Seonghee Park

Globotriaosylceramide leads to KCa3.1 channel dysfunction: a new insight into endothelial dysfunction in Fabry disease

AIMS Excessive endothelial globotriaosylceramide (Gb3) accumulation is associated with endothelial dysfunction and impaired endothelium-dependent relaxation in Fabry disease. In endothelial cells, K(Ca)3.1 channels contribute to endothelium-dependent relaxation. However, the effect of Gb3 on K(Ca)3.1 channels and the underlying mechanisms of Gb3-induced dysfunction are unknown. Herein, we hypothesized that Gb3 accumulation induces K(Ca)3.1 channel dysfunction and aimed to clarify the underlying mechanisms. METHODS AND RESULTS The animal model of Fabry disease, α-galactosidase A (Gla) knockout mice, displayed age-dependent K(Ca)3.1 channel dysfunction. K(Ca)3.1 current and the channel expression were significantly reduced in mouse aortic endothelial cells (MAECs) of aged Gla knockout mice, whereas they were not changed in MAECs of wild-type and young Gla knockout mice. In addition, K(Ca)3.1 current and the channel expression were concentration-dependently reduced in Gb3-treated MAECs. In both Gb3-treated and aged Gla knockout MAECs, extracellular signal-regulated kinase (ERK) and activator protein-1 (AP-1) were down-regulated and repressor element-1 silencing transcription factor (REST) was up-regulated. Gb3 inhibited class III phosphoinositide 3-kinase and decreased intracellular levels of phosphatidylinositol 3-phosphate [PI(3)P]. In addition, endothelium-dependent relaxation was significantly attenuated in Gb3-treated mouse aortic rings. CONCLUSION Gb3 accumulation reduces K(Ca)3.1 channel expression by down-regulating ERK and AP-1 and up-regulating REST and the channel activity by decreasing intracellular levels of PI(3)P. Gb3 thereby evokes K(Ca)3.1 channel dysfunction, and the channel dysfunction in vascular endothelial cells may contribute to vasculopathy in Fabry disease.

Mechanism and Synergism in Epithelial Fluid and Electrolyte Secretion

A central function of epithelia is the control of the volume and electrolyte composition of bodily fluids through vectorial transport of electrolytes and the obligatory H2O. In exocrine glands, fluid and electrolyte secretion is carried out by both acinar and duct cells, with the portion of fluid secreted by each cell type varying among glands. All acinar cells secrete isotonic, plasma-like fluid, while the duct determines the final electrolyte composition of the fluid by absorbing most of the Cl− and secreting HCO3−. The key transporters mediating acinar fluid and electrolyte secretion are the basolateral Na+/K+ /2Cl− cotransporter, the luminal Ca2+-activated Cl− channel ANO1 and basolateral and luminal Ca2+-activated K+ channels. Ductal fluid and HCO3− secretion are mediated by the basolateral membrane Na+-HCO3− cotransporter NBCe1-B and the luminal membrane Cl−/HCO3− exchanger slc26a6 and the Cl− channel CFTR. The function of the transporters is regulated by multiple inputs, which in the duct include major regulation by the WNK/SPAK pathway that inhibit secretion and the IRBIT/PP1 pathway that antagonize the effects of the WNK/SPAK pathway to both stimulate and coordinate the secretion. The function of these regulatory pathways in secretory glands acinar cells is yet to be examined. An important concept in biology is synergism among signaling pathways to generate the final physiological response that ensures regulation with high fidelity and guards against cell toxicity. While synergism is observed in all epithelial functions, the molecular mechanism mediating the synergism is not known. Recent work reveals a central role for IRBIT as a third messenger that integrates and synergizes the function of the Ca2+ and cAMP signaling pathways in activation of epithelial fluid and electrolyte secretion. These concepts are discussed in this review using secretion by the pancreatic and salivary gland ducts as model systems.

cAMP and Ca2+ signaling in secretory epithelia: Crosstalk and synergism

The Ca(2+) and cAMP/PKA pathways are the primary signaling systems in secretory epithelia that control virtually all secretory gland functions. Interaction and crosstalk in Ca(2+) and cAMP signaling occur at multiple levels to control and tune the activity of each other. Physiologically, Ca(2+) and cAMP signaling operate at 5-10% of maximal strength, but synergize to generate the maximal response. Although synergistic action of the Ca(2+) and cAMP signaling is the common mode of signaling and has been known for many years, we know very little of the molecular mechanism and mediators of the synergism. In this review, we discuss crosstalk between the Ca(2+) and cAMP signaling and the function of IRBIT (IP3 receptors binding protein release with IP3) as a third messenger that mediates the synergistic action of the Ca(2+) and cAMP signaling.

