STAT (signal transducers and activators of transcription) proteins are transcription factors that are activated by phosphorylation on tyrosine residues upon stimulation by cytokines. Seven members of the STAT family are known, including the closely related STAT5A and STAT5B, which are activated by various cytokines. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5 activation in various systems are not clear. PCR-driven random mutagenesis and a retrovirus-mediated expression screening system were applied to identify constitutively active forms of STAT5. By this strategy, a constitutively active STAT5 mutant has been identifed that has two amino acid substitutions; one is located upstream of the putative DNA binding domain (H299R), and the other is located in the transactivation domain (S711F). The mutant STAT5 is constitutively phosphorylated on tyrosine residues, localized in the nucleus, and is transcriptionally active. Expression of the mutant STAT5 partially dispenses with interleukin 3 (IL-3) as a growth stimulant of IL-3-dependent cell lines. Further analyses of the mutant STAT5 have demonstrated that both of the mutations are required for nuclear localization, efficient transcriptional activation, and induction of IL-3-independent growth of an IL-3-dependent cell line, Ba/F3, and have indicated that the stability of the phosphorylated form of the mutant STAT5 is the molecular basis for the constitutive activation (Onishi, 1998).

Prolactin (Prl)-induced phosphorylation of Stat (signal transducer and activator of transcription) 5 is considered a key event in functional mammary development and differentiation. Both alveolar proliferation and functional differentiation are impaired in the absence of Stat5a, and the underlying defect is autonomous to the epithelium. Not only Prl, but also growth hormone (GH) and epidermal growth factor (EGF), can activate Stat5 in mammary tissue. The roles of these hormones in mammary development were investigated using mice in which the respective receptors had been inactivated. Although Prl receptor (PrlR)-null mice are infertile, pregnancies could be maintained in a few mice by treatment with progesterone. Mammary tissue in these mice is severely underdeveloped and exhibits limited differentiation as assessed by the phosphorylation status of Stat5 and the expression of milk protein genes. PrlR +/- mice show impaired mammary development and alveolar differentiation during pregnancy -- this corresponds with reduced phosphorylation levels of Stat5a and 5b, and impaired expression of milk protein genes. Development of the glands in these mice is arrested at around day 13 of pregnancy. While Prl activates Stat5 only in the epithelium, GH and EGF activate Stat5 preferentially in the stroma. To assess the relevance of the GH receptor (GHR) in the mammary gland, GHR-null epithelium were transplanted into cleared fat pads of wild-type mice. These experiments demonstrate that the GHR in the epithelium is not required for functional mammary development. Similarly, the EGFR in the epithelium is not required for alveolar development. In contrast, epithelial PrlR is required for mammary development and milk protein gene expression during pregnancy. Although GH is not required for alveolar development, its lactogenic function could be demonstrated in cultured mammary epithelium from PrlR-null mice. However, ductal development in GHR-null mice is impaired, supporting the notion that GH signals through the stromal compartment. These findings demonstrate that GH, Prl, and EGF activate Stat5 in separate compartments, which in turn reflects their specific roles in ductal and alveolar development and differentiation (Gallego, 2001).

The signal transducer and activator of transcription, STAT5b, has been implicated in signal transduction pathways for a number of cytokines and growth factors, including growth hormone (GH). Pulsatile (but not continuous) GH exposure activates liver STAT5b by tyrosine phosphorylation, leading to dimerization, nuclear translocation, and transcriptional activation of the STAT, thought to play a key role in regulating the sexual dimorphism of liver gene expression induced by pulsatile plasma GH. The importance of STAT5b for the physiological effects of GH pulses has been evaluated using a mouse gene knockout model. STAT5b gene disruption leads to a major loss of multiple, sexually differentiated responses associated with the sexually dimorphic pattern of pituitary GH secretion. Male-characteristic body growth rates and male-specific liver gene expression are decreased to wild-type female levels in STAT5b-/- males, while female-predominant liver gene products are increased to a level intermediate between wild-type male and female levels. Although these responses are similar to those observed in GH-deficient Little mice, STAT5b-/- knockout mice are not GH-deficient, suggesting that they may be GH pulse-resistant. Indeed, the dwarfism, elevated plasma GH, low plasma insulin-like growth factor I, and development of obesity seen in STAT5b-/- mice are all characteristics of Laron-type dwarfism, a human GH-resistance disease generally associated with a defective GH receptor. The requirement of STAT5b to maintain sexual dimorphism of body growth rates and liver gene expression suggests that STAT5b may be the major, if not the sole, STAT protein that mediates the sexually dimorphic effects of GH pulses in liver and perhaps other target tissues. STAT5b thus has unique physiological functions for which, surprisingly, the highly homologous STAT5a is unable to substitute (Udy, 1997).

Prolactin (PRL) induces transcriptional activation of not only growth-related genes, such as interferon regulatory factor-1 (IRF-1), but also differentiation-specific genes, such as beta-casein, through a signaling cascade consisting of Janus kinases and Stat (signal transducer and activator of transcription) factors. To understand better the role of Stats in PRL signaling, rat Stat5b was cloned from a PRL-responsive T cell line Nb2. A Stat5b-specific peptide antibody was generated. In PRL receptor-reconstituted COS cells cotransfected with Stat5b or Stat5a, both Stat5 proteins become tyrosine phosphorylated and bind to the IRF-1 GAS (interferon-gamma activation sequence) element in a PRL-inducible manner. Unexpectedly, both Stat5b and Stat5a inhibit PRL induction of the IRF-1 promoter, but they mediate PRL stimulation of the beta-casein promoter. Stat5-mediated inhibition is observed only at the native IRF-1 promoter and not at the isolated IRF-1 GAS element linked to a heterologous thymidine kinase promoter. Mutational analyses shows that the DNA binding activity of Stat5b is not required, but the carboxyl-terminal transactivation domain is essential for Stat5b to inhibit PRL induction of the IRF-1 promoter. These results suggest that Stat5b mediates inhibition via protein-protein interactions. In contrast, both DNA binding and transactivation domains of Stat5b are required to mediate PRL induction of the beta-casein promoter. A carboxyl-terminal truncated dominant negative Stat5b can reverse Stat5b inhibition at the IRF-1 promoter. These studies suggest that Stat proteins can act as not only positive but also negative regulator of gene transcription. Further, Stat5 can modulate gene expression without binding to DNA but via protein-protein interactions (G. Luo, 1997).

Prolactin and glucocorticoid hormone are signals that regulate the transcription of milk protein genes in mammary epithelial cells. Molecular mechanisms by which these hormones cooperate in the induction of transcription were investigated. Both hormones activate latent transcription factors in the cytoplasm of mammary epithelial cells. Prolactin exerts its effect through binding to the extracellular domain of the prolactin receptor and through receptor dimerization. This leads to the activation of a protein tyrosine kinase (Jak2), which is noncovalently associated with the cytoplasmic domain of the prolactin receptor. Jak2 phosphorylates the signal transducer and transcription activator (Stat5) that causes its dimerization and nuclear translocation. In the nucleus, Stat5 specifically binds to sequence elements in the promoter regions of milk protein genes. In contrast, the glucocorticoid receptor is activated by a lipophilic steroid ligand in the cytoplasm, which causes allosteric changes in the molecule, dimerization, and nuclear localization. It has been demonstrated that Stat5 and the glucocorticoid receptor form a molecular complex that cooperates in the induction of transcription of the beta-casein gene. The DNA sequence requirements have been defined for this cooperative mechanism and the functional domains in Stat5 and the glucocorticoid receptor have been delimited that are necessary for the functional interaction. The Stat5 response element (Stat5RE) within the beta-casein gene promoter is sufficient to elicit the cooperative action of Stat5 and the glucocorticoid receptor on transcription. Activation of Stat5 through phosphorylation of tyrosine 694 is an absolute prerequisite for transcription. Deletion of the transactivation domain of Stat5 results in a molecule which cannot mediate transactivation by itself but can still cooperate with the glucocorticoid receptor. Mutated variants of the glucocorticoid receptor with a nonfunctional DNA binding domain or a DNA binding domain contributed by the estrogen receptor are still able to cooperate with Stat5 in transcriptional induction. Deletion of the ligand binding domain of the glucocorticoid receptor does not impede cooperation with Stat5, whereas deletion of the AF-1 transactivation domain does prevent cooperation. These results indicate that the glucocorticoid receptor acts as a ligand-dependent coactivator of Stat5 independent of its DNA binding function (Stoecklin, 1997).

During development of CD4+ T lymphocytes in the periphery, differential expression of cytokine genes, such as those of interleukin (IL)-2 and IL-4, occurs in distinct T-cell subsets. IL-4 is a cytokine produced by T-helper 2 (Th2) cells, and the IL-4 receptor (IL-4R)-mediated signaling pathway is thought to be required for commitment to the Th2 phenotype. However, the molecular basis for development of the Th subset-specific production of IL-4 remains unclear. The IL-4 promoter is functional in Th1 and B cells which do not normally form IL-4 transcripts as well as in IL-4-producing T cells. Based on studies of the effect of several different upstream and downstream regions of the IL-4 gene on IL-4 promoter activity, a Th1-specific IL-4 silencer element was identified in the 3'-untranslated region. The silencer region contains a consensus sequence for a transcriptional factor that is normally regulated by the IL-4 R signaling pathway, STAT6. Nuclear expression of STAT6 protein, which was shown to bind to the silencer region, is observed in Th2 cells but not in Th1 cells. Deletion of the STAT6-binding site from the silencer region and inhibition of STAT6 function results in the appearance of silencing function even in Th2 cells. These results provide evidence that the silencer element, and the binding of STAT6 to this element, play a permissive role in determining the commitment into Th2 phenotype (Kubo, 1997).

Interleukin (IL)-4-mediated nuclear signaling by Stat6 has been implicated in lymphoid cell proliferation and the transcriptional activation of genes encoding major histocompatability complex (MHC) class II molecules and Fc receptors. To investigate IL-4-mediated transcriptional events, two naturally occurring human Stat6 isoforms were cloned (Stat6b and Stat6c) that encode (respectively) an NH2-terminal truncation and an SH2 domain deletion, respectively. Stat6 variant mRNAs are differentially expressed in many human tissues. To elucidate the biologic role of each isoform, the consequences of overexpression were examined in IL-4-responsive FDC-P2 cells. Stat6 and Stat6b (to a lesser extent) enhance DNA synthesis, up-regulate endogenous MHC class II and Fcgamma receptors, and become tyrosine phosphorylated in response to IL-4 stimulation. In contrast, Stat6c, which lacks functionally critical SH2 domain residues, unexpectedly inhibits IL-4-mediated mitogenesis and cell surface antigen expression and is not tyrosine phosphorylated. Although Stat6c only modestly diminishes endogenous Stat6 tyrosine phosphorylation, it abolishes endogenous Stat6 FcgammaRI and Iepsilon DNA binding activity and FcgammaRI-luciferase reporter transcriptional activation. These results indicate that the molecular mechanism of inhibition by Stat6c is due to suppression of endogenous Stat6 dimer formation. Thus, Stat6b and Stat6c are naturally occurring attenuated and dominant negative Stat6 variants (respectively) that affect IL-4-mediated biologic responses through differential transcriptional regulation (Patel, 1998).

