STAT3 DNA-binding Activity Research Articles

STAT3 Contributes to the Mitogenic Response of Hepatocytes during Liver Regeneration

STAT3 is rapidly induced during liver regeneration in an interleukin 6 (IL-6)-dependent fashion, and IL-6 is required for normal liver regeneration. We wanted to know whether STAT3 was also required for liver regeneration but disruption of the STAT3 gene during embryonic stages causes lethality. Therefore, an albumin promoter-driven Cre-loxP recombination system was used to create a STAT3 deletion in the adult mouse liver to study the role of STAT3 in liver regeneration. After partial hepatectomy, there was virtually no STAT3 RNA or protein induction in Alb(+) STAT3(fl/fl) livers. STAT3 DNA binding activity was also absent in Alb(+) STAT3(fl/fl) livers. Unlike in control livers, STAT1 was activated in STAT3 conditional-mutant livers posthepatectomy. Hepatocyte DNA synthesis at 40 h posthepatectomy in Alb(+) STAT3(fl/fl) livers was reduced to approximately one-third of the control. Alb(+) STAT3(fl/fl) livers had abnormalities in immediate-early gene activation that largely correlated with but were not identical to those seen in IL-6-/- livers. G(1) phase cyclins including cyclins D1 and E had lower expression levels in Alb(+) STAT3(fl/fl) livers, indicating an abnormal G(1) to S phase transition. Therefore, STAT3 accounts for part of the DNA synthetic response of the hepatocytes during liver regeneration, which cannot be compensated for by induction of STAT1. Normal activation of the MAPK pathway in Alb(+) STAT3(fl/fl) livers reinforces the fact that at least part of the effect of IL-6 on hepatocyte proliferation is not mediated by STAT3. This study provides the first in vivo evidence that STAT3 promotes cell cycle progression and cell proliferation under physiological growth conditions.

Methamphetamine activates DNA binding of specific redox-responsive transcription factors in mouse brain.

Cellular oxidative stress and alterations in redox status can be implicated in methamphetamine (METH)-induced neurotoxicity. To elucidate the molecular signaling pathways of METH-induced neurotoxicity, we investigated the effects of a single intraperitoneal injection of METH (1.0, 10, or 20 mg/kg) on DNA-binding activity of specific redox-sensitive transcription factors in mouse brain. Transcription factors studied included activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB), cAMP-responsive element-binding protein (CREB), SP-1, and signal transducers and activators of transcription (STAT1 and STAT3). Significant and dose-dependent inductions of AP-1 and CREB DNA-binding activities were observed in four different regions (striatum, frontal cortex, hippocampus, and cerebellum) isolated from the brains of mice injected with METH. However, injections with METH did not affect DNA binding activities of NF-kappaB, SP-1, STAT1, and STAT3. These results suggest that METH-induced oxidative stress may trigger the molecular signaling pathways via specific and selective activation of AP-1 and CREB.

Cytoplasmic transport of Stat3 by receptor-mediated endocytosis.

Signal transducer and activator of transcription (STAT) proteins are cytoplasmic transcription factors that translocate to the nucleus and regulate gene expression upon activation of cytokine or growth factor receptors. While this translocation event is essential for gene regulation by STATs, their mechanism of transport through the cytoplasm to the nucleus has remained elusive. We now report that cytoplasmic transport of Stat3 is an active process that requires receptor-mediated endocytosis. Stat3 co-localizes with endocytic vesicles in transit from the cell membrane to the perinuclear region in response to growth factor stimulation. Consistent with a role for receptor endocytosis in growth factor signaling, disruption of endocytosis with specific inhibitors blocks Stat3 nuclear translocation and Stat3-dependent gene regulation. These results indicate that receptor-mediated endocytosis may be a general mechanism of transport through the cytoplasm for a subset of cytoplasmic signaling proteins destined for the nucleus.

STAT3 activation is required for interleukin-6 induced transformation in tumor-promotion sensitive mouse skin epithelial cells.