The ER/PM microdomain, PI(4,5)P2 and the regulation of STIM1–Orai1 channel function

All forms of cell signaling occur in discreet cellular microdomains in which the ER is the main participant and include microdomains formed by the ER with lysosomes, endosomes, the nucleus, mitochondria and the plasma membrane. In the microdomains the two opposing organelles transfer and exchange constituents including lipids and ions. As is the case for other forms of signaling pathways, many components of the receptor-evoked Ca(2+) signal are clustered at the ER/PM microdomain, including the Orai1-STIM1 complex. This review discusses recent advances in understanding the molecular components that tether the ER and plasma membrane to form the ER/PM microdomains in which PI(4,5)P2 is enriched, and how dynamic targeting of the Orai1-STIM1 complex to PI(4,5)P2-poor and PI(4,5)P2-rich microdomains controls the activity of Orai1 and its regulation by Ca(2+) that is mediated by SARAF.

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 &agr;-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.

NADPH oxidase 2-derived superoxide downregulates endothelial KCa3.1 in preeclampsia

Endothelial dysfunction is associated with KCa3.1 dysfunction and contributes to the development of hypertension in preeclampsia. However, evidence of endothelial KCa3.1 dysfunction in the vascular system from women with preeclampsia is still lacking. Therefore, we examined whether endothelial KCa3.1 dysfunction occurs in vessels from women with preeclampsia. We compared KCa3.1 and NADPH oxidase (NOX) expression in umbilical vessels and primary cultured human umbilical vein endothelial cells (HUVECs) from normal (NP; n=17) and preeclamptic pregnancy (PE; n=19) and examined the effects of plasma from NP or PE on KCa3.1 and NOX2 expression in primary cultured HUVECs from NP or human uterine microvascular endothelial cells. The endothelial KCa3.1 was downregulated, and NOX2 was upregulated, in umbilical vessels and HUVECs from PE, compared with those from NP. In addition, HUVECs from PE showed a significant decrease in KCa3.1 current. Plasma from PE induced KCa3.1 down regulation, NOX2 upregulation, phosphorylated-p38 mitogen-activated protein kinase downregulation, and superoxide generation, and these effects were prevented by antioxidants (tempol or tiron), NOX2 inhibition, or anti-lectin-like oxidized low-density lipoprotein (LDL) receptor 1 (LOX1) antibody. Oxidized LDL and the superoxide donor xanthine/xanthine oxidase mixture induced KCa3.1 downregulation. In contrast, plasma from PE did not generate hydrogen peroxide, and the hydrogen peroxide donor tert-butylhydroperoxide induced KCa3.1 upregulation. These results provide the first evidence that plasma from PE generates superoxide via a LOX1-NOX2-mediated pathway and downregulates endothelial KCa3.1, which may contribute to endothelial dysfunction and vasculopathy in preeclampsia. This suggests KCa3.1as a novel target for patients with preeclampsia.

Lyso-globotriaosylceramide downregulates KCa3.1 channel expression to inhibit collagen synthesis in fibroblasts.

Fabry disease is an X-linked lysosomal storage disorder that is caused by a deficiency of α-galactosidase A. The disease ultimately manifests as multiple organ dysfunctions owing to excessive accumulation of globotriaosylceramide (Gb3). Among the several complications of Fabry disease, ascending thoracic aortic aneurysm is relatively common, which is classically associated with connective tissue disorders characterized by abnormal defects or deficiencies in structural proteins such as collagen and elastin. Although an elevated Gb3 level is regarded as a prerequisite for the manifestations of Fabry disease, only this excess accumulation cannot explain the pathophysiology of these complications. Recently, an increased plasma level of lyso-Gb3 was suggested as a new biomarker in Fabry disease. Therefore, the aim of this study was to assess the effects of lyso-Gb3 on the pathogenesis of thoracic ascending aortic aneurysms in Fabry disease, with a particular focus on the responses related to aortic remodeling by fibroblasts. We found that lyso-Gb3 inhibited the growth of fibroblasts, as well as their differentiation into myofibroblasts, and collagen expression. Moreover, all of these compromised responses could be attributed to the effects of lyso-Gb3 on downregulation of KCa3.1 channel expression, and these impairments could be rescued when activating the KCa3.1 channel or increasing intracellular Ca(2+) concentration. This study provides new evidence that lyso-Gb3 inhibits the differentiation into myofibroblasts and collagen synthesis of fibroblasts owing to decreased Ca(2+) levels by KCa3.1 channel dysfunction. These findings suggest that the KCa3.1 channel can serve as a new target to attenuate and prevent development of ascending thoracic aortic aneurysm in Fabry disease.

