STAT3 DNA-binding Activity Research Articles

Cooperative regulation of Mcl-1 by Janus kinase/stat and phosphatidylinositol 3-kinase contribute to granulocyte-macrophage colony-stimulating factor-delayed apoptosis in human neutrophils.

Polymorphonuclear neutrophils (PMN) are phagocytic cells constitutively programmed for apoptotic cell death. Exposure to GM-CSF delays apoptosis as measured by annexin-V staining and cell morphological change. We found that STAT5B, STAT1, and STAT3 DNA-binding activity was induced by GM-CSF. We also detected activation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway after GM-CSF treatment which was inhibited by treatment with the PI 3-kinase inhibitors, wortmannin and LY294002. We investigated whether STAT or PI 3-kinase activity was necessary for the pro-survival response of GM-CSF in PMN. Exposure of PMN to GM-CSF in the presence of either AG-490, antisense STAT3 oligonucleotides, or wortmannin resulted in a partial inhibition of GM-CSF-mediated pro-survival activity. GM-CSF induced a time-dependent increase in the mRNA and protein expression of the anti-apoptotic Bcl-2-family protein, Mcl-1. We examined the hypothesis that Janus kinase/STAT and PI 3-kinase regulation of Mcl-1 contributed to GM-CSF-delayed apoptosis. Using either AG-490 or wortmannin alone, we observed a dose-dependent inhibition of GM-CSF-induced Mcl-1 expression. Using suboptimal doses of AG-490 and wortmannin, we found that both drugs together had an additive effect on delayed apoptosis and Mcl-1 expression. These data suggest that cooperative regulation of Mcl-1 by the Janus kinase/STAT and PI 3-kinase pathways contribute to GM-CSF-delayed apoptosis.

Differential Activation of the STAT Pathway by Angiotensin II via Angiotensin Type 1 and Type 2 Receptors in Cultured Human Fetal Mesangial Cells

The vasoactive peptide angiotensin II is the principal effector of the renin-angiotensin system. It exerts mitogenic and growth-inhibiting effects in many target tissues, including renal mesangial cells. To investigate mechanisms of angiotensin II signaling in human mesangial cells, we explored the signal transducer and activator of transcription (STAT) pathway as a possible regulator of angiotensin II receptor-specific signaling. We tested whether angiotensin II could induce STAT activation and nuclear translocation of STAT proteins in human mesangial cells by electromobility shift assays and by immunostaining and confocal microscopy. We found that fetal human mesangial cells express STAT1,2,3,5, and 6 and that stimulation of these cells by angiotensin II results in rapid induction of STAT1 and STAT5 DNA-binding activity. This DNA-binding activity was identified as STAT5 for angiotensin receptor type 1 activation and STAT1 for angiotensin receptor type 2-mediated activation, as induction of STAT-DNA binding by angiotensin II could be differentially blocked by the angiotensin receptor type 1 blocker losartan and by angiotensin II receptor type 2 blocker PD 123,319. Angiotensin II also induced STAT1 and STAT5 tyrosine phosphorylation and nuclear translocation of activated STATs in a receptor subtype-specific manner. STAT activation thus appears to provide an important signaling pathway for angiotensin II-induced cellular responses.

Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells.

Constitutive activation of signal transducer and activator of transcription (STAT) proteins has been detected in a wide variety of human primary tumor specimens and tumor cell lines including blood malignancies, head and neck cancer, and breast cancer. We have previously demonstrated a high frequency of Stat3 DNA-binding activity that is constitutively-induced by an unknown mechanism in human breast cancer cell lines possessing elevated EGF receptor (EGF-R) and c-Src kinase activities. Using tyrosine kinase selective inhibitors, we show here that Src and JAK family tyrosine kinases cooperate to mediate constitutive Stat3 activation in the absence of EGF stimulation in model human breast cancer cell lines. Inhibition of Src or JAKs results in dose-dependent suppression of Stat3 DNA-binding activity, which is accompanied by growth inhibition and induction of programmed cell death. In addition, transfection of a dominant-negative form of Stat3 leads to growth inhibition involving apoptosis of breast cancer cells. These results indicate that the biological effects of the Src and JAK tyrosine kinase inhibitors are at least partially mediated by blocking Stat3 signaling. While EGF-R kinase activity is not required for constitutive Stat3 activation in breast cancer cells, EGF stimulation further increases STAT DNA-binding activity, consistent with an important role for EGF-R in STAT signaling and malignant progression. Analysis of primary breast tumor specimens from patients with advanced disease revealed that the majority exhibit elevated STAT DNA-binding activity compared to adjacent non-tumor tissues. Our findings, taken together, suggest that tyrosine kinases transduce signals through Stat3 protein that contribute to the growth and survival of human breast cancer cells in culture and potentially in vivo.

