Class IIb HDAC6 regulates endothelial cell migration and angiogenesis by deacetylation of cortactin
2011; Springer Nature; Volume: 30; Issue: 20 Linguagem: Inglês
10.1038/emboj.2011.298
ISSN1460-2075
AutoresDavid Kaluza, Jens Krøll, Sabine Gesierich, Tso-Pang Yao, Reinier A. Boon, Eduard Hergenreider, Marc Tjwa, Lothar Rössig, Edward Seto, Hellmut G. Augustin, Andreas M. Zeiher, Stefanie Dimmeler, Carmen Urbich,
Tópico(s)Protein Degradation and Inhibitors
ResumoArticle16 August 2011free access Source Data Class IIb HDAC6 regulates endothelial cell migration and angiogenesis by deacetylation of cortactin David Kaluza David Kaluza Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Jens Kroll Jens Kroll Department of Vascular Biology and Tumor Angiogenesis, Center for Biomedicine and Medical Technology Mannheim, Mannheim, Germany Division of Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Sabine Gesierich Sabine Gesierich Division of Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Tso-Pang Yao Tso-Pang Yao Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Reinier A Boon Reinier A Boon Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Eduard Hergenreider Eduard Hergenreider Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Marc Tjwa Marc Tjwa Institute for Transfusion Medicine, Blutspendedienst, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Lothar Rössig Lothar Rössig Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Edward Seto Edward Seto Department of Molecular Oncology, H Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA Search for more papers by this author Hellmut G Augustin Hellmut G Augustin Department of Vascular Biology and Tumor Angiogenesis, Center for Biomedicine and Medical Technology Mannheim, Mannheim, Germany Division of Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Andreas M Zeiher Andreas M Zeiher Department of Cardiology, Internal Medicine III, Frankfurt University, Frankfurt, Germany Search for more papers by this author Stefanie Dimmeler Corresponding Author Stefanie Dimmeler Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Carmen Urbich Carmen Urbich Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, University of Frankfurt, Frankfurt, Germany Search for more papers by this author David Kaluza David Kaluza Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Jens Kroll Jens Kroll Department of Vascular Biology and Tumor Angiogenesis, Center for Biomedicine and Medical Technology Mannheim, Mannheim, Germany Division of Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Sabine Gesierich Sabine Gesierich Division of Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Tso-Pang Yao Tso-Pang Yao Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Reinier A Boon Reinier A Boon Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Eduard Hergenreider Eduard Hergenreider Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Marc Tjwa Marc Tjwa Institute for Transfusion Medicine, Blutspendedienst, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Lothar Rössig Lothar Rössig Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Edward Seto Edward Seto Department of Molecular Oncology, H Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA Search for more papers by this author Hellmut G Augustin Hellmut G Augustin Department of Vascular Biology and Tumor Angiogenesis, Center for Biomedicine and Medical Technology Mannheim, Mannheim, Germany Division of Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Andreas M Zeiher Andreas M Zeiher Department of Cardiology, Internal Medicine III, Frankfurt University, Frankfurt, Germany Search for more papers by this author Stefanie Dimmeler Corresponding Author Stefanie Dimmeler Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Carmen Urbich Carmen Urbich Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, University of Frankfurt, Frankfurt, Germany Search for more papers by this author Author Information David Kaluza1, Jens Kroll2,3, Sabine Gesierich3, Tso-Pang Yao4, Reinier A Boon1, Eduard Hergenreider1, Marc Tjwa5, Lothar Rössig1, Edward Seto6, Hellmut G Augustin2,3, Andreas M Zeiher7, Stefanie Dimmeler 1 and Carmen Urbich1 1Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, University of Frankfurt, Frankfurt, Germany 2Department of Vascular Biology and Tumor Angiogenesis, Center for Biomedicine and Medical Technology Mannheim, Mannheim, Germany 3Division of Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ), Heidelberg, Germany 4Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA 5Institute for Transfusion Medicine, Blutspendedienst, University of Frankfurt, Frankfurt, Germany 6Department of Molecular Oncology, H Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA 7Department of Cardiology, Internal Medicine III, Frankfurt University, Frankfurt, Germany *Corresponding author. Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, University of Frankfurt, Theodor Stern-Kai 7, 60590 Frankfurt, Germany. Tel.: +49 69 6301 7113; Fax: +49 69 6301 7440; E-mail: [email protected] The EMBO Journal (2011)30:4142-4156https://doi.org/10.1038/emboj.2011.298 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Histone deacetylases (HDACs) deacetylate histones and non-histone proteins, thereby affecting protein activity and gene expression. The regulation and function of the cytoplasmic class IIb HDAC6 in endothelial cells (ECs) is largely unexplored. Here, we demonstrate that HDAC6 is upregulated by hypoxia and is essential for angiogenesis. Silencing of HDAC6 in ECs decreases sprouting and migration in vitro and formation of functional vascular networks in matrigel plugs in vivo. HDAC6 regulates zebrafish vessel formation, and HDAC6-deficient mice showed a reduced formation of perfused vessels in matrigel plugs. Consistently, overexpression of wild-type HDAC6 increases sprouting from spheroids. HDAC6 function requires the catalytic activity but is independent of ubiquitin binding and deacetylation of α-tubulin. Instead, we found that HDAC6 interacts with and deacetylates the actin-remodelling protein cortactin in ECs, which is essential for zebrafish vessel formation and which mediates the angiogenic effect of HDAC6. In summary, we show that HDAC6 is necessary for angiogenesis in vivo and in vitro, involving the interaction and deacetylation of cortactin that regulates EC migration and sprouting. Introduction Histone deacetylases (HDACs) are known repressors of gene transcription by deacetylating histone proteins, favouring condensation of chromatin structure (for review see Narlikar et al (2002)). Interestingly, HDACs remove acetyl groups from lysine residues of both histone and non-histone proteins, thereby affecting protein stability, activity and binding affinity as recently described (Bolden et al, 2006; Choudhary et al, 2009; Spange et al, 2009). The HDAC family consists of four classes: class I (HDAC1, 2, 3 and 8), class II (class IIa: HDAC4, 5, 7 and 9; class IIb: HDAC6 and 10), class III (sirtuin 1–7) and class IV (HDAC11). Broad-spectrum inhibitors of class I and II HDACs inhibit angiogenesis in vitro and in animal models (Kim et al, 2001; Rossig et al, 2002; Qian et al, 2006b). In addition, class I and II HDAC inhibitors abrogate endothelial differentiation of progenitor cells (Rossig et al, 2005). Because of their repressive effects on tumour-driven angiogenesis, HDAC inhibitors meanwhile represent promising anti-cancer agents in early phase clinical trials (Carew et al, 2008; Mottet and Castronovo, 2010). On the basis of these findings, recent studies addressed the specific function of individual HDAC isoenzymes for endothelial cells (ECs) and angiogenesis (Chang et al, 2006; Mottet et al, 2007; Ha et al, 2008; Martin et al, 2008; Wang et al, 2008; Urbich et al, 2009). In particular, class IIa HDAC7 is an essential regulator of embryonic blood vessel development (Chang et al, 2006). Moreover, HDAC7 controls endothelial angiogenic functions, such as tube formation, migration and proliferation in vitro (Mottet et al, 2007; Martin et al, 2008; Margariti et al, 2010). Recent studies further indicate that protein kinase D-dependent phosphorylation and nuclear export (or cytoplasmic accumulation) of HDAC5 and HDAC7 plays an important role in VEGF-induced angiogenesis in vitro (Ha et al, 2008; Wang et al, 2008). In our previous study, we identified HDAC5 as a repressor of angiogenic gene expression in EC and angiogenesis (Urbich et al, 2009). In contrast to class IIa HDACs, the role of class IIb HDACs for angiogenesis is largely unexplored. HDAC6 is localized predominantly in the cytoplasm and is the only member of the HDAC family that harbours a full duplication of its deacetylase homology region, followed by a specific ubiquitin-binding domain at the C-terminal end (Valenzuela-Fernandez et al, 2008). HDAC6 interacts with misfolded ubiquitinated proteins (Seigneurin-Berny et al, 2001; Hook et al, 2002), concentrates toxic protein aggregates and facilitates the aggresome-dependent clearance of these proteins (Kawaguchi et al, 2003; Boyault et al, 2007; Lee et al, 2010). Thus, the HDAC6-dependent clearance of misfolded proteins plays a role in the pathogenesis of neurodegenerative proteinopathies (Pandey et al, 2007). Consistent with its localization in the cytoplasm, the activities of HDAC6 are mainly independent of histones, but instead involve the deacetylation of cytoplasmic substrates such as α-tubulin, Hsp90 and cortactin (Hubbert et al, 2002; Matsuyama et al, 2002; Zhang et al, 2003, 2007; Kovacs et al, 2005). HDAC6 is a major determinant in the control of cell motility by regulation of the tubulin as well as the actin network (Gao et al, 2007; Zhang et al, 2007). HDAC6 regulates the binding of cortactin to actin in a deacetylation-dependent manner, and thereby controls the branching of the actin network at the leading edge of cells (Zhang et al, 2007). Beyond deacetylation, HDAC6 also modulates cell migration by deacetylase-independent mechanisms (Cabrero et al, 2006; Zilberman et al, 2009). Recently, it has been shown that HDAC6 and HDAC10 play an important role in Hsp-mediated regulation of VEGFR in cancer cells (Park et al, 2008). Moreover, HDAC6 associates with HIF-1α to increase its stability and transcriptional activity in cancer cells (Qian et al, 2006a). During revision of this manuscript, one study demonstrates that HDAC6 promotes angiogenesis by regulating the polarization and migration of vascular ECs in a microtubule end-binding protein 1-dependent manner (Li et al, 2011). Here, we assessed the role of HDAC6 for EC migration and sprout formation in vitro and angiogenesis in vivo, and explored the underlying mechanism involving the deacetylation of cortactin as a target of HDAC6 in ECs. Results HDAC6 is required for angiogenesis in vitro To study the role of HDAC6 for EC functions and angiogenesis, we first addressed the expression and localization of HDAC6 in EC. HDAC6 mRNA is expressed in ECs from different sources, such as human umbilical vein endothelial cells (HUVECs), microvascular endothelial cells and coronary artery endothelial cells, as well as in non-ECs such as coronary artery smooth muscle cells and cardiomyocytes (Supplementary Figure 1A). HDAC6 protein expression was detectable in ECs (Figure 1A, left panel and Supplementary Figure 1B), and HDAC6 protein is mainly localized in the cytoplasm of HUVECs (Figure 1A, right panel). To assess whether HDAC6 contributes to angiogenic functions in ECs, we performed different in vitro angiogenesis assays. Downregulation of HDAC6 in HUVECs with three independent siRNA oligonucleotides efficiently decreases HDAC6 expression, without affecting the expression of other HDACs, such as HDAC9 (Supplementary Figure 1C). Silencing of HDAC6 does not affect cell viability (Supplementary Figure 1D), but profoundly reduced sprouting in a three-dimensional spheroid assay as measured by the cumulative sprout length (Figure 1B and C), number of sprouts (Supplementary Figure 1E) and number of branch points (Supplementary Figure 1F) per spheroid. Moreover, downregulation of HDAC6 in HUVECs decreases tube formation in a matrigel assay (Supplementary Figure 1G), reduced migration of HUVECs in a scratched wound assay (Supplementary Figure 1H) and significantly inhibits cell migration in a transwell migration assay (Figure 1D). Figure 1. Knockdown of HDAC6 decreases endothelial cell sprouting and migration in vitro. (A) Western blot analysis of HDAC6 expression in cultured endothelial and non-endothelial cells (left panel). α-Tubulin serves as loading control. Immunofluorescence analysis of HDAC6 (shown in green) localization in HUVECs (right panel). Nuclei are stained with Hoechst 33342 in blue. (B) Capillary-like sprouting from spheroids was measured after HDAC6 silencing with three independent HDAC6 siRNAs compared to two different control siRNAs, untreated and vehicle (only transfection reagent)-treated cells. The spheroid assay was performed 24 h after siRNA transfection. Data are shown as mean cumulative sprout length per spheroid (*P<0.05 versus Scr I and Scr II, n=3–10). (C) Representative pictures are shown. (D) HUVECs were transfected with HDAC6 or control siRNA for 48 h, and cell migration was monitored in a transwell migration assay for 4 h under basal conditions. Migrated cells were counted by staining the nuclei with DAPI (n=4). HEK, human embryonic kidney cell. Download figure Download PowerPoint Since spheroid sprouting was most efficiently suppressed by HDAC6 silencing and sprouting angiogenesis requires the specification of the ECs to tip or stalk cells (Gerhardt et al, 2003), we determined whether HDAC6 silencing alters the position of ECs within the sprouts. In competitive spheroid assays, in which control or HDAC6-silenced HUVECs were labelled with the fluorescent proteins CFP or YFP, the HDAC6-deficient ECs showed a similar positioning in the growing sprouts compared to the control cells (Supplementary Figure 2A and B), indicating that HDAC6 depletion does not influence the ability of ECs to become tip or stalk cells. Hypoxia transcriptionally upregulates HDAC6 Because the physiological regulation of HDAC6 is largely unknown, we tested several stimuli that are known to control endothelial responses. Whereas prolonged shear stress for 72 h does not affect HDAC6 mRNA expression in HUVECs (Supplementary Figure 2C), HDAC6 mRNA expression at 24 h and HDAC6 protein expression at 36 h is increased by hypoxia (Supplementary Figure 2D–G). HDAC6 contributes to vessel formation during zebrafish embryonic development Having shown that knockdown of HDAC6 decreases in vitro angiogenesis, we determined whether HDAC6 contributes to endothelial sprouting and vessel formation in vivo. Therefore, we studied the effect of HDAC6 silencing during zebrafish embryonic development in a tg(fli1:EGFP) zebrafish line expressing EGFP in the developing vessels under the control of the fli-1 promoter. PCR of FACS-sorted EGFP+ ECs from the developing zebrafish revealed that HDAC6 mRNA is present in ECs yielding a band of 253 bp (Supplementary Figure 3A and B). Knockdown of HDAC6 was achieved by injection of morpholinos (Mos) targeting the ATG-start codon for translation-blocking (HDAC6 TB-Mo) or by injection of a splice-blocking Mo (HDAC6 SB-Mo), resulting in an aberrant splicing of HDAC6 mRNA (Figure 2A and Supplementary Figure 3C). PCR and western Blot analyses confirmed a robust silencing of HDAC6 with both Mos (Figure 2A and B). Confocal fluorescence microscopy shows defects in intersegmental vessels (ISVs) and dorsal longitudinal anastomotic vessels (DLAVs) for the HDAC6 morphants at 48 h after fertilization (Figure 2C), without other obvious morphological defects. For quantification of vessel defects, zebrafish embryos were stained against EGFP by whole-mount antibody staining (Figure 2D). Silencing of HDAC6 significantly increases the number of defects for ISVs and DLAVs for both tested Mos compared to a control Mo (Figure 2E and F). For the translation-blocking Mo, a similar increase of defects for the ISVs and the DLAVs was observed, which was dose dependent. In summary, these data indicate that HDAC6 contributes to vessel formation in zebrafish. Figure 2.Silencing of HDAC6 impairs embryonic vessel formation in zebrafish. (A) Aberrant splicing of Danio rerio HDAC6 mRNA after HDAC6 splice-blocking Mo injection by PCR. Injection of the HDAC6 SB-Mo generated at 24 h post fertilization a morphant signal of 338 bp, whereas the HDAC6 wt signal completely disappeared (253 bp), showing the functionality of the Mo. Whole-zebrafish embryo mRNA was isolated 24 h after Mo injection and subjected to RT–PCR. Actin mRNA expression serves as loading control. (B) HDAC6 protein expression was analysed in whole-zebrafish embryo lysate at 24 h after injection of HDAC6 translation-blocking or splice-blocking Mo. Protein lysates were subjected to western blotting with HDAC6-specific antibody. Actin was used as loading control. C–F phenotyping of HDAC6 morphants 48 h post fertilization. (C) Representative confocal fluorescence pictures of vessel in the anterior part of tg(fli1:EGFP) zebrafish embryos after injection of HDAC6 translation-blocking or control Mo. Arrows indicate vessel defects. (D–F) For quantification of vessel defects, HDAC6 Mo- or control Mo-treated zebrafish embryos were stained for GFP using anti-GFP antibody. (D) Representative overview pictures and higher magnification of two regions of the anterior part of control-Mo-injected and HDAC6 TB-Mo-injected embryos are shown. Arrows indicate vessel defects. (E) Quantification of defects in ISVs and DLAVs for HDAC6 and control morphants. Statistical significance was calculated for the respective Mo concentration (n=22–30). (F) Penetration of vessel defects for HDAC6 or control Mo. Numbers represent the number of animals and percentage of animals with at least one ISV or DLAV defect. DLAV, dorsal longitudinal anastomotic vessel; ISV, intersegmental vessels; PAV, parachordal vessel. Figure source data link available below. Download figure Download PowerPoint HDAC6 contributes to human vessel maturation and perfusion in vivo As a model for human vessel formation, we used an in vivo spheroid assay in mice as previously described (Alajati et al, 2008). Therefore, we transduced HUVEC with control or HDAC6 shRNA lentiviral vectors, allowing long-term silencing. Knockdown of HDAC6 was assessed by western blot (Supplementary Figure 3D), and the specificity of HDAC6 shRNA was further confirmed by demonstrating that overexpression of a non-targeting HDAC6 (HDAC6 wild-type (wt) lacking the binding site for shRNA within the 3′UTR) rescued the effect of HDAC6 silencing on sprouting in vitro (Supplementary Figure 3E). For the in vivo assay, spheroids of the transduced cells were mixed with matrigel and injected subcutaneously into mice for 3 weeks (Supplementary Figure 3F). Immunohistological analysis shows that silencing of HDAC6 in human ECs does not affect the overall microvascular density (Figure 3A and D), but significantly reduces the perfusion of the vasculature compared to control (Figure 3B and D). Furthermore, the vessels of the developing vasculature were significantly smaller in vessel size compared to control vessels (Figure 3C and D). To further assess the in vivo relevance of HDAC6, neovascularization was determined in implanted matrigel plugs in HDAC6 knockout mice. The number of perfused vessels that invaded into the implanted matrigel plugs in vivo is decreased in HDAC6 knockout mice (Figure 3E and F). Additionally, the number of invaded cells measured by H&E staining is significantly decreased (Figure 3G). Furthermore, 14 days after induction of hind limb ischaemia HDAC6 knockout mice tend to have a reduced capillary density in the thigh muscle (Figure 3H) and show a significant reduction of capillary density in the lower leg (Figure 3I) compared to wt mice. However, in contrast to zebrafish, we did not observe developmental vessel defects