STAT3 Ser727 Phosphorylation Research Articles

Abstract PR-06: PKC∈-activated Stat3 and subsequent AR interaction are potential molecular targets for prevention of late-stage prostate cancer

Abstract Prostate cancer (PCa) is the second most common malignancy in men and the second leading cause of cancer-related deaths in the Western World. Androgen ablation therapy remains the gold standard for the treatment of PCa. Despite the initial success of androgen ablation therapy, resistance to anti-androgen therapy manifests by progression to castration-resistant prostate cancer (CRPC). CRPC is the end stage that accounts for the majority of PCa patient deaths. An understanding of the mechanisms linked to the emergence of CRPC is the key to the prevention of this disease. Overwhelming evidence supports the concept that development of CRPC is usually related to continued activation of androgen receptor (AR). Several mechanisms are proposed for androgen-dependent (AD) and androgen-independent (AI) activation of AR in CRPC. Gene amplification and mutations in AR are frequently observed in recurrent PCa, which may account for the hypersensitivity of the AR to low castrate level of androgens and altered ligand specificity. In the AR-dependent pathway, the functions of AR are modified significantly and involve AR interaction with many co-activators and co-repressors. Increased AR activity in CRPC is perhaps caused by cross talk of AR with multiple intracellular signaling cascades. We present here that PKC∈ and Stat3, which correlate with the aggressiveness of PCa, may play roles in both AI AR activation and promotion of PCa cell survival for emergence and progression of CRPC. Stat3 has two conserved amino acid residues (Tyr705 and Ser727), which have to be phosphorylated for maximum transcriptional activity. PKC∈, which is constitutively activated in PCa, mediates phosphorylation of both Stat3Tyr705 and Stat3Ser727. Specific observations are our finding that PKC∈ deletion in TRAMP mice, which prevents PCa development and metastasis, inhibits the expression levels of IL-6, pStat3Ser727 and pStat3Tyr705. PKC∈ overexpression is sufficient to transform AD LNCaP cells into an AI variant. We further explored the possibility that PKC∈− activated Stat3 is a non-androgen activator of AR. In this experiment, human PCa LNCaP (AD) cells were infected with adenoviral vector containing PKC∈ or empty vector; 48 h later, cells were harvested and whole cell lysates were prepared. The effect of PKC∈ overexpression on the interaction of Stat3 with AR was determined by performing reciprocal immunoprecipitation/blotting experiments. Overexpression of PKC∈ increased the interaction of Stat3 with AR. We also found similar interaction of Stat3 with AR in human PCa C4–2 (AI) cells treated with IL-6. To determine the link of PKC∈ in Stat3 interaction with AR and the growth of C4–2 cells, we used plumbagin (PL). PL is a unique plant-derived chemopreventive agent, which selectively inhibits expression of PKC∈. PL administration delayed the growth of C4–2 cells xenograft tumors as determined by both tumor weight and volume. In a reciprocal immunoprecipation/blotting experiments, PL inhibited Stat3-AR interaction. We conclude that: 1) PKC∈-mediated Stat3Ser727 phosphorylation and subsequent Stat3-AR interaction are essential components of the emergence and progression of CRPC, 2) Progression of CRPC is the result of cross-talk of PKC∈-Stat3 signaling with other signaling pathways (AKT and MAPK) which activate AR as well as promote cell survival and 3) PKC∈-activated Stat3 and subsequent AR interaction are potential molecular targets for prevention of late stage PCa. Support: NIH Grant CA138761. Citation Information: Cancer Prev Res 2011;4(10 Suppl):PR-06.

Abstract 5204: PP2A- STAT3 pathway mediates suspension survival in colon cancer stem cells

Abstract Cancer stem or initiating cells (CSCs) which share similarity with normal stem cells are a small population of cells in tumor with the abilities of self-renewal, multi- potent differentiation, and tumor initiation. CSCs also contribute to the resistance of tumor to chemotherapy or irradiation therapy. Therefore, targeting CSCs could be a hopeful way in treating cancer. Involvement of constitutive phosphorylation of STAT3 at Tyr705 in tumorigenesis of CSCs was implicated in many types of cancers. However, the role of Ser727 phosphorylation in STAT3 activation and CSCs tumorigenesis remains unknown. In this study, we are trying to figure out whether Ser727 phosphorylation of STAT3 plays a role in the characteristic properties of colon CSCs. Efforts will also be made to shed light on the upper signaling pathway and downstream targets of Ser727 phosphorylation of STAT3. Our preliminary data demonstrate enriched CSCs increase in the capacity for suspension survival both in vitro and in vivo. After enrichment of CSC population by spheroid culture, the phosphorylation of STAT3 at Ser727 but not Tyr705 was increased and localized at nucleus. The increase of Ser727 activation is also associated with down-regulation of PP2A activity, a serine/threonine phosphatase candidate of STAT3. We further proved PP2A formed immunoprecipitated complex with STAT3 and regulated STAT3 activation at Ser727 by treating the cells with PP2A inhibitor in normal culture or PP2A activator in spheroid culture. Overexpression of wild-type PP2A or Ser727 point mutated STAT3 reduced the survival ability in CSC. In contrast, knock down of PP2A activity or gain of function mutation at Ser727 of STAT3 increased the survival ability in nCSC. Taken together, our current data indicate enriched CSCs increased the capacity of suspension survival through the activation of PP2A-STAT3 pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5204. doi:10.1158/1538-7445.AM2011-5204

Ultraviolet radiation and 12‐O‐tetradecanoylphorbol‐13‐acetate‐induced interaction of mouse epidermal protein kinase Cε with Stat3 involve integration with ERK1/2

We have reported that protein kinase C epsilon (PKCε) expression level in epidermis dictates the susceptibility of mice to the development of squamous cell carcinomas (SCC) elicited either by repeated exposure to ultraviolet radiation (UVR) or by the DMBA-TPA tumor promotion protocol. To find clues about the mechanism by which PKCε mediates susceptibility to UVR-induced development of SCC, we found that PKCε-over-expressing transgenic mice, as compared to their wild-type littermates, when exposed to UVR, elicit enhanced phosphorylation of Stat3 at Ser727 residues. Stat3 is constitutively activated in SCC and UVR fails to induce SCC in Stat3 mutant mice. Stat3Ser727 phosphorylation is essential for Stat3 transcriptional activity (Cancer Res. 67: 1385, 2007). We now present several novel findings including that PKCε integrates with its downstream partner ERK1/2 to phosphorylate Stat3Ser727. In these experiments, mice were either exposed to UVR (2 kJ/m(2)/dose) emitted by Kodacel-filtered FS-40 sun lamps or treated with TPA (5 nmol). Both UVR and TPA treatment stimulated PKCε-Stat3 interaction, Stat3Ser727 phosphorylation and Stat3-regulated gene COX-2 expression. PKCε-Stat3 interaction and Stat3Ser727 phosphorylation was also observed in SCC elicited by repeated UVR exposures of mice. PKCε-Stat3 interaction was PKCε specific. UVR or TPA-stimulated Stat3Ser727 phosphorylation accompanied interaction of PKCε with ERK1/2 in intact mouse skin in vivo. Deletion of PKCε in wild-type mice attenuated both TPA and UVR-induced expression of phosphoforms of ERK1/2 and Stat3Ser727. These results indicate that PKCε integrates with ERK1/2 to mediate both TPA and UVR-induced epidermal Stat3Ser727 phosphorylation. PKCε and Stat3 may be potential molecular targets for SCC prevention.

A murine model of inducible, cardiac-specific deletion of STAT3: Its use to determine the role of STAT3 in the upregulation of cardioprotective proteins by ischemic preconditioning

Pharmacological studies have shown that signal transducers and activators of transcription (STATs) are necessary for the delayed cardioprotection of ischemic preconditioning (PC). However, pharmacologic STAT inhibitors are not specific; furthermore, the individual role of STAT3 in late PC remains unknown. The objectives of the study were (i) to create an inducible, cardiac-specific STAT3 knockout mouse; (ii) to verify whether STAT3 deletion has any adverse effects in the short term (~ 1 month); and (iii) to use this novel tool to evaluate the role of STAT3 in the PC-induced upregulation of cardioprotective and anti-apoptotic proteins. We created an inducible, cardiomyocyte-restricted STAT3 deficient mouse ( MCM TG: STAT3 flox/flox ) by interbreeding STAT3 flox/flox mice and tamoxifen-inducible MCM TG mice. Treatment of MCM TG: STAT3 flox/flox mice with tamoxifen resulted in deletion of STAT3 specifically in cardiac myocytes, concomitant with abrogation of ischemic PC-induced Tyr-705 and Ser-727 phosphorylation of STAT3 and increased STAT3 DNA-binding activity. In vehicle-treated MCM TG: STAT3 flox/flox mice, ischemic PC increased the expression of cardioprotective (COX-2 and HO-1) and anti-apoptotic (e.g., Mcl-1, Bcl-x L, c-FLIP L, c-FLIP S) proteins 24 h later; in contrast, in tamoxifen-treated MCM TG: STAT3 flox/flox mice this increase was completely absent. Deletion of STAT3 had no apparent adverse effects on LV structure or function after 35 days. We have developed a novel inducible, cardiomyocyte-restricted STAT3 deficient mouse that can be used to specifically interrogate the role of this transcription factor in cardiovascular pathophysiology in vivo. Our data demonstrate, for the first time, that recruitment of STAT3 plays an obligatory role in the upregulation of cardioprotective and anti-apoptotic proteins and suggest that STAT3 activation is important in inhibiting both the death receptor pathway (which is modulated by c-FLIP L and c-FLIP S) and the mitochondrial pathway (which is mediated by Mcl-1 and Bcl-x L).

