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J Biol Chem. Oct 22, 2010; 285(43): 33082–33091.
Published online Aug 12, 2010. doi:  10.1074/jbc.M110.142034
PMCID: PMC2963370

A New Role for PTEN in Regulating Transient Receptor Potential Canonical Channel 6-mediated Ca2+ Entry, Endothelial Permeability, and Angiogenesis*An external file that holds a picture, illustration, etc.
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Abstract

Phosphatase and tensin homologue (PTEN) is a dual lipid-protein phosphatase that catalyzes the conversion of phosphoinositol 3,4,5-triphosphate to phosphoinositol 4,5-bisphosphate and thereby inhibits PI3K-Akt-dependent cell proliferation, migration, and tumor vascularization. We have uncovered a previously unrecognized role for PTEN in regulating Ca2+ entry through transient receptor potential canonical channel 6 (TRPC6) that does not require PTEN phosphatase activity. We show that PTEN tail-domain residues 394–403 permit PTEN to associate with TRPC6. The inflammatory mediator thrombin promotes this association. Deletion of PTEN residues 394–403 prevents TRPC6 cell surface expression and Ca2+ entry. However, PTEN mutant, C124S, which lacks phosphatase activity, did not alter TRPC6 activity. Thrombin failed to increase endothelial monolayer permeability in the endothelial cells, transducing the Δ394–403 PTEN mutant. Paradoxically, we also show that thrombin failed to induce endothelial cell migration and tube formation in cells transducing the Δ394–403 PTEN mutant. Our results demonstrate that PTEN, through residues 394–403, serves as a scaffold for TRPC6, enabling cell surface expression of the channel. Ca2+ entry through TRPC6 induces an increase in endothelial permeability and directly promotes angiogenesis. Thus, PTEN is indicated to play a role beyond suppressing PI3K signaling.

Keywords: Calcium, Calcium Channels, Phosphatase, TRP Channels, Tumor Suppressor, Endothelial Barrier Function, PTEN, TRPC6

Introduction

Phosphatase and tensin homologue (PTEN),2 a dual lipid-protein phosphatase, is composed of an N-terminal phosphatase domain, a C2 domain, and a C-terminal tail domain that has a PDZ (post-synaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and zonula occludens-1 protein (ZO1)) domain binding sequence (1, 2). The phosphatase domain specifically dephosphorylates the D3 inositol head group of phosphoinositol 3,4,5-triphosphate (PIP3) leading to generation of phosphoinositol 4,5-bisphosphate (PIP2) (1, 2). Thus, through this domain PTEN negatively regulates PI3K-Akt-dependent signaling and thereby controls diverse cellular responses such as neointima formation, neutrophil migration and chemotaxis, angiogenesis, and tumor formation (3,9). PIP2 is also the key source for generating cellular diacylglycerol (DAG) and inositol triphosphate, both of which increase intracellular Ca2+ (10). DAG increases intracellular Ca2+ by directly activating plasmalemmal Ca2+ entry through receptor-operated Ca2+ (ROC) channels (11,13). Inositol triphosphate mobilizes Ca2+ from endoplasmic reticulum (ER) stores (11,14). Depletion of ER stores activates stromal interacting molecule 1 (STIM1), which induces Ca2+ entry from store-operated Ca2+ channels (15, 16). The domain structure of PTEN is consistent with the possibility that PTEN may have a role beyond acting as a phosphatase. In fact, PTEN has been shown to serve as a scaffold for MAGI (membrane-associated guanylate kinase) and Na+/H+ exchanger regulatory factor (17,20). This has been suggested to contribute to the stabilization of intercellular junctions and Ca2+ signaling (19, 20). In the present study we delineate the heretofore unknown role of PTEN in regulating endothelial Ca2+ entry and thereby its role in regulating endothelial permeability and angiogenesis.

Transient receptor potential canonical (TRPC) channels are non-selective Ca2+ channels in endothelial cells (12, 14, 21,24). TRPC6, TRPC3, and TRPC7 are activated by DAG and thereby mediate ROC entry (11,13, 25). We have shown that the TRPC6 channel is a critical determinant of endothelial cell contraction in response to permeability-increasing and pro-inflammatory mediators such as thrombin, which by binding to protease activating receptor 1 (PAR1), induces the formation of interendothelial gaps and thereby increases endothelial permeability (12, 13).

Here, we show that PTEN serves as a scaffold for TRPC6, which is essential for cell surface expression of TRPC6 enabling Ca2+ entry through the channel. This function of PTEN occurs independently of its phosphatase activity. Moreover, PTEN-induced TRPC6 activity increases endothelial permeability. Paradoxically, these findings show that PTEN regulation of TRPC6 activity is also crucial in stimulating endothelial migration and tube formation.

EXPERIMENTAL PROCEDURES

Materials

Human pulmonary arterial endothelial (HPAE) cells and endothelial growth medium (EBM-2) were obtained from Lonza (Walkerville, MD). Human α-thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). The Nucleofactor HCAEC kit and electroporation system were from Amaxa (Gaithersburg, MD). Anti-TRPC6, horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG) antibodies, and protein A/G beads were purchased from Santa Cruz Biotechnology (San Diego). Alexa-labeled 546 goat anti-rabbit secondary antibody, 4′,6′-diamidino-2-phenylindole (DAPI), and ProLong Gold antifade were from Molecular Probes (Eugene, OR). PTEN siRNA was custom synthesized from Sigma (St. Louis, MO). Scrambled siRNA with no sequence homology to human genome was purchased from Ambion, Applied Biosystems (Austin, TX). Anti-phospho-Akt (Ser-473) and anti-PTEN antibody were purchased from Cell Signaling Technology (Danvers, MA). diC8-PIP3 was purchased from Echelon (Salt Lake City, UT), and Biomol Green Reagent was purchased from Biomol (Plymouth Meeting, PA). PTEN mutants Δ394–403 PTEN (Addgene plasmid 10767), Δ354–403 PTEN (Addgene plasmid 10765), and C124S PTEN (Addgene plasmid 10744) were purchased from Addgene (Cambridge, MA).

Cell Culture

HPAE cells were cultured as described (13, 26, 27).

Cell Transfection

PTEN siRNA (5′-AACCCACCACAGCUAGAACUU-3′) (28) or scrambled siRNA (2.4 μg) were transfected in HPAE cells as described (13, 26, 27). The cells were used after 48 h transfection when there was clear evidence of PTEN suppression. For transduction of cDNA, 3.5 μg of cDNA was electroporated in HPAE cells using the Amaxa electroporation system as described (13, 26, 27). COS7 or CHO cells were transfected with indicated cDNA using Lipofectamine 2000 following the manufacturer's protocol.

Endothelial Permeability

Endothelial permeability was determined by measuring the transendothelial flux of Evans Blue-labeled albumin as described (26, 27, 29).

Immunoprecipitation

Cells lysed in immunoprecipitation assay buffer were immunoprecipitated with the appropriate Ab overnight at 4 °C followed by incubation with protein A/G-agarose for 6 h at 4 °C as described (13, 26, 27).

Phosphatase Assay

PTEN phosphatase activity was determined in 50 μl of assay buffer containing 100 mm Tris-HCl, pH 8, 10 mm DTT, and 200 μm water-soluble diC8-PIP3 as a substrate (Echelon) (30). HPAE cells were lysed after stimulation with thrombin at indicated times, and PTEN was immunoprecipitated using anti-PTEN antibody. PTEN immunoprecipitated on beads was washed twice in a low stringency buffer and once in phosphatase assay buffer lacking PIP3. Lysates immunoprecipitated with isotype-matching control antibody were used as a control. The reactions were performed for 40 min at 37 °C. The released phosphate was quantified colorimetrically using the Biomol Green Reagent (Biomol) in accordance with the instructions of the manufacturer.

Cytosolic Ca2+

An increase in intracellular Ca2+ was measured using the Ca2+-sensitive fluorescent dye Fura 2-AM as described (13).

Immunofluorescence

Cells were stimulated with thrombin for specified period of time and fixed with 2% paraformaldehyde for 20 min. The cells were then permeabilized and immunostained for PTEN or TRPC6 using appropriate primary antibodies followed by Alexa 546 secondary antibodies.

Fluorescence Resonance Energy Transfer (FRET)

HPAE cells cotransfected with CFP-tagged PTEN (donor) and YFP-tagged TRPC6 (acceptor) were stimulated with thrombin for specified times and fixed. Cells were viewed with a Zeiss LSM-510 META confocal imaging system equipped with a 50-milliwatt argon laser and 63× objective. Cells that expressed either PTEN-CFP or TRPC6-YFP were imaged with a Zeiss META detector. An image stack was generated with the 458-nm laser line spanning an emission wavelength range from 462.9 to 602 nm with bandwidths of 10.7 nm (pinhole of 1.66 Airy units and a vertical (Z) resolution of <2.0 μm). These spectra served as CFP and YFP reference emission signatures. We used them in a linear unmixing algorithm (Zeiss AIM software) to separate the fluorescence contribution of CFP and YFP (on a pixel by pixel basis) in images of cells that coexpressed PTEN-CFP and TRPC6-YFP. Prebleach CFP and YFP images were collected using the argon laser with a 458/514-nm dual dichroic. A selected region of interest on the plasma membrane was irradiated with the 514-nm laser line (100% intensity) for 55 s (200 iterations) to photobleach YFP. Postbleach images were captured immediately. FRET in the region of interest was evidenced by an increase in CFP fluorescence intensity (donor dequenching) after YFP (acceptor) photobleaching (31).

Fluorescence-activated Cell Sorting (FACS)

HPAE cells were transfected with 3.5 μg of either control vector (CFP) or CFP-tagged Δ394–403 PTEN mutant. After 24 h cells were stimulated with thrombin for 5 min, trypsinized, and washed with PBS. Cells were then incubated with 2 μg of anti-TRPC6 antibody for 20 min on ice in the dark followed by 1 μg of a Fluor 488 secondary antibody for 20 min on ice. Cell surface expression of TRPC6 was determined using Beckman Coulter Cyn II flow cytometer (32).

Endothelial Cell Migration

Motility was assessed using a Boyden chamber assay as previously described (33,35). HPAE cells (2 × 105) suspended in 500 μl of serum-free EBM-2 media were plated in the upper chamber of transwell inserts containing polycarbonate filters of 10-mm diameter and a pore size of 8 μm (Fisher). The bottom sides of these filters were coated with Matrigel (BD Biosciences). Thrombin (50 nm) was added in the bottom chamber of a 24-well plate. Cells were allowed to migrate across the filter for 9 h at 37 °C in 5% CO2 after which they were fixed on the filter with methanol. Cells that had not migrated through the filter were removed using cotton swabs. The migrated cells were stained with DAPI and mounted on coverslips (Vectashield mounting media with DAPI). Migration was quantified by counting the nuclei in 10 random microscopic fields with a florescence microscope at 20× magnification. The data represent ±S.D. of three individual experiments.

Endothelial Tube Formation

HPAE cells (5 × 104) were suspended in 500 μl of serum-free EBM-2 media with or without 10 nm thrombin on a 24-well plate pre-coated with Matrigel. After 6 h of incubation at 37 °C and 5% CO2, pictures of the vascular tube-like structures were taken under 20× magnification. The number of polygonal areas formed by these tube-like structures was counted for each field (34, 36).

Statistical Analysis

Comparisons between experimental groups were made by one-way analysis of variance and post-hoc tests. Differences in mean values were considered significant at p < 0.05.

RESULTS

PTEN Regulates Receptor-operated Ca2+ Entry

To determine the role of PTEN in regulating intracellular Ca2+ rise, we suppressed endogenous PTEN using siRNA. We observed that PTEN siRNA significantly reduced endogenous PTEN expression 48 h post-transfection (Fig. 1A, top). However, PTEN siRNA did not alter the expression of SHIP2 (Src homology 2-containing 5′-inositol phosphatase), another PIP3 phosphatase (37) (Fig. 1A, middle). Thrombin increases intracellular Ca2+ in two phases; that is, a rapid phase that is due to the release of Ca2+ from the ER stores and a slower phase representing Ca2+ entry (11, 13, 23). Both phases were present in cells transducing PTEN siRNA, although the slower phase was depressed in PTEN siRNA-transfected cells (Fig. 1B). To assess whether PTEN affects the Ca2+ release or Ca2+ entry components of Ca2+ signaling, we stimulated cells with thrombin in the absence of Ca2+ to mobilize Ca2+ from the ER stores followed by the re-addition of 2 mm Ca2+ to induce Ca2+ entry through calcium channels. We observed that the knockdown of PTEN did not alter the Ca2+ release from ER but markedly suppressed Ca2+ entry (Fig. 1C).

FIGURE 1.
PTEN regulates Ca2+ entry. A, immunoblot of siRNA-induced inhibition of PTEN expression is shown. HPAE cells were transduced with 2.4 μg of either scrambled (siSc) or PTEN siRNA (siPTEN). After 48 h cell lysates were immunoblotted with anti-PTEN, ...

We next examined whether PTEN-induced PIP2 formation is required for Ca2+ entry. Di-potassium bisperoxo (picolinato) oxovanadate (bpV(pic)) is a specific inhibitor of PTEN phosphatase activity (38, 39). We pretreated HPAE cells with 1 μm bpV(pic) and determined the effect of the inhibition of PTEN phosphatase activity on thrombin-induced Ca2+ entry. We observed that PTEN is basally active in unstimulated HPAE cells (Fig. 2A). Thrombin transiently increased PTEN lipid phosphatase activity by ~2.5-fold within 1 min, which remained elevated for 5 min. Pretreatment of HPAE cells with bpV(pic) slightly reduced basal PTEN activity (Fig. 2A). Thrombin failed to induce PTEN activity in cells pretreated with bpV(pic) (Fig. 2A). Inhibiting PTEN activity also decreased Akt phosphorylation (data not shown), which requires PIP3 as a substrate (40,42). However, inhibiting PTEN phosphatase activity had no effect on thrombin-induced Ca2+ entry (Fig. 2B). To further corroborate these findings, we transduced phosphatase activity-deficient PTEN mutant (C124S PTEN mutant) (43,45) into HPAE cells. As expected, thrombin failed to stimulate PTEN phosphatase activity in HPAE cells transducing the C124S PTEN mutant (Fig. 2C). However, C124S PTEN mutant did not alter thrombin-induced Ca2+ entry (Fig. 2D). These findings demonstrate that PTEN regulates Ca2+ entry independently of PTEN catalytic activity.

FIGURE 2.
PTEN regulates Ca2+ entry independent of lipid phosphatase activity. HPAE cells were either pretreated with 1 μm bpV(pic) for 30 min (A and B) or transduced with 3.5 μg HA-tagged phosphatase defective PTEN mutant (C124S PTEN) (C and D). ...

Thapsigargin and DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) activate Ca2+ entry independent of ligand receptor G protein-coupled receptors (11, 13, 14, 22, 23). Thus, we used thapsigargin and OAG to assess which of these Ca2+ entry mechanisms is regulated by PTEN. We observed that knockdown of PTEN had no effect on thapsigargin-induced Ca2+ entry (Fig. 3, A and C), but inhibited OAG-induced Ca2+ entry (Fig. 3, B and C). These findings identify a critical role of PTEN in regulating OAG-mediated Ca2+ entry.

FIGURE 3.
PTEN regulates ROC-mediated Ca2+ entry. HPAE cells were transduced with either scrambled (siSc) or PTEN siRNA (siPTEN). After 48 h post-transfection cells were labeled with Fura 2-AM for 15 min and then stimulated with 2 μm thapsigargin (Thap) ...

PTEN Interacts with TRPC6

Both TRPC6 and TRPC3 are constituents of ROC channels (11, 13, 23). However, TRPC6 has been shown to largely account for DAG-activated Ca2+ entry in endothelial cells (11, 13, 22, 23, 46). Consistent with our previous reports (13) we failed to detect TRPC3 expression in HPAE cells at both mRNA and protein levels (supplemental Fig. 1A). We also showed that knockdown of TRPC6 markedly suppressed OAG-induced Ca2+ entry (Fig. 4A). However, knockdown of TRPC1, another prominent TRPC expressed in human endothelial cells (11, 13, 14, 23), had no effect (Fig. 4A). These results demonstrate that TRPC6 predominantly regulates OAG-mediated Ca2+ entry in endothelial cells. Because PTEN has a PDZ binding domain sequence (1, 47), we reasoned that PTEN may regulate OAG-mediated Ca2+ entry by functioning as an adaptor for TRPC6 (47). Thus, we determined whether increased ROC activity in endothelial cells requires complex formation between PTEN and TRPC6. Lysates from endothelial cells post-thrombin challenge were immunoprecipitated with an anti-PTEN antibody and immunoblotted with an anti-TRPC6 antibody to assess complex formation. We found that TRPC6 co-immunoprecipitated with PTEN under basal conditions, and the association increased further after thrombin stimulation (Fig. 4B). PTEN failed to interact with TRPC1, TRPC3, or TRPC4, indicating that PTEN specifically interacts with TRPC6 (supplemental Fig. 1). To corroborate these findings further, we also co-expressed wild type (WT) PTEN with FLAG-tagged TRPC6 or FLAG-tagged TRPC1 in COS7 cells to determine complex formation. We observed that PTEN associated with TPRC6 but not with TRPC1 (Fig. 4C).

FIGURE 4.
PTEN associates with TRPC6. A, HPAE cells transfected with siSc, siTRPC6, or TRPC1 were loaded with Fura2 AM, and ratiometric measurements of intracellular Ca2+ were determined after stimulation with OAG. Lysates from these cells were immunoblotted ( ...

To map the domain in PTEN that mediates PTEN interaction with TRPC6, we co-transduced FLAG-tagged TRPC6 with WT-PTEN or a PTEN mutant that lacked the tail domain (Δ354–403 PTEN) in COS7 cells. We also co-transduced cells with FLAG-tagged TRPC6 and a PTEN mutant that lacked the final 10 amino acids in the tail domain (Δ394–403 PTEN), as this motif contains a PDZ domain binding sequence that is shown to mediate protein interactions (19, 20, 48). We found that WT-PTEN co-immunoprecipitated with TRPC6 (Fig. 4D). However, Δ354–403 PTEN mutant or Δ394–403 PTEN mutant failed to interact with TRPC6 (Fig. 4D), thereby identifying that 394–403 residues within PTEN are required to mediate interaction between PTEN and TRPC6.

PTEN Interaction with TRPC6 Stabilizes TRPC6 at the Endothelial Cell Surface

We next determined whether the observed association between PTEN and TRPC6 is required for cell-surface expression of TRPC6. We transduced YFP-tagged TRPC6 in HPAE cells and assessed the cellular localization of YFP-TRPC6 and endogenous PTEN before and after thrombin stimulation. We observed membrane and cytoplasmic expression of PTEN and YFP-TRPC6 under basal conditions, both of which increased markedly after thrombin stimulation (Fig. 5A). Inhibiting endogenous expression of PTEN markedly suppressed TRPC6 immunostaining at the membrane (Fig. 5B).

FIGURE 5.FIGURE 5.
PTEN maintains cell surface expression of TRPC6. A, PTEN and TRPC6 co-localize at the plasma membrane after thrombin challenge. HPAE cells transducing YFP-TRPC6 mutant were stimulated with thrombin for the indicated times, fixed, and stained with anti-PTEN ...

Next, we used FRET to detect interaction between TRPC6 and PTEN as FRET can assess interaction at a separation distance of 10 nm or less. HPAE cells were co-transfected with either YFP-TRPC6 and WT-CFP-PTEN or YFP-TRPC6 and CFP-Δ394–403 PTEN mutant. FRET was determined without or with thrombin stimulation. Fig. 5C shows a representative example of FRET after 5 min of thrombin stimulation. We observed that thrombin significantly increased FRET between YFP-TRPC6 and CFP-PTEN in a time-dependent manner (Fig. 5, C, iv-vi, and D). However, no FRET signal was observed between TRPC6 and PTEN in cells transducing Δ394–403 PTEN mutant (Fig. 5, C, vii-xii, and D). TRPC6 expression was similar in endothelial cells transducing WT-PTEN mutant or Δ394–403 PTEN mutant (Fig. 5D, inset).

We also performed FACS analysis in HPAE cells transducing Δ394–403 PTEN to determine the requirement for these residues in maintaining cell surface expression of TRPC6. Consistently, we found that thrombin increased the cell surface expression of TRPC6 in HPAE cells transducing control vector (Fig. 5E). However, thrombin failed to induce cell surface expression of TRPC6 in HPAE cells transduced with the Δ394–403 PTEN mutant (Fig. 5E). Altogether, these findings demonstrate that PTEN 394–403 residues are required for cell surface expression of TRPC6.

PTEN Association with TRPC6 Is Required for Inducing Ca2+ Entry

To address the functional role of PTEN-TRPC6 interaction in regulating TRPC6 mediated Ca2+ entry, HPAE cells transduced with Δ394–403 PTEN mutant were stimulated with OAG or thrombin. We observed that OAG failed to induce Ca2+ entry in HPAE cells transduced with the Δ354–403 PTEN mutant (Fig. 6, A and B). Similar results were obtained in cells transducing Δ394–403 PTEN mutant (Fig. 6, A and B). However, inhibition of PTEN phosphatase activity had no effect on OAG-induced Ca2+ entry (Fig. 6B). Because CHO cells express TRPC6 (49) (supplemental Fig. 1B), we also determined OAG-induced Ca2+ entry in CHO cells transducing either empty HA vector or Δ394–403 PTEN mutant. As expected, OAG increased Ca2+ entry in CHO cells transducing control vector. However, OAG failed to induce Ca2+ entry in Δ394–403 PTEN-expressing CHO cells (Fig. 6C). Δ394–403 PTEN mutant expression had no effect on PTEN phosphatase activity (basal 0.9 ± 0.01 v/s 0.84 ± 0.03; thrombin 1.54 ± 0.02 versus 1.32 ± 0.07). We also determined the effect of Δ354–403 PTEN mutant on Ca2+ entry induced after PAR1 activation by thrombin. HPAE cells transducing Δ354–403 PTEN mutant were stimulated with thrombin in the absence of Ca2+ to mobilize Ca2+ from the ER stores. This was followed by the re-addition of 2 mm Ca2+ to invoke Ca2+ entry through calcium channels. We observed that Δ354–403 PTEN mutant did not alter Ca2+ release from ER but markedly suppressed Ca2+ entry (Fig. 6D). Collectively, these results along with the findings above indicate that PTEN via residues 394–403 retains TRPC6 at the cell surface and is, therefore, crucial in maintaining TRPC6 activity.

FIGURE 6.
PTEN-induced TRPC6 cell surface expression is required for channel activity. HPAE cells (A, B, and D) or CHO cells (C) expressing control vector (HA) or indicated PTEN mutants were subjected to ratiometric measurements of intracellular Ca2+ in response ...

Role of PTEN-induced TRPC6 Activity in Regulating Endothelial Permeability and Endothelial Network Formation

Ca2+ entry plays a critical role in regulating endothelial permeability, gene transcription, and angiogenesis (12, 14, 22, 25, 50, 51). Paradoxically, PTEN, via regulating PI3K signaling, inhibits cell migration and proliferation, tumor formation, and angiogenesis (5, 30, 52,55). We, therefore, impaired PTEN and TRPC6 interaction using Δ354–403 PTEN mutant, which retains phosphatase activity, to address the specific role of PTEN-TRPC6-mediated Ca2+ entry in regulating endothelial permeability and angiogenesis. Endothelial permeability was determined by measuring the transendothelial clearance of Evans Blue albumin (26, 27, 29). We determined angiogenesis by assaying endothelial cell migration and network formation (56, 57). HPAE cells transfected with the empty vector (control), Δ354–403, or Δ394–403PTEN mutants were stimulated with thrombin. As expected, thrombin increased transendothelial albumin flux; however, thrombin did not induce albumin flux in cells transducing Δ354–403 or Δ394–403 PTEN mutants (Fig. 7A). We also found that thrombin increased endothelial cell migration and tube formation in cells transducing control vector but failed to induce these responses in cells transducing Δ394–403 PTEN mutant (Fig. 7, B and C). Thus, we have identified a novel role of PTEN residues 394–403 in triggering TRPC6-mediated Ca2+ entry, which plays a key role in regulating endothelial barrier function as well as angiogenesis.

FIGURE 7.
PTEN-mediated TRPC6 activity regulates endothelial permeability and angiogenesis. A, HPAE cells expressing control HA vector or indicated PTEN mutants were seeded on transwell plate, and transendothelial flux of Evans Blue-tagged albumin (EBA) was determined ...

DISCUSSION

Our results show that amino acids 394–403 residing in the non-catalytic domain of PTEN enable PTEN to associate with TRPC6, a well known Ca2+ entry pathway in endothelial cells activated downstream of growth factors and permeability increasing agonists (13, 51, 58, 59). We show that this association is required for cell surface expression of TRPC6, which in turn increases endothelial permeability and stimulates angiogenesis by inducing Ca2+-entry. Thus, our results indicate that PTEN, through its influence on TRPC6, has a role beyond functioning as a phosphatase for suppressing PI3K-mediated cellular responses.

PTEN dephosphorylates PIP3 to PIP2, which is catalyzed by PLC to generate DAG and inositol triphosphate for inducing an increase in intracellular Ca2+ (10, 60, 61). We showed that thrombin induced PTEN catalytic activity. We, therefore, predicted that impairing PTEN lipid producing activity would inhibit the increase in intracellular Ca2+. Knockdown of PTEN impaired Ca2+ entry after the ER store was depleted after activation of PAR1 by thrombin without altering ER Ca2+ release. Moreover, suppression of PTEN expression prevented OAG-induced Ca2+ entry but failed to affect the thapsigargin-induced Ca2+ entry, indicating that PTEN specifically regulates ROC activity. We further showed that inhibition of PTEN lipid-producing activity had no effect on thrombin-induced Ca2+ entry. These studies demonstrated that PTEN regulation of intracellular Ca2+ occurs independently of its lipid-producing activity.

DAG is the known endogenous activator of TRPC3 and TRPC6 (11,13). However, our findings show that TRPC6 predominantly regulates ROC-mediated Ca2+ entry in endothelial cells, consistent with previous reports (13, 14, 25, 51, 58, 59). We, therefore, postulated that PTEN must be required for inducing TRPC6 activity. In agreement with this assumption, we showed that inhibition of PTEN expression blocked OAG-induced Ca2+ entry. Evidence indicates that scaffold proteins such as receptor for activated protein kinase C-1 (RACK1) or Na+/H+ exchanger regulatory factor associates with TRPC (18, 62). Because PTEN catalytic activity was not required for TRPC6 activity, these results implied that a physical interaction between PTEN and TRPC6 is necessary for TRPC6 activity. Indeed, we showed that PTEN associated with TRPC6, and thrombin further stimulated this association. However, PTEN failed to interact with TRPC1, TRPC3, or TRPC4. Using COS7 cells as a heterologous expression system, we showed that WT (full-length) PTEN co-immunoprecipitated with TRPC6 but not with TRPC1, demonstrating that PTEN specifically associates with TRPC6. We mapped PTEN 394–403 residues that mediate its interaction with TRPC6. This motif contains the PDZ binding sequence (TRL) through which PTEN associates with PDZ domain-containing proteins such as MAGI-1 and -2 (19, 20, 54, 63) and Na+/H+ exchanger regulatory factor (17). However, TRPC6 does not contain a PDZ domain (18, 64). Thus, the possibility exists that additional PDZ domain-containing proteins are involved in mediating the PTEN and TRPC6 interaction. Sequence alignment software predicted homology between TRPC6 residues 145–149 and PTEN residues 394–403, raising the possibility that PTEN directly interacts with TRPC6.

Exocytosis of the TRPC6 channel to the cell surface is a known requirement for channel activity (65). We showed that knockdown of PTEN impaired TRPC6 localization at the cell membrane. Furthermore, expression of a PTEN mutant lacking amino acid residues 394–403 abrogated TRPC6 localization at the cell surface. Consistent with previous reports, we observed that deletion of 394–403 PTEN residues had no effect on PTEN phosphatase activity (2, 66). PTEN C2 and tail domains are shown to be crucial for PTEN stabilization at the membrane (1, 2, 66). A likely scenario may be that the Δ394–403 PTEN mutant competes with endogenous PTEN, thereby impairing its ability to interact with binding partners. Accordingly, we showed that the Δ394–403 PTEN mutant did not bind TRPC6 and impaired cell surface expression of TRPC6, resulting thereby in loss of thrombin and OAG-induced Ca2+ entry responses. Moreover, the Δ394–403 PTEN mutant prevented OAG-induced Ca2+ entry in CHO cells, indicating that PTEN plays a generalized role in regulating ROC-induced Ca2+ entry. Immunoblot analysis ruled out degradation of TRPC6 in the experiments, i.e. TRPC6 protein expression was the same whether or not PTEN was deleted. We conclude that PTEN by associating with the TRPC6 channel stabilizes its cell surface expression that is required for channel activity.

We showed previously that TRPC6 mediated Ca2+ entry is required for thrombin-induced cytoskeletal reorganization in endothelial cells, leading to increased endothelial permeability (13, 23, 25, 46). Herein, we showed that PTEN interacted with TRPC6 and PTEN interaction with TRPC6 was required for thrombin-induced increase in endothelial permeability. Thus, our results identify PTEN as an upstream regulator of TRPC6 and thereby in the resultant increase in endothelial permeability.

PTEN is a well known tumor suppressor (5, 52). TRPC6, on the other hand, is shown to be required for angiogenesis (67). Paradoxically, we show that PTEN residues 394–403 via regulating TRPC6 activity play an important stimulatory role in angiogenesis. These findings raise the intriguing possibility that PTEN may regulate both pathological and normal angiogenesis of vessels.

The mechanism by which thrombin stimulates PTEN translocation is not clear. Evidence indicates that Gα12/13 activates PTEN in endothelial cells (6). Thrombin induces endothelial permeability increase via PAR1-mediated activation of Gαq and Gα12/13 pathways (12, 13). Further investigation will be needed to determine the mechanism of PTEN activation in endothelial cells. In summary, we showed that thrombin activates PTEN, enabling it to interact with TRPC6 via PTEN residues 394–403. PTEN interaction with TRPC6 is essential for cell surface expression of TRPC6 and channel activity. This in turn induces endothelial barrier disruption and angiogenesis. We demonstrate that PTEN regulation of TRPC6 activity does not require PTEN phosphatase activity. Thus, our study points to the PTEN 394–403 peptide sequence as a target for developing potential therapeutic substances selectively suppressing PTEN-TRPC6 interaction and thereby limiting endothelial permeability increase and angiogenesis without affecting the PTEN phosphatase function.

Supplementary Material

Supplemental Data:

Acknowledgments

We acknowledge Drs. Asrar B. Malik and Stephen M. Vogel (University of Illinois at Chicago) for constructive criticism during progression of this work and preparation of this manuscript. We thank Drs. Koteshwara Rao Chava for help with endothelial migration and tube formation assays and Richard Minshall and Zhenlong Chen (University of Illinois at Chicago) for input with FRET analysis. We also thank Dr. William Sellers (Harvard Medical School, Boston) for the PTEN constructs and Dr. Tiffany Sharma and Mrs. Debra Salvi (University of Illinois at Chicago) for their help with YFP tagged TRPC6 and CFP-PTEN constructs.

*This work was supported, in whole or in part, by National Institutes of Health Grants HL71794 and HL84153.

An external file that holds a picture, illustration, etc.
Object name is sbox.jpgThe on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

2The abbreviations used are:

PTEN
phosphatase and tensin homolog
PAR1
protease-activated receptor-1
TRPC
transient receptor potential channel
HPAE
human pulmonary arterial endothelial cells
EBM-2
endothelial basal medium-2
OAG
oleoyl-2-acetyl-sn-glycerol
DAG
diacylglycerol
ROC
receptor-operated Ca2+
ER
endoplasmic reticulum
SHIP2
Src homology-containing 5′-inositol phosphatase
MAGI
membrane-associated guanylate kinase 1
bpV(pic)
di-potassium bisperoxo (picolinato) oxovanadate
PIP3
phosphoinositol 3,4,5-triphosphate
PIP2
phosphoinositol 4,5-bisphosphate
CFP
cyan fluorescent protein
YFP
yellow fluorescent protein.

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