Adenovirus-mediated hypoxia-inducible factor 1alpha double-mutant promotes differentiation of bone marrow stem cells to cardiomyocytes.
The hypoxia-inducible factor 1alpha (HIF-1alpha) regulates transcriptional genes involved in cell proliferation, survival, and differentiation. Under normoxia, HIF-1alpha has a short half-life (t((1/2)) approximately 5 min) and low transcriptional activity. An HIF-1alpha mutant, produced by substitution of alanine (Ala) for proline (Pro) at position 564 and asparagine (Asp) at position 803, can prevent HIF-1alpha hydroxylation and results in a highly active form of HIF-1alpha (HIF-1alpha-Ala564-Ala803). We hypothesized that adenovirus (Ad)-mediated transfer of the active form of HIF-1alpha (pAd-HIF-1alpha-Ala564-Ala803) could effectively occur in bone marrow stem cells (MSCs) and promote MSC differentiation under normoxia. PCR-based site-specific mutagenesis was used to construct the Ad vector expressing HIF-1alpha-Ala564-Ala803. RT-PCR and immunostaining were used to study whether pAd-HIF-1alpha-Ala564-Ala803 affected MSC differentiation to cardiomyocyte (CMC). pAd-HIF-1alpha-Ala564-Ala803 exhibited higher transcriptional activity and stable HIF-1alpha protein expression. Under normoxia, an MSC-CMC co-culture treated with pAd-HIF1a-Ala564-Ala803 augmented TGF-beta(1), Smad4, NKx2.5, and GATA4 expression. Higher expression of cTnT and alpha-actinin was observed by immunostaining in MSCs, compared with the control and contrast groups. Adenovirus-mediated hypoxia-inducible factor 1alpha double-mutant, pAd-HIF-1alpha-Ala564-Ala803, can stably express HIF-1alpha and promote its downstream genes and MSC differentiation to CMC in the MSC-CMC co-culture system under normoxia.
Wang
Yesong
Y
Department of Cardiology, The First Affiliated Hospital of Sun Yat-sen University, 510080, Guangzhou, China. yesong_wang@yahoo.com.cn
Feng
Chong
C
Xue
Jiaojie
J
Sun
Aijiao
A
Li
Jun
J
Wu
Juekan
J
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics*; D002469:Cell Separation; D002478:Cells, Cultured; D005269:Femur / Q000166:cytology; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D032383:Myocytes, Cardiac / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013234:Stem Cells / Q000378:metabolism*; D013977:Tibia / Q000166:cytology; D014162:Transfection
D015415:Biomarkers; D051795:Hypoxia-Inducible Factor 1, alpha Subunit