C-Jun NH2-terminal kinase is required for lineage-specific differentiation but not stem cell self-renewal.
ABSTRACT: The c-Jun NH(2)-terminal kinase (JNK) is implicated in proliferation. Mice with a deficiency of either the Jnk1 or the Jnk2 genes are viable, but a compound deficiency of both Jnk1 and Jnk2 causes early embryonic lethality. Studies using conditional gene ablation and chemical genetic approaches demonstrate that the combined loss of JNK1 and JNK2 protein kinase function results in rapid senescence. To test whether this role of JNK was required for stem cell proliferation, we isolated embryonic stem (ES) cells from wild-type and JNK-deficient mice. We found that Jnk1(-/-) Jnk2(-/-) ES cells underwent self-renewal, but these cells proliferated more rapidly than wild-type ES cells and exhibited major defects in lineage-specific differentiation. Together, these data demonstrate that JNK is not required for proliferation or self-renewal of ES cells, but JNK plays a key role in the differentiation of ES cells.
Project description:Mitogen-activated protein kinases, including c-Jun NH2-terminal kinase (JNK), play an important role in the development and function of a large variety of tissues. The skeletal phenotype of JNK1 and JNK2 double-knockout (dKO) mice (JNK1<sup>fl/fl</sup>Col2-Cre/JNK2<sup>-/-</sup>) and control genotypes were analyzed at different embryonic and postnatal stages. JNK1/2 dKO mice displayed a severe scoliotic phenotype beginning during development that was grossly apparent around weaning age. Alcian blue staining at embryonic day 17.5 showed abnormal fusion of the posterior spinal elements. In adult mice, fusion of vertebral bodies and of spinous and transverse processes was noted by micro-computed tomography, Alcian blue/Alizarin red staining, and histology. The long bones developed normally, and histologic sections of growth plate and articular cartilage revealed no significant abnormalities. Histologic sections of the vertebral column at embryonic days 15.5 and 17.5 revealed an abnormal organization of the annulus fibrosus in the dKOs, with chondrocyte-like cells and fusion of dorsal processes. Spinal sections in 10-week-old dKO mice showed replacement of intervertebral disk structures (annulus fibrosus and nucleus pulposus) by cartilage and bone tissues, with cells staining for markers of hypertrophic chondrocytes, including collagen X and runt-related transcription factor 2. These findings demonstrate a requirement for both JNK1 and JNK2 in the normal development of the axial skeleton. Loss of JNK signaling results in abnormal endochondral bone formation and subsequent severe scoliosis.
Project description:A major link between inflammation and cancer is provided by NF-kappaB transcription factors. Ikkbeta(Deltahep) mice, which specifically lack IkappaB kinase beta (IKKbeta), an activator of NF-kappaB, in hepatocytes, are unable to activate NF-kappaB in response to proinflammatory stimuli, such as TNF-alpha. Surprisingly, Ikkbeta(Deltahep) mice are hypersusceptible to diethylnitrosamine (DEN)-induced hepatocarcinogenesis. Because defective NF-kappaB activation promotes sustained c-Jun N-terminal kinase (JNK) activation in cells exposed to TNF-alpha, whose expression is induced by DEN, and JNK activity is required for normal hepatocyte proliferation, we examined whether increased susceptibility to DEN-induced hepatocarcinogenesis in Ikkbeta(Deltahep) mice requires JNK activation. Hepatocytes express both JNK1 and JNK2, but previous studies indicate that JNK1 is more important for hepatocyte proliferation. We therefore investigated this hypothesis using mice homozygous for a JNK1 deficiency either in wild-type or Ikkbeta(Deltahep) backgrounds. In both cases, mice lacking JNK1 were much less susceptible to DEN-induced hepatocarcinogenesis. This impaired tumorigenesis correlated with decreased expression of cyclin D and vascular endothelial growth factor, diminished cell proliferation, and reduced tumor neovascularization. Whereas hepatocyte-specific deletion of IKKbeta augmented DEN-induced hepatocyte death and cytokine-driven compensatory proliferation, disruption of JNK1 abrogated this response. In addition to underscoring the importance of JNK1-mediated hepatocyte death and compensatory proliferation, these results strongly suggest that the control of tissue renewal through the IKK and JNK pathways plays a key role in liver carcinogenesis.
Project description:Overexpression and activation of c-Jun N-terminal kinases (JNKs) have been observed in multiple cancer cell lines and tumor samples. Various JNK isoforms have been reported to promote lung and liver cancer, as well as keratinocyte transformation, suggesting an important role of JNK signaling in promoting tumor development. However, there are three JNK isoforms, and it is unclear how each individual isoform, especially the ubiquitously expressed JNK1 and JNK2, functions in melanoma. Our previous study found that C116S mutations in both JNK1 and JNK2 rendered them insensitive to the covalent pan-JNK inhibitor JNK-IN-8 while retaining kinase activity. To delineate the specific roles of JNK1 and JNK2 in melanoma cell proliferation and invasiveness, we expressed the wild type (WT) and C116S mutants in melanoma cell lines and used JNK-IN-8 to enable chemical-genetic dissection of JNK1 and JNK2 activity. We found that the JNK2C116S allele consistently enhanced colony proliferation and cell invasiveness in the presence of JNK-IN-8. When cells individually expressing WT or C116S JNK1/2 were subcutaneously implanted into immunodeficient mice, we again found that bypass of JNK-IN-8-mediated inhibition of JNK signaling by expression of JNK2C116S specifically resulted in enhanced tumor growth in vivo. In addition, we observed a high level of JNK pathway activation in some human BRAF inhibitor (BRAFi) resistant melanoma cell lines relative to their BRAFi sensitive isogenic counterparts. JNK-IN-8 significantly enhanced the response to dabrafenib in resistant cells overexpressing JNK1WT, JNK2WT, and JNK1C116S but had no effect on cells expressing JNK2C116S, suggesting that JNK2 signaling is also crucial for BRAFi resistance in a subset of melanomas. Collectively, our data show that JNK2 activity is specifically required for melanoma cell proliferation, invasiveness, and BRAFi resistance and that this activity is most important in the context of JNK1 suppression, thus providing a compelling rationale for the development of JNK2 selective inhibitors as a potential therapy for the treatment of melanoma.
Project description:cJun NH(2)-terminal kinase (JNK) signaling has been implicated in the developmental morphogenesis of epithelial organs. In this study, we employed a compound deletion of the murine Jnk1 and Jnk2 genes in the mammary gland to evaluate the requirement for these ubiquitously expressed genes in breast development and tumorigenesis. JNK1/2 was not required for breast epithelial cell proliferation or motility. However, JNK1/2 deficiency caused increased branching morphogenesis and defects in the clearance of lumenal epithelial cells. In the setting of breast cancer development, JNK1/2 deficiency significantly increased tumor formation. Together, these findings established that JNK signaling is required for normal mammary gland development and that it has a suppressive role in mammary tumorigenesis.
Project description:Targeted inhibition of the c-Jun N-terminal kinases (JNKs) has shown therapeutic potential in intrahepatic cholangiocarcinoma (CCA)-related tumorigenesis. However, the cell-type-specific role and mechanisms triggered by JNK in liver parenchymal cells during CCA remain largely unknown. Here, we aimed to investigate the relevance of JNK1 and JNK2 function in hepatocytes in two different models of experimental carcinogenesis, the dethylnitrosamine (DEN) model and in nuclear factor kappa B essential modulator (NEMO)hepatocyte-specific knockout (?hepa) mice, focusing on liver damage, cell death, compensatory proliferation, fibrogenesis, and tumor development. Moreover, regulation of essential genes was assessed by reverse transcription polymerase chain reaction, immunoblottings, and immunostainings. Additionally, specific Jnk2 inhibition in hepatocytes of NEMO?hepa/JNK1?hepa mice was performed using small interfering (si) RNA (siJnk2) nanodelivery. Finally, active signaling pathways were blocked using specific inhibitors. Compound deletion of Jnk1 and Jnk2 in hepatocytes diminished hepatocellular carcinoma (HCC) in both the DEN model and in NEMO?hepa mice but in contrast caused massive proliferation of the biliary ducts. Indeed, Jnk1/2 deficiency in hepatocytes of NEMO?hepa (NEMO?hepa/JNK?hepa) animals caused elevated fibrosis, increased apoptosis, increased compensatory proliferation, and elevated inflammatory cytokines expression but reduced HCC. Furthermore, siJnk2 treatment in NEMO?hepa/JNK1?hepa mice recapitulated the phenotype of NEMO?hepa/JNK?hepa mice. Next, we sought to investigate the impact of molecular pathways in response to compound JNK deficiency in NEMO?hepa mice. We found that NEMO?hepa/JNK?hepa livers exhibited overexpression of the interleukin-6/signal transducer and activator of transcription 3 pathway in addition to epidermal growth factor receptor (EGFR)-rapidly accelerated fibrosarcoma (Raf)-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade. The functional relevance was tested by administering lapatinib, which is a dual tyrosine kinase inhibitor of erythroblastic oncogene B-2 (ErbB2) and EGFR signaling, to NEMO?hepa/JNK?hepa mice. Lapatinib effectively inhibited cystogenesis, improved transaminases, and effectively blocked EGFR-Raf-MEK-ERK signaling. Conclusion: We define a novel function of JNK1/2 in cholangiocyte hyperproliferation. This opens new therapeutic avenues devised to inhibit pathways of cholangiocarcinogenesis.
Project description:The c-Jun NH(2)-terminal kinase (JNK) signaling cascade has been implicated in a wide range of diseases, including cancer. It is unclear how different JNK proteins contribute to human cancer. Here, we report that JNK2 is activated in more than 70% of human squamous cell carcinoma (SCC) samples and that inhibition of JNK2 pharmacologically or genetically impairs tumorigenesis of human SCC cells. Most importantly, JNK2, but not JNK1, is sufficient to couple with oncogenic Ras to transform primary human epidermal cells into malignancy with features of SCC. JNK2 prevents Ras-induced cell senescence and growth arrest by reducing the expression levels of the cell cycle inhibitor p16 and the activation of NF-kappaB. On the other hand, JNK, along with phosphoinositide 3-kinase, is essential for Ras-induced glycolysis, an energy-producing process known to benefit cancer growth. These data indicate that JNK2 collaborates with other oncogenes, such as Ras, at multiple molecular levels to promote tumorigenesis and hence represents a promising therapeutic target for cancer.
Project description:As a target, the JNK pathway has been implicated in roles including cell death, proliferation, and inflammation in variety of contexts which span cardiovascular disease, neurodegenerative pathologies, and cancer. JNK1 and JNK2 have recently been demonstrated to function independently, highlighting a new parameter in the study of the JNK pathway. In order for JNK1 and JNK2-specific roles to be defined, better tools need to be employed. Previous studies have relied upon the broad spectrum JNK inhibitor, SP600125, to characterize the role of JNK signaling in a number of cell lines, including the breast cancer cell line MCF-7. In line with previous literature, our study has demonstrated that SP600125 treatment inhibited c-Jun and JNK phosphorylation and MCF-7 proliferation. However, in addition to targeting JNK1, JNK2, and JNK3, SP600125 has been previously demonstrated to suppress the activity of a number of other serine/threonine kinases, making SP600125 an inadequate tool for JNK isoform-specific roles to be determined. In this study, lentiviral shRNA was employed to selectively knockdown JNK1, JNK2, and JNK1/2 in MCF-7 cells. Using this approach, JNK phosphorylation was fully inhibited following stable knockdown of respective JNK isoforms. Interestingly, despite suppression of JNK phosphorylation, MCF-7 cell proliferation, cell cycle progression, or cell death remained unaffected. These findings raise the question of whether JNK phosphorylation really is pivotal in MCF-7 cell growth and death or if suppression of these events is a result of one of the many off-targets cited for SP600125.
Project description:The c-Jun NH2-terminal kinases (JNK) are regulated by a wide variety of cellular stresses and have been implicated in apoptotic signaling. Macrophages express 2 JNK isoforms, JNK1 and JNK2, which may have different effects on cell survival and atherosclerosis.To dissect the effect of macrophage JNK1 and JNK2 on early atherosclerosis, Ldlr(-/-) mice were reconstituted with wild-type, Jnk1(-/-), and Jnk2(-/-) hematopoietic cells and fed a high cholesterol diet. Jnk1(-/-)?Ldlr(-/-) mice have larger atherosclerotic lesions with more macrophages and fewer apoptotic cells than mice transplanted with wild-type or Jnk2(-/-) cells. Moreover, genetic ablation of JNK to a single allele (Jnk1(+/-)/Jnk2(-/-) or Jnk1(-/-)/Jnk2(+/-)) in marrow of Ldlr(-/-) recipients further increased atherosclerosis compared with Jnk1(-/-)?Ldlr(-/-) and wild-type?Ldlr(-/-) mice. In mouse macrophages, anisomycin-mediated JNK signaling antagonized Akt activity, and loss of Jnk1 gene obliterated this effect. Similarly, pharmacological inhibition of JNK1, but not JNK2, markedly reduced the antagonizing effect of JNK on Akt activity. Prolonged JNK signaling in the setting of endoplasmic reticulum stress gradually extinguished Akt and Bad activity in wild-type cells with markedly less effects in Jnk1(-/-) macrophages, which were also more resistant to apoptosis. Consequently, anisomycin increased and JNK1 inhibitors suppressed endoplasmic reticulum stress-mediated apoptosis in macrophages. We also found that genetic and pharmacological inhibition of phosphatase and tensin homolog abolished the JNK-mediated effects on Akt activity, indicating that phosphatase and tensin homolog mediates crosstalk between these pathways.Loss of Jnk1, but not Jnk2, in macrophages protects them from apoptosis, increasing cell survival, and this accelerates early atherosclerosis.
