Masashi Isshiki

Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts.

Signaling by RANKL is essential for terminal differentiation of monocytes/macrophages into osteoclasts. The TRAF6 and c-Fos signaling pathways both play important roles downstream of RANKL. We show here that RANKL selectively induces NFATc1 expression via these two pathways. RANKL also evokes Ca(2+) oscillations that lead to calcineurin-mediated activation of NFATc1, and therefore triggers a sustained NFATc1-dependent transcriptional program during osteoclast differentiation. We also show that NFATc1-deficient embryonic stem cells fail to differentiate into osteoclasts in response to RANKL stimulation, and that ectopic expression of NFATc1 causes precursor cells to undergo efficient differentiation without RANKL signaling. Thus, NFATc1 may represent a master switch for regulating terminal differentiation of osteoclasts, functioning downstream of RANKL.

Impaired flow-dependent control of vascular tone and remodeling in P2X4-deficient mice

The structure and function of blood vessels adapt to environmental changes such as physical development and exercise. This phenomenon is based on the ability of the endothelial cells to sense and respond to blood flow; however, the underlying mechanisms remain unclear. Here we show that the ATP-gated P2X4 ion channel, expressed on endothelial cells and encoded by P2rx4 in mice, has a key role in the response of endothelial cells to changes in blood flow. P2rx4−/− mice do not have normal endothelial cell responses to flow, such as influx of Ca2+ and subsequent production of the potent vasodilator nitric oxide (NO). Additionally, vessel dilation induced by acute increases in blood flow is markedly suppressed in P2rx4−/− mice. Furthermore, P2rx4−/− mice have higher blood pressure and excrete smaller amounts of NO products in their urine than do wild-type mice. Moreover, no adaptive vascular remodeling, that is, a decrease in vessel size in response to a chronic decrease in blood flow, was observed in P2rx4−/− mice. Thus, endothelial P2X4 channels are crucial to flow-sensitive mechanisms that regulate blood pressure and vascular remodeling.

Dietary Phosphorus Acutely Impairs Endothelial Function

Excessive dietary phosphorus may increase cardiovascular risk in healthy individuals as well as in patients with chronic kidney disease, but the mechanisms underlying this risk are not completely understood. To determine whether postprandial hyperphosphatemia may promote endothelial dysfunction, we investigated the acute effect of phosphorus loading on endothelial function in vitro and in vivo. Exposing bovine aortic endothelial cells to a phosphorus load increased production of reactive oxygen species, which depended on phosphorus influx via sodium-dependent phosphate transporters, and decreased nitric oxide production via inhibitory phosphorylation of endothelial nitric oxide synthase. Phosphorus loading inhibited endothelium-dependent vasodilation of rat aortic rings. In 11 healthy men, we alternately served meals containing 400 mg or 1200 mg of phosphorus in a double-blind crossover study and measured flow-mediated dilation of the brachial artery before and 2 h after the meals. The high dietary phosphorus load increased serum phosphorus at 2 h and significantly decreased flow-mediated dilation. Flow-mediated dilation correlated inversely with serum phosphorus. Taken together, these findings suggest that endothelial dysfunction mediated by acute postprandial hyperphosphatemia may contribute to the relationship between serum phosphorus level and the risk for cardiovascular morbidity and mortality.

Suppression of arthritic bone destruction by adenovirus-mediated csk gene transfer to synoviocytes and osteoclasts

Rheumatoid arthritis (RA) is characterized by a chronic inflammation of the synovial joints resulting from hyperplasia of synovial fibroblasts and infiltration of lymphocytes, macrophages, and plasma cells, all of which manifest signs of activation. Recent studies have revealed the essential role of osteoclasts in joint destruction in RA. Src family tyrosine kinases are implicated in various intracellular signaling pathways, including mitogenic response to growth factors in fibroblasts, activation of lymphocytes, and osteoclastic bone resorption. Therefore, inhibiting Src activity can be a good therapeutic strategy to prevent joint inflammation and destruction in RA. We constructed an adenovirus vector carrying the csk gene, which negatively regulates Src family tyrosine kinases. Csk overexpression in cultured rheumatoid synoviocytes remarkably suppressed Src kinase activity and reduced their proliferation rate and IL-6 production. Bone-resorbing activity of osteoclasts was strongly inhibited by Csk overexpression. Furthermore, local injection of the virus into rat ankle joints with adjuvant arthritis not only ameliorated inflammation but suppressed bone destruction. In conclusion, adenovirus-mediated direct transfer of the csk gene is useful in repressing bone destruction and inflammatory reactions, suggesting the involvement of Src family tyrosine kinases in arthritic joint breakdown and demonstrating the feasibility of intervention in the kinases for gene therapy in RA. off

Function of caveolae in Ca2+ entry and Ca2+-dependent signal transduction.

