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* Department of Research, Cardiovascular Laboratories, Basel University Hospital, Basel, Switzerland; and
Division of Cardiology, Luzern Kantonsspital, Luzern, Switzerland
1Correspondence: Cardiovascular Laboratories, Basel University Hospital, Hebelstrasse 20, CH 4031 Basel, Switzerland. E-mail: therese-j.resink{at}unibas.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: GPI-anchored protein • signal transduction • active ß-catenin accumulation • proliferation • survival
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, restenosis after balloon angioplasty (2)
, and tumor neovascularization (3)
. Accumulating data support that T-cad participates in several processes (e.g., differentiation, migration, proliferation, and survival) that occur during vascular remodeling and angiogenesis. In vitro, homophilic ligation of T-cad on the endothelial cell (EC) surface with soluble recombinant T-cad or with agonistic antibodies induces the motile phenotype via activation of Rho and Rac pathways (4
, 5)
, facilitates cell migration (5)
, and stimulates in-gel outgrowth of endothelial sprouts in 3-dimensional EC-spheroid and heart tissue models of angiogenesis (6)
. In vivo, myoblast-mediated delivery of recombinant soluble T-cad to mouse skeletal muscle facilitates VEGF-induced angiogenesis (6)
. Moreover, T-cad expression in EC and aortic smooth muscle cells (SMC) is dynamically regulated during the cell cycle, and adenoviral-mediated overexpression of T-cad in EC and SMC promotes cell cycle progression and proliferation (7)
. Up-regulation of T-cad in EC occurs in vitro in response to oxidative stress, and adenoviral-mediated overexpression protects against oxidative stress-induced apoptosis (8)
.
Signal transduction pathways mediating the effects of T-cad on vascular cell differentiation, migration, proliferation, and survival are not well delineated. T-cad overexpression is accompanied by activation of the PI3-kinase/Akt/mTOR/p70S6 kinase axis (8)
. T-cad ligation, which in EC induces cell polarization and angiogenic switching and increases cell motility (5)
, also leads to activation of Akt (6)
. Ligation-dependent effects of T-cad on EC motility require activation of RhoA/ROCK and Rac (4)
. T-cad-promoted survival was found to be associated with concomitant PI3K-dependent activation of the PI3K/Akt/mTOR survival signal pathway and suppression of the p38 MAPK proapoptotic pathway (8)
; this might reflect a crosstalk between p38 MAPK and Akt (9)
or involvement of other upstream regulators of p38 MAPK [e.g., small GTPases (10)
and integrin-linked kinase (ILK) (11)
].
The multiple effects of T-cad on EC behavior presuppose signal pathway intersection and networking. GSK3ß is a multifunctional serine/threonine kinase that serves as a pivotal point of convergent signaling pathways in EC to control angiogenic responses, including proliferation, migration, and survival (12
, 13)
. Akt-catalyzed phosphorylation of Ser-9 on GSK3ß leads to its inhibition and in a cascade affects the functionality of diverse GSK3ß substrates, including metabolic and signaling proteins, structural proteins, and transcription factors (14
, 15)
. Several other kinases including protein kinase A, protein kinase C, and p70S6 kinase can modulate activity of GSK3ß by phosphorylation on Ser-9 (14
, 15)
. ILK, although lacking critical consensus sequences of Ser/Thr kinases in the ILK kinase domain, has also been shown to have PI3K-dependent kinase activity toward GSK3ß on Ser-9 (16
, 17)
. ILK can associate with some components of the Wnt signaling pathway (18
, 19)
. This association can modulate acute canonical Wnt signaling and ß-catenin stabilization in a PI3K-GSK3ß-independent manner (19)
. On the other hand, prolonged Wnt signaling was proposed to lead to growth factor activation of the PI3K-ILK-GSK3ß signaling axis (19)
.
How T-cad might transduce its signals to intracellular effectors is currently unknown. T-cad shares the general molecular organization of cadherin extracellular domains but lacks transmembrane and cytoplasmic domains and is attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor (20)
. Signaling by GPI-anchored proteins is thought to require their interaction with adaptor molecules within plasma membrane lipid rafts (21
, 22)
, where GPI-anchored proteins, including T-cad (23)
are preferentially localized.
In this study, we aimed to further delineate proliferation and survival signal transduction mechanisms activated by T-cad in EC and to identify a membrane proximal molecule that could mediate inward signal transmission by GPI-anchored T-cad.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was cultured in the same medium supplemented with 10% FCS.
Reagents and antibodies
PI3K inhibitor LY294002 was from Sigma-Aldrich (Buchs, Switzerland). GSK3 fusion protein was from Cell Signaling Technology Inc. (Danvers, MA, USA). Transfection of short-interfering RNA (siRNA) was carried out using siPORT Lipid [Ambion (Europe), Cambridgeshire, UK]. Protein G-Sepharose beads were from Amersham Biosciences (Little Chalfont, UK). The following antibodies were used: polyclonal antibody against the first extracellular domain of T-cad generated in our laboratory (25)
, anti-Akt, antiphospho-(Ser-473)Akt, anti-GSK3ß and antiphospho-(Ser-9)GSK3ß (Cell Signaling, New England Biolabs GMBH, Frankfurt, Germany), anti-ß-catenin, anti-active ß-catenin (ABC; Upstate Cell Signaling Solutions, Lake Placid, NY, USA), anti-ILK (clone 65.1, Sigma-Aldrich and clone 65.1.9, Upstate), anti-cyclin D1 (BD Biosciences, San Jose, CA, USA), and anti-ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Anti-rabbit and anti-mouse secondary antibodies coupled to horseradish peroxidase were from Southern Biotechnologies (BioReba AG, Reinach, Switzerland). Secondary anti-species Cy-2- and Cy3-labeled IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Amersham ECL, Pierce (Rockford, IL, USA) Enhanced Luminescence System, or Pierce SuperSignal West Dura were variously used for detection of immunoreactive proteins.
Adenoviral infection
Adenoviral vectors encoding wild-type GSK3ß (wt-GSK3ß) was a kind gift of Dr. Morris Birnbaum (University of Pennsylvania, Philadelphia, PA, USA). Catalytically inactive GSK3ß (KM-GSK3ß) and nonphosphorylatable constitutively active mutant of GSK3ß (S9A-GSK3ß) were a kind gift of Dr. Kenneth Walsh (Tufts University, Boston, MA, USA). Adenoviral vector encoding T-cad has been described previously (5
, 7)
. All constructs were amplified in HEK293 cells and purified by ultracentrifugation. Viral titers were determined as plaque-forming units (5
, 7)
. For infection, HUVEC were typically incubated with adenovirus at a multiplicity of infection (MOI) 50–100 for 12 h. Under these conditions, the transfection efficiency was >90%.
siRNA transfection
siRNA for T-cad (5'-GGACCAGUCAAUUCUAAACtt; ref. 26
) and ILK (5'-CCCGGCUCAGGAUUUUCUCtt; ref. 27
) were purchased from Microsynth (Balgach, Switzerland). Equal amounts of complementary RNA oligonucleotides were combined to a final concentration of 20 mM and annealed according to the protocol supplied by the manufacturer. RNA oligos 5'CUCUGUUCGUCCAUGCACGga and 5'AAGGUGGUUGUUUUGUUCAtt were used as negative controls for T-cad and ILK siRNA, respectively; neither of these siRNAs affected expression of either T-cad or ILK at both transcriptional and protein levels. Transfection of HUVEC with siRNA was performed using Si-PORT Lipid (Ambion) according to manufacturer’s recommendations, and unless otherwise stated, cells were analyzed 48–72 h posttransfection. The efficiency of ILK and T-cad silencing, routinely determined by immunoblotting, ranged between 70–80%.
Reporter gene analysis
Reporter plasmids for Lef/Tcf transcription factors (Bat-Lux) were a kind gift from Dr. Stefano Piccolo (University of Padua, Padua, Italy). Bat-Lux plasmid constitutes seven repeats of Tcf binding element and siamois minimal, cloned upstream of Luciferase gene in pGL3 backbone. Sixteen hours after infection of HMECs with empty and T-cad-containing adenovirus cells were co-transfected with either pGL3 or Bat-Lux constructs and pRLTK vector as an internal transfection control using Lipofectamine 2000. Luciferase activity was determined after 48 h using Dual Luciferase Assay Kit (Promega, Madison, WI, USA).
RNA extraction and reverse transcription-polymerase chain reaction
Isolation of mRNA, reverse transcription (RT), and real-time polymerase chain reaction (PCR) analysis were performed as described previously (6)
. Cyclin D1 expression was normalized to the expression of GAPDH gene. Primer sequences were as follows: 5'-CGTGGCCTCTAAGATGAAGGA-3' forward and 5'-CGGTGTAGATGCACAGCTTCTC-3' reverse for cyclin D1; 5'-TCATGACCACAGTCCATGCC-3' forward and 5'-GCCATCCACAGTCTTCTGGGT-3' reverse for GAPDH.
Cell proliferation and survival assays
Proliferation was determined by cell enumeration as described previously (7)
. Survival response to serum deprivation (6 h culture in medium containing 0.1% BSA instead of 2% FCS) was assayed using a fluorimetric caspase assay kit (homogeneous caspases assay; Roche Diagnostics GmbH, Mannheim, Germany) as detailed previously (8)
. Caspase activity, measured as arbitrary fluorescence units (au) per 2 x 104 cells, is expressed relative to activity in respectively control-transduced cells.
Immunofluorescence microscopy
Immunofluorescence techniques have been described in detail previously (4
, 28)
. Briefly, HUVEC were plated at a density of 105 cells/well onto 0.5% gelatin-precoated round 12 mm glass coverslips in 24-well plates. Unless otherwise specified stainings were performed on fixed (4% paraformaldehyde) and permeabilized (0.1% Triton X-100) cells. Cells were sequentially incubated with primary antibodies (anti-ABC, anti-ILK, or anti-T-cad) and secondary anti-species Cy-2- and Cy3-labeled IgG. For nonspecific controls, nonimmune species IgG substituted primary antibodies. Coverslips were mounted upside-down on slides using FluorSave reagent (Calbiochem, Darmstadt, Germany). Single-stained samples were studied under a Zeiss Axiophot fluorescent microscope (Zeiss), and photos were taken using a digital camera and AnalySIS software (Soft Imaging System GmbH, Munich, Germany). Double-stained samples were examined using a laser scanning confocal microscope LSM 5410 (Zeiss, Feldbach, Switzerland). Images were processed and analyzed for colocalization on an O2 Workstation (Silicon Graphics Computer Systems, Mountain View, CA, USA) using Imaris 3.0 and Colocalization Bitplanes Software (Bit Plane AG, Zurich, Switzerland). Micrographs present typical images.
Coimmunoprecipitation
Subconfluent HUVEC in 10 cm dishes were infected with empty or T-cad-containing adenovirus. After 48 h, cells were washed twice with PBS and lysed by incubation for 2 h at 4°C in buffer (0.7 ml/dish) containing 100 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1% Triton X-114, 0.2% SDS, 5 mM CaCl2, and Complete Mini Protease Inhibitor Cocktail (Roche). Lysates were centrifuged (16,000 g, 10 min, 4°C), and supernatants were precleared with mouse nonimmune IgG (50 µg/ml) overnight at 4°C. Nonimmune IgG were precipitated by being mixed with protein G-Sepharose 4 FastFlow beads (30 µl/sample) for 2 h at 4°C and centrifuged for 30 s at 16,000 g. Supernatants were incubated with anti-ILK or nonimmune IgG (as control) (15 µg/ml) overnight at 4°C after precipitation with protein G-Sepharose beads and centrifugation as described above. Supernatants were discarded, agarose beads were washed three times with 1 ml of PBS, and 80 µl of 2x Laemmli sample buffer were added to the pelleted beads. Samples were analyzed for T-cad by immunoblotting.
In vitro ILK assay
ILK pull-down was performed as described previously (16
, 17)
with minor modifications. Cell lysis was performed directly on the plates with lysis buffer [150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 50 mM HEPES, pH 7.5, and proteases mixture inhibitor (Roche)]. Equivalent lysate concentrations (determined using the Lowry method) were precleared with nonspecific IgG as above, and supernatants were immunoprecipitated with anti-ILK antibody overnight at 4°C. Protein G-Sepharose was added, and after 4 h, ILK immune complexes were collected and washed twice with lysis buffer and twice with kinase wash buffer (10 mM MgCl2, 10 mM MnCl2, 50 mM HEPES, pH 7.5, 0.1 mM sodium orthovanadate, and 1 mM dithiothreitol). The washed pellet was suspended in 25 µl kinase assay buffer (10 mM MgCl2, 10 mM MnCl2, 5 mM HEPES, pH 7.5, 1 mM sodium orthovanadate, 2 mM NaF, and 200 µM ATP), containing 2.5 µg of GSK-3 fusion protein as substrate (29)
, and incubated for 30 min at 30°C. The reaction was terminated by adding an equal volume of 2x SDS loading buffer, and phosphorylation of the substrate was detected by immunoblotting using antiphospho-Ser-9-GSK3ß.
Immunoblotting
The method of immunoblotting has been described previously (8
, 25)
. Lysis buffer was PBS containing 1% SDS, 1 mM PMSF, 2 µg/ml pepstatin, 20 µg/ml aprotinin, 30 µg/ml bacitracin, 1 mM orthovanadate, and 5 mM NaFl. Protein concentrations were determined using the Lowry method, and after appropriate dilution into Laemmli buffer, equal amounts of protein/lane were loaded for resolution by SDS-PAGE and subsequent transfer onto nitrocellulose. Proteins were immunoblotted with specific primary antibodies and detected using horseradish peroxidase-conjugated secondary antibodies and chemiluminescence. Equivalence of protein loading was routinely controlled by both Ponceau S staining and immunoblotting with anti-ß-actin. Scanned images of immunoblots were analyzed using AIDA Image or Scion (National Institutes of Health, Bethesda, MD, USA) Image software. Representative images of immunoblots are shown.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, one of the main signaling cascades modulating GSK3ß activity in mammalian cells (14
, 15)
. To examine participation of GSK3ß in T-cad-mediated signaling in HUVEC, we first determined the effect of T-cad overexpression on phosphorylation of GSK3ß on Ser-9 (pSer-9GSK3ß). Adenoviral-mediated overexpression of T-cad in HUVEC (T-cad+) results in hyperphosphorylation of GSK3ß (Fig. 1
A); this was true for both HUVEC cultured under normal growth conditions and after serum-deprivation, although in the latter case levels of pSer-9GSK3ß were markedly lower (Fig. 1A
). As reported for Akt (8)
, and included herein as experimental control, the effects of T-cad on pSer-9GSK3ß were dependent on PI3K (Fig. 1B
). Inclusion of PI3K inhibitor LY294002 under normal culture conditions reduced pSer-9GSK3ß to comparably low levels in empty-vector (E)- and T-cad (T+)-transduced cells. Sensitivity of T-cad-dependent Ser-9GSK3ß phosphorylation to PI3K inhibition indicates that the ability of T-cad to modulate GSK3ß activity is independent of canonical Wnt signaling.
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T-cad expression level affects accumulation of active ß-catenin
Because T-cad overexpression increases cell cycle progression and proliferation of HUVEC (7)
and because ß-catenin has been shown to be one of the characteristic mediators of GSK3ß actions in the nucleus, we hypothesized that T-cad might affect the activity of ß-catenin. We performed immunoblotting using anti-catenin antibodies that distinguish between total cellular ß-catenin or active (unphosphorylated) ß-catenin (30)
. Whereas total ß-catenin levels were similar between T-cad+- and E-HUVECs, levels of active ß-catenin were elevated in T-cad+-HUVEC (Fig. 2
A). Immunofluorescence microscopy using anti-active ß-catenin (ABC) revealed a higher degree of cells positive for nuclear ß-catenin in T-cad+-HUVEC as compared to E-HUVEC (35 vs. 6%; P<0.001; Fig. 2B
).
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To confirm the specificity of T-cad effects on GSK3ß phosphorylation and nuclear accumulation of active ß-catenin, we examined HUVEC in which T-cad expression was either depleted (siRNA-mediated) or overexpressed (adenoviral-mediated). Akt phosphorylation on Ser-473 (pSer-473Akt) in T-cad-depleted HUVEC was additionally evaluated since we previously examined only consequences of T-cad overexpression (8)
. In T-cad-siRNA-transduced HUVEC, levels of pSer-473Akt, pSer-9GSK3ß, and active ß-catenin were significantly reduced compared with control siRNA-transduced HUVEC (Fig. 3
A). This profile is reciprocal to that observed in T-cad-overexpressing HUVEC in which levels of pSer-9GSK3ß, active ß-catenin, and pSer-473Akt were significantly higher than in control empty-vector-transduced HUVEC (Fig. 3B
). Incubation of HUVEC with agonistic anti-T-cad antibodies against the first domain, mimicking homophilic ligation (5)
, also increased levels of pSer-473Akt, pSer-9GSK3ß and active ß-catenin (Fig. 3C
). Levels of total ß-catenin, GSK3ß, and Akt were not affected by depletion or overexpression of T-cad or by inclusion of antibodies. Taken together these data unambiguously demonstrate modulation of Akt and GSK3ß axis signaling and levels of active ß-catenin by T-cad.
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To examine the dependence of T-cad-induced active ß-catenin accumulation on GSK3ß inactivation, we compared HUVEC after transduction with T-cad-containing (T-cad+) or empty adenovectors (E) and/or with adenovectors carrying kinase mutant GSK3ß (KM), constitutively active GSK3ß (S9A), or wild-type GSK3ß (wt). Cell lysates were immunoblotted for total and active ß-catenin and for total and pSer-9GSK3ß (Fig. 4
A). Equivalent signal intensities for total GSK3ß in KM-, S9A-, or wt-transduced HUVEC indicated equivalent expression of GSK3ß protein. Compared with E-HUVEC, and as expected (13
, 31)
, levels of active ß-catenin were elevated in KM- and wt-transduced HUVEC and reduced in S9A-transduced HUVEC. Levels of active ß-catenin in T-cad+-, KM-, and wt-transduced HUVEC were comparable. In T-cad++KM and T-cad++wt cotransduced HUVEC, the levels of active ß-catenin and pSer-9GSK3ß were higher than those in singly transduced HUVEC (Fig. 4B
C). These additive effects were not present in E+KM or E+wt cotransduced HUVEC. Most importantly, cotransduction of T-cad+-HUVEC with S9A abrogated the stimulatory effects of T-cad on both pSer-9GSK3ß and active ß-catenin and (c.f., T-cad++S9A vs. T-cad+; Fig. 4B, C
). These data support that GSK3ß underlies the accumulation of active ß-catenin induced by T-cad.
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To determine whether the effect of T-cad on nuclear accumulation of active ß-catenin is functionally relevant, we performed reporter gene analysis using a BAT-lux plasmid, a ß-catenin specifically driven lymphocyte enhancer-binding protein/T cell factor (Lef/Tcf) reporter construct (32)
. T-cad- and empty-adenovector-infected HUVEC were cotransfected with BAT-lux or empty plasmids and with pRLTK plasmid constitutively expressing Renilla luciferase as internal control. Lef/Tcf promoter activity in T-cad+-HUVEC was greater than in control E-HUVEC (Fig. 5
A). To obtain additional evidence that T-cad increases ß-catenin activation of Lef/Tcf transcription factors, we investigated the expression of cyclin D1, a key transcriptional target of ß-catenin/Lef/Tcf (33)
. Immunoblotting and quantitative RT-PCR experiments demonstrated elevated levels of cyclin D1 protein (Fig. 5B
) and cyclin D1 mRNA (Fig. 5C
), respectively, in T-cad+-HUVEC.
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T-cad effects on proliferation and survival are dependent on GSK3ß inhibition
We have previously reported that T-cad up-regulation increases cell cycle progression and proliferation under normal culture conditions (7)
and also enhances survival under conditions of stress induced by serum-deprivation (8)
. Here the possible role of GSK3ß in these functional responses was investigated using HUVEC transduced with T-cad and/or the various GSK3ß vectors. Figure 6
A shows proliferation profiles of singly transduced HUVEC; whereas T-cad transduction increased proliferation (vs. empty), GSK3ß-KM or GSK3ß-wt had no effect and constitutively active GSK3ß-S9A was inhibitory. Cotransduction with both T-cad and constitutively active GSK3ß-S9A genes completely abrogated the stimulatory effects of T-cad on cell proliferation (Fig. 6B
; c.f., T-cad++S9A vs. T-cad+). Apoptosis induced by serum deprivation was similarly reduced in GSK3ß-KM-, GSK3ß-wt-, and T-cad+-HUVEC (Fig. 6C
; vs. empty). Cotransduction with both T-cad and constitutively active GSK3ß-S9A abolished the ability of T-cad to protect cells from apoptosis induced by serum deprivation (Fig. 6C
; c.f., T-cad++S9A vs. T-cad+). Proliferation and survival parameters in empty+GSK3ß-KM, empty+GSK3ß-wt, or empty+GSK3ß-S9A cotransduced HUVEC were not different than those in HUVEC singly transfected with GSK3ß-KM, GSK3ß-wt, or GSK3ß-S9A, respectively (data not shown). Taken together, these data indicate that the ability of T-cad to induce inhibition of GSK3ß is requisite for its ability to stimulate proliferation and protect from apoptosis.
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ILK is a mediator of T-cad signaling
In vitro, ILK can serve as a membrane-proximal upstream PI3K-dependent modulator of key signaling molecules crucial for cell survival, cell proliferation, and the process of angiogenesis, including Akt and GSK3ß (17
, 34
35
36)
. Since T-cad expression affects both Akt and GSK3ß activities, and in a PI3K-dependent manner, we considered the possible involvement of ILK in T-cad-induced effects.
We first examined ILK-activity in E-HUVEC and T-cad+-HUVEC by pull-down (anti-ILK) kinase assays with GSK3ß fusion protein as substrate and detection of pSer-9GSKß3 by Western blotting. Phosphorylation of GSK3ß on Ser-9 was markedly greater with ILK-immunoprecipitates from T-cad+-HUVEC than E-HUVEC; pSer-9GSKß3 was not detected when ATP was omitted from the assay buffer, thus excluding possible contribution from endogenous pSer-9GSKß3 (Fig. 7
A). The differential in ILK activity between T-cad+-HUVEC and E-HUVEC is also unlikely to be explained by any difference in levels of ILK protein, since ILK immunoreactivty in whole cell lysates was similar. These experiments indicate that T-cad can "activate" ILK.
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We next examined the consequences of ILK knock-down on downstream signaling pathways affected by T-cad. Western blot analysis revealed that transfection of HUVEC with ILK siRNA suppressed ILK expression up to 90% after 72 h compared to control siRNA transfected cells (Fig. 7B
). Silencing of ILK expression resulted in a striking reduction in levels of pSer-473Akt, pSer-9GSKß3, and active ß-catenin in both E- and T-cad+-HUVEC, and a loss of any differential in these parameters between E- and T-cad+-HUVEC (Fig. 7B
). Additionally, silencing of ILK expression abolished the ability of T-cad to protect cells from apoptosis induced by serum deprivation (Fig. 7C
). These data show that ILK mediates T-cad-induced signaling and is an essential component of the T-cad signaling cascade.
T-cad associates with ILK
Since both T-cad (23)
and ILK (37)
are located within raft membrane domains, the ILK-dependent activation of signaling by T-cad may involve some association or colocalization. To investigate this possibility, we performed coimmunoprecipitation studies on E- and T-cad+-HUVEC. T-cad was found to be specifically present in anti-ILK immune complexes and at higher levels in T-cad+-HUVEC than in E-HUVEC (Fig. 8
A). Confirmation of the putative association between ILK and T-cad by reverse immunoprecipitation with anti-T-cad antibodies was not possible since neither our own affinity purified anti-T-cad antibodies nor commercially available anti-T-cad antibodies were efficient in immunoprecipitating T-cad. Confocal microscopy was applied to further examine the association between ILK and T-cad. Immunofluorescence was performed after wounding of subconfluent monolayers in order to be able to examine protein localization in both spread and migrating cells. Two different anti-ILK antibodies (clone 65.1, Sigma-Aldrich and clone 65.1.9, Upstate) were tested. The typical staining pattern for T-cad in HUVEC, namely a global and punctuate localization over the entire cell body and an enrichment within lamellipodia of migrating cells (Fig. 8B
, middle) has been described previously (28)
. In HUVEC the staining pattern for ILK using either of the anti-ILK antibodies was mostly fibrillar over the cell body and with punctuate localization at sites of focal adhesion (Fig. 8B
, left). In our analyses, we additionally observed a pronounced enrichment of ILK within lamellipodia of migrating cells where it colocalized with T-cad (Fig. 8B
, right).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unequivocal signaling of T-cad through Akt, GSK3ß, and subsequently ß-catenin in HUVEC was demonstrated using a variety of experimental approaches, including pharmacological inhibition of PI3K, adenoviral-mediated overexpression of T-cad, and inhibition of T-cad expression by RNA interference. Collectively, the experiments show that alterations (increase or decrease) in T-cad expression in HUVEC modify (increase or decrease, respectively) the phosphorylation status of Akt and GSK3ß and the level of active ß-catenin and that the effect of T-cad on these signaling effectors is dependent on PI3K activity. Complete abolition of T-cad-induced active ß-catenin accumulation after cotransduction with constitutively active GSK3ß mutant (GSK3ß-S9A) demonstrates that the stimulatory effect of T-cad on active ß-catenin accumulation in HUVEC was dependent on GSK3ß inhibition.
Phosphorylation of Akt and stabilization and nuclear translocation of ß-catenin as a consequence of GSK3ß phosphorylation are the two main pathways that regulate cell survival and proliferation (14
, 15
, 38
39
40
41
42)
. We have previously proposed that up-regulation of T-cad under conditions of oxidative stress functions to protect HUVEC from apoptosis via enhanced survival signaling through Akt/mTOR (8)
. Here we show that transduction of HUVEC with constitutively active GSK3ß mutant (GSK3ß-S9A) completely abrogated the pro-proliferative and anti-apoptotic effects of T-cad, indicating that signal transmission via GSK3ß inactivation is also important for T-cad modulation of EC growth and survival functions.
