Calreticulin couples calcium release and calcium influx in integrin-mediated calcium signaling

The engagement of integrin alpha7 in E63 skeletal muscle cells by laminin or anti-alpha7 antibodies triggered transient elevations in the intracellular free Ca(2+) concentration that resulted from both inositol triphosphate-evoked Ca(2+) release from intracellular stores and extracellular Ca(2+) influx through voltage-gated, L-type Ca(2+) channels. The extracellular domain of integrin alpha7 was found to associate with both ectocalreticulin and dihydropyridine receptor on the cell surface. Calreticulin appears to also associate with cytoplasmic domain of integrin alpha7 in a manner highly dependent on the cytosolic Ca(2+) concentration. It appeared that intracellular Ca(2+) release was a prerequisite for Ca(2+) influx and that calreticulin associated with the integrin cytoplasmic domain mediated the coupling of between the Ca(2+) release and Ca(2+) influx. These findings suggest that calreticulin serves as a cytosolic activator of integrin and a signal transducer between integrins and Ca(2+) channels on the cell surface.     

Air bubble contact with endothelial cells in vitro induces calcium influx and IP3-dependent release of calcium stores

Gas embolism is a serious complication of decompression events and clinical procedures, but the mechanism of resulting injury remains unclear. Previous work has demonstrated that contact between air microbubbles and endothelial cells causes a rapid intracellular calcium transient and can lead to cell death. Here we examined the mechanism responsible for the calcium rise. Single air microbubbles (50-150 μm), trapped at the tip of a micropipette, were micromanipulated into contact with individual human umbilical vein endothelial cells (HUVECs) loaded with Fluo-4 (a fluorescent calcium indicator). Changes in intracellular calcium were then recorded via epifluorescence microscopy. First, we confirmed that HUVECs rapidly respond to air bubble contact with a calcium transient. Next, we examined the involvement of extracellular calcium influx by conducting experiments in low calcium buffer, which markedly attenuated the response, or by pretreating cells with stretch-activated channel blockers (gadolinium chloride or ruthenium red), which abolished the response. Finally, we tested the role of intracellular calcium release by pretreating cells with an inositol 1,4,5-trisphosphate (IP3) receptor blocker (xestospongin C) or phospholipase C inhibitor (neomycin sulfate), which eliminated the response in 64% and 67% of cases, respectively. Collectively, our results lead us to conclude that air bubble contact with endothelial cells causes an influx of calcium through a stretch-activated channel, such as a transient receptor potential vanilloid family member, triggering the release of calcium from intracellular stores via the IP3 pathway.     

Inhibition of integrin-mediated adhesion and signaling disrupts retinal development

Integrins are the major family of cell adhesion receptors that mediate cell adhesion to the extracellular matrix (ECM). Integrin-mediated adhesion and signaling play essential roles in neural development. In this study, we have used echistatin, an RGD-containing short monomeric disintegrin, to investigate the role of integrin-mediated adhesion and signaling during retinal development in Xenopus. Application of echistatin to Xenopus retinal-derived XR1 glial cells inhibited the three stages of integrin-mediated adhesion: cell attachment, cell spreading, and formation of focal adhesions and stress fibers. XR1 cell attachment and spreading increased tyrosine phosphorylation of paxillin, a focal adhesion associated protein, while echistatin significantly decreased phosphorylation levels of paxillin. Application of echistatin or beta(1) integrin function blocking antibody to the embryonic Xenopus retina disrupted retinal lamination and produced rosette structures with ectopic photoreceptors in the outer retina. These results indicate that integrin-mediated cell-ECM interactions play a critical role in cell adhesion, migration, and morphogenesis during vertebrate retinal development.     

The role of mitochondria in the regulation of calcium influx into Jurkat cells.

