Mikami Makoto, Sadahira Yoshito, Haga Arayo, Otsuki Takemi, Wada Hideho, Sugihara Takashi
Division of Hematology, Department of Medicine, Kawasaki Medical School, Kurashiki, Japan.
Exp Hematol. 2005 May;33(5):531-41. doi: 10.1016/j.exphem.2005.01.013.
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.
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.
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.
Under hypoxic conditions, erythroid progenitors may exhibit active migration in the bone marrow and the opportunity for contact with stromal cells increases, inhibiting apoptosis.
众所周知，低氧应激可强烈增强红细胞生成，但低氧对红系祖细胞的影响尚未得到精确研究。在本研究中，我们使用具有红系祖细胞特征的促红细胞生成素依赖性细胞系UT-7/Epo，研究了低氧在红细胞生成中的新作用。
将UT-7/Epo和其他四种造血及淋巴细胞系（HL-60、THP-1、Raji和CEM）在20%、5%或1%氧气条件下培养。在相差显微镜下观察形态。使用Transwell迁移试验评估细胞运动性。分别使用蛋白质印迹法和免疫细胞化学法分析缺氧诱导因子-1（HIF-1）α和自分泌运动因子（AMF）的蛋白质水平。进行逆转录聚合酶链反应以评估AMF mRNA的表达。将人骨髓基质细胞与UT-7/Epo共培养。使用抗活性形式的半胱天冬酶3抗体通过免疫细胞化学法检测UT-7/Epo的凋亡。
在这五种细胞系中，UT-7/Epo在低氧（1%氧气）条件下表现出活跃的伪足伸展，细胞运动性增加。HL-60、THP-1、Raji和CEM即使在1%氧气条件下也未显示细胞运动性增加。此外，HIF-1的α亚基表达在低氧条件下被激活，由HIF-1诱导的AMF的mRNA和蛋白质表达增加，从而促进细胞运动性。添加HIF-1抑制剂氯化镉（CdCl2）或α-酮戊二酸可降低AMF mRNA表达，而AMF抑制剂4-磷酸赤藓糖可降低细胞运动性。当UT-7/Epo与人骨髓来源的基质细胞共培养时，基质细胞可显著抑制UT-7/Epo的凋亡，在低氧条件下，UT-7/Epo在基质细胞下的迁移（伪足环绕）增加。
在低氧条件下，红系祖细胞可能在骨髓中表现出活跃的迁移，与基质细胞接触的机会增加，从而抑制凋亡。
