The endoplasmic reticulum stress sensor IRE1α modulates macrophage metabolic function during Brucella abortus infection.

Endoplasmic reticulum (ER) stress plays a major role in several inflammatory disorders. ER stress induces the unfolded protein response (UPR), a conserved response broadly associated with innate immunity and cell metabolic function in various scenarios. Brucella abortus, an intracellular pathogen, triggers the UPR via Stimulator of interferon genes (STING), an important regulator of macrophage metabolism during B. abortus infection. However, whether ER stress pathways underlie macrophage metabolic function during B. abortus infection remains to be elucidated. Here, we showed that the UPR sensor inositol-requiring enzyme 1α (IRE1α) is as an important component regulating macrophage immunometabolic function. In B. abortus infection, IRE1α supports the macrophage inflammatory profile, favoring M1-like macrophages. IRE1α drives the macrophage metabolic reprogramming in infected macrophages, contributing to the reduced oxidative phosphorylation and increased glycolysis. This metabolic reprogramming is probably associated with the IRE1α-dependent expression and stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), an important molecule involved in cell metabolism that sustains the inflammatory profile in B. abortus-infected macrophages. Accordingly, we demonstrated that IRE1α favors the generation of mitochondrial reactive oxygen species (mROS) which has been described as an HIF-1α stabilizing factor. Furthermore, in infected macrophages, IRE1α drives the production of nitric oxide and the release of IL-1ß. Collectively, these data unravel a key mechanism linking the UPR and the immunometabolic regulation of macrophages in Brucella infection and highlight IRE1α as a central pathway regulating macrophage metabolic function during infectious diseases.

Galectin-3 regulates proinflammatory cytokine function and favours Brucella abortus chronic replication in macrophages and mice.

In this study, we provide evidence that galectin-3 (Gal-3) plays an important role in Brucella abortus infection. Our results showed increased Gal-3 expression and secretion in B. abortus infected macrophages and mice. Additionally, our findings indicate that Gal-3 is dispensable for Brucella-containing vacuoles disruption, inflammasome activation and pyroptosis. On the other hand, we observed that Brucella-induced Gal-3 expression is crucial for induction of molecules associated to type I IFN signalling pathway, such as IFN-ß: Interferon beta (IFN-ß), C-X-C motif chemokine ligand 10 (CXCL10) and guanylate-binding proteins. Gal-3 KO macrophages showed reduced bacterial numbers compared to wild-type cells, suggesting that Gal-3 facilitates bacterial replication in vitro. Moreover, priming Gal-3 KO cells with IFN-ß favoured B. abortus survival in macrophages. Additionally, we also observed that Gal-3 KO mice are more resistant to B. abortus infection and these animals showed elevated production of proinflammatory cytokines when compared to control mice. Finally, we observed an increased recruitment of macrophages, dendritic cells and neutrophils in spleens of Gal-3 KO mice compared to wild-type animals. In conclusion, this study demonstrated that Brucella-induced Gal-3 is detrimental to host and this molecule is implicated in inhibition of recruitment and activation of immune cells, which promotes B. abortus spread and aggravates the infection. TAKE AWAYS: Brucella abortus infection upregulates galectin-3 expression Galectin-3 regulates guanylate-binding proteins expression but is not required for Brucella-containing vacuole disruption Galectin-3 modulates proinflammatory cytokine production during bacterial infection Galectin-3 favours Brucella replication.

STING regulates metabolic reprogramming in macrophages via HIF-1α during Brucella infection.

Macrophages metabolic reprogramming in response to microbial insults is a major determinant of pathogen growth or containment. Here, we reveal a distinct mechanism by which stimulator of interferon genes (STING), a cytosolic sensor that regulates innate immune responses, contributes to an inflammatory M1-like macrophage profile upon Brucella abortus infection. This metabolic reprogramming is induced by STING-dependent stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), a global regulator of cellular metabolism and innate immune cell functions. HIF-1α stabilization reduces oxidative phosphorylation and increases glycolysis during infection with B. abortus and, likewise, enhances nitric oxide production, inflammasome activation and IL-1ß release in infected macrophages. Furthermore, the induction of this inflammatory profile participates in the control of bacterial replication since absence of HIF-1α renders mice more susceptible to B. abortus infection. Mechanistically, activation of STING by B. abortus infection drives the production of mitochondrial reactive oxygen species (mROS) that ultimately influences HIF-1α stabilization. Moreover, STING increases the intracellular succinate concentration in infected macrophages, and succinate pretreatment induces HIF-1α stabilization and IL-1ß release independently of its cognate receptor GPR91. Collectively, these data demonstrate a pivotal mechanism in the immunometabolic regulation of macrophages during B. abortus infection that is orchestrated by STING via HIF-1α pathway and highlight the metabolic reprogramming of macrophages as a potential treatment strategy for bacterial infections.

Impact of STING Inflammatory Signaling during Intracellular Bacterial Infections.

The early detection of bacterial pathogens through immune sensors is an essential step in innate immunity. STING (Stimulator of Interferon Genes) has emerged as a key mediator of inflammation in the setting of infection by connecting pathogen cytosolic recognition with immune responses. STING detects bacteria by directly recognizing cyclic dinucleotides or indirectly by bacterial genomic DNA sensing through the cyclic GMP-AMP synthase (cGAS). Upon activation, STING triggers a plethora of powerful signaling pathways, including the production of type I interferons and proinflammatory cytokines. STING activation has also been associated with the induction of endoplasmic reticulum (ER) stress and the associated inflammatory responses. Recent reports indicate that STING-dependent pathways participate in the metabolic reprogramming of macrophages and contribute to the establishment and maintenance of a robust inflammatory profile. The induction of this inflammatory state is typically antimicrobial and related to pathogen clearance. However, depending on the infection, STING-mediated immune responses can be detrimental to the host, facilitating bacterial survival, indicating an intricate balance between immune signaling and inflammation during bacterial infections. In this paper, we review recent insights regarding the role of STING in inducing an inflammatory profile upon intracellular bacterial entry in host cells and discuss the impact of STING signaling on the outcome of infection. Unraveling the STING-mediated inflammatory responses can enable a better understanding of the pathogenesis of certain bacterial diseases and reveal the potential of new antimicrobial therapy.

Lack of Interleukin-6 Affects IFN-γ and TNF-α Production and Early In Vivo Control of Brucella abortus Infection.

Interleukin-6 (IL-6) is a pleiotropic cytokine promptly produced in response to infections, which contributes to host defense through the stimulation of acute phase immune responses. Brucella abortus is an intracellular bacterium that causes chronic disease in humans and domestic animals and triggers a robust immune response, characterized by the production of inflammatory cytokines. However, the mechanisms of IL-6-related immune responses in the context of Brucella infections are not completely understood. In this report, we describe an increased susceptibility of IL-6 knockout (KO) mice in the early phase of Brucella infection. Furthermore, we demonstrate that IL-6 is required for interferon (IFN)-γ and tumor necrosis factor (TNF)-α induction by infected splenocytes, indicating a protective role for IL-6 against B. abortus that parallels with Th1 type of immune response. Additionally, IL-6 KO mice exhibited reduced splenomegaly during the early phase of the infection. Corroborating this result, IL-6 KO mice displayed reduced numbers of macrophages, dendritic cells, and neutrophils in the spleen and reduced myeloperoxidase activity in the liver compared to wild-type infected mice. However, we demonstrate that IL-6 is not involved in B. abortus intracellular restriction in mouse macrophages. Taken together, our findings demonstrate that IL-6 contributes to host resistance during the early phase of B. abortus infection in vivo, and suggest that its protective role maybe partially mediated by proinflammatory immune responses and immune cell recruitment.

Canonical and Non-canonical Inflammasome Activation by Outer Membrane Vesicles Derived From Bordetella pertussis.

Outer Membrane Vesicles (OMVs) derived from different Gram-negative bacteria have been proposed as an attractive vaccine platform because of their own immunogenic adjuvant properties. Pertussis or whooping cough is a highly contagious vaccine-preventable respiratory disease that resurged during the last decades in many countries. In response to the epidemiological situation, new boosters have been incorporated into vaccination schedules worldwide and new vaccine candidates have started to be designed. Particularly, our group designed a new pertussis vaccine candidate based on OMVs derived from Bordetella pertussis (BpOMVs). To continue with the characterization of the immune response induced by our OMV based vaccine candidate, this work aimed to investigate the ability of OMVs to activate the inflammasome pathway in macrophages. We observed that NLRP3, caspase-1/11, and gasdermin-D (GSDMD) are involved in inflammasome activation by BpOMVs. Moreover, we demonstrated that BpOMVs as well as transfected B. pertussis lipooligosaccharide (BpLOS) induce caspase-11 (Casp11) and guanylate-binding proteins (GBPs) dependent non-canonical inflammasome activation. Our results elucidate the mechanism by which BpOMVs trigger one central pathway of the innate response activation that is expected to skew the adaptive immune response elicited by BpOMVs vaccination.

NLRP6 Plays an Important Role in Early Hepatic Immunopathology Caused by Schistosoma mansoni Infection.

Schistosomiasis is a debilitating parasitic disease that affects more than 200 million people worldwide and causes approximately 280,000 deaths per year. Inside the definitive host, eggs released by Schistosoma mansoni lodge in the intestine and especially in the liver where they induce a granulomatous inflammatory process, which can lead to fibrosis. The molecular mechanisms initiating or promoting hepatic granuloma formation remain poorly understood. Inflammasome activation has been described as an important pathway to induce pathology mediated by NLRP3 receptor. Recently, other components of the inflammasome pathway, such as NLRP6, have been related to liver diseases and fibrotic processes. Nevertheless, the contribution of these components in schistosomiasis-associated pathology is still unknown. In the present study, using dendritic cells, we demonstrated that NLRP6 sensor is important for IL-1ß production and caspase-1 activation in response to soluble egg antigens (SEA). Furthermore, the lack of NLRP6 has been shown to significantly reduce periovular inflammation, collagen deposition in hepatic granulomas and mRNA levels of α-SMA and IL-13. Livers of Nlrp6-/- mice showed reduced levels of CXCL1/KC, CCL2, CCL3, IL-5, and IL-10 as well as Myeloperoxidase (MPO) and Eosinophilic Peroxidase (EPO) enzymatic activity. Consistently, the frequency of macrophage and neutrophil populations were lower in the liver of NLRP6 knockout mice, after 6 weeks of infection. Finally, it was further demonstrated that the onset of hepatic granuloma and collagen deposition were also compromised in Caspase-1-/- , IL-1R-/- and Gsdmd-/- mice. Our findings suggest that the NLRP6 inflammasome is an important component for schistosomiasis-associated pathology.

The role of the adaptor molecule STING during Schistosoma mansoni infection.

Schistosomiasis is a human parasitic disease responsible for serious consequences for public health, as well as severe socioeconomic impacts in developing countries. Here, we provide evidence that the adaptor molecule STING plays an important role in Schistosoma mansoni infection. S. mansoni DNA is sensed by cGAS leading to STING activation in murine embryonic fibroblasts (MEFs). Sting-/- and C57BL/6 (WT) mice were infected with schistosome cercariae in order to assess parasite burden and liver pathology. Sting-/- mice showed worm burden reduction but no change in the number of eggs or granuloma numbers and area when compared to WT animals. Immunologically, a significant increase in IFN-γ production by the spleen cells was observed in Sting-/- animals. Surprisingly, Sting-/- mice presented an elevated percentage of neutrophils in lungs, bronchoalveolar lavage, and spleens. Moreover, Sting-/- neutrophils exhibited increased survival rate, but similar ability to kill schistosomula in vitro when stimulated with IFN-γ when compared to WT cells. Finally, microbiota composition was altered in Sting-/- mice, revealing a more inflammatory profile when compared to WT animals. In conclusion, this study demonstrates that STING signaling pathway is important for S. mansoni DNA sensing and the lack of this adaptor molecule leads to enhanced resistance to infection.

Guanylate-binding proteins at the crossroad of noncanonical inflammasome activation during bacterial infections.

The immune system is armed with a broad range of receptors to detect and initiate the elimination of bacterial pathogens. Inflammasomes are molecular platforms that sense a diverse range of microbial insults to develop appropriate host response. In that context, noncanonical inflammasome arose as a sensor for Gram-negative bacteria-derived LPS leading to the control of infections. This review describes the role of caspase-11/gasdermin-D-dependent immune response against Gram-negative bacteria and presents an overview of guanylate-binding proteins (GBPs) at the interface of noncanonical inflammasome activation. Indeed, caspase-11 acts as a receptor for LPS and this interaction elicits caspase-11 autoproteolysis that is required for its optimal catalytic activity. Gasdermin-D is cleaved by activated caspase-11 generating an N-terminal domain that is inserted into the plasmatic membrane to form pores that induce pyroptosis, a cell death program involved in intracellular bacteria elimination. This mechanism also promotes IL-1ß release and potassium efflux that connects caspase-11 to NLRP3 activation. Furthermore, GBPs display many features to allow LPS recognition by caspase-11, initiating the noncanonical inflammasome response prompting the immune system to control bacterial infections. In this review, we discuss the recent findings and nuances related to this mechanism and its biological functions.

Brucella abortus Cyclic Dinucleotides Trigger STING-Dependent Unfolded Protein Response That Favors Bacterial Replication.

Brucella abortus is a facultative intracellular bacterium that causes brucellosis, a prevalent zoonosis that leads to abortion and infertility in cattle, and undulant fever, debilitating arthritis, endocarditis, and meningitis in humans. Signaling pathways triggered by B. abortus involves stimulator of IFN genes (STING), which leads to production of type I IFNs. In this study, we evaluated the pathway linking the unfolded protein response (UPR) and the endoplasmic reticulum-resident transmembrane molecule STING, during B. abortus infection. We demonstrated that B. abortus infection induces the expression of the UPR target gene BiP and XBP1 in murine macrophages through a STING-dependent pathway. Additionally, we also observed that STING activation was dependent on the bacterial second messenger cyclic dimeric GMP. Furthermore, the Brucella-induced UPR is crucial for induction of multiple molecules linked to type I IFN signaling pathway, such as IFN-ß, IFN regulatory factor 1, and guanylate-binding proteins. Furthermore, IFN-ß is also important for the UPR induction during B. abortus infection. Indeed, IFN-ß shows a synergistic effect in inducing the IRE1 axis of the UPR. In addition, priming cells with IFN-ß favors B. abortus survival in macrophages. Moreover, Brucella-induced UPR facilitates bacterial replication in vitro and in vivo. Finally, these results suggest that B. abortus-induced UPR is triggered by bacterial cyclic dimeric GMP, in a STING-dependent manner, and that this response supports bacterial replication. In summary, association of STING and IFN-ß signaling pathways with Brucella-induced UPR unravels a novel link between innate immunity and endoplasmic reticulum stress that is crucial for bacterial infection outcome.

Guanylate-binding protein 5 licenses caspase-11 for Gasdermin-D mediated host resistance to Brucella abortus infection.

Innate immune response against Brucella abortus involves activation of Toll-like receptors (TLRs) and NOD-like receptors (NLRs). Among the NLRs involved in the recognition of B. abortus are NLRP3 and AIM2. Here, we demonstrate that B. abortus triggers non-canonical inflammasome activation dependent on caspase-11 and gasdermin-D (GSDMD). Additionally, we identify that Brucella-LPS is the ligand for caspase-11 activation. Interestingly, we determine that B. abortus is able to trigger pyroptosis leading to pore formation and cell death, and this process is dependent on caspase-11 and GSDMD but independently of caspase-1 protease activity and NLRP3. Mice lacking either caspase-11 or GSDMD were significantly more susceptible to infection with B. abortus than caspase-1 knockout or wild-type animals. Additionally, guanylate-binding proteins (GBPs) present in mouse chromosome 3 participate in the recognition of LPS by caspase-11 contributing to non-canonical inflammasome activation as observed by the response of Gbpchr3-/- BMDMs to bacterial stimulation. We further determined by siRNA knockdown that among the GBPs contained in mouse chromosome 3, GBP5 is the most important for Brucella LPS to be recognized by caspase-11 triggering IL-1ß secretion and LDH release. Additionally, we observed a reduction in neutrophil, dendritic cell and macrophage influx in spleens of Casp11-/- and Gsdmd-/- compared to wild-type mice, indicating that caspase-11 and GSDMD are implicated in the recruitment and activation of immune cells during Brucella infection. Finally, depletion of neutrophils renders wild-type mice more susceptible to Brucella infection. Taken together, these data suggest that caspase-11/GSDMD-dependent pyroptosis triggered by B. abortus is important to infection restriction in vivo and contributes to immune cell recruitment and activation.

miR-181a-5p Regulates TNF-α and miR-21a-5p Influences Gualynate-Binding Protein 5 and IL-10 Expression in Macrophages Affecting Host Control of Brucella abortus Infection.

Brucella abortus is a Gram-negative intracellular bacterium that causes a worldwide zoonosis termed brucellosis, which is characterized as a debilitating infection with serious clinical manifestations leading to severe complications. In spite of great advances in studies involving host-B. abortus interactions, there are many gaps related to B. abortus modulation of the host immune response through regulatory mechanisms. Here, we deep sequenced small RNAs from bone marrow-derived macrophages infected with B. abortus, identifying 69 microRNAs (miRNAs) that were differentially expressed during infection. We further validated the expression of four upregulated and five downregulated miRNAs during infection in vitro that displayed the same profile in spleens from infected mice at 1, 3, or 6 days post-infection. Among these miRNAs, mmu-miR-181a-5p (upregulated) or mmu-miR-21a-5p (downregulated) were selected for further analysis. First, we determined that changes in the expression of both miRNAs induced by infection were dependent on the adaptor molecule MyD88. Furthermore, evaluating putative targets of mmu-miR-181a-5p, we demonstrated this miRNA negatively regulates TNF-α expression following Brucella infection. By contrast, miR-21a-5p targets included a negative regulator of IL-10, programmed cell death protein 4, and several guanylate-binding proteins (GBPs). As a result, during infection, miR-21a-5p led to upregulation of IL-10 expression and downregulation of GBP5 in macrophages infected with Brucella. Since GBP5 and IL-10 are important molecules involved in host control of Brucella infection, we decided to investigate the role of mmu-miR-21a-5p in bacterial replication in macrophages. We observed that treating macrophages with a mmu-miR-21a-5p mimic enhanced bacterial growth, whereas transfection of its inhibitor reduced Brucella load in macrophages. Taken together, the results indicate that downregulation of mmu-miR-21a-5p induced by infection increases GBP5 levels and decreases IL-10 expression thus contributing to bacterial control in host cells.

Brucella abortus Triggers a cGAS-Independent STING Pathway To Induce Host Protection That Involves Guanylate-Binding Proteins and Inflammasome Activation.

Immunity against microbes depends on recognition of pathogen-associated molecular patterns by innate receptors. Signaling pathways triggered by Brucella abortus DNA involves TLR9, AIM2, and stimulator of IFN genes (STING). In this study, we observed by microarray analysis that several type I IFN-associated genes, such as IFN-ß and guanylate-binding proteins (GBPs), are downregulated in STING knockout (KO) macrophages infected with Brucella or transfected with DNA. Additionally, we determined that STING and cyclic GMP-AMP synthase (cGAS) are important to engage the type I IFN pathway, but only STING is required to induce IL-1ß secretion, caspase-1 activation, and GBP2 and GBP3 expression. Furthermore, we determined that STING but not cGAS is critical for host protection against Brucella infection in macrophages and in vivo. This study provides evidence of a cGAS-independent mechanism of STING-mediated protection against an intracellular bacterial infection. Additionally, infected IFN regulatory factor-1 and IFNAR KO macrophages had reduced GBP2 and GBP3 expression and these cells were more permissive to Brucella replication compared with wild-type control macrophages. Because GBPs are critical to target vacuolar bacteria, we determined whether GBP2 and GBPchr3 affect Brucella control in vivo. GBPchr3 but not GBP2 KO mice were more susceptible to bacterial infection, and small interfering RNA treated-macrophages showed reduction in IL-1ß secretion and caspase-1 activation. Finally, we also demonstrated that Brucella DNA colocalizes with AIM2, and AIM2 KO mice are less resistant to B. abortus infection. In conclusion, these findings suggest that the STING-dependent type I IFN pathway is critical for the GBP-mediated release of Brucella DNA into the cytosol and subsequent activation of AIM2.

Modulation of Microtubule Dynamics Affects Brucella abortus Intracellular Survival, Pathogen-Containing Vacuole Maturation, and Pro-inflammatory Cytokine Production in Infected Macrophages.

The microtubule (MT) cytoskeleton regulates several cellular processes related to the immune system. For instance, an intricate intracellular transport mediated by MTs is responsible for the proper localization of vesicular receptors of innate immunity and its adaptor proteins. In the present study, we used nocodazole to induce MTs depolymerization and paclitaxel or recombinant (r) TIR (Toll/interleukin-1 receptor) domain containing protein (TcpB) to induce MT stabilization in bone marrow-derived macrophages infected with Brucella abortus. Following treatment of the cells, we evaluated their effects on pathogen intracellular replication and survival, and in pro-inflammatory cytokine production. First, we observed that intracellular trafficking and maturation of Brucella-containing vesicles (BCVs) is affected by partial destabilization or stabilization of the MTs network. A typical marker of early BCVs, LAMP-1, is retained in late BCVs even 24 h after infection in the presence of low doses of nocodazole or paclitaxel and in the presence of different amounts of rTcpB. Second, microscopy and colony forming unit analysis revealed that bacterial load was increased in infected macrophages treated with lower doses of nocodazole or paclitaxel and with rTcpB compared to untreated cells. Third, innate immune responses were also affected by disturbing MT dynamics. MT depolymerization by nocodazole reduced IL-12 production in infected macrophages. Conversely, rTcpB-treated cells augmented IL-12 and IL-1ß secretion in infected cells. In summary, these findings demonstrate that modulation of MTs affects several crucial steps of B. abortus pathogenesis, including BCV maturation, intracellular survival and IL-12 secretion in infected macrophages.

TLR7 and TLR3 Sense Brucella abortus RNA to Induce Proinflammatory Cytokine Production but They Are Dispensable for Host Control of Infection.

Brucella abortus is a Gram-negative, facultative intracellular bacterium that causes brucellosis, a worldwide zoonotic disease leading to undulant fever in humans and abortion in cattle. The immune response against this bacterium relies on the recognition of microbial pathogen-associated molecular patterns, such as lipoproteins, lipopolysaccharides, and DNA; however, the immunostimulatory potential of B. abortus RNA remains to be elucidated. Here, we show that dendritic cells (DCs) produce significant amounts of IL-12, IL-6, and IP-10/CXCL10, when stimulated with purified B. abortus RNA. IL-12 secretion by DCs stimulated with RNA depends on TLR7 while IL-6 depends on TLR7 and partially on TLR3. Further, only TLR7 plays a role in IL-12 production induced by B. abortus infection. Moreover, cytokine production in DCs infected with B. abortus or stimulated with bacterial RNA was reduced upon pretreatment with MAPK/NF-κB inhibitors. By confocal microscopy, we demonstrated that TLR7 is colocalized with B. abortus in LAMP-1+Brucella-containing vacuoles. Additionally, type I IFN expression and IP-10/CXCL10 secretion in DCs stimulated with bacterial RNA were dependent on TLR3 and TLR7. Our results suggest that TLR3 and TLR7 are not required to control Brucella infection in vivo, but they play an important role on sensing B. abortus RNA in vitro.

Calcium signalling from the type I inositol 1,4,5-trisphosphate receptor is required at early phase of liver regeneration.

