Table of contents
The adverse effects of lipopolysaccharide (LPS) are mediated primarily by tumor necrosis factor alpha (TNF-alpha). TNF-alpha production by LPS-stimulated macrophages is regulated at the levels of both transcription and translation. Several mitogen-activated protein kinases (MAPKs) are activated in response to LPS. LPS activates MAPK family members extracellular-signal-regulated kinases 1 and 2 (ERK1 and ERK2), p38, and Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), as well as the immediate upstream MAPK activators MAPK/ERK kinases 1 and 4 (MEK1 and MEK4). LPS also activates MEK2, MEK3, and MEK6. Dexamethasone, which inhibits the production of cytokines, including TNF-alpha, significantly inhibits LPS induction of JNK/SAPK activity but not that of p38, ERK1 and ERK2, or MEK3, MEK4, or MEK6. Dexamethasone also blocks the sorbitol but not anisomycin stimulation of JNK/SAPK activity. A kinase-defective mutant of SAPKbeta, SAPKbeta K-A, blocks translation of TNF-alpha, as determined by using a TNF-alpha translational reporting system. Overexpression of wild-type SAPKbeta is able to overcome the dexamethasone-induced block of TNF-alpha translation. These data confirm that three MAPK family members and their upstream activators are stimulated by LPS and demonstrate that JNK/SAPK is required for LPS-induced translation of TNF-alpha mRNA. A novel mechanism by which dexamethasone inhibits translation of TNF-alpha is also revealed (Swantek, 1997).
Activation of the recently identified c-Jun N-terminal kinases (JNKs) typically results in programmed cell death (apoptosis) in neurons and other cell types grown in culture. However, the effects of JNK activation in the central nervous system in vivo are unknown. At baseline, JNK activity in mice is on average 17-fold higher in brain than in peripheral organs, whereas JNK protein levels are similar. In brain, JNK is expressed primarily in neurons. Restraining mice or allowing them to explore a novel environment rapidly increases JNK activity 3- to 15-fold in various brain regions, but these manipulations do not increase brain activity of the extracellular signal-regulated kinase. Because noninvasive environmental stimuli that do not induce neurodegeneration elicit prominent increases in JNK activity in the brain, it is concluded that acute activation of the JNK cascade in central nervous system neurons does not induce neuronal apoptosis in vivo. In contrast, the high baseline activity of JNK in the brain and the activation of the JNK cascade by environmental stimuli suggest that this kinase may play an important physiological role in neuronal function (Xu, 1997).
Tumor necrosis factor alpha (TNF alpha) has multiple biological functions including the prolonged activation of the collagenase and c-Jun genes, which are regulated via their AP-1 binding sites. Incubating human fibroblasts with TNF alpha induces prolonged activation of JNK, the c-Jun kinase, which phosphorylates the transactivation domain of c-Jun. Furthermore, an immune complex kinase assay specifically demonstrates that TNF alpha stimulates the activity of JNK1. TNF alpha also produces a small and transient increase in extracellular signal-regulated kinase (ERK) activity, but no measured increase in Raf-1 kinase activity. In contrast, epidermal growth factor causes a prolonged activation of Raf-1 kinase and ERK activity and a smaller, more transient activation of JNK, whereas the phorbol ester causes a small stimulation of Raf-1 kinase and a pronounced stimulation of ERK activity. The activation of JNK by TNF alpha does not correlate with Raf-1 or ERK activity. The kinetics of Raf-1, ERK, and JNK induction by epidermal growth factor, phorbol 12-myristate 13-acetate, or TNF alpha indicate distinct mechanisms of activation in human fibroblasts (Westwick, 1994).
Tumor necrosis factor alpha (TNF alpha) activates the SAPKs ( also known as Jun nuclear kinases or JNKs), resulting in the stimulation of AP-1-dependent gene transcription and induces the translocation of NF kappa B to the nucleus. This results in the stimulation of NF kappa B-dependent gene transcription. A potential second messenger for these signaling pathways is ceramide, which is generated when TNF alpha activates sphingomyelinases. Treatment of HL-60 human promyelocytic cells with exogenous sphingomyelinase leads to rapid stimulation of JNK/SAPK activity, an effect not mimicked by treatment with phospholipase A2, C, or D. Further, JNK/SAPK activity is stimulated 2.7- and 2.8-fold, respectively, in cells exposed to C2-ceramide (5 microM) or TNF alpha (10 ng/ml). The prolonged stimulation of this kinase activity by C2-ceramide is similar to that of TNF alpha. In contrast, the related mitogen-activated protein kinases ERK1 and ERK2 are weakly stimulated following TNF alpha treatment (1.5-fold) and are inhibited by C2-ceramide treatment. TNF alpha also potently stimulates NF-kappa B DNA binding activity and transcriptional activity, but these effects are not mimicked by the addition of C2-ceramide or sphingomyelinase to intact cells. Furthermore, TNF alpha, sphingomyelinase, and C2-ceramide all induce c-Jun, a gene that is stimulated by the ATF-2 and c-Jun transcription factors. These data suggest that ceramide may act as a second messenger for a subset of TNF alpha's biochemical and biological effects (Westwick, 1995).