STAT3, a member of signal transducers and activators of transcription (STATs) originally discovered as mediators in cytokine signaling pathways, plays an active role in oncogenesis. However, the function of STAT3 in signaling multistage carcinogenesis, especially in transformation of tumor-promotion sensitive epithelial cells has not been elucidated. The present study demonstrates that STAT3 is activated in interleukin-6 induced transformation in mouse skin epithelial cells. DNA binding and transcriptional activities of STAT3 were significantly increased by interleukin-6. This induced anchorage-independent transformation in tumor-promotion sensitive JB6 mouse skin P+ cells but not in the resistant variant P- cells. Two forms of dominant negative STAT3 (mutant of transcriptional domain, mF, or DNA-binding domain, mD) were stably transfected into P+ cells. Activation of STAT3 was abolished and importantly, interleukin-6 induced anchorage-independent growth was absent in both mutant STAT3 transfectants. To determine the genes targeted by STAT3, three matrix metalloproteinase proteins linked with carcinogenesis of epithelial cells were analysed. Both basal and interleukin-6 induced expression of collagenase I and stromelysin I, but not gelatinase A, were inhibited in the mutant STAT3 transfectants. Furthermore, transfection of a wild type STAT3 restored STAT3 transactivation and response to interleukin-6 induced transformation in mutant STAT3 transfectants, which up-regulated collagenase I and stromelysin I as well. Together, these results provide the first evidence that STAT3 activation is required in the progression of multistage carcinogenesis of mouse skin epithelial cells, and matrix metalloproteinases are actively involved in STAT3-mediated cell transformation.

The intranuclear prolactin/cyclophilin B complex as a transcriptional inducer.

The nuclear translocation of peptide hormones, such as the somatolactogenic hormone prolactin, after receptor internalization has been widely reported. Prolactin has been demonstrated to interact with cyclophilin B, a member of the immunophilin family of proteins. Cyclophilin B interaction with prolactin potentiated prolactin-induced proliferation, cell growth, and the nuclear retrotransport of prolactin. These effects could be abrogated by the removal of the peptidyl-prolyl isomerase activity of cyclophilin B. Our findings indicate that the intranuclear prolactin/cyclophilin B complex acts as a transcriptional inducer by interacting directly with Stat5, resulting in the removal of the Stat-repressor protein inhibitor of activated Stat 3 (PIAS3), thereby enhancing Stat5 DNA-binding activity and prolactin-induced, Stat5-mediated gene expression.

Association of the chaperone glucose-regulated protein 58 (GRP58/ER-60/ERp57) with Stat3 in cytosol and plasma membrane complexes.

Glucose-regulated protein 58 (GRP58/ER-60/ERp57), best known as a chaperone in the endoplasmic reticulum lumen, was previously identified by us as one of several accessory proteins in the S100 cytosol fraction of human hepatoma Hep3B cells that was differentially coshifted by anti-Stat3 antibody in an antibody-subtracted differential protein display assay. In the present study, the association between GRP58 and Stat3 in different cytoplasmic compartments was evaluated using cross-immunoprecipitation and cell-fractionation techniques. In the S100 cytosol fraction, three different anti-GRP58 polyclonal antibodies (pAb) cross-immunoprecipitated Stat3 (but not Stat1), and, conversely, anti-Stat3 pAb cross-immunoprecipitated GRP58. Both cytosolic Stat3 and GRP58 eluted during Superose-6 gel-filtration chromatography in complexes of size 200-400 kDa (statosome I), and anti-Stat3 pAb cross-immunoprecipitated GRp58 from these FPLC elution fractions. Using differential sedimentation and density equilibrium flotation methods, Stat3 and GRP58 were observed to be coassociated with cytoplasmic membranes enriched for the plasma membrane marker 5' nucleotidase but not with those containing the endoplasmic reticulum marker BiP/GRP78. The Stat3 and GRP58-containing plasma membrane fraction also contained Stat1, Stat5b, and gp130. Stat activation by orthovanadate caused the accumulation of PY-Stat3 in the GRP58-containing plasma membrane fraction. However, this PY-Stat3 was DNA-binding deficient. Likewise, excess exogenous recombinant human GRP58 prepared using a baculovirus expression system preferentially inhibited Stat3 DNA-binding activity in the S100 cytosol, suggesting that GRP58 may sequester activated Stat3. The new data confirm the association between GRP58 and Stat3 in cytosolic 200-400-kDa statosome I complexes and show that both GRP58 and Stat family members coassociate in the plasma membrane compartment. We suggest that the chaperone GRP58 may regulate signaling by sequestering inactive and activated Stat3.

