Striatin knock out induces a gain of function of INa and impaired Ca2+ handling in mESC-derived cardiomyocytes.

AIM: Striatin (Strn) is a scaffold protein expressed in cardiomyocytes (CMs) and alteration of its expression are described in various cardiac diseases. However, the alteration underlying its pathogenicity have been poorly investigated. METHODS: We studied the role(s) of cardiac Strn gene (STRN) by comparing the functional properties of CMs, generated from Strn-KO and isogenic WT mouse embryonic stem cell lines. RESULTS: The spontaneous beating rate of Strn-KO CMs was faster than WT cells, and this correlated with a larger fast INa conductance and no changes in If. Paced (2-8 Hz) Strn-KO CMs showed prolonged action potential (AP) duration in comparison with WT CMs and this was not associated with changes in ICaL and IKr. Motion video tracking analysis highlighted an altered contraction in Strn-KO CMs; this was associated with a global increase in intracellular Ca2+, caused by an enhanced late Na+ current density (INaL) and a reduced Na+/Ca2+ exchanger (NCX) activity and expression. Immunofluorescence analysis confirmed the higher Na+ channel expression and a more dynamic microtubule network in Strn-KO CMs than in WT. Indeed, incubation of Strn-KO CMs with the microtubule stabilizer taxol, induced a rescue (downregulation) of INa conductance toward WT levels. CONCLUSION: Loss of STRN alters CMs electrical and contractile profiles and affects cell functionality by a disarrangement of Strn-related multi-protein complexes. This leads to impaired microtubules dynamics and Na+ channels trafficking to the plasma membrane, causing a global Na+ and Ca2+ enhancement.

Autophagy suppresses Ras-driven epithelial tumourigenesis by limiting the accumulation of reactive oxygen species.

This corrects the article DOI: 10.1038/onc.2017.175.

Autophagy suppresses Ras-driven epithelial tumourigenesis by limiting the accumulation of reactive oxygen species.

Activation of Ras signalling occurs in ~30% of human cancers; however, activated Ras alone is not sufficient for tumourigenesis. In a screen for tumour suppressors that cooperate with oncogenic Ras (RasV12) in Drosophila, we identified genes involved in the autophagy pathway. Bioinformatic analysis of human tumours revealed that several core autophagy genes, including GABARAP, correlate with oncogenic KRAS mutations and poor prognosis in human pancreatic cancer, supporting a potential tumour-suppressive effect of the pathway in Ras-driven human cancers. In Drosophila, we demonstrate that blocking autophagy at any step of the pathway enhances RasV12-driven epithelial tissue overgrowth via the accumulation of reactive oxygen species and activation of the Jun kinase stress response pathway. Blocking autophagy in RasV12 clones also results in non-cell-autonomous effects with autophagy, cell proliferation and caspase activation induced in adjacent wild-type cells. Our study has implications for understanding the interplay between perturbations in Ras signalling and autophagy in tumourigenesis, which might inform the development of novel therapeutics targeting Ras-driven cancers.

Mice haploinsufficient for Map2k7, a gene involved in neurodevelopment and risk for schizophrenia, show impaired attention, a vigilance decrement deficit and unstable cognitive processing in an attentional task: impact of minocycline.

RATIONALE: Members of the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein (MAP) kinases, and the upstream kinase MKK7, have all been strongly linked with synaptic plasticity and with the development of the neocortex. However, the impact of disruption of this pathway on cognitive function is unclear. OBJECTIVE: In the current study, we test the hypothesis that reduced MKK7 expression is sufficient to cause cognitive impairment. METHODS: Attentional function in mice haploinsufficient for Map2k7 (Map2k7 +/- mice) was investigated using the five-choice serial reaction time task (5-CSRTT). RESULTS: Once stable performance had been achieved, Map2k7 +/- mice showed a distinctive attentional deficit, in the form of an increased number of missed responses, accompanied by a more pronounced decrement in performance over time and elevated intra-individual reaction time variability. When performance was reassessed after administration of minocycline-a tetracycline antibiotic currently showing promise for the improvement of attentional deficits in patients with schizophrenia-signs of improvement in attentional performance were detected. CONCLUSIONS: Overall, Map2k7 haploinsufficiency causes a distinctive pattern of cognitive impairment strongly suggestive of an inability to sustain attention, in accordance with those seen in psychiatric patients carrying out similar tasks. This may be important for understanding the mechanisms of cognitive dysfunction in clinical populations and highlights the possibility of treating some of these deficits with minocycline.

Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors.

Grb2-associated binder 2 (Gab2) serves as a critical amplifier in the signaling network of Bcr-Abl, the driver of chronic myeloid leukemia (CML). Despite the success of tyrosine kinase inhibitors (TKIs) in CML treatment, TKI resistance, caused by mutations in Bcr-Abl or aberrant activity of its network partners, remains a clinical problem. Using inducible expression and knockdown systems, we analyzed the role of Gab2 in Bcr-Abl signaling in human CML cells, especially with respect to TKI sensitivity. We show for the first time that Gab2 signaling protects CML cells from various Bcr-Abl inhibitors (imatinib, nilotinib, dasatinib and GNF-2), whereas Gab2 knockdown or haploinsufficiency leads to increased TKI sensitivity. We dissected the underlying molecular mechanism using various Gab2 mutants and kinase inhibitors and identified the Shp2/Ras/ERK and the PI3K/AKT/mTOR axes as the two critical signaling pathways. Gab2-mediated TKI resistance was associated with persistent phosphorylation of Gab2 Y452, a PI3K recruitment site, and consistent with this finding, the protective effect of Gab2 was completely abolished by the combination of dasatinib with the dual PI3K/mTOR inhibitor NVP-BEZ235. The identification of Gab2 as a novel modulator of TKI sensitivity in CML suggests that Gab2 could be exploited as a biomarker and therapeutic target in TKI-resistant disease.

CBL-B is required for leukemogenesis mediated by BCR-ABL through negative regulation of bone marrow homing.

BCR-ABL induces chronic myeloid leukemia (CML) through the aberrant regulation of multiple signaling substrates. Previous research has shown that BCR-ABL mediates down-modulation of CBL-B protein levels. A murine bone marrow transplantation (BMT) study was performed to assess the contribution of Cbl-b to BCR-ABL-induced disease. The predominant phenotype in the Cbl-b(-/-) recipients was a CML-like myeloproliferative disease (MPD) similar to that observed in the wild-type animals, but with a longer latency, diminished circulating leukocyte numbers and reduced spleen weights. Despite the decreased leukemic burden in comparison to their wild-type counterparts, the Cbl-b(-/-) animals displayed enhanced numbers of Gr-1(+)/Mac-1(+) spleen cells and neutrophilia. On the basis of prior evidence of CBL-B-dependent motility toward SDF-1α, we hypothesized that Cbl-b deficiency might impair bone marrow localization during transplantation. Homing experiments showed reduced migration of Cbl-b(-/-) cells to the bone marrow. Intrafemoral transplantation of BCR-ABL-transduced Cbl-b(-/-) cells revealed equivalent latency of disease development to the wild-type transplants, supporting the conclusion that Cbl-b deficiency diminishes homing of leukemic cells to the bone marrow, and perturbs the proliferation of BCR-ABL-expressing malignant clones during CML development.

Angiotensin converting enzyme 2 abrogates bleomycin-induced lung injury.

Despite substantial progress, mortality and morbidity of the acute respiratory distress syndrome (ARDS), a severe form of acute lung injury (ALI), remain unacceptably high. There is no effective treatment for ARDS/ALI. The renin-angiotensin system (RAS) through Angiotensin-converting enzyme (ACE)-generated Angiotensin II contributes to lung injury. ACE2, a recently discovered ACE homologue, acts as a negative regulator of the RAS and counterbalances the function of ACE. We hypothesized that ACE2 prevents Bleomycin (BLM)-induced lung injury. Fourteen to 16-week-old ACE2 knockout mice-male (ACE2(-/y)) and female (ACE2(-/-))-and age-matched wild-type (WT) male mice received intratracheal BLM (1.5U/kg). Male ACE2(-/y) BLM injured mice exhibited poorer exercise capacity, worse lung function and exacerbated lung fibrosis and collagen deposition compared with WT. These changes were associated with increased expression of the profibrotic genes α-smooth muscle actin (α-SMA) and Transforming Growth Factor ß1. Compared with ACE2(-/y) exposed to BLM, ACE2(-/-) exhibited better lung function and architecture and decreased collagen deposition. Treatment with intraperitoneal recombinant human (rh) ACE2 (2 mg/kg) for 21 days improved survival, exercise capacity, and lung function and decreased lung inflammation and fibrosis in male BLM-WT mice. Female BLM WT mice had mild fibrosis and displayed a possible compensatory upregulation of the AT2 receptor. We conclude that ACE2 gene deletion worsens BLM-induced lung injury and more so in males than females. Conversely, ACE2 protects against BLM-induced fibrosis. rhACE2 may have therapeutic potential to attenuate respiratory morbidity in ALI/ARDS.

Jun and JunD-dependent functions in cell proliferation and stress response.

Jun is essential for fetal development, as fetuses lacking Jun die at mid-gestation with multiple cellular defects in liver and heart. Embryos expressing JunD in place of Jun (Jun(d/d)) can develop to term with normal fetal livers, but display cardiac defects as observed in fetuses lacking Jun. Jun(d/d) mouse embryonic fibroblasts (MEFs) exhibit early senescence, which can be rescued by EGF and HB-EGF stimulation, probably through activation of Akt signaling. Thus, JunD cannot functionally replace Jun in regulating fibroblast proliferation. In Jun(-/-) fetal livers, increased hydrogen peroxide levels are detected and expression of Nrf1 and Nrf2 (nuclear erythroid 2-related transcription factors) is downregulated. Importantly, increased oxidative stress as well as expression of Nrf1 and Nrf2 is rescued by JunD in Jun(d/d) fetal livers. These data show that Jun is of critical importance for cellular protection against oxidative stress in fetal livers and fibroblasts, and Jun-dependent cellular senescence can be restored by activation of the epidermal growth factor receptor pathway.

ESCI award lecture: from a little mouse to rationale medicine for bone loss.

Completion of the human genome is one of the many significant milestones in the new era of systems biology. The current phase of genomic studies is focused upon parsing this new found genetic data with respect to scientific interest, and economic and health impact applications. As the sequences are now available and whole genome single nucleotide polymorphism maps for multiple human diseases will be available with the advent of modern genomics, the big challenge is to determine the function of these genes in the context of the entire organism. The emphasis is therefore on functional genomic analysis that represents the new front-line and limiting factor for realizing potential benefits of genome-based science. Defined gene targeting has been proven to be particularly useful as loss of expression mutants can reveal essential functions of molecules and the pathogenesis of disease. Using gene-targeted mice, my group has over the years identified genes that control heart and lung functions; apoptosis; lymphocyte activation; cancer; pain; diabetes; fertility or wound healing . In this study, I would like to review our work on RANKL in more detail.

SARS-coronavirus modulation of myocardial ACE2 expression and inflammation in patients with SARS.

BACKGROUND: Angiotensin converting enzyme 2 (ACE2), a monocarboxylase that degrades angiotensin II to angiotensin 1-7, is also the functional receptor for severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) and is highly expressed in the lungs and heart. Patients with SARS also suffered from cardiac disease including arrhythmias, sudden cardiac death, and systolic and diastolic dysfunction. MATERIALS AND METHODS: We studied mice infected with the human strain of the SARS-CoV and encephalomyocarditis virus and examined ACE2 mRNA and protein expression. Autopsy heart samples from patients who succumbed to the SARS crisis in Toronto (Canada) were used to investigate the impact of SARS on myocardial structure, inflammation and ACE2 protein expression. RESULTS: Pulmonary infection with the human SARS-CoV in mice led to an ACE2-dependent myocardial infection with a marked decrease in ACE2 expression confirming a critical role of ACE2 in mediating SARS-CoV infection in the heart. The SARS-CoV viral RNA was detected in 35% (7/20) of autopsied human heart samples obtained from patients who succumbed to the SARS crisis during the Toronto SARS outbreak. Macrophage-specific staining showed a marked increase in macrophage infiltration with evidence of myocardial damage in patients who had SARS-CoV in their hearts. The presence of SARS-CoV in the heart was also associated with marked reductions in ACE2 protein expression. CONCLUSIONS: Our data show that SARS-CoV can mediate myocardial inflammation and damage associated with down-regulation of myocardial ACE2 system, which may be responsible for the myocardial dysfunction and adverse cardiac outcomes in patients with SARS.

The molecular archaeology of a mitochondrial death effector: AIF in Drosophila.

Apoptosis-inducing factor (AIF) is a phylogenetically conserved redox-active flavoprotein that contributes to cell death and oxidative phosphorylation in Saccharomyces cerevisiae, Caenorhabditis elegans, mouse and humans. AIF has been characterized as a caspase-independent death effector that is activated by its translocation from mitochondria to the cytosol and nucleus. Here, we report the molecular characterization of AIF in Drosophila melanogaster, a species in which most cell deaths occur in a caspase-dependent manner. Interestingly, knockout of zygotic D. melanogaster AIF (DmAIF) expression using gene targeting resulted in decreased embryonic cell death and the persistence of differentiated neuronal cells at late embryonic stages. Although knockout embryos hatch, they undergo growth arrest at early larval stages, accompanied by mitochondrial respiratory dysfunction. Transgenic expression of DmAIF misdirected to the extramitochondrial compartment (DeltaN-DmAIF), but not wild-type DmAIF, triggered ectopic caspase activation and cell death. DeltaN-DmAIF-induced death was not blocked by removal of caspase activator Dark or transgenic expression of baculoviral caspase inhibitor p35, but was partially inhibited by Diap1 overexpression. Knockdown studies revealed that DeltaN-DmAIF interacts genetically with the redox protein thioredoxin-2. In conclusion, we show that Drosophila AIF is a mitochondrial effector of cell death that plays roles in developmentally regulated cell death and normal mitochondrial function.

The phosphoinositide-3 kinase gamma-Akt pathway mediates renal tubular injury in cisplatin nephrotoxicity.

Nephrotoxicity is a frequent complication of cisplatin-based chemotherapy often limiting its use. In this study, we attempted to the role of the phosphoinositide-3 kinase (PI3K)-gamma-Akt pathway in this form of acute kidney injury. Using PI3K-gamma knockout mice, we found that a conventional dose of cisplatin was more lethal in the knockout mice where the blood urea nitrogen and serum creatinine were significantly higher in them than in wild-type mice. Phosphorylation of Akt in the renal tubules was abrogated in the knockout mice with the severity of renal dysfunction and numbers of TUNEL (terminal deoxynucleotidyl transferase (TdT) mediated nick-end labeling)-positive renal tubule cells being higher in the knockout than in wild-type mice. Cisplatin treatment significantly increased. Caspase-3 activity, histone-associated DNA fragments, and number of annexin V-positive cells was significantly higher in cisplatin-treated primary cultured renal tubular epithelial cells of knockout mice. Transfection of dominant-active forms of Akt and PI3K-gamma ameliorated apoptosis of the tubule epithelial cells derived from the knockout mice. Our results suggest that the PI3K-gamma-Akt pathway lessens apoptosis and plays a critical role in the maintenance of renal function in cisplatin-induced acute kidney injury.

Antagonistic control of cell fates by JNK and p38-MAPK signaling.

During the development and organogenesis of all multicellular organisms, cell fate decisions determine whether cells undergo proliferation, differentiation, or aging. Two independent stress kinase signaling pathways, p38-MAPK, and JNKs, have evolved that relay developmental and environmental cues to determine cell responses. Although multiple stimuli can activate these two stress kinase pathways, the functional interactions and molecular cross-talks between these common second signaling cascades are poorly elucidated. Here we report that JNK and p38-MAPK pathways antagonistically control cellular senescence, oncogenic transformation, and proliferation in primary mouse embryonic fibroblasts (MEFs). Similarly, genetic inactivation of the JNK pathway results in impaired proliferation of fetal hepatoblasts in vitro and defective adult liver regeneration in vivo, which is rescued by inhibition of the p38-MAPK pathway. Thus, the balance between the two stress-signaling pathways, MKK7-JNK and MKK3/6-p38-MAPK, determines cell fate and links environmental and developmental stress to cell cycle arrest, senescence, oncogenic transformation, and adult tissue regeneration.

Angiotensin-converting enzyme 2 in acute respiratory distress syndrome.

Angiotensin-converting enzyme (ACE) and ACE2 are highly homologous metalloproteases that provide essential catalytic functions in the renin-angiotensin system (RAS). Angiotensin II is one key effector peptide of the RAS, inducing vasoconstriction and exerting multiple biological functions. ACE cleaves angiotensin I to generate angiotensin II, whereas ACE2 reduces angiotensin II levels. Accumulating evidence has demonstrated a physiological and pathological role of ACE2 in the cardiovascular systems. Intriguingly, the SARS coronavirus, the cause of severe acute respiratory syndrome (SARS), utilizes ACE2 as an essential receptor for cell fusion and in vivo infections. Moreover, recent studies have demonstrated that ACE2 protects murine lungs from acute lung injury as well as SARS-Spike protein-mediated lung injury, suggesting a dual role of ACE2 in SARS infections and protection from ARDS.

Phosphatidylinositol 3-kinase facilitates bile acid-induced Ca(2+) responses in pancreatic acinar cells.

Bile acids are known to induce Ca(2+) signals in pancreatic acinar cells. We have recently shown that phosphatidylinositol 3-kinase (PI3K) regulates changes in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) elicited by CCK by inhibiting sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). The present study sought to determine whether PI3K regulates bile acid-induced [Ca(2+)](i) responses. In pancreatic acinar cells, pharmacological inhibition of PI3K with LY-294002 or wortmannin inhibited [Ca(2+)](i) responses to taurolithocholic acid 3-sulfate (TLC-S) and taurochenodeoxycholate (TCDC). Furthermore, genetic deletion of the PI3K gamma-isoform also decreased [Ca(2+)](i) responses to bile acids. Depletion of CCK-sensitive intracellular Ca(2+) pools or application of caffeine inhibited bile acid-induced [Ca(2+)](i) signals, indicating that bile acids release Ca(2+) from agonist-sensitive endoplasmic reticulum (ER) stores via an inositol (1,4,5)-trisphosphate-dependent mechanism. PI3K inhibitors increased the amount of Ca(2+) in intracellular stores during the exposure of acinar cells to bile acids, suggesting that PI3K negatively regulates SERCA-dependent Ca(2+) reloading into the ER. Bile acids inhibited Ca(2+) reloading into ER in permeabilized acinar cells. This effect was augmented by phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), suggesting that both bile acids and PI3K act synergistically to inhibit SERCA. Furthermore, inhibition of PI3K by LY-294002 completely inhibited trypsinogen activation caused by the bile acid TLC-S. Our results indicate that PI3K and its product, PIP(3), facilitate bile acid-induced [Ca(2+)](i) responses in pancreatic acinar cells through inhibition of SERCA-dependent Ca(2+) reloading into the ER and that bile acid-induced trypsinogen activation is mediated by PI3K. The findings have important implications for the mechanism of acute pancreatitis since [Ca(2+)](i) increases and trypsinogen activation mediate key pathological processes in this disorder.

Chlamydia and antigenic mimicry.

Chlamydial infections are among the most common human infections. Every year, in millions of humans, they cause infections of the eyes, the respiratory tract, the genital tract, joints, and the vasculature. Chlamydiae are obligate intracellular prokaryotic pathogens. Chlamydiae promote, in susceptible host cells that include mucosal epithelial cells, vascular endothelial cells, smooth muscle cells, and monocytes and macrophages, their survival while causing disease of varying clinical importance and consequence in their hosts. Chlamydia infections often precede the initiation of autoimmune diseases, and Chlamydiae are often found within autoimmune lesions. Thus, they have been suspected in the etiology and pathogenesis of autoimmune diseases. Autoimmune diseases have many causes. Genes, notably genes encoding cell-surface proteins that display peptides for immune recognition, the major histocompatibility complex (MHC), the environment, and the microbial diversity within the human body determine the susceptibility to autoimmune diseases. One mechanism by which infection is linked to the initiation of autoimmunity is termed molecular mimicry. Molecular mimicry describes the phenomenon of protein products from dissimilar genes sharing similar structures that elicit an immune response to both self and microbial proteins. Molecular mimicry might thus be a mechanism by which infections trigger autoimmune diseases. For the purpose of this chapter, we will focus on chlamydial proteins that mimic host self-proteins and thus contribute to initiation and maintenance of autoimmune diseases. Thus far, the strongest cases for molecular mimicry seem to have been made for chlamydial heat shock proteins 60, the DNA primase of Chlamydia trachomatis, and chlamydial OmcB proteins.

Developmentally regulated expression of the regulator of G-protein signaling gene 2 (Rgs2) in the embryonic mouse pituitary.

During the development of the anterior pituitary gland, five distinct hormone-producing cell types emerge in a spatially and temporally regulated pattern from an invagination of oral ectoderm termed Rathke's Pouch. Evidence from mouse knockout and ectopic expression studies indicates that 12.5 days post coitum (dpc) to 14.5 dpc is a critical period for the expansion of the progenitor cell pool and the determination of most hormone-secreting cell types. While signaling proteins and transcription factors have been identified as having key roles in pituitary cell differentiation, little is known about the identity and function of proteins that mediate signal transduction in progenitor cells. To identify genes that are enriched in the embryonic pituitary gland, we compared gene expression in 14.5 dpc pituitary and 14.5 dpc embryo minus pituitary tissues using the NIA 15K microarray. Analysis of the data using the R program revealed that the Regulator of G Protein Signaling 2 (Rgs2) gene was 3.9-fold more abundant in the 14.5 dpc pituitary. In situ hybridisation confirmed this finding, and showed that Rgs2 expression in midline tissues was restricted to the pituitary and discrete regions of the nervous system. Within the pituitary, Rgs2 was expressed in undifferentiated cells, and was downregulated at the completion of the hormone cell differentiation. To investigate Rgs2 function in the pituitary, we examined hormone cell differentiation in Rgs2 null neonate mice. Pituitary cell differentiation and morphology appeared normal in the Rgs2 mutant animals, suggesting that other Rgs family members with similar activities may be present in the developing pituitary.

Physiological roles of angiotensin-converting enzyme 2.

Angiotensin-converting enzyme 2 (ACE2) is a recently discovered homologue of the key enzyme of the renin-angiotensin system, the angiotensin-converting enzyme. The ACE2 enzyme is mainly expressed in cardiac blood vessels and tubular epithelia of the kidneys. Together with ACE2's unique metallocarboxypeptidase activity, the restricted tissue distribution suggests a distinctive physiological function in blood pressure, blood flow and fluid regulation. The ace2 gene was mapped to quantitative trait loci affecting susceptibility to hypertension in rats. Furthermore, ACE2 appears to be a negative regulator of ACE in the heart. ACE2 messenger RNA and protein levels are substantially regulated in the kidney of diabetic and pregnant rats. The mechanism of ACE2 function and its physiologic significance are not yet fully understood; however, as ACE2 differs in its specificity and physiological role from ACE, this opens a new potential venue for drug discovery aimed at cardiovascular disease, hypertension and diabetic complications.