Abstract
The biology of the alpha subunits of hypoxia-inducible factors (HIFalpha) has expanded from their role in angiogenesis to their current position in the self-renewal and differentiation of stem cells. The results reported in this article show the discovery of FM19G11, a novel chemical entity that inhibits HIFalpha proteins that repress target genes of the two alpha subunits, in various tumor cell lines as well as in adult and embryonic stem cell models from rodents and humans, respectively. FM19G11 inhibits at nanomolar range the transcriptional and protein expression of Oct4, Sox2, Nanog, and Tgf-alpha undifferentiating factors, in adult rat and human embryonic stem cells, FM19G11 activity occurs in ependymal progenitor stem cells from rats (epSPC), a cell model reported for spinal cord regeneration, which allows the progression of oligodendrocyte cell differentiation in a hypoxic environment, has created interest in its characterization for pharmacological research. Experiments using small interfering RNA showed a significant depletion in Sox2 protein only in the case of HIF2alpha silencing, but not in HIF1alpha-mediated ablation. Moreover, chromatin immunoprecipitation data, together with the significant presence of functional hypoxia response element consensus sequences in the promoter region of Sox2, strongly validated that this factor behaves as a target gene of HIF2alpha in epSPCs. FM19G11 causes a reduction of overall histone acetylation with significant repression of p300, a histone acetyltransferase required as a co-factor for HIF-transcription activation. Arrays carried out in the presence and absence of the inhibitor showed the predominant involvement of epigenetic-associated events mediated by the drug.
Highlights
Hypoxia-inducible transcription factors (HIFs) is a heterodimer consisting of an oxygen-regulated ␣ subunit (1␣, 2␣, or 3␣) and a constitutively expressed  subunit, or ARNT
HIF activity is mainly regulated at the protein level, due to the hydroxylation of key proline residues present in the oxygen-dependent degradation domain of the ␣ subunits by the prolyl-hydroxylases (PHDs) triggering polyubiquitination and rapid degradation of the HIF␣ proteins dermal growth factor; hESC, human embryonic stem cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Small Interfering RNA (siRNA), small interfering RNA; TRE, 12-Otetradecanoylphorbol-13-acetate-responsive element; GFAP, glial fibrillary acidic protein
The specific inhibition of HIF␣ proteins by FM19G11 reduces the transcriptional activation of the expression of pluripotency markers Sox2 and Oct4 and the corresponding target genes Tgf-␣ and Nanog in ependymalderived stem/progenitor cells (epSPC), driving cell differentiation to oligodendrocytes in a process that may favor the design of pharmacological strategies for spinal cord regeneration
Summary
HIF is a heterodimer consisting of an oxygen-regulated ␣ subunit (1␣, 2␣, or 3␣) and a constitutively expressed  subunit, or ARNT. The specific inhibition of HIF␣ proteins by FM19G11 reduces the transcriptional activation of the expression of pluripotency markers Sox2 and Oct4 and the corresponding target genes Tgf-␣ and Nanog in epSPC, driving cell differentiation to oligodendrocytes in a process that may favor the design of pharmacological strategies for spinal cord regeneration.
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