The critical role of SRC homology domain 2-containing tyrosine phosphatase-1 in recombinant human erythropoietin hyporesponsive anemia in chronic hemodialysis patients.
The molecular mechanism of anemia that is hyporesponsive to recombinant human erythropoietin (rHuEPO) in hemodialysis patients without underlying causative factors has not been investigated fully in hematopoietic stem cell system. Circulating CD34+ cells (1 x 10(4)) were isolated from rHuEPO hyporesponsive hemodialysis patients (EPO-H; n = 9), patients who were responsive to rHuEPO (EPO-R; n = 9), and healthy control subjects (n = 9). The patients with known causes of EPO hyporesponsiveness were eliminated from the current study. The cells were cultured in STEM PRO 34 liquid medium, supplemented with rHuEPO, IL-3, stem cell factor, and granulocyte-macrophage colony stimulating factor for 7 d and then transferred to a semisolid methylcellulose culture medium for performing burst forming unit-erythroid (BFU-E) colony assay. Expression of src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1), phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 5 (p-STAT5) was assessed with Western blot analysis. In EPO-H patients, SHP-1 antisense or scrambled S-oligos were included in the culture medium, and its effects were evaluated. The number of circulating CD34+ cells was not statistically different among the three groups, and their proliferation rates were similar for 7 d in culture. However, BFU-E colonies were significantly decreased in EPO-H patients compared with EPO-R and control groups. The mRNA and protein expression of SHP-1 and p-SHP-1 was significantly increased, whereas that of p-STAT5 was reduced in EPO-H patients. The inclusion of SHP-1 antisense S-oligo in culture suppressed SHP-1 protein expression associated with p-STAT5 upregulation, increase in p-STAT5-regulated genes, and partial recovery of BFU-E colonies. In EPO-H hemodialysis patients, the EPO signaling pathway is attenuated as a result of dephosphorylation of STAT5 via upregulation of SHP-1 phosphatase activity, and SHP-1 may be a novel target molecule to sensitize EPO action in these patients.
Akagi
Shigeru
S
Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
Ichikawa
Haruo
H
Okada
Tatsuo
T
Sarai
Ai
A
Sugimoto
Taro
T
Morimoto
Hisanori
H
Kihara
Takashi
T
Yano
Ai
A
Nakao
Kazushi
K
Nagake
Yoshio
Y
Wada
Jun
J
Makino
Hirofumi
H
D000740:Anemia / Q000188:drug therapy*; D018952:Antigens, CD34 / Q000378:metabolism; D015153:Blotting, Western; D002455:Cell Division; D003470:Culture Media / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D004921:Erythropoietin / Q000494:pharmacology*; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D053614:Janus Kinase 2; D007676:Kidney Failure, Chronic / Q000150:complications* / Q000628:therapy; D008894:Milk Proteins / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011994:Recombinant Proteins; D006435:Renal Dialysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D019089:Stem Cell Factor / Q000494:pharmacology; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains
D018952:Antigens, CD34; D003470:Culture Media; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008894:Milk Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D050799:STAT5 Transcription Factor; D019089:Stem Cell Factor; D015534:Trans-Activators; D004921:Erythropoietin; D014443:Tyrosine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D054645:Protein Phosphatase 1; C509249:PTPN6 protein, human; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases