VEGF stimulates HDAC7 phosphorylation and cytoplasmic accumulation modulating matrix metalloproteinase expression and angiogenesis - PubMed (original) (raw)

VEGF stimulates HDAC7 phosphorylation and cytoplasmic accumulation modulating matrix metalloproteinase expression and angiogenesis

Chang Hoon Ha et al. Arterioscler Thromb Vasc Biol. 2008 Oct.

Abstract

Objective: Histone acetylation/deacetylation plays an important role in the control of gene expression, tissue growth, and development. In particular, histone deacetylases 7 (HDAC7), a member of class IIa HDACs, is crucial in maintaining vascular integrity. However, whether HDAC7 is involved in the processes of vascular endothelial signaling and angiogenesis remains unclear. Here, we investigated the role of HDAC7 in vascular endothelial growth factor (VEGF) signaling and angiogenesis.

Methods and results: We show for the first time that VEGF stimulated phosphorylation of HDAC7 at the sites of Ser178, Ser344, and Ser479 in a dose- and time-dependent manner, which leads to the cytoplasmic accumulation of HDAC7. Using pharmacological inhibitors, siRNA, and adenoviruses carrying dominant-negative mutants, we found that phospholipase Cgamma/protein kinase C/protein kinase D1 (PKD1)-dependent signal pathway mediated HDAC7 phosphorylation and cytoplasmic accumulation by VEGF. Infection of ECs with adenoviruses encoding a mutant of HDAC7 specifically deficient in PKD1-dependent phosphorylation inhibited VEGF-induced angiogenic gene expression, including matrix metalloproteinases MT1-matrix metalloproteinase (MMP) and MMP10. Moreover, HDAC7 and its targeting genes were involved in VEGF-stimulated endothelial cell migration, tube formation, and microvessel sprouting.

Conclusions: Our results demonstrate that VEGF stimulates PKD1-dependent HDAC7 phosphorylation and cytoplasmic accumulation in endothelial cells modulating gene expression and angiogenesis.

PubMed Disclaimer

Figures

Figure 1

Figure 1. VEGF stimulates HDAC7 phosphorylation in endothelial cells

(A) BAECs were exposed to VEGF (20 ng/ml) for various times as indicated. (B) BAECs were exposed to VEGF for 15 min with the indicated concentrations. HDAC7 phosphorylation and expression in cell lysates were determined as described in Materials and Methods. Representative immunoblots and quantitative data of HAC7 phosphorylation normalized with the level of HDAC7 were shown (n=4). *, # and &, p < 0.05 in comparison to value from control group without VEGF treatment (0 min).

Figure 1

Figure 1. VEGF stimulates HDAC7 phosphorylation in endothelial cells

(A) BAECs were exposed to VEGF (20 ng/ml) for various times as indicated. (B) BAECs were exposed to VEGF for 15 min with the indicated concentrations. HDAC7 phosphorylation and expression in cell lysates were determined as described in Materials and Methods. Representative immunoblots and quantitative data of HAC7 phosphorylation normalized with the level of HDAC7 were shown (n=4). *, # and &, p < 0.05 in comparison to value from control group without VEGF treatment (0 min).

Figure 2

Figure 2. PKD1 mediates HDAC7 phosphorylation by VEGF

(A) BAECs were pretreated with the potent PKD inhibitor Go6976 at indicated concentration for 30 min, and then exposed to VEGF (20 ng/ml) for 15 min. (B) HUVECs were transfected with scrambled siRNA (100 nM, control) or PKD1 siRNA (100 nM) for 48 h, and then exposed to VEGF for 15 min. (C) BAECs were infected with adenoviruses encoding GFP (Ad-GFP, control), or Ad-GFP-PKD1-KN at 100 MOI for 24 h, and then exposed to VEGF for 15 min. HDAC7 phosphorylation and expression in cell lysates were determined. PKD1 expression levels were also analyzed to confirm the knockdown effect of PKD1 siRNA (B) and overexpression of GFP-PKD1-KN in ECs (C). Representative immunoblots and quantitative data were shown (n=3).

Figure 2

Figure 2. PKD1 mediates HDAC7 phosphorylation by VEGF

(A) BAECs were pretreated with the potent PKD inhibitor Go6976 at indicated concentration for 30 min, and then exposed to VEGF (20 ng/ml) for 15 min. (B) HUVECs were transfected with scrambled siRNA (100 nM, control) or PKD1 siRNA (100 nM) for 48 h, and then exposed to VEGF for 15 min. (C) BAECs were infected with adenoviruses encoding GFP (Ad-GFP, control), or Ad-GFP-PKD1-KN at 100 MOI for 24 h, and then exposed to VEGF for 15 min. HDAC7 phosphorylation and expression in cell lysates were determined. PKD1 expression levels were also analyzed to confirm the knockdown effect of PKD1 siRNA (B) and overexpression of GFP-PKD1-KN in ECs (C). Representative immunoblots and quantitative data were shown (n=3).

