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 Cγ-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. 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 Cγ-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. Angiogenesis, the formation of new blood capillaries, is an important component of embryonic vascular development, wound healing, and organ regeneration, as well as the pathological processes such as diabetic retinopathies, atherosclerosis, and tumor growth (1Folkman J. Nat. Med. 1995; 1: 27-31Crossref J. Nat. Med. Nat. Med. Vascular endothelial growth factor (VEGF) VEGF, vascular endothelial growth endothelial endothelial of protein kinase protein kinase histone myocyte enhancer endothelial VEGF, vascular endothelial growth endothelial endothelial of protein kinase protein kinase histone myocyte enhancer endothelial is essential for processes in normal and pathological Nat. Med. VEGF and are in to endothelial cells of VEGF to and phosphorylation of the to the activation of signaling such as protein kinase protein and J. expression by VEGF in plays an important role in angiogenesis. However, the VEGF signaling to gene regulation kinase D a protein in the regulation of a of including J. we that VEGF activation of PKD the and that PKD is involved in VEGF-induced protein kinase signaling and endothelial cell J. that PKD gene transcription the of histone in and in cells J. J. Med. However, PKD in vascular is as a for the of gene expression J. J. and to transcriptional and to signaling J. J. J. J. and vascular by HDAC5 and as of However, is the role of in VEGF we that VEGF PKD-dependent phosphorylation of HDAC5 in to HDAC5 nuclear and myocyte enhancer factor-2 transcriptional PKD-HDAC5 pathway a VEGF-induced specific subset of gene expression, cell migration, and These findings a critical role for PKD and HDAC5 in regulation of gene transcription and angiogenesis by and and J. as and VEGF and and of the and endothelial cells in as J. J. J. and in as the and specifically to and in for the by stimulated and to the the and J. VEGF and kinase mutant by J. and as and and J. the as a the of for and by the of VEGF J. and and to J. by the of J. and and cells stimulated VEGF in the of the protein kinase of the by and the and by the a the by for are in as an the of cell wound J. and to a cell of and and VEGF in a a of endothelial cells that the formation for in vitro angiogenesis. the in vitro angiogenesis J. a of 5 of a in and for by and a of the an as J. and the are as of the by a by the of HDAC5 in the role of HDAC5 in VEGF we HDAC5 phosphorylation in in response to stimulated VEGF as and the phosphorylation of HDAC5 in cell by HDAC5 HDAC5 and VEGF HDAC5 phosphorylation the activation a and the of VEGF the of HDAC5 expression in cells not Moreover, VEGF HDAC5 phosphorylation in a a as as and a of the activation phosphorylation of HDAC5 by VEGF is not VEGF stimulated HDAC5 phosphorylation in and in not VEGF-induced HDAC5 and as well as we that VEGF-induced HDAC5 phosphorylation is mediated by not further pathways involved in VEGF-induced HDAC5 HDAC5 phosphorylation the not VEGF-induced HDAC5 phosphorylation Moreover, the and inhibited HDAC5 phosphorylation in a and These that the pathway HDAC5 phosphorylation in we that the pathway not involved in HDAC5 phosphorylation in by VEGF pathway VEGF-induced HDAC5 as and for and to VEGF for HDAC5 phosphorylation and expression in cell are the VEGF HDAC5 by that VEGF stimulated PKD phosphorylation and activation in a J. the role of PKD in VEGF-induced HDAC5 we inhibited the PKD a PKD inhibited VEGF-induced HDAC5 phosphorylation in a expression in by J. 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Med. we the PKD-HDAC5 pathway through regulation of gene expression is in the processes of angiogenesis. the J. VEGF migration, to overexpression of phosphorylation and activation not as well as HDAC5 phosphorylation and nuclear export and the VEGF-induced VEGF-mediated and wound and VEGF-induced inhibited by and endothelial cell is by the PKD-HDAC5 pathway. and and for in response to VEGF in wound and are in to and that the VEGF the of in vitro the of to and by the of the J. J. VEGF stimulated formation in and VEGF-induced formation and However, the of in VEGF-induced formation and of VEGF-induced formation and a critical role for HDAC5 and in regulation of VEGF-induced in vitro HDAC5 phosphorylation is for VEGF-induced in vitro angiogenesis. and and in and for in vitro angiogenesis in response to VEGF formation and the of and the in to and that the VEGF findings of are that VEGF PKD-dependent HDAC5 phosphorylation and nuclear export in and that the pathway is involved in VEGF-induced gene expression, migration, and transcription and NR4A1 gene expression are in angiogenesis in vitro and in J. Med. suggest that the PKD-HDAC5 pathway is to an important role in VEGF signaling and angiogenesis in and pathological of and transcription is of a signaling that is involved in the of gene expression J. J. showed that as of further an important role of HDAC5 as a VEGF of transcriptional activation in We showed that VEGF phosphorylation of in we that VEGF HDAC5 the to the VEGF of and transcriptional of to in response to mutant inhibited the in transcriptional NR4A1 expression, and and that the gene of HDAC5 in is by the HDAC5 phosphorylation and nuclear export in response to as a of the as a in We that is in and that activation is of the signaling in in response to VEGF J. we that is a kinase to HDAC5 phosphorylation and nuclear export in VEGF We that HDAC5 phosphorylation and nuclear export is mediated through the pathway. including and the mutant of further a critical role of in VEGF-induced HDAC5 phosphorylation and nuclear overexpression of HDAC5 phosphorylation and nuclear export as well as cell migration, to activation of we that overexpression of in cells stimulated phosphorylation and activation not in phosphorylation and nuclear export of HDAC5 cells and in J. J. However, in VEGF signaling in we found that the pathway is not involved in pathways the of VEGF, including of cell migration, vascular and angiogenesis J. that PKD the pathway to activation and J. the we further found that PKD a role in VEGF regulation of transcriptional activation overexpression of and by inhibited transcriptional activation in transcription in blood and vascular J. including NR4A1 and that NR4A1 VEGF-induced and formation and angiogenesis in J. Med. we that VEGF stimulated the of NR4A1 in in a PKD-HDAC5 overexpression of and VEGF-induced NR4A1 the of NR4A1 the HDAC5 nuclear export of VEGF for is that the HDAC5 transcription activation to the as by that that kinase signaling the in the of the HDAC5 nuclear export Moreover, VEGF-mediated and formation inhibited by expression of and NR4A1 is important for VEGF-induced is that in angiogenesis by the PKD-HDAC5 pathway. we found that the PKD-HDAC5 pathway a subset of gene including NR4A1, and VEGF-induced growth including and are not by PKD-HDAC5 that VEGF HDAC5 phosphorylation and nuclear export in through the pathway. a role of PKD and HDAC5 in VEGF-induced transcriptional NR4A1 expression, migration, and in vitro angiogenesis. the findings a critical role of HDAC5 as a PKD in VEGF and suggest that the PKD-HDAC5 pathway is important for angiogenesis by new to and pathological angiogenesis in and Angiogenesis, the formation of new blood capillaries, is an important component of embryonic vascular development, wound healing, and organ regeneration, as well as the pathological processes such as diabetic retinopathies, atherosclerosis, and tumor growth (1Folkman J. Nat. Med. 1995; 1: 27-31Crossref J. Nat. Med. Nat. Med. Vascular endothelial growth factor (VEGF) VEGF, vascular endothelial growth endothelial endothelial of protein kinase protein kinase histone myocyte enhancer endothelial VEGF, vascular endothelial growth endothelial endothelial of protein kinase protein kinase histone myocyte enhancer endothelial is essential for processes in normal and pathological Nat. Med. VEGF and are in to endothelial cells of VEGF to and phosphorylation of the to the activation of signaling such as protein kinase protein and J. expression by VEGF in plays an important role in angiogenesis. However, the VEGF signaling to gene regulation kinase D a protein in the regulation of a of including J. we that VEGF activation of PKD the and that PKD is involved in VEGF-induced protein kinase signaling and endothelial cell J. that PKD gene transcription the of histone in and in cells J. J. Med. However, PKD in vascular is as a for the of gene expression J. J. and to transcriptional and to signaling J. J. J. J. and vascular by HDAC5 and as of However, is the role of in VEGF we that VEGF PKD-dependent phosphorylation of HDAC5 in to HDAC5 nuclear and myocyte enhancer factor-2 transcriptional PKD-HDAC5 pathway a VEGF-induced specific subset of gene expression, cell migration, and These findings a critical role for PKD and HDAC5 in regulation of gene transcription and angiogenesis by and and J. as and VEGF and and of the and endothelial cells in as J. J. J. and in as the and specifically to and in for the by stimulated and to the the and J. VEGF and kinase mutant by J. and as and and J. the as a the of for and by the of VEGF J. and and to J. by the of J. and and cells stimulated VEGF in the of the protein kinase of the by and the and by the a the by for are in as an the of cell wound J. and to a cell of and and VEGF in a a of endothelial cells that the formation for in vitro angiogenesis. the in vitro angiogenesis J. a of 5 of a in and for by and a of the an as J. and the are as of the by a by the of and and J. as and VEGF and and of the and endothelial cells in as J. J. J. and in as the and specifically to and in for the by stimulated and to the the and J. VEGF and kinase mutant by J. and as and and J. the as a the of for and by the of VEGF J. and and to J. by the of J. and and cells stimulated VEGF in the of the protein kinase of the by and the and by the a the by for are in as an the of cell wound J. and to a cell of and and VEGF in a a of endothelial cells that the formation for in vitro angiogenesis. the in vitro angiogenesis J. a of 5 of a in and for by and a of the an as J. and the are as of the by a by the of HDAC5 in the role of HDAC5 in VEGF we HDAC5 phosphorylation in in response to stimulated VEGF as and the phosphorylation of HDAC5 in cell by HDAC5 HDAC5 and VEGF HDAC5 phosphorylation the activation a and the of VEGF the of HDAC5 expression in cells not Moreover, VEGF HDAC5 phosphorylation in a a as as and a of the activation phosphorylation of HDAC5 by VEGF is not VEGF stimulated HDAC5 phosphorylation in and in not VEGF-induced HDAC5 and as well as we that VEGF-induced HDAC5 phosphorylation is mediated by not further pathways involved in VEGF-induced HDAC5 HDAC5 phosphorylation the not VEGF-induced HDAC5 phosphorylation Moreover, the and inhibited HDAC5 phosphorylation in a and These that the pathway HDAC5 phosphorylation in we that the pathway not involved in HDAC5 phosphorylation in by VEGF HDAC5 by that VEGF stimulated PKD phosphorylation and activation in a J. the role of PKD in VEGF-induced HDAC5 we inhibited the PKD a PKD inhibited VEGF-induced HDAC5 phosphorylation in a expression in by J. 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Med. we the PKD-HDAC5 pathway through regulation of gene expression is in the processes of angiogenesis. the J. VEGF migration, to overexpression of phosphorylation and activation not as well as HDAC5 phosphorylation and nuclear export and the VEGF-induced VEGF-mediated and wound and VEGF-induced inhibited by and endothelial cell is by the PKD-HDAC5 pathway. and and for in response to VEGF in wound and are in to and that the VEGF the of in vitro the of to and by the of the J. J. VEGF stimulated formation in and VEGF-induced formation and However, the of in VEGF-induced formation and of VEGF-induced formation and a critical role for HDAC5 and in regulation of VEGF-induced in vitro HDAC5 phosphorylation is for VEGF-induced in vitro angiogenesis. and and in and for in vitro angiogenesis in response to VEGF formation and the of and the in to and that the VEGF VEGF HDAC5 in the role of HDAC5 in VEGF we HDAC5 phosphorylation in in response to stimulated VEGF as and the phosphorylation of HDAC5 in cell by HDAC5 HDAC5 and VEGF HDAC5 phosphorylation the activation a and the of VEGF the of HDAC5 expression in cells not Moreover, VEGF HDAC5 phosphorylation in a a as as and a of the activation phosphorylation of HDAC5 by VEGF is not VEGF stimulated HDAC5 phosphorylation in and in not VEGF-induced HDAC5 and as well as we that VEGF-induced HDAC5 phosphorylation