Interestingly, when wtGSK3ß was cotransduced with T-cad the measured parameters for active ß-catenin and proliferation resembled those found in HUVEC cotransduced with T-cad and GSK3ß kinase mutant KM, indicating a similar decrease in kinase activity. This would be consistent with the high ratio of pser9GSK3ß/total level of phosphorylated GSK3ß after overexpression of wt GSK3ß. Furthermore, since parameters in the case of wtGSK3ß/empty vector-cotransduced HUVEC were not different from HUVEC transduced with only wtGSK3ß, it is possible to speculate on either an additive effect of endogenous and transduced GSK3ß phosphorylation or a higher recruitment and inactivation GSK3ß via the T-cad/ILK/Akt signal relay in T-cad overexpressing cells. Reported effects of overexpression of wt GSK3ß are variable, and it is unclear whether one can predict increased enzymatic activity (like constitutively active mutant S9A) or decreased activity (like dominant negative mutant KM) on the basis of changes in GSK3ß phosphorylation status. It has been shown that while overexpression leads to increased GSK3ß phosphorylation, parameters of GSK3ß enzymatic activity are increased (31
, 43
, 44)
. On the other hand, it has also been shown that overexpression of wt GSK3ß is accompanied by a decrease in GSK3ß phosphorylation and an increase of parameters of GSK3ß enzymatic activity (45)
. A further study did not examine phosphorylation but found functional equivalence between control and wt-GSK3ß –transfected cells (43)
. Additional discrepancies also evidently exist with respect to levels of total GSK3ß (vs. control empty vector) whereby Rossig et al., (44)
and Gong et al., (43)
reported increased levels (as expected), whereas Hashimoto et al., (31)
and Salas et al., (45)
reported no change.
That T-cad expression could affect levels of active ß-catenin was unexpected. Classical cadherins, including VE- and N-cadherin in endothelial cells, are recognized to function as modulators of ß-catenin signaling by regulating the availability of free ß-catenin for nuclear translocation (40
41
42
, 46)
. The intracellular domains of classical cadherins such as N- and VE- cadherin interact with ß-catenin, 
-catenin, and p120ctn to assemble the cytoplasmic cell adhesion complex that is critical for the formation of extracellular cell-cell adhesion. ß-Catenin and -catenin bind directly to 
-catenin, which links the cytoplasmic cell adhesion complex to the actin cytoskeleton. Disruption (via loss of cadherin) or alterations (via phosphorylation) of interactions between ß-catenin and cadherins not only alter intercellular adhesions but can also regulate levels of the availability of ß-catenin for nuclear translocation (40
41
42
, 46)
. GPI-anchored protein T-cad does not possess the necessary cytoplasmic domain to effect a direct physical association with either ß-catenin or other intracellularly located molecules (e.g., GSK3ß) that may regulate ß-catenin availability (20)
. Thus, T-cad effects on accumulation and nuclear translocation of active ß-catenin must necessarily proceed via some proximal molecular mediator that couples T-cad-induced signal transmission to intracellular effectors.
ILK is a ubiquitously expressed protein composed of four ankyrin repeats, a pleckstrin homology phospholipid-binding motif, and a C-terminal Ser/Thr kinase domain. A key function of ILK is to provide a molecular scaffold for the assembly of proteins involved in connecting integrins and growth factor receptors to the actin cytoskeleton (47
48
49
50
51
52)
. Furthermore, and although controversy exists as to whether ILK is a bona fide kinase, it can also regulate the activities of key signaling components (e.g., Akt and GSK3ß) involved in control of cell survival, cell cycle progression, cell adhesion, and ECM modification (37
, 47
48
49
, 53
54
55)
. We considered ILK as a candidate molecular mediator for T-cad for a number of reasons. ILK activity is regulated in a PI3K manner (17)
, it is an upstream regulator of Akt phosphorylation on Ser-473 (17
, 35
, 36
, 55
56
57)
and GSK3ß phosphorylation on Ser-9 (17
, 58
, 59)
, and it has been shown to regulate nuclear ß-catenin and cyclin D1 levels (58)
, all these properties being common to T-cad-induced signaling in HUVEC. Functionally, ILK has been demonstrated to be an important regulator of EC phenotype and differentiation, adhesion, survival, proliferation, and angiogenesis (27
, 34
, 35
, 57
, 60
61
62)
, processes also affected by T-cad in HUVEC (4
5
6
7
8
, 28)
. Furthermore, both ILK and T-cad are located within plasma membrane raft domains (23
, 37)
, which would facilitate the proximity necessary for ILK to serve as a mediator for T-cad. Raft domains function as assembly platforms for the dynamic formation of complexes between surface receptors, signal transduction regulators, and scaffolding proteins, and many of the molecular components regulating cell polarization, motility, adhesion, proliferation, and survival are associated with plasma membrane rafts (21
, 22)
.
Several lines of evidence in our study support that T-cad indeed utilizes ILK to elicit signaling in HUVEC. First, in vitro pull-down assay for ILK (with exogenous GSK3ß fusion protein as substrate) showed increased activity in immunoprecipitates from HUVEC overexpressing T-cad. Second, and more importantly, siRNA-mediated ILK depletion completely abolished the stimulatory effects of T-cad on Akt and GSK3ß phosphorylation and accumulation of active ß-catenin. Third, T-cad was present in anti-ILK immune complexes. Fourth, confocal microscopy demonstrated colocalization of T-cad and ILK within lamellipodial extensions of migrating cells.
A number of studies have examined the role of ILK in modulating EC function (27
, 34
, 35
, 57
, 60
61
62)
. EC-specific deletion in mice and antisense-mediated silencing in zebra fish were both shown to confer lethal vascular morphogenic insufficiency in vivo; in vitro analysis of ILK-deleted murine EC demonstrated a disruption of integrin-matrix interactions, increased apoptosis, and a reduction of phosphorylation of Akt on Ser-473 without a change in phosphorylation of GSK3ß on Ser-9 (34)
. An in vitro study using bovine aortic EC showed that siRNA-mediated silencing of ILK increased cell adhesivity to a variety of specific matrix substratum components (collagen I, vitronectin, fibronectin, and fibrinogen) but impaired cell spreading, migration, and capillary morphogenesis; these effects were attributed to destabilization of cell matrix through alterations in surface distribution of vß3 and 5ß1 integrins and were not accompanied by changes in Ser-473Akt or Ser-9GSK3ß phosphorylation (27)
. In HUVEC, ILK was shown to associate with VEGF receptor (57)
, whereas transduction with dominant negative, kinase deficient ILK, or pharmacological inhibition of ILK resulted in inhibition of a number of VEGF-induced responses, including adherence to collagen 1, phosphorylation of Ser-473Akt, migration, survival, proliferation, and capillary morphogenesis (35
, 57
, 60)
. In both HUVEC and endothelial progenitor cells, ILK plays a key role in cell survival signaling responses (Ser-473Akt or Ser-9GSK3ß phosphorylation) to anchorage deprivation, nutrient deprivation or hypoxia; furthermore, with the use of a model of mouse hind limb ischemia, ILK was demonstrated to promote endothelial progenitor cell recruitment and neovascularization of ischemic tissue (61
, 62)
. Thus, from the above, there is a growing consensus that ILK plays a key role in the complex process of vascular morphogenesis by influencing different mechanisms.
The data herein support a model (Fig. 9
) for T-cad-induced signal transduction whereby ILK serves as an essential proximal downstream regulator for T-cad to enable inward-directed signals via intracellular effectors Akt/GSKß and subsequent modulation of proliferation and survival responses. "Activation" of ILK by T-cad through proximal association is an appealing mediator paradigm for PI3K/Akt/GSK3ß axis signaling by other GPI-anchored proteins. Whether T-cad and ILK constitute part of a larger signaling complex, including other transmembrane adapter proteins and cytoplasmic signaling molecules, requires further investigations.
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ACKNOWLEDGMENTS |
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Received for publication November 21, 2007. Accepted for publication April 5, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Growth of HUVEC transduced with empty vector alone was comparable to empty/KM and empty/wt cotransduced HUVEC (individual profiles not shown; –X– in B). For caspase assay (C) subconfluent HUVEC were passaged into 96-well plates at 2 x 104cells/well after overnight transduction. After 24 h, apoptosis was induced in HUVEC by culture for 6 h under conditions of serum deprivation; caspase activity was measured using homogenous caspases assay kit (Roche Diagnostics). Data are mean ± SD (n=3); **significant difference (P<0.001) between T-cad+, and T-cad+ + S9A.
