Reduction of AHSP synthesis in hemin-induced K562 cells and EPO-induced CD34(+) cells leads to alpha-globin precipitation, impairment of normal hemoglobin production, and increased cell death.
OBJECTIVE
alpha-Hemoglobin stabilizing protein (AHSP) binds alpha-hemoglobin (Hb), avoiding its precipitation and its pro-oxidant activity. In the presence of betaHb, the alphaHb-AHSP complex is dismembered and betaHb displaces AHSP to generate the quaternary structure of Hb. The relationship between Hb formation and alterations in AHSP expression, which may affect human erythropoiesis, has not yet been described in human cells. Hence, in this study, we examined the effects of AHSP knockdown in hemin-induced K562 and erythropoietin-induced CD34(+) cells with particular reference to cellular aspects and gene expression.


METHODS
Short-hairpin RNA expression vectors aimed at the AHSP mRNA target sequence were cloned and transfected into K562 and CD34(+) cells. K562 and CD34(+) cells were stimulated to erythroid differentiation. Cells were examined in terms of gene expression using quantitative real-time polymerase chain reaction; reactive oxygen species (ROS) production, apoptosis, and Hb production through flow cytometry assays; and immunofluorescence assays for globin chains.


RESULTS
RNA interference-mediated knockdown of AHSP expression resulted in considerable alphaHb precipitation, as well as in a significant decrease in HbF formation. AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis.


CONCLUSIONS
These findings strengthen the hypothesis that AHSP stabilizes the alphaHb chain, avoiding its precipitation and its ability to generate ROS, which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.
Pinho
Flavia Oliveira
FO
Center of Hematology and Hemotherapy, State University at Campinas, São Paulo, Brazil.
de Albuquerque
Dulcineia Martins
DM
Olalla Saad
Sara Teresinha
ST
Costa
Fernando Ferreira
FF
D018952:Antigens, CD34 / Q000096:biosynthesis / Q000187:drug effects*; D017209:Apoptosis / Q000187:drug effects; D001798:Blood Proteins / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D003001:Cloning, Molecular; D015672:Erythroid Precursor Cells / Q000187:drug effects; D004921:Erythropoietin / Q000494:pharmacology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D005914:Globins / Q000187:drug effects* / Q000378:metabolism; D006427:Hemin / Q000494:pharmacology*; D006454:Hemoglobins / Q000096:biosynthesis*; D006801:Humans; D020014:K562 Cells; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
C468768:AHSP protein, human; D018952:Antigens, CD34; D001798:Blood Proteins; D006454:Hemoglobins; D018832:Molecular Chaperones; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D004921:Erythropoietin; D006427:Hemin; D005914:Globins