is mediated by not We further pathways involved in VEGF-induced HDAC5 HDAC5 phosphorylation the not VEGF-induced HDAC5 phosphorylation Moreover, the and inhibited HDAC5 phosphorylation in a and These that the pathway HDAC5 phosphorylation in we that the pathway not involved in HDAC5 phosphorylation in by VEGF PKD HDAC5 by that VEGF stimulated PKD phosphorylation and activation in a J. the role of PKD in VEGF-induced HDAC5 we inhibited the PKD a PKD inhibited VEGF-induced HDAC5 phosphorylation in a expression in by J. VEGF-induced HDAC5 phosphorylation VEGF-induced HDAC5 the of HDAC5 phosphorylation demonstrate an essential role of for VEGF-induced HDAC5 we that and HDAC5 by VEGF in in VEGF HDAC5 the of HDAC5 phosphorylation in VEGF-mediated signaling we the of VEGF HDAC5 HDAC5 HDAC5 and to of VEGF, in the of response to VEGF in the cells a export of HDAC5 of VEGF, and a in the for and the a VEGF VEGF of the the the VEGF-induced HDAC5 to HDAC5 the for and VEGF-induced HDAC5 nuclear export the and HDAC5 nuclear export by VEGF the critical role of PKD in VEGF-induced HDAC5 the PKD and to VEGF-induced HDAC5 nuclear export and Moreover, in an HDAC5 VEGF stimulated nuclear export of by of nuclear export of the VEGF not of nuclear export of HDAC5 in response to VEGF in the and cell is a J. HDAC5 for PKD-dependent phosphorylation of HDAC5 is for HDAC5 nuclear we of the in VEGF for the in the VEGF and the that PKD-dependent phosphorylation of HDAC5 is critical for nuclear PKD-HDAC5 in VEGF-induced and to transcription of the and an important role in the of gene expression J. we the of and VEGF-induced transcriptional activation in the the cells and VEGF transcriptional of such an of transcriptional activation by VEGF involved in the inhibited the VEGF-induced transcriptional activation the role of PKD and HDAC5 in VEGF regulation of we the expression of NR4A1 a response gene and orphan nuclear receptor transcription factor in tumor and growth and VEGF stimulated NR4A1 in a and inhibited the of NR4A1 by VEGF We further the of in regulation of VEGF for and VEGF for we found that and a subset of including NR4A1, factor and However, the of including and not by and PKD-HDAC5 for VEGF-induced and NR4A1 and are involved in VEGF-induced angiogenesis J. Med. we the PKD-HDAC5 pathway through regulation of gene expression is in the processes of angiogenesis. the J. VEGF migration, to overexpression of phosphorylation and activation not as well as HDAC5 phosphorylation and nuclear export and the VEGF-induced VEGF-mediated and wound and VEGF-induced inhibited by and the of in vitro the of to and by the of the J. J. VEGF stimulated formation in and VEGF-induced formation and However, the of in VEGF-induced formation and of VEGF-induced formation and a critical role for HDAC5 and in regulation of VEGF-induced in vitro angiogenesis. findings of are that VEGF PKD-dependent HDAC5 phosphorylation and nuclear export in and that the pathway is involved in VEGF-induced gene expression, migration, and transcription and NR4A1 gene expression are in angiogenesis in vitro and in J. Med. suggest that the PKD-HDAC5 pathway is to an important role in VEGF signaling and angiogenesis in and pathological of and transcription is of a signaling that is involved in the of gene expression J. J. showed that as of further an important role of HDAC5 as a VEGF of transcriptional activation in We showed that VEGF phosphorylation of in we that VEGF HDAC5 the to the VEGF of and transcriptional of to in response to mutant inhibited the in transcriptional NR4A1 expression, and and that the gene of HDAC5 in is by the HDAC5 phosphorylation and nuclear export in response to as a of the as a in We that is in and that activation is of the signaling in in response to VEGF J. we that is a kinase to HDAC5 phosphorylation and nuclear export in VEGF We that HDAC5 phosphorylation and nuclear export is mediated through the pathway. including and the mutant of further a critical role of in VEGF-induced HDAC5 phosphorylation and nuclear overexpression of HDAC5 phosphorylation and nuclear export as well as cell migration, to activation of we that overexpression of in cells stimulated phosphorylation and activation not in phosphorylation and nuclear