Activation of TAK1 by Sef-S induces apoptosis in 293T cells

Sef (similar expression to fgf genes, also named IL-17RD) was identified as a negative regulator of fibroblast growth factor signaling. Sef-S, an alternative splice isoform of Sef, inhibits FGF-induced NIH3T3 cell proliferation. Here we report that Sef-S physically interacts with TAK1, induces Lys63-linked TAK1 polyubiquitination on lysine 209 and TAK1-mediated JNK and p38 activation. Co-overexpression of TAK1 WT, K34R, K150R, K158R mutants with Sef-S induces Lys63-linked TAK1 polyubiquitination whereas TAK1 K63R and K209R mutants fail. Furthermore, co-overexpression of Sef-S and TAK1 induce 293T cells apoptosis. These results reveal Sef-S actives Lys63-linked TAK1 polyubiquitination on lysine 209, induces TAK1-mediated JNK and p38 activation and also results apoptosis in 293T cells.     

A role for extracellular and transmembrane domains of Sef in Sef-mediated inhibition of FGF signaling

Sef (similar expression to fgf genes) is a member of the fibroblast growth factor (FGF) synexpression group that negatively regulates FGF receptor (FGFR) signaling in zebrafish during early embryonic development and in mammalian cell culture systems. The mechanism by which Sef exerts its inhibitory effect remains controversial. It has been reported that Sef functions either through binding to and inhibiting FGFR1 activation or by acting downstream of FGF receptors at the level of MEK/ERK kinases. In both cases, the intracellular domain of Sef was found to play a role in the inhibitory function of Sef. Here we demonstrated that both extracellular and transmembrane domains of Sef contributed to Sef-mediated negative regulation of FGF signaling. In fact, over-expression studies in NIH3T3 cells showed that a truncated mutant of Sef, which lacks the intracellular domain (SefECTM), exerted the inhibitory activity on FGF signaling by inhibiting FGFR1 tyrosine phosphorylation and subsequent activation of the Raf/MEK/ERK signaling cascade. We also showed that SefECTM associated with FGFR1, and inhibited FGF-induced ERK activation in HEK293T cells. Furthermore, we demonstrated that the over-expression of SefECTM was able to inhibit the function of a constitutively activated form of FGFR1, FGFR1-C289R, but not FGFR1-K562E. Finally, we found that SefECTM reduced cell viability when over-expressed in human umbilical vein endothelial cells (HUVEC). These data provide additional insight into the structure-activity relationship in the mechanism of inhibitory action of Sef on FGFR1-mediated signaling.     

Sef inhibits fibroblast growth factor signaling by inhibiting FGFR1 tyrosine phosphorylation and subsequent ERK activation

Signaling through fibroblast growth factor receptors (FGFRs) is essential for many cellular processes including proliferation and migration as well as differentiation events such as angiogenesis, osteogenesis, and chondrogenesis. Recently, genetic screens in Drosophila and gene expression screens in zebrafish have resulted in the identification of several feedback inhibitors of FGF signaling. One of these, Sef (similar expression to fgf genes), encodes a transmembrane protein that belongs to the FGF synexpression group. Here we show that like zebrafish Sef (zSef), mouse Sef (mSef) interacts with FGFR1 and that the cytoplasmic domain of mSef mediates this interaction. Overexpression of mSef in NIH3T3 cells results in a decrease in FGF-induced cell proliferation associated with a decrease in Tyr phosphorylation of FGFR1 and FRS2. As a consequence, there is a reduction in the phosphorylation of Raf-1 at Ser(338), MEK1/2 at Ser(217) and Ser(221), and ERK1/2 at Thr(202) and Tyr(204). Furthermore, mSef inhibits ERK activation mediated by a constitutively activated FGFR1 but not by a constitutively active Ras and decreases FGF but not PDGF-mediated activation of Akt. These results indicate that Sef exerts its inhibitory effects at the level of FGFR and upstream of Ras providing an additional level of negative regulation of FGF signaling.     

hSef inhibits PC-12 cell differentiation by interfering with Ras-mitogen-activated protein kinase MAPK signaling