Figure 3

Figure 3. VEGF promotes cytoplasmic accumulation of HDAC7 through PKD1-dependent phosphorylation

(A-B) BAECs were infected with Ad-YFP-HDAC7 at 50 MOI for 24 h, and then exposed to VEGF (20 ng/ml) for various times as indicated (A); or then pretreated with PKD inhibitor Go6976 (1 μM) for 30 min followed by the exposure of VEGF for 3 h (B). (C) HUVECs were transfected with scrambled siRNA (100 nM, control) or PKD1 siRNA (100 nM) for 24 h, and then infected with Ad-YFP-HDAC7 at 50 MOI for 24 h, followed by the exposure of VEGF (20 ng/ml) for 3 h. (D) BAECs were infected with Ad-YFP-HDAC7-WT or Ad- YFP-HDAC7-S/A at 50 MOI for 24 h, and then stimulated with VEGF for 3 h. (E) BAECs were infected with Ad-HA-HDAC7-WT or Ad- HA-HDAC7-S/A at 50 MOI for 24 h, and then stimulated with VEGF (20 ng/ml) for 3 h. The cells were fixed and YFP-HDAC7 or HA-HDAC7 distribution in the cell was analyzed by fluorescence microscopy. Representative images (magnification, x40 for (A) or x60 for (B-E)) were shown (n=3). Nuclei stained with DAPI (A) were also analyzed by fluorescence microscopy.

Figure 3

Figure 3. VEGF promotes cytoplasmic accumulation of HDAC7 through PKD1-dependent phosphorylation

(A-B) BAECs were infected with Ad-YFP-HDAC7 at 50 MOI for 24 h, and then exposed to VEGF (20 ng/ml) for various times as indicated (A); or then pretreated with PKD inhibitor Go6976 (1 μM) for 30 min followed by the exposure of VEGF for 3 h (B). (C) HUVECs were transfected with scrambled siRNA (100 nM, control) or PKD1 siRNA (100 nM) for 24 h, and then infected with Ad-YFP-HDAC7 at 50 MOI for 24 h, followed by the exposure of VEGF (20 ng/ml) for 3 h. (D) BAECs were infected with Ad-YFP-HDAC7-WT or Ad- YFP-HDAC7-S/A at 50 MOI for 24 h, and then stimulated with VEGF for 3 h. (E) BAECs were infected with Ad-HA-HDAC7-WT or Ad- HA-HDAC7-S/A at 50 MOI for 24 h, and then stimulated with VEGF (20 ng/ml) for 3 h. The cells were fixed and YFP-HDAC7 or HA-HDAC7 distribution in the cell was analyzed by fluorescence microscopy. Representative images (magnification, x40 for (A) or x60 for (B-E)) were shown (n=3). Nuclei stained with DAPI (A) were also analyzed by fluorescence microscopy.

Figure 3

Figure 3. VEGF promotes cytoplasmic accumulation of HDAC7 through PKD1-dependent phosphorylation

(A-B) BAECs were infected with Ad-YFP-HDAC7 at 50 MOI for 24 h, and then exposed to VEGF (20 ng/ml) for various times as indicated (A); or then pretreated with PKD inhibitor Go6976 (1 μM) for 30 min followed by the exposure of VEGF for 3 h (B). (C) HUVECs were transfected with scrambled siRNA (100 nM, control) or PKD1 siRNA (100 nM) for 24 h, and then infected with Ad-YFP-HDAC7 at 50 MOI for 24 h, followed by the exposure of VEGF (20 ng/ml) for 3 h. (D) BAECs were infected with Ad-YFP-HDAC7-WT or Ad- YFP-HDAC7-S/A at 50 MOI for 24 h, and then stimulated with VEGF for 3 h. (E) BAECs were infected with Ad-HA-HDAC7-WT or Ad- HA-HDAC7-S/A at 50 MOI for 24 h, and then stimulated with VEGF (20 ng/ml) for 3 h. The cells were fixed and YFP-HDAC7 or HA-HDAC7 distribution in the cell was analyzed by fluorescence microscopy. Representative images (magnification, x40 for (A) or x60 for (B-E)) were shown (n=3). Nuclei stained with DAPI (A) were also analyzed by fluorescence microscopy.

