STAT family of transcription factors in cytokine-mediated biological responses

Cytokine & Growth Factor Reviews 11 (2000) 199±207

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Mini review

STAT family of transcription factors in cytokine-mediated biological responses Kiyoshi Takeda*, Shizuo Akira Department of Host Defense, Research Institute for Microbial Diseases, and CREST of Japan Science and Technology Corporation, Osaka University, 3-1 Yamada-oka, Suita, Osaka, 565-0871, Japan

Abstract STAT (signal transducers and activators of transcription) family, consisting of seven members, is involved in cytokine signal transduction. Biological roles of each STAT family protein have now been elucidated through studies of gene targeted mice. Stat1 knockout mice are defective in interferon-mediated functions. Stat4 and Stat6 knockout mice show defective responses to IL-12 and IL-4, respectively. Analyses of Stat5a and Stat5b knockout mice reveal important roles in prolactin-mediated mammary gland development and growth hormone-mediated induction of sexual dimorphism, respectively. Conditional knockout study of Stat3 demonstrates its critical roles in cytokine-mediated functions in several tissues, including T cells, macrophages, skin, and mammary gland. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Cytokine; Signal transduction; STAT; Knockout mice

1. Introduction Immune and hematopoietic systems are ®nely regulated through the control of growth, di€erentiation, and activation of cells by soluble factors called cytokines. Cytokines exert multiple biological functions through interactions with their speci®c receptors. The binding of cytokines to their receptors induces homoor hetero-dimerization of the receptors and triggers activation of intracellular signaling cascades [1]. The earliest event is activation of Janus kinase (Jak) families, which are constitutively associated with the receptor [2,3]. Activated Jak phosphorylates several substrates critical for cytokine signal transduction pathways. One Abbreviations: STAT, signal transducers and activators of transcription; Jak, Janus kinase; SOCS, suppressor of cytokine signaling; PIAS, protein inhibitors of activated STAT. * Corresponding author. Tel.: +81-6-6879-8303; fax: +81-6-68798305. E-mail address: [email protected] (K. Takeda).

of the most important substrates is a latent cytoplasmic transcription factor, termed STAT (signal transducers and activators of transcription). STAT transcription factors are recruited to the phosphorylated tyrosine residue of the cytoplasmic region of the receptors, and activated through tyrosine phosphorylation. Activated STATs dimerize and rapidly translocate into the nucleus, where they induce gene expressions [4,5]. This Jak-STAT pathway is a simple signaling cascade that directly links the cell membrane (receptor) and the nucleus (gene expression). So far, seven members of mammalian STAT family proteins have been identi®ed and each member has been shown to be activated by distinct cytokines. Therefore, the Jak-STAT pathway was expected to be a key pathway by which we can explain the speci®c action of cytokines. Now, the roles of STATs in cytokine signaling pathways have been clearly demonstrated through generation and analysis of gene targeted mice. In this review, we focus on the biological roles of STAT families revealed by gene targeting.

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2. Structure of STAT transcription factors STAT proteins were ®rst discovered in interferon (IFN) signaling pathways in the beginning of 1990s. Subsequently, several family proteins were isolated. Until now, seven mammalian STAT proteins have been identi®ed as Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b, and Stat6. The genes encoding these seven STATs have been shown to be localized in three chromosomal clusters. The genes for Stat1 and Stat4 map to mouse chromosome 1; Stat2 and Stat6 map to mouse chromosome 2; Stat3, Stat5a, and Stat5b map to mouse chromosome 11 [6]. These suggest an existence of a common ancestral gene that was initially duplicated, followed by dispersion of the linked loci to di€erent chromosomes. The di€erent clustering localization is re¯ected in some features of STAT proteins. Stat2 and Stat6 proteins consist of approximately 850 amino acid residues, whereas the other ®ve members are 750-800 amino acids in length. Stat2 and Stat6 lack conserved serine residue in the carboxyl terminal portions, whereas the other ®ve members have the serine residue [7]. Stat3, Stat5a, and Stat5b are activated by a variety of cytokines, whereas each of the other

®ve members is activated by speci®c cytokines. In addition to mammalian STATs, invertebrate STATs were identi®ed in Drosophila and Anopheles gambiae. The Drosophila homologue of STAT has been shown to be critical for embryonic development of ¯ies [8,9]. Anopheles gambiae STAT has been shown to participate in anti-bacterial immune response in mosquito [10]. Furthermore, STAT genes have been identi®ed in Caenorhabditis elegans, and even in Dictyostelium [11,12]. Thus, the STAT gene is evolutionarily conserved. Each of the STAT proteins has several conserved domains critical for STAT functions (Fig. 1). DNA binding domain is observed in the central region of each STAT protein [13,14]. The DNA binding domain of each STAT protein has been suggested to regulate DNA binding speci®city [14]. There is a conserved SH2 domain in the region between 600 and 700 amino acid residues. A conserved tyrosine residue exists near the carboxy-terminus. STAT proteins are activated through phosphorylation of this tyrosine residue by Jak. Once phosphorylated, STAT proteins form homoor hetero-dimers through interactions of phosphorylated tyrosine of one STAT and SH2 domain of another, then translocate into the nucleus.

Fig. 1. Structure of STAT proteins. STAT family proteins are about 700±850 amino acid in length (The amino acid lengths are indicated at the right side). STAT proteins have a DNA binding domain in the middle portion. SH2 domain mediates docking to the receptor and STAT dimerization. A conserved tyrosine around 700 amino acid residue is responsible for interaction with SH2 domain of another STAT protein, when phosphorylated. Serine residue localized carboxy-terminal to tyrosine residue is phosphorylated in Stat1, Stat3, Stat4, and Stat5a/b. Cytokines and growth factors that activate STAT proteins are shown.

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The SH2 domain has been shown to regulate the speci®city of cytokine-induced STAT activation. In general, STAT protein is recruited to the docking site of the receptor, where tyrosine residue is phosphorylated by Jak. Binding of STAT proteins with the receptor occurs through interaction of SH2 domain and phosphorylated tyrosine, respectively. The SH2 domain of each STAT protein has been shown to recognize distinct phosphorylated docking sites. Stat6 binds to the docking site in IL-4 receptor a chain [15]. Similarly, Stat1 binds to the IFN-g receptor, whereas Stat5a and Stat5b to IL-2 receptor b chain and IL-7 receptor a chain [16,17]. However, this mechanism is not applicable to all cases. In the case of the IFN-a receptor, both Stat1 and Stat2 are activated. However, Stat1 does not bind to the receptor, but Stat2 does. Stat2 interacts with non-phosphorylated IFN-a receptor. When IFN-a binds to the receptor, Stat2 and the receptor are tyrosine phosphorylated. Then, Stat1 docks to the phosphorylated tyrosine residue of Stat2 protein [18]. In addition, Stat5 has been shown to be activated by Jak2 through direct interaction with Jak2 [19]. 3. Regulation of activity of STAT proteins STAT proteins are activated by not only cytokine receptor family, but also several receptors. These include the receptors for growth factors such as epidermal growth factor (EGF) [20], hepatocyte growth factor (HGF) [21], platelet-derived growth factor (PDGF) [22], and colony stimulating factor 1 (CSF-1) [23], and G-protein coupled receptors such as the receptors for angiotensin II [24] and serotonin 5-HT2A [25]. The receptor tyrosine kinases like the receptors for growth factors do not require Jak activation for phosphorylation of STAT proteins. The activity of STAT proteins is negatively regulated by several mechanisms. First, dephosphorylation of the critical tyrosine residue by tyrosine phosphatase has been shown to lead to inactivation of Stat1 protein [26]. Second, ubiquitin-proteasome pathway-mediated degradation has been shown to be involved in inactivation of Stat1 protein [27]. Third, two distinct novel families of negative regulators for the Jak/STAT pathway have been identi®ed. One is a large family, consisting of eight members, termed suppressor of cytokine signaling (SOCS)/ Jak binding protein (JAB)/ STAT-induced STAT inhibitor (SSI) [28±30]. The SOCS family proteins possess an SH2 domain and their expressions are enhanced by cytokine stimulation. Among these family members, SOCS1 and SOCS3 directly bind to the kinase domain of Jak, and inhibit the kinase activity. Biological roles of SOCS family have recently been delineated through generation of

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transgenic and knockout mice. Transgenic mice of CIS1, an originally identi®ed member of SOCS family, displayed severely impaired Stat5-mediated functions, indicating that CIS1 is a speci®c negative regulator for Stat5 activity [31]. Studies with SOCS1 knockout mice have revealed its important role in negative regulation of IFN-g action [32±34]. SOCS3 has been revealed to be important in negative regulation of erythropoietinmediated fetal liver hematopoiesis [35]. The other is a family termed protein inhibitors of activated STAT (PIAS), consisting of PIAS1 and PIAS3 [36,37]. PIAS3 interacts with phosphorylated Stat3, leading to inhibition of Stat3 DNA binding activity. Similarly, PIAS1 inhibits Stat1-induced gene expression. Thus, in addition to dephosphorylation or degradation of STAT proteins, activity of STAT proteins is negatively regulated by several inhibitory proteins. SOCS family modulates the Jak/STAT pathway at the level of Jak, whereas PIAS family seems to regulate STAT activity. Several reports indicate that STAT proteins interact with several signaling or transcription adapter molecules. First, p300/CBP (CREB binding protein) has been shown to interact with Stat1 and Stat2 [38±40]. Second, Stat3 has been shown to interact with Smad1 via p300 [41]. Smad1 is a member of another signaling molecule family essential for TGF-b family signaling. These suggest an existence of a crosstalk between distinct signaling cascades to induce biological responses more e€ectively. As mentioned above, activation of STAT proteins is transient through several negative regulations in normal state. However, constitutive activation of STAT proteins is observed in several cells transformed by several types of oncoproteins and tumor viruses. These include cells infected with human T-cell lymphotropic virus type I (HTLV-I) [42], and abl/myc viruses [43], and cells transformed by v-Src [44±46], and v-abl [47]. Constitutive activation of STAT proteins, especially Stat3, has also been observed in several human cancer cells [48±52]. Dominant negative form of Stat3 protein has been shown to inhibit transformation of ®broblasts by Src oncoprotein [46]. Furthermore, it has recently been reported that introduction of constitutively active Stat3 protein induced cellular transformation, indicating that Stat3 by itself can mediate transformation [53]. Thus, increasing evidences suggest that STAT protein, especially Stat3, is involved in oncogenesis. 4. Knockout study of STAT family proteins Biological functions of STAT family proteins have now been revealed clearly through generation of gene targeted mice. So far, all STAT family members, except for Stat2, have been knocked out, and their phenotypes have been reported. Knockout mice of

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each member of STAT family displayed impaired cytokine-mediated functions (Table 1). 4.1. Stat1 knockout mice Stat1 has been shown to be activated by IFN-a/b and IFN-g. IFNs are known to be important for macrophage activation and host defense response to microbial pathogens. Both IFN-a and IFN-g induced mRNA expression of several genes, such as interferon regulatory factor 1 (IRF1), guanylate binding protein 1 (GBP-1), and complement protein C3 in normal macrophages. In contrast, IFN-induced mRNA expression of these genes was not observed in Stat1 knockout mice. IFN-a and IFN-g also enhanced surface expression of MHC class II and B7-2 expression in normal macrophages. However, Stat1 knockout mice did not show any IFN-induced augmentation of these molecules. Treatment of normal macrophages with IFNa or IFNg in the presence of lipopolysaccharide (LPS) induced secretion of nitric oxide (NO). In contrast, IFN-induced NO production was not observed in Stat1 knockout macrophages. Thus, macrophages from Stat1 knockout mice showed impaired responses to IFNa and IFNg. Accordingly, Stat1 knockout mice were very sensitive to infection with viral and microbial pathogens, such as vesicular stomatitis virus, mouse hepatitis virus, and Listeria monocytogenes. Although several in vitro data indicated that Stat1 was activated by several cytokines, Stat1 knockout mice showed normal response to these cytokines, such as growth hormone, epidermal growth factor, and IL-10. Taken together, analysis of Stat1 knockout mice revealed that Stat1 is essential for IFNmediated functions in vivo [54,55]. 4.2. Stat4 and Stat6 knockout mice Naive CD4+ helper T cells, when stimulated, develop into two cell types, designated type 1 helper T (Th1) and Th2 cells. Th1 cells producing IFN-g modulate cell-mediated immunity to infections by intracellular pathogens, whereas Th2 cells producing IL-4, IL-5, and IL-10 modulate humoral immunity to helminth