In electrically nonexcitable cells the activity of the plasma membrane calcium channels is controlled by events occurring in mitochondria, as well as in the lumen of the endoplasmic reticulum. Thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+-ATPase, produces the release of calcium from the endoplasmic reticulum and thus, activation of store-operated calcium channels in the plasma membrane. However, thapsigargin failed to produce significant activation of the channels in Jurkat cells that had been pretreated with mitochondria-directed agents: an uncoupler (carbonyl cyanide m-chlorophenylhydrazone) and oligomycin. This is in spite of the fact that Jurkat cells pretreated with carbonyl cyanide m-chlorophenylhydrazone plus oligomycin are otherwise energetically competent, due to a high rate of glycolysis and the inhibition of mitochondrial F1Fo-ATPase by oligomycin. The pool of intracellular ATP was found not to be influenced by the pretreatments of cells with oligomycin or with oligomycin plus carbonyl cyanide m-chlorophenylhydrazone. In the control cells, we found that the ATP pool amounted to 23.2 +/- 1.9 nmoles per 107 cells (n = 4). In cells pretreated with oligomycin the level of ATP was 21.8 +/- 1.9 nmoles per 107 cells (n = 4), and in cells pretreated with both oligomycin and an uncoupler the level of ATP was 22.1 +/- 0.2 nmoles per 107 cells (n = 3). Moreover, in cells pretreated with oligomycin plus carbonyl cyanide m-chlorophenylhydrazone and suspended in a nominally calcium-free medium, thapsigargin produces transient increases in cytosolic calcium identical to those in the control cells. Thus, this pretreatment does not modify either the content of intracellular calcium stores and/or the activity of calcium ATPase in the plasma membrane. Similar results were obtained when Jurkat cells were challenged by myxothiazol, a potent inhibitor of mitochondrial cytochrome bc1 oxidoreductase. Thapsigargin, although producing calcium release from intracellular stores, was ineffective in triggering the activation of calcium channels in the plasma membrane in the case of cells pretreated with myxothiazol and oligomycin. Our results suggest that coupled mitochondria participate directly in the control of calcium channel activity in the plasma membrane of Jurkat cells. When the mitochondrial protonmotive force is collapsed, either by carbonyl cyanide m-chlorophenylhydrazone or myxothiazol, the channel remains inactive even under conditions of empty intracellular calcium stores.     

Alteration of integrin-dependent adhesion and signaling in EMT-like MDCK cells established through overexpression of calreticulin

Calreticulin (CRT) is a multi-functional Ca(2+) -binding molecular chaperone in the endoplasmic reticulum. We previously reported that kidney epithelial cell-derived Madin-Darby Canine Kidney cells were transformed into mesenchymal-like cells by gene transfection of CRT. In this study, we investigated the altered characteristics of cell adhesion in these epithelial-mesenchymal transition (EMT)-like cells. Several extracellular matrix substrata were tested, and cell adhesion to fibronectin was found to be specifically increased in the CRT-overexpressing cells compared to controls. The expression of integrins was significantly up-regulated in subunits α5 and αV, resulting in an increase in the formation of complexes such as α5β1 and αVβ3. These integrins also contributed to the enhanced binding of fibronectin. In the CRT-overexpressing cells, the phosphorylation of Akt, a downstream target of integrin-linked kinase (ILK), was up-regulated on attachment to fibronectin or collagen IV. Integrin-associated signaling through ILK was also promoted on attachment to fibronectin, suggesting some of the correlation between ILK and Akt in the CRT-overexpressing cells. Furthermore, on treatment with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester, a membrane-permeable Ca(2+) chelator, the enhanced Akt signaling was suppressed with a concomitant decrease in the formation of complexes between integrins and ILK in the CRT-overexpressing cells. In conclusion, these findings demonstrate that CRT regulates cell-substratum adhesion by modulating integrin-associated signaling through altered Ca(2+) homeostasis in the CRT-overexpressing EMT-like cells, suggesting a novel regulatory role for CRT in EMT.     

