Specific DNA binding of Stat5, but not of glucocorticoid receptor, is required for their functional cooperation in the regulation of gene transcription

Prolactin and glucocorticoid hormone are signals which regulate the transcription of milk protein genes in mammary epithelial cells. We have investigated the molecular mechanisms by which these hormones cooperate in the induction of transcription. 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) which causes its dimerization and nuclear translocation where Stat5 specifically binds to sequence elements in the promoter regions of milk protein genes. In comparison, 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 which cooperates in the induction of transcription of the beta-casein gene. We have defined the DNA sequence requirements for this cooperative mechanism and have delimited the functional domains in Stat5 and the glucocorticoid receptor that are necessary for the functional interaction. We find that 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. Our results indicate that the glucocorticoid receptor acts as a ligand-dependent coactivator of Stat5 independently of its DNA binding function.

[1]  J. Darnell,et al.  Tyrosine-phosphorylated Stat1 and Stat2 plus a 48-kDa protein all contact DNA in forming interferon-stimulated-gene factor 3. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. Rosen,et al.  Nuclear factor I and mammary gland factor (STAT5) play a critical role in regulating rat whey acidic protein gene expression in transgenic mice , 1995, Molecular and cellular biology.

[3]  B. Groner,et al.  Hormonal regulation of transcription factor activity in mammary epithelial cells , 1994, Molecular and Cellular Endocrinology.

[4]  P. Kelly,et al.  The prolactin/growth hormone receptor family. , 1991, Endocrine reviews.

[5]  Thorsten Heinzel,et al.  A CBP Integrator Complex Mediates Transcriptional Activation and AP-1 Inhibition by Nuclear Receptors , 1996, Cell.

[6]  K. Horwitz,et al.  Nuclear receptor coactivators and corepressors. , 1996, Molecular endocrinology.

[7]  P. Herrlich,et al.  Interference between pathway‐specific transcription factors: glucocorticoids antagonize phorbol ester‐induced AP‐1 activity without altering AP‐1 site occupation in vivo. , 1992, The EMBO journal.

[8]  B. Groner,et al.  Interaction of two sequence-specific single-stranded DNA-binding proteins with an essential region of the beta-casein gene promoter is regulated by lactogenic hormones , 1993, Molecular and cellular biology.

[9]  A. Ray,et al.  Physical association and functional antagonism between the p65 subunit of transcription factor NF-kappa B and the glucocorticoid receptor. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[10]  B. Groner,et al.  Activation of Stat5 by interleukin 2 requires a carboxyl-terminal region of the interleukin 2 receptor beta chain but is not essential for the proliferative signal transmission. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[11]  B. Groner,et al.  Lactogenic Hormone Activation of Stat5 and Transcription of the β-Casein Gene in Mammary Epithelial Cells Is Independent of p42 ERK2 Mitogen-activated Protein Kinase Activity* , 1996, The Journal of Biological Chemistry.

[12]  B. Groner,et al.  Prolactin-mediated gene activation in mammary epithelial cells. , 1995, Current opinion in genetics & development.

[13]  D. Cantrell,et al.  Interleukin‐2 activation of STAT5 requires the convergent action of tyrosine kinases and a serine/threonine kinase pathway distinct from the Raf1/ERK2 MAP kinase pathway. , 1996, The EMBO journal.

[14]  B. Groner,et al.  Hormone-dependent phosphorylation of the glucocorticoid receptor occurs mainly in the amino-terminal transactivation domain. , 1990, The Journal of biological chemistry.

[15]  Stephen K Burley,et al.  Architectural Transcription Factors: Proteins That-Remodel DNA , 1997, Cell.

[16]  B. Groner,et al.  Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[17]  B. Groner,et al.  Functional interactions between Stat5 and the glucocorticoid receptor , 1996, Nature.

[18]  T. Hirano,et al.  IL-6-inducible complexes on an IL-6 response element of the junB promoter contain Stat3 and 36 kDa CRE-like site binding protein(s). , 1996, Oncogene.

[19]  A. Munck,et al.  Kinetics of glucocorticoid receptor phosphorylation in intact cells. Evidence for hormone-induced hyperphosphorylation after activation and recycling of hyperphosphorylated receptors. , 1993, The Journal of biological chemistry.

[20]  H. Erdjument-Bromage,et al.  Interleukin‐3 signals through multiple isoforms of Stat5. , 1995, The EMBO journal.

[21]  J. Ihle.  STATs: Signal Transducers and Activators of Transcription , 1996, Cell.

[22]  K. Yamamoto,et al.  The basic region of AP-1 specifies glucocorticoid receptor activity at a composite response element. , 1992, Genes & development.

[23]  J. Gustafsson,et al.  Mechanism of gene expression by the glucocorticoid receptor: Role of protein‐protein interactions , 1997, BioEssays : news and reviews in molecular, cellular and developmental biology.

[24]  L. Hennighausen,et al.  Stat5a is mandatory for adult mammary gland development and lactogenesis. , 1997, Genes & development.

[25]  B. Raught,et al.  Regulation of mammary gland factor/Stat5a during mammary gland development. , 1995, Molecular endocrinology.

[26]  L. Hennighausen,et al.  Activation of Stat5a and Stat5b by tyrosine phosphorylation is tightly linked to mammary gland differentiation. , 1996, Molecular endocrinology.

[27]  B. Groner,et al.  Prolactin regulation of beta‐casein gene expression and of a cytosolic 120‐kd protein in a cloned mouse mammary epithelial cell line. , 1988, The EMBO journal.

[28]  Miguel Beato,et al.  Steroid hormone receptors: Many Actors in search of a plot , 1995, Cell.

[29]  T. Welte,et al.  CCAAT/Enhancer-binding Protein Isoforms and Are Expressed in Mammary Epithelial Cells and Bind to Multiple Sites in the -Casein Gene Promoter (*) , 1995, The Journal of Biological Chemistry.

[30]  F. Moreau-Gachelin,et al.  Involvement of the transcription factor PU.1/Spi-1 in myeloid cell-restricted expression of an interferon-inducible gene encoding the human high-affinity Fc gamma receptor , 1994, Molecular and cellular biology.

[31]  J. Cidlowski,et al.  Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors , 1995, Molecular and cellular biology.

[32]  P. Chambon,et al.  Functional domains of the human estrogen receptor , 1987, Cell.

[33]  B. Raught,et al.  Developmentally and hormonally regulated CCAAT/enhancer-binding protein isoforms influence beta-casein gene expression. , 1995, Molecular endocrinology.

[34]  S. Rusconi,et al.  Interference and synergism of glucocorticoid receptor and octamer factors. , 1991, The EMBO journal.

[35]  J. Darnell,et al.  Two contact regions between Stat1 and CBP/p300 in interferon γ signaling , 1996 .

[36]  P. Angel,et al.  The glucocorticoid receptor synergizes with Jun homodimers to activate AP-1-regulated promoters lacking GR binding sites. , 1995, Chemical senses.

[37]  D. Levy,et al.  Thrombopoietin activates a STAT5‐like factor in hematopoietic cells. , 1995, The EMBO journal.

[38]  B. Groner,et al.  Deletion of the carboxyl-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype , 1996, Molecular and cellular biology.

[39]  J. Darnell,et al.  Two contact regions between Stat1 and CBP/p300 in interferon gamma signaling. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[40]  J. Gustafsson,et al.  Glucocorticoid receptor binding sites in the promoter region of milk protein genes , 1993, The Journal of Steroid Biochemistry and Molecular Biology.

[41]  J. Ihle.  Signaling by the cytokine receptor superfamily in normal and transformed hematopoietic cells. , 1996, Advances in cancer research.

[42]  M. Ptashne,et al.  Transcriptional activation by recruitment , 1997, Nature.

[43]  G. Chrousos,et al.  Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids. , 1996, Endocrine reviews.

[44]  S. Bhattacharya,et al.  Cooperation of Stat2 and p300/CBP in signalling induced by interferon-α , 1996, Nature.

[45]  K. Struhl,et al.  Mechanisms of transcriptional activation in vivo: two steps forward. , 1996, Trends in genetics : TIG.

[46]  M. Stallcup,et al.  GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[47]  B. Groner,et al.  Developmental and environmental regulation of a mammary gland-specific nuclear factor essential for transcription of the gene encoding beta-casein. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[48]  A. Miyajima,et al.  Suppression of interleukin‐3‐induced gene expression by a C‐terminal truncated Stat5: role of Stat5 in proliferation. , 1996, The EMBO journal.

[49]  B. Groner,et al.  Prolactin and glucocorticoid hormones synergistically induce expression of transfected rat beta-casein gene promoter constructs in a mammary epithelial cell line. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[50]  T Lagrange,et al.  The general transcription factors of RNA polymerase II. , 1996, Genes & development.

[51]  A. Miyajima,et al.  Interleukin‐3, granulocyte‐macrophage colony stimulating factor and interleukin‐5 transduce signals through two STAT5 homologs. , 1995, The EMBO journal.

[52]  W. Doppler.  Regulation of gene expression by prolactin. , 1994, Reviews of physiology, biochemistry and pharmacology.