infection. Thus, the balance between Th1 and Th2 cell functions is a major factor in the regulation of immune responses. The development of Th1 and Th2 cells is regulated by cytokines, IL-12 and IL-4, respectively [56,57]. Stat4 and Stat6 have been shown to be activated by IL-12 and IL-4, respectively. In Stat4 knockout mice, IL-12-mediated increases in IFN-g production, cellular proliferation, and NK cell cytotoxic activity of lymphocytes were impaired. In addition, IL-12-induced development of Th1 cells was not observed in Stat4 knockout mice. Thus, Stat4 knockout mice showed impaired IL-12-mediated functions [58,59]. In Stat6 knockout mice, IL-4-mediated increases in surface expression of MHC class II and IL-4 receptor a chain, cellular proliferation, and IgE class switching were impaired. In addition, IL-4-induced development of Th2 cells was also impaired [60±62]. Stat6 knockout mice also showed impaired response to IL-13, a closely related cytokine to IL-4 which shares IL-4 receptor a chain as the receptor complex [63]. Thus, Stat6 knockout mice were defective in IL-4- and IL-13-mediated functions. In accord with the impaired IL-4 signaling, Stat6 knockout mice showed impaired Th2 response in several models of infection such as Nippostrongylus brasiliensis, Leishmania mexicana and Schistosoma mansoni [60,64±66]. Several lines of evidence indicate that the imbalance between Th1 and Th2 response causes outbreak of several immune disorders. For example, excessive Th1 response leads to development of autoimmune diseases accompanied by tissue damage. These include type 1 diabetes mellitus, arthritis, and enterocolitis. In contrast, dominant Th2 response triggers allergic diseases [56]. The involvement of Stat4 and Stat6 in the Th1/ Th2 related disorders has been assessed. Adoptive transfer of T cells into immunode®cient mice is shown to induce enterocolitis. In this experimental colitis model, Th1 cells are key mediators in the development of diseases. Transfer of T cells from Stat4 knockout mice has been shown to induce signi®cantly milder colitis than wild-type, indicating that a Stat4-mediated pathway is involved in the pathogenesis of colitis [67]. Involvement of Stat6 in allergic diseases has been

Table 1 STAT family knockout mice Knockout mice Phenotypes Stat1 Stat3 Stat4 Stat5a Stat5b Stat6

Responsible ligands

Defective macrophage activity; high sensitivity to viral infections IFN-g, IFN-a/b Embryonic lethality To be determined Impaired Th1 response IL-12 Impaired mammary gland development and lactation; partial defect in T cell growth Prolactin; IL-2 Impaired expression of sexual dimorphism; defect in T cell growth; defect in NK cell development Growth hormone; IL-2; IL-15 Impaired Th2 response IL-4; IL-13

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demonstrated in allergen-induced airway in¯ammation models. Ovalbumin-treated Stat6 knockout mice did not show any in¯ammatory change, such as eosinophilia in bronchoalveolar lavage, peribronchial pathological change, and increased airway reactivity [68±70]. 4.3. Stat5a and Stat5b knockout mice Stat5a and Stat5b are highly related proteins with 96% similarity in amino acid level. In contrast to Stat4 and Stat6, both Stat5a and Stat5b are activated by a variety of cytokines, including prolactin, growth hormone, erythropietin (Epo), thrompbopoietin (Tpo), granulocyte-macrophage colony stimulating factor (GM-CSF), IL-2, IL-3, IL-5, IL-7, IL-9, and IL-15. Stat5a was originally identi®ed in mammary glands as a transcription factor activated by prolactin. Therefore, the role of Stat5a in mammary glands was ®rst analyzed in Stat5a knockout mice [71]. Impaired lobuloalveolar outgrowth during pregnancy and defective lactation was observed in Stat5a knockout females, indicating that Stat5a is indispensable for mammopoiesis and lactogenesis. Although Stat5b knockout females did not show signi®cant impairment in mammopoiesis, Stat5b knockout males showed a loss of sexually dimorphic pattern as a result of impaired growth hormone response [72,73]. Further, female mice lacking both Stat5a and Stat5b were infertile due to the impaired development of functional copora lutea in the ovary. Female infertility was not observed in Stat5a or Stat5b single knockout females, indicating that Stat5 proteins have redundant functions [73]. Although Stat5 proteins are activated by Epo and Tpo, adult Stat5a/Stat5b double knockout mice did not show any signi®cant change in the number of red cells and platelets [73]. However, a recent report showed that Stat5a/Stat5b double knockout embryos were anemic. Erythroid progenitor cells from Stat5a/ Stat5b double knockout embryos were highly apoptotic due to the reduced responsiveness to Epo [74]. These ®ndings indicate that Stat5 proteins are involved in Epo signaling, although not essential. Study with Stat5a/Sat5b knockout mice also indicated an existence of Stat5-independent pathway in response to Epo. Stat5-independent pathway critical for Epo-mediated erythropoiesis remains to be revealed in the future. The immunological role of Stat5 proteins was also assessed. Splenocytes from Stat5a knockout mice showed a partial impairment in IL-2-induced proliferation. This was due to the partial impairment in IL-2induced expression of IL-2 receptor a chain [75]. Splenocytes from Stat5b knockout mice showed more severe impairment in IL-2-induced proliferation than Stat5a knockout mice [76]. IL-2-induced proliferation was profoundly reduced in splenocytes from Stat5a/ Stat5b double knockout mice [77]. Stat5a/Stat5b

