, Immediate-Early / Q000235:genetics; D006151:Guanosine / Q000494:pharmacology; D007166:Immunosuppressive Agents / Q000494:pharmacology; D050527:Mesangial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D009173:Mycophenolic Acid / Q000494:pharmacology*; D010982:Platelet-Derived Growth Factor / Q000096:biosynthesis* / Q000494:pharmacology; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism
D017931:DNA Primers; D003848:Deoxyguanine Nucleotides; D051766:Early Growth Response Protein 1; C486110:Egr1 protein, rat; D007166:Immunosuppressive Agents; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D006151:Guanosine; D000077214:Becaplermin; C029603:deoxyguanosine triphosphate; D020797:Receptor, Platelet-Derived Growth Factor beta; D009173:Mycophenolic Acid