Figure 4

Figure 4. PKD1-dependent HDAC7 phosphorylation modulates VEGF-induced MMP10 and MT1-MMP expression in endothelial cells

(A) HUVECs were stimulated with VEGF (20 ng/ml) for various times as indicated. (B) HUVECs were infected with Ad-GFP (control), Ad-YFP-HDAC7-S/A or GFP-PKD1-KN (100 MOI) for 24 h, and then stimulated with VEGF (20 ng/ml) for 1 h. The mRNA was extracted from the cell lysates, and RT-PCR with the primers of MMP1, MT1-MMP, and GADPH (internal control) were performed as described in Materials and Methods. The representative images and quantitative data were shown (n=4).

Figure 5

Figure 5. HDAC7 and MT1-MMP are involved in VEGF-induced endothelial cell migration and tube formation

(A) BAECs were infected with Ad-LacZ (control), or Ad-HA-HDAC7-S/A and then measured for cell migration in response to VEGF in wound closure assay (n=4). (B) HUVECs were transfected with control siRNA or human MT1-MMP1 siRNA and then measured for cell migration in response to VEGF in wound closure assay. The representative images and quantitative data were shown (n=4). (C) HUVECs infected with Ad-LacZ (control), or Ad-HA-HDAC7-S/A were cultured in Matrigel and measured for in vitro angiogenesis in response to VEGF with tube formation assay (n=4). (D) HUVECs transfected with control siRNA or human MT1-MMP1 siRNA were cultured in Matrigel and measured for in vitro angiogenesis in response to VEGF with tube formation assay. The length of tube-like structure were quantified and shown. *, p < 0.05 in comparison to value from control; #, p < 0.05 vs. that from the group treated with VEGF along with Ad-lacZ or control siRNA.

Figure 5

Figure 5. HDAC7 and MT1-MMP are involved in VEGF-induced endothelial cell migration and tube formation

(A) BAECs were infected with Ad-LacZ (control), or Ad-HA-HDAC7-S/A and then measured for cell migration in response to VEGF in wound closure assay (n=4). (B) HUVECs were transfected with control siRNA or human MT1-MMP1 siRNA and then measured for cell migration in response to VEGF in wound closure assay. The representative images and quantitative data were shown (n=4). (C) HUVECs infected with Ad-LacZ (control), or Ad-HA-HDAC7-S/A were cultured in Matrigel and measured for in vitro angiogenesis in response to VEGF with tube formation assay (n=4). (D) HUVECs transfected with control siRNA or human MT1-MMP1 siRNA were cultured in Matrigel and measured for in vitro angiogenesis in response to VEGF with tube formation assay. The length of tube-like structure were quantified and shown. *, p < 0.05 in comparison to value from control; #, p < 0.05 vs. that from the group treated with VEGF along with Ad-lacZ or control siRNA.

Figure 6

Figure 6. PKD1-dependent HDAC7 phosphorylation is required for VEGF-induced microvessel sprouting in mouse aorta ring assay

(A) Mouse aorta rings were isolated form mice and infected Ad-LacZ (control), Ad-HA-HDAC7-S/A or Ad-GFP-PKD1-KN, and then the aorta ring assay for ex vivo angiogenesis was performed. The representative images were shown (n=4). (B) Schema for potential role and pathways of PKD1 and HDAC7 for VEGF-induced angiogenesis (see the text in detail).

Figure 6

Figure 6. PKD1-dependent HDAC7 phosphorylation is required for VEGF-induced microvessel sprouting in mouse aorta ring assay

(A) Mouse aorta rings were isolated form mice and infected Ad-LacZ (control), Ad-HA-HDAC7-S/A or Ad-GFP-PKD1-KN, and then the aorta ring assay for ex vivo angiogenesis was performed. The representative images were shown (n=4). (B) Schema for potential role and pathways of PKD1 and HDAC7 for VEGF-induced angiogenesis (see the text in detail).

Comment in

Similar articles

Cited by

References

    1. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995;1:27–31. - PubMed
    1. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9:669–76. - PubMed
    1. Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003;9:653–60. - PubMed
    1. Takahashi T, Yamaguchi S, Chida K, Shibuya M. A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells. Embo J. 2001;20:2768–78. - PMC - PubMed
    1. Zachary I. VEGF signalling: integration and multi-tasking in endothelial cell biology. Biochem Soc Trans. 2003;31:1171–7. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources