Voltage-dependent anion channel 2 modulates resting Ca²+ sparks, but not action potential-induced Ca²+ signaling in cardiac myocytes

Voltage-dependent anion channels (VDACs) are pore forming proteins predominantly found in the outer mitochondrial membrane and are thought to transport Ca(2+). In this study, we have investigated the possible role of type 2 VDAC (VDAC2) in cardiac Ca(2+) signaling and Ca(2+) sparks using a lentiviral knock-down (KD) technique and two-dimensional confocal Ca(2+) imaging in immortalized autorhythmic adult atrial cells, HL-1. We confirmed high expression of VDAC2 protein in ventricular, atrial, and HL-1 cells using Western blot analysis. Infection of HL-1 cells with VDAC2-targeting lentivirus reduced the level of VDAC2 protein to ～10%. Comparisons of autorhythmic Ca(2+) transients between wild-type (WT) and VDAC2 KD cells showed no significant change in the magnitude, decay, and beating rate of the Ca(2+) transients. Caffeine (10mM)-induced Ca(2+) release, which indicates sarcoplasmic reticulum (SR) Ca(2+) content, was not altered by VDAC2 KD. Interestingly, however, the intensity, width, and duration of the individual Ca(2+) sparks were significantly increased by VDAC2 KD in resting conditions, with no change in the frequency of sparks. VDAC2 KD significantly delayed mitochondrial Ca(2+) uptake during artificial Ca(2+) pulses in permeabilized HL-1 cells. These results suggest that VDAC2 may facilitate mitochondrial Ca(2+) uptake and restrict Ca(2+) spark expansion without regulating activations of sparks under resting conditions, thereby providing evidence on the functional role of VDAC2 in cardiac local Ca(2+) signaling.     

How strict is the correlation between STIM1 and Orai1 expression, puncta formation, and ICRAC activation?

Stromal interaction molecule 1 (STIM1) and Orai1 have been identified as crucial elements of the store-operated Ca(2+) entry (SOCE) pathway, but the mechanism of their functional interaction remains controversial. It is now well established that, upon depletion of the stores, both molecules can accumulate and colocalize in specific areas (puncta) where the endoplasmic reticulum comes in close proximity to the plasma membrane. Some models propose a direct interaction between STIM1 and Orai1 as the most straightforward mechanism for signal transduction from the stores to the plasma membrane. To test some of the predictions of a conformational coupling model, we assessed how tight the relationships are between STIM1 and Orai1 expression, puncta formation, and SOCE activation. Here we present evidence that STIM1 accumulates in puncta equally well in the presence or absence of Orai1 expression, that STIM1 accumulation is not sufficient for Orai1 accumulation in the same areas, and that normal Ca(2+) release-activated Ca(2+) current (I(CRAC)) can be activated in STIM1-deficient cells. These data challenge the idea of direct conformational coupling between STIM1 and Orai1 as a viable mechanism of puncta formation and SOCE activation and uncover greater complexity in their relationship, which may require additional intermediate elements.     

Hypochlorite-oxidized low-density lipoprotein upregulates CD36 and PPARγ mRNA expression and modulates SR-BI gene expression in murine macrophages

Huntington's disease (HD) is associated with expansion of polyglutamine tract in a protein named huntingtin (htt) that is expressed in virtually all body tissues. Thus mutated htt (HD-htt) might affect all organs, although clinical manifestations of HD are associated with selective loss of corticostriatal neurons of the brain. In this work we studied how HD-htt affects mitochondria in human peripheral blood cells. We compared various functions of mitochondria isolated from cultured lymphoblastoid cells derived from three HD patients with juvenile onset of the disease (HD-LBM) and three age-matched control (C-LBM) individuals. Respiratory parameters in different metabolic states, with succinate and glutamate plus malate were the same for all control and HD cell lines. State 4 membrane potential in HD-LBM was slightly lower than in C-LBM. The calcium retention capacity (CRC) of mitochondria was estimated using simultaneously several methods to register permeability transition (PT). We found that LBM do not undergo swelling upon Ca2+-induced PT, and do not increase CRC in the presence of ADP + oligomycin. Although each cell line had different CRC values, qualitatively PT was different in C-LBM and HD-LBM. With C-LBM cyclosporin A (CsA) increased CRC significantly, while with HD-LBM CsA was ineffective. In C-LBM depolarization of mitochondria and a large pore opening (PT) always occurred simultaneously. In HD-LBM depolarization occurred at 20-50% lower Ca2+ loads than PT. We suggest that HD-htt promotes low H+ conductance of the mitochondria by interacting with proteins at the contacts sites without directly promoting PT or hampering mitochondrial oxidative phosphorylation.     

Thiol modulation of TNFα and IL-1 induced MnSOD gene expression and activation of NF-κB

TNF and IL-1 each can activate NF-B and induce gene expression of manganese superoxide dismutase (MnSOD), a mitochondrial matrix enzyme which can provide critical protection against hyperoxic lung injury. The regulation of MnSOD gene expression is not well understood. Since redox status can modulate NF-B and potential B site(s) exist in the MnSOD promoter, the effect of thiols (including NAC, DTT and 2-ME) on TNF and IL-1 induced activation of NF-B and MnSOD gene expression was investigated. Activation of NF-kB and increased MnSOD expression were potentiated by thiol reducing agents. In contrast, thiol oxidizing or alkylating agents inhibited both NF-B activation and elevated MnSOD expression in response to TNF or IL-1. Since protease inhibitors TPCK and TLCK can inhibit NF-B activation, we also investigated the effect of these compounds on MnSOD expression and NF-B activation. TPCK and TLCK each inhibited MnSOD gene expression and NF-B activation. Since the MnSOD promoter also contains anAP-1 binding site, the effect of thiols and thiol modifying agents on AP-1 activation was investigated. Thiols had no consistent effect onAP-1 activation. Likewise, some of the thiol modifying compounds inhibited AP-1 activation by TNF or IL-1, whereas others did not. Since diverse agents had similar effects on activation of NF-B and MnSOD gene expression, we have demonstrated that activation of NF-B and MnSOD gene expression are closely associated and that reduced sulfhydryl groups are required for cytokine mediation of both processes.Abbreviations O₂ Superoxide radical - H₂O₂ Hydrogen peroxide - NAC N-acetyl L-cysteine - DTT Dithiothreitol - 2-ME 2-Mercaptoethanol - MnSOD Manganese superoxide dismutase - NF-B Nuclear factor kappa B - AP-1 Activator protein-1 - NBT Nitroblue tetrazolium - CAT Chloramphenicol acetyltransferase - TPCK N-tosyl-L-phenylalanine chloromethyl ketone - TLCK Na-p-tosyl-L-lysine chloromethyl ketone - TAME N--p-tosyl-L-arginine methyl ester - NEM N-ethyl maleimide - DEM Diethyl maleate - CDNB 1-chloro-2,4-dinitrobenzene - DTT_OX Oxidized dithiothreitol     

Polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca²⁺ signaling by differentially modulating STIM1 and STIM2

Early epithelial restitution occurs as a consequence of intestinal epithelial cell (IEC) migration after wounding, and its defective regulation is implicated in various critical pathological conditions. Polyamines stimulate intestinal epithelial restitution, but their exact mechanism remains unclear. Canonical transient receptor potential-1 (TRPC1)-mediated Ca(2+) signaling is crucial for stimulation of IEC migration after wounding, and induced translocation of stromal interaction molecule 1 (STIM1) to the plasma membrane activates TRPC1-mediated Ca(2+) influx and thus enhanced restitution. Here, we show that polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca(2+) signaling by altering the ratio of STIM1 to STIM2. Increasing cellular polyamines by ectopic overexpression of the ornithine decarboxylase (ODC) gene stimulated STIM1 but inhibited STIM2 expression, whereas depletion of cellular polyamines by inhibiting ODC activity decreased STIM1 but increased STIM2 levels. Induced STIM1/TRPC1 association by increasing polyamines enhanced Ca(2+) influx and stimulated epithelial restitution, while decreased formation of the STIM1/TRPC1 complex by polyamine depletion decreased Ca(2+) influx and repressed cell migration. Induced STIM1/STIM2 heteromers by polyamine depletion or STIM2 overexpression suppressed STIM1 membrane translocation and inhibited Ca(2+) influx and epithelial restitution. These results indicate that polyamines differentially modulate cellular STIM1 and STIM2 levels in IECs, in turn controlling TRPC1-mediated Ca(2+) signaling and influencing cell migration after wounding.     

Local Ca²+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca²+ signals required for specific cell functions

Store-operated Ca²⁺ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca²⁺ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent I_SOC, activated in response to Ca²⁺ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated I_CRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(⁶⁸⁴EE⁶⁸⁵). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca²⁺ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd³⁺, removal of extracellular Ca²⁺, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca²⁺-containing, but not Ca²⁺-free, medium. Consistent with this, I_CRAC is activated in cells pretreated with thapsigargin in Ca²⁺-free medium while I_SOC is activated in cells pretreated in Ca²⁺-containing medium. Significantly, TRPC1 function is required for sustained K_Ca activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca²⁺ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca²⁺ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE.     

Resveratrol induces insulin gene expression in mouse pancreatic α-cells

Background

Type 1 and type 2 diabetes are characterized by loss of β-cells; therefore, β-cell regeneration has become one of the primary approaches to diabetes therapy. Resveratrol, a naturally occurring polyphenolic compound, has been shown to improve glycaemic control in diabetic patients, but its action on pancreatic α-cells is not well understood.

