Study on the signalling pathway of inhibitory effect of adreno-medullin on the growth of cultured glomerular mesangial cells.
BACKGROUND
Adrenomedullin (ADM), a potent hypotensive small peptide, was recently found to inhibit the proliferation of glomerular mesangial cells (MsC) in vitro and to attenuate glomerular lesions in vivo, however the mechanisms remain poorly understood. In this study, we attempted to elucidate them using molecular signal transduction.


METHODS
Cultured rat MsC were treated with ADM and several inhibitors of signalling molecules. Methyl thiazoleterazolium (MTT) assay and BrdU incorporation method were employed for examining MsC proliferation. Western blot analysis was used for detecting total mitogen activated protein kinases (t-MAPKs) and phosphorylated MAPKs (p-MAPKs) proteins.


RESULTS
ADM suppressed MsC proliferation in a concentration- and time-dependent fashion. This response was inhibited by ADM receptor antagonist CGRP8-37 and a potent protein kinase-A (PKA) inhibitor, H89. Forskolin, a direct adenylate cyclase activator, also significantly inhibited MsC proliferation. SB203580, a P38MAPK inhibitor, and U0126, a MEK inhibitor, both completely blocked ADM mediated responses in MsC. However, curcumin, a SAPK/JNK inhibitor, and GF109203X, a potent protein kinase-C (PKC) inhibitor, had no effect on MsC growth. Western blot analysis showed that ADM did not change the expression of t-MAPKs but increased p-SAPK/JNK and p-P38MAPK levels and decreased p-ERK level. These responses were inhibited by CGRP8-37. All these kinase phosphorylations, except for the increase in p-SAPK/JNK, could be stimulated using forskolin. In addition, only ADM mediated changes in ERK and P38MAPK phosphorylations were inhibited by H89. GF109203X did not affect ADM induced changes in three p-MAPKs expressions.


CONCLUSIONS
ADM inhibits MsC proliferation possibly through cAMP-PKA pathway. Both phosphorylations of ERK and P38MAPK pathways were necessary in mediating the antiproliferative response of ADM. It does not preclude the involvement of cAMP independent pathways in the ADM mediated responses.
Liu
Xue-guang
XG
Department of Pathology, Shanghai Medical College, Fudan University, Shanghai 200032, China.
Dai
Lu
L
Yang
Chen
C
Zhao
Zhong-hua
ZH
Zhang
Xiu-rong
XR
Zhang
Zhi-gang
ZG
Guo
Mu-yi
MY
D053607:Adrenomedullin; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects*; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology; D010455:Peptides / Q000494:pharmacology*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology
D010455:Peptides; D053607:Adrenomedullin; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases