Klotho is associated with VEGF receptor-2 and the transient receptor potential canonical-1 Ca²⁺ channel to maintain endothelial integrity

Abstract

Klotho is a circulating protein, and Klotho deficiency disturbs endothelial integrity, but the molecular mechanism is not fully clarified. We report that vascular endothelium in Klotho-deficient mice showed hyperpermeability with increased apoptosis and down-regulation of vascular endothelial (VE)-cadherin because of an increase in VEGF-mediated internal calcium concentration ([Ca²⁺]i) influx and hyperactivation of Ca²⁺-dependent proteases. Immunohistochemical analysis, the pull-down assay using Klotho-fixed agarose, and FRET confocal imaging confirmed that Klotho protein binds directly to VEGF receptor 2 (VEGFR-2) and endothelial, transient-receptor potential canonical Ca²⁺ channel 1 (TRPC-1) and strengthens the association to promote their cointernalization. An in vitro mutagenesis study revealed that the second hydrolase domain of Klotho interacts with sixth and seventh Ig domains of VEGFR-2 and the third extracellular loop of TRPC-1. In Klotho-deficient endothelial cells, VEGF-mediated internalization of the VEGFR-2/TRPC-1 complex was impaired, and surface TRPC-1 expression increased 2.2-fold; these effects were reversed by supplementation of Klotho protein. VEGF-mediated elevation of [Ca²⁺]i was sustained at higher levels in an extracellular Ca²⁺-dependent manner, and normalization of TRCP-1 expression restored the abnormal [Ca²⁺]i handling. These findings provide evidence that Klotho protein is associated with VEGFR-2/TRPC-1 in causing cointernalization, thus regulating TRPC-1–mediated Ca²⁺ entry to maintain endothelial integrity.

The Klotho gene was identified by insertion mutagenesis in mice (1). The homogenous mutant mouse exhibited a phenotype of accelerated aging, including extensive vascular calcification with hyperphosphatemia. Klotho is abundantly expressed in the kidney and, to a lesser extent, in other organs, including the aorta (1). Interestingly, patients with chronic renal failure have low concentrations of Klotho protein in the serum (2) and vascular calcification with hyperphosphatemia, suggesting that a decrease in circulating Klotho may contribute to vascular lesions in the patients with chronic renal failure.

We previously reported that Klotho expression is induced by statin and attenuated by angiotensin II through the regulation of Ras homolog gene family, member A (3). VEGF-mediated angiogenesis is impaired in Klotho-deficient mice, in which reduced release of endothelial NO was reported (4). Klotho gene delivery was shown to improve endothelial dysfunction through an NO-dependent pathway (5) and to extend the survival of rats with glomerulonephritis (6) or angiotensin II-induced renal failure (7). Thus, Klotho is likely to be a kidney-derived vasoprotective protein.

The molecular function of Klotho has been partly deciphered. Klotho can act as a glycosidase (8) that hydrolyses sugar residues on the epithelial Ca²⁺ channel, the transient receptor potential (TRP) vanilloid receptor-related channel TRPV5 (9). Furthermore, Klotho binds directly to multiple FGF receptors (FGFRs), to FGF23 (10), and also to insulin receptor (11) but not to EGF or PDGF receptors (11), and the Klotho/FGF23 complex binds to FGFR1 with higher affinity than FGF23 and significantly enhances the ability of FGF23 to induce FGFR phosphorylation (10). However, the action of Klotho on VEGF receptor (VEGFR)-mediated signals remains to be determined. Ca²⁺ signals regulate various biological functions in endothelial cells (ECs), such as proliferation, migration, and apoptosis (12). Upon ligand binding to receptors on ECs, the internal calcium concentration ([Ca²⁺]i) increases via inositol triphosphate-mediated Ca²⁺ release from the endoplasmic reticulum (ER), causing subsequent Ca²⁺ entry triggered by the depletion of Ca²⁺ stores in the ER (12). One of the types of ion channel participating in store depletion-operated Ca²⁺ entry is the TRP family, which is divided further into four subfamilies, TRPV, TRPC (canonical or classical), TRPM (melastatin-related), and TRPP (protein kinase D subtype) (13). The TRPC family consists of seven isoforms, each of which shows specific cellular distribution and functions (13, 14). TRPC-1, TRPC-3, TRPC-4, and TRPC-6 are expressed in the ECs, and these subtypes are closely involved in various vascular functions: TRPC-1 in VEGF-mediated Ca²⁺ entry (13–15), TRPC-4 in hypoxia-induced vascular remodeling, and TRPC-3 and TRPC-4 in oxidative stress-induced responses (16). The association with Klotho protein has not yet been studied.

In the present study, we examined whether Klotho protein maintains endothelial integrity in association with VEGFR and endothelial TRPC-1. We report that vascular endothelium in Klotho-deficient mice is hyperpermeable because of enhanced apoptosis and decreased surface expression of vascular endothelial (VE)-cadherin, and Ca²⁺-dependent calpain/caspase-3 are hyperactivated. Klotho binds directly to both VEGFR-2 and TRPC-1, but not to other TRPC subtypes, and the complex is internalized in response to VEGF stimulation, thus regulating VEGFR/TRPC-1–mediated Ca²⁺ influx to maintain endothelial biological homeostasis.

Results

Vascular Hyperpermeability and Increased Apoptosis in Klotho-Deficient Aorta.

To study the endothelial function in Klotho-deficient mice, we first evaluated the vascular apoptosis. TUNEL staining of the aorta showed that CD31⁺ ECs and alpha-smooth muscle actin (αSMA)-positive vascular smooth muscle cells (VSMCs) already were TUNEL-positive in 4-wk-old Klotho-deficient mice in which vascular calcification was not apparent but were barely detectable in WT littermates (Fig. 1A).

Fig. 1.

Endothelial apoptosis and hyperpermeability. (A) Immunostaining of the aorta with TUNEL, anti-CD31, and anti-αSMA antibodies. Double-immunofluorescent cells are indicated by arrows. Images are representative of similar results observed in 4-wk-old mice (n = 7). (B) Evans Blue dye was injected 10 min before 2-wk-old mice were killed (n = 7). The aorta was excised longitudinally (Left), and cross-sectioned aorta was stained with H&E (Right). Hyperpermeabilized areas (dark blue, arrows) were measured with computed morphometry (n = 7). (Scale bars: 500 μm.) K^−/−, Klotho-deficient mice.

Serum concentrations of Ca²⁺ and phosphate are elevated in Klotho-deficient mice (1). We hypothesized that Klotho deficiency may cause endothelial hyperpermeability, leading to exudation of Ca²⁺/phosphate-rich plasma into the vessel wall. To evaluate vascular permeability, Evans Blue dye was administered to 2-wk-old mice 10 min before they were killed. In the aorta of WT mice, dark blue-stained hyperpermeable lesions were barely detectable on the luminal surface of longitudinally excised aorta (Fig. 1B, Left) and in the intimal region (Fig.1B, Right); such lesions were patchily detected in the aorta of Klotho-deficient mice.

Increase in Calpain/Caspase-3 Activities and Acceleration of Apoptosis.

Hyperactivation of calpain causes damage to renal epithelial cells in Klotho-deficient mice (17). We observed that calpain and caspase-3 activities against exogenous substrate in the aorta from Klotho-deficient mice were 130% and 35% higher, respectively, than in WT mice (Fig. 2A). Calpain activities were evaluated further by determining the endogenous level of αII-spectrin, a cleaved substrate fragment that is highly sensitive to calpain (17). αII-spectrin is cleaved at particular site by calpain, yielding a 136-kDa fragment (N-terminal cleaved products) and a 148-kDa fragment (C-terminal cleaved products). Because αII-spectrin is a plasma membrane-bound cytoskeletal protein, membrane fraction was isolated from the aorta and analyzed by SDS/PAGE and Western blotting using an anti-αII-spectrin antibody reactive to the 148-kDa cleaved fragment and to full-length αII-spectrin. The relative intensity of the 148-kDa cleaved fragment to full-length αII spectrin was 4.1-fold higher in the Klotho-deficient aorta than in WT aorta (P < 0.005; n = 5) (Fig. 2B).

Fig. 2.

VEGF-mediated apoptosis of Klotho-deficient ECs through Ca²⁺/calpain/caspase-3. (A) Calpain and caspase-3 activity was measured in the aortas of 4-wk-old mice (n = 5). (B) Membrane fraction of the aorta of 4-wk-old mice was subjected to Western blot analysis using anti-αII-spectrin antibody that recognizes the C-terminal lesion of the calpain-cleaved (148 kDa) and full-length (288 kDa) αII-spectrin. The relative intensity of the cleaved to the full-length αII-spectrin was evaluated (n = 5, *P < 0.005 vs. WT). (C and D) ECs were stimulated with VEGF (100 ng/mL) for 30 min in medium containing 1.5 mM or 0 mM Ca²⁺. Calpain/caspase-3 activities (C) and TUNEL-positive apoptotic EC (arrowheads) (D) were evaluated. *P < 0.05 and **P < 0.01 vs. baseline (n = 10). (Scale bar: 50 μm.)

Although basal calpain and caspase-3 activities did not differ significantly between WT and Klotho-deficient ECs, the activities after VEGF stimulation were markedly higher in Klotho-deficient ECs (1.8-fold and 3.2-fold, respectively) than in WT ECs (Fig. 2C). In Ca²⁺-free medium, no significant increase was observed in VEGF-induced calpain and caspase-3 activities in either WT or Klotho-deficient ECs (Fig. 2C).

Calpain is activated by an increase in [Ca²⁺]i, suggesting that regulation of [Ca²⁺]i may be altered in Klotho-deficient cells. We therefore examined Ca²⁺-mediated apoptosis in Klotho-deficient ECs (Fig. 2D). Basal numbers of TUNEL-positive ECs did not differ significantly between WT and Klotho-deficient ECs. The number of apoptotic cells increased markedly in Klotho-deficient ECs (5.3-fold, P < 0.05) 3 h after VEGF stimulation, whereas no change was observed in WT ECs. In Ca²⁺-free medium, no significant increase was observed in either group.

Hyperactivated Calpain Degrades p120 Catenin, Leading to Internalization and Proteolysis of VE-Cadherin.

VE-cadherin plays a crucial role in maintaining endothelial integrity (18). We observed that VEGF stimulation significantly reduced the VE-cadherin level in Klotho-deficient ECs (Fig. 3A). VE-cadherin was localized on the plasma membrane in both groups, but the expression level was significantly lower in Klotho-deficient ECs after VEGF stimulation (Fig. 3A). We therefore examined the mechanism for VEGF-mediated down-regulation of VE-cadherin. Treatment of Klotho-deficient ECs with the μ-calpain inhibitor acetyl-leucyl-leucyl-norleucinal (ALLN) (20 μM) blocked the VEGF-mediated decrease in VE-cadherin expression toward the WT level (Fig. 3A), suggesting that μ-calpain is involved in the mechanism down-regulating VE-cadherin. Therefore, we immunoprecipitated VE-cadherin from WT EC lysates and incubated it with purified μ-calpain; however, VE-cadherin protein was not cleaved (Fig. 3B). It has been reported that p120-catenin (p120^ctn) associated with VE-cadherin prevents internalization and subsequent lysosomal degradation of VE-cadherin (19). We confirmed that p120^ctn is associated with VE-cadherin in WT ECs (Fig. 3B) and found that incubation of immunoprecipitated p120^ctn with μ-calpain markedly cleaved the p120^ctn (76% reduction) (Fig. 3B). Although basal p120^ctn levels are similar in WT and Klotho-deficient ECs, VEGF treatment caused a significant decrease in p120^ctn expression in Klotho-deficient ECs (52% lower than in WT ECs; P < 0.01), but pretreatment with ALLN blocked the VEGF-mediated decrease (Fig. 3C). The expression of p120^ctn was localized mainly on the cell surface in both WT and Klotho-deficient ECs. VEGF stimulation markedly decreased surface expression of p120^ctn in Klotho-deficient ECs but not in WT ECs (Fig. 3C).

Fig. 3.