Findings

Using mouse α-cells (αTC9), we showed that resveratrol induces expression of pancreatic β-cell genes such as Pdx1 and Ins2 in a SirT1-dependent manner. The mRNA and protein levels of insulin were further increased by histone deacetylase (HDAC) inhibition.

Conclusion

In summary, we provide new mechanistic insight into the anti-diabetic action of resveratrol through its ability to express β-cell genes in α-cells.

     

Enhanced store-operated Ca²+ entry and TRPC channel expression in pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats

Pulmonary hypertension (PH) is associated with profound vascular remodeling and alterations in Ca(2+) homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Previous studies show that canonical transient receptor potential (TRPC) genes are upregulated and store-operated Ca(2+) entry (SOCE) is augmented in PASMCs of chronic hypoxic rats and patients of pulmonary arterial hypertension (PAH). Here we further examine the involvement of TRPC and SOCE in PH with a widely used rat model of monocrotaline (MCT)-induced PAH. Rats developed severe PAH, right ventricular hypertrophy, and significant increase in store-operated TRPC1 and TRPC4 mRNA and protein in endothelium-denuded pulmonary arteries (PAs) 3 wk after MCT injection. Contraction of PA and Ca(2+) influx in PASMC evoked by store depletion using cyclopiazonic acid (CPA) were enhanced dramatically, consistent with augmented SOCE in the MCT-treated group. The time course of increase in CPA-induced contraction corresponded to that of TRPC1 expression. Endothelin-1 (ET-1)-induced vasoconstriction was also potentiated in PAs of MCT-treated rats. The response was partially inhibited by SOCE blockers, including Gd(3+), La(3+), and SKF-96365, as well as the general TRPC inhibitor BTP-2, suggesting that TRPC-dependent SOCE was involved. Moreover, the ET-1-induced contraction and Ca(2+) response in the MCT group were more susceptible to the inhibition caused by the various SOCE blockers. Hence, our study shows that MCT-induced PAH is associated with increased TRPC expression and SOCE, which are involved in the enhanced vascular reactivity to ET-1, and support the hypothesis that TRPC-dependent SOCE is an important pathway for the development of PH.     

Cyclic stretch induces cyclooxygenase-2 gene expression in vascular endothelial cells via activation of nuclear factor kappa-β

Vascular endothelial cells respond to biomechanical forces, such as cyclic stretch and shear stress, by altering gene expression. Since endothelial-derived prostanoids, such as prostacyclin and thromboxane A₂, are key mediators of endothelial function, we investigated the effects of cyclic stretch on the expression of genes in human umbilical vein endothelial cells controlling prostanoid synthesis: cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS) and thromboxane A₂ synthase (TXAS). COX-2 and TXAS mRNAs were upregulated by cyclic stretch for 24 h. In contrast, PGIS mRNA was decreased and stretch had no effect on COX-1 mRNA expression. We further show that stretch-induced upregulation of COX-2 is mediated by activation of the NF-κβ signaling pathway.     

Lysophosphatidic acid induces increased BACE1 expression and Aβ formation

The abnormal production and accumulation of β-amyloid peptide (Aβ), which is produced from amyloid precursor protein (APP) by the sequential actions of β-secretase and γ-secretase, are thought to be the initial causative events in the development of Alzheimer's disease (AD). Accumulating evidence suggests that vascular factors play an important role in the pathogenesis of AD. Specifically, studies have suggested that one vascular factor in particular, oxidized low density lipoprotein (oxLDL), may play an important role in regulating Aβ formation in AD. However, the mechanism by which oxLDL modulates Aβ formation remains elusive. In this study, we report several new findings that provide biochemical evidence suggesting that the cardiovascular risk factor oxLDL may contribute to Alzheimer's disease by increasing Aβ production. First, we found that lysophosphatidic acid (LPA), the most bioactive component of oxLDL induces increased production of Aβ. Second, our data strongly indicate that LPA induces increased Aβ production via upregulating β-secretase expression. Third, our data strongly support the notion that different isoforms of protein kinase C (PKC) may play different roles in regulating APP processing. Specifically, most PKC members, such as PKCα, PKCβ, and PKCε, are implicated in regulating α-secretase-mediated APP processing; however, PKCδ, a member of the novel PKC subfamily, is involved in LPA-induced upregulation of β-secretase expression and Aβ production. These findings may contribute to a better understanding of the mechanisms by which the cardiovascular risk factor oxLDL is involved in Alzheimer's disease.     

