Jun N-terminal Protein Kinase Signaling Research Articles

Role of Fibroblast Growth Factor Receptor 2b in the Cross Talk between Autophagy and Differentiation: Involvement of Jun N-Terminal Protein Kinase Signaling.

ABSTRACTFibroblast growth factor receptor 2b (FGFR2b) is a receptor tyrosine kinase expressed exclusively in epithelial cells. We previously demonstrated that FGFR2b induces autophagy and that this process is required for the triggering of FGFR2b-mediated early differentiation of keratinocytes. However, the molecular mechanisms regulating this interplay remain to be elucidated. Since we have also recently shown that Jun N-terminal protein kinase 1 (JNK1) signaling is involved in FGFR2b-induced autophagy and a possible role of the JNK pathway in epidermal differentiation has been suggested (though it is still debated), we investigated here the cross talk between FGFR2b-mediated autophagy and differentiation, focusing on the downstream JNK signaling. Biochemical, molecular, and immunofluorescence approaches in 2-dimensional (2-D) keratinocyte cultures and three-dimensional (3-D) organotypic skin equivalents confirmed that FGFR2b overexpression increased both autophagy and early differentiation. The use of FGFR2b substrate inhibitors and the silencing of JNK1 highlighted that this signaling is required not only for autophagy but also for the triggering of early differentiation. In contrast, the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway did not appear to be involved in the two processes, and AKT signaling, whose activation contributes to the FGFR2b-mediated onset of keratinocyte differentiation, was not required for the triggering of autophagy. Overall, our results point to JNK1 as a signaling hub that regulates the interplay between FGFR2b-induced autophagy and differentiation.

Identification of Signaling Pathways by Which CD40 Stimulates Autophagy and Antimicrobial Activity against Toxoplasma gondii in Macrophages.

CD40 is an important stimulator of autophagy and autophagic killing of Toxoplasma gondii in host cells. In contrast to autophagy induced by nutrient deprivation or pattern recognition receptors, less is known about the effects of cell-mediated immunity on Beclin 1 and ULK1, key regulators of autophagy. Here we studied the molecular mechanisms by which CD40 stimulates autophagy in macrophages. CD40 ligation caused biphasic Jun N-terminal protein kinase (JNK) phosphorylation. The second phase of JNK phosphorylation was dependent on autocrine production of tumor necrosis factor alpha (TNF-α). TNF-α and JNK signaling were required for the CD40-induced increase in autophagy. JNK signaling downstream of CD40 caused Ser-87 phosphorylation of Bcl-2 and dissociation between Bcl-2 and Beclin 1, an event known to stimulate the autophagic function of Beclin 1. However, TNF-α alone was unable to stimulate autophagy. CD40 also stimulated autophagy via a pathway that included calcium/calmodulin-dependent kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), and ULK1. CD40 caused AMPK phosphorylation at its activating site, Thr-172. This effect was mediated by CaMKKβ and was not impaired by neutralization of TNF-α. CD40 triggered AMPK-dependent Ser-555 phosphorylation of ULK1. CaMKKβ, AMPK, and ULK1 were required for CD40-induced increase in autophagy. CD40-mediated autophagic killing of Toxoplasma gondii is known to require TNF-α. Knockdown of JNK, CaMKKβ, AMPK, or ULK1 prevented T. gondii killing in CD40-activated macrophages. The second phase of JNK phosphorylation-Bcl-2 phosphorylation-Bcl-2-Beclin 1 dissociation and AMPK phosphorylation-ULK1 phosphorylation occurred simultaneously at ∼4 h post-CD40 stimulation. Thus, CaMKKβ and TNF-α are upstream molecules by which CD40 acts on ULK1 and Beclin 1 to stimulate autophagy and killing of T. gondii.

Preclinical Evaluation of Liposomal C8 Ceramide as a Potent anti-Hepatocellular Carcinoma Agent.

Hepatocellular carcinoma (HCC) remains a global health threat. The search for novel anti-HCC agents is urgent. In the current study, we synthesized a liposomal C8 ceramide, and analyzed its anti-tumor activity in pre-clinical HCC models. The liposomal C8 (ceramide) potently inhibited HCC cell (HepG2, SMMC-7721 and Huh-7 lines) survival and proliferation, more efficiently than free C8 ceramide. Yet, non-cancerous HL7702 human hepatocytes were resistant to the liposomal C8 treatment. Liposomal C8 activated caspase-dependent apoptosis in HCC cells, and HCC cytotoxicity by liposomal C8 was significantly attenuated with co-treatment of caspase inhibitors. At the molecular level, we showed that liposomal C8 activated ASK1 (apoptosis signal-regulating kinase 1)-JNK (Jun N-terminal protein kinase) signaling in HCC cells. On the other hand, JNK pharmacological inhibition or dominant negative mutation, as well as ASK1 shRNA-knockdown remarkably inhibited liposomal C8-induced apoptosis in HCC cells. Further studies showed that liposomal C8 inhibited AKT-mTOR (mammalian target of rapamycin) activation in HCC cells. Restoring AKT-mTOR activation by introducing a constitutively-active AKT alleviated HepG2 cytotoxicity by liposomal C8. In vivo, intravenous (i.v.) injection of liposomal C8 significantly inhibited HepG2 xenograft growth in severe combined immuno-deficient (SCID) mice, and mice survival was significantly improved. These preclinical results suggest that liposomal C8 could be further studied as a valuable anti-HCC agent.

A Genome-Wide RNA Interference Screen Identifies Caspase 4 as a Factor Required for Tumor Necrosis Factor Alpha Signaling

Tumor necrosis factor alpha (TNF-α) is a potent inflammatory cytokine secreted upon cellular stress as well as immunological stimuli and is implicated in the pathology of inflammatory diseases and cancer. The therapeutic potential of modifying TNF-α pathway activity has been realized in several diseases, and antagonists of TNF-α have reached clinical applications. While much progress in the understanding of signaling downstream of the TNF-α receptor complex has been made, the compendium of factors required for signal transduction is still not complete. In order to find novel regulators of proinflammatory signaling induced by TNF-α, we conducted a genome-wide small interfering RNA screen in human cells. We identified several new candidate modulators of TNF-α signaling, which were confirmed in independent experiments. Specifically, we show that caspase 4 is required for the induction of NF-κB activity, while it appears to be dispensable for the activation of the Jun N-terminal protein kinase signaling branch. Taken together, our experiments identify caspase 4 as a novel regulator of TNF-α-induced NF-κB signaling that is required for the activation of IκB kinase. We further provide the genome-wide RNA interference data set as a compendium in a format compliant with minimum information about an interfering RNA experiment (MAIRE).

Toxoplasma gondii Induces B7-2 Expression through Activation of JNK Signal Transduction

Toxoplasma gondii is a globally distributed parasite pathogen that infects virtually all warm-blooded animals. A hallmark of immunity to acute infection is the production of gamma interferon (IFN-γ) and interleukin-12 (IL-12), followed by a protective T cell response that is critical for parasite control. Naïve T cell activation requires both T-cell receptor (TCR) stimulation and the engagement of costimulatory receptors. Because of their important function in activating T cells, the expression of costimulatory ligands is believed to be under tight control. The molecular mechanisms governing their induction during microbial stimulation, however, are not well understood. We found that all three strains of T. gondii (types I, II, and III) upregulated the expression of B7-2, but not B7-1, on the surface of mouse bone marrow-derived macrophages. Additionally, intraperitoneal infection of mice with green fluorescent protein (GFP)-expressing parasites resulted in enhanced B7-2 levels specifically on infected, GFP(+) CD11b(+) cells. B7-2 induction occurred at the transcript level, required active parasite invasion, and was not dependent on MyD88 or TRIF. Functional assays demonstrated that T. gondii-infected macrophages stimulated naïve T cell proliferation in a B7-2-dependent manner. Genome-wide transcriptional analysis comparing infected and uninfected macrophages revealed the activation of mitogen-activated protein kinase (MAPK) signaling in infected cells. Using specific inhibitors against MAPKs, we determined that parasite-induced B7-2 is dependent on Jun N-terminal protein kinase (JNK) but not extracellular signal-regulated kinase (ERK) or p38 signaling. We also observed that T. gondii-induced B7-2 expression on human peripheral blood monocytes is dependent on JNK signaling, indicating that a common mechanism of B7-2 regulation by T. gondii may exist in both humans and mice.

Interleukin(IL)-4 promotion of CXCL-8 gene transcription is mediated by ERK1/2 pathway in human pulmonary artery endothelial cells

Interleukin-4 is central to allergic pulmonary inflammatory responses, but its contribution to airway neutrophilia remains controversial. The endothelium plays a critical role in regulating leukocyte recruitment and migration during inflammation. However, its response to IL-4 is reported to either increase or decrease the production of neutrophil chemotactic factors. We hypothesized that these conflicting findings may be due to the tissue and the size of the vessels from which endothelial cells have been derived. The expression of CXCL-8 by human primary culture umbilical veins endothelial cells (HUVECs), human pulmonary artery endothelial cells (HPAECs), and human pulmonary microvascular endothelial cells (HPMECs) when stimulated with recombinant human IL-4 (rhIL-4) was studied. The chemoattractant property of the cells’ supernatants for neutrophils was evaluated using Boyden chambers. The role of the nuclear factor-κB (NF-κB), and mitogen-activated protein kinases (MAPK) in IL-4-induced HPAECs was studied using Western blotting and electrophoretic mobility shift assay (EMSA). We demonstrated that IL-4 increased the mRNA expression and the protein production of CXCL-8 in HPAECs, but not in HUVECs and HPMECs. The supernatants of HAPECs stimulated by IL-4 significantly promoted neutrophils migration in a dose-dependent manner, and was significantly attenuated by an inhibitor of CXCL-8. We also found that extracellular-regulated protein kinase1/2 (ERK1/2) is activated by IL-4 in HPAECs, but not JUN-N-terminal protein kinase (JNK) or p38 MAPK pathway. Furthermore, NF-κB–DNA binding activity, phosphorylation of IκBα and p65 levels were not affected by rhIL-4 in HAPECs. These findings indicate marked functional differences in the response of micro and macro-ECs to IL-4. ERK1/2, rather than NF-κB, JNK and p38 MAPK signaling, plays a role in IL-4 induced chemokine activation. Our results suggest that inhibition of ERK1/2 may be a possible target for airway neutrophilia in allergic lung diseases.

Herpes Simplex Virus Type 1 ICP27 Induces p38 Mitogen-Activated Protein Kinase Signaling and Apoptosis in HeLa Cells

The herpes simplex virus type 1 (HSV-1) protein ICP27 has been implicated in a variety of functions important for viral replication including host shutoff, viral gene expression, activation of mitogen-activated protein kinases p38 and Jun N-terminal protein kinase (JNK), and apoptosis inhibition. In the present study we sought to examine the functions of ICP27 in the absence of viral infection by creating stable HeLa cell lines that inducibly express ICP27. Here, we characterize two such cell lines and show that ICP27 expression is associated with a cellular growth defect. The observed defect is caused at least in part by the induction of apoptosis as indicated by caspase-3 activation, annexin V staining, and characteristic changes in cellular morphology. In an effort to identify the function of ICP27 responsible for inducing apoptosis, we show that ICP27 expression is sufficient to activate p38 signaling to a level that is similar to that observed during wild-type HSV-1 infection. However, ICP27 expression alone is unable to lead to a strong activation of JNK signaling. Using chemical inhibitors, we show that the ICP27-mediated activation of p38 signaling is responsible for the observed induction of apoptosis in the induced cell lines. Our findings suggest that during viral infection, ICP27 activates p38 and JNK signaling pathways via two distinct mechanisms. ICP27 directly activates p38 signaling, leading to stimulation of the host cell apoptotic pathways. In contrast, robust activation of JNK signaling by ICP27 requires one or more delayed early or late viral gene products and may be associated with the inhibition of apoptosis.

Unique Signaling Properties of CTAR1 in LMP1-Mediated Transformation

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene is considered the EBV oncogene as it is necessary for EBV-mediated transformation of B lymphocytes and itself transforms rodent fibroblasts. LMP1 activates the NF-kappaB, phosphatidylinositol 3-kinase (PI3K)-Akt, mitogen-activated protein kinase, and Jun N-terminal protein kinase signaling pathways through its two signaling domains, carboxyl-terminal activating regions 1 and 2 (CTAR1 and CTAR2). CTAR1 and CTAR2 induce signal transduction pathways through their direct (CTAR1) or indirect (CTAR2) recruitment of tumor necrosis factor receptor-associated factors (TRAFs). CTAR1 is necessary for LMP1-mediated transformation as well as activation of PI3K signaling and induction of cell cycle markers associated with G(1)/S transition. In this study, activation of PI3K-Akt signaling and deregulation of cell cycle markers were mapped to the TRAF-binding domain within CTAR1 and to the residues between CTAR1 and CTAR2. LMP1 CTAR1 also activated the MEK1/2-extracellular signal-regulated kinase 1/2 signaling pathway, and this activation was necessary for LMP1-induced transformation of Rat-1 fibroblasts. Dominant-negative forms of TRAF2 and TRAF3 inhibited but did not fully block LMP1-mediated transformation. These findings identify a new signaling pathway that is uniquely activated by the TRAF-binding domain of LMP1 and is required for transformation.

Inhibition of the Jun N-terminal protein kinase pathway by SHIP-1, a lipid phosphatase that interacts with the adaptor molecule Dok-3.

Dok-3 is a Dok-related adaptor expressed in B cells and macrophages. Previously, we reported that Dok-3 is an inhibitor of B-cell activation in A20 B cells and that it associates with SHIP-1, a 5' inositol-specific lipid phosphatase, as well as Csk, a negative regulator of Src kinases. Here, we demonstrate that Dok-3 suppresses B-cell activation by way of its interaction with SHIP-1, rather than Csk. Our biochemical analyses showed that the Dok-3-SHIP-1 complex acts by selectively inhibiting the B-cell receptor (BCR)-evoked activation of the Jun N-terminal protein kinase (JNK) cascade without affecting overall protein tyrosine phosphorylation or activation of previously described SHIP-1 targets like Btk and Akt/PKB. Studies of B cells derived from SHIP-1-deficient mice showed that BCR-triggered activation of JNK is enhanced in the absence of SHIP-1, implying that the Dok-3-SHIP-1 complex (or a related mechanism) is a physiological negative regulator of the JNK cascade in normal B cells. Together, these data elucidate the mechanism by which Dok-3 inhibits B-cell activation. Furthermore, they provide evidence that SHIP-1 can be a negative regulator of JNK signaling in B cells.