Calpain-mediated proteolysis of p120^ctn induces internalization and degradation of VE-cadherin. (A, C, and E) ECs were stimulated with VEGF (100 ng/mL) for 30 min in the presence or absence of calpain inhibitor (ALLN, 20 μm). Cell lysates were analyzed by Western blotting with antibodies against VE-cadherin, p120^ctn, or α-tubulin. Cells also were immunostained with anti-VE-cadherin or anti-p120^ctn antibodies (arrowheads). (Scale bars: 5 μm.) Cell lysates were immunoprecipitated and analyzed by Western blotting with anti-p120^ctn or anti-VE-cadherin antibodies (C) and VE-cadherin, VEGFR-2m, and TRPC-1 (E). *P < 0.01 vs. WT baseline. (B) VE-cadherin and p120^ctn were immunoprecipitated from EC lysates. Immunocomplexes were incubated with purified μ-calpain (Calbiochem), final concentration 0.1 U/mL, under 100 μM of Ca²⁺ for 60 min at 37 °C and then were analyzed by Western blotting. *P < 0.01 and **P < 0.005 vs. baseline. (D) ECs were incubated with VEGF, ALLN, or chlorpromazine, an inhibitor for internalization (5 μg/mL). The plasma membrane fraction was isolated as described in SI Materials and Methods and analyzed by Western blotting with antibodies against VE-cadherin, p120^ctn, β1-integrin, EEA-1 (the early endosomal marker), or MPR (the late endosomal marker). *P < 0.01 vs. WT baseline.

To evaluate p120^ctn/VE-cadherin expressions on plasma membrane quantitatively, we isolated plasma membrane fractions containing no early endosomal antigen 1 (EEA), the early endosomal marker protein, or mannose-6-phospate receptor (MPR), the late endosomal marker proteins (Fig. 3E, Right). The expression levels of VE-cadherin and 120^ctn after VEGF simulation were 60% and 76% lower, respectively, in Klotho-deficient ECs than in WT ECs, although baseline levels in the two groups were similar (Fig. 3D). Pretreating Klotho-deficient ECs with ALLN blocked the VEGF-mediated decrease in VE-cadherin and p120^ctn expression. An inhibitor of internalization (chlorpromazine, 5 μg/mL) also inhibited the VEGF-mediated decrease in VE-cadherin but did not affect p120^ctn (Fig. 3D). Taken together, these data suggest that hyperactivated calpain in Klotho-deficient ECs causes p120^ctn degradation, leading to enhanced internalization of VE-cadherin. The association between VE-cadherin and VEGFR-2/Klotho was barely detectable (Fig. 3E), suggesting that VE-cadherin is not cointernalized with VEGFR-2/Klotho. Furthermore, siRNA-mediated TRPC-1 knockdown in Klotho-deficient ECs blocked VEGF-mediated decreases in p120^ctn and VE-cadherin expression (Fig. S1B), indicating that TRPC-1–mediated Ca²⁺ entry is involved in VEGF-mediated down-regulation of p120^ctn/VE-cadherin.

Direct Interactions Among Klotho, VEGFR-2, and TRPC-1.

The TRPC-1, TRPC-3, TRPC-4, and TRPC-6 members of the TRP family are expressed in ECs (13, 14), and TRPC-1 promotes VEGF-mediated Ca²⁺ entry, leading to increase vascular permeability (14). To evaluate interactions among Klotho, VEGFR-2, and TRPCs, we overexpressed Klotho by Klotho gene recombinant adenovirus (3 × 10⁷ pfu) in human umbilical vein endothelial cell (HUVECs) (Fig. S2A) and then stimulated the HUVECs with VEGF for 30 min. (We used HUVECs, rather than WT ECs, in this experiment to achieve more efficient infection.) As shown in Fig. 4A, Klotho bound constitutively to TRPC-1 and VEGFR-2, and VEGF stimulation did not affect the association, whereas Klotho was not associated with TRPC-3, TRPC-4, or TRPC-6, although Homer-1, a positive control interactor (20), bound to all TRPCs (Fig. S2A).

Fig. 4.

Klotho binds to VEGFR-2 and TRPC1. (A) HUVECs were infected with the Klotho gene or LacZ gene recombinant adenovirus (3 × 10⁷ pfu). Forty-eight hours after infection, cells were stimulated with VEGF (100 ng/mL) for 30 min, and the lysates were immunoprecipitated, followed by Western blotting with antibodies against VEGFR-2 or Klotho. (B) His-tag–fused, Klotho-bound agarose was incubated with HUVEC-derived lysates, followed by Western blotting with anti-VEGFR-2 or TRPC-1 antibody. HUVEC lysates also were analyzed by Western blotting.

To confirm the interactions among Klotho, VEGFR-2, and TRPC-1, we overexpressed histidine (His)-tagged Klotho protein in COS cells and extracted the Klotho protein from cell lysates by fixing it to Ni-nitrilotriacetate (Ni-NTA)-affinity agarose beads (Fig. S2B). Then lysates from HUVEC were incubated with Klotho-fixed agarose. Protein bound to this agarose was analyzed by Western blotting. The result showed that both TRPC-1 and VEGFR-2 were specifically detectable in the Klotho-fixed agarose (Fig. 4B).

FRET microscopy is an ideal technique to highlight in vivo protein–protein interactions. To obtain the FRET signal between Klotho and VEGFR-2 or TRPC-1, EYFP was fused to the C terminus of Klotho, and ECFP was fused to the C terminus of VEGFR-2 and TRPC-1. Recombinant cDNA plasmids coding Klotho-EYFP/VEGFR-2-ECFP or Klotho-EYFP/TRPC-1-ECFP were coexpressed in 293 HEK cells. Klotho was colocalized with VEGFR-2 or TRPC-1, and FRET signals were obtained between EYFP-Klotho and ECFP-VEGFR-2 or ECFP-TRPC-1, whereas coexpression of ECFP and EYFP-Klotho protein did not produce a FRET signal (Fig. 3C). Furthermore, no FRET signal was detected in cells in which only ECFP-TRPC-1 or only ECFP-VEGFR-2 was overexpressed (Fig. 3D), indicating that Klotho actually binds to TRPC-1 or VEGFR-2 in the living cells.

Klotho Strengthens Association Between VEGFR-2 and TRPC-1 and Promotes VEGF-Mediated Internalization of the VEGFR-2/TRPC-1 Complex.

We studied the association between VEGFR-2 and TRPC-1 by an immunoprecipitation experiment. The result showed that there is a very weak association between VEGFR-2 and TRPC-1 in the WT ECs, and this association was attenuated in Klotho-deficient ECs (Fig. 5A). Addition of Klotho markedly promoted the association of VEGFR-2 with TRPC-1 in both WT and Klotho-deficient ECs (Fig. 5A). Because Klotho mRNA is detectable in the steady state of WT ECs (Fig. 5B), it is likely that endogenously released Klotho induces the basal weak association of VEGFR-2 and TRPC-1 in the WT ECs. These findings suggested that Klotho induces a cross-link between VEGFR-2 and TRPC-1, forming a ternary complex. Because the size of the intracellular region of Klotho is too small to interact with other proteins (1), the extracellular domain of Klotho (secreted form) may bind to VEGFR-2/TRPC-1. We obtained the secreted form of Klotho from conditioned medium (CM) of NIH 3T3-cells infected with Klotho gene recombinant adenovirus (3 × 10⁷pfu) (Fig. S2C). The concentration of secreted Klotho in the CM was ∼250 ng/mL, and it was added to the EC with 25% volume (vol/vol). The addition of Klotho protein-rich CM to VEGF-stimulated ECs markedly strengthened the association (3.3-fold, P < 0.01) in both WT and Klotho-deficient ECs (Fig. 5A).

Fig. 5.

Klotho enhances VEGF-mediated internalization of TRPC-1 and VEGFR-2. (A and C) ECs were incubated with VEGF (100 g/mL) for 30 min. In some samples, Klotho-rich CM (final concentration of Klotho protein ∼250 ng/mL) was added 12 h before VEGF treatment. (A) Cell lysates were analyzed with immunoprecipitation, followed by Western blotting with anti-TRPC-1 or anti-VEGFR-2 antibodies. (C) Cells were immunostained with anti-VEGFR-2 antibody, followed by FITC-conjugated secondary antibody and Alexa Fluor 568-preconjugated anti-TRPC-1 antibody. White arrowheads and yellow arrows indicate VEGFR-2/TRPC-1 localized on the cell surface and in the cytoplasm, respectively. (B) siRNA for Klotho or nonsilencing (Cont.) RNA (50 nM final concentration) were introduced into ECs, and then cells were stimulated with VEGF (100 g/mL) for 30 min. Total RNA was analyzed by real-time PCR to measure the level of Klotho mRNA. ECs also were immunoprecipitated and followed by Western blotting with anti-VEGFR-2 or anti-TRPC-1. *P < 0.005.

Furthermore, TRPC-1 and VEGFR-2 were colocalized on the cell surface of WT ECs. The addition of VEGF to WT ECs caused the marked internalization of TRPC-1 and VEGFR-2, whereas such internalization was barely detected in Klotho-deficient ECs (Fig. 5C). Total protein contents of TRPC-1 and VEGFR-2 were not changed in ECs of either genotype at baseline or after VEGF stimulation (Fig. S4), indicating a lack of internalization of TRPC-1 and VEGFR-2 in Klotho-deficient ECs. The addition of Klotho protein-rich CM to VEGF-stimulated, Klotho-deficient ECs restored the lack of VEGF-mediated internalization of TRPC1 and VEGFR-2 to the normal level (Fig. 5C), but the addition of Klotho protein-rich CM to VEGF-stimulated WT ECs did not affect the internalization patterns of TRPC-1 and VEGFR-2 (Fig. 5C).

Determination of the Binding Site of Klotho and VEGFR-2/TRPC-1.

We determined the domains responsible for interactions between Klotho and VEGFR-2 or TRPC-1. Recombinant cDNA plasmids of Klotho mutants deleting hydrolase domains 1 or 2 (ΔHy1 or ΔHy2) were coexpressed with full-length cDNA plasmids of VEGF-R2 or TRPC-1 to HEK293 cells. We found that the ΔHy2 deletion mutant failed to bind to TRPC-1 or VEGFR-2 (Fig. 5A). Also, cDNA plasmids of VEGF-R2 mutants deleting Ig domains (ΔIg1–7, ΔIg1–4, ΔIg5–7, ΔIg5-6, ΔIg6-7, ΔIg7) (Fig. 5B) or TRPC-1 mutant plasmids deleting N-terminal loop (L) domains (ΔL 1-2, ΔL 1–3, ΔL 1–4, ΔL 1–5) (Fig. 5C) were cotransfected with full-length Klotho cDNA. VEGFR-2 mutants lacking the sixth and seventh domains of Ig (ΔIg1–7, ΔIg5–7, ΔIg6-7) or TRPC-1 mutants lacking the third extracellular loop (ΔL1–5) did not bind to Klotho. To confirm that these domains are responsible for association, the cDNA plasmid of Klotho-Hy-2 (Klotho-Hy2) was overexpressed in HEK293 cells, and cell lysates were incubated with GST-fused VEGF-R2-Ig6/7, with GST-fused TRPC-1-L5-domains, or with GST control fixed to glutathione Sepharose beads. Protein bound to the beads was analyzed by Western blotting. The result showed that Klotho-Hy2 bound directly to both GST-fused proteins but did not bind to control GST protein (Fig. 5D). Furthermore, we found that in 293 HEK cells coexpressing the EYFP-fused Klotho-Hy2 and ECFP-fused VEGF-R2-Ig6/7 or ECFP-fused TRPC-1-L5 domains, Klotho-Hy-2 was colocalized with VEGFR-2-Ig6/7 and TRPC-1-L5, and FRET signals were obtained between EYFP-Klotho-Hy2 and ECFP-VEGFR-2-Ig6/7 or ECFP-TRPC-1-L5 (Fig. S6 A and B). These findings indicate that the second hydrolase domain of Klotho interacts directly with the sixth/seventh Ig domains of VEGFR-2 or the fifth loop (third extracellular loop) domain of TRPC-1 (Fig. S5E).

Sustained [Ca²⁺]i Elevation After EGF Stimulation in Klotho-Deficient Cells.

Enhanced expression of TRPC-1 on the plasma membrane because of the lack of internalization may augment Ca²⁺ influx in Klotho-deficient ECs. We therefore evaluated [Ca²⁺]i in Fura-2–loaded aortic ECs. One minute after stimulation with VEGF in buffer containing 1.5 mM Ca²⁺, [Ca²⁺]i increased to a peak (2.1-fold elevation from the baseline) in WT ECs, followed by a prompt decrease toward the baseline. By contrast, in Klotho-deficient ECs, after reaching a peak comparable to that in WT ECs, the elevation of [Ca²⁺]i was more sustained and did not return to the baseline level even after 10 min. However, the sustained elevation of [Ca²⁺]i in Klotho-deficient ECs was abolished in Ca²⁺-free buffer (Fig. 6A).

Fig. 6.

Sustained Ca²⁺ elevation through TRPC-1 in Klotho-deficient ECs. (A) Fura-2–loaded ECs were stimulated with VEGF (100 ng/mL) in buffer containing 1.5 mM Ca²⁺ (1.5 mM) or Ca²⁺-free buffer. (B) Fura-2–loaded ECs were incubated with thapsigargin in Ca²⁺-free buffer and then were exposed to 1.5 mM Ca²⁺. Averaged [Ca²⁺]i is presented in the graph. *P < 0.05 vs. WT ECs at the same time points. (C) Klotho^−/− ECs were introduced with siRNA against TRPC-1 or nonsilencing (control) RNA. SiRNA-incorporated cells were identified by cotransfection with Alexa Fluor red fluorescent oligo-RNA (Left, arrowheads). They were fura-2–loaded, and the VEGF (100 ng/mL)-induced increase in [Ca²⁺]i was evaluated. Representative data are shown (Right) (n = 6).

Sustained [Ca²⁺]i elevation after a sharp peak is mediated through a store-operated Ca²⁺ channel including TRPCs (15). Therefore, ECs were treated with thapsigargin (1 μM) in Ca²⁺-free medium to deplete the intracellular Ca²⁺ store and thereafter were exposed to 1.5 mM Ca²⁺ to induce store-operated Ca²⁺ current. We observed that the elevation of [Ca²⁺]i apparently was more prolonged in Klotho-deficient ECs than in WT ECs (Fig. 6B). To confirm that the exaggerated Ca²⁺ influx in Klotho-deficient cells is mediated by TRPC-1, we knocked down TRPC-1 by siRNA (78% reduction in protein level). In most siRNA-treated cells, TRPC-1 expression on the plasma membrane was barely detectable (Fig. S1A). We labeled siRNA-transfected cells with Alexa Fluor red fluorescent oligo-RNA to test the effect of TRPC-1 inhibition on VEGF-mediated Ca²⁺ influx (Fig. 6C). VEGF-induced peak [Ca²⁺]i levels were markedly reduced (62 ± 2% reduction vs. control), and the subsequent [Ca²⁺]i elevation was restored to the baseline level, whereas ATP (1 mM)-induced [Ca²⁺]i levels were similar in siRNA and control cells. Furthermore, siRNA-mediated TRPC-1 knockdown in Klotho-deficient ECs restored the VEGF-mediated decrease in p120^ctn/VE-cadherin expression to the WT level (Fig. S1B) and significantly decreased the activities of caspase 3 and calpain (51% and 57% reduction, respectively; P < 0.01) (Fig. S1C) and the number of apoptotic cells (69% reduction; P < 0.01) (Fig. S1D). Thus, enhanced Ca²⁺ influx via TRPC-1 activated calpain/caspase-3/apoptosis in Klotho-deficient ECs.

Discussion

The present study demonstrates that the disruption of endothelial integrity in Klotho-deficient mice results from endothelial hyperpermeability caused by abnormal Ca²⁺ handling. We also provide evidence that VEGF-mediated Ca²⁺ signaling is tightly regulated by interaction with Klotho and that Klotho plays an important role in VEGF-mediated vascular action as well as in the maintenance of endothelial function. Furthermore, Klotho strengthens the association between VEGFR-2 and TRPC-1 and causes cointernalization of VEGFR-2 and TRPC-1, thus regulating the expression level of TRPC-1 on the plasma membrane. In contrast, a lack of Klotho results in the impairment of TRPC-1 internalization, leading to increased expression of TRPC-1 that enhances Ca²⁺ influx. We also found that calpain hyperactivated by increased Ca²⁺ influx cleaves p120^ctn, leading to subsequent down-regulation of VE-cadherin in Klotho-deficient ECs. These findings suggest that Klotho deficiency causes a sustained increase in [Ca²⁺]i and hyperactivity of Ca²⁺-dependent proteases, resulting in vascular hyperpermeability caused by endothelial damage and, eventually, extensive vascular calcification.

Klotho was shown to bind to receptor-type tyrosine kinases, including FGFRs (10), although no interaction was reported in receptors for EGF or PDGF (11). Klotho protein also was associated with an epithelial TRP family Ca²⁺ channel, TRPV5 (9) and with Na⁺/K⁺-ATPase (21). The present study demonstrates the direct association of Klotho with both VEGFR-2 and an endothelial major TRP family Ca²⁺ channel, TRPC-1. To transmit the VEGF-mediated Ca²⁺ signals effectively, VEGFR-2 and TRPC-1 need to be associated in the plasma membrane. Indeed, our mutagenesis analyses showed that the second hydrolase domain of Klotho interacts directly with the extracellular sixth/seventh Ig domains of VEGFR-2 and the fifth loop (third extracellular loop) domain of TRPC-1 (Fig. 6E). ECs endogenously released Klotho, which constitutively induced a basal cross-link between VEGFR-2 and TRPC-1, forming a ternary complex (Fig. 5), suggesting that Klotho is constitutively associated with TRPC-1 and VEGFR-2 and effectively transmits the physiological VEGF signal to TRPC-1 that causes Ca²⁺ influx.

We found that VSMCs as well as ECs in the aorta from Klotho-deficient mice showed apoptotic changes, although VEGFR-2 is expressed predominantly in endothelium. The mechanism(s) by which Klotho promotes VSMC apoptosis through VEGFR-2 remain to be determined. It has been reported that endothelium-derived NO has an antiproliferative influence on VSMCs, and the release of endothelial NO is impaired in Klotho-deficient mice (5). Given that VSMCs in proliferative condition are more vulnerable to apoptosis (22), a decrease in endothelial NO in Klotho-deficient ECs might cause VSMC apoptosis.

In conclusion, the present study presents evidence that Klotho interacts directly with the extracellular domain of VEGFR-2 and TRPC-1 to regulate VEGF-mediated Ca²⁺ entry and the hyperactivity of Ca²⁺-dependent proteases, thus maintaining endothelial integrity. Klotho deficiency causes a sustained increase in intracellular [Ca²⁺]i, resulting in vascular hyperpermeability caused by endothelial apoptosis or loss of endothelial integrity and, eventually, extensive vascular calcification. Klotho expression is reduced in patients with diabetes mellitus or renal failure (2), and the circulating level of Klotho might be involved in the development of vascular damage in patients with these diseases. Klotho might be a therapeutic target for preventing cardiovascular disease that complicates various diseases such as chronic kidney disease.

Materials and Methods

Materials, animal experiments, primary culture of endothelial cells, measurement of intracellular calcium concentration, RT-PCR, TUNEL staining assay, measurements of caspase-3 and calpain activity, in vitro proteolysis of VE-cadherin and p120ctn by purified calpain, permeability of thoracic aorta, immunocytochemistry, adenovirus-mediated gene transfer, Western blotting, knockdown of TRPC-1 and Klotho, construct of deletion mutants, binding assays, FRET analysis, and statistical data analysis are described in SI Materials and Methods. Four-week-old male Klotho homozygous mutant mice and their wild-type littermates (C57Bl/6 strain) were purchased from Nihon Clea Co., Ltd (Shizuoka, Japan).