Tumor necrosis factor alpha (TNF alpha) a pro-inflammatory cytokine is an endogenous mediator of septic shock, inflammation, anti-viral responses and apoptotic cell death. TNF alpha elicits its complex biological responses through the individual or cooperative action of two TNF receptors of mol. wt 55 kDa (TNF-RI) and mol. wt 75 kDa (TNF-RII). To determine signaling events specific for TNF-RII the extracellular domain of the mouse CD4 antigen was fused to the intracellular domain of TNF-RII. Crosslinking of the chimeric receptor using anti-CD4 antibodies initiates exclusively TNF-RII-mediated signals. TNF-RII is able to activate two members of the MAP kinase family: extracellular regulated kinase (ERK) and c-jun N-terminal kinase (JNK). TRAF2, a molecule that binds TNF-RII and associates indirectly with TNF-RI, is sufficient to activate JNK upon overexpression. Dominant-negative TRAF2 blocks TNF alpha-mediated JNK activation and TRAF2 signals the activation of JNK and NF-kappaB through different pathways. These findings suggest that TNF alpha-mediated JNK activation in fibroblasts is independent of the cell death pathway (see Drosophila Caspase1) and that TRAF2 occupies a key role in TNF receptor signaling to JNK (Reinhard, 1997).
A key step by which tumor necrosis factor (TNF) signals the activation of nuclear factor-kappaB (NF-kappaB: Drosophila homolog Dorsal) and the stress-activated protein kinase (SAPK, also called c-Jun N-terminal kinase or JNK) is the recruitment to the TNF receptor of TNF receptor-associated factor 2 (TRAF2). However, the subsequent steps in TRAF2-induced SAPK and NF-kappaB activation remain unresolved. The identification of a TNF-responsive serine/threonine protein kinase termed GCK related (GCKR) is reported that likely signals via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase kinase 1 (MEKK1) to activate the SAPK pathway. TNF, TRAF2, and ultraviolet (UV) light, which in part uses the TNF receptor signaling pathway, all increase GCKR activity. A TRAF2 mutant, which inhibits both TRAF2-induced NF-kappaB and SAPK activation, blocks TNF-induced GCKR activation. Finally, interference with GCKR expression impedes TRAF2- and TNF-induced SAPK activation but not that of NF-kappaB. This suggests a divergence in the TNF signaling pathway that leads to SAPK and NF-kappaB activation. The divergence occurs downstream of TRAF2 but upstream of GCKR (Shi, 1997).
Tumor necrosis factor (TNF) elicits a diverse array of inflammatory responses through engagement of its type-1 receptor (TNFR1). Many of these responses require de novo gene expression mediated by the activator protein-1 (AP-1) transcription factor. The mechanism was investigated by which TNFR1 recruits the stress-activated protein kinases (SAPKs) and the p38s, two mitogen-activated protein kinase (MAPK) families that together regulate AP-1. The human SPS1 homologue germinal center kinase (GCK) can interact in vivo with the TNFR1 signal transducer TNFR-associated factor-2 (TRAF2) and with MAPK/ERK kinase kinase 1 (MEKK1), a MAPK kinase kinase (MAPKKK) upstream of the SAPKs, thereby coupling TRAF2 to the SAPKs. Receptor interacting protein (RIP) is a second TNFR signal transducer that can bind TRAF2. RIP activates both p38 and SAPK; and TRAF2 activation of p38 requires RIP. The RIP noncatalytic intermediate domain associates in vivo with an endogenous MAPKKK that can activate the p38 pathway in vitro. Thus, TRAF2 initiates SAPK and p38 activation by binding two proximal protein kinases: GCK and RIP. GCK and RIP, in turn, signal by binding MAPKKKs upstream of the SAPKs and p38s (Yuasa, 1998).
TRAF2 is believed to mediate the activation of NF-kappaB and JNK induced by the tumor necrosis factor receptor (TNFR) superfamily, which elicits pleiotropic responses in lymphocytes. The physiological roles of TRAF2 in these processes have been investigated by expressing a lymphocyte-specific dominant negative form of TRAF2, thereby blocking this protein's effector function. The TNFR superfamily signals require TRAF2 for activation of JNK but not NF-kappaB. In addition, TRAF2 induces NF-kappaB-independent antiapoptotic pathways during TNF-induced apoptosis. Inhibition of TRAF2 leads to splenomegaly, lymphadenopathy, and an increased number of B cells. These findings indicate that TRAF2 is involved in the regulation of lymphocyte function and growth in vivo (Lee, 1997).
JNK protein kinases are distantly related to mitogen-activated protein kinases (ERKs) and are activated by dual phosphorylation on Tyr and Thr. The JNK protein kinase group includes the 46-kDa isoform JNK1. A second member of the JNK group is the 55-kDa protein kinase JNK2. The activities of both JNK isoforms are markedly increased by exposure of cells to UV radiation. Furthermore, JNK protein kinase activation is observed in cells treated with tumor necrosis factor. Although both JNK isoforms phosphorylate the NH2-terminal activation domain of the transcription factor c-Jun, the activity of JNK2 is approximately 10-fold greater than that of JNK1. This difference in c-Jun phosphorylation correlates with increased binding of c-Jun to JNK2 as compared with JNK1. The distinct in vitro biochemical properties of these JNK isoforms suggests that they may have different functions in vivo. Evidence in favor of this hypothesis has been obtained from the observation that JNK1, but not JNK2, complements a defect in the expression of the mitogen-activated protein kinase HOG1 in the yeast S. cerevisiae. Together, these data indicate a role for the JNK group of protein kinases in the signal transduction pathway initiated by proinflammatory cytokines and UV radiation (Sluss, 1994).
One of insulin's many biological effects is the increased transcription of AP-1-regulated genes. cJun is the principal component of the AP-1 transcription complex, which is regulated by the newly discovered members of the MAPK superfamily referred to either as cJun NH2-terminal kinases (JNKs) or stress-activated protein kinases (SAPKs). Insulin stimulates a dose- and time-dependent increase in JNK activity in Rat fibroblasts that overexpress human insulin receptors. JNK activation occurs 15 min after insulin addition, resulting in a 2.5-fold increase in cJun phosphorylation over unstimulated controls. Maximal JNK activation correlates with the onset of AP-1 DNA binding activity. Both insulin-stimulated JNK activity and insulin-induced AP-1 transcriptional activity are Ras-dependent. These data suggest that in these rat fibroblasts, JNK activation may play a role in insulin-regulated AP-1 transcriptional activity leading to a mitogenic response (Miller, 1996).
Activation of macrophages by bacterial lipopolysaccharide (LPS) induces transcription of genes that encode for proinflammatory regulators of the immune response. Previous work has suggested that activation of the transcription factor activator protein 1 (AP-1) is one LPS-induced event that mediates this response. Consistent with this notion, LPS stimulates AP-1-mediated transcription in a murine macrophage cell line. LPS treatment of macrophages results in rapid activation of the p46 and p54 isoforms of JNK. Treatment with wild-type and rough mutant forms of LPS and synthetic lipid A results in JNK activation, while pretreatment with the tyrosine kinase inhibitor herbimycin A inhibits this response. Binding of LPS-LPS binding protein (LBP) complexes to CD14, a surface receptor that mediates many LPS responses, was found to be crucial: pretreatment of THP-1 cells with the monoclonal antibody 60 (which blocks this binding) inhibits JNK activation. These results suggest that LPS activation of JNK in monocyte/macrophage cells is a CD14- and protein tyrosine phosphorylation-dependent event that may mediate the early activation of AP-1 in regulating LPS-triggered gene induction (Hambleton, 1996).
Harmful conditions, including heat shock, oxidative stress and UV, cause programmed cell death, whose triggering requires activation of the Jun N-terminal kinase, JNK. High levels of Hsp72, a heat-inducible member of Hsp70 family, protect cells against a variety of stresses by a mechanism that is unclear at present. Elevated levels of Hsp72 inhibit a signal transduction pathway leading to programmed cell death by preventing stress-induced activation of JNK. Stress-induced activation of another stress-kinase, p38 (HOG1), is also blocked when the level of Hsp72 is increased. Similarly, addition of a purified recombinant Hsp72 to a crude cell lysate reduces p38 kinase activation, while depletion of the whole family of Hsp70 proteins with a monoclonal antibody enhances such activation. Accumulation of abnormal proteins in cells upon incubation with amino acid analogs causes activation of JNK and p38 kinases; such activation can be prevented by overproduction of Hsp72. Taken together, these data suggest that, in regulation of JNK and p38 kinases, Hsp70 serves as a "sensor" of the build-up of abnormal proteins after heat shock and other stresses. The inhibitory effect of an increased level of Hsp70 on JNK appears to be a major contributor to acquired thermotolerance in mammalian cells (Gabai, 1997).
Resistance to stress-induced apoptosis was examined in cells in which the expression of hsp70 was either constitutively elevated or inducible by a tetracycline-regulated transactivator. Heat-induced apoptosis is blocked in hsp70-expressing cells; this is associated with reduced cleavage of the common death substrate protein poly(ADP-ribose) polymerase (PARP). Heat-induced cell death is correlated with the activation of the stress-activated protein kinase SAPK/JNK (c-Jun N-terminal kinase). Activation of SAPK/JNK is strongly inhibited in cells in which hsp70 is induced to a high level, indicating that hsp70 is able to block apoptosis by inhibiting signaling events upstream of SAPK/JNK activation. In contrast, SAPK/JNK activation is not inhibited by heat shock in cells with constitutively elevated levels of hsp70. Cells that constitutively overexpress hsp70 resist apoptosis induced by ceramide, a lipid signaling molecule generated by apoptosis-inducing treatments and linked to SAPK/JNK activation. Similar to heat stress, resistance to ceramide-induced apoptosis occurs in spite of strong SAPK/JNK activation. Therefore, hsp70 is also able to inhibit apoptosis at some point downstream of SAPK/JNK activation. Since PARP cleavage is prevented in both cell lines, these results suggest that hsp70 is able to prevent the effector steps of apoptotic cell death. Processing of the CED-3-related protease caspase-3 (CPP32/Yama/apopain) is inhibited in hsp70-expressing cells; however, the activity of the mature enzyme is not affected by hsp70 in vitro. Caspase processing may represent a critical heat-sensitive target leading to cell death that is inhibited by the chaperoning function of hsp70. The inhibition of SAPK/JNK signaling and apoptotic protease effector steps by hsp70 likely contributes to the resistance to stress-induced apoptosis seen in transiently induced thermotolerance (Mosser, 1997).
Monofunctional alkylating agents like methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are potent inducers of cellular stress leading to chromosomal aberrations, point mutations, and cell killing. These agents induce a specific cellular stress response program, which includes the activation of Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPKs), p38 mitogen-activated protein kinase, and the upstream kinase SEK1/MKK4 and which depends on the reaction mechanism of the alkylating agent in question. The intracellular glutathione (GSH) level is critical for JNK/SAPK activation by MMS: enhancing the GSH level by pretreatment of the cells with GSH or N-acetylcysteine is inhibitory, whereas depletion of the cellular GSH pool causes hyperinduction of JNK/SAPK activity by MMS. In light of the JNK/SAPK-dependent induction of c-jun and c-fos transcription, and the Jun/Fos-induced transcription of xenobiotic-metabolizing enzymes, these data provide evidence for a critical role for JNK/SAPK and p38 in the induction of a cellular defense program against cytotoxic xenobiotics such as MMS (Wilhelm, 1997).
Hsp72, a major inducible member of the heat shock protein family, can protect cells against many cellular stresses including heat shock. Pretreatment of NIH 3T3 cells with mild heat shock (43°C for 20 min) suppresses UV-stimulated c-Jun N-terminal kinase 1 (JNK1) activity. Constitutively overexpressed Hsp72 also inhibits JNK1 activation in NIH 3T3 cells, whereas it does not affect either SEK1 or MEKK1 activity. Both in vitro binding and kinase studies indicate that Hsp72 bind to JNK1 and that the peptide binding domain of Hsp72 is important to the binding and inhibition of JNK1. In vivo binding between endogenous Hsp72 and JNK1 in NIH 3T3 cells was confirmed by co-immunoprecipitation. Hsp72 also inhibits JNK-dependent apoptosis. Hsp72 antisense oligonucleotides block Hsp72 production in NIH 3T3 cells in response to mild heat shock and concomitantly abolishes the suppressive effect of mild heat shock on UV-induced JNK activation and apoptosis. Collectively, these data suggest strongly that Hsp72 can modulate stress-activated signaling by directly inhibiting JNK (Park, 2001).
Cells respond to stresses such as osmotic shock and heat shock by activating stress-activated protein kinases (SAPKs), including c-Jun N-terminal kinase (JNK). Activation of JNK requires phosphorylation of threonine and tyrosine residues in the TPY activation loop motif and can be reversed by the removal of either phosphate group. Numerous JNK phosphatases including dual-specificity phosphatases, have been identified. Many stimuli activate JNK by increasing its rate of phosphorylation; however, JNK dephosphorylation is inhibited in cells after heat shock, suggesting that a JNK phosphatase(s) is inactivated. M3/6 is a dual-specificity phosphatase selective for JNK. M3/6 has been expressed in the mouse bone marrow cell line BAF3 in order to show that JNK activation by IL-3 is necessary for cell survival and proliferation. M3/6 dissociates from JNK and appears in an insoluble fraction after heat shock. These data identify M3/6 as a JNK phosphatase that is inactivated by heat shock and provide a molecular mechanism for the activation of JNK by heat shock (Palacios, 2001).
Forkhead transcription factors of the FOXO class (see Drosophila Foxo) are negatively regulated by PKB/c-Akt in response to insulin/IGF signalling, and are involved in regulating cell cycle progression and cell death. In contrast to insulin signalling, low levels of oxidative stress generated by treatment with H2O2 induce the activation of FOXO4. Upon treatment of cells with H2O2, the small GTPase Ral is activated and this results in a JNK-dependent phosphorylation of FOXO4 on threonine 447 and threonine 451. This Ral-mediated, JNK-dependent phosphorylation is involved in the nuclear translocation and transcriptional activation of FOXO4 after H2O2 treatment. In addition, it is shown that this signalling pathway is also employed by tumor necrosis factor alpha to activate FOXO4 transcriptional activity. FOXO members have been implicated in cellular protection against oxidative stress via the transcriptional regulation of manganese superoxide dismutase and catalase gene expression. The results reported here, therefore, outline a homeostasis mechanism for sustaining cellular reactive oxygen species that is controlled by signalling pathways that can convey both negative (PI-3K/PKB) and positive (Ras/Ral) inputs (Essers, 2004).
Mechanical forces affect all body tissues. Experiments conducted mainly on cultured cells have established that altering these forces influences cell behaviors, including migration, differentiation, apoptosis, and proliferation. The transcriptional coactivator YAP has been identified as a nuclear relay of mechanical signals, but the molecular mechanisms that lead to YAP activation were not identified. YAP is the main transcriptional effector of the Hippo signaling pathway, a major growth regulatory pathway within metazoa, but at least in some instances, the influence of mechanical strain on YAP was reported to be independent of Hippo signaling. This study identified a molecular pathway that can promote the proliferation of cultured mammary epithelial cells in response to cyclic or static stretch. These mechanical stimuli are associated with increased activity of the transcriptional coactivator YAP, which is due at least in part to inhibition of Hippo pathway activity. Much of this influence on Hippo signaling can be accounted for by the activation of c-Jun N-terminal kinase (JNK) activity by mechanical strain and subsequent inhibition of Hippo signaling by JNK. LATS1 (see Drosophila Warts) is a key negative regulator of YAP within the Hippo pathway, and this study further shows that cyclic stretch is associated with a JNK-dependent increase in binding of a LATS inhibitor, LIMD1 (Drosophila homolog: Ajuba LIM protein), to the LATS1 kinase and that reduction of LIMD1 expression suppresses the activation of YAP by cyclic stretch. Together, these observations establish a pathway for mechanical regulation of cell proliferation via JNK-mediated inhibition of Hippo signaling (Codelia, 2014).
Obesity is closely associated with insulin resistance and establishes the leading risk factor for type 2 diabetes mellitus, yet the molecular mechanisms of this association are poorly understood. The c-Jun amino-terminal kinases (JNKs) can interfere with insulin action in cultured cells and are activated by inflammatory cytokines and free fatty acids, molecules that have been implicated in the development of type 2 diabetes. JNK activity is shown to be abnormally elevated in obesity. Furthermore, an absence of JNK results in decreased adiposity, significantly improved insulin sensitivity and enhanced insulin receptor signaling capacity in two different models of mouse obesity. Thus, JNK is a crucial mediator of obesity and insulin resistance and a potential target for therapeutics (Hirosumi, 2002).
This study provides evidence that obesity is associated with abnormally elevated JNK activity, predominantly provided by JNK1. Importantly, the absence of JNK1 results in substantial protection from obesity-induced insulin resistance. Abnormal production of inflammatory cytokines such as TNF-a and increased concentrations of free fatty acids (FFAs) are crucial players in obesity-induced insulin resistance. Induction of insulin resistance by these mediators involves inhibitory serine phosphorylation of IRS-1. Both TNF-a and FFAs are potent activators of JNK, which in turn phosphorylates IRS-1 at Ser 307. These studies provide strong evidence that JNK1 is indeed a crucial component of the biochemical pathway responsible for obesity-induced insulin resistance in vivo. There is also genetic evidence demonstrating that increased JNK activity caused by loss-of-function mutations in the JNK scaffold protein JIP1 is causal to type 2 diabetes in humans. It is therefore suggested that a selective interference with JNK1 activity presents an attractive opportunity for the treatment of human obesity, insulin resistance and type 2 diabetes (Hirosumi, 2002).
Activation of the c-Jun N-terminal kinase (JNK) by proinflammatory cytokines inhibits insulin signaling, at least in part, by stimulating phosphorylation of rat/mouse insulin receptor substrate 1 (Irs1) at Ser(307) [Ser(312) in human IRS1]. JNK mediated feedback inhibition of the insulin signal has been demonstrated in mouse embryo fibroblasts, 3T3-L1 adipocytes, and 32D(IR) cells. Insulin stimulation of JNK activity requires phosphatidylinositol 3-kinase and Grb2 signaling. Moreover, activation of JNK by insulin is inhibited by a cell-permeable peptide that disrupts the interaction of JNK with cellular proteins. However, the direct binding of JNK to Irs1 is not required for its activation by insulin, whereas direct binding is required for Ser(307) phosphorylation of Irs1. Insulin-stimulated Ser(307) phosphorylation was reduced 80% in cells lacking JNK1 and JNK2 or in cells expressing a mutant Irs1 protein lacking the JNK binding site. Reduced Ser(307) phosphorylation is directly related to increased insulin-stimulated tyrosine phosphorylation, Akt phosphorylation, and glucose uptake. These results support the hypothesis that JNK is a negative feedback regulator of insulin action by phosphorylating Ser(307) in Irs1 (Lee, 2003).
Microtubules (MTs) play an important role in elaboration and maintenance of axonal and dendritic processes. MT dynamics are modulated by MT-associated proteins (MAPs), whose activities are regulated by protein phosphorylation. A member of the c-Jun NH2-terminal protein kinase (JNK) subgroup of MAP kinases, JNK1, is involved in regulation of MT dynamics in neuronal cells. Jnk1-/- mice exhibit disrupted anterior commissure tract formation and a progressive loss of MTs within axons and dendrites. MAP2 and MAP1B polypeptides are hypophosphorylated in Jnk1-/- brains, resulting in compromised ability to bind MTs and promote their assembly. These results suggest that JNK1 is required for maintaining the cytoskeletal integrity of neuronal cells and is a critical regulator of MAP activity and MT assembly (Chang, 2003).
MAP2 is composed of three different domains: the central domain (CD) at the N-terminal region, unique to HMWMAP2; a proline-rich domain (PRD), and a tubulin binding domain (TBD) at the C-terminal region, which are present in both HMWMAP2 and LMWMAP2. Examination of the MAP2 amino acid sequence reveals more than 20 canonical MAPK phosphorylation sites located mainly within the CD. This is consistent with phosphopeptide mapping, which shows that most of the sites phosphorylated by JNK1 in vitro are located within the CD. Although the loss of JNK1 results in a marked decrease in HMWMAP2 phosphorylation, decreased phosphorylation of LMWMAP2 is found in Jnk1-/- brains at P6, the stage at which LMWMAP2 expression is still abundant. Thus, some JNK1 phosphorylation sites are outside the CD. Loss of JNK1-dependent phosphorylation is associated with decreased HMWMAP2 binding to MTs. In vitro, JNK1-dependent phosphorylation clearly enhances the ability of HMWMAP2 to bind MT and promote their assembly. It should also be noted that reduced MAP2 phosphorylation precedes any overt signs of neurodegeneration. Thus, it is rather unlikely that hypophosphorylation of MAP2 is a consequence of neurodegeneration (Chang, 2003).
JNK2 shares 83% amino acid sequence identity with JNK1. Surprisingly, JNK2 does not phosphorylate MAP2 as efficiently as JNK1 and its loss has no effect on neuronal MT organization. Although it is not yet possible to explain the basis for the different activity of these two closely related kinases to phosphorylate MAP2, it should be noted that alternatively spliced forms of JNK differ in their ability to phosphorylate c-Jun due to the presence of different substrate docking sequences. The observed differences in MAP2 phosphorylation and the absence of JNK2 from MT preparations that contain JNK1 are consistent with the finding that JNK1 is uniquely required for MAP2 phosphorylation and maintaining neuronal MT integrity (Chang, 2003).
The coordinated migration of neurons is a pivotal step for functional architectural formation of the mammalian brain. To elucidate its molecular mechanism, gene transfer by means of in utero electroporation was applied in the developing murine brain, revealing the crucial roles of Rac1, its activators, STEF/Tiam1, and its downstream molecule, c-Jun N-terminal kinase (JNK), in the cerebral cortex. Functional repression of these molecules results in inhibition of radial migration of neurons without affecting their proper differentiation. Interestingly, distinct morphological phenotypes were observed; suppression of Rac1 activity causes loss of the leading process, whereas repression of JNK activity does not, suggesting the complexity of the signaling cascade. In cultured neurons from the intermediate zone, activated JNK was detected along microtubules in the processes. Application of a JNK inhibitor caused irregular morphology and increased stable microtubules in processes, and decreased phosphorylation of microtubule associated protein 1B, raising a possibility of the involvement of JNK in controlling tubulin dynamics in migrating neurons. These data thus provide important clues for understanding the intracellullar signaling machinery for cortical neuronal migration (Kawauchi, 2003).
Mammalian preimplantation development involves several crucial events, such as compaction and blastocyst formation, but little is known about essential genes that regulate this developmental process. This study focuses on MAP kinase signaling pathways as potential regulatory pathways for the process. The results show that inhibition of the JNK pathway or of the p38 MAP kinase pathway, but not of the ERK pathway, results in inhibition of cavity formation, and that JNK and p38 are active during mouse preimplantation development. Subsequent microarray analyses shows that, of about 39,000 transcripts analyzed, the number of those genes whose expression level is sensitive to the inhibition of the JNK or the p38 pathway, but insensitive to the inhibition of the ERK pathway, is only 156. Moreover, of the 156 genes, expression of 10 genes (two genes upregulated and eight genes downregulated) is sensitive to either inhibition of the JNK or p38 pathways. These 10 genes include several genes known for their function in axis and pattern formation. Downregulation of some of the 10 genes simultaneously using siRNA leads to abnormality in cavity formation. Thus, this study has successfully narrowed down candidate genes of interest, detailed analysis of which will probably lead to elucidation of the molecular mechanism of preimplantation development (Maekawa, 2005).
In ascidian tadpoles, metamorphosis is triggered by a polarized wave of apoptosis, via mechanisms that are largely unknown. The MAP kinases ERK and JNK are both required for the wave of apoptosis and metamorphosis. By employing a gene-profiling-based approach, the network was identified of genes controlled by either ERK or JNK activity that stimulate the onset of apoptosis. This approach identified a gene network involved in hormonal signalling, in innate immunity, in cell-cell communication and in the extracellular matrix. Through gene silencing, it was show that Ci-sushi, a cell-cell communication protein controlled by JNK activity, is required for the wave of apoptosis that precedes tail regression. Ci-sushi encodes a protein containing domains known as complement control protein (CCP) modules, or short consensus repeats (SCR), which exist in a wide variety of complement and adhesion proteins. These observations have lead to proposal of a model of metamorphosis whereby JNK activity in the CNS induces apoptosis in several adjacent tissues that compose the tail by inducing the expression of genes such as Ci-sushi (Chambon, 2007).