Exposure of cells to protein tyrosine phosphatase (PTP) inhibitors causes an increase in the phosphotyrosine content of many cellular proteins. However, the level at which the primary signaling event is affected is still unclear. Jaks are activated by tyrosine phosphorylation in cells that are briefly exposed to the PTP inhibitor pervanadate (PV), resulting in tyrosine phosphorylation and functional activation of Stat6 (in addition to other Stats). Mutant cell lines that lack Jak1 activity fail to support PV-mediated (or interleukin 4 [IL-4]-dependent) activation of Stat6 but can be rescued by complementation with functional Jak1. The docking sites for both Jak1 and Stat6 reside in the cytoplasmic domain of the IL-4 receptor alpha-chain (IL-4Ralpha). The glioblastoma-derived cell lines T98G, GRE, and M007, which do not express the IL-4Ralpha chain, fail to support Stat6 activation in response to either IL-4 or PV. Complementation of T98G cells with the IL-4Ralpha restores both PV-mediated and IL-4-dependent Stat6 activation. Murine L929 cells, which do not express the gamma common chain of the IL-4 receptor, support PV-mediated (but not IL-4-dependent) Stat6 activation. Thus, Stat6 activation by PV is an IL-4Ralpha-mediated, Jak1-dependent event that is independent of receptor dimerization. It is proposed that receptor-associated constitutive PTP activity functions to down-regulate persistent, receptor-linked kinase activity. Inhibition or deletion of PTP activity results in constitutive activation of cytokine signaling pathways (Haque, 1997).

The proliferation of lymphocytes in response to cytokine stimulation is essential for a variety of immune responses. Studies of Stat6-deficient mice have demonstrated that this protein is required for the normal proliferation of lymphocytes in response to interleukin-4 (IL-4). The impaired IL-4-induced proliferative response of Stat6-deficient lymphocytes is not due to an inability to activate alternate signaling pathways, such as those involving insulin receptor substrates, or to a failure to upregulate IL-4 receptor levels. Cell cycle analysis shows that the percentage of Stat6-deficient lymphocytes that transit from the G1 to the S phase of the cell cycle following IL-4 stimulation is lower than that of control lymphocytes. Although the regulation of many genes involved in the control of cytokine-induced proliferation is normal in Stat6-deficient lymphocytes, protein levels of the cdk inhibitor p27Kip1 are markedly dysregulated. p27Kip1 is expressed at significantly higher levels in Stat6-deficient lymphocytes than in control cells following IL-4 stimulation. The higher level of p27Kip1 expression seen in IL-4-stimulated Stat6-deficient lymphocytes correlates with decreased cdk2-associated kinase activity and is the result of the increased accumulation of protein rather than altered mRNA expression. Similarly, higher levels of p27Kip1 protein expression are also seen following IL-12 stimulation of Stat4-deficient lymphocytes than are seen following stimulation of control cells. These data suggest that Stat proteins may control the cytokine-induced proliferative response of activated T cells by regulating the expression of cell cycle inhibitors so that cyclin-cdk complexes may function to promote transition from the G1 to the S phase of the cell cycle (Kaplan, 1998).

Signal transducer and activator of transcription 6 (Stat6) and NF-kappaB are widely distributed transcription factors that are induced by different stimuli and bind to distinct DNA sequence motifs. Interleukin-4 (IL-4), which activates Stat6, synergizes with activators of NF-kappaB to induce IL-4-responsive genes, but the molecular mechanism of this synergy is poorly understood. Using glutathione S-transferase pulldown assays and coimmunoprecipitation techniques, it has been found that NF-kappaB and tyrosine-phosphorylated Stat6 can directly bind each other in vitro and in vivo. An IL-4-inducible reporter gene containing both cognate binding sites in the promoter is synergistically activated in the presence of IL-4 when Stat6 and NF-kappaB proteins are coexpressed in human embryonic kidney 293 (HEK 293) cells. The same IL-4-inducible reporter gene is also synergistically activated by the endogenous Stat6 and NF-kappaB proteins in IL-4-stimulated I.29mu B lymphoma cells. Furthermore, Stat6 and NF-kappaB bind cooperatively to a DNA probe containing both sites; the presence of a complex formed by their cooperative binding correlates with the synergistic activation of the promoter by Stat6 and NF-kappaB. It is concluded that the direct interaction between Stat6 and NF-kappaB may provide a basis for synergistic activation of transcription by IL-4 and activators of NF-kappaB (Shen, 1998).

