Protein Kinase D-dependent Phosphorylation and Nuclear Export of Histone Deacetylase 5 Mediates Vascular Endothelial Growth Factor-induced Gene Expression and Angiogenesis

Abstract

Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase C␥-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis.

Protein kinase D in vascular biology and angiogenesis

“…Nurr77 transcription is regulated by the MEF2 transcriptional factor and Ha et al, showed that MEF2 transcriptional activity was induced by VEGF. Furthermore, this induction was prevented by the overexpression of either a kinase dead PKD1 mutant or a constitutively active HDAC5 mutant (10). Nurr77 and another immediate-early gene, Egr3, are strongly and rapidly induced by VEGF in HUVECs (30), and PKD knockdown by siRNA reduced the VEGF-induced transcription of these genes, though Nurr77 transcription was only affected by PKD2 knockdown (31).…”

“…The reason for the discrepancy between our findings and those of Ha et al is unclear, but it is possible that overexpression of the kinase dead PKD1 mutant may have had an adverse effect on PKD2 activity. Both Egr3 and Nurr77 are required for effective VEGF-mediated migration and tube formation in HUVECs, suggesting that PKD may regulate angiogenic responses in part by mediating transcription of these genes (10,31). HDAC7 also regulates Nurr77 transcription via MEF2, and another gene, RCAN2, which stimulates angiogenesis via the calcineurin-NFAT pathway (29,32).…”

“…In a recent study, CREB dependent transcription of the immediate-early gene, Nurr1, was found to be important for in vivo angiogenesis in a mouse model using matrigel plugs (27). Furthermore, PKD can also phosphorylate several members of the Class IIa histone deacetylase (HDAC) family, notably HDAC5 and HDAC7 (10,28,29). HDACs repress transcription by reducing the level of histone acetylation, whereas phosphorylation of HDACs induces their export from the nucleus, thereby facilitating the transcription of target genes.…”

“…PKDs 1 and 2 are expressed in endothelial cells and recent evidence indicates that PKD activation is required for biological responses to vascular endothelial growth factor (VEGF or VEGF-A) in endothelial cells important for angiogenesis, such as migration (9)(10)(11), proliferation (11) and tubulogenesis in vitro (9)(10)(11). Less is known about the role of PKD3 in endothelial cell signaling, but work to date suggests it is expressed at low levels in these cells and plays no significant role in endothelial migration (9).…”

PKA phosphorylates histone deacetylase 5 and prevents its nuclear export, leading to the inhibition of gene transcription and cardiomyocyte hypertrophy

“…In particular, class IIa HDACs, such as HDAC5, acting as transcriptional repressors, have been implicated in cardiac hypertrophy, skeletal muscle differentiation, and angiogenesis (5)(6)(7)(8)(9)(10). Dynamic nucleocytoplasmic shuttling has been proposed as a fundamental mechanism regulating the function of class IIa HDACs (1,(11)(12)(13).…”