Epidermal growth factor receptor-dependent regulation of integrin-mediated signaling and cell cycle entry in epithelial cells

Integrin-mediated adhesion of epithelial cells to extracellular matrix (ECM) proteins induces prolonged tyrosine phosphorylation and partial activation of epidermal growth factor receptor (EGFR) in an integrin-dependent and EGFR ligand-independent manner. Integrin-mediated activation of EGFR in epithelial cells is required for multiple signal transduction events previously shown to be induced by cell adhesion to matrix proteins, including tyrosine phosphorylation of Shc, Cbl, and phospholipase Cgamma, and activation of the Ras/Erk and phosphatidylinositol 3'-kinase/Akt signaling pathways. In contrast, activation of focal adhesion kinase, Src, and protein kinase C, adhesion to matrix proteins, cell spreading, migration, and actin cytoskeletal rearrangements are induced independently of EGFR kinase activity. The ability of integrins to induce the activation of EGFR and its subsequent regulation of Erk and Akt activation permitted adhesion-dependent induction of cyclin D1 and p21, Rb phosphorylation, and activation of cdk4 in epithelial cells in the absence of exogenous growth factors. Adhesion of epithelial cells to the ECM failed to efficiently induce degradation of p27, to induce cdk2 activity, or to induce Myc and cyclin A synthesis; subsequently, cells did not progress into S phase. Treatment of ECM-adherent cells with EGF, or overexpression of EGFR or Myc, resulted in restoration of late-G(1) cell cycle events and progression into S phase. These results indicate that partial activation of EGFR by integrin receptors plays an important role in mediating events triggered by epithelial cell attachment to ECM; EGFR is necessary for activation of multiple integrin-induced signaling enzymes and sufficient for early events in G(1) cell cycle progression. Furthermore, these findings suggest that EGFR or Myc overexpression may provoke ligand-independent proliferation in matrix-attached cells in vivo and could contribute to carcinoma development.     

Apical macropinocytosis in polarized MDCK cells: regulation by N-ethylmaleimide-sensitive proteins

In cells tested so far endocytosis seems to be dependent on N-ethylmaleimide (NEM)-sensitive proteins, and treatment with NEM results in a complete block of endocytosis. We here demonstrate that treatment of polarized MDCK I cells with NEM strongly increased endocytosis of ricin and horseradish peroxidase at the apical side, and electron microscopy revealed NEM-induced formation of large macropinosomes at the apical pole. The NEM-stimulated apical endocytosis seemed to involve phosphatidylinositol-3 kinase, protein kinase C and phospholipase D and it was dependent on ATP. Moreover, in contrast to endocytosis in nonpolarized cells ricin endocytosis at the basolateral side continued in the presence of NEM whereas endocytosis of transferrin was blocked. Furthermore, recycling of ricin endocytosed in the absence of NEM was not inhibited on either side upon addition of NEM demonstrating the existence of a NEM-resistant fusion machinery. The results suggest that the fusogenic property of both the apical and the basolateral plasma membrane of MDCK cells differs from that typically observed in cells unable to polarize.     

Regulation of EGF signaling by cell polarity in MDCK kidney epithelial cells.

Although the presence of a dominant basolateral sorting signal ensures that the majority of newly synthesized epidermal growth factor (EGF) receptors are delivered directly to the basolateral surface in polarized epithelial cells, a fraction of the receptors are also delivered to the apical surface. Similar to most basolateral membrane proteins, the EGF receptor has an additional signal(s) that selectively targets molecules lacking a dominant basolateral signal to the apical surface. Although the physiological relevance of signal hierarchy is not known, alternative targeting may occur in different epithelial cell types or during development. The goal of this study, therefore, was to determine the effect of membrane domain location on EGF receptor function, focusing on EGF-induced MAP kinase signaling and DNA synthesis. Whereas ligand responsiveness was restricted to the basolateral domain in Madin-Darby canine kidney (MDCK) cells expressing a normal complement of receptors, apical ligand was effective if apical receptor density was increased by overexpression of an exogenous wild-type human gene. Unexpectedly, cells expressing apically localized, cytoplasmically truncated receptors, which behave as dominant negative mutations in other cell types, were also responsive to apical EGF. The cytoplasmically truncated molecules appear to have at least two effects: first, to increase the local concentration of ligand at the apical cell surface; and second, to facilitate activation of the relatively few native EGF receptors normally located at the apical surface. These results indicate that cell context is a critical determinant of receptor mutant protein phenotype.     

Dynamic regulation of IP3 receptor clustering and activity by IP3

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are intracellular Ca²⁺ channels. Their regulation by both IP₃ and Ca²⁺ allows interactions between IP₃Rs to generate a hierarchy of intracellular Ca²⁺ release events. These can progress from openings of single IP₃R, through near-synchronous opening of a few IP₃Rs within a cluster to much larger signals that give rise to regenerative Ca²⁺ waves that can invade the entire cell. We have used patch-clamp recording from excised nuclear membranes of DT40 cells expressing only IP₃R3 and shown that low concentrations of IP₃ rapidly and reversibly cause IP₃Rs to assemble into small clusters. In addition to bringing IP₃Rs close enough to allow Ca²⁺ released by one IP₃R to regulate the activity of its neighbors, clustering also retunes the regulation of IP₃Rs by IP₃ and Ca²⁺. At resting cytosolic [Ca²⁺], lone IP₃R are more sensitive to IP₃ and the mean channel open time (~10ms) is twice as long as for clustered IP₃R. When the cytosolic free [Ca²⁺] is increased to 1µM, to mimic the conditions that might prevail when an IP₃R within a cluster opens, clustered IP₃R are no longer inhibited and their gating becomes coupled. IP₃, by dynamically regulating IP₃R clustering, both positions IP₃R for optimal interactions between them and it serves to exaggerate the effects of Ca²⁺ within a cluster. During the course of these studies, we have observed that nuclear IP₃R stably express one of two single channel K ⁺ conductances (γ_K ~120 or 200pS). Here we demonstrate that for both states of the IP₃R, the effects of IP₃ on clustering are indistinguishable. These observations reinforce our conclusion that IP₃ dynamically regulates assembly of IP₃Rs into clusters that underlie the hierarchical recruitment of elementary Ca²⁺ release events.     

A role for caveolin and the urokinase receptor in integrin-mediated adhesion and signaling

The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with beta1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with beta1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes beta1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, beta1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with beta1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal beta1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.     

Cytosolic and nuclear calcium signaling in atrial myocytes: IP3-mediated calcium release and the role of mitochondria

In rabbit atrial myocytes Ca signaling has unique features due to the lack of transverse (t) tubules, the spatial arrangement of mitochondria and the contribution of inositol-1,4,5-trisphosphate (IP₃) receptor-induced Ca release (IICR). During excitation-contraction coupling action potential-induced elevation of cytosolic [Ca] originates in the cell periphery from Ca released from the junctional sarcoplasmic reticulum (j-SR) and then propagates by Ca-induced Ca release from non-junctional (nj-) SR toward the cell center. The subsarcolemmal region between j-SR and the first array of nj-SR Ca release sites is devoid of mitochondria which results in a rapid propagation of activation through this domain, whereas the subsequent propagation through the nj-SR network occurs at a velocity typical for a propagating Ca wave. Inhibition of mitochondrial Ca uptake with the Ca uniporter blocker Ru360 accelerates propagation and increases the amplitude of Ca transients (CaTs) originating from nj-SR. Elevation of cytosolic IP₃ levels by rapid photolysis of caged IP₃ has profound effects on the magnitude of subcellular CaTs with increased Ca release from nj-SR and enhanced CaTs in the nuclear compartment. IP₃ uncaging restricted to the nucleus elicites ‘mini’-Ca waves that remain confined to this compartment. Elementary IICR events (Ca puffs) preferentially originate in the nucleus in close physical association with membrane structures of the nuclear envelope and the nucleoplasmic reticulum. The data suggest that in atrial myocytes the nucleus is an autonomous Ca signaling domain where Ca dynamics are primarily governed by IICR.     

Rap1 is activated by erythropoietin or interleukin-3 and is involved in regulation of beta1 integrin-mediated hematopoietic cell adhesion

The CrkL adaptor protein is involved in signaling from the receptor for erythropoietin (Epo) as well as interleukin (IL)-3 and activates beta(1) integrin-mediated hematopoietic cell adhesion through its interaction with C3G, a guanine nucleotide exchange factor for Rap1. We demonstrate here that Epo as well as IL-3 activates Rap1 in an IL-3-dependent hematopoietic cell line, 32D, expressing the Epo receptor. The cytokine-induced activation of Rap1 was augmented in cells that inducibly overexpress CrkL or C3G. The CrkL-mediated enhancement of cell adhesion was inhibited by expression of a dominant negative mutant of Rap1, Rap1A-17N, whereas an activated mutant of Rap1, Rap1A-63E, activated beta(1) integrin-dependent adhesion of hematopoietic cells. In 32D cells, Rap1 was also activated by phorbol 12-myristate 13-acetate and ionomycin, which also enhanced cell adhesion to fibronectin, whereas, an inhibitor of phospholipase C, inhibited both cytokine-induced activation of Rap1 and cell adhesion. It was also demonstrated that Rap1 as well as CrkL is involved in signaling from the EpoR endogenously expressed in a human leukemic cell line, UT-7. These results suggest that Epo and IL-3 activate Rap1 at least partly through the CrkL-C3G complex as well as through additional pathways most likely involving phospholipase Cgamma and strongly implicate Rap1 in regulation of beta(1) integrin-mediated hematopoietic cell adhesion.     

Autocrine regulation of calcium influx and gonadotropin-releasing hormone secretion in hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult rats, cultured fetal hypothalamic cells, and immortalized GnRH-secreting neurons (GT1 cells). Their activation by GnRH agonists leads to an overall increase in the extracellular Ca2+-dependent pulsatile release of GnRH. Electrophysiological studies showed that GT1 cells exhibit spontaneous, extracellular Ca2+-dependent action potentials, and that their inward currents include Na+, T-type and L-type Ca2+ components. Several types of potassium channels, including apamin-sensitive Ca2+-controlled potassium (SK) channels, are also expressed in GT1 cells. Activation of GnRH receptors leads to biphasic changes in intracellular Ca2+ concentration ([Ca2+]i), with an early and extracellular Ca2+-independent peak and a sustained and extracellular Ca2+-dependent plateau phase. During the peak [Ca2+]i response, electrical activity is abolished due to transient hyperpolarization that is mediated by SK channels. This is followed by sustained depolarization and resumption of firing with increased spike frequency and duration. The agonist-induced depolarization and increased firing are independent of [Ca2+]i and are not mediated by inhibition of K+ currents, but by facilitation of a voltage-insensitive and store depletion-activated Ca2+-conducting inward current. The dual control of pacemaker activity by SK and store depletion-activated Ca2+ channels facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process accounts for the autoregulatory action of GnRH on its release from hypothalamic neurons.     

Mechanisms of hypotonicity-induced calcium signaling and integrin activation by arachidonic acid-derived inflammatory mediators in B cells

We previously characterized the initial steps in the activation of novel (calcium-permeant) nonselective cation channels (NSCCs) and calcium release-activated calcium channels in primary murine B lymphocytes. Phospholipase C products, namely diacylglycerol and d-myo-inositol 1,4,5-trisphosphate, were identified as proximal intracellular agonists of these respective channels following mechanical stimulation of B cells. However, neither the distal steps in NSCC activation nor the contribution of these channels to sustained mechanical signaling were defined in these previous studies. In this study, single cell measurements of intracellular Ca(2+) were used to define the mechanisms of NSCC activation and demonstrate a requirement for arachidonic acid liberated from diacylglycerol. Several arachidonic acid-derived derivatives were identified that trigger Ca(2+) entry into B cells, including the lipoxygenase product 5-hydroperoxyeicosatetranenoic acid and the cytochrome P450 hydroxylase product 20-hydroxyeicosatetraenoic; however, the cytochrome P450 epoxygenase product 5,6-epoxyeicosatrienoic acid is primarily responsible for hypotonicity-induced responses. In addition to regulating calcium entry, our data suggest that eicosanoid-activated NSCCs have a separate and direct role in regulating the avidity of integrins on B cells for extracellular matrix proteins, including ICAM-1 and VCAM-1. Thus, in addition to defining a novel osmotically activated signal transduction pathway in B cells, our results have broad implications for understanding how inflammatory mediators dynamically and rapidly regulate B cell adhesion and trafficking.     

