Phosphorylation of the androgen receptor by PIM1 in hormone refractory prostate cancer.

Integration of cellular signaling pathways with androgen receptor (AR) signaling can be achieved through phosphorylation of AR by cellular kinases. However, the kinases responsible for phosphorylating the AR at numerous sites and the functional consequences of AR phosphorylation are only partially understood. Bioinformatic analysis revealed AR serine 213 (S213) as a putative substrate for PIM1, a kinase overexpressed in prostate cancer. Therefore, phosphorylation of AR serine 213 by PIM1 was examined using a phosphorylation site-specific antibody. Wild-type PIM1, but not catalytically inactive PIM1, specifically phosphorylated AR but not an AR serine-to-alanine mutant (S213A). In vitro kinase assays confirmed that PIM1 can phosphorylate AR S213 in a ligand-independent manner and cell type-specific phosphorylation was observed in prostate cancer cell lines. Upon PIM1 overexpression, AR phosphorylation was observed in the absence of hormone and was further increased in the presence of hormone in LNCaP, LNCaP-abl and VCaP cells. Moreover, phosphorylation of AR was reduced in the presence of PIM kinase inhibitors. An examination of AR-mediated transcription showed that reporter gene activity was reduced in the presence of PIM1 and wild-type AR, but not S213A mutant AR. Androgen-mediated transcription of endogenous PSA, Nkx3.1 and IGFBP5 was also decreased in the presence of PIM1, whereas IL6, cyclin A1 and caveolin 2 were increased. Immunohistochemical analysis of prostate cancer tissue microarrays showed significant P-AR S213 expression that was associated with hormone refractory prostate cancers, likely identifying cells with catalytically active PIM1. In addition, prostate cancers expressing a high level of P-AR S213 were twice as likely to be from biochemically recurrent cancers. Thus, AR phosphorylation by PIM1 at S213 impacts gene transcription and is highly prevalent in aggressive prostate cancer.

PEST-domain-enriched tyrosine phosphatase and glucocorticoids as regulators of anaphylaxis in mice.

BACKGROUND: PEST-domain-enriched tyrosine phosphatase (PEP) is a protein tyrosine phosphatase exclusively expressed in hematopoietic cells. It is a potent negative regulator of T-cell receptor signalling that acts on receptor-coupled protein tyrosine kinases. PEST-domain-enriched tyrosine phosphatase is also expressed in mast cell and is positively regulated by glucocorticoids, but its function is unknown. In this communication, the function of PEP is analysed in mast cells. METHODS: Signal transduction cascades following IgE receptor cross-linking were compared in bone marrow-derived mast cells (BMMC) from PEP(-/-) and PEP(+/+) mice. Furthermore, antigen-induced passive systemic anaphylaxis (PSA) was analysed in PEP(+/+) and PEP(-/-) mice. RESULTS: Bone marrow-derived mast cells from PEP(-/-) mice showed impaired PLCγ1 phosphorylation and Ca(2+) mobilization. Additionally, mice deficient in PEP showed impaired mast cell degranulation and were less susceptible to PSA. Treatment of wild-type BMMC or mice with an Au(I)-phosphine complex that selectively inhibits PEP activity produced defects in Ca(2+) signalling pathway and reduced anaphylaxis similar to that caused by the deletion of the PEP gene. Glucocorticoid that negatively regulates a wide range of mast cell action increased PEP expression and only partially inhibited anaphylaxis. However, glucocorticoid potently inhibited anaphylaxis when combined with the PEP inhibitor. CONCLUSIONS: PEST-domain-enriched tyrosine phosphatase is an important positive regulator of anaphylaxis. Pharmacological inhibition of its activity together with glucocorticoid administration provide an effective rescue for PSA in mice.

Antidepressants increase human hippocampal neurogenesis by activating the glucocorticoid receptor.

Antidepressants increase adult hippocampal neurogenesis in animal models, but the underlying molecular mechanisms are unknown. In this study, we used human hippocampal progenitor cells to investigate the molecular pathways involved in the antidepressant-induced modulation of neurogenesis. Because our previous studies have shown that antidepressants regulate glucocorticoid receptor (GR) function, we specifically tested whether the GR may be involved in the effects of these drugs on neurogenesis. We found that treatment (for 3-10 days) with the antidepressant, sertraline, increased neuronal differentiation via a GR-dependent mechanism. Specifically, sertraline increased both immature, doublecortin (Dcx)-positive neuroblasts (+16%) and mature, microtubulin-associated protein-2 (MAP2)-positive neurons (+26%). This effect was abolished by the GR-antagonist, RU486. Interestingly, progenitor cell proliferation, as investigated by 5'-bromodeoxyuridine (BrdU) incorporation, was only increased when cells were co-treated with sertraline and the GR-agonist, dexamethasone, (+14%) an effect which was also abolished by RU486. Furthermore, the phosphodiesterase type 4 (PDE4)-inhibitor, rolipram, enhanced the effects of sertraline, whereas the protein kinase A (PKA)-inhibitor, H89, suppressed the effects of sertraline. Indeed, sertraline increased GR transactivation, modified GR phosphorylation and increased expression of the GR-regulated cyclin-dependent kinase-2 (CDK2) inhibitors, p27(Kip1) and p57(Kip2). In conclusion, our data suggest that the antidepressant, sertraline, increases human hippocampal neurogenesis via a GR-dependent mechanism that requires PKA signaling, GR phosphorylation and activation of a specific set of genes. Our data point toward an important role for the GR in the antidepressant-induced modulation of neurogenesis in humans.