Phytochemical Inhibition of Interleukin-4-Activated Stat6 and Expression of VCAM-1

Cellular functions induced by cytokine interleukin (IL)-4 and IL-4 signaling through signal transducer and activator of transcription (Stat)6 typify a Th2-type immune response. We investigated the inhibitor effect of the NFκB blocker parthenolide in the late-phase, Th2-type immune response. Parthenolide blocked by 90.6 ± 7.4% the IL-4-induced expression of the endothelial vascular cell adhesion molecule (VCAM)-1, a hallmark of extravasation of very late antigen-4-positive leukocytes. The noncytotoxic concentrations of 10 μM parthenolide left a section of the IL-4 receptor signal transduction intact. Parthenolide did not interfere with the immediate IL-4-induced phosphorylation of endothelial Stat6 on its tyrosine residue Y641 and with tyrosine phosphorylation of the adapter molecule, Jak2—both processes are obligatory for dimerization and nuclear translocation of Stat6. But parthenolide inhibited the Stat6 DNA-binding activity in IL-4-stimulated endothelial cells and inhibited the IL-4-driven activation of a luciferase reporter gene under the control of Stat6-responsive elements (IC50 5.11 ± 0.67 μM). Together, these data suggest an anti-chronic disease profile for the sesquiterpene lactone parthenolide.

Regulation of Cell Proliferation, Apoptosis, and Transcription Factor Activities during the Promotion of Liver Carcinogenesis by Polychlorinated Biphenyls

Polychlorinated biphenyls (PCBs) are environmental pollutants that are complete carcinogens and tumor promoters in the liver. The mechanisms of their promoting activities are not clear, but one possible mechanism is the induction of oxidative stress. In the present study we evaluated the ability of two PCB congeners to activate the oxidative stress-responsive transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), as well as hepatocyte cell proliferation and apoptosis, which are influenced by activation of these transcription factors, in rat liver. Two transcription factors not activated by oxidative stress, signal transducers and activators of transcription 3 and 5 (STAT3 and STAT5), were also examined. All the animals in this study received a single dose of diethylnitrosamine (150 mg/kg) followed by four biweekly injections of 3,3′,4,4′-tetrachlorobiphenyl (PCB-77) or 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB-153) (100 or 300 μmol/kg), or both PCBs (100 μmol/kg each). Ten days after the last PCB injection, all animals were euthanized; 3 days before euthanasia all animals were implanted with Alzet osmotic pumps containing 5-bromo-2′-deoxyuridine (BrdU). The number of placental glutathione S-transferase (PGST)-positive foci were increased in rats administered PCBs, with the highest increase seen in rats administered PCB-77. The number of foci in rats administered both PCBs was intermediate between the numbers seen with either PCB-77 or PCB-153, indicating that a synergistic effect did not occur. There was a significant increase in NF-κB and AP-1 binding activities in hepatic nuclear extracts from rats receiving the high dose of PCB-77 or PCB-153 and in rats receiving both PCBs. In contrast, the DNA binding activities of STAT3 and STAT5 were decreased in rats administered PCBs. Cell proliferation in both focal and nonfocal hepatocytes was increased by PCB-77 but was not affected by PCB-153. Apoptotic indexes, as quantified by the TUNEL method, were increased in both focal and nonfocal hepatocytes by PCB-77 but were decreased in focal hepatocytes by PCB-153. This study shows that both PCBs alone or in combination can increase the DNA binding activities of NF-κB and AP-1, whereas the DNA binding activities of STAT3 and STAT5 are decreased. The induction of altered hepatic foci appears to be related to compensatory cell proliferation in PCB-77-treated rats, whereas the inhibition of apoptosis appears to be important in PCB-153-treated rats.

STAT3, but not ERKs, mediates the IL-6–induced proliferation of renal cancer cells, ACHN and 769P

Although interleukin-6 (IL-6) has been suggested to function as an autocrine growth factor in renal cell carcinoma (RCC), the underlying mechanism responsible for the IL-6-induced proliferation of RCC has not been defined. The aim of this study was to characterize the signaling cascades mediating IL-6-induced proliferation and to investigate the use of effective novel interventions to block the IL-6-induced autocrine growth of renal cancer cells. IL-6-induced proliferation and intracellular signaling cascades were analyzed in four human renal cancer cell lines Caki-1, ACHN, 769P and A498. IL-6-induced activation of STAT3 (signal transducer and activator of transcription-1) and extracellular signal-regulated kinases (ERKs), and the effects of anti-IL-6 neutralizing antibody, Jak inhibitor AG 490, and MEK1 inhibitor PD 98059 were analyzed by Western blotting using phospho-specific antibodies. The DNA-binding activities of STATs were analyzed by EMSA. Apoptosis was determined by using nuclear staining and the TUNEL assay. Changes in the apoptosis-related proteins, bcl-2, bcl-xL, and bax were analyzed by Western blotting. IL-6 induced tyrosine phosphorylation and increased the DNA binding activity of STAT3 and, to a lesser extent, STAT1 in all cell lines except for Caki-1, which did not express the IL-6 receptor subunit gp130. ERKs were constitutively activated in all cell lines and the activation level was not up-regulated further by exogenously added IL-6 nor down-regulated by anti-IL-6 neutralizing antibody. IL-6-induced STAT3 tyrosine phosphorylation and DNA binding activity was inhibited by treatment with Jak specific inhibitor AG 490; however, it was not affected by the MEK1 inhibitor PD 98059. Moreover, treatment with AG 490 inhibited IL-6-induced proliferation of ACHN and 769P cells and induced apoptosis with the down-regulation of bcl-2 and the up-regulation of bax. This study identified STAT3, but not ERKs, to be a major mediator of IL-6-induced proliferation of renal cancer cells. Although ERKs were constitutively activated, ERKs were not found to be essential for the IL-6-induced proliferation and modulation of the STAT3 activity. Because the Jak specific inhibitor AG 490 effectively inhibited the IL-6-induced STAT3 activity and induced apoptosis, the blockade of the STAT3 signaling pathways is considered to be potentially useful as a novel therapeutic approach for RCC.

Thrombopoietin Regulates Bcl-xL Gene Expression through Stat5 and Phosphatidylinositol 3-Kinase Activation Pathways

Thrombopoietin (TPO), an essential factor for megakaryopoiesis and thrombopoiesis, works as a survival factor for megakaryocytic lineage cells. However, little is known about the molecular mechanism in detail. We show here that TPO supports the survival of TPO-dependent leukemia cell line UT-7/TPO and normal megakaryocytic progenitors via the induction of Bcl-xL, an anti-apoptotic member of the Bcl-2 family. We further analyzed the signal transduction pathways required for TPO-induced Bcl-xL gene expression. A reporter assay with various lengths of Bcl-x gene promoter revealed that both Stat- and nuclear factor kappa B-binding sites are prerequisites for TPO-induced promoter activity. Consistent with these results, TPO induced the binding of Stat5 and subunits of nuclear factor kappa B, p50, and c-Rel to the Bcl-x gene promoter. AG490, a specific inhibitor for Jak2, and LY294002, a specific inhibitor for phosphatidylinositol (PI) 3-kinase, reduced the protein level of Bcl-xL in UT-7/TPO cells, accompanied by an increase in the ratio of apoptotic cells. Interestingly, LY294002 enhanced the TPO-induced DNA binding activity of Stat5 without affecting the Jak2 activation and tyrosine phosphorylation of Stat5. Concomitantly, confocal microscopy revealed that LY294002 clearly inhibited the nuclear export of Stat5, suggesting that PI 3-kinase regulates the subcellular localization of Stat5. Taken together, our results suggest that both Jak-Stat and PI 3-kinase activation pathways regulate the TPO-induced survival of megakaryocytic cells via Bcl-xL gene expression. In addition, our data suggest possible cross-talk between these two signaling pathways.