in HDAC6 knockout mice as assessed by the vascular outgrowth of the retina (Supplementary Figure 3G and H). Taken together, HDAC6 contributes to the formation and perfusion of mature human and mouse vessels in vivo in adult mice. Figure 3. HDAC6 contributes to human vessel maturation and perfusion in an in vivo mouse model. (A–C) Immunohistological analysis of human neovessel formation after subcutaneous injection of HUVEC spheroids in a matrigel–fibrin matrix in mice. Human vessels were stained with hCD34 antibody. (A) Quantification of microvascular density. (B) Perfusion was analysed as percentage of TRIC-lectin-positive vessels in relation to hCD34-positive vessels. (C) Average fragment size of human vessel was determined measuring the total outline surface of hCD34-positive vessels in relation to the total number of human vessels using ImageJ. (A–C) shControl, n=5; shHDAC6, n=4. *P<0.05. (D) Representative pictures of the human vasculature (shown in green) and perfusion (shown in red). Nuclei are shown in blue. (E) HDAC6 knockout mice (HDAC6−/−, n=6) and wild-type (HDAC6+/+, n=5) mice were subjected to a matrigel plug assay. Lectin-positive structures were counted manually. *P<0.05. (F) Representative images of invaded lectin-positive vessels into the matrigel plug. (G) Quantification of invaded cells into the matrigel plug by H&E staining (right panel, n=5 each). Left panel shows representative images. *P<0.05. (H, I) HDAC6 knockout mice (HDAC6−/−) and wild-type (HDAC6+/+) mice were subjected to hind limb ischaemia. Capillary density was examined histologically in the thigh (H; n=4 for HDAC6+/+ versus n=6 for HDAC6−/−) and the lower leg (I; n=5 each). Left panels show representative images of the capillaries stained for lectin and myofibres stained for laminin. Right panels show the quantification of number of capillaries per myofibre. *P<0.05. Download figure Download PowerPoint HDAC6 does not contribute to tumour vascularization in a Lewis lung carcinoma model Broad-spectrum HDAC inhibitors abrogate tumour growth, in part, by inhibition of tumour vascularization (Kim et al, 2001). Having shown that HDAC6 is required for neovascularization in adult mice, we asked whether HDAC6 might contribute to tumour vascularization. Therefore, we injected wt Lewis lung carcinoma cells subcutaneously into male HDAC6 knockout mice and control mice. Unexpectedly, male HDAC6 knockout mice show an increased tumour growth (Supplementary Figure 4A–C) compared to control mice, whereas tumour vascularization was not affected (Supplementary Figure 4D). Since HDAC6 was implicated in oestrogen signalling and breast cancer (Azuma et al, 2009), we additionally reproduced this study in female mice with similar results (Supplementary Figure 4E–I). These data suggest that in mice lacking HDAC6, the growth of subcutaneously injected Lewis lung carcinoma cells is promoted, whereas angiogenesis is not affected. HDAC6 promotes angiogenesis independently of α-tubulin deacetylation and ubiquitin binding HDAC6-dependent regulation of cell migration was attributed, in part, to its ability to interact with the tubulin cytoskeleton (Hubbert et al, 2002). The stability of a dynamic pool of tubulin fibres depends on the acetylation state of α-tubulin. Thus, deacetylated α-tubulin is less stable and facilitates migration by an increased fibre turnover (Matsuyama et al, 2002). In ECs, silencing of HDAC6 by two independent siRNAs increases the acetylation of α-tubulin (Figure 4A). To determine whether the control of angiogenesis by HDAC6 depends on its α-tubulin deacetylation activity, we used the HDAC6-specific inhibitor tubacin, which mainly blocks the α-tubulin deacetylation activity, whereas the HDAC activity is mainly unaffected (Haggarty et al, 2003). Application of 10 μM tubacin increases α-tubulin acetylation about 25-fold in HUVECs (Supplementary Figure 5A and B), but does not affect EC sprouting, migration and network formation (Supplementary Figure 5C), indicating that