α5β1 integrin induces the expression of noncartilaginous procollagen gene expression in articular chondrocytes cultured in monolayers

Introduction

Articular chondrocytes undergo an obvious phenotypic change when cultured in monolayers. During this change, or dedifferentiation, the expression of type I and type III procollagen is induced where normal chondrocytes express little type I and type III procollagen. In this study, we attempted to determine the mechanism(s) for the induction of such procollagen expression in dedifferentiating chondrocytes.

Methods

All experiments were performed using primary-cultured human articular chondrocytes under approval of institutional review boards. Integrin(s) responsible for the induction of type I and type III procollagen expression were specified by RNAi experiments. The signal pathway(s) involved in the induction were determined by specific inhibitors and RNAi experiments. Adenovirus-mediated experiments were performed to identify a small GTPase regulating the activity of integrins in dedifferentiating chondrocytes. The effect of inhibition of integrins on dedifferentiation was investigated by experiments using echistatin, a potent disintegrin. The effect of echistatin was investigated first with monolayer-cultured chondrocytes, and then with pellet-cultured chondrocytes.

Results

In dedifferentiating chondrocytes, α5β1 integrin was found to be involved in the induction of type I and type III procollagen expression. The induction was known to be mediated by v-akt murine thymoma viral oncogene homolog (AKT) signaling. Among the three AKT isoforms, AKT1 seemed to be most involved in the signaling. Elated RAS viral (r-ras) oncogene homolog (RRAS) was considered to regulate the progression of dedifferentiation by modulating the affinity and avidity of α5β1 integrin to ligands. Echistatin inhibited dedifferentiation of monolayer-cultured chondrocytes. Furthermore, the matrix formed by pellet-cultured chondrocytes more closely resembled that of normal cartilage compared with the controls.

Conclusions

The result of this study has shown, for the first time, that α5β1 integrin may be responsible for the induction of non-cartilaginous collagen expression in chondrocytes undergoing dedifferentiation. Again, this study has shown that the inhibition of ligand ligation to integrins may be an effective strategy to inhibit phenotypic change of cultured chondrocytes, and to improve the quality of matrix synthesized by primary cultured chondrocytes.     

The predominant role of IP₃ type 1 receptors in activation of store-operated Ca²+ entry in liver cells

Physiologically, hormone induced release of Ca2+ from intracellular stores occurs in response to inositol 1,4,5-trisphosphate (IP?) binding to its receptors expressed on the membranes of intracellular organelles, mainly endoplasmic reticulum. These IP? receptors act as channels, releasing Ca2+ into the cytoplasmic space where it is responsible for regulating a host of distinct cellular processes. The depletion of intracellular Ca2+ stores leads to activation of store-operated Ca2+ channels on the plasma membrane which replenishes lost Ca2+ and sustain Ca2+ signalling. There are three isoforms of IP? receptor, each exhibiting distinctive properties, however, little is known about the role of each isoform in the activation of store-operated Ca2+ entry. Recent evidence suggest that at least in some cell types the endoplasmic reticulum is not a homogeneous Ca2+ store, and there might be a sub-compartment specifically linked to the activation of store-operated Ca2+ channels, and Ca2+ release activated Ca2+ (CRAC) channel in particular. Furthermore, this sub-compartment might express only certain types of IP? receptor but not the others. Here we show that H4IIE liver cells express all three types of IP? receptor, but only type 1 and to a lesser extent type 3, but not type 2, participate in the activation of CRAC current (I(CRAC)), while type 1 and type 2, but not type 3, participate in observed Ca2+ release in response to receptor stimulation. Presented results suggest that in H4IIE rat liver cells the sub-compartment of intracellular Ca2+ store linked to the activation of I(CRAC) predominantly expresses type 1 IP? receptors.     

Synaptotagmin increases the dynamic range of synapses by driving Ca²+-evoked release and by clamping a near-linear remaining Ca²+ sensor

Ca2+-evoked transmitter release shows a high dynamic range over spontaneous release. We investigated the role of the Ca2+ sensor protein, Synaptotagmin2 (Syt2), in both spontaneous and Ca2+-evoked release under direct control of presynaptic [Ca2+](i), using an in vivo rescue approach at the calyx of Held. Re-expression of Syt2 rescued the highly Ca2+ cooperative release and suppressed the elevated spontaneous release seen in Syt2 KO synapses. This latter release clamping function was partially mediated by the poly-lysine motif of the C?B domain. Using an aspartate mutation in the C?B domain (D364N) in which Ca2+ triggering was abolished but release clamping remained intact, we show that Syt2 strongly suppresses the action of another, near-linear Ca2+ sensor that mediates release over a wide range of [Ca2+](i). Thus, Syt2 increases the dynamic range of synapses by driving release with a high Ca2+ cooperativity, as well as by suppressing a remaining, near-linear Ca2+ sensor.