Jun N‐terminal kinase activity is required for invagination but not differentiation of the sea urchin archenteron

Although sea urchin gastrulation is well described at the cellular level, our understanding of the molecular changes that trigger the coordinated cell movements involved is not complete. Jun N‐terminal kinase (JNK) is a component of the planar cell polarity pathway and is required for cell movements during embryonic development in several animal species. To study the role of JNK in sea urchin gastrulation, embryos were treated with JNK inhibitor SP600125 just prior to gastrulation. The inhibitor had a limited and specific effect, blocking invagination of the archenteron. Embryos treated with 2 μM SP600125 formed normal vegetal plates, but did not undergo invagination to form an archenteron. Other types of cell movements, specifically ingression of the skeletogenic mesenchyme, were not affected, although the development and pattern of the skeleton was abnormal in treated embryos. Pigment cells, derived from nonskeletogenic mesenchyme, were also present in SP600125‐treated embryos. Despite the lack of a visible archenteron in treated embryos, cells at the original vegetal plate expressed several molecular markers for endoderm differentiation. These results demonstrate that JNK activity is required for invagination of the archenteron but not its differentiation, indicating that in this case, morphogenesis and differentiation are under separate regulation. genesis 53:762–769, 2015. © 2015 Wiley Periodicals, Inc.     

Specific association of c‐Jun‐like immunoreactivity but not c‐Jun p39 with normal and induced programmed cell death in the chick embryo

We have examined c‐Jun protein expression by immunocytochemistry in normal and pathologically induced cell death by focusing primarily on the developing neuromuscular system of the chick embryo. Several commercially available antibodies against c‐Jun were used in combination with the TUNEL technique or propidium iodide staining for detection of cells undergoing programmed cell death (PCD). Among these, a rabbit polyclonal antibody raised against the amino acids 91‐105 mapping to the amino terminal domain of mouse c‐Jun p39 (c‐Jun/sc45) transiently immunostained the cytoplasm of dying spinal cord motoneurons at a time coincident with naturally occurring motoneuron death. Late apoptotic bodies were devoid of c‐Jun/sc45 immunoreactivity. A monoclonal antibody directed against a region corresponding to the amino acids 26‐175 of c‐Jun p39 (c‐Jun/mAB) did not specifically immunostain dying neurons, but, rather, showed nuclear immunolabeling in almost all healthy motoneurons. Experimentally induced programmed death of motoneurons by means of early limb bud ablation, axotomy, or in ovo injection of the neurotoxin β‐bungarotoxin increased the number of dying cells showing positive c‐Jun/sc45 immunoreactivity. Immunoelectron microscopy with c‐Jun/sc45 antibody showed that the signal was present in the cytoplasm without a specific association with organelles, and was also present in large lysosome‐like dense bodies inside neuritic profiles. Similar findings were obtained in different types of cells undergoing normal or experimentally induced PCD. These include dorsal root ganglion neurons, Schwann cells, muscle cells, neural tube and neural crest cells during the earliest stages of spinal cord development, and interdigital mesenchymal cells of hindlimbs. In all these cases, cells showed morphological and histochemical characteristics of apoptotic‐like PCD. By contrast, motoneurons undergoing necrotic cell death induced by the excitotoxin N‐methyl‐D ‐aspartate did not show detectable c‐Jun/sc45 immunoreactivity, although they displayed an increase in nuclear c‐Jun/mAB immunostaining. In Western blot analysis of spinal cord extracts, c‐Jun/sc45 antibody weakly detected a 39‐kD band, corresponding to c‐Jun, and more strongly detected two additional bands of 66 and 45 kD which followed developmental changes coincident with naturally occurring or experimentally stimulated apoptotic motoneuron death. By contrast, c‐Jun/mAB only recognized a single p39 band as expected for c‐Jun, and did not display changes associated with neuronal apoptosis. From these data, we conclude that the c‐Jun/sc45 antibody recognizes apoptosis‐related proteins associated with the early stages of morphological PCD in a variety of neuronal and nonneuronal cells, and that c‐Jun/sc45 is a reliable marker for a variety of developing cells undergoing programmed cell death. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 171–190, 1999     

Nuclear morphology and c‐Jun N‐terminal kinase 1 expression differentiate serum‐starved oxidative stress signalling from hydrogen peroxide‐induced apoptosis in retinal neuronal cell line

Oxidative stress induced by serum starvation and H₂O₂ exposure, both triggers apoptosis in retinal neuronal cell line RGC‐5 (retinal ganglion cell‐5). We have examined whether, despite excess generation of ROS (reactive oxygen species) and apoptosis induction, there is any dissimilarity in nuclear morphology and apoptotic signalling pathway in RGC‐5 under these conditions. Sub‐confluent cells were treated either with H₂O₂ or maintained in SFM (serum‐free medium). ROS level was detected along with nuclear morphology and ultrastructural analysis. Generation of excess intracellular ROS, nuclear localization of Bax and caspase 3 activation along with decrease of cellular viability, confirmed apoptosis induction in RGC‐5 by 72 h serum starvation and 500 M H₂O₂ exposure for 1 h. Nuclear swelling as supported by nuclear cytoplasmic ratio and conspicuous black spots with nuclear remodelling were observed only upon SFM, but not with H₂O₂ treatment. Serum starvation did not alter JNK1 (c‐Jun N‐terminal kinase 1) expression, although nuclear translocation and higher level of pJNK (phospho‐JNK) was evident. Conversely, H₂O₂ exposure blocked the expression and activation of JNK1 to phospho‐JNK as a negligible level of pJNK was present in the cytoplasm. Despite similar ROS generation in both the conditions, difference in nuclear morphology and JNK1 expression leads to the hypothesis that RGC‐5 cells may follow different signalling pathways when challenged with serum starvation and H₂O₂.     

Involvement of c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase 1/2 in EGF‐induced angiogenesis

Angiogenesis is a process during which endothelial cells divide and migrate to form new capillaries from the preexisting blood vessels. The present study was designed to investigate whether MAPKs (mitogen‐activated protein kinases) play crucial roles in regulating EGF (epidermal growth factor)‐induced endothelial cell angiogenesis. Our results showed that EGF stimulated HUVEC (human umbilical vein endothelial cells) proliferation in a concentration‐dependent manner, of which the maximum effective concentration of EGF was 10 ng/ml. Western blot analysis showed that EGF at 10 ng/ml significantly induced the phosphorylation of ERK1/2 (extracellular signal‐regulated kinase 1 and 2) and p38 kinase at 5 min, while it induced the phosphorylation of JNK/SAPK (c‐Jun N‐terminal kinase/stress‐activated protein kinase) at 15 min. Further results showed that a JNK/SAPK inhibitor, SP600125, and a specific siRNA JNK/SAPK could both significantly inhibit EGF‐induced tube formation in HUVEC cells, and an ERK1/2 inhibitor PD098059 could also block the tube formation in some content, while a p38 inhibitor SB203580 failed to do so. Furthermore, only SP600125 significantly inhibited EGF‐induced HUVEC cell proliferation under no cytotoxic concentration, so did JNK/SAPK siRNA. In conclusion, JNK/SAPK and ERK1/2 signals therefore play critical roles in EGF‐mediated HUVEC cell angiogenesis.     

Comparative effects of a vasopeptidase inhibitor vs. an angiotensin convertin enzyme inhibitor on cardiomyocyte apoptosis in rats with heart failure

Apoptosis is involved in ventricular remodeling after myocardial infarction (MI). We investigated the effects of the vasopeptidase inhibitor (VPI) omapatrilat on cardiomyocyte apoptosis and compared it to the angiotensin converting enzyme inhibitor (ACEI) captopril in the rat post-MI model and in cultured neonatal rat cardiomyocytes. Wistar males rats surviving 4 h post-MI were assigned to omapatrilat (40 or 80 mg/kg/day), captopril (160 mg/kg/day) or no treatment. After 56 days, hemodynamic measurements were performed (n = 96) and rats were sacrificed. One group had assessment of cardiac remodeling and detection of DNA fragments by in situ end labelling method (ISEL), while the other had morphologic measurements and DNA laddering assessed. In addition, cultured neonatal rat cardiomyocytes (n = 6) were treated for 72 h with vehicle, captopril or omapatrilat in the presence or absence of the apoptosis inducing agent H₂O₂. Omapatrilat and captopril resulted in similar improvements of hemodynamic measurements, ventricular weight and dilatation, cardiac fibrosis and myocardial cell cross-section in large MI rats. Omapatrilat increased scar thickness more than did captopril. All sham-operated groups had little evidence of apoptosis. In the large MI group, there was a significant increase in ISEL-positive cells in the control (0.095 ± 0.016%) and captopril (0.124 ± 0.024%) groups in comparison with control sham-operated (0.006 ± 0.006%), but this increase was limited to the peri-MI area. Omapatrilat (0.012 ± 0.012% for both doses) prevented the increase in apoptosis in the peri-MI area. Also, omapatrilat but not captopril reduced DNA laddering in large MI. Moreover, in cultured neonatal rat cardiomyocytes, omapatrilat but not captopril reduced apoptosis as assessed by DNA laddering. The VPI omapatrilat, with its combination of NEP and ACE inhibition, suppresses cardiomyocyte apoptosis post-MI and in neonatal cultured rat cardiomyocytes more than the ACEI captopril, but this does not result in significant hemodynamic or morphologic differences between omapatrilat and captopril.     

Role of brain c‐Jun N‐terminal kinase 2 in the control of the insulin receptor and its relationship with cognitive performance in a high‐fat diet pre‐clinical model

Insulin resistance has negative consequences on the physiological functioning of the nervous system. The appearance of type 3 diabetes in the brain leads to the development of the sporadic form of Alzheimer's disease. The c‐Jun N‐terminal kinases (JNK), a subfamily of the Mitogen Activated Protein Kinases, are enzymes composed by three different isoforms with differential modulatory activity against the insulin receptor (IR) and its substrate. This research focused on understanding the regulatory role of JNK2 on the IR, as well as study the effect of a high‐fat diet (HFD) in the brain. Our observations determined how JNK2 ablation did not induce compensatory responses in the expression of the other isoforms but led to an increase in JNKs total activity. HFD‐fed animals also showed an increased activity profile of the JNKs. These animals also displayed endoplasmic reticulum stress and up‐regulation of the protein tyrosine phosphatase 1B (PTP1B) and the suppressor of cytokine signalling 3 protein. Consequently, a reduction in insulin sensitivity was detected and it is correlated with a decrease on the signalling of the IR. Moreover, cognitive impairment was observed in all groups but only wild‐type genotype animals fed with HFD showed neuroinflammatory responses. In conclusion, HFD and JNK2 absence cause alterations in normal cognitive activity by altering the signalling of the IR. These affectations are related to the appearance of endoplasmic reticulum stress and an increase in the levels of inhibitory proteins like PTP1B and suppressor of cytokine signalling 3 protein.

     

c‐Jun N‐terminal kinase is largely involved in the regulation of tricellular tight junctions via tricellulin in human pancreatic duct epithelial cells

Tricellulin (TRIC) is a tight junction protein at tricellular contacts where three epithelial cells meet, and it is required for the maintenance of the epithelial barrier. To investigate whether TRIC is regulated via a c‐Jun N‐terminal kinase (JNK) pathway, human pancreatic HPAC cells, highly expressed at tricellular contacts, were exposed to various stimuli such as the JNK activators anisomycin and 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), and the proinflammatory cytokines IL‐1β, TNFα, and IL‐1α. TRIC expression and the barrier function were moderated by treatment with the JNK activator anisomycin, and suppressed not only by inhibitors of JNK and PKC but also by siRNAs of TRIC. TRIC expression was induced by treatment with the PKC activator TPA and proinflammatory cytokines IL‐1β, TNFα, and IL‐1α, whereas the changes were inhibited by a JNK inhibitor. Furthermore, in normal human pancreatic duct epithelial cells using hTERT‐transfected primary cultured cells, the responses of TRIC expression to the various stimuli were similar to those in HPAC cells. TRIC expression in tricellular tight junctions is strongly regulated together with the barrier function via the JNK transduction pathway. These findings suggest that JNK may be involved in the regulation of tricellular tight junctions including TRIC expression and the barrier function during normal remodeling of epithelial cells, and prevent disruption of the epithelial barrier in inflammation and other disorders in pancreatic duct epithelial cells. J. Cell. Physiol. 225: 720–733, 2010. © 2010 Wiley‐Liss, Inc.     

Apoptosis inhibitor 5 is an endogenous inhibitor of caspase‐2

Caspases are key enzymes responsible for mediating apoptotic cell death. Across species, caspase‐2 is the most conserved caspase and stands out due to unique features. Apart from cell death, caspase‐2 also regulates autophagy, genomic stability and ageing. Caspase‐2 requires dimerization for its activation which is primarily accomplished by recruitment to high molecular weight protein complexes in cells. Here, we demonstrate that apoptosis inhibitor 5 (API5/AAC11) is an endogenous and direct inhibitor of caspase‐2. API5 protein directly binds to the caspase recruitment domain (CARD) of caspase‐2 and impedes dimerization and activation of caspase‐2. Interestingly, recombinant API5 directly inhibits full length but not processed caspase‐2. Depletion of endogenous API5 leads to an increase in caspase‐2 dimerization and activation. Consistently, loss of API5 sensitizes cells to caspase‐2‐dependent apoptotic cell death. These results establish API5/AAC‐11 as a direct inhibitor of caspase‐2 and shed further light onto mechanisms driving the activation of this poorly understood caspase.     

Dual specificity phosphatase16 is a negative regulator of c‐Jun NH2‐terminal kinase activity in T cells

Activation of MAPK is negatively regulated by DUSP, which dephosphorylate the phosphothreonine and phosphotyrosine residues. We have identified a novel JNK‐specific DUSP, DUSP16, from murine macrophages. Its involvement in T cells has not yet been defined. In the present study, we found expression of DUSP16 in thymocytes and activated T cells but not in naive T cells. To elucidate the roles of DUSP16 in T cells, transgenic mice expressing a dominant negative form of DUSP16 specifically in T cells were generated (dnDUSP16 Tg). JNK activity was selectively augmented in the thymocytes of these dnDUSP16 Tg mice. CD4 T cells in dnDUSP16 Tg mice showed normal levels of proliferation and IL‐2 production after TCR triggering, while they produced increased IFN‐γ but reduced Th2 cytokines compared with wild type CD4 T cells. On the other hand CD8 T cells in dnDUSP16 Tg mice produced an increased amount of IL‐2, which resulted in enhanced proliferation and IFN‐γ production. These results suggest that DUSP16 is an important regulator of JNK activity and effector functions of CD4 and CD8 T cells.