Stat5 phosphorylation status and DNA-binding activity in the bovine and murine mammary glands

The transcription factors Stat5a and Stat5b are mediators of prolactin signalling in mammary epithelial cells, and are thought to play a role in lactogenesis. In cultured cells, activation of Stat5 activity through phosphorylation results in Stat5 binding to the promoters of at least some of the milk protein genes, thereby stimulating their transcription. However, the mammary biology of Stat5 differs between species, and the role of Stat5 in the bovine mammary gland is not fully understood. We have generated an antibody that specifically recognises the phosphorylated forms of Stat5a and Stat5b and used it to compare the levels of phosphorylated Stat5 with Stat5 DNA-binding activity in bovine and murine mammary tissue. Both Stat5 DNA-binding activity and phosphorylation status in the bovine mammary gland were at near-maximal levels at late pregnancy (27–35 days prior to calving), when at least three of the major milk proteins are not highly expressed. In addition, these studies revealed significant animal-to-animal variation in the level of Stat5 activity in both species. The results are consistent with a role in terminal differentiation of mammary epithelial cells. They also suggest that the stimulation of high-level expression of milk protein genes in the bovine mammary gland is not through activation of the prolactin receptor-Jak2-Stat5 pathway.

Methylation Regulates STAT

Cytokine receptors like the interferon (IFN) receptor cause activation of STAT (signal transducers and activators of transcription) transcription factors. STAT proteins are regulated by phosphorylation on tyrosine and serine phosphorylation. Mowen et al. now show that STAT1 is also posttranslationally modified by methylation on a highly conserved arginine residue. Protein arginine methyl-transferase PRMT1 associates with the interferon-α/β receptor in yeast two-hybrid screen and inhibition of PRMT1 expression with antisense oligonucleotides decreases the effects of type I interferons. Mowen et al. show that STAT1 can be methylated in vitro. Furthermore, methylation of STAT1 (detected with antibodies to dimethylarginine) was increased in mammalian cell lines treated with IFN-α and the methylation was blocked by 5'-methyl-thioadenosine (MTA), a protein methyltransferase inhibitor. MTA also inhibited interferon-induced gene expression. The authors also observed that treatment of cells with MTA reduced DNA-binding activity of STAT1, an effect that correlated with increased association of STAT1 with PIAS1 (the protein inhibitor of activated STATs). MTA is a metabolic intermediate that is a substrate for methyl-thioadenosine phospohorylase (MTAP), and the MTAP gene is deleted in some human cancer cells. Mowen et al. show that accumulation of MTA in cells lacking MTAP can inhibit methylation of STAT1 and appears to make such cells resistant to the effects of interferon. K. A. Mowen, J. Tang, W. Zhu, B. T. Schurter, K. Shuai, H. R. Herschman, M. David, Arginine methylation of STAT1 modulates IFNα/β-induced transcription. Cell 104 , 731-741 (2001). [Online Journal]

Oncostatin M-induced matrix metalloproteinase and tissue inhibitor of metalloproteinase-3 genes expression in chondrocytes requires Janus kinase/STAT signaling pathway.

Oncostatin M (OSM), a member of the IL-6 superfamily of cytokines, is elevated in patients with rheumatoid arthritis and, in synergy with IL-1, promotes cartilage degeneration by matrix metalloproteinases (MMPs). We have previously shown that OSM induces MMP and tissue inhibitor of metalloproteinase-3 (TIMP-3) gene expression in chondrocytes by protein tyrosine kinase-dependent mechanisms. In the present study, we investigated signaling pathways regulating the induction of MMP and TIMP-3 genes by OSM. We demonstrate that OSM rapidly stimulated phosphorylation of Janus kinase (JAK) 1, JAK2, JAK3, and STAT1 as well as extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase 1/2 mitogen-activated protein kinases in primary bovine and human chondrocytes. A JAK3-specific inhibitor blocked OSM-stimulated STAT1 tyrosine phosphorylation, DNA-binding activity of STAT1 as well as collagenase-1 (MMP-1), stromelysin-1 (MMP-3), collagenase-3 (MMP-13), and TIMP-3 RNA expression. In contrast, a JAK2-specific inhibitor, AG490, had no impact on these events. OSM-induced ERK1/2 activation was also not affected by these inhibitors. Similarly, curcumin (diferuloylmethane), an anti-inflammatory agent, suppressed OSM-stimulated STAT1 phosphorylation, DNA-binding activity of STAT1, and c-Jun N-terminal kinase activation without affecting JAK1, JAK2, JAK3, ERK1/2, and p38 phosphorylation. Curcumin also inhibited OSM-induced MMP-1, MMP-3, MMP-13, and TIMP-3 gene expression. Thus, OSM induces MMP and TIMP-3 genes in chondrocytes by activating JAK/STAT and mitogen-activated protein kinase signaling cascades, and interference with these pathways may be a useful approach to block the catabolic actions of OSM.

Crucial role of cytokines in sex steroid formation in normal and tumoral tissues

There is evidence suggesting that local intracrine formation of sex steroids from inactive precursors, dehydroepiandrosterone (DHEA), its sulfate (DHEA-S) and 4-androstenedione (4-DIONE) plays an important role in the regulation of growth and function of peripheral target tissues. Moreover, human solid tumors are often infiltrated by stromal/immune cells secreting a wide spectra of cytokines. These cytokines might in turn regulate the activity of both immune and neoplastic cells. Our data demonstrate that the potent regulatory effects of interleukin-4 (IL-4) and IL-6 on both estrogenic and androgenic 17β-HSD/KSR activities in breast cancer cells depend on the cell-specific gene expression of various types of 17β-HSD/KSR enzymes. However, in both estrogen-receptor (ER)-positive (ZR-75-1, T-47D) and ER-negative (MDA-MB-231, BT-20) human breast cancer cells, exposure to IL-4 and IL-13 caused a rapid and potent induction of 3β-HSD type 1 gene expression. Such an induction was also observed in normal human mammary and prostate epithelial cells in primary culture as well as in human HaCaT immortalized keratinocytes, ME-180 cervix cancer cells, and HT-29 colon cancer cells. The DNA-binding activity of Stat6, a member of the Signal Transducers and Activators of Transcription gene family, was activated after a 30 min exposure to IL-4 in all the cell types where IL-4 induced 3β-HSD expression, but not in those that failed to respond to IL-4. Our data therefore suggest that IL-4 and IL-13 may play a role in the biosynthesis of active sex steroids from the inactive adrenal steroid DHEA, not only in breast cells but also in various cell types derived from peripheral target tissues.

LIF transduces contradictory signals on capillary outgrowth through induction of stat3 and (P41/43)MAP kinase.

The signaling pathways regulating blood vessel growth and development are not well understood. In the present report, an in vitro model was used to identify signaling pathways regulating capillary formation in embryonic endothelial cells. Basic fibroblast growth factor (bFGF) plus leukemia inhibitory factor (LIF) optimally stimulate the formation of capillary-like structures of the embryonic endothelial cell line IEM. LIF stimulation of IEM cells leads to activation of the Stat3 as well as the (P41/43)mitogen-activated protein kinase ((P41/43)MAPK) cascade, while bFGF does not activate Stat3 but does induce the (P41/43)MAPK cascade. Inhibition of Stat3 DNA-binding activity by expression of a dominant inhibitory Stat3 mutant increases the capillary outgrowth of the IEM cells induced by LIF. Increased Stat3 activity by overexpression of the wild-type Stat3 greatly reduced capillary outgrowth. In contrast, inhibition of the (P41/43)MAPK cascade using a MEK-1 inhibitor dramatically inhibits the LIF-induced capillary outgrowth. Moreover, the increased formation of capillary-like structures of the IEM cells mediated by Stat3 inhibition does not overcome the requirement for activation of the (P41/43)MAPK pathway for capillary outgrowth. Stat3 activity correlates with the LIF-induced expression of the negative feedback regulators of the Janus (JAK) family of tyrosine kinases, SOCS-1 and SOCS-3. These results provide evidence that Stat3 acts as a negative regulator of capillary outgrowth, possibly by increasing SOCS-1 or SOCS-3 expression. The contradictory signals stimulated by LIF could be necessary to control the intensity of the response leading to capillary outgrowth in vivo.

