Involvement of erythropoietin-induced cytosolic free calcium mobilization in activation of mitogen-activated protein kinase and DNA synthesis in vascular smooth muscle cells.
OBJECTIVE
Human recombinant erythropoietin (rHuEPO) induces cytosolic free calcium ([Ca2+]i) mobilization, an activation of mitogen-activated protein (MAP) kinase and DNA synthesis in several tissues. We explored the mechanism of rHuEPO-induced [Ca2+]i mobilization and its role in the activation of MAP kinase and DNA synthesis in vascular smooth muscle cells (VSMC).


METHODS
[Ca2+]i concentrations were measured by fura-2. MAP kinase activation was analyzed using an immunocomplex kinase assay and Western blotting. DNA synthesis was measured as an incorporation of 5-bromo-2'-deoxyuridine.


RESULTS
Although addition of rHuEPO significantly increased [Ca2+]i, either in the presence or absence of extracellular Ca2+, the peak level and sustained elevation of [Ca2+]i were significantly reduced in the absence of extracellular Ca2+. Pretreatment with genistein completely blocked the elevation of [Ca2+]i in both conditions. Calphostin C and staurosporine did not completely block the elevation of [Ca2+]i. Staurosporine reduced its peak level in a dose-dependent manner, whereas calphostin C reduced its peak level at concentrations over 1 nmol/l in the presence of extracellular Ca2+. Similar results to those with staurosporine were observed with nifedipine. In the absence of extracellular Ca2+, their dose-dependent effects disappeared even though rHuEPO increased [Ca2+]i. rHuEPO activated MAP kinase and DNA synthesis, both of which were significantly suppressed by the chelation of intracellular Ca2+.


CONCLUSIONS
These findings suggest that rHuEPO increases [Ca2+]i by both Ca2+ influx and Ca2+ release from intracellular stores. Tyrosine phosphorylation is critical in the regulation of [Ca2+]i, but protein kinase C activation is important only in the regulation of Ca2+ influx. Dihydropyridine-sensitive L-type Ca2+ channels seem to be involved in rHuEPO-induced Ca2+ influx. In addition, increase of [Ca2+]i by rHuEPO stimulates MAP kinase activation and DNA synthesis in VSMC.
Akimoto
T
T
Department of Nephrology, Jichi Medical School, Minamikawachi, Tochigi, Japan.
Kusano
E
E
Ito
C
C
Yanagiba
S
S
Inoue
M
M
Amemiya
M
M
Ando
Y
Y
Asano
Y
Y
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation; D004921:Erythropoietin / Q000494:pharmacology*; D019833:Genistein / Q000494:pharmacology; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017467:Receptors, Erythropoietin / Q000502:physiology; D011994:Recombinant Proteins
D009281:Naphthalenes; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin; D004247:DNA; D019833:Genistein; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; C058819:calphostin C; D009543:Nifedipine; D002118:Calcium