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knockout mice exhibited reduced number of NK cells, splenomegaly, and activated phenotype of T cells, like the case in IL-2 receptor b chain knockout mice, which lack IL-2 response [77]. Stat5b knockout splenocytes also showed impaired NK cell cytotoxic activity due to the impaired IL-15-mediated signaling [76]. Thus, Stat5 proteins are involved in IL-2 and IL-15 signaling. 4.4. Stat3 knockout mice Unlike other STAT family knockout mice, Stat3 knockout mice were embryonic lethal [78]. Stat3 knockout embryos showed rapid degeneration between 6.5 days post coitum (dpc) and 7.5 dpc. Expression of Stat3 mRNA was observed exclusively in visceral endoderm from 6.0 dpc. Visceral endoderm has an important function in metabolic exchange between embryo and maternal blood. The coincidence of the onset of degeneration of Stat3 knockout embryos with Stat3 mRNA expression indicated that Stat3 knockout embryos died due to the impaired functions of visceral endoderm such as nutritional insuciency. Although Stat3 is activated by a variety of cytokines, including IL-6 family cytokines (IL-6, IL-11, leukemia inhibitory factor, oncostatin M, ciliary neurotropic factor, and cardiotropin-1), growth factors (EGF, CSF-1, and PDGF), IFN-g, IL-10, and IL-2, a cytokine which activates Stat3 and accounts for embryonic development remains unclear. By the classical gene targeting of Stat3, the role of Stat3 in cytokine-mediated functions was not delineated due to the early embryonic lethality. Therefore, in an attempt to assess the role in adult tissues, Stat3 has been conditionally knocked out in a tissue- or cellspeci®c manner using Cre-loxP recombination system. So far, the roles of Stat3 in T cells, macrophages, skin, and mammary gland have been analyzed (Table 2). 4.5. Stat3 in T cells Using transgenic mice expressing Cre recombinase under the control of Lck promoter, Stat3 has been knocked out speci®cally in T cells [79]. Stat3-de®cient T cells showed severely impaired IL-6-induced cell proliferation. This was due to the lack of IL-6-mediated prevention of apoptosis of T cells. This study demonstrated that Stat3 is responsible for anti-apoptotic signal, leading to enhanced T cell proliferation. In addition, Stat3-de®cient T cells showed partially impaired IL-2-induced cell proliferation [80]. This was due to the partial defect in IL-2-mediated augmentation of IL-2 receptor a chain like the case in Stat5a knockout mice. Thus, studies of T cell-speci®c Stat3 knockout mice revealed an indirect involvement of Stat3 in IL-2- and IL-6-induced T cell proliferation.

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4.6. Stat3 in macrophages and neutrophils The role of Stat3 in macrophages and neutrophils has been revealed through generation of mice lacking Stat3 in these cells by using the mice expressing Cre proteins under the control of lysozyme M promoter [81]. The mutant mice were highly susceptible to endotoxin shock with increased serum concentration of in¯ammatory cytokines such as tumor necrosis factor-a (TNF-a ), IL-6, IL-1b, and IFN-g. Stat3-de®cient macrophages showed abnormally activated phenotypes, such as increased production of the in¯ammatory cytokines in response to endotoxin, and enhanced expression of MHC class II and B7-1. IL-10 is known to suppress the activity of macrophages. In Stat3-de®cient macrophages and neutrophils, IL-10-mediated reduction in production of in¯ammatory mediators was not observed, indicating that IL-10-induced Stat3 activation is responsible for anti-in¯ammatory responses in macrophages and neutrophils. Abnormal activity of macrophages and neutrophils resulted in development of chronic enterocolitis with enhanced IFN-g production from CD4+ T cells in Stat3 mutant mice. Thus, Stat3 activation in macrophages and neutrophils has been shown to be indispensable for prevention of chronic in¯ammation in mice. 4.7. Stat3 in skin In addition to the immune system, the role of Stat3 in the skin has been assessed using Cre recombinase driven by keratin 5 promoter [82]. While the development of epidermis and hair follicles appeared normal, hair cycle and wound healing were severely compromised in Stat3 mutant mice. The proliferation and di€erentiation of keratinocytes are regulated by several growth factors and cytokines. These include EGF, TGF-a, keratinocyte growth factor, HGF, and IL-6, all of which have a potential to phosphorylate Stat3. Stat3-de®cient keratinocytes showed normal proliferative response to these growth factors, however, in vitro migration in response to these factors was severely impaired. These results indicate that Stat3 is essential for skin remodeling such as hair cycle and wound heal-