Lactation defect in mice lacking the helix-loop-helix inhibitor Id2.

Id proteins are thought to be negative regulators of cell differentiation and positive regulators of cell proliferation. Mammary glands of Id2(-/-) female mice reveal severely impaired lobulo-alveolar development during pregnancy. Id2(-/-) mammary epithelia show no precocious maturation, but instead exhibit intrinsic defects in both cell proliferation and cell survival, implying that the role of Id2 in pregnant mammary epithelia is mainly stimulation of cell proliferation and support of cell viability. Expression studies of genes required for mammary gland development suggest Id2 to be a downstream or parallel factor of these genes. A decrease in the DNA binding activity of Stat5 was also observed in Id2(-/-) mammary glands at 7 days post-coitus. Our results indicate an indispensable role of Id2 in pregnant mammary glands.

In vitro analysis of STAT5 activation by granulocyte-macrophage colony-stimulating factor.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor activates multiple and complex signalling pathways in response to GM-CSF stimulation. Biochemical studies suggested that signalling pathways are transmitted through protein/protein interactions, but how these biochemical cascades are initiated and transmitted in response to cytokine stimulation is largely unknown. To investigate these events biochemically, we established an in vitro system leading to the GM-CSF-dependent activation of Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 5 in cell homogenates prepared from BA/F3 cells expressing the GM-CSF receptor. Activation of STAT5 DNA binding ability requires both membrane and cytoplasmic fractions while phosphorylation of JAK2 requires only the membrane fraction. Since the addition of anti-betac or phosphotyrosine antibodies inhibited GM-CSF induced STAT5 DNA binding activity, we examined the role of tyrosine residues of betac for in vitro activation of STAT5. Addition of synthetic tyrosine-phosphorylated peptides derived from betac cytoplasmic tyrosines prior to GM-CSF stimulation inhibited the in vitro activation of STAT5. The association between these tyrosine-phosphorylated peptides and STAT5 was observed by using peptide-coupling beads and BA/F3 lysates. We established a GM-CSF-dependent in vitro system. In cases of STAT5 activation, each phosphorylated tyrosine residue of betac can act as a docking site and enhance STAT5 activation.

Activation of the Jak/Stat signal transduction pathway in GH-treated rat osteoblast-like cells in culture

In this study, activation of the Jak/Stat signaling pathway was followed upon growth hormone (GH) stimulation, using the rat osteosarcoma cell-line UMR-106.01 that expresses high affinity GH receptors. The results show a GH-induced and sustained phosphorylation of Jak2 and Stat5 on tyrosine residues. The tyrosine phosphorylation status of Jak2 was increased in a dose-dependent manner. In contrast to Jak2, tyrosine phosphorylation of Stat5, also elicited at 42 ng/ml GH, remained unchanged when GH concentration was raised up to 4200 ng/ml. DNA binding activity of Stat5 was also observed in response to GH. However, GH was unable to cause transactivation of reporter gene constructs harboring Stat5 binding sites (the GHREII from the rat spi 2.1 gene promoter, and the LHRE from the rat β-casein gene promoter), except in cells transiently transfected with either Stat5 cDNAs or the rat GHR cDNA. Altogether the results suggest that UMR-106.01 cells exhibit original features of the GH-dependent Jak/Stat signaling pathway.

Plasma growth hormone pulse activation of hepatic JAK-STAT5 signaling: developmental regulation and role in male-specific liver gene expression.

The intracellular signaling molecule STAT5 is activated in rat liver by the intermittent male plasma GH pattern to a 10-fold higher level than by the more continuous pattern of plasma GH stimulation seen in females. Individual adult male rats are presently shown to exhibit large differences in liver STAT5 DNA-binding activity, which correlates with the presence of significant levels of GH in plasma at the time of liver excision. Examination of STAT5 activity as a function of postnatal development revealed that these intermittent pulses of liver STAT5 activity are first observed at 5 weeks of age, when plasma GH pulsation first begins and expression of male-specific, GH pulse-activated liver genes, including CYP2C11, first occurs. Prepubertal rats exhibited low liver STAT5 activity, likely a consequence of the absence of high plasma GH pulses in these animals. Proteins required for GH activation of STAT5 are expressed in liver before puberty, and correspondingly, STAT5 can be precociously activated by exogenous administration of GH pulses given to 2-week-old rats, albeit with a lower sensitivity to GH than is seen in hypophysectomized adult rats. However, this precocious activation of STAT5, via twice daily administration of GH for 7 days, did not lead to CYP2C11 expression or masculinization of hepatic enzyme profiles, unlike in GH pulse-stimulated hypophysectomized adult rats. Based on these findings we conclude: 1) liver STAT5 is repeatedly activated in adult male rats in direct response to the intermittent pattern of plasma GH stimulation; 2) the developmental onset of this STAT5 activation pattern supports the proposed requirement of STAT5 transcriptional activity for male-specific, GH pulse-regulated hepatic gene expression; and 3) the activation of STAT5 is, by itself, not sufficient to impart the adult male pattern of liver gene expression, suggesting a requirement for additional liver factors that are absent in prepubertal rats.

AP-1 and STAT mediate hyperoxia-induced gene transcription of heme oxygenase-1.

We have previously shown marked induction of the stress-inducible gene heme oxygenase-1 (HO-1) in vivo and in vitro after hyperoxia. In RAW 264.7 cells, HO-1 induction is transcriptionally regulated and dependent on cooperation between the HO-1 gene promoter and the 5' distal enhancer element SX2. In our present study, further deletional and mutational analyses demonstrate that signal transducer and activator of transcription (STAT) DNA binding sites located in the promoter of HO-1 and activator protein (AP)-1 DNA binding sites in the distal enhancer element SX2 are necessary for optimal HO-1 gene activation after hyperoxia. Interestingly, a second 5' distal enhancer element, AB1, located 10 kb upstream from the HO-1 promoter, alone is activated after hyperoxia but cannot confer maximal hyperoxia-induced HO-1 gene transcription. Mutational analysis of the AB1 enhancer shows that AP-1 is essential for AB1-mediated HO-1 gene transcription after hyperoxia. Electromobility shift assays show increased STAT1, STAT3, STAT5, and AP-1 DNA binding activity in RAW 264.7 cells after hyperoxia. Taken together, our data suggest that the 5' distal enhancer elements of the HO-1 gene in concert with the promoter regulate HO-1 gene induction and highlight the complexity of HO-1 gene transcription in response to hyperoxia.

Induced nitric oxide inhibits IL-6-induced stat3 activation and type II acute phase mRNA expression.

Inducible nitric oxide synthase (iNOS) can be coexpressed with acute phase reactants in hepatocytes; however, it is unknown if NO can regulate the acute phase response. We tested the hypothesis that iNOS-derived nitric oxide (NO) attenuates the acute phase response by inhibiting IL-6-enhanced Stat3 DNA-binding activity and type II acute phase mRNA expression. iNOS was overexpressed in cultured rat hepatocytes via transduction with a replication defective adenovirus containing cDNA for human iNOS (AdiNOS), and Stat3 DNA-binding activity was determined by electrophoretic mobility shift assay (EMSA). EMSAs demonstrated that AdiNOS inhibits IL-6-induced Stat3 activation and that this inhibition is reversible in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMA). The induction of beta-fibrinogen mRNA by IL-6, a Stat3 dependent process, is attenuated in AdiNOS-transduced cells and partially reversed by L-NMA. Thus, iNOS overexpression suppresses IL-6-induced Stat3 activation and type II acute phase mRNA expression in cultured hepatocytes. This suppression may represent a mechanism by which NO down-regulates the acute phase response.

Cooperation among Stat1, glucocorticoid receptor, and PU.1 in transcriptional activation of the high-affinity Fc gamma receptor I in monocytes.