[53]  J. Ihle,et al.  Naturally occurring dominant negative variants of Stat5 , 1996, Molecular and cellular biology.

[54]  L. Sanders,et al.  Cooperative transcriptional activity of Jun and Stat3 beta, a short form of Stat3. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[55]  A. Cato,et al.  Molecular mechanisms of anti‐inflammatory action of glucocorticoids , 1996, BioEssays : news and reviews in molecular, cellular and developmental biology.

[56]  B. Groner,et al.  Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. , 1994, The EMBO journal.

[57]  J A Gustafsson,et al.  Negative cross-talk between RelA and the glucocorticoid receptor: a possible mechanism for the antiinflammatory action of glucocorticoids. , 1995, Molecular endocrinology.

[58]  D. Levy,et al.  Prolactin, growth hormone, erythropoietin and granulocyte‐macrophage colony stimulating factor induce MGF‐Stat5 DNA binding activity. , 1995, The EMBO journal.

[59]  J. Lammers,et al.  STAT3β, a Splice Variant of Transcription Factor STAT3, Is a Dominant Negative Regulator of Transcription* , 1996, The Journal of Biological Chemistry.

[60]  W. Leonard,et al.  An IL‐2 response element in the human IL‐2 receptor alpha chain promoter is a composite element that binds Stat5, Elf‐1, HMG‐I(Y) and a GATA family protein. , 1996, The EMBO journal.

[61]  D. Taverna,et al.  Epidermal growth factor receptor, platelet-derived growth factor receptor, and c-erbB-2 receptor activation all promote growth but have distinctive effects upon mouse mammary epithelial cell differentiation. , 1991, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[62]  A. Munck,et al.  Control by basal phosphorylation of cell cycle-dependent, hormone-induced glucocorticoid receptor hyperphosphorylation. , 1997, Molecular endocrinology.

[63]  P. Mellon,et al.  Mutual cross-interference between glucocorticoid receptor and CREB inhibits transactivation in placental cells. , 1992, The New biologist.

[64]  B. Groner,et al.  Cloning and expression of Stat5 and an additional homologue (Stat5b) involved in prolactin signal transduction in mouse mammary tissue. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[65]  P. Herrlich,et al.  A distinct modulating domain in glucocorticoid receptor monomers in the repression of activity of the transcription factor AP‐1. , 1994, The EMBO journal.

[66]  J. Blenis,et al.  Requirement of serine phosphorylation for formation of STAT-promoter complexes. , 1995, Science.

[67]  B. Groner,et al.  The nuclear factor YY1 participates in repression of the beta-casein gene promoter in mammary epithelial cells and is counteracted by mammary gland factor during lactogenic hormone induction , 1994, Molecular and cellular biology.

[68]  Dwight C. Look,et al.  Stat1 Depends on Transcriptional Synergy with Sp1 (*) , 1995, The Journal of Biological Chemistry.

[69]  C. Glass,et al.  Nuclear integration of JAK/STAT and Ras/AP-1 signaling by CBP and p300. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[70]  B. O’Malley,et al.  Sequence and Characterization of a Coactivator for the Steroid Hormone Receptor Superfamily , 1995, Science.

[71]  I. Dusanter-Fourt,et al.  The structure, regulation and function of the Janus kinases (JAKs) and the signal transducers and activators of transcription (STATs). , 1997, European journal of biochemistry.

[72]  J. Darnell,et al.  Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. , 1995, Annual review of biochemistry.

[73]  B. Groner,et al.  Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription. , 1994, The EMBO journal.

[74]  C. Schindler,et al.  Differentiation-regulated serine phosphorylation of STAT1 promotes GAF activation in macrophages , 1995, Molecular and cellular biology.

[75]  Allard Kaptein,et al.  Dominant Negative Stat3 Mutant Inhibits Interleukin-6-induced Jak-STAT Signal Transduction (*) , 1996, The Journal of Biological Chemistry.

[76]  J. Darnell,et al.  Maximal activation of transcription by statl and stat3 requires both tyrosine and serine phosphorylation , 1995, Cell.

[77]  R. Evans,et al.  Functional domains of the human glucocorticoid receptor , 1986, Cell.

[78]  B. Groner,et al.  Beta-casein gene promoter activity is regulated by the hormone-mediated relief of transcriptional repression and a mammary-gland-specific nuclear factor , 1991, Molecular and cellular biology.

[79]  B. Groner,et al.  Down-regulation and phosphorylation of glucocorticoid receptors in cultured cells. Investigations with a monospecific antiserum against a bacterially expressed receptor fragment. , 1989, The Journal of biological chemistry.