ing through growth factor-dependent regulation of keratinocyte motility. 4.8. Stat3 in mammary gland The study with Stat5a knockout mice revealed that Stat5a is responsible for mammary gland development and lactation during pregnancy. Indeed, Stat5a is activated during pregnancy and lactation. However, expression of Stat5a protein is rapidly down-regulated during involution. In contrast, Stat3 is activated at the start of involution. Thus, Stat3 and Stat5a are reciprocally activated during mammary gland development. The role of Stat3 in mammary glands was analyzed in the mice lacking Stat3 in mammary glands [83]. Involution of mammary glands is characterized by extensive apoptosis of the epithelial cells, accompanied by increase in expression of insulin-like growth factor binding protein 5 (IGFBP5), which is responsible for induction of apoptosis. Stat3 mutant mice showed decreased apoptosis of epithelial cells and delayed involution after weaning. IGFBP5 expression was severely reduced in mammary glands of Stat3 mutant mice. Thus, Stat3 is responsible for induction of apoptosis in mammary glands during involution. One of the interesting points to be revealed in the future is identi®cation of a ligand which activates Stat3 during involution. 5. Biological importance of STAT family proteins Knockout studies of STAT family proteins have clearly elucidated their essential roles in cytokinemediated biological responses. Now it is well established that biological actions of cytokines are exhibited through activation of each STAT protein. In particular, it is noteworthy that the fate of cells is determined by di€erential activation of STAT proteins. For example, naive CD4+ T cells are di€erentially destined through activation of Stat4 or Stat6. IL-12-induced Stat4 activation leads to di€erentiation into Th1 cells, whereas IL-4-induced Stat6 activation to Th2 cells. Macrophage activity is also regulated through activity

Table 2 Conditional knockout of Stat3 Cells or tissues lacking Stat3

Phenotypes

Responsible ligands

T cells

Impaired prevention of apoptosis of T cells; impaired IL-2 receptor a chain expression of T cells Increased in¯ammatory responses of macrophages; enhanced Th1 activity; develpment of chronic enterocolitis Impaired hair cycle and wound healing due to defective keratinocyte motility Delayed involution due to impaired epithelial cell apoptosis

IL-6; IL-2

Macrophages Keratinocytes Mammary gland

IL-10 EGF, KGF, TGF-a To be determined

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of two STAT proteins, Stat1 and Stat3. IFN-induced Stat1 phosphorylation is responsible for activation of macrophages, whereas IL-10-induced Stat3 activation suppresses macrophage activity. Stat1 knockout mice showed impaired macrophage activity and they were highly sensitive to viral infections. In contrast, mice lacking Stat3 in macrophages showed abnormally enhanced macrophage activity and developed chronic in¯ammation. Thus, the well-balanced activation of both Stat1 and Stat3 ®nely regulates macrophage activity to maintain the homeostasis of immune response in vivo. Finally, mammary gland development is regulated by two STAT proteins, Stat5a and Stat3. Stat5a is responsible for development of mammary gland during pregnancy, whereas Stat3 is for involution after weaning. 6. Conclusion Biological importance of STAT family proteins has now been clearly characterized through generation of knockout mice. Cytokines are involved in several aspects of immune disorders and diseases. Therefore, involvement of STAT proteins in these diseases may be a major interest to be revealed in the future. We expect that development of new drugs targeted to regulation of STAT activity will be therapeutically useful in several diseases in the future.

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We thank T. Aoki for secretarial assistance. This work was in part supported by grants from the Naito Foundation and the Ministry of Education of Japan.

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