export of HDAC5 cells and in J. J. However, in VEGF signaling in we found that the pathway is not involved in pathways the of VEGF, including of cell migration, vascular and angiogenesis J. that PKD the pathway to activation and J. the we further found that PKD a role in VEGF regulation of transcriptional activation overexpression of and by inhibited transcriptional activation in transcription in blood and vascular J. including NR4A1 and that NR4A1 VEGF-induced and formation and angiogenesis in J. Med. we that VEGF stimulated the of NR4A1 in in a PKD-HDAC5 overexpression of and VEGF-induced NR4A1 the of NR4A1 the HDAC5 nuclear export of VEGF for is that the HDAC5 transcription activation to the as by that that kinase signaling the in the of the HDAC5 nuclear export Moreover, VEGF-mediated and formation inhibited by expression of and NR4A1 is important for VEGF-induced is that in angiogenesis by the PKD-HDAC5 pathway. we found that the PKD-HDAC5 pathway a subset of gene including NR4A1, and VEGF-induced growth including and are not by PKD-HDAC5 that VEGF HDAC5 phosphorylation and nuclear export in through the pathway. a role of PKD and HDAC5 in VEGF-induced transcriptional NR4A1 expression, migration, and in vitro angiogenesis. the findings a critical role of HDAC5 as a PKD in VEGF and suggest that the PKD-HDAC5 pathway is important for angiogenesis by new to and pathological angiogenesis in and findings of are that VEGF PKD-dependent HDAC5 phosphorylation and nuclear export in and that the pathway is involved in VEGF-induced gene expression, migration, and transcription and NR4A1 gene expression are in angiogenesis in vitro and in J. Med. suggest that the PKD-HDAC5 pathway is to an important role in VEGF signaling and angiogenesis in and pathological of and transcription is of a signaling that is involved in the of gene expression J. J. showed that as of further an important role of HDAC5 as a VEGF of transcriptional activation in We showed that VEGF phosphorylation of in we that VEGF HDAC5 the to the VEGF of and transcriptional of to in response to mutant inhibited the in transcriptional NR4A1 expression, and and that the gene of HDAC5 in is by the HDAC5 phosphorylation and nuclear export in response to as a of the as a in We that is in and that activation is of the signaling in in response to VEGF J. we that is a kinase to HDAC5 phosphorylation and nuclear export in VEGF We that HDAC5 phosphorylation and nuclear export is mediated through the pathway. including and the mutant of further a critical role of in VEGF-induced HDAC5 phosphorylation and nuclear overexpression of HDAC5 phosphorylation and nuclear export as well as cell migration, to activation of we that overexpression of in cells stimulated phosphorylation and activation not in phosphorylation and nuclear export of HDAC5 cells and in J. J. However, in VEGF signaling in we found that the pathway is not involved in pathways the of VEGF, including of cell migration, vascular and angiogenesis J. that PKD the pathway to activation and J. the we further found that PKD a role in VEGF regulation of transcriptional activation overexpression of and by inhibited transcriptional activation in transcription in blood and vascular J. including NR4A1 and that NR4A1 VEGF-induced and formation and angiogenesis in J. Med. we that VEGF stimulated the of NR4A1 in in a PKD-HDAC5 overexpression of and VEGF-induced NR4A1 the of NR4A1 the HDAC5 nuclear export of VEGF for is that the HDAC5 transcription activation to the as by that that kinase signaling the in the of the HDAC5 nuclear export Moreover, VEGF-mediated and formation inhibited by expression of and NR4A1 is important for VEGF-induced is that in angiogenesis by the PKD-HDAC5 pathway. we found that the PKD-HDAC5 pathway a subset of gene including NR4A1, and VEGF-induced growth including and are not by PKD-HDAC5 pathway. that VEGF HDAC5 phosphorylation and nuclear export in through the pathway. a role of PKD and HDAC5 in VEGF-induced transcriptional NR4A1 expression, migration, and in vitro angiogenesis. the findings a critical role of HDAC5 as a PKD in VEGF and suggest that the PKD-HDAC5 pathway is important for angiogenesis by new to and pathological angiogenesis in and We for critical of the