Abstract P5-03-02: Progestin Activation of p42/p44 MAPK Induces Stat3 Phosphorylation at Serine 727 Promoting Breast Cancer Growth

Abstract We have previously demonstrated that the synthetic progestin medroxyprogesterone acetate (MPA) induces Stat3 phosphorylation at Tyr705 leading to Stat3 transcriptional activation, which is an absolute requirement for progestin stimulation of in vitro and in vivo breast cancer growth. These studies were performed in C4HD cells from an experimental model of hormonal carcinogenesis in which MPA induced mammary adenocarcinomas in Balb/c mice, and in the human breast cancer cell line T47D. Besides tyrosine phosphorylation, Stat3 can also be phosphorylated on a single serine residue (Ser727) leading to a full transcriptional activation of Stat3. Here, we explored the effect of progestins on Stat3 Ser727 phosphorylation and its biological significance. MPA treatment of C4HD and T47D cells for 5 to 10 min induced phosphorylation of Stat3 on Ser727. The 727 Ser residue in Stat3 is situated in a conserved PMSP motive which resembles the consensus PxS/TP motive for mitogen-activated protein kinase (MAPK) targets. To address whether MPA activation of p42/p44 MAPK signaling pathway is directly involved in Ser727 Stat3 phosphorylation, we pretreated C4HD cells with U0126, a p42/p44 MAPK inhibitor, which suppressed phosphorylation of Stat3 on Ser727. To strengthen the finding that Ser phosphorylation of Stat3 proceeds in a p42/p44 MAPK-dependent manner, we performed a cold in vitro phosphorylation assay. We observed that immunoprecipitated p42/p44MAPK from C4HD cells activated with MPA was able to phosphorylate Stat3 at Ser727 obtained from non treated cells. Neither p42/p44 MAPK obtained from control cells nor p42/p44 MAPK inactivated by U0126 increased Stat3 Ser727 phosphorylation. Next, we explored the relevance of MPA-induced Stat3 Ser727 phosphorylation on cyclin D1 promoter activation, as it contains Stat3 response elements, named GAS sites. C4HD and T47D cells transiently co-transfected with a cyclin D1 promoter luciferase construct and a Stat3 expression vector, showed an enhanced transcriptional activity upon MPA treatment. Co-transfection of Ser727 to alanine-mutated Stat3 (Stat3S727-A) expression vector resulted in abrogation of MPA-induced cyclin D1 promoter activation. Moreover, MPA-induced cyclin D1 protein expression in C4HD and T47D cells was inhibited in cells transfected with Stat3S727-A. To further investigate the correlation between MPA-induced Stat3 Ser727 phosphorylation and cell growth, C4HD and T47D cells were transiently transfected with Stat3S727-A expression vector. Expression of Stat3S727-A had an inhibitory effect on in vitro MPA-induced growth of C4HD and T47D cells. Finally we explored the requirement of Stat3 Ser727 for in vivo progestin-driven breast cancer growth. Transfection of C4HD cells with Stat3S727-A significantly inhibited the ability of these cells to grow in syngeneic mice. Our findings have for the first time demonstrated that progestins are able to induce Stat3 Ser727 phosphorylation through a mechanism requiring p42/p44 MAPK activity in both mouse and human mammary tumor cells. We also found Stat3 phosphorylated at Ser727 to be a requisite for progestin-induced cyclin D1 promoter activation and protein up-regulation, as well as for stimulation of breast cancer growth in vitro and in vivo Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-03-02.

Abstract 5107: HDL and sphingosine-1-phosphate activate signal transducer and activator of transcription 3 in prostate cancer DU145 cells via ERK1/2 and S1P receptors

Abstract (a) Introduction and Objective: Signal transducer and activator of transcription 3 (Stat3) has been implicated in the promotion of migration, invasion and growth of prostate cancer cells. Recently, HDL, which transports the bioactive lipid sphingosine-1-phosphate (S1P), was reported to activate Stat3 in cardiomyocytes and is a known mitogen for several cell types. S1P acts by binding to several G protein-coupled receptors, namely S1P1, S1P2 and S1P3, which are expressed in prostate cancer cell lines. Androgen deprivation therapy in men with prostate cancer leads to a significant increase of HDL-C, but the effect of HDL on prostate cancer is unknown. In this study, we examined the effect of HDL and S1P on Stat3 activation in prostate cancer cells and the involvement of ERK1/2, Akt, and S1P receptors in this process in 3 prostate cancer cell lines (PC-3, LNCaP and DU145). (b) Methods: We used HDL isolated from health volunteer men. Discordial reconstituted(r) HDL containing POPC and apoA-1, (POPC/A-1)rHDL or POPC, S1P and A-1, (POPC/S1P/A-1)rHDL were prepared by the cholate dialysis method. The phosphorylations of Stat3, ERK1/2 and Akt were detected by Western blot analysis. mRNA expressions of PC cells were evaluated by quantitative real-time PCR. Cell viability, migration and invasion were determined by MTS assay, would healing assay and matrigel invasion chamber assay, respectively. (c) Results: HDL increased a dose and a time dependent Ser727 phosphorylation of Stat3, but not Tyr705 in DU145 cells but not in the other two cell lines. S1P and (POPC/S1P/A-1)rHDL also induced the phosphorylation, but not rHDL made without S1P (POPC/A-1). They also induced DU145 cells proliferation, migration, invasion and phosphorylations of ERK1/2 and Akt. PD98059, a MEK inhibitor, and pertussis toxin, a Gi inhibitor, attenuated HDL-, S1P- and (POPC/S1P/A-1)rHDL-induced Stat3 phosphorylation, whereas LY294002, a PI3K inhibitor, had no effect. Concerning S1P receptors, S1P1 expression was much lower than S1P2 and S1P3 in DU145 cells. Both JTE013, a S1P2 antagonist, and VPC23019, a S1P1/S1P3 antagonist, attenuated HDL-, S1P- and (POPC/S1P/A-1)rHDL-induced Stat3 phosphorylations and cell migrations. (d) Conclusions: HDL was shown to induce Ser727 phosphorylation of Stat3 via its S1P constituent and involved both S1P2 and S1P3 receptors in DU145 cells. Moreover, ERK1/2 activation is involved in cell activation by HDL. These results suggest that the change in HDL plasma levels by androgen deprivation therapy HDL may alter prostate cancer growth and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5107.

Abstract 3780: Plumbagin a non-toxic natural agent, induces apoptosis and inhibits the growth of prostate cancer LNCaP and C4-2 cells via blocking of both androgen and non-androgen activation of androgen receptor signaling

Abstract Prostate cancer (PCa) is one of the most commonly diagnosed cancers and leading cause of cancer related death in men in Western countries including the Unites States. Androgen receptor (AR)-mediated signaling plays a critical role in the development and progression of PCa. Most androgen independent (AI) prostate tumors continue to express AR as well as its target gene PSA, which indicates that these tumor cells maintain functional AR signaling. Evidence suggests that AR signaling is activated through androgen and androgen bypass mechanism. We believe that identification of non-toxic natural agents capable of blocking both androgen and non-androgen activation of AR signaling could be effective for the treatment of PCa. We have previously shown that a novel plant derived napthoquinone plumbagin (PL) induces apoptosis and inhibits growth of PCa cells in in vitro as well as in in vivo model systems (Cancer Research 2008, PMID: 18974148). We present here that PL inhibits both androgen and non-androgen activation of AR signaling in PCa cells. We observed that PL inhibits synthetic androgen methyltrienolone (R1881) induced growth of androgen-dependent (AD) LNCaP and AI C4-2 cells. PL treatment was found to inhibit protein levels of AR and AR-target gene PSA and accelerates AR degradation. To analyze the effect of PL on decreased AR transactivation, transient transfections using androgen responsive reporters PSA-luc were carried out. Treatment of LNCaP and C4-2 cells with R1881 led to induction of PSA reporter in these cells. Co-treatment of LNCaP and C4-2 cells with PL led to a dose-dependent decrease in AR transactivation as reflected by a decrease in the PSA reporter activity. These results suggest that PL interferes with androgen-induced transactivation function of AR. It has been reported that IL-6 signaling acts as a ligand for the activation of AR signaling. IL-6 induced phosphorylated Stat3 interacts with the N-terminal domain of AR and activates its target genes. We have previously shown that PL treatment inhibits Stat3 phosphorylation in PCa DU-145 cells xenograft tumor tissues (Cancer Research 2008, PMID: 18974148). Therefore, we hypothesized that PL treatment will inhibit Stat3 and AR N-terminal domain interaction in PCa cells. Reciprocal immunoprecipitation/blotting experiments revealed that PL treatment inhibited IL-6 induced interaction of Stat3 with AR N-terminal domain in both the PCa LNCaP and C4-2 cells. PL treatment also inhibited IL-6 induced Tyr 705 and Ser727 phosphorylation of Stat3. These results establish that PL has the potential to block the androgen and non-androgen activation of AR signaling in PCa and provide further evidence that PL could be used as a chemopreventive natural agent in ongoing anti-hormone therapy of PCa. (Support: NIH grants, CA35368). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3780.

Abstract 1478: Role of Stat3 Ser727 phosphorylation in mouse hepatocarcinogenesis

Abstract Inappropriate activation of signal transducer and activator of transcription 3 (Stat3) occurs with extreme frequency in a wide variety of cancers including HCC, and constitutive Stat3 activation in hepatocytes was demonstrated to accelerate hepatocarcinogenesis, while repression of Stat3 activation suppresses hepatocarcinogenesis. Diverse factors such as cytokines, growth factors and oncoproteins can activate Stat3, where phosphorylation of the conserved tyrosine residue at the C-terminus (Y705) results in the Stat3 homodimer or the heterodimer with Stat1, and phosphorylation of the serine residue on the transactivation domain (S727) contributes to its maximal transcriptional activity. The activated Stat dimers bind to specific DNA-response elements in the promoter of target genes and thereby induce unique gene expression programs cooperating with other transcription factors and cofactors. We investigated Stat3 activation in mouse hepatocarcinogenesis induced by neonatal treatment with diethylnitrosamine (DEN) and found that Stat3 phosphorylation occurred by two steps, where S727 phosphorylation was seen in the preneoplastic stage, whereas Y705 phosphorylation was further associated with malignant progression. In mouse HCC cells, serum treatment induced Ser727 phosphorylation via MAPK activation, associating with cell proliferation and Stat3 nuclear translocation, whereas IL-6 induced Tyr705 phosphorylation via JAK activation without stimulating cell proliferation and the Stat3 nuclear translation. On the other hand, the Ser727 phosphorylation dead Stat3 (S727A), in which the serine residue was replaced by alanine, reduced the cell proliferation and Stat3 nuclear translocation, while the Ser727-phosphorylation mimic Stat3 (S727D), in which the serine residue was replaced by aspartic acid, enhanced the cell proliferation and Stat3 nuclear translocation. To further investigate the role of Stat3 Ser727 phosphorylation, the Stat3 knockdown HCC cell lines were established by introducing the Stat3 siRNA into the mouse HCC cells. These cell lines showed retarded growth and less attachment to the substrate forming spheroidal aggregates, whereas the parent cells showed rapid proliferation and attached to the substrate with extended morphology. cDNA array analysis revealed that the Stat3 knockdown cells showed variety of gene expression changes relating to cell survival and growth, and RT-PCR demonstrated that the expression of EGFR, FGFR and E-cadherin was markedly reduced in the Stat3 knockdown cells. Our findings indicate that the Ser727-phosphorylated Stat3 plays an important role from the early stage in mouse hepatocarcinogenesis, presumably by controlling cellular growth and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1478.

Protein kinase Cɛ mediates Stat3Ser727 phosphorylation, Stat3-regulated gene expression, and cell invasion in various human cancer cell lines through integration with MAPK cascade (RAF-1, MEK1/2, and ERK1/2)

Protein kinase C epsilon (PKCε), a novel calcium-independent PKC isoform, has been shown to be a transforming oncogene. PKCε-mediated oncogenic activity is linked to its ability to promote cell survival. However, the mechanisms by which PKCε signals cell survival remain elusive. We found that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, is a protein partner of PKCε. Stat3 has two conserved amino acid (Tyr705 and Ser727) residues, which are phosphorylated during Stat3 activation. PKCε interacts with Stat3α isoform which has Ser727 and not with Stat3β isoform which lacks Ser727. PKCε-Stat3 interaction and Stat3Ser727 phosphorylation was initially observed during induction of squamous cell carcinomas and in prostate cancer. Now we present that: 1) PKCε physically interacts with Stat3α isoform in various human cancer cells: skin melanomas (MeWo and WM266-4), gliomas (T98G and MO59K), bladder (RT-4 and UM-UC-3), colon (Caco-2), lung (H1650), pancreatic (PANC-1), and breast (MCF-7 and MDA:MB-231). 2) Inhibition of PKCε expression using specific siRNA inhibits Stat3Ser727 phosphorylation, Stat3-DNA binding, Stat3-regulated gene expression as well as cell invasion. 3) PKCε mediates Stat3Ser727 phosphorylation via integration with the MAPK cascade (RAF-1, MEK1/2, and ERK1/2). The results indicate that PKCε-mediated Stat3Ser727 phosphorylation is essential for constitutive activation of Stat3 and cell invasion in various human cancers.

Inhibition of ERK1/2 does not affect LIF-induced STAT3 ser727 phosphorylation in trophoblastic cells

Inhibition of ERK1/2 does not affect LIF-induced STAT3 ser727 phosphorylation in trophoblastic cells

Differential Regulation of Foxp3 and IL-17 Expression in CD4 T Helper Cells by IRAK-1

Host immune responses are finely regulated by the opposing effects of Th17 and T regulatory (Treg) cells. Treg cells help to dampen inflammatory processes and Th17 cells facilitate various aspects of immune activation. The differentiation of Th cells depends on a unique combination of stimulants and subsequent activation of diverse transcription factors. In particular, cooperative activation of NFAT and Smad3 leads to the induction of Treg cells, and cooperation among STAT3 and Smad3 switches to the induction of Th17 cells. We have previously shown that the IL-1 receptor associated kinase 1 (IRAK-1) selectively activates STAT3 and inactivates NFAT. Physiological studies have shown that IRAK-1(-/-) mice are protected from developing various inflammatory diseases, including experimental autoimmune encephalomyelitis and atherosclerosis with unknown mechanism. In this study, we demonstrate that IRAK-1 plays a critical modulatory role in the differentiation of Th17 and Treg cells. Following stimulation with TCR agonists and TGFbeta, IRAK-1(-/-) CD4 Th cells display elevated nuclear NFATc2 levels and increased interaction of NFATc2 and Smad3, resulting in increased expression of Foxp3, a key marker for Treg cells. IRAK-1(-/-) mice have constitutively higher populations of Treg cells. In contrast, when stimulated with TCR agonists together with IL-6 and TGF-beta, IRAK-1(-/-) CD4 Th cells exhibit attenuated STAT3 Ser727 phosphorylation and reduced expression of IL-17 and RORgamma t compared with wild-type cells. Correspondingly, IRAK-1 deletion results in decreased IL-17 expression and dampened inflammatory responses in acute and chronic inflammatory mice models. Our data provides mechanistic explanation for the anti-inflammatory phenotypes of IRAK-1(-/-) mice.

Inhibition of Ataxia Telangiectasia Mutated Kinase Activity Enhances TRAIL-Mediated Apoptosis in Human Melanoma Cells

The aim of the present study was to elucidate the effects of ataxia telangiectasia mutated (ATM) kinase on the regulation of the extrinsic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2/DR5-mediated death pathway in human melanoma cells. We revealed that total ATM protein levels were high in some human melanoma lines compared with normal cells. The basal levels of active form ATM phospho-Ser(1981) were also detectable in many melanoma lines and could be further up-regulated by gamma-irradiation. Pretreatment of several melanoma lines just before gamma-irradiation with the inhibitor of ATM kinase KU-55933 suppressed p53 and nuclear factor-kappaB (NF-kappaB) activation but notably increased radiation-induced DR5 surface expression, down-regulated cFLIP (caspase-8 inhibitor) levels, and substantially enhanced exogenous TRAIL-induced apoptosis. Furthermore, gamma-irradiation in the presence of KU-55933 rendered TRAIL-resistant HHMSX melanoma cells susceptible to TRAIL-mediated apoptosis. In addition, suppression of ATM expression by the specific short hairpin RNA also resulted in down-regulation of cFLIP levels, up-regulation of surface DR5 expression, and TRAIL-mediated apoptosis in melanoma cells. Besides p53 and NF-kappaB, crucial regulators of DR5 expression, transcription factor STAT3 is known to negatively regulate DR5 expression. Suppression of Ser(727) and Tyr(705) phosphorylation of STAT3 by KU-55933 reduced STAT3 transacting activity accompanied by elevation in DR5 expression. Dominant-negative STAT3beta also efficiently up-regulated the DR5 surface expression and down-regulated cFLIP levels in melanoma cells in culture and in vivo. Taken together, our data show the existence of an ATM-dependent STAT3-mediated antiapoptotic pathway, which on suppression sensitizes human melanoma cells to TRAIL-mediated apoptosis.

A critical role of IRAK1 in the differential regulation of Th17 and T regulatory cells (135.8)

Abstract ObjectiveIRAK-1 has been linked to several human inflammatory diseases including autoimmune diseases. Loss of IRAK1 confers resistance to EAE, however, the exact mechanism is not understood. Given the recent discovery of Th17 lineage and its link to diverse autoimmune diseases, we tested the hypothesis that IRAK1 may contribute to the progression of autoimmune diseases via modulating the differentiation of Th17 and T regulatory cells. MethodsNaïve CD4+ T cells were isolated from splenocytes of wild type (WT) and IRAK1-/- mice. In addition to anti-CD3 and anti-CD28, CD4+ T cells were stimulated with TGF β or TGF β plus IL6. Target proteins and mRNAs were harvested and analyzed via Western blot and RT-PCR. ChIP assays were used to demonstrate in vivo binding of transcription factors to the promoter region of corresponding genes. ResultsIn response to TGF β and IL6, loss of IRAK1 reduced IL-17A and RORγt expression in naïve CD4+ T cells contrary to WT. Also, IRAK1 knockout impaired TGFβ and IL-6 induced STAT3 Ser727 phosphorylation. ChIP assays indicated reduced in vivo STAT3 binding to the promoter of IL-17 in IRAK1-/- cells. In contrast, the levels of Foxp3 and NFAT were highly induced in IRAK1-/- cells treated with TCR agonists and TGFβ, indicating higher percentage of Treg cells. In vivo binding assays with Smad3 antibodies demonstrated increased binding to the Foxp3 promoter in IRAK1-/- cells. ConclusionOur data supports a novel role of IRAK1 in the regulation of Th17 and Treg differentiation. These findings may have significant implications in the pathogenesis of autoimmune diseases. This work is supported by NIH grant R01 AI64414.

Noncytolytic Clearance of Sindbis Virus Infection from Neurons by Gamma Interferon Is Dependent on Jak/Stat Signaling

The alphavirus Sindbis virus (SINV) causes encephalomyelitis in mice by infecting neurons of the brain and spinal cord. The outcome is age dependent. Young animals develop fatal disease, while older animals recover from infection. Recovery requires noncytolytic clearance of SINV from neurons, and gamma interferon (IFN-gamma) is an important contributor to clearance in vivo. IFN-gamma-dependent clearance has been studied using immortalized CSM14.1 rat neuronal cells that can be differentiated in vitro. Previous studies have shown that differentiated, but not undifferentiated, cells develop prolonged SINV replication and respond to IFN-gamma treatment with noncytolytic clearance of virus preceded by suppression of genomic viral RNA synthesis and reactivation of cellular protein synthesis. To determine the signaling mechanisms responsible for clearance, the responses of SINV-infected differentiated neurons to IFN-gamma were examined. IFN-gamma treatment of SINV-infected differentiated CSM14.1 cells, AP-7 olfactory neuronal cells, and primary dorsal root ganglia neurons triggered prolonged Stat-1 Tyr(701) phosphorylation, Stat-1 Ser(727) phosphorylation, and transient Stat-5 phosphorylation. Inhibition of Jak kinase activity with Jak inhibitor I completely reversed the neuroprotective and antiviral activities of IFN-gamma in differentiated cells. We conclude that activation of the Jak/Stat pathway is the primary mechanism for IFN-gamma-mediated clearance of SINV infection from mature neurons.

Protein Kinase Cε Interacts with Signal Transducers and Activators of Transcription 3 (Stat3), Phosphorylates Stat3Ser727, and Regulates Its Constitutive Activation in Prostate Cancer

Prostate cancer is the most common type of cancer in men and ranks second only to lung cancer in cancer-related deaths. The management of locally advanced prostate cancer is difficult because the cancer often becomes hormone insensitive and unresponsive to current chemotherapeutic agents. Knowledge about the regulatory molecules involved in the transformation to androgen-independent prostate cancer is essential for the rational design of agents to prevent and treat prostate cancer. Protein kinase Cepsilon (PKCepsilon), a member of the novel PKC subfamily, is linked to the development of androgen-independent prostate cancer. PKCepsilon expression levels, as determined by immunohistochemistry of human prostate cancer tissue microarrays, correlated with the aggressiveness of prostate cancer. The mechanism by which PKCepsilon mediates progression to prostate cancer remains elusive. We present here for the first time that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, including prostate cancer, interacts with PKCepsilon. The interaction of PKCepsilon with Stat3 was observed in human prostate cancer, human prostate cancer cell lines (LNCaP, DU145, PC3, and CW22rv1), and prostate cancer that developed in transgenic adenocarcinoma of mouse prostate mice. In reciprocal immunoprecipitation/blotting experiments, prostatic Stat3 coimmunoprecipitated with PKCepsilon. Localization of PKCepsilon with Stat3 was confirmed by double immunofluorescence staining. The interaction of PKCepsilon with Stat3 was PKCepsilon isoform specific. Inhibition of PKCepsilon protein expression in DU145 cells using specific PKCepsilon small interfering RNA (a) inhibited Stat3Ser727 phosphorylation, (b) decreased both Stat3 DNA-binding and transcriptional activity, and (c) decreased DU145 cell invasion. These results indicate that PKCepsilon activation is essential for constitutive activation of Stat3 and prostate cancer progression.

Endothelial Nitric Oxide Synthase Plays an Obligatory Role in the Late Phase of Ischemic Preconditioning by Activating the Protein Kinase Cε–p44/42 Mitogen-Activated Protein Kinase–pSer-Signal Transducers and Activators of Transcription1/3 Pathway

The role of endothelial nitric oxide synthase (eNOS) in ischemic preconditioning (PC) and cardioprotection is poorly understood. We addressed this issue using a genetic, rather than pharmacological, approach. In the nonpreconditioned state, eNOS-/- mice exhibited infarct sizes similar to those of wild-type mice. A sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles (ischemic PC) induced late PC in wild-type mice; genetic deletion of eNOS abrogated the cardioprotection induced by late PC. In wild-type mice, ischemic PC induced membranous translocation of protein kinase C (PKC) epsilon and an increase in pSer-MEK-1/2 and pTyr-p44/42 mitogen-activated protein kinase, nuclear pSer-signal transducers and activators of transcription (STAT)1 and pSer-STAT3, and nuclear STAT1/3 DNA binding activity, followed by upregulation of cyclooxygenase-2 protein and activity 24 hours later. All of these changes were abrogated in eNOS-/- mice. The NO donor diethylenetriamine/NO recapitulated the effects of ischemic PC. In contrast to previous reports, we found that basal eNOS activity does not modulate infarct size in the nonpreconditioned state. However, eNOS is obligatorily required for the development of the cardioprotective effects of late PC and acts as the trigger of this process by activating the PKC epsilon-MEK-1/2-p44/42 mitogen-activated protein kinase pathway, leading to Ser-727 phosphorylation of STAT1 and STAT3 and consequent upregulation of STAT-dependent genes such as cyclooxygenase-2. The effects of eNOS-derived NO are reproduced by exogenous NO (NO donors), implying that nitrates can upregulate cardiac cyclooxygenase-2.

Protein kinase Cε interacts with Stat3 and regulates its activation that is essential for the development of skin cancer

Protein kinase C (PKC) represents a large family of phosphatidylserine (PS)-dependent serine/threonine protein kinases. At least six PKC isoforms (alpha, delta, epsilon, eta, micro, and zeta) are expressed in epidermis. PKC is a major intracellular receptor for 12-O-tetradecanoylphorbol-13-acetate (TPA) and is also activated by a variety of stress factors including ultraviolet radiation (UVR). PKC isozymes (alpha, delta, epsilon, and eta), exhibit specificities to the development of skin cancer. PKCepsilon, a calcium-insensitive PKC isoform, is linked to the development of squamous cell carcinoma (SCC) elicited either by the 7,12-Dimethylbenzanthracene (DMBA)-TPA protocol or by repeated exposures to UVR. PKCepsilon overexpressing transgenic mice, when treated either with TPA or exposed to UVR, elicit similar responses such as inhibition of apoptosis, promotion of cell survival, and development of SCC. PKCepsilon overexpression increases Stat3 activation after either TPA treatment or UVR exposure. Both PKCepsilon and signal transducers and activators of transcription-3 (Stat3) are implicated in the development of SCC. However, the link between PKCepsilon and Stat3 remains elusive. We found that PKCepsilon interacts with Stat3. PKCepsilon interaction with Stat3 was dependent upon UVR treatment. In reciprocal immunoprecipitation/blotting experiments, Stat3 coimmunoprecipitated with PKCepsilon. Colocalization of PKCepsilon with Stat3 was confirmed by double immunofluorescence staining. PKCepsilon interaction with Stat3 was PKCepsilon isoform specific and was not observed with other protein kinases. As observed in vitro with immunocomplex kinase assay with immunopurified PKCepsilon and Stat3, PKCepsilon phosphorylated Stat3 at the serine 727 residue. PKCepsilon depletion prevented Stat3Ser727 phosphorylation, Stat3 DNA binding, and transcriptional activity. The results presented indicate that PKCepsilon mediates Stat3 activation.

Phosphorylation of Stats at Ser727 in renal proximal tubular epithelial cells exposed to cadmium

The effects of cadmium exposure on serine phosphorylation of signal transducers and activators of transcription (Stats) and an upstream kinase were examined in renal proximal tubular cells. In porcine LLC-PK1 cells treated with cadmium, Stat1 and Stat3 proteins were phosphorylated at Ser727 without changing total Stat protein levels. While phosphorylated forms of the members of mitogen-activated protein kinases (MAPKs) increased in response to cadmium exposure, treatment with a p38 inhibitor, SB203580 reduced Ser727 phosphorylation of Stat1 and Stat3 markedly in LLC-PK1 cells. The expression of human matrix metalloproteinase-3 (MMP-3), a Stats-inducible gene, was found to be up-regulated in human HK-2 cells exposed to cadmium, and suppressed by preincubation with SB203580. These results suggest that cadmium might induce the phosphorylation of Stat1 and Stat3 at Ser727 via the p38 pathway at least in part, and modulate gene expression in these proximal tubular cells.

Ras/ERK1/2-mediated STAT3 Ser727 Phosphorylation by Familial Medullary Thyroid Carcinoma-associated RET Mutants Induces Full Activation of STAT3 and Is Required for c-fos Promoter Activation, Cell Mitogenicity, and Transformation

The precise role of STAT3 Ser(727) phosphorylation in RET-mediated cell transformation and oncogenesis is not well understood. In this study, we have shown that familial medullary thyroid carcinoma (FMTC) mutants RET(Y791F) and RET(S891A) induced, in addition to Tyr(705) phosphorylation, constitutive STAT3 Ser(727) phosphorylation. Using inhibitors and dominant negative constructs, we have demonstrated that RET(Y791F) and RET(S891A) induce STAT3 Ser(727) phosphorylation via a canonical Ras/ERK1/2 pathway and that integration of the Ras/ERK1/2/ELK-1 and STAT3 pathways was required for up-regulation of the c-fos promoter by FMTC-RET. Moreover, inhibition of ERK1/2 had a more severe effect on cell proliferation and cell phenotype in HEK293 cells expressing RET(S891A) compared with control and RET(WT)-transfected cells. The transforming activity of RET(Y791F) and RET(S891A) in NIH-3T3 cells was also inhibited by U0126, indicating a role of the ERK1/2 pathway in RET-mediated transformation. To investigate the biological significance of Ras/ERK1/2-induced STAT3 Ser(727) phosphorylation for cell proliferation and transformation, N-Ras-transformed NIH-3T3 cells were employed. These cells displayed elevated levels of activated ERK1/2 and Ser(727)-phosphorylated STAT3, which were inhibited by treatment with U0126. Importantly, overexpression of STAT3, in which the Ser(727) was mutated into Ala (STAT3(S727A)), rescued the transformed phenotype of N-Ras-transformed cells. Immunohistochemistry in tumor samples from FMTC patients showed strong nuclear staining of phosphorylated ERK1/2 and Ser(727) STAT3. These data show that FMTC-RET mutants activate a Ras/ERK1/2/STAT3 Ser(727) pathway, which plays an important role in cell mitogenicity and transformation.

Monocyte 15-Lipoxygenase Expression Is Regulated by a Novel Cytosolic Signaling Complex with Protein Kinase C δ and Tyrosine-Phosphorylated Stat3

Our previous studies demonstrated that the IL-13-induced 15-lipoxygenase expression in primary human monocytes is regulated by the activation of both Stat1 and Stat3 and by protein kinase C (PKC)delta. IL-13 stimulated the phosphorylation of Stat3 on both Tyr705 and Ser727. In this study we show that IL-13 induces the association of PKCdelta with Stat3, not with Stat1, and is required for Stat3 Ser727 phosphorylation. We found a novel IL-13-dependent cytosolic signaling complex of PKCdelta and tyrosine-phosphorylated Stat3. A tyrosine kinase inhibitor blocked PKCdelta association with Stat3 as well as Stat3 Ser727 phosphorylation. We therefore hypothesized that tyrosine phosphorylation was required for Stat3 interaction with PKCdelta and subsequent PKCdelta-dependent phosphorylation of Stat3 Ser727. We developed an efficient transfection protocol for human monocytes. Expression of Stat3 containing a mutation in Tyr705 inhibited the association of PKCdelta with Stat3 and blocked Stat3 Ser727 phosphorylation, whereas transfection with wild-type Stat3 did not. Furthermore, by transfecting monocytes with Stat3 containing mutations in Tyr705 or Ser727 or with wild-type Stat3, we demonstrated that both Stat3 tyrosine and serine phosphorylations are required for optimal binding of Stat3 with DNA and maximal expression of 15-lipoxygenase, an important regulator of inflammation and apoptosis.