ABSTRACT
Partial endothelial-to-mesenchymal transition (EndMT) is an intermediate phenotype observed in endothelial cells (ECs) undergoing a transition toward a mesenchymal state to support neovascularization during (patho)physiological angiogenesis. Here, we investigated the occurrence of partial EndMT in ECs under hypoxic/ischemic conditions and identified general transcription factor IIH subunit 4 (GTF2H4) as a positive regulator of this process. In addition, we discovered that GTF2H4 collaborates with its target protein excision repair cross-complementation group 3 (ERCC3) to co-regulate partial EndMT. Furthermore, by using phosphorylation proteomics and site-directed mutagenesis, we demonstrated that GTF2H4 was involved in the phosphorylation of receptor coactivator 3 (NCOA3) at serine 1330, which promoted the interaction between NCOA3 and p65, resulting in the transcriptional activation of NF-κB and the NF-κB/Snail signaling axis during partial EndMT. In vivo experiments confirmed that GTF2H4 significantly promoted partial EndMT and angiogenesis after ischemic injury. Collectively, our findings reveal that targeting GTF2H4 is promising for tissue repair and offers potential opportunities for treating hypoxic/ischemic diseases.
ABSTRACT
BACKGROUND: SIRT6, an important NAD+ -dependent protein, protects endothelial cells from inflammatory and oxidative stress injuries. However, the role of SIRT6 in cardiac microvascular endothelial cells (CMECs) under ischemia-reperfusion injury (IRI) remains unclear. METHODS: The HUVECs model of oxygen-glucose deprivation/reperfusion (OGD/R) was established to simulate the endothelial IRI in vitro. Endoplasmic reticulum oxidase 1 alpha (Ero1α) mRNA and protein levels in SIRT6-overexpressing or SIRT6-knockdown cells were measured by qPCR and Western blotting. The levels of H2 O2 and mitochondrial reactive oxygen species (ROS) were detected to evaluate the status of oxidative stress. The effects of SIRT6 deficiency and Ero1α knockdown on cellular endoplasmic reticulum stress (ERS), inflammation, apoptosis and barrier function were detected by a series of molecular biological experiments and functional experiments in vitro. Chromatin immunoprecipitation, Western blotting, qPCR, and site-specific mutation experiments were used to examine the underlying molecular mechanisms. Furthermore, endothelial cell-specific Sirt6 knockout (ecSirt6-/- ) mice were subjected to cardiac ischemia-reperfusion surgery to investigate the effects of SIRT6 in CMECs in vivo. RESULTS: The expression of Ero1α was significantly upregulated in SIRT6-knockdown endothelial cells, and high Ero1α expression correlated with the accumulation of H2 O2 and mitochondrial ROS. In addition, SIRT6 deficiency increased ERS, inflammation, apoptosis and endothelial permeability, and these effects could be significantly attenuated by Ero1α knockdown. The deacetylase catalytic activity of SIRT6 was important in regulating Ero1α expression and these biological processes. Mechanistically, SIRT6 inhibited the enrichment of HIF1α and p300 at the Ero1α promoter through deacetylating H3K9, thereby antagonizing HIF1α/p300-mediated Ero1α expression. Compared with SIRT6-wild-type (SIRT6-WT) cells, cells expressing the SIRT6-H133Y-mutant and SIRT6-R65A-mutant exhibited increased Ero1α expression. Furthermore, ecSirt6-/- mice subjected to ischemia-reperfusion surgery exhibited increased Ero1α expression and ERS in CMECs and worsened injuries to microvascular barrier function and cardiac function. CONCLUSIONS: Our results revealed an epigenetic mechanism associated with SIRT6 and Ero1α expression and highlighted the therapeutic potential of targeting the SIRT6-HIF1α/p300-Ero1α axis.