Selective inhibition of STAT3 phosphorylation by sodium salicylate in cardiac fibroblasts

The effects of salicylate on the phosphorylation and nuclear translocation of signal transducers and activators of transcription (STATs) induced by interferon-γ (IFN-γ) were studied in rat cardiac fibroblasts as a possible model for the anti-inflammatory effects of salicylate on this signaling pathway. Salicylate inhibited the tyrosine phosphorylation of both STAT1 and STAT3, but had a more pronounced effect on STAT3 activation. Salicylate pretreatment prevented both the nuclear translocation and the DNA-binding activity of STAT1 and STAT3, assessed by immunoblotting and gel shift assays, respectively. In addition to causing phosphorylation at tyrosine residues, IFN-γ also phosphorylated STAT3 and STAT1 at serine 727. Salicylate attenuated both tyrosine and serine phosphorylations of STAT3, and also suppressed extracellular signal-regulated kinase (ERK) activation, implicating the effect of salicylate on ERK as a possible mechanism for attenuating STAT3 activation. The possibility that salicylate might affect signaling cascades by altering the redox state of the cells was examined, and its effects differed from those of other reducing agents. Salicylate did attenuate the effects of hydrogen peroxide on STAT phosphorylation, consistent with a mechanism involving an interaction between salicylate and reactive oxygen species within the cell.

A New Role for the STAT3 Inhibitor, PIAS3: A REPRESSOR OF MICROPHTHALMIA TRANSCRIPTION FACTOR

In vitro and in vivo evidence suggest that microphthalmia transcription factor (MITF) plays a key regulatory role in tissue-specific gene regulation in several cell types, including melanocytes, osteoclasts, and mast cells. A yeast two-hybrid search, using a portion of a nonmutated MITF gene as the bait in the screening of a mast cell library, resulted in the isolation of the STAT3 inhibitor, PIAS3. PIAS3 is a transcriptional inhibitor that acts by specifically inhibiting STAT3's DNA binding activity. We found that it can directly associate with MITF using an in vitro pull-down assay. Immunoprecipitation of MITF from rat basophilic leukemic cells or mouse melanocytes resulted in the specific co-immunoprecipitation of PIAS3. Co-transfection of MITF with PIAS3 in NIH 3T3 fibroblasts containing an mMCP-6 promoter-luciferase reporter demonstrated up to 94% inhibition of MITF-mediated transcriptional activation. Using a gel-shift assay, it was shown that PIAS3 can block DNA binding activity. It was also found that STAT3 does not interfere, either in vitro or in vivo, with the interaction between PIAS3 and MITF. These data suggest that PIAS3 functions in vivo as a key molecule in supressing the transcriptional activity of MITF, a role of considerable importance in mast cell and melanocyte development.

Serine phosphorylation of GH-activated signal transducer and activator of transcription 5a (STAT5a) and STAT5b: impact on STAT5 transcriptional activity.