The correct spatial and temporal control of Ca2+ signaling is essential for such cellular activities as fertilization, secretion, motility, and cell division. There has been a long‐standing interest in the role of caveolae in regulating intracellular Ca2+ concentration. In this review we provide an updated view of how caveolae may regulate both Ca2+ entry into cells and Ca2+‐dependent signal transduction

An essential role of myosin light-chain kinase in the regulation of agonist- and fluid flow-stimulated Ca2+ influx in endothelial cells

Cytosolic Ca2+ ([Ca2+];) plays an important role in endothelial cell signaling. Although it has been suggested that the influx of Ca2+ can be triggered by depletion of intracellular Ca2+ stores, the mechanism (or mechanisms) underlying this phenomenon needs further elaboration. In the present study, involvement of myosin light‐chain kinase (MLCK) in the regulation of Ca2+ signaling was investigated in agonist‐ and fluid flow‐stimulated endothelial cells loaded with Ca2+‐sensitive dyes. Bradykinin (BK) and thapsigargin caused an increase in [Ca2+]i followed by a sustained rise due to Ca2+ influx from extracellular space and shifted total myosin light‐chain (MLC) from the unphosphorylated to the diphosphorylated form. ML‐9 (100 pM), an inhibitor of MLCK, abolished Ca2+ influx and prevented MLC diphosphorylation in BK‐ and thapsigargin‐treated cells, but did not affect Ca2+ mobilization from internal stores. Fluid flow stimulation (shear stress=5 dynes/cm2) increased [Ca2+]i and enhanced MLC phosphorylation. ML‐9 also inhibited Ca2+ response and MLC phosphorylation in fluid flow‐stimulated cells. The Ca2+ influx in response to BK was linearly correlated with the diphosphorylation of MLC in ML‐9 treated cells. Effects of ML‐5 and ML‐7, analogs of ML‐9, to inhibit Ca2+ influx paralleled their potencies to inhibit MLCK activity. These findings demonstrate that MLCK plays an essential role in regulating the plasmalemmal Ca2+ influx in agonist‐ and fluid flow‐stimulated endothelial cells. This study is the first to report the close relationship between Ca2+ influx and MLC diphosphorylation.—Watanabe, H., Takahashi, R., Zhang, X.‐X., Goto, Y., Hayashi, H., Ando, J., Isshiki, M., Seto, M., Hidaka, H., Niki, I., Ohno, R. An essential role of myosin light‐chain kinase in the regulation of agonist‐ and fluid flow‐stimulated Ca2+ influx in endothelial cells. FASEB J. 12, 341–348 (1998)

Subcortical Ca2+ Waves Sneaking Under the Plasma Membrane in Endothelial Cells

Subplasmalemmal Ca2+, dynamically equilibrated with extracellular Ca2+, affects numerous signaling molecules, effectors, and events within this restricted space. We demonstrated the presence of a novel Ca2+ wave propagating beneath the plasma membrane in response to acute elevation of extracellular [Ca2+], by targeting a Ca2+ sensor, cameleon, to the endothelial plasmalemma. These subcortical waves, spatially distinct from classical cytosolic Ca2+ waves, originated in localized regions and propagated throughout the subplasmalemma. Translocation of an expressed GFP fused with a PH domain of PLC&dgr; from the plasma membrane to the cytosol accompanied these subcortical waves, and U73122 attenuated not only the GFP-PH&dgr; translocation, but also the peak amplitude of the subcortical Ca2+ waves; this finding suggests the involvement of local IP3 production through PLC-mediated PIP2 hydrolysis in the initiation of these waves. Changes in NO production as well as PKC&bgr;-GFP translocation from the cytosol to the plasma membrane, but not of GFP-PLA2 to perinuclear endomembranes, were associated with the subplasmalemmal Ca2+ changes. Thus, extracellular Ca2+ maintains the basal PLC activity of the plasma membrane, is involved in the initiation of compartmentalized subcortical Ca2+ waves, and regulates Ca2+-dependent signaling molecules residing in or translocated to the plasma membrane. The full text of this article is available online at http://circres.ahajournals.org.

Aldosterone enhances ligand-stimulated nitric oxide production in endothelial cells.