Growth factor signaling by receptor tyrosine kinases regulates several cell fates, such as proliferation and differentiation. Sef was genetically identified as a negative regulator of fibroblast growth factor (FGF) signaling. Using bioinformatic methods and rapid amplification of cDNA ends-PCR, we isolated both the mouse and the human Sef genes, which encoded the Sef protein and Sef-S isoform that was generated through alternative splicing. We provide evidence that the Sef gene products were located mainly on the cell membrane. Co-immunoprecipitation and immunostaining experiments indicate that hSef interacts with FGFR1 and FGFR2 but not FGFR3. Our results demonstrated that stably expressed hSef strongly inhibits FGF2- or nerve growth factor-induced PC-12 cell differentiation. The intracellular domain of hSef is necessary for the inhibitory effect on FGF2-induced PC-12 cell differentiation. Furthermore, our data suggested Sef exerted the negative effect on FGF2-induced PC-12 cell differentiation through the prevention of Ras-mitogen-activated protein kinase signaling, possibly functioning upstream of the Ras molecule. These findings suggest that Sef may play an important role in the regulation of PC-12 cell differentiation.     

Regulation of myostatin signaling by c-Jun N-terminal kinase in C2C12 cells

Myostatin, a member of the transforming growth factor beta (TGF-beta) superfamily, is a negative regulator of skeletal muscle growth. We found that myostatin could activate c-Jun N-terminal kinase (JNK) signaling pathway in both proliferating and differentiating C2C12 cells. Using small interfering RNA (siRNA) mediated activin receptor type IIB (ActRIIB) knockdown, the myostatin-induced JNK activation was significantly reduced, indicating that ActRIIB was required for JNK activation by myostatin. Transfection of C2C12 cells with TAK1-specific siRNA reduced myostatin-induced JNK activation. In addition, JNK could not be activated by myostatin when the expression of MKK4 was suppressed with MKK4-specific siRNA, suggesting that TAK1-MKK4 cascade was involved in myostatin-induced JNK activation. We also found that blocking JNK signaling pathway by pretreatment with JNK specific inhibitor SP600125, attenuated myostatin-induced upregulation of p21 and downregulation of the differentiation marker gene expression. Furthermore, it was also observed that the presence of SP600125 almost annulled the growth inhibitory role of myostatin. Our findings provide the first evidence to reveal the involvement of JNK signaling pathway in myostatin's function as a negative regulator of muscle growth.     

Transforming growth factor-beta-activated kinase 1 regulates natural killer cell-mediated cytotoxicity and cytokine production

Carma1, a caspase recruitment domain-containing membrane-associated guanylate kinase, initiates a unique signaling cascade via Bcl10 and Malt1 in NK cells. Carma1 deficiency results in reduced phosphorylation of JNK1/2 and activation of NF-κB that lead to impaired NK cell-mediated cytotoxicity and cytokine production. However, the precise identities of the downstream signaling molecules that link Carma1 to these effector functions were not defined. Here we show that transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is abundantly present in NK cells, and activation via NKG2D results in its phosphorylation. Lack of Carma1 considerably reduced TAK1 phosphorylation, demonstrating the dependence of TAK1 on Carma1 in NKG2D-mediated NK cell activations. Pharmacological inhibitor to TAK1 significantly reduced NK-mediated cytotoxicity and its potential to generate IFN-γ, GM-CSF, MIP-1α, MIP-1β, and RANTES. Conditional in vivo knockdown of TAK1 in NK cells from Mx1Cre(+)TAK1(fx/fx) mice resulted in impaired NKG2D-mediated cytotoxicity and cytokine/chemokine production. Inhibition or conditional knockdown of TAK1 severely impaired the NKG2D-mediated phosphorylation of ERK1/2 and JNK1/2 and activation of NF-κB and AP1. Our results show that TAK1 links Carma1 to NK cell-mediated effector functions.     

Alternate FGF2-ERK1/2 signaling pathways in retinal photoreceptor and glial cells in vitro

Basic fibroblast growth factor (FGF2) stimulates photoreceptor survival in vivo and in vitro, but the molecular signaling mechanism(s) involved are unknown. Immunohistochemical and immunoblotting analyses of pure photoreceptors, inner retinal neurons, and Müller glial cells (MGC) in vitro revealed differential expression of the high affinity FGF receptors (FGFR1-4), as well as many cytoplasmic signaling intermediates known to mediate the extracellular signal-regulated kinase (ERK1/2) pathway. FGF2-induced tyrosine phosphorylation in vitro exhibited distinct profiles for each culture type, and FGF2-induced ERK1/2 activation was observed for all three preparations. Whereas U0126, a specific inhibitor of ERK kinase (MEK), completely abolished FGF2-induced ERK1/2 tyrosine phosphorylation and survival in cultured photoreceptors, persistent ERK1/2 phosphorylation was observed in cultured inner retinal cells and MGC. Furthermore U0126 treatment entirely blocked nerve growth factor-induced ERK1/2 activation in MGC, as well as FGF2-induced ERK1/2 activation in cerebral glial cells. Taken together, these data indicate that FGF2-induced ERK1/2 activation is entirely mediated by MEK within photoreceptors, which is responsible for FGF2-stimulated photoreceptor survival. In contrast, inner retina/glia possess alternative, cell type, and growth factor-specific MEK-independent ERK1/2 activation pathways. Hence signaling and biological effects elicited by FGF2 within retina are mediated by cell type-specific pathways.     

Sef is a negative regulator of fiber cell differentiation in the ocular lens

Growth factor signaling, mediated via receptor tyrosine kinases (RTKs), needs to be tightly regulated in many developmental systems to ensure a physiologically appropriate biological outcome. At one level this regulation may involve spatially and temporally ordered patterns of expression of specific RTK signaling antagonists, such as Sef (similar expression to fgfs). Growth factors, notably FGFs, play important roles in development of the vertebrate ocular lens. FGF induces lens cell proliferation and differentiation at progressively higher concentrations and there is compelling evidence that a gradient of FGF signaling in the eye determines lens polarity and growth patterns. We have recently identified the presence of Sef in the lens, with strongest expression in the epithelial cells. Given the important role for FGFs in lens developmental biology, we employed transgenic mouse strategies to determine if Sef could be involved in regulating lens cell behaviour. Over-expressing Sef specifically in the lens of transgenic mice led to impaired lens and eye development that resulted in microphthalmia. Sef inhibited primary lens fiber cell elongation and differentiation, as well as increased apoptosis, consistent with a block in FGFR-mediated signaling during lens morphogenesis. These results are consistent with growth factor antagonists, such as Sef, being important negative regulators of growth factor signaling. Moreover, the lens provides a useful paradigm as to how opposing gradients of a growth factor and its antagonist could work together to determine and stabilise tissue patterning during development and growth.     

TAK1 participates in c-Jun N-terminal kinase signaling during Drosophila development

Transforming growth factor beta (TGF-beta)-activated kinase 1 (TAK1) is a member of the MAPKKK superfamily and has been characterized as a component of the TGF-beta/bone morphogenetic protein signaling pathway. TAK1 function has been extensively studied in cultured cells, but its in vivo function is not fully understood. In this study, we isolated a Drosophila homolog of TAK1 (dTAK1) which contains an extensively conserved NH(2)-terminal kinase domain and a partially conserved COOH-terminal domain. To learn about possible endogenous roles of TAK1 during animal development, we generated transgenic flies which express dTAK1 or the mouse TAK1 (mTAK1) gene in the fly visual system. Ectopic activation of TAK1 signaling leads to a small eye phenotype, and genetic analysis reveals that this phenotype is a result of ectopically induced apoptosis. Genetic and biochemical analyses also indicate that the c-Jun amino-terminal kinase (JNK) signaling pathway is specifically activated by TAK1 signaling. Expression of a dominant negative form of dTAK during embryonic development resulted in various embryonic cuticle defects including dorsal open phenotypes. Our results strongly suggest that in Drosophila melanogaster, TAK1 functions as a MAPKKK in the JNK signaling pathway and participates in such diverse roles as control of cell shape and regulation of apoptosis.     