Signal transducer and activator of transcription 5b (STAT5b), the major liver-expressed STAT5 form, is phosphorylated on both tyrosine and serine in GH-stimulated cells. Although tyrosine phosphorylation is known to be critical for the dimerization, nuclear translocation, and activation of STAT5b DNA-binding and transcriptional activities, the effect of STAT5b serine phosphorylation is uncertain. Presently, we identify Ser730 as the site of STAT5b serine phosphorylation in GH-stimulated liver cells. We additionally show that the serine kinase inhibitor H7 partially blocks the GH-stimulated formation of (Ser,Tyr)-diphosphorylated STAT5b without inhibiting STAT5b nuclear translocation. Evaluation of the functional consequences of STAT5b serine phosphorylation by mutational analysis revealed an approximately 50% decrease in GH-stimulated luciferase reporter gene activity regulated by an isolated STAT5-binding site when STAT5b Ser730 was mutated to alanine and under conditions where STAT5 DNA-binding activity was not diminished. No decrease in GH-stimulated reporter activity was seen with the corresponding STAT5a-Ser725Ala mutant; however, a decrease in reporter activity occurred when the second established STAT5a serine phosphorylation site, serine 779, was additionally mutated to alanine. Unexpectedly, STAT5a-Ser725,779Ala and STAT5b-Ser730Ala displayed approximately 2-fold higher GH- or PRL-stimulated transcriptional activity compared with wild-type STAT5b when assayed using an intact beta-casein promoter-luciferase reporter. Finally, STAT5b-stimulated gene transcription was abolished in cells treated with H7, but in a manner unrelated to the inhibitory effects of H7 on STAT5b Ser730 phosphorylation. These findings suggest that the effects of STAT5b and STAT5a serine phosphorylation on STAT-stimulated gene transcription can be modulated by promoter context. Moreover, in the case of STAT5a, phosphorylation of serine 779, but not serine 725, may serve to regulate target gene transcriptional activity.

Constitutive activation of STAT-3 and downregulation of SOCS-3 expression induced by adrenalectomy.

Removal of adrenal steroids by adrenalectomy (ADX) slows or reverses the development of many forms of obesity in rodents, including those that are leptin or leptin receptor deficient. Obesity is associated with hyperleptinemia and leptin resistance. We hypothesized that glucocorticoids impair leptin receptor signaling and that removal thereof would activate the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling pathway. The inhibitory effect of leptin (2.5 microg icv) on food intake was enhanced in ADX rats. A combination of ribonuclease protection assays, RT-PCR, Western blots, and mobility shift assays was used to evaluate the leptin signaling pathway in whole hypothalami from sham-operated, ADX and corticosterone-replaced ADX (ADX-R) Sprague-Dawley rats that were treated acutely with either saline vehicle or leptin intracerebroventricularly. ADX increased the expression of leptin receptor mRNA, increased STAT-3 mRNA and protein levels, induced constitutive STAT-3 phosphorylation and DNA binding activity, and also reduced suppressor of cytokine signaling-3 (SOCS-3) mRNA and protein levels. ADX and leptin treatment increased STAT-3 phosphorylation, but with no concomitant increase in DNA binding activity. Leptin and ADX decreased NPY mRNA expression, but their combination did not further decrease NPY mRNA. Corticosterone supplementation of ADX rats partially reversed many of these effects. In conclusion, ADX through activation of STAT-3 and inhibition of SOCS-3 activates the JAK-STAT signaling pathway. These effects most probably explain the ability to prevent the development of obesity by removal of adrenal steroids.

Inhibition of constitutively active Stat3 suppresses growth of human ovarian and breast cancer cells.

Signal transducers and activators of transcription (STATs) are transcription factors activated in response to cytokines and growth factors. Constitutively active Stat3 has been shown to mediate oncogenic transformation in cultured cells and induce tumor formation in mice. An increasing number of tumor-derived cell lines as well as samples from human cancer have been reported to express constitutively active Stat3 protein. We previously demonstrated that ovarian cancer cell lines express high levels of constitutively active Stat3. In this study, we show that inhibition of the Stat3 signaling pathway using the Janus Kinase-selective inhibitor, AG490, and a dominant negative Stat3 (Stat3beta) significantly suppresses the growth of ovarian and breast cancer cell lines harboring constitutively active Stat3. In the ovarian cancer cell lines, AG490 also diminished the phosphorylation of Stat3, Stat3 DNA binding activity, and the expression of Bcl-x(L). Further, AG490 induced significant apoptosis in ovarian and breast cancer cell lines expressing high levels of constitutively active Stat3 but had a less profound effect on normal cells lacking constitutively active Stat3. AG490 also enhanced apoptosis induced by cisplatin in ovarian cancer cells. These results suggest that inhibition of Stat3 signaling may provide a potential therapeutic approach for treating ovarian and breast cancers.