acetylation of α-tubulin does not directly regulate HUVEC migration. To further confirm these results and to determine whether the deacetylation activity of HDAC6 is necessary for angiogenesis, we overexpressed different HDAC6 constructs exhibiting single mutations in either one (HDAC6 H216A and HDAC6 H611A) or both deacetylation domains (HDAC6 H216/611A). The contribution of the single HDAC domains for catalytic activity is currently controversially discussed (Grozinger et al, 1999; Haggarty et al, 2003; Zhang et al, 2003; Zou et al, 2006) (for details see Supplementary Figure 5D). However, all studies consistently indicate that mutations in both HDAC domains abolish catalytic activity. Under basal conditions, overexpression of these constructs only modestly affect α-tubulin acetylation (Figure 4B), probably because of the low levels of acetylated α-tubulin in HUVECs. To increase the sensitivity of our experimental readout, we acetylated α-tubulin in HUVECs by treatment with tubacin and monitored the activity of the constructs by ELISA after washout of the inhibitor. We observed an increased α-tubulin deacetylation in HDAC6 wt and HDAC6 H216A-overexpressing cells compared to mock-transduced cells (Figure 4C and Supplementary Figure 5E), demonstrating the α-tubulin deacetylation activity of these constructs. In contrast, HDAC6 H611A- and HDAC6 H216/611A-overexpressing cells show an even higher acetylation of α-tubulin compared to mock-transduced cells (Figure 4C and Supplementary Figure 5E), indicating that these constructs exhibit no tubulin deacetylation activity and might compete with endogenous HDAC6 for α-tubulin. Under basal conditions, overexpression of HDAC6 wt increases EC migration (Figure 4D and Supplementary Figure 5F) and sprout formation (Figure 4E), whereas the deacetylation-deficient construct HDAC6 H216/611A shows no effect. Additionally, overexpression of the single-mutation constructs HDAC6 H216A and HDAC6 H611A increases endothelial sprouting, although the HDAC6 H611A mutant shows no α-tubulin deacetylation activity. In summary, these data indicate that HDAC6 regulates cell migration and angiogenesis dependent on deacetylation activity, but independent of α-tubulin deacetylation. Figure 4. HDAC6-mediated endothelial sprouting depends on catalytic activity, but is independent of α-tubulin deacetylation and ubiquitin binding. (A) Western blot of HUVEC protein lysate showing the level of acetylated α-tubulin at 72 h after transfection with two different HDAC6 siRNAs. α-Tubulin serves as loading control (n=3). (B–E) HUVECs were transduced with virus encoding HDAC6 wt and mutated HDAC6 constructs possessing single-point mutations in either one (HDAC6 H216A and HDAC6 H611A) or both deacetylation domains (HDAC6 H216/611A). As controls serve either untransduced, GFP or mock (empty pLenti4 virus)-transduced cells. (B) HUVECs were transduced with different HDAC6 constructs for 72 h. Overexpression of the constructs and level of the α-tubulin acetylation were measured by western blot. Total level of α-tubulin serves as loading control (n=3). (C) HUVEC overexpressing different HDAC6 constructs were treated for 4 h with 2.5 μM tubacin following washout of the inhibitor. Cell lysates were taken 90 min after washout, and levels of acetylated α-tubulin and total tubulin were measured by ELISA. Data are presented as the ratio of acetylated α-tubulin to total α-tubulin (n=3). (D) HUVECs were transduced with different HDAC6 constructs for 6 days, followed by scratched wound migration assay for 24 h. Data are presented as migrated distance in percentage (%) mock control (n=4). (E) HUVECs were transduced with different HDAC6 constructs for 24 h followed by spheroid assay (*P<0.05 versus untransduced control, GFP and mock control, n=3). (F–H) HUVECs were transduced with virus encoding HDAC6 wt and HDAC6 C-terminal deletion constructs. The HDAC6 Δubiquitin binding constructs lacks the ZnF-UBP domain, w
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