BACKGROUND & AIMS: Liver regeneration is a multistage process that unfolds gradually, with different mediators acting at different stages of regeneration. Calcium (Ca(2+) ) signalling is essential for liver regeneration. In hepatocytes, Ca(2+) signalling results from the activation of inositol 1,4,5-trisphosphate receptors (InsP3 R) of which two of the three known isoforms are expressed (InsP3 R-I and InsP3 R-II). Here, we investigated the role of the InsP3 R-I-dependent Ca(2+) signals in hepatic proliferation during liver regeneration. METHODS: Partial hepatectomy (HX) in combination with knockdown of InsP3 R-I (AdsiRNA-I) was used to evaluate the role of InsP3 R-I on liver regeneration and hepatocyte proliferation, as assessed by liver to body mass ratio, PCNA expression, immunoblots and measurements of intracellular Ca(2+) signalling. RESULTS: AdsiRNA-I efficiently infected the liver as demonstrated by the expression of ß-galactosidase throughout the liver lobules. Moreover, this construct selectively and efficiently reduced the expression of InsP3 R-I, as evaluated by immunoblots. Expression of AdsiRNA-I in liver decreased peak Ca(2+) amplitude induced by vasopressin in isolated hepatocytes 2 days after HX. Reduced InsP3 R-I expression prior to HX also delayed liver regeneration, as measured by liver to body weight ratio, and reduced hepatocyte proliferation, as evaluated by PCNA staining, at the same time point. At later stages of regeneration, control hepatocytes showed a decreased expression of InsP3 R, as well as reduced InsP3 R-mediated Ca(2+) signalling, events that did not affect liver growth. CONCLUSION: Together, these results show that InsP3 R-I-dependent Ca(2+) signalling is an early triggering pathway required for liver regeneration.

Decoding calcium signaling across the nucleus.

Calcium (Ca(2+)) is an important multifaceted second messenger that regulates a wide range of cellular events. A Ca(2+)-signaling toolkit has been shown to exist in the nucleus and to be capable of generating and modulating nucleoplasmic Ca(2+) transients. Within the nucleus, Ca(2+) controls cellular events that are different from those modulated by cytosolic Ca(2+). This review focuses on nuclear Ca(2+) signals and their role in regulating physiological and pathological processes.

The insulin receptor translocates to the nucleus to regulate cell proliferation in liver.

UNLABELLED: Insulin's metabolic effects in the liver are widely appreciated, but insulin's ability to act as a hepatic mitogen is less well understood. Because the insulin receptor (IR) can traffic to the nucleus, and Ca(2+) signals within the nucleus regulate cell proliferation, we investigated whether insulin's mitogenic effects result from activation of Ca(2+)-signaling pathways by IRs within the nucleus. Insulin-induced increases in Ca(2+) and cell proliferation depended upon clathrin- and caveolin-dependent translocation of the IR to the nucleus, as well as upon formation of inositol 1,4,5,-trisphosphate (InsP3) in the nucleus, whereas insulin's metabolic effects did not depend on either of these events. Moreover, liver regeneration after partial hepatectomy also depended upon the formation of InsP3 in the nucleus, but not the cytosol, whereas hepatic glucose metabolism was not affected by buffering InsP3 in the nucleus. CONCLUSION: These findings provide evidence that insulin's mitogenic effects are mediated by a subpopulation of IRs that traffic to the nucleus to locally activate InsP3 -dependent Ca(2+)-signaling pathways. The steps along this signaling pathway reveal a number of potential targets for therapeutic modulation of liver growth in health and disease.

Nucleoplasmic calcium signaling and cell proliferation: calcium signaling in the nucleus.

Calcium (Ca2+) is an essential signal transduction element involved in the regulation of several cellular activities and it is required at various key stages of the cell cycle. Intracellular Ca2+ is crucial for the orderly cell cycle progression and plays a vital role in the regulation of cell proliferation. Recently, it was demonstrated by in vitro and in vivo studies that nucleoplasmic Ca2+ regulates cell growth. Even though the mechanism by which nuclear Ca2+ regulates cell proliferation is not completely understood, there are reports demonstrating that activation of tyrosine kinase receptors (RTKs) leads to translocation of RTKs to the nucleus to generate localized nuclear Ca2+ signaling which are believed to modulate cell proliferation. Moreover, nuclear Ca2+ regulates the expression of genes involved in cell growth. This review will describe the nuclear Ca2+ signaling machinery and its role in cell proliferation. Additionally, the potential role of nuclear Ca2+ as a target in cancer therapy will be discussed.