Erythropoietin regulates vascular smooth muscle cell apoptosis by a phosphatidylinositol 3 kinase-dependent pathway.
BACKGROUND
Recent studies have shown that several cytokines could induce apoptosis in vascular smooth muscle cells (VSMCs) via the induction of nitric oxide (NO). In the present study, we explored whether human recombinant erythropoietin (rHuEPO) has a modulatory effect of apoptosis on interleukin-1beta (IL-1beta) or NO donor sodium nitroprusside (SNP)-induced apoptosis in rat cultured VSMCs.


METHODS
The quantitation of apoptosis among VSMCs was assessed by nuclear morphological analysis with fluorescent DNA-binding dye Hoechst 33258. Apoptotic changes were also confirmed by the detection of DNA fragmentation. The expression of EPO receptor (EpoR), cellular protein tyrosine phosphorylation, including EpoR and Janus kinase (JAK) 2, and the association of p85 subunit of phosphatidylinositol 3 kinase (PI3-kinase) to tyrosine-phosphorylated proteins, including EpoR, were explored by using Western blotting analysis combined in part with immunoprecipitation.


RESULTS
rHuEPO inhibited the apoptosis induced by IL-1beta or SNP in a dose- and time-dependent manner. The anti-apoptotic effects of rHuEPO were diminished in the presence of a tyrosine kinase (TK) inhibitor genistein or anti-EpoR antibody. After stimulation with rHuEPO, EpoR and JAK 2 were tyrosine phosphorylated, and p85 subunits were associated with EpoR. Also, rHuEPO induced phosphorylation of Akt through a PI3-kinase-dependent pathway. The phosphorylation of Akt and the anti-apoptotic effects of rHuEPO were diminished in the presence of a PI3-kinase inhibitor, wortmannin.


CONCLUSIONS
Our present study demonstrates that rHuEPO inhibites IL-1beta or SNP-induced VSMC apoptosis. The TK-dependent pathway, particularly the PI3-kinase-dependent pathway, seems to be critical to the countervailing effect of rHuEPO on IL-1beta and SNP-induced VSMC apoptosis.
Akimoto
T
T
Departments of Nephrology and Molecular Biology, Jichi Medical School, Tochigi, Japan.
Kusano
E
E
Inaba
T
T
Iimura
O
O
Takahashi
H
H
Ikeda
H
H
Ito
C
C
Ando
Y
Y
Ozawa
K
K
Asano
Y
Y
D000740:Anemia / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000276:immunology; D019281:Dimerization; D004921:Erythropoietin / Q000494:pharmacology*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D053614:Janus Kinase 2; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism; D009599:Nitroprusside / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism
D000959:Antihypertensive Agents; D004921:Erythropoietin; D007375:Interleukin-1; D053614:Janus Kinase 2; D009569:Nitric Oxide; D009599:Nitroprusside; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D014443:Tyrosine; C507924:JAK2 protein, human; C507927:Jak2 protein, rat; C494918:AKT1 protein, human; C494920:Akt1 protein, rat