HIF-1α modulates β-catenin pathway to enhance the survival and angiogenesis of PBMSCs under hypoxia environment

Figure 4 Hypoxia-inducible factor-1α regulates β-catenin transcription. A-D: The experiments were categorized into the following groups: Transfection of vehicle in normoxia, transfection of vehicle in hypoxia, hypoxia-inducible factor-1α (HIF-1α) overexpression (oeHIF-1α), HIF-1α knockdown (shHIF-1α), oeHIF-1α plus shHIF-1α (oe + shHIF-1α) [or β-catenin overexpression (oeβ-catenin), β-catenin knockdown (shβ-catenin), and β-catenin overexpression plus β-catenin knockdown (oe + shβ-catenin)] in hypoxia. Hypoxia enhanced the expression of HIF-1α and β-catenin, with oeHIF-1α increasing the expressions of HIF-1α and β-catenin in peripheral blood mesenchymal stem cells (PBMSCs) under hypoxic conditions (A and B). Hypoxia enhanced the expression of HIF-1α; shβ-catenin and oeβ-catenin did not increase the expression of HIF-1α under hypoxic conditions (C and D); E: Chromatin immunoprecipitation assay for the binding of HIF-1α to the β-catenin promoter. Anti-immunoglobulin G was employed as a negative control, and anti-RNA-polymerase II was utilized as a positive control; F: Representative immunofluorescence results of the HIF-1α and β-catenin in PBMSCs treated with normoxia (vehicle) or hypoxia (vehicle, oeHIF-1α, oeβ-catenin, shHIF-1α + oeβ-catenin, and oeHIF-1α + shβ-catenin). HIF-1α and β-catenin were stained green and red, respectively. The nuclei of PBMSCs were counterstained blue using 4,6-diamino-2-phenylindole. Scale bars = 20 μm. HIF-1α was predominantly localized in the nucleus. β-catenin was mainly expressed in the cytoplasm and partially in the cell nucleus. All graphical data are presented as the means ± SEMs. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ^dP < 0.0001. n = 3 in each group. HIF-1α: Hypoxia-inducible factor-1α; Normoxia-Con: Transfection of vehicle in normoxia; Hypoxia-Con: Transfection of vehicle in hypoxia; oeHIF-1α: Hypoxia-inducible factor-1α overexpression; shHIF-1α: Hypoxia-inducible factor-1α knockdown; oe + shHIF-1α: Hypoxia-inducible factor-1α overexpression plus hypoxia-inducible factor-1α knockdown in hypoxia; oeβ-catenin: Β-catenin overexpression; shβ-catenin: Β-catenin knockdown; oe + shβ-catenin: Β-catenin overexpression plus β-catenin knockdown in hypoxia; PCR: Polymerase chain reaction; IgG: Immunoglobulin G; DAPI: 4,6-diamino-2-phenylindole.