Homocysteine activates NADH/NADPH oxidase through ceramide-stimulated Rac GTPase activity in rat mesangial cells.
BACKGROUND
We recently demonstrated that homocysteine (Hcys) increases superoxide (O2-) production via NADH/NADPH oxidase in renal mesangial cells. This O2- production contributes to increased expression of tissue inhibitor of metalloproteinase (TIMP-1) and consequent deposition of collagen in response to Hcys. However, the mechanism by which Hcys activates NADH/NADPH oxidase remains unknown. Given that ceramide is an intracellular activator of this oxidase in several cell types, the present study tests the hypothesis that Hcys activates NADH/NADPH oxidase through a ceramide-mediated signaling pathway in rat mesangial (MG) cells, resulting in O2- production.


METHODS
Rat MG cells were incubated with L-homocysteine (L-Hcys) to determine the mechanism by which Hcys activates NADH/NADPH oxidase. Thin layer chromatography (TLC), Western blot analysis, Rac GTPase activity pull down assay, and NADH/NADPH oxidase activity measurements were performed.


RESULTS
TLC analysis demonstrated that L-Hcys increased de novo production of ceramide in MG cells. L-Hcys and increased ceramide did not alter the amount of NADH/NADPH oxidase subunit p47phox and p67phox in both membrane and cytosolic fractions from MG cells. However, L-Hcys or ceramide markedly increased the level of GTP-bound Rac, which was accompanied by enhanced activity of NADH/NADPH oxidase. These Hcys or ceramide-induced actions were substantially blocked by a Rac GTPase inhibitor, GDPbetaS, and a de novo ceramide synthesis inhibitor, fumonisin B1 (FB1).


CONCLUSIONS
These results indicate that Hcys activates NADH/NADPH oxidase by stimulating de novo ceramide synthesis, and subsequently enhancing Rac GTPase activity in rat MG cells. This ceramide-Rac GTPase signaling pathway may mediate Hcys-induced oxidative stress in these glomerular cells.
Yi
Fan
F
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Zhang
Andrew Y
AY
Janscha
Jennifer L
JL
Li
Pin-Lan
PL
Zou
Ai-Ping
AP
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D002518:Ceramides / Q000096:biosynthesis / Q000502:physiology*; D002855:Chromatography, Thin Layer; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D037341:Fumonisins / Q000494:pharmacology; D005920:Glomerular Mesangium / Q000166:cytology / Q000201:enzymology*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D006710:Homocysteine / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D051381:Rats; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D002518:Ceramides; D004791:Enzyme Inhibitors; D037341:Fumonisins; D007527:Isoenzymes; D009097:Multienzyme Complexes; D013873:Thionucleotides; D006710:Homocysteine; D006153:Guanosine Diphosphate; C056933:fumonisin B1; C023427:guanosine 5'-O-(2-thiodiphosphate); C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D013108:Sphingomyelin Phosphodiesterase; D020744:rac GTP-Binding Proteins