Activation of mesangial cell MAPK in response to homocysteine.
BACKGROUND
Alteration in mesangial cell function is central to the progression of glomerular disease in numerous models of chronic renal failure (CRF). Animal models of chronic glomerular disease are characterized by mesangial cell proliferation and elaboration of extracellular matrix protein (ECM), resulting in glomerulosclerosis. Elevated plasma levels of homocysteine (Hcy) are seen in both animal models and humans with CRF, and have been proposed to contribute to the high prevalence of vascular disease in this group. Some of the pathogenetic effects of Hcy are thought to be mediated via the induction of endoplasmic reticulum stress. Thus, Hcy effects on mesangial cells could contribute to the progression of CRF. Previous work has shown Hcy- mediated induction of Erk mitogen-activated protein kinase (MAPK) in vascular smooth muscle cells (VSMCs). Erk induces increases in activator protein-1 (AP-1) transcription factor activity which may augment mesangial cell proliferation and ECM protein production. Consequently, we studied the effect of Hcy on mesangial cell Erk signaling.


METHODS
Mesangial cells were exposed to Hcy after 24 hours of serum starvation and Erk activity assessed. Nuclear translocation of phospho-Erk was visualized by confocal microscopy. AP-1 nuclear protein binding was measured in response to Hcy by mobility shift assay. Hcy-induced mesangial cell calcium flux was measured in Fura-2 loaded cells. Mesangial cell DNA synthesis in response to Hcy was assessed by [3H]-thymidine incorporation and proliferation by Western blotting for proliferating cell nuclear antigen (PCNA). Expression of endoplasmic reticulum stress response genes were determined by Northern and Western analysis.


RESULTS
Hcy led to an increase in Erk activity that was maximal at 50 micromol/L and 20 minutes of treatment. Subsequent experiments used this concentration and time point. Erk activity in response to Hcy was insensitive to n-acetylcysteine and catalase, indicating oxidative stress did not play a role. However, Hcy50 micromol/L induced a brief increase in intracellular mesangial cell calcium within 5 minutes, and the calcium ionophores A23187 and ionomycin increased Erk activity while chelation of intracellular calcium with BAPTA-AM abrogated the Erk response to Hcy. Confocal microscopy of activated Erk nuclear translocation mirrored these results as did mesangial cell nuclear protein binding to AP-1 consensus sequences. Hcy- induced increases in thymidine incorporation and PCNA expression at 24 hours were Erk dependent. The expression of endoplasmic reticulum stress response genes was significantly elevated by Hcy in an Erk-dependent manner.


CONCLUSIONS
Hcy increases Erk activity in mesangial cells via a calcium-dependent mechanism, resulting in increased AP-1 nuclear protein binding, cell DNA synthesis and proliferation and induction of endoplasmic reticulum stress. These observations suggest potential mechanisms by which Hcy may contribute to progressive glomerular injury.
Ingram
Alistair J
AJ
Department of Medicine, McMaster University, Hamilton, Ontario. ingrama@mcmaster.ca
Krepinsky
Joan C
JC
James
Leighton
L
Austin
Richard C
RC
Tang
Damu
D
Salapatek
Anne Marie
AM
Thai
Kerri
K
Scholey
James W
JW
D000818:Animals; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005920:Glomerular Mesangium / Q000166:cytology / Q000201:enzymology*; D006710:Homocysteine / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D018808:Transcription Factor AP-1 / Q000378:metabolism
D018808:Transcription Factor AP-1; D006710:Homocysteine; D004247:DNA; D020928:Mitogen-Activated Protein Kinases