Epoprostenol treatment for idiopathic pulmonary arterial hypertension in pregnancy.

Idiopathic pulmonary arterial hypertension is a rare condition associated with significant maternal mortality. We report the management of a 37-year-old multigravida with severe disease using epoprostenol, a multidisciplinary approach, and a planned delivery. Although the patient survived the pregnancy, her pulmonary function significantly worsened. Epoprostenol, a pulmonary vasodilator, should be considered when indicated during pregnancy. Neither fetal nor neonatal harm was identified.

Androgen stimulated cellular proliferation in the human prostate cancer cell line LNCaP is associated with reduced retinoblastoma protein expression.

To elucidate the mechanism of androgen-dependent cellular proliferation in prostate cancer, androgen-dependent alterations of individual cell cycle regulatory proteins in the androgen-sensitive prostate cancer cell line LNCaP were evaluated. LNCaP cells were deprived of androgens by culture in steroid-depleted media for 5 days, which resulted in the maximal accumulation of cells in G(0)/G(1) phase of the cell cycle. The mitogenic concentration of the synthetic androgen R1881 was established as 0.1 nM using cell proliferation assay. Protein and mRNA levels of particular cyclins, cyclin-dependent kinases (Cdks), cyclin-dependent kinase inhibitors (Ckis), and the retinoblastoma proteins (Rb) were assessed. Androgen stimulation resulted in a post-transcriptional reduction in Rb protein levels, an increase in Rb phosphorylation at serine 780 and an accumulation of high molecular weight Rb protein species. Androgen stimulation also induced the expression of the Cdk2 and Cdk1 as well as their regulatory partners, cyclin A and cyclin B, resulting in a corresponding increase in cyclin A/Cdk2 activity in vitro. Pulse-chase showed decreased Rb protein stability in androgen-treated LNCaP cells. Collectively, our findings suggest a novel mechanism of androgen-dependent prostate cancer growth in which androgen stimulation results in decreased Rb protein expression in LNCaP cells. The observation of decreased Rb protein stability in the setting of increased phosphorylation supports the concept of phosphorylation mediated protein degradation. We propose that the observed reduction in Rb protein level occurs through Rb degradation via the ubiquitin/proteasome pathway, and is preceded by selective Rb phosphorylation by cyclin A/Cdk2 and cyclin B/Cdk1.

Rho GTPases as modulators of the estrogen receptor transcriptional response.

The estrogen receptor alpha (ER) is a ligand-dependent transcription factor that plays a critical role in the development and progression of breast cancer, in part, by regulating target genes involved in cellular proliferation. To identify novel components that affect the ER transcriptional response, we performed a genetic screen in yeast and identified RDI1, a Rho guanine nucleotide dissociation inhibitor (Rho GDI), as a positive regulator of ER transactivation. Overexpression of the human homologue of RDI1, Rho GDIalpha, increases ERalpha, ERbeta, androgen receptor, and glucocorticoid receptor transcriptional activation in mammalian cells but not activation by the unrelated transcription factors serum response factor and Sp1. In contrast, expression of constitutively active forms of RhoA, Rac1, and Cdc42 decrease ER transcriptional activity, suggesting that Rho GDI increases ER transactivation by antagonizing Rho function. Inhibition of RhoA by expression of either the Clostridium botulinum C3 transferase or a dominant negative RhoA resulted in enhanced ER transcriptional activation, thus phenocopying the effect of Rho GDI expression on ER transactivation. Together, these findings establish the Rho GTPases as important modulators of ER transcriptional activation. Since Rho GTPases regulate actin polymerization, our findings suggest a link between the major regulators of cellular architecture and steroid receptor transcriptional response.

Selective activation of the glucocorticoid receptor by steroid antagonists in human breast cancer and osteosarcoma cells.

Steroid hormones regulate the transcription of numerous genes via high affinity receptors that act in concert with chromatin remodeling complexes, coactivators and corepressors. We have compared the activities of a variety of glucocorticoid receptor (GR) antagonists in breast cancer and osteosarcoma cell lines engineered to stably maintain the mouse mammary tumor virus promoter. In both cell types, GR activation by dexamethasone occurs via the disruption of mouse mammary tumor virus chromatin structure and the recruitment of receptor coactivator proteins. However, when challenged with a variety of antagonists the GR displays differential ability to activate transcription within the two cell types. For the breast cancer cells, the antagonists fail to activate the promoter and do not promote the association of the GR with either remodeling or coactivator proteins. In contrast, in osteosarcoma cells, the antiglucocorticoids, RU486 and RU43044, exhibit partial agonist activity. The capacity of these antagonists to stimulate transcription in the osteosarcoma cells is reflected