Stat5 is activated by multiple receptors of hematopoietic cytokines. To study its role during hematopoiesis, primary chicken myeloblasts expressing different dominant-negative (dn) alleles of Stat5 were generated. These alleles cause a striking inability to generate mature cells, due to massive apoptosis during differentiation. Bcl-2 is able to rescue differentiating cells expressing dnStat5 from apoptosis, suggesting that during cytokine-dependent differentiation the main function of the protein is to ensure cell survival. The findings with dnStat5-expressing chicken myeloblasts were confirmed with primary hematopoietic cells from Stat5a/Stat5b-deficient mice. Bone marrow cells from these animals display a strong increase in apoptotic cell death during GM-CSF-dependent functional maturation in vitro. In avian myeloblasts, the antiapoptotic protein Bcl-2 is able to prevent cells expressing dnStat5 from apoptosis during differentiation. However, Bcl-2 is not induced by cMGF, indicating that it is not a Stat5 target gene. The antiapoptotic protein Bcl-x is induced by GM-CSF and IL-3 in a Stat5-dependent fashion. Ectopic expression of Bcl-x rescues Stat5-deficient bone marrow cells from apoptosis, indicating that Stat5 promotes the survival of myeloid progenitor cells through its ability to induce transcription of the bcl-x gene. The recruitment of myeloid cells to inflammatory sites is strongly impeded in Stat5-deficient mice. Taken together, these findings suggest that Stat5 may promote cytokine-dependent survival and proliferation of differentiating myeloid progenitor cells in stress or pathological situations, such as inflammation (Kieslinger, 2000).

STATs play a central role in cytokine signaling. Activating and repressing gene transcription is a dynamic process involving chromatin remodeling by histone acetylases and deacetylases, yet the role of this process in STAT-dependent transcription remains largely unknown. In a search for STAT5-interacting proteins by yeast two-hybrid screening, the nuclear receptor co-repressor SMRT (silencing mediator for retinoic acid receptor and thyroid hormone receptor) was identified as a potential STAT5-binding partner. SMRT binds to both STAT5A and 5B, and strongly represses STAT5-dependent transcription in vitro. SMRT binds to the N-terminal coiled-coil domain of STAT5 and a mutation within this region previously found to render STAT5 hyperactive in response to cytokines abolished the interaction with SMRT. Overexpression of SMRT suppresses the induction of STAT5 target genes by interleukin-3, whereas the histone deacetylase inhibitor trichostatin A effectively enhances and prolongs their expression. Together, these findings illuminate the potential role of SMRT in down-regulating STAT5 activity, with a consequent reduction of STAT5 target gene expression (Nakajima, 2001).

STAT5 is constitutively activated by BCR/ABL, the oncogenic tyrosine kinase responsible for chronic myelogenous leukemia. The BCR/ABL SH3 and SH2 domains interact with hematopoietic cell kinase (Hck), leading to the stimulation of Hck catalytic activity. Active Hck phosphorylates STAT5B on Tyr699, which represents an essential step in STAT5B stimulation. Moreover, a kinase-dead Hck mutant and Hck inhibitor PP2 abrogates BCR/ABL-dependent activation of STAT5 and elevation of expression of STAT5 downstream effectors A1 and pim-1. These data identify a novel BCR/ABL-Hck-STAT5 signaling pathway, which plays an important role in BCR/ABL-mediated transformation of myeloid cells (Klejman, 2002).

STAT1 and STAT3