Project description:The relative contributions of the JNK subtypes in inflammatory ?-cell failure and apoptosis are unclear. The JNK protein family consists of JNK1, JNK2, and JNK3 subtypes, encompassing many different isoforms. INS-1 cells express JNK1?1, JNK1?2, JNK1?1, JNK1?2, JNK2?1, JNK2?2, JNK3?1, and JNK3?2 mRNA isoform transcripts translating into 46 and 54?kDa isoform JNK proteins. Utilizing Lentiviral mediated expression of shRNAs against JNK1, JNK2, or JNK3 in insulin-producing INS-1 cells, we investigated the role of individual JNK subtypes in IL-1?-induced ?-cell apoptosis. JNK1 knockdown prevented IL-1?-induced INS-1 cell apoptosis associated with decreased 46?kDa isoform JNK protein phosphorylation and attenuated Myc expression. Transient knockdown of Myc also prevented IL-1?-induced apoptosis as well as caspase 3 cleavage. JNK2 shRNA potentiated IL-1?-induced apoptosis and caspase 3 cleavage, whereas JNK3 shRNA did not affect IL-1?-induced ?-cell death compared to nonsense shRNA expressing INS-1 cells. In conclusion, JNK1 mediates INS-1 cell death associated with increased Myc expression. These findings underline the importance of differentiated targeting of JNK subtypes in the development of inflammatory ?-cell failure and destruction.
Project description:BACKGROUND & AIMS:Inclusion of liver grafts from cardiac death donors (CDD) would increase the availability of donor livers but is hampered by a higher risk of primary non-function. Here, we seek to determine mechanisms that contribute to primary non-function of liver grafts from CDD with the goal to develop strategies for improved function and outcome, focusing on c-Jun-N-terminal kinase (JNK) activation and mitochondrial depolarization, two known mediators of graft failure. METHODS:Livers explanted from wild-type, inducible nitric oxide synthase knockout (iNOS(-/-)), JNK1(-/-) or JNK2(-/-) mice after 45-min aorta clamping were implanted into wild-type recipients. Mitochondrial depolarization was detected by intravital confocal microscopy in living recipients. RESULTS:After transplantation of wild-type CDD livers, graft iNOS expression and 3-nitrotyrosine adducts increased, but hepatic endothelial NOS expression was unchanged. Graft injury and dysfunction were substantially higher in CDD grafts than in non-CDD grafts. iNOS deficiency and inhibition attenuated injury and improved function and survival of CDD grafts. JNK1/2 and apoptosis signal-regulating kinase-1 activation increased markedly in wild-type CDD grafts, which was blunted by iNOS deficiency. JNK inhibition and JNK2 deficiency, but not JNK1 deficiency, decreased injury and improved function and survival of CDD grafts. Mitochondrial depolarization and binding of phospho-JNK2 to Sab, a mitochondrial protein linked to the mitochondrial permeability transition, were higher in CDD than in non-CDD grafts. iNOS deficiency, JNK inhibition and JNK2 deficiency all decreased mitochondrial depolarization and blunted ATP depletion in CDD grafts. JNK inhibition and deficiency did not decrease 3-nitrotyrosine adducts in CDD grafts. CONCLUSION:The iNOS-JNK2-Sab pathway promotes CDD graft failure via increased mitochondrial depolarization, and is an attractive target to improve liver function and survival in CDD liver transplantation recipients.