IFN-gamma and glucocorticoids regulate inflammatory and immune responses through Stat1 and glucocorticoid receptor (GR) transcription factors, respectively. The biological responses to these polypeptides are determined by integration of various signaling pathways in a cell-type and promoter-dependent manner. In this study we have characterized the molecular basis for the functional cooperation between IFN-gamma and dexamethasone (Dex) in the induction of the high-affinity Fc gamma receptor I (Fc gamma RI) in monocytes. Dex did not affect IFN-gamma-induced Stat1 DNA binding activity or induce novel DNA-binding complexes to the Fc gamma RI promoter. By using cell systems lacking functional GR or Stat1, we showed that GR stimulated Stat1-dependent transcription in a ligand-dependent manner, while Stat1 did not influence GR-dependent transcription. The cooperation required phosphorylation of Tyr701, DNA binding, and the trans-activation domain of Stat1, but did not involve Ser727 phosphorylation of Stat1 or physical interaction between GR and Stat1. The costimulatory effect of Dex was not dependent on a consensus glucocorticoid response element in the Stat1-responsive promoters, but required the DNA-binding and trans-activation functions of GR, and Dex-induced protein synthesis. GR activated the natural Fc gamma RI promoter construct, and this response required both Stat1 and the Ets family transcription factor PU.1. Previously, physical association between GR and Stat5 has been shown to enhance Stat5-dependent and suppress GR-dependent transcription. The results shown here demonstrate a distinct, indirect mechanism of cross-modulation between cytokine and steroid receptor signaling that integrates Stat1 and GR pathways with cell type-specific PU.1 transcription factor in the regulation of Fc gamma RI gene transcription.

Inhibition of Signal Transducer and Activator Transcription Factor 3 in Rats with Acute Hepatic Failure

In fulminant hepatic failure, survival is not possible without recovery of sufficient hepatocyte mass. Remarkably, only a few studies exist that provide insight into the mechanisms that control proliferation of residual hepatocytes after extensive hepatocyte loss. In this regard, the role of growth-regulatory factors, including pro-inflammatory cytokines such as interleukin-6 (IL-6), is not well understood. In the present study we show that in rats with critically low (10%) hepatocyte mass, whether with or without ongoing liver cell necrosis, inhibition of liver regeneration is associated with early and sustained increase in blood IL-6 levels. Under these conditions, the signal transducer and activator of transcription (Stat3) DNA binding activity was lowered at the time of G1/S cell-cycle transition. We further demonstrate that the protein inhibitor of activated Stat3 (PIAS3) and the suppressor of cytokine signaling (SOCS-1) were up-regulated early after induction of liver failure (6–12 h). In vitro, IL-6 induced PIAS3 expression in HGF stimulated rat hepatocytes. These findings suggest that after massive hepatocyte loss, an early and rapid rise in blood IL-6 levels may weaken the hepatic regenerative response through up-regulation of Stat3 inhibitors PIAS3 and SOCS-1.

Activation of Stat3 and Stat1 DNA binding and transcriptional activity in human brain tumour cell lines by gp130 cytokines

In this study we examine the activation of the latent Stat family of transcription factors by the gp130 family of cytokines in cell lines derived from human brain tumours. Of the cytokines tested, oncostatin M resulted in the most dramatic induction of Stat1 and Stat3 in all cell lines analysed, as assessed by the formation of protein/DNA complexes. Interleukin-6, leukemia inhibitory factor, and ciliary neurotrophic factor also induced Stat complexes more selectively and to a lesser magnitude than oncostatin M. The kinetics of Stat1 and Stat3 activation was rapid and transient; the nuclear accumulation of DNA binding-proficient Stat protein was detected in the nucleus within minutes of cytokine induction. The transcriptional potential of the oncostatin M-activated Stat molecules was demonstrated in two glioma cell lines (U87-MG, SNB-19) by transient transfection experiments using a Stat-responsive reporter plasmid. Oncostatin M-dependent transcription from this reporter plasmid was reduced to uninduced levels by the inclusion of a dominant-negative Stat3 molecule, demonstrating that Stat molecules were responsible for the induction. These studies demonstrate that oncostatin M is the most potent activator of Stat molecules in a variety of brain tumour-derived cell lines, an observation that could have implications affecting the balance between proliferation/apoptosis of these cells.

