Globotriaosylceramide Induces Lysosomal Degradation of Endothelial KCa3.1 in Fabry Disease
Objective—Globotriaosylceramide (Gb3) induces KCa3.1 downregulation in Fabry disease (FD). We investigated whether Gb3 induces KCa3.1 endocytosis and degradation.
Approach and Results—KCa3.1, especially plasma membrane–localized KCa3.1, was downregulated in both Gb3-treated mouse aortic endothelial cells (MAECs) and human umbilical vein endothelial cells. Gb3-induced KCa3.1 downregulation was prevented by lysosomal inhibitors but not by a proteosomal inhibitor. Endoplasmic reticulum stress–inducing agents did not induce KCa3.1 downregulation. Gb3 upregulated the protein levels of early endosome antigen 1 and lysosomal-associated membrane protein 2 in MAECs. Compared with MAECs from age-matched wild-type mice, those from aged α-galactosidase A (Gla)-knockout mice, an animal model of FD, showed downregulated KCa3.1 expression and upregulated early endosome antigen 1 and lysosomal-associated membrane protein 2 expression. In contrast, no significant difference was found in early endosome antigen 1 and lysosomal-associated membrane protein 2 expression between young Gla-knockout and wild-type MAECs. In aged Gla-knockout MAECs, clathrin was translocated close to the cell border and clathrin knockdown recovered KCa3.1 expression. Rab5, an effector of early endosome antigen 1, was upregulated, and Rab5 knockdown restored KCa3.1 expression, the current, and endothelium-dependent relaxation.
Conclusions —Gb3 accelerates the endocytosis and lysosomal degradation of endothelial KCa3.1 via a clathrin-dependent process, leading to endothelial dysfunction in FD.
Fabry disease (MIM 301500) is an X-linked recessive lysosomal storage disease caused by α-galactosidase A (Gla) deficiency, which leads to the accumulation of glycosphingolipids, particularly globotriaosylceramide (Gb3).1 The incidence of Fabry disease (FD) has been estimated at 1 in 40 000 to 117 000, and FD shows greater severity in males than in females.1,2 In this disease, Gb3 accumulates in excess in endothelial cells (ECs) and causes endothelial dysfunction.
See accompanying editorial on page 2
Because the endothelium influences vascular smooth muscle tone and therefore vascular diameter, endothelial dysfunction in the cardiovascular system causes perfusion defects and dysfunction of multiple organs, including the kidneys and brain, in individuals with FD.1,2
ECs release autacoids such as NO, prostaglandins, and a factor that causes relaxation through smooth muscle hyperpolarization, whereby vascular diameter is controlled. Intermediate-conductance Ca2+-activated K+ channel (KCa3.1) plays a crucial role in endothelium-dependent responses, including endothelium-dependent hyperpolarization. The impairment of this process through mutation of KCa3.1 affects endothelium-dependent control of vascular contractility, leading to a predisposition to vascular diseases including hypertension and atherosclerosis.3,4 Furthermore, we have reported that KCa3.1 downregulation contributes to endothelial dysfunction in the animal model of FD, Gla-knockout mice,5 and patients with preeclampsia.6
The density of membrane proteins in the plasma membrane is determined by a balance between forward trafficking from the endoplasmic reticulum (ER), endocytosis, and recycling/degradation.7,8 Recent studies demonstrate that various K+ channel proteins undergo endocytosis and degradation, that is, the ATP-sensitive K+ channel,9 or KCa3.1.10,11 However, K+ channel degradation has been reported in HEK cells in which K+ channels are not endogenously expressed but exogenously expressed by transfection; future studies are required to confirm these results on endogenously expressed K+ channels in native cells. Our previous study showed that excessive Gb3 impairs the endothelial expression and activity of KCa3.1, and that Gb3-induced KCa3.1 downregulation is at least in part caused by a defect in the synthesis of this channel protein.5 Furthermore, KCa3.1 downregulation may be caused by Gb3-induced degradation of the KCa3.1 protein, but this hypothesis has not been proven till date.