Chronic and acute actions of aldosterone have been shown recently to directly affect the cardiovascular system. However, it is unclear whether the acute effects of aldosterone on vasculature are constrictive or dilatory. Here, to clarify the nongenomic effects of aldosterone on endothelial function, we examined the effects of aldosterone on nitric oxide (NO) production in cultured endothelial cells (ECs) and on vascular tone. The intracellular NO production of bovine aortic ECs loaded with DAF-2 was determined using confocal microscopy. Accumulated NO in the culture medium was quantified by a microplate reader using membrane-impermeable DAF-2. Phosphorylation of endothelial NO synthase (eNOS) at Ser1179 was assessed by Western blotting. Changes in intracellular Ca2+ ([Ca2+]i) were determined by confocal microscopy in ECs doubly loaded with fluo-4 and Fura Red. The effects of aldosterone, acetylcholine (ACh), and other signaling molecules on the tension of phenylephrine (PE)-contracted aortas of Sprague-Dawley rats were examined in an ex vivo organ bath chamber system. Short-term pre-exposure to aldosterone (1 × 10−7 mol/L) enhanced ATP-induced NO production in ECs with increased phosphorylation of eNOS at Ser1179. These effects were blocked by eplerenone, a mineralocorticoid receptor (MR) antagonist, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Notably, aldosterone alone did not affect ATP-induced [Ca2+]i changes or the Ser1179 phosphorylation. Similarly, aldosterone (1 × 10−8 to 1 × 10−7 mol/L) did not affect the tone of rat aortas precontracted by PE, but enhanced ACh-induced vasorelaxation, which was again reversed by eplerenone or LY29400. In contrast, sodium nitroprusside–induced vasorelaxation in endothelium-denuded aortas was not affected by aldosterone. Thus, aldosterone acutely enhances ligand-mediated endothelial NO production by eplerenone-sensitive mechanisms involving a PI3K that may synergize Ca2+-dependent eNOS phosphorylation at Ser1179.

In vitro and in vivo suppression of osteoclast function by adenovirus vector-induced csk gene.

The proto‐oncogene c‐src, which encodes a non–receptor‐type tyrosine kinase c‐Src, has been shown to be essential for osteoclastic bone resorption by the finding that the targeted disruption of the c‐src gene induced osteopetrosis in mice. The csk (C‐terminal Src family kinase) gene encodes a cytoplasmic protein‐tyrosine kinase that specifically phosphorylates the negative regulatory site of c‐Src (Tyr‐527), thereby inhibiting its kinase activity. To regulate osteoclast function by modulating the kinase activity of c‐Src, we constructed an adenovirus vector that carries this gene. The recombinant adenovirus vector carrying csk cDNA induced Csk expression in mouse osteoclast‐like cells formed in vitro and clearly reduced c‐Src kinase activity in a dose‐dependent manner. The expression of Csk caused cytoskeletal disorganization of osteoclast‐like cells and strongly suppressed pit‐forming activity of the cells in vitro. In addition, the viral vector carrying csk gene dramatically suppressed interleukin‐1α–induced bone resorption in vivo. Conversely, kinase‐inactive Csk caused an increase in c‐Src kinase activity and bone resorbing activity of the cells both in vitro and in vivo, acting as a dominant negative molecule against intrinsic Csk. These findings indicate that the inhibition of c‐Src activity by adenovirus vector‐mediated csk expression offers an efficient means for inhibiting pathological bone resorption by suppressing osteoclast function.

Targeting of neutral cholesterol ester hydrolase to the endoplasmic reticulum via its N-terminal sequence.

Neutral cholesterol ester hydrolase (NCEH) accounts for a large part of the nCEH activity in macrophage foam cells, a hallmark of atherosclerosis, but its subcellular localization and structure-function relationship are unknown. Here, we determined subcellular localization, glycosylation, and nCEH activity of a series of NCEH mutants expressed in macrophages. NCEH is a single-membrane-spanning type II membrane protein comprising three domains: N-terminal, catalytic, and lipid-binding domains. The N-terminal domain serves as a type II signal anchor sequence to recruit NCEH to the endoplasmic reticulum (ER) with its catalytic domain within the lumen. All of the putative N-linked glycosylation sites (Asn270, Asn367, and Asn389) of NCEH are glycosylated. Glycosylation at Asn270, which is located closest to the catalytic serine motif, is important for the enzymatic activity. Cholesterol loading by incubation with acetyl-LDL does not change the ER localization of NCEH. In conclusion, NCEH is targeted to the ER of macrophages, where it hydrolyzes CE to deliver cholesterol for efflux out of the cells.