Silencing of VAMP3 inhibits cell migration and integrin-mediated adhesion

Integrins are transmembrane receptors for cell adhesion to the extracellular matrix. In cell migration, integrins are endocytosed from the plasma membrane or the cell surface, transported in vesicles and exocytosed actively at the cell front. In the present study, we examined the roles of VAMP3, a SNARE protein that mediates exocytosis, in cell migration and integrin trafficking. Small interfering RNA (siRNA)-induced silencing of VAMP3 inhibited chemotactic cell migration by more than 60% without affecting cell proliferation. VAMP3 silencing reduced the levels of β1 integrin at the cell surface but had no effect on total cellular β1 integrin, indicating that VAMP3 is required for trafficking of β1 integrin to the plasma membrane. Furthermore, VAMP3 silencing diminished cell adhesion to laminin but not to fibronectin or collagen. Taken together, these data suggest that VAMP3-dependent integrin trafficking is crucial in cell migration and cell adhesion to laminin.     

Effects of integrin-mediated cell adhesion on plasma membrane lipid raft components and signaling

Anchorage dependence of cell growth, which is mediated by multiple integrin-regulated signaling pathways, is a key defense against cancer metastasis. Detachment of cells from the extracellular matrix triggers caveolin-1-dependent internalization of lipid raft components, which mediates suppression of Rho GTPases, Erk, and phosphatidylinositol 3-kinase in suspended cells. Elevation of cyclic adenosine monophosphate (cAMP) following cell detachment is also implicated in termination of growth signaling in suspended cells. Studies of integrins and lipid rafts, however, examined mainly ganglioside GM1 and glycosylphosphatidylinositol-linked proteins as lipid raft markers. In this study, we examine a wider range of lipid raft components. Whereas many raft components internalized with GM1 following cell detachment, flotillin2, connexin43, and Gα(s) remained in the plasma membrane. Loss of cell adhesion caused movement of many components from the lipid raft to the nonraft fractions on sucrose gradients, although flotillin2, connexin43, and H-Ras were resistant. Gα(s) lost its raft association, concomitant with cAMP production. Modification of the lipid tail of Gα(s) to increase its association with ordered domains blocked the detachment-induced increase in cAMP. These data define the effects of that integrin-mediated adhesion on the localization and behavior of a variety of lipid raft components and reveal the mechanism of the previously described elevation of cAMP after cell detachment.     

Mechanisms of adhesion among cells of the early chick blastoderm. Role of calcium ions in the adhesion of extraembryonic endoderm cells

Summary Cells from the extraembryonic endoderm of the gastrulating chick embryo adhere to one another in the absence of divalent cations. The addition of Mg²⁺ ions to the medium has no effect on the aggregation kinetics but the addition of Ca²⁺ ions increases the number of cells which aggregate and also stabilizes adhesion. Some aggregation also occurs when cells are suspended in saline devoid of Ca²⁺ and Mg²⁺ ions and supplemented with EGTA, a Ca²⁺ ion complexing agent, but adhesion is not stabilized. Shear sensitive and shear resistant bonds form in Ca-containing as well as in EGTA-containing saline. These results suggest that extraembryonic endoderm cells have Ca²⁺ indepedent and Ca²⁺ dependent mechanisms of adhesion.