X-linked inhibitor of apoptosis (XIAP) inhibits c-Jun N-terminal kinase 1 (JNK1) activation by transforming growth factor beta1 (TGF-beta1) through ubiquitin-mediated proteosomal degradation of the TGF-beta1-activated kinase 1 (TAK1)

Active NF-kappaB renders malignant hepatocytes refractory to the growth inhibitory and pro-apoptotic properties of transforming growth factorbeta1 (TGF-beta1). NF-kappaB counteracts TGF-beta1-induced apoptosis through up-regulation of downstream target genes, such as XIAP and Bcl-X(L), which in turn inhibit the intrinsic pathway of apoptosis. In addition, induction of NF-kappaB by TGF-beta1 inhibits JNK signaling, thereby attenuating TGF-beta1-induced cell death of normal hepatocytes. However, the mechanism involved in the negative cross-talk between the NF-kappaB and JNK pathways during TGF-beta1 signaling has not been determined. In this study, we have identified the XIAP gene as one of the critical mediators of NF-kappaB-mediated suppression of JNK signaling. We show that NF-kappaB plays a role in the up-regulation of XIAP gene expression in response to TGF-beta1 treatment and forms a TGF-beta1-inducible complex with TAK1. Furthermore, we show that the RING domain of XIAP mediates TAK1 polyubiquitination, which then targets this molecule for proteosomal degradation. Down-regulation of TAK1 protein expression inhibits TGF-beta1-mediated activation of JNK and apoptosis. Conversely, silencing of XIAP promotes persistent JNK activation and potentiates TGF-beta1-induced apoptosis. Collectively, our findings identify a novel mechanism for the regulation of JNK activity by NF-kappaB during TGF-beta1 signaling and raise the possibility that pharmacologic inhibition of the NF-kappaB/XIAP signaling pathway might selectively abolish the pro-oncogenic activity of TGF-beta1 in advanced hepatocellular carcinomas (HCCs) without affecting the pro-apoptotic effects of TGF-beta1 involved in normal liver homeostasis.     

Hematopoietic progenitor kinase 1 is a component of transforming growth factor beta-induced c-Jun N-terminal kinase signaling cascade

The c-Jun N-terminal kinase (JNK) signaling pathway is involved in transforming growth factor beta (TGF-beta) signaling in a variety of cell systems. We report here that hematopoietic progenitor kinase 1 (HPK1), a novel Ste20-like protein serine/threonine kinase, serves as an upstream mediator for the TGF-beta-activated JNK1 cascade in 293T cells. TGF-beta treatment resulted in a time-dependent activation of HPK1, which was accompanied by similar kinetics of JNK1 activation. The activation of JNK1 by TGF-beta was abrogated by a kinase-defective HPK1 mutant but not by a kinase-defective mutant of kinase homologous to Ste20/Sps1. This result indicates that HPK1 is specifically required for TGF-beta-induced activation of JNK1. We also found that TGF-beta-induced JNK1 activation was blocked by a kinase-defective mutant of TGF-beta-activated kinase 1 (TAK1). In addition, interaction between HPK1 and TAK1 was observed in transient transfection assays, and this interaction was enhanced by TGF-beta treatment. Both stress-activated protein kinase/extracellular signal-regulated kinase kinase (SEK) and mitogen-activated protein kinase kinase 7 (MKK7) are immediate upstream activators of JNK1. Although SEK and MKK7 acted downstream of TAK1, only a kinase-defective SEK mutant blocked TGF-beta-induced activation of JNK1, indicating that the TGF-beta signal is relayed solely through SEK, but not MKK7, in vivo. Furthermore, TGF-beta-induced activating protein 1 activation was blocked by a HPK1 mutant, as well as by TAK1 and SEK mutants. Taken together, these studies establish a potential cascade of TGF-beta-activated interacting kinases beginning with HPK1, a Ste20 homolog, and ending in JNK1 activation: HPK1 --> TAK1 --> SEK --> JNK1.     

Osmotic stress activates the TAK1-JNK pathway while blocking TAK1-mediated NF-kappaB activation: TAO2 regulates TAK1 pathways

Osmotic stress activates MAPKs, including JNK and p38, which play important roles in cellular stress responses. Transforming growth factor-beta-activated kinase 1 (TAK1) is a member of the MAPK kinase kinase (MAPKKK) family and can activate JNK and p38. TAK1 can also activate IkappaB kinase (IKK) that leads to degradation of IkappaB and subsequent NF-kappaB activation. We found that TAK1 is essential for osmotic stress-induced activation of JNK but is not an exclusive mediator of p38 activation. Furthermore, we found that although TAK1 was highly activated upon osmotic stress, it could not induce degradation of IkappaB or activation of NF-kappaB. These results suggest that TAK1 activity is somehow modulated to function specifically in osmotic stress signaling, leading to the activation of JNK but not of IKK. To elucidate the mechanism underlying this modulation, we screened for potential TAK1-binding proteins. We found that TAO2 (thousand-and-one amino acid kinase 2) associates with TAK1 and can inhibit TAK1-mediated activation of NF-kappaB but not of JNK. We observed that TAO2 can interfere with the interaction between TAK1 and IKK and thus may regulate TAK1 function. TAK1 is activated by many distinct stimuli, including cytokines and stresses, and regulation by TAO2 may be important to activate specific intracellular signaling pathways that are unique to osmotic stress.     

Angiotensin II increases CTGF expression via MAPKs/TGF-β1/TRAF6 pathway in atrial fibroblasts

The activation of transforming growth factor-β1(TGF-β1)/Smad signaling pathway and increased expression of connective tissue growth factor (CTGF) induced by angiotensin II (AngII) have been proposed as a mechanism for atrial fibrosis. However, whether TGFβ1/non-Smad signaling pathways involved in AngII-induced fibrogenetic factor expression remained unknown. Recently tumor necrosis factor receptor associated factor 6 (TRAF6)/TGFβ-associated kinase 1 (TAK1) has been shown to be crucial for the activation of TGF-β1/non-Smad signaling pathways. In the present study, we explored the role of TGF-β1/TRAF6 pathway in AngII-induced CTGF expression in cultured adult atrial fibroblasts. AngII (1 μM) provoked the activation of P38 mitogen activated protein kinase (P38 MAPK), extracellular signal-regulated kinase 1/2(ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). AngII (1 μM) also promoted TGFβ1, TRAF6, CTGF expression and TAK1 phosphorylation, which were suppressed by angiotensin type I receptor antagonist (Losartan) as well as p38 MAPK inhibitor (SB202190), ERK1/2 inhibitor (PD98059) and JNK inhibitor (SP600125). Meanwhile, both TGFβ1 antibody and TRAF6 siRNA decreased the stimulatory effect of AngII on TRAF6, CTGF expression and TAK1 phosphorylation, which also attenuated AngII-induced atrial fibroblasts proliferation. In summary, the MAPKs/TGFβ1/TRAF6 pathway is an important signaling pathway in AngII-induced CTGF expression, and inhibition of TRAF6 may therefore represent a new target for reversing Ang II-induced atrial fibrosis.     

Disabled-2 (Dab2) mediates transforming growth factor beta (TGFbeta)-stimulated fibronectin synthesis through TGFbeta-activated kinase 1 and activation of the JNK pathway

The multifunctional cytokine transforming growth factor beta (TGFbeta) exerts many of its effects through its regulation of extracellular matrix components, including fibronectin (FN). Although expression of both TGFbeta and FN are essential for embryonic development and wound healing in the adult, overexpression leads to excessive deposition of extracellular matrix observed in many fibroproliferative disorders. We previously have demonstrated that TGFbeta-stimulated FN induction requires activation of the c-Jun N-terminal kinase (JNK) pathway; however, the signaling molecules that link the TGFbeta receptors to the JNK pathway remain unknown. We show here that the cytosolic adaptor protein disabled-2 (Dab2) directly stimulates JNK activity, whereas stable small interfering RNA-mediated ablation of Dab2 in NIH3T3 mouse fibroblasts and A10 rat aortic smooth muscle cells demonstrates that its expression is required for TGFbeta-mediated FN induction. We demonstrate that TGFbeta treatment stimulates the association of Dab2 with the mitogen-activated protein kinase kinase kinase, TAK1. Attenuation of cellular TAK1 levels by transient double-stranded RNA oligonucleotide transfection as well as overexpression of kinase-deficient TAK1 leads to abrogation of TGFbeta-stimulated FN induction. Furthermore, cell migration, another JNK-dependent response, is attenuated in NIH3T3-siDab2-expressing clones. We, therefore, delineate a signaling pathway proceeding from the TGFbeta receptors to Dab2 and TAK1, leading to TGFbeta-stimulated JNK activation, FN expression, and cell migration.