Activation of JAK2 by the oxidative stress generated with oxidized low-density lipoprotein

Atherosclerosis includes a series of cellular and molecular responses characteristic of an inflammatory disease. We provide evidence that cupric-ion-oxidized LDL (CuLDL) or endothelial cell-oxidized LDL (ELDL) induced the activation by Tyr-phosphorylation of JAK2, one of the Janus kinase involved upstream of STATs in the JAK/STAT pathway of cytokine transduction. Oxidized LDL (OxLDL) also initiated STAT1 and STAT3 Tyr-phosphorylation and translocation to the nucleus, with a more marked effect for the extensively modified CuLDL. Genistein, a nonspecific Tyr-kinase inhibitor, and AG490, a specific inhibitor of JAKs, markedly prevented the CuLDL-induced enhancement of STAT1 and STAT3 Tyr-phosphorylation and DNA-binding activity, suggesting that JAKs are the main kinases involved in STATs’ activation by oxidized LDL. In addition, the lipid extract of CuLDL increased the intracellular levels of lipid peroxidation products and the Tyr-phosphorylation of JAK2, STAT1, and STAT3, whereas the antioxidant vitamin E prevented all these effects. These results demonstrate that OxLDL induces the activation by Tyr-phosphorylation of JAK2, STAT1, and STAT3 by generation of an intracellular oxidative stress by means of its lipid peroxidation products, and thus include JAK2 within the range of oxidative stress-activated kinases.

Phosphotyrosyl Peptides Block Stat3-mediated DNA Binding Activity, Gene Regulation, and Cell Transformation

Signal transducers and activators of transcription (STATs) comprise a family of cytoplasmic signaling proteins that participates in normal cellular responses to cytokines and growth factors. Frequently, however, constitutive activation of certain STAT family members, particularly Stat3, has accompanied a wide variety of human malignancies. To identify small molecule inhibitors of Stat3, we investigated the ability of the Stat3 SH2 domain-binding peptide, PY*LKTK (where Y* represents phosphotyrosine), to disrupt Stat3 activity in vitro. The presence of PY*LKTK, but not PYLKTK or PFLKTK, in nuclear extracts results in significant reduction in the levels of DNA binding activities of Stat3, to a lesser extent of Stat1, and with no effect on that of Stat5. Analyses of alanine scanning mutagenesis and deletion derivatives of PY*LKTK reveal that the Leu residue at the Y+1 position and a substituent at the Y-1 position (but not necessarily Pro) are essential for the disruption of active Stat3, thereby mapping the minimum active sequence to the tripeptide, XY*L. Studies involving bead-coupled PY*LKTK peptide demonstrate that this phosphopeptide directly complexes with Stat3 monomers in vitro, suggesting that PY*LKTK disrupts Stat3:Stat3 dimers. As evidence for the functional importance of peptide-directed inhibition of Stat3, PY*LKTK-mts (mts, membrane translocating sequence) selectively inhibits constitutive and ligand-induced Stat3 activation in vivo. Furthermore, PY*LKTK-mts suppresses transformation by the Src oncoprotein, which has been shown previously to require constitutive Stat3 activation. Altogether, we have identified a minimal peptide that inhibits Stat3 signaling and provides the conceptual basis for use of this peptide as a lead for novel peptidomimetic drug design.

Temporal relationship between the sexually dimorphic spontaneous GH secretory profiles and hepatic STAT5 activity.