GATA-1 blocks IL-6-induced macrophage differentiation and apoptosis through the sustained expression of cyclin D1 and Bcl-2 in a murine myeloid cell line M1

Cytokines exert pleiotropic effects on target cells in a manner dependent on the cell type or stage of differentiation. To determine how instinctive cell properties affect biological effects of cytokine, we introduced an erythroid/megakaryocyte lineage-specific transcription factor, GATA-1, into a murine myeloid cell line M1, which is known to undergo macrophage differentiation in response to interleukin 6 (IL-6). Overexpression of GATA-1 changed the phenotype of M1 cells from myeloid to megakaryocytic lineage. Furthermore, GATA-1 blocked both IL-6-induced macrophage differentiation and apoptosis of M1 cells. Although STAT3 is essential for IL-6-induced macrophage differentiation of M1 cells, GATA-1 had little or no effect on tyrosine phosphorylation, DNA binding, and transcriptional activities of STAT3 in Western blot analysis, electropholic mobility shift assay (EMSA), and luciferase assays. During IL-6-induced macrophage differentiation of M1 cells, IL-6 down-regulated cyclin D1 expression and induced p19(INK4D) expression, leading to reduction in cdk4 activities. In contrast, sustained expression of cyclin D1 and a significantly lesser amount of p19(INK4D) induction were observed in IL-6-treated M1 cells overexpressing GATA-1. Furthermore, although bcl-2 expression was severely reduced by IL-6 in M1 cells, it was sustained in GATA-1-introduced M1 cells during the culture with IL-6. Both IL-6-induced macrophage differentiation and apoptosis were significantly abrogated by coexpression of cyclin D1 and bcl-2, whereas overexpressions of cyclin D1 or bcl-2 inhibited only differentiation or apoptosis, respectively. These results suggested that GATA-1 may not only reprogram the lineage phenotype of M1 cells but also disrupt the biologic effects of IL-6 through the sustained expression of cyclin D1 and bcl-2. (Blood. 2000;95:1264-1273)

Targeted disruption of Stat6 DNA binding activity by an oligonucleotide decoy blocks IL-4–driven TH2 cell response

The transcription factor, signal transducer and activator of transcription (Stat) 6, regulates T(H)2-lymphocyte activity by controlling the expression and responsiveness to interleukin (IL)-4, which plays a key role in numerous allergic maladies. Therefore, we sought to use a phosphorothiolate cis-element decoy to target disruption of Stat6 transcriptional activity. Here we showed that the Stat6 decoy potently ablated the messenger RNA expression and production of IL-4, but not of several other cytokines. The Stat6 decoy functionally disrupted IL-4-inducible cell proliferation of murine T(H)2 cells and primary human CD4(+) T lymphocytes. Specificity of the decoy was demonstrated by its ability to directly block Stat6 binding to a cis-element probe and transactivation, but not affect Stat6 tyrosine phosphorylation or expression of the IL-4 receptor chains. Moreover, the decoy failed to inhibit non-Stat6-dependent signaling pathways since IL-2 was competent to induce cell proliferation and activation of Stats 1, 3, and 5a/b. With the use of laser scanning confocal microscopy, fluorescently tagged Stat6 decoy was detectable in the cytoplasm and nucleus; however, greater levels of oligonucleotide were present in the latter following IL-4 treatment. Taken together, these data suggest that IL-4-driven T(H)2 cell activity can be preferentially restricted via targeted disruption of Stat6 by a novel and specific decoy strategy that may possess gene therapeutic potential. (Blood. 2000;95:1249-1257)

Linkage between α1 Adrenergic Receptor and the Jak/STAT Signaling Pathway in Vascular Smooth Muscle Cells

The Jak/STAT pathway is activated following stimulation of the type I angiotensin II receptor. To examine whether this pathway is shared among other G-protein-coupled receptors, we studied the linkage between the α1 adrenergic receptor and this pathway. The α1 agonist phenylephrine induced tyrosine phosphorylation of Jak2, Tyk2, and STAT1 in vascular smooth muscle cells. The phosphorylation of Jak2 was prevented by the α1 receptor antagonists prazosin and chloroethylclonidine, but not by WB4101, and that of STAT1 was inhibited by prazosin and the Jak2 inhibitor AG490. After stimulation with phenylephrine, Jak2 and STAT1 were found to associate with α1B receptor. Phenylephrine stimulated the DNA binding activity of STAT1. Protein synthesis promoted by phenylephrine was inhibited by prazosin, AG490, and the introduction of a decoy oligonucleotide for STAT1. These results suggested that α1 receptor is linked to the Jak/STAT pathway and that this pathway mediates α1 agonist-induced smooth muscle hypertrophy.