In the present study, using human umbilical vein ECs (HUVECs) and mouse aortic ECs (MAECs) from wild-type and Gla-knockout mice, we showed that Gb3 reduces KCa3.1 expression, especially in the plasma membrane, and that the mechanism underlying the observed decrease is lysosomal degradation of KCa3.1 via a clathrin-dependent process after Gb3 accumulation.
Materials and Methods
Materials and Methods are available in the online-only Supplement.
Exogenously Added Gb3 Decreases the Level of Plasma Membrane–Localized KCa3.1
To investigate whether exogenously added Gb3 downregulates endothelial KCa3.1, primary cultured MAECs or HUVECs were incubated in Gb3-containing culture media for 24 hours, and KCa3.1 expression was examined in the cells (Figure 1). Gb3 significantly reduced KCa3.1 expression in MAECs compared with the vehicle-treated control (Figure 1A). Next, we examined whether Gb3 also reduces KCa3.1 expression in HUVECs. KCa3.1 expression was significantly reduced in Gb3-treated HUVECs in a concentration-dependent manner (Figure 1B). Thus, endothelial KCa3.1 seems to be negatively regulated by Gb3, which is consistent with our previous result.5 We then examined the effect of Gb3 on plasma membrane–localized KCa3.1 in HUVECs (Figure 1C). KCa3.1 protein was biotinylated at the cell surface and labeled with horseradish peroxidase–conjugated streptavidin. Gb3 reduced the level of plasma membrane–localized KCa3.1 by 80.3%. In addition, immunocytochemical analysis targeting plasma membrane–localized KCa3.1 in KCa3.1-expressing HEK 293 cells12 was performed using anti-KCa3.1 serum (Figure 1D). The green fluorescence of KCa3.1 was strong along the border of vehicle-treated control cells and overlapped greatly with the red fluorescence of cell membrane, indicating that the anti-KCa3.1 serum successfully bound to the membrane-localized KCa3.1. The level of plasma membrane–localized KCa3.1 was gradually decreased by Gb3 in a time-dependent manner. These results suggest that Gb3 facilitates KCa3.1 internalization and degradation.
Exogenously Added Gb3 Induces the Lysosomal Degradation of KCa3.1
Next, we investigated whether intracellular endosomes (lysosomes and proteosomes) are involved in Gb3-induced KCa3.1 downregulation, using specific blockers for lysosomes or proteosomes (Figure 2A). The lysosomal inhibitor, bafilomycin A1 or (2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester (E64d)/pepstatin A, prevented Gb3-induced KCa3.1 downregulation, whereas the proteosomal blocker lactacystin did not. The former result by bafilomycin A1 was confirmed by immunocytochemical analysis of KCa3.1 in KCa3.1-expressing HEK 293 cells. Gb3 suppressed KCa3.1 expression, but this suppression was recovered to a large extent in cells pretreated with bafilomycin A1 (Figure 2B). Collectively, these data support the view that Gb3 induces the lysosomal degradation of KCa3.1.
The expression of plasma membrane–localized KCa3.1 may be suppressed by defective modification of protein synthesis because the accumulation of unfolded or misfolded protein in the ER induces ER stress13 followed by ER-associated degradation of the unfolded or misfolded proteins.14 To investigate whether ER-associated degradation is involved in the Gb3-induced suppression of plasma membrane–localized KCa3.1, the mRNA level of glucose-related protein 78 kDa was examined in Gb3-treated HUVECs (Figure 2C) because glucose-related protein 78 kDa is a response element for ER stress. Gb3 did not induce a significant change in the mRNA level of glucose-related protein 78 kDa, whereas the ER-stress–inducing antibiotic tunicamycin (TM) did. Furthermore, the level of phosphorylated eukaryotic initiation factor 2-α was measured in HUVECs treated with Gb3 for 24 hours (Figure 2D) because the intracellular phosphorylated eukaryotic initiation factor 2-α content increases under conditions of ER stress. Gb3 did not induce a change in the level of phosphorylated eukaryotic initiation factor 2-α, whereas the ER-stress–inducing agents TM and 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ) did. To further clarify the relevance of ER stress in KCa3.1 degradation, we examined whether TM or BHQ affects KCa3.1 expression (Figure 2E). Unlike Gb3, these agents did not affect the level of KCa3.1. These results indicate that ER stress or ER-associated degradation is not involved in KCa3.1 degradation.