The genes identified were separated for convenience as either upregulated or downregulated by JNK or ERK activity into four categories. There are genes involved in innate immunity, hormone signalling, metabolism of the extracellular matrix (ECM) or coding components of the ECM, and the remaining genes into one category that was term diverse genes. The screen identified genes that had previously been shown to be specifically expressed during ascidian metamorphosis, such as Ci-meta5, which is downregulated in response to JNK-inhibitor treatment. Ci-meta5 has been identified by differential screening of a cDNA library of swimming larvae and metamorphosing juveniles. Three genes (glutathione S-transferase, Cytochrome p450 and Gluthathione-requiring prostaglandin D synthase) were identified that are under the control of Ci-ERK and are orthologues of or closely related to genes expressed in papillae of the ascidians (Chambon, 2007).
Gene expression during early ascidian metamorphosis requires signalling by Hemps, an EGF-like protein. Although it is not known whether Hemps activates ERK, it is tempting to speculate that it does, because activation of the Ras/Raf/ERK pathway by EGF is well described in many species. Moreover, the identification of genes controlled by Ci-ERK in papillae, the observation that metamorphosis does not occur with MEK inhibition, and data on Hemps that shows that it induces settlement and metamorphosis are consistent with that observations that one of the effects of the Hemps pathway is to activate the ERK cascade in papillae cells (Chambon, 2007).
Among the genes identified that are controlled by the JNK pathway, two are interesting: Ci-GNRH and Ci-oatp, which are involved, respectively, in the reproductive and thyroid axes. The mouse Oatp14 (also known as Slco1c1) was described in the transport of thyroxine across the blood-brain barrier. The role of thyroid hormones in metamorphosis had been reported previously in ascidians, and also in amphibians and lamprey. Moreover, in four ascidian species, thyroxin is present in larval mesenchyme and seems to be involved in the control of metamorphosis. The expression of Ci-oatp via JNK activation in the CNS may enhance thyroid signalling in larvae. Concerning GNRH, no report describes any function of this hormone in invertebrate metamorphosis. However, it is possible that GNRH may have a role in lamprey metamorphosis because, in sea lamprey, the level of GNRH increases throughout the stage of spontaneous metamorphosis (Chambon, 2007).
In addition to identifying genes involved in the immune system and hormonal signalling, a number of genes were identified coding for proteins involved in the composition or processing of the ECM. For example, Ci-LyOx, which is responsible for the cross linking and deposition of collagen fibres, elastin fibres and Ci-Mx, a matrix metalloprotease was identified. The regulation of matrix metalloprotease and the ECM remodelling have been shown to affect apoptosis in different systems, including the apoptotic remodelling of the intestine during Xenopus laevis metamorphosis and post-lactation involution of the mouse mammary gland. Anoikis is apoptosis induced by the loss of, or inappropriate, cell adhesion. It is tempting to hypothesize that one of the inductive signals from Ci-JNK in the CNS controls apoptosis by changing ECM composition. The role of JNK in ECM degradation has already been reported in rat aortic walls. In the tail of the tadpole, nerve corde is surrounded by matrix, which leads us to speculate that remodelling the ECM could provide a means to coordinate the response of tail cells in promoting either cell death or survival. In support of such a scenario, it was reported that, after a modification in ECM components, activation of the MAPK ERK leads to anoikis-type death. Because Ci-ERK activation precedes apoptosis in tail cells, this cell death could be regulated by JNK-controlled anoikis in the tail of ascidian tadpoles (Chambon, 2007).
The iterative formation of nephrons during embryonic development relies on continual replenishment of progenitor cells throughout nephrogenesis. Defining molecular mechanisms that maintain and regulate this progenitor pool is essential to understanding nephrogenesis in developmental and regenerative contexts. Maintenance of nephron progenitors is absolutely dependent on BMP7 signaling, and Bmp7-null mice exhibit rapid loss of progenitors. However, the signal transduction machinery operating downstream of BMP7 as well as the precise target cell remain undefined. Using a novel primary progenitor isolation system, signal transduction and biological outcomes elicited by BMP7 were investigated. It was found that BMP7 directly and rapidly activates JNK signaling in nephron progenitors resulting in phosphorylation of Jun and ATF2 transcription factors. This signaling results in the accumulation of cyclin D3 and subsequent proliferation of PAX2(+) progenitors, inversely correlating with the loss of nephron progenitors seen in the Bmp7-null kidney. Activation of Jun and ATF2 is severely diminished in Bmp7-null kidneys, providing an important in vivo correlate. BMP7 thus promotes proliferation directly in nephron progenitors by activating the JNK signaling circuitry (Blank, 2009).
Using a BMP-reporter mouse, it has been shown that the nephrogenic zone (NZ) is essentially unresponsive to SMAD-mediated transcription in vivo. Owing to the strong effect of Bmp7 inactivation on nephron progenitor maintenance, this surprising finding prompted an investigation of whether BMP7 acts through a SMAD-independent signaling mechanism in the NZ. The most extensively described alternative pathway downstream of TGFβ and BMP is activation of MAPK signaling through TGFβ-activated kinase 1 (TAK1). Activation of p38 and JNK pathways have both been reported downstream of TAK1, and immortalized Tak1 mutant embryonic fibroblasts exhibited impaired phosphorylation of JNK. Mice deficient in Tak1 die at mid-gestation, precluding analysis of TAK1-mediated signaling in metanephric kidney development. However, TAK1 is expressed in the NZ of the developing kidney, suggesting that activation of MAPK signaling downstream of BMPs might occur in this region of the kidney. Previous work has indicate that BMP7 can activate p38 signaling in collecting duct cells in vitro. Similarly, conditional deletion of Bmp7 using a podocyte-specific Cre, resulted in defective p38 signaling, but no effect on phosphorylation of SMAD1/SMAD5/SMAD8 was observed. Using primary cell purification system, this study found no evidence that BMP7 activates p38 in the NZ. Instead, several JNK isoforms were rapidly phosphorylated in response to BMP7, resulting in downstream phosphorylation of both Jun and ATF2. Importantly, it was shown that TAK1 is required for efficient activation of JNK in response to BMP7. SIX2 immunostaining shows that JNK activation takes place directly in the nephron progenitor compartment, indicating that these cells indeed do respond directly to BMP7. Interestingly, phosphorylated forms of the JNK-activated transcription factors Jun and ATF2 are both localized to cap mesenchyme in the developing E17.5 kidney. Furthermore, E12.5 Bmp7-null kidneys displayed sharply reduced JNK-Jun-ATF2 signaling in metanephric mesenchyme compared with the wild type, demonstrating that Bmp7 is required for activation of this signaling axis in nephron progenitors in vivo. Interestingly, phosphorylation of Jun or ATF2 does not appear to be significantly reduced in collecting duct cells of Bmp7-deficient embryos, suggesting that other growth factors function to activate the pathway in this cellular compartment (Blank, 2009).
The vertebrate limb is a classical model for understanding patterning of three-dimensional structures during embryonic development. Although decades of research have elucidated the tissue and molecular interactions within the limb bud required for patterning and morphogenesis of the limb, the cellular and molecular events that shape the limb bud itself have remained largely unknown. This study shows that the mesenchymal cells of the early limb bud are not disorganized within the ectoderm as previously thought but are instead highly organized and polarized. Using time-lapse video microscopy, it was demonstrated that cells move and divide according to this orientation. The combination of oriented cell divisions and movements drives the proximal-distal elongation of the limb bud necessary to set the stage for subsequent morphogenesis. These cellular events are regulated by the combined activities of the WNT and FGF pathways. WNT5A/JNK is necessary for the proper orientation of cell movements and cell division. In contrast, the FGF/MAPK signaling pathway, emanating from the apical ectodermal ridge, does not regulate cell orientation in the limb bud but instead establishes a gradient of cell velocity enabling continuous rearrangement of the cells at the distal tip of the limb. Together, these data shed light on the cellular basis of vertebrate limb bud morphogenesis and uncover new layers to the sequential signaling pathways acting during vertebrate limb development (Gros, 2010).
The related small GTPases Ras and Rap1 are important for signaling synaptic AMPA receptor (-R) trafficking during long-term potentiation (LTP) and long-term depression (LTD), respectively. Rap2, which shares 60% identity to Rap1, is present at excitatory synapses, but its functional role is unknown. This study reports that Rap2 activity, stimulated by NR2A-containing NMDA-R activation, depresses AMPA-R-mediated synaptic transmission via activation of JNK rather than Erk1/2 or p38 MAPK. Moreover, Rap2 controls synaptic removal of AMPA-Rs with long cytoplasmic termini during depotentiation. Thus, Rap2-JNK pathway, which opposes the action of the NR2A-containing NMDA-R-stimulated Ras-ERK1/2 signaling and complements the NR2B-containing NMDA-R-stimulated Rap1-p38 MAPK signaling, channels the specific signaling for depotentiating central synapses (Zhu, 2005).
The cJun N-terminal kinase 1 (JNK1) is implicated in diet-induced obesity. Indeed, germline ablation of the murine Jnk1 gene prevents diet-induced obesity. This study demonstrates that selective deficiency of JNK1 in the murine nervous system is sufficient to suppress diet-induced obesity. The failure to increase body mass is mediated, in part, by increased energy expenditure that is associated with activation of the hypothalamic-pituitary-thyroid axis. Disruption of thyroid hormone function prevents the effects of nervous system JNK1 deficiency on body mass. These data demonstrate that the hypothalamic-pituitary-thyroid axis represents an important target of metabolic signaling by JNK1 (Sabio, 2010).
The thyroid hormone pathway is negatively regulated by JNK1. The increased amount of T4 and T3 in the blood of JNK1-deficient mice compared with WT mice correlates with increased expression of hypothalamic TRH and pituitary gland TSH. These changes in TRH and TSH expression were unexpected because thyroid hormone exerts powerful negative feedback control of TRH and TSH expression. The association of increased T4 and T3 in the blood with increased expression of TRH and TSH in JNK1-deficient mice indicates that JNK1 deficiency in the brain disrupts the normal negative feedback control of the hypothalamic-pituitary-thyroid axis. An important goal for future studies will be to determine the molecular mechanism of JNK1 regulation of the hypothalamic-pituitary-thyroid axis (Sabio, 2010).
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