STAT5 transduces transcriptional responses to GH in liver and other tissues and is proposed to mediate the sexually dimorphic effects of plasma GH secretory profiles on rodent liver gene expression. Previous studies have suggested that STAT5 undergoes repeated activation in direct response to successive GH pulses in adult male rats, with STAT5 activation being desensitized in females by their more persistent pattern of GH exposure. These findings, however, were based on in vitro studies or single blood samples analyzed for GH in vivo. In view of the highly pulsatile nature of rat GH secretion, we presently examined these hypotheses by concurrent monitoring of spontaneous GH secretory profiles and hepatic STAT5 activity in conscious, free-moving adult male and female rats. Rats were killed at times associated with spontaneous peaks or troughs of the GH rhythm; livers were removed and analyzed for STAT5 DNA-binding activity. In males, liver STAT5 activity was highest during the initial phase (15-60 min) of a GH secretory episode (mean +/- SE relative STAT5 activity = 86.5 +/- 11.4; plasma GH = 146.7 +/- 22.4 ng/ml) and was significantly lower (P < 0.01) during the downswing of a pulse, 45-75 min after the GH peak (STAT5 = 26.1 +/- 1.7; GH = 33.3 +/- 13.1 ng/ml), consistent with a time-dependent down-regulation of GH signaling to STAT5. The lowest STAT5 activity was observed during the subsequent GH trough period (STAT5 = 3.6 +/- 1.1; GH = 2.6 +/- 0.1 ng/ml). In females, liver STAT5 activity was significantly lower (P < 0.05) than peak male levels during the initial phase of a GH secretory burst (STAT5 = 35.1 +/- 15.9; GH = 68.1 +/- 31.6 ng/ml) although similar to that of males during a plasma GH nadir (STAT5 = 11.0 +/- 2.6; GH = 8.4 +/- 2.2 ng/ml). We conclude that: 1) liver STAT5 is repeatedly activated by successive, spontaneous GH secretory episodes in intact adult male rats at approximately 3- to 3.5-h intervals; 2) time-dependent down-regulation of GH signaling to hepatic STAT5 in vivo begins by 45 min after GH peak stimulation; and 3) the lower level of liver STAT5 activation seen in adult female rats, compared with males, is a consequence of the sex-dependent differences in GH secretory patterns that characterize these animals (i.e. lower-amplitude GH pulses and lack of prolonged interpulse nadir of GH in the feminine, compared with masculine profile).

Induction of Apoptosis by Protein Inhibitor of Activated Stat1 through c-Jun NH2-terminal Kinase Activation

Members of the protein inhibitor of activated signal transducer and activator of transcription (STAT) family (PIAS family) of proteins act as negative regulators of STATs in cytokine signaling. We report here that PIAS proteins have proapoptotic activity. PIAS1 induced apoptosis in both human 293T cells and human osteosarcoma U2OS cells. PIAS1 is localized in the nucleus as distinct nuclear dots. Ectopic expression of PIAS1 in U2OS cells activated JNK1 (c-Jun NH(2)-terminal kinase). A dominant-negative JNK1, capable of inhibiting PIAS1-induced JNK1 activation, blocked PIAS1-mediated apoptosis. Furthermore, a mutant PIAS1, lacking the first 9 amino acid residues, failed to repress Stat1-mediated gene activation although it retained its ability to activate JNK and to trigger apoptosis. Our results identify a novel function of PIAS1 in the induction of JNK-dependent apoptosis, independent of the previously known inhibitory activity of PIAS1 in STAT-mediated gene activation.

Constitutive and Interleukin-7- and Interleukin-15-Stimulated DNA Binding of STAT and Novel Factors in Cutaneous T Cell Lymphoma Cells

On testing cutaneous T cell lymphoma cell lines and skin lesions, we found that the transcription factors STAT2, STAT3, STAT5, and STAT6 (STAT, signal transducer and activator of transcription) were present in the nuclei of these cells and that the binding to their specific DNA binding sites was stimulated by interleukin-7 and interleukin-15. DNA binding studies also revealed the presence of three additional DNA factors in cutaneous T cell lymphoma cells that bound to the same sequences and could also be stimulated by interleukin-7 and interleukin-15. One of these novel factors was also present in the adult T cell leukemia cell line Jurkat and malignant T cells from the blood of Sézary syndrome patients, but not in normal peripheral blood lymphocytes. It may therefore be a marker of T cell leukemia. It seems to interfere with the binding of STAT1 to the sis inducible element, suggesting that the DNA binding activity of STAT1 in cutaneous T cell lymphoma cells is disturbed.