Gb3 Upregulates Lysosomal-Associated Membrane Protein 2 and Early Endosome Antigen 1 Expression in MAECs
To determine whether endocytic mediators are involved in the lysosomal degradation of KCa3.1, we examined the effect of Gb3 on the levels of lysosomal-associated membrane protein 2 (LAMP2) and early endosome antigen 1 (EEA1). First, we measured the mRNA level of LAMP2 as a lysosomal marker in HUVECs (Figure IA in the online-only Data Supplement). Gb3 treatment increased the mRNA level of LAMP2 in a concentration-dependent manner. The protein levels of LAMP2 (Figure 3A) and EEA1 (Figure 3B) were also examined in MAECs exposed to Gb3 for 24 hours. Gb3 significantly upregulated the protein levels of LAMP2 and EEA1 in MAECs. These results support the view that an EEA1-enriched endosome-mediated lysosomal pathway is involved in Gb3-induced KCa3.1 degradation. Next, we examined whether similar changes as those induced by exogenous Gb3 occur in the ECs of Gla-knockout mice. The mRNA level of LAMP2 was markedly increased in Gla-knockout MAECs compared with wild-type MAECs (Figure IB in the online-only Data Supplement). We then compared the expression levels of EEA1, LAMP2, and KCa3.1 in MAECs (Figure 3C and 3D) and aortas (Figure IIA in the online-only Data Supplement) from aged Gla-knockout and age-matched wild-type mice. The protein levels of LAMP2 and EEA1 were markedly increased in MAECs and aortas from the Gla-knockout mice compared with those from wild-type mice. In contrast, KCa3.1 expression was markedly decreased in aortas from the Gla-knockout mice compared with those from wild-type mice (Figure IIA in the online-only Data Supplement, top). In addition, we examined KCa3.1 expression along with that of LAMP2 (Figure 3E) or EEA1 (Figure 3F) using immunocytochemical analysis. An inverse relationship between KCa3.1 expression and LAMP2/EEA1 expression was found. LAMP2 fluorescence was mainly found in the cytosol. Thus, the LAMP2 and KCa3.1 fluorescence signals overlapped in the cytosol (Figure 3E, right, boxed area). On the contrary, KCa3.1 fluorescence overlapped greatly with that of EEA1 (Figure 3F), especially in Gla-knockout MAECs, indicating the colocalization of these proteins. In aged Gla-knockout MAECs, the green fluorescence faded away along the cell border, whereas spots of dense green and red fluorescence appeared in the cytosol (Figure 3F, right, boxed area). In addition, immunohistochemical analysis targeting KCa3.1, EEA1, and LAMP2 was performed in aortic tissues from 36-week-old Gla-knockout and age-matched wild-type mice (Figure IIB and IIC in the online-only Data Supplement). The fluorescence signal of KCa3.1 was weak in aortic tissues from the Gla-knockout mice, whereas it was strong in those from wild-type mice (Figure IIB in the online-only Data Supplement). In contrast, the fluorescence signals of LAMP2 (Figure IIB in the online-only Data Supplement) and EEA1 (Figure IIC in the online-only Data Supplement) seem much stronger in tissues from the Gla-knockout mice than in aortic tissues from wild-type mice. These data indicate that KCa3.1 expression is inversely related to EEA1 and LAMP2 expression. Collectively, these results confirm that changes in EEA1, LAMP2, and KCa3.1 expression similar to those induced by exogenous Gb3 occur in the ECs of aged Gla-knockout mice, and suggest that plasma membrane–localized KCa3.1 was transported into the cytosol via an EEA1-enriched endosome-mediated process.
We then examined LAMP2 (Figure IIIA in the online-only Data Supplement) and EEA1 (Figure IIIB in the online-only Data Supplement) expression in MAECs exposed to Gb3 for 2 hours. In contrast to the 24-hour exposure, the 2-hour exposure did not upregulate LAMP2 and EEA1, suggesting that the EEA1-enriched endosome-mediated process does not occur during the 2-hour exposure. In addition, no significant differences were found in the protein levels of EEA1 (Figure IIIC in the online-only Data Supplement) and LAMP2 (Figure IIID in the online-only Data Supplement) between MAECs from 4-week-old Gla-knockout and age-matched wild-type mice. These results suggest that the changes in the expression levels of EEA1, LAMP2, and KCa3.1 develop via an age-dependent process, and that Gb3 accumulation leads to KCa3.1 degradation and EEA1/LAMP2 upregulation in ECs.
Role of Clathrin and Rab5 in Gb3-Induced KCa3.1 Degradation
We examined whether clathrin is involved in Gb3-induced KCa3.1 degradation. The intracellular clathrin level was slightly increased in Gb3-treated (Figure 4A) and aged Gla-knockout (Figure 4C) MAECs, but the difference was not significant. Because clathrin heavy chain plays an essential role in the endocytotic pathway,15 we inhibited clathrin by transfecting with siRNA against the clathrin heavy chain and examined whether clathrin knockdown affects KCa3.1 expression.
No significant change in KCa3.1 expression was found with clathrin knockdown in Gb3-untreated wild-type MAECs (Figure 4B), although the transfection significantly decreased clathrin expression (Figure 4A). In contrast, in aged Gla-knockout MAECs, clathrin knockdown decreased clathrin expression and recovered the decreased KCa3.1 expression (Figure 4C).
In addition, clathrin knockdown recovered KCa3.1 expression in wild-type MAECs treated with Gb3 for 24 hours (data not shown). Furthermore, KCa3.1 and clathrin were labeled with anti-KCa3.1 serum and anticlathrin antibody to determine the distribution of KCa3.1 in relation to clathrin (Figure 4D). Along the cell margin in wild-type MAECs, the green fluorescence of KCa3.1 was strong, whereas the red fluorescence of clathrin was not found (Figure 4D, upper, boxed area), suggesting that clathrin does not colocalize with KCa3.1 in this region. In contrast, in aged Gla-knockout MAECs, the green fluorescence overlapped with the red fluorescence along the cell margin, suggesting that clathrin colocalized with KCa3.1 in this region (Figure 4D, middle, boxed area). On the contrary, in aged Gla-knockout MAECs transfected with siRNA against clathrin (Figure 4D, lower), the red fluorescence signal was markedly decreased, whereas the green fluorescence signal was markedly increased, suggesting an inverse relationship between KCa3.1 and clathrin. These results suggest that Gb3 induces the translocation of clathrin close to the plasma membrane, and that plasma membrane–localized KCa3.1 is internalized via a clathrin-dependent process.
The small GTPase Rab5, whose effector protein is EEA1,16 is known to play a rate-limiting role in membrane docking or fusion in the early endocytic pathway.17 We thus examined the mRNA level of Rab5 in MAECs. Compared with age-matched wild-type MAECs, aged Gla-knockout MAECs showed a significantly higher mRNA level of Rab5 (Figure 5A). In addition, the mRNA level of Rab5 was increased by Gb3 treatment, whereas those of Rab2, Rab4, Rab9, and Rab18 were not increased by the treatment (Figure IIIE in the online-only Data Supplement). Rab5 has 3 isoforms—Rab5A, Rab5B, and Rab5C—of which Rab5C is the most engaged in the early endosomes.18 Therefore, we examined whether Rab5C is important for Gb3-induced KCa3.1 degradation in MAECs. First, we examined whether Rab5C knockdown affects KCa3.1 expression in Gb3-untreated wild-type MAECs. No significant change in KCa3.1 expression was found in MAECs by treatment with siRNA against Rab5C (Figure 5D). Then, we examined the effect of Rab5C knockdown on KCa3.1 expression in Gb3-treated MAECs (Figure 5B). Gb3 treatment hampered KCa3.1 expression but enhanced Rab5C expression in MAECs. In contrast, in MAECs transfected with siRNA against Rab5C, Gb3 treatment did not induce Rab5 upregulation and KCa3.1 downregulation. Furthermore, changes similar to those shown in Gb3-treated MAECs were found in MAECs (Figure 5C) and aortas (Figure IVA in the online-only Data Supplement) from aged Gla-knockout mice. MAECs and aortas from aged Gla-knockout mice showed upregulated Rab5C and downregulated KCa3.1 compared with those from age-matched wild-type mice. When the Gla-knockout MAECs and aortas were transfected with siRNA against Rab5C, KCa3.1 and Rab5C expression was reversed to the levels in age-matched wild-type MAECs, that is, the control levels. Furthermore, downregulated KCa3.1 and upregulated Rab5C were shown in fibroblasts from a patient with FD compared with those from a healthy control (Figure IVB in the online-only Data Supplement). However, siRNA against Rab5C did not reverse downregulated pERK and activator protein-1, upregulated repressor element-1 silencing transcription factor, downregulated class III phosphoinositide 3-kinase, and decreased phosphatidylinositol 3-phosphate, in aged Gla-knockout MAECs (Figure V in the online-only Data Supplement), suggesting that reduced KCa3.1 synthesis and the channel activity by Gb35 are not recovered by Rab5C knockdown. These results suggest that Rab5 is necessary for the induction of Gb3-induced KCa3.1 degradation in Gb3-treated and aged Gla-knockout ECs.
Gb3-Induced Endothelial Dysfunction Is Recovered by Inhibition of Rab5
In our previous study, we reported that the KCa3.1 current and endothelium-dependent relaxation (EDR) are decreased in Gb3-treated or aged Gla-knockout MAECs and aortic rings, respectively.5 Thus, we investigated whether KCa3.1 recovery by Rab5C knockdown leads to the recovery of the KCa3.1 current in aged Gla-knockout MAECs (Figure 6A) and in EDR of aortic ring segments from aged Gla-knockout mice (Figure 6B). Compared with age-matched wild-type MAECs, aged Gla-knockout MAECs showed markedly reduced KCa3.1 current. However, this decreased current was restored by transfection with Rab5C siRNA in aged Gla-knockout MAECs. Then, we examined the effect of Rab5C knockdown on EDR (Figure 6B and 6C). Compared with aortic rings from age-matched wild-type mice, those from aged Gla-knockout mice showed markedly attenuated acetylcholine (ACh)–induced EDR. However, ACh-induced EDR was significantly improved by transfection with siRNA against Rab5C in aged Gla-knockout aortic ring segments. In contrast, sodium nitroprusside–induced relaxation was not changed by Rab5C knockdown in endothelium-denuded aortic rings (Figure 6C, right), indicating that Gb3 and Rab5C knockdown do not affect NO-induced relaxation of vascular smooth muscle cells. These results suggest that KCa3.1 recovery by Rab5C knockdown leads to the recovery of Gb3-induced endothelial dysfunction.
The results of our study showed that the glycosphingolipid Gb3 modulates KCa3.1 expression in HUVECs and MAECs via clathrin-dependent and EEA1-enriched endosome-mediated lysosomal degradation (Figure VI in the online-only Data Supplement). Specifically, inhibition of Gb3-induced KCa3.1 degradation by clathrin knockdown suggested that the KCa3.1 protein is internalized by Gb3 via a clathrin-dependent process. Furthermore, Gb3-induced KCa3.1 degradation was prevented by the lysosome blockers, suggesting that Gb3 induces lysosomal degradation of KCa3.1. To our knowledge, these findings represent the first direct evidence for the mechanism of Gb3-induced KCa3.1 degradation, which may be implicated in the endothelial dysfunction observed in FD.
To prove the hypothesis that accumulated Gb3 induces endothelial KCa3.1 degradation in Gla-knockout mice, we measured age-dependent changes in EEA1, LAMP2, and KCa3.1 expression in ECs from Gla-knockout mice and compared them with those induced by exogenously added Gb3. We found similar changes in the expression of KCa3.1, EEA1, Rab5, and LAMP2 consistently in both aged Gla-knockout and Gb3-treated wild-type MAECs. This finding indicated that the mechanisms for the regulation of KCa3.1 internalization and degradation were similar in these sets of MAECs and that the changes in EEA1, LAMP2, and KCa3.1 expression in MAECs from aged Gla-knockout mice are caused by accumulated Gb3. The plasma concentration of Gb3 ranges from 6.7 to 15.4 μmol/L in patients with classic FD,19 and the concentration used in this study (≤15 μmol/L) was within this range. Moreover, Gb3 induced similar responses in the human ECs HUVECs. Therefore, we suggest that accumulated Gb3 is adequate to induce endothelial KCa3.1 degradation in Gla-knockout mice and subjects with FD.
KCa3.1 degradation has been reported in cells in which KCa3.1 was not endogenously expressed but exogenously expressed by transfection. The exogenously expressed KCa3.1 proteins were rapidly endocytosed from the plasma membrane to endosomes10 and degraded in lysosomes.11 However, the regulation of the internalization and degradation/recycling of the endogenous KCa3.1 in native cells has not been studied yet. To our knowledge, this is the first study that provides evidence of the inducible downregulation of endogenous KCa3.1 in native ECs.
Because clathrin knockdown prevented KCa3.1 degradation, KCa3.1 internalization might occur via a clathrin-dependent mechanism, which is consistent with a previous result in migrating transformed renal epithelial Mardin-Darby canine kidney-focus cells.20 Although Gb3 did not increase intracellular clathrin content, it may increase KCa3.1 internalization by affecting the intracellular distribution of clathrin. Clathrin was found to have translocated close to the plasma membrane in aged Gla-knockout MAECs in which EEA1 expression was markedly enhanced. Therefore, KCa3.1 internalization and transportation to early endosomes might be facilitated by the interaction of clathrin with EEA1. Rab5 also seems necessary for Gb3-induced KCa3.1 degradation. Rab5 is mainly localized to the cytosolic face of the plasma membrane, clathrin-coated vesicles, and early endosomes. It regulates the formation of clathrin-coated vesicles at the plasma membrane,21 and their subsequent fusion with early endosomes.22 On the contrary, EEA1 has been reported to provide directionality to vesicular transport from the plasma membrane to the early endosomes.23 Thus, an increase in the expression levels of these 2 molecules by Gb3 might control intracellular vesicle traffic from the plasma membrane to early endosomes, which explains the mechanism of Gb3-induced transport of KCa3.1 to the early endosomes. Collectively, Gb3 might induce KCa3.1 internalization via a clathrin-dependent mechanism, after which the KCa3.1 protein is targeted to early endosomes characterized by the molecular markers EEA1 and Rab5.
The inverse relationship between KCa3.1 expression and EEA1/LAMP2 expression and the colocalization of endocytosed KCa3.1 and EEA1 indicated that early endosomes and lysosomes are involved in Gb3-induced KCa3.1 degradation. Gb3 treatment in the presence of lysosomal inhibitors prevented the decrease in KCa3.1 protein, confirming the involvement of lysosomes in Gb3-induced KCa3.1 degradation. In contrast, Gb3 treatment in the presence of a proteosomal inhibitor failed to prevent the decrease in KCa3.1 protein, suggesting that proteosomes are not involved in KCa3.1 degradation. In addition, ER-associated degradation might be not involved in Gb3-induced KCa3.1 degradation because the level of glucose-related protein 78 kDa and phosphorylated eukaryotic initiation factor 2-α did not increase after treatment with Gb3, and ER-stress–inducing reagents did not affect KCa3.1 expression. These results conclusively indicate that the internalized KCa3.1 proteins are transported to lysosomes and degraded there, consistent with the results of Balut et al.11,24
The expression levels of EEA1 and LAMP2 did not increase after Gb3 treatment for 2 hours but did after treatment for 24 hours. Therefore, it takes >2 hours to evoke Gb3-induced KCa3.1 degradation, a result that is consistent with those of the study performed by Gao et al,10 who showed that the levels of exogenously expressed KCa3.1 protein initially decreased slowly ≤6 hours and then rapidly between 6 and 12 hours.
The amount of KCa3.1 was not changed by clathrin or Rab5C knockdown in Gb3-untreated wild-type MAECs, suggesting that the amount of newly synthesized KCa3.1 protein is too small to detect using Western blotting in MAECs where KCa3.1 internalization is not facilitated by Gb3. There are ≥3 possibilities in this finding. First, in native ECs, KCa3.1 internalization and degradation might occur slowly and thus the increase in the amount of KCa3.1 on inhibiting KCa3.1 internalization and degradation might be too small to detect. Second, almost all of the internalized KCa3.1 is recycled; therefore, new protein synthesis is not necessary to maintain the level of KCa3.1 in plasma membrane. Third, the process of KCa3.1 internalization and degradation might rapidly affect the synthesis process, and thus the inhibition of the internalization and degradation process might rapidly inhibit the synthesis process. However, further studies are required to clarify the processes of KCa3.1 recycling, degradation, and synthesis in native ECs in physiological conditions.
Abnormalities in arterial blood flow were reported in patients with FD.25 The abnormalities might at least partially be explained by Gb3-induced impairment of endothelium-dependent vasodilation. Endothelial dysfunction leads to a decrease in the endothelium-dependent vasodilating response of blood vessels, and endothelial KCa3.1 dysfunction contributes to impaired EDR25 because KCa3.1 activation induces endothelium-dependent hyperpolarization of vascular smooth muscle and increases the Ca2+ driving force in ECs. An increase in the Ca2+ driving force increases Ca2+ influx and thereby NO synthesis by activating Ca2+-dependent endothelial NO synthase. Furthermore, KCa3.1 recovery by Rab5C knockdown leads to the recovery of Gb3-induced endothelial dysfunction (Figure 6). Thus, Gb3-induced KCa3.1 degradation might explain the mechanism underlying endothelial dysfunction in aged Gla-knockout mice, a mouse model of FD, and in FD itself.
Our findings have potential clinical implications. We identified clathrin and Rab5C as a critical component in KCa3.1 degradation. Inhibiting Rab5 with siRNA against Rab5C has demonstrated to improve endothelial function by inhibiting KCa3.1 degradation, suggesting that siRNA against Rab5C can be used to treat FD. The present findings suggest that plasma membrane–localized KCa3.1 is targeted for lysosomal degradation via clathrin-dependent and EEA1-enriched endosome-mediated processes, and the processes for the degradation of endothelial KCa3.1 are caused by accumulated Gb3. To the best of our knowledge, this is the first study to show the mechanisms underlying the degradation of endogenous KCa3.1 in freshly cultured ECs. The identification of KCa3.1 as a target for Gb3 raises important questions about the contribution of this interaction to the progression of FD.
Sources of Funding
This research was supported by Basic Science Research Program through the Nation Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R01-2010-000-10466-0) and by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2010-220-E00001).
Globotriaosylceramide induces endothelial KCa3.1 downregulation in FD, but little is known about the underlying mechanisms of the globotriaosylceramide-induced downregulation. Here, we report that the endocytosis and lysosomal degradation of endothelial KCa3.1 is accelerated in globotriaosylceramide-treated ECs or in mouse aortic endothelial cells from aged α-galactosidase A–knockout mice, suggesting that KCa3.1 degradation might be implicated in the process of FD. Surprisingly, Rab5 knockdown using siRNA against Rab5C recovered KCa3.1 expression, the current, and acetylcholine-induced endothelium-dependent relaxation in α-galactosidase A–knockout mouse aortic endothelial cells, suggesting that globotriaosylceramide-induced endothelial dysfunction might be recovered by Rab5 knockdown. Our study suggests that endothelial KCa3.1 is targeted for lysosomal degradation via clathrin-dependent and early endosome antigen 1–enriched endosome-mediated processes. This is the first study to show the mechanisms underlying the degradation of endogenous KCa3.1 in freshly cultured endothelial cells, and that endothelial KCa3.1 is a therapeutic target for FD.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.302200/-/DC1.
- Nonstandard Abbreviations and Acronyms
- bafilomycin A1
- endothelial cell
- endothelium-dependent relaxation
- early endosome antigen 1
- endoplasmic reticulum
- α-galactosidase A
- human umbilical vein endothelial cell
- Ca2+-activated K+ channel
- lysosomal-associated membrane protein 2
- mouse aortic endothelial cells
- Received July 12, 2013.
- Accepted October 9, 2013.
- © 2013 American Heart Association, Inc.
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