The effect of erythropoietin on interleukin-1beta mediated increase in nitric oxide synthesis in vascular smooth muscle cells.
OBJECTIVE
Recently, we observed that recombinant human erythropoietin (rHuEPO) inhibits the interleukin (IL)-1beta induced nitric oxide (NO) production and inducible NO synthase (iNOS) expression in cultured rat vascular smooth muscle cells (VSMC). The mechanisms of these inhibitory effects of rHuEPO were evaluated.


METHODS
Reverse transcription-polymerase chain reaction (RT-PCR) was performed to identify a specific erythropoietin receptor (EpoR). Tyrosine phosphorylation of phospholipase C (PLC) was analyzed by combination of immunoprecipitation and Western blotting. Protein kinase C (PKC) activities were analyzed by phosphorylation assay of myelin basic protein (MBP4-14). VSMC were incubated with test agents for 24 h and nitrite as a stable NO metabolite was measured. iNOS mRNA and protein expression was analyzed by Northern and Western blotting, respectively.


RESULTS
RT-PCR analysis revealed that EpoR m-RNA was expressed; furthermore, it might be alternatively spliced in VSMC. rHuEPO induced tyrosine phosphorylation of PLC-gamma1 and activation of PKC. rHuEPO inhibited not only IL-1beta induced nitrite production, but also the expression of iNOS mRNA and protein. These inhibitory effects of rHuEPO were reversed in the presence of PKC inhibitors, calphostin C (1 pmol/l) or staurosporine (10 nmol/l). PKC activation by phorbol myristate acetate inhibited nitrite production. The inhibitory effect of rHuEPO on IL-1beta induced nitrite production was also eliminated in PKC depleted cells or in the existence of anti-EpoR antibody.


CONCLUSIONS
rHuEPO inhibits IL-1beta induced NO production by suppressing iNOS mRNA and protein expressions through EpoR, and the PLC-gamma1 and PKC pathway may be involved.
Akimoto
T
T
Department of Nephrology, Jichi Medical School, Tochigi, Japan.
Kusano
E
E
Muto
S
S
Fujita
N
N
Okada
K
K
Saito
T
T
Komatsu
N
N
Ono
S
S
Ebata
S
S
Ando
Y
Y
Homma
S
S
Asano
Y
Y
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D004921:Erythropoietin / Q000494:pharmacology*; D006801:Humans; D007375:Interleukin-1 / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D051966:Phospholipase C gamma; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017467:Receptors, Erythropoietin / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins; D010738:Type C Phospholipases / Q000378:metabolism
D000906:Antibodies; D007375:Interleukin-1; D007527:Isoenzymes; D012333:RNA, Messenger; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; C103998:epoetin beta; D004921:Erythropoietin; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D011493:Protein Kinase C; D010738:Type C Phospholipases; D051966:Phospholipase C gamma