Hypoxia-inducible factor-1 drives the motility of the erythroid progenitor cell line, UT-7/Epo, via autocrine motility factor.
OBJECTIVE
It is well known that hypoxic stress strongly enhances erythropoiesis, but the effect of hypoxia on erythroid progenitors has not been examined precisely. In the present study, using the erythropoietin-dependent cell line UT-7/Epo, which has characteristics of erythroid progenitors, we investigated a novel role of hypoxia in erythropoiesis.


METHODS
UT-7/Epo and four other hematopoietic and lymphoid cell lines (HL-60, THP-1, Raji, and CEM) were cultured in 20%, 5%, or 1% O2. Morphology was observed under a phase-contrast microscope. Cell motility was evaluated using the Transwell migration assay. An analysis of the protein level of hypoxia-inducible factor-1 (HIF-1) alpha and autocrine motility factor (AMF) was conducted using Western blotting and immunocytochemistry, respectively. Reverse transcription polymerase chain reaction was performed to evaluate the expression of AMF mRNA. Human bone marrow stromal cells were used in cocultures with UT-7/Epo. Apoptosis of UT-7/Epo was examined by immunocytochemistry using an antiactive form of caspase 3 antibody.


RESULTS
Among the five cell lines, UT-7/Epo exhibited active pseudopodial extension in hypoxia (1% O2), and cell motility was increased. HL-60, THP-1, Raji, and CEM did not show an increase in cell motility even in 1% O2. In addition, expression of the alpha-subunit of HIF-1 was activated by hypoxia, and expression of the mRNA and protein of AMF induced by HIF-1, increasing cell motility, was promoted. The addition of an HIF-1 inhibitor, cadmium chloride (CdCl2), or alpha-ketoglutarate (2-oxoglutarate) decreased the AMF mRNA expression, and an AMF inhibitor, erythrose 4-phosphate, decreased the cell motility. When UT-7/Epo was cocultured with human bone marrow-derived stromal cells that significantly inhibit the apoptosis of UT-7/Epo, the migration of UT-7/Epo under the stromal cells (pseudoemperipolesis) was increased in hypoxia.


CONCLUSIONS
Under hypoxic conditions, erythroid progenitors may exhibit active migration in the bone marrow and the opportunity for contact with stromal cells increases, inhibiting apoptosis.
Mikami
Makoto
M
Division of Hematology, Department of Medicine, Kawasaki Medical School, Kurashiki, Japan.
Sadahira
Yoshito
Y
Haga
Arayo
A
Otsuki
Takemi
T
Wada
Hideho
H
Sugihara
Takashi
T
D015153:Blotting, Western; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D018920:Coculture Techniques; D004268:DNA-Binding Proteins / Q000502:physiology*; D005434:Flow Cytometry; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007150:Immunohistochemistry; D009687:Nuclear Proteins / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000502:physiology*
D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D005956:Glucose-6-Phosphate Isomerase