Nucleoplasmic calcium is required for cell proliferation

Ca(2+) signals regulate cell proliferation, but the spatial and temporal specificity of these signals is unknown. Here we use selective buffers of nucleoplasmic or cytoplasmic Ca(2+) to determine that cell proliferation depends upon Ca(2+) signals within the nucleus rather than in the cytoplasm. Nuclear Ca(2+) signals stimulate cell growth rather than inhibit apoptosis and specifically permit cells to advance through early prophase. Selective buffering of nuclear but not cytoplasmic Ca(2+) signals also impairs growth of tumors in vivo. These findings reveal a major physiological and potential pathophysiological role for nucleoplasmic Ca(2+) signals and suggest that this information can be used to design novel therapeutic strategies to regulate conditions of abnormal cell growth.     

Differential codes for free Ca(2+)-calmodulin signals in nucleus and cytosol

BACKGROUND: Many targets of calcium signaling pathways are activated or inhibited by binding the Ca(2+)-liganded form of calmodulin (Ca(2+)-CaM). Here, we test the hypothesis that local Ca(2+)-CaM-regulated signaling processes can be selectively activated by local intracellular differences in free Ca(2+)-CaM concentration. RESULTS: Energy-transfer confocal microscopy of a fluorescent biosensor was used to measure the difference in the concentration of free Ca(2+)-CaM between nucleus and cytoplasm. Strikingly, short receptor-induced calcium spikes produced transient increases in free Ca(2+)-CaM concentration that were of markedly higher amplitude in the cytosol than in the nucleus. In contrast, prolonged increases in calcium led to equalization of the nuclear and cytosolic free Ca(2+)-CaM concentrations over a period of minutes. Photobleaching recovery and translocation measurements with fluorescently labeled CaM showed that equalization is likely to be the result of a diffusion-mediated net translocation of CaM into the nucleus. The driving force for equalization is a higher Ca(2+)-CaM-buffering capacity in the nucleus compared with the cytosol, as the direction of the free Ca(2+)-CaM concentration gradient and of CaM translocation could be reversed by expressing a Ca(2+)-CaM-binding protein at high concentration in the cytosol. CONCLUSIONS: Subcellular differences in the distribution of Ca(2+)-CaM-binding proteins can produce gradients of free Ca(2+)-CaM concentration that result in a net translocation of CaM. This provides a mechanism for dynamically regulating local free Ca(2+)-CaM concentrations, and thus the local activity of Ca(2+)-CaM targets. Free Ca(2+)-CaM signals in the nucleus remain low during brief or low-frequency calcium spikes, whereas high-frequency spikes or persistent increases in calcium cause translocation of CaM from the cytoplasm to the nucleus, resulting in similar concentrations of nuclear and cytosolic free Ca(2+)-CaM.     

Distinct calcium signaling pathways regulate calmodulin gene expression in tobacco

Cold shock and wind stimuli initiate Ca(2+) transients in transgenic tobacco (Nicotiana plumbaginifolia) seedlings (named MAQ 2.4) containing cytoplasmic aequorin. To investigate whether these stimuli initiate Ca(2+) pathways that are spatially distinct, stress-induced nuclear and cytoplasmic Ca(2+) transients and the expression of a stress-induced calmodulin gene were compared. Tobacco seedlings were transformed with a construct that encodes a fusion protein between nucleoplasmin (a major oocyte nuclear protein) and aequorin. Immunocytochemical evidence indicated targeting of the fusion protein to the nucleus in these plants, which were named MAQ 7.11. Comparison between MAQ 7.11 and MAQ 2.4 seedlings confirmed that wind stimuli and cold shock invoke separate Ca(2+) signaling pathways. Partial cDNAs encoding two tobacco calmodulin genes, NpCaM-1 and NpCaM-2, were identified and shown to have distinct nucleotide sequences that encode identical polypeptides. Expression of NpCaM-1, but not NpCaM-2, responded to wind and cold shock stimulation. Comparison of the Ca(2+) dynamics with NpCaM-1 expression after stimulation suggested that wind-induced NpCaM-1 expression is regulated by a Ca(2+) signaling pathway operational predominantly in the nucleus. In contrast, expression of NpCaM-1 in response to cold shock is regulated by a pathway operational predominantly in the cytoplasm.     

Hepatocyte growth factor and c-Met promote dendritic maturation during hippocampal neuron differentiation via the Akt pathway

During central nervous system development, growth factors and their associated receptor protein tyrosine kinases regulate many neuronal functions such as neurite extension and dendrite maturation. Hepatocyte growth factor (HGF) and its receptor, c-Met, can promote formation of neurites and enhance elaboration of dendrites in mature neurons, but their effects on the early stages of dendrite maturation in hippocampal neurons and the signaling pathways by which they promote dendrite formation have not been studied. Exogenous HGF treatment effectively enhanced the phosphorylation and activation of c-Met in cultured hippocampal neurons at 4 days in vitro. HGF treatment increased the number of dendrites and promoted dendrite elongation in these neurons. Consistent with these results, HGF activated Akt, which phosphorylates glycogen synthase kinase-3beta (GSK-3beta) to inactivate it, and reduced phosphorylation of microtubule-associated protein 2 (MAP2), which can promote microtubule polymerization and dendrite elongation when dephosphorylated. Conversely, pharmacological inhibition of c-Met with its specific inhibitor, PHA-665752, or genetic knock-down of c-Met with short hairpin RNAs (shRNAs) suppressed HGF-induced phosphorylation of Akt and GSK-3beta, increased phosphorylation of MAP2, and reduced dendrite number and length in cultured hippocampal neurons. Moreover, suppressing c-Met with PHA-665752 or by shRNA decreased MAP2 expression. Inhibiting Akt activity with the phosphoinositide-3-kinase inhibitor LY294002 or Akt inhibitor X suppressed HGF-induced phosphorylation of GSK-3beta, increased MAP2 phosphorylation, and blocked the ability of HGF to enhance dendritic length. These observations indicate that HGF and c-Met can regulate the early stages of dendrite maturation via activation of the Akt/GSK-3beta pathway.     

Mitochondria adjust Ca(2+) signaling regime to a pattern of stimulation in salivary acinar cells

The salivary acinar cells have unique Ca(2+) signaling machinery that ensures an extensive secretion. The agonist-induced secretion is governed by Ca(2+) signals originated from the endoplasmic reticulum (ER) followed by a store-operated Ca(2+) entry (SOCE). During tasting and chewing food a frequency of parasympathetic stimulation increases up to ten fold, entailing cells to adapt its Ca(2+) machinery to promote ER refilling and ensure sustained SOCE by yet unknown mechanism. By employing a combination of fluorescent Ca(2+) imaging in the cytoplasm and inside cellular organelles (ER and mitochondria) we described the role of mitochondria in adjustment of Ca(2+) signaling regime and ER refilling according to a pattern of agonist stimulation. Under the sustained stimulation, SOCE is increased proportionally to the degree of ER depletion. Cell adapts its Ca(2+) handling system directing more Ca(2+) into mitochondria via microdomains of high [Ca(2+)] providing positive feedback on SOCE while intra-mitochondrial tunneling provides adequate ER refilling. In the absence of an agonist, the bulk of ER refilling occurs through Ca(2+)-ATPase-mediated Ca(2+) uptake within subplasmalemmal space. In conclusion, mitochondria play a key role in the maintenance of sustained SOCE and adequate ER refilling by regulating Ca(2+) fluxes within the cell that may represent an intrinsic adaptation mechanism to ensure a long-lasting secretion.     

肝细胞生长因子及受体传递的调节与内吞作用

肝细胞生长因子(HGF)是一种具有多重功能的细胞调控因子。HGF与其受体Met酪氨酸激酶(c-Met)的结合可激发多种生物学反应,从而调节细胞的增殖、分化、形态发生和侵袭运动等。有多种因素参与了HGF/c-Met信号传导的调控,从而防止信号的过度放大,其中Cbl1、Rab、泛素化激酶和HGF/c-Met的内吞等发挥了重要的作用。因此,对HGF/c-Met内吞过程的研究,了解内吞对于HGF/c-Met的信号传导及其调控的影响,探讨HGF/c-Met信号传导通路的调控机理和相互作用模式,可进一步阐明HGF/c-Met信号传导的调控机制,从而验证肝细胞中内吞作用直接调节HGF/c-Met信号通路的作用机制。     

Ca2+-dependent monomer and dimer formation switches CAPRI Protein between Ras GTPase-activating protein (GAP) and RapGAP activities

CAPRI is a member of the GAP1 family of GTPase-activating proteins (GAPs) for small G proteins. It is known to function as an amplitude sensor for intracellular Ca(2+) levels stimulated by extracellular signals and has a catalytic domain with dual RasGAP and RapGAP activities. Here, we have investigated the mechanism that switches CAPRI between its two GAP activities. We demonstrate that CAPRI forms homodimers in vitro and in vivo in a Ca(2+)-dependent manner. The site required for dimerization was pinpointed by deletion and point mutations to a helix motif that forms a hydrophobic face in the extreme C-terminal tail of the CAPRI protein. Deletion of this helix motif abolished dimer formation but did not affect translocation of CAPRI to the plasma membrane upon cell stimulation with histamine. We found that dimeric and monomeric CAPRI coexist in cells and that the ratio of dimeric to monomeric CAPRI increases upon cell stimulation with histamine. Free Ca(2+) at physiologically relevant concentrations was both necessary and sufficient for dimer formation. Importantly, the monomeric and dimeric forms of CAPRI exhibited differential GAP activities in vivo; the wild-type form of CAPRI had stronger RapGAP activity than RasGAP activity, whereas a monomeric CAPRI mutant showed stronger RasGAP than RapGAP activity. These results demonstrate that CAPRI switches between its dual GAP roles by forming monomers or homodimers through a process regulated by Ca(2+). We propose that Ca(2+)-dependent dimerization of CAPRI may serve to coordinate Ras and Rap1 signaling pathways.     

Simulating calcium influx and free calcium concentrations in yeast

Yeast can proliferate in environments containing very high Ca(2+) primarily due to the activity of vacuolar Ca(2+) transporters Pmc1 and Vcx1. Yeast mutants lacking these transporters fail to grow in high Ca(2+) environments, but growth can be restored by small increases in environmental Mg(2+). Low extracellular Mg(2+) appeared to competitively inhibit novel Ca(2+) influx pathways and to diminish the concentration of free Ca(2+) in the cytoplasm, as judged from the luminescence of the photoprotein aequorin. These Mg(2+)-sensitive Ca(2+) influx pathways persisted in yvc1 cch1 double mutants. Based on mathematical models of the aequorin luminescence traces, we propose the existence in yeast of at least two Ca(2+) transporters that undergo rapid feedback inhibition in response to elevated cytosolic free Ca(2+) concentration. Finally, we show that Vcx1 helps return cytosolic Ca(2+) toward resting levels after shock with high extracellular Ca(2+) much more effectively than Pmc1 and that calcineurin, a protein phosphatase regulator of Vcx1 and Pmc1, had no detectable effects on these factors within the first few minutes of its activation. Therefore, computational modeling of Ca(2+) transport and signaling in yeast can provide important insights into the dynamics of this complex system.     

TRPV channels and modulation by hepatocyte growth factor/scatter factor in human hepatoblastoma (HepG2) cells

Using patch clamp and Ca(2+) imaging techniques, we have studied Ca(2+) entry pathways in human hepatoblastoma (HepG2) cells. These cells express the mRNA of TRPV1, TRPV2, TRPV3 and TRPV4 channels, but not those of TRPV5 and TRPV6. Functional assessment showed that capsaicin (10 microM), 4alpha-phorbol-12,13-didecanoate (4alphaPDD, 1 microM), arachidonic acid (10 microM), hypotonic stress, and heat all stimulated increases in [Ca(2+)](i) within minutes. The increase in [Ca(2+)](i) depended on extracellular Ca(2+) and on the transmembrane potential, which indicated that both driving forces affected Ca(2+) entry. Capsaicin also stimulated an increase in [Ca(2+)](i) in nominally Ca(2+)-free solutions, which was compatible with the receptor functioning as a Ca(2+) release channel. Hepatocyte growth factor/scatter factor (HGF/SF) modulated Ca(2+) entry. Ca(2+) influx was greater in HepG2 cells incubated with HGF/SF (20 ng/ml for 20 h) compared with non-stimulated cells, but this occurred only in those cells with a migrating phenotype as determined by presence of a lamellipodium and trailing footplate. The effect of capsaicin on [Ca(2+)](i) was greater in migrating HGF/SF-treated cells, and this was inhibited by capsazepine. The difference between control and HGF/SF-treated cells was not found in Ca(2+)-free solutions. 4alphaPDD also had no greater effect on HGF/SF-treated cells. We conclude that TRPV1 and TRPV4 channels provide Ca(2+) entry pathways in HepG2 cells. HGF/SF increases Ca(2+) entry via TRPV1, but not via TRPV4. This rise in [Ca(2+)](i) may constitute an early response of a signalling cascade that gives rise to cell locomotion and the migratory phenotype.     

657WTAPELL663 motif of the photoreceptor ROS-GC1: a general phototransduction switch

This study documents the identity of an intriguing transduction mechanism of the [Ca(2+)](i) signals by the photoreceptor ROS-GC1. Despite their distal residences and operational modes in phototransduction, the two GCAPs transmit and activate ROS-GC1 through a common Ca(2+) transmitter switch (Ca(2+)TS). A combination of immunoprecipitation, fluorescent spectroscopy, mutational analyses and reconstitution studies has been used to demonstrate that the structure of this switch is (657)WTAPELL(663). The two Ca(2+) signaling GCAP pathways converge in Ca(2+)TS, get transduced, activate ROS-GC1, generate the LIGHT signal second messenger cyclic GMP and yet functionally perform divergent operations of the phototransduction machinery. The findings define a new Ca(2+)-modulated photoreceptor ROS-GC transduction model; it is depicted and discussed for its application to processing the different shades of LIGHT.     

CD44-independent activation of the Met signaling pathway by HGF and InlB

Listeria monocytogenes is a facultative intracellular Gram-positive bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The L. monocytogenes surface protein InlB interacts with c-Met, the hepatocyte growth factor (HGF) receptor, inducing bacterial internalization in numerous non-phagocytic cells. As InlB and HGF are known to trigger similar signaling pathways upon c-Met activation, we investigated the role of CD44, and more specifically its isoform CD44v6, in bacterial internalization in non-phagocytic cells. Indeed, CD44, the hyaluronic acid transmembrane receptor, and more specifically its isoform CD44v6 have been reported as necessary for the activation of c-Met upon the interaction with either the endogenous ligand HGF or the L. monocytogenes surface protein InlB. Our results demonstrate that, in the cell lines that we used, CD44 receptors play no role in the activation of c-Met, neither during L. monocytogenes entry, nor upon HGF activation. Furthermore, none of the CD44 isoforms was recruited at the L. monocytogenes entry site, and depletion by siRNA of total CD44 or of CD44v6 isoform did not reduce bacterial infections. Conversely, the overexpression of CD44 or CD44v6 had no significant effect on L. monocytogenes internalization. Together our results reveal that the activation of c-Met can be largely CD44-independent.