Role of protein kinase A and class II phosphatidylinositol 3-kinase C2β in the downregulation of KCa3.1 channel synthesis and membrane surface expression by lyso-globotriaosylceramide.

The intermediate conductance calcium-activated potassium channel (KCa3.1) mediates proliferation of many cell types including fibroblasts, and is a molecular target for intervention in various cell proliferative diseases. Our previous study showed that reduction of KCa3.1 channel expression by lyso-globotriaosylceramide (lyso-Gb3) inhibits differentiation into myofibroblasts and collagen synthesis, which might lead to development of ascending thoracic aortic aneurysm secondary to Fabry disease. However, how lyso-Gb3 downregulates KCa3.1 channel expression is unknown. Therefore, we aimed to investigate the underlying mechanisms of lyso-Gb3-mediated KCa3.1 channel downregulation, focusing on the cAMP signaling pathway. We found that lyso-Gb3 increased the intracellular cAMP concentration by upregulation of adenylyl cyclase 6 and inhibited ERK 1/2 phosphorylation through the protein kinase A (PKA) pathway, leading to the inhibition of KCa3.1 channel synthesis, not the exchange protein directly activated by cAMP (Epac) pathway. Moreover, lyso-Gb3 suppressed expression of class II phosphatidylinositol 3-kinase C2β (PI3KC2β) by PKA activation, which reduces the production of phosphatidylinositol 3-phosphate [PI(3)P], and the reduced membrane surface expression of KCa3.1 channel was recovered by increasing the intracellular levels of PI(3)P. Consequently, our findings that lyso-Gb3 inhibited both KCa3.1 channel synthesis and surface expression by increasing intracellular cAMP, and controlled surface expression through changes in PI3KC2β-mediated PI(3)P production, suggest that modulation of PKA and PI3KC2β activity to control of KCa3.1 channel expression can be an alternative important target to attenuate ascending thoracic aortic aneurysms in Fabry disease.

Orai1 and STIM1 in ER/PM junctions: roles in pancreatic cell function and dysfunction

Membrane contact sites (MCS) are critical junctions that form between the endoplasmic reticulum (ER) and membranes of various organelles, including the plasma membrane (PM). Signaling complexes, including mediators of Ca(2+) signaling, are assembled within MCS, such as the ER/PM junction. This is most evident in polarized epithelial cells, such as pancreatic cells. Core Ca(2+) signaling proteins cluster at the apical pole, the site of inositol 1,4,5-trisphosphate-mediated Ca(2+) release and Orai1/transient receptor potential canonical-mediated store-dependent Ca(2+) entry. Recent advances have characterized the proteins that tether the membranes at MCS and the role of these proteins in modulating physiological and pathological intracellular signaling. This review discusses recent advances in the characterization of Ca(2+) signaling at ER/PM junctions and the relation of these junctions to physiological and pathological Ca(2+) signaling in pancreatic acini.

Modulation of nonselective cation current by oxidized LDL and lysophosphatidylcholine and its inhibitory contribution to endothelial damage

AIMS This study examined the effects of oxidized low-density lipoprotein (LDL) and its major lipid constituent lysophosphatidylcholine (LPC) on nonselective cation (NSC) current and its inhibitory contribution to LPC-induced cytotoxicity in cultured human umbilical endothelial cells (HUVECs). MAIN METHODS Patch-clamp technique and the resazurin-based cell viability assay were used. KEY FINDINGS In voltage-clamped cells, oxidized LDL or LPC slowly activated NSC current. NSC current was also activated by loading cells with Ca(2+) solution buffered at various concentrations using a patch pipette or by applying the sarcoplasmic reticulum Ca(2+) pump blocker 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), the metabolic inhibitor CN(-) or the hydroperoxide donor tert-butyl hydroperoxide (TBHP). On the contrary, when intracellular Ca(2+) was strongly buffered with 12mM BAPTA or cells were loaded with superoxide dismutase using a patch pipette, LPC or BHQ did not activate NSC current. Furthermore, NSC current activated by LPC, TBHP or CN(-) was inhibited by the antioxidant tempol or extracellular Ca(2+) depletion and NSC current activated by intracellular Ca(2+) was further augmented by oxidized LDL or LPC. LPC or oxidized LDL released Ca(2+) from intracellular stores and further enhanced store-operated Ca(2+) entry. LPC-induced cytotoxicity was augmented by inhibiting Ca(2+) influx and NO synthesis. SIGNIFICANCE Oxidized LDL or its main component LPC activated Ca(2+)-permeable NSC current via releasing Ca(2+) from intracellular stores and producing ROS and thereby increased Ca(2+) influx. Ca(2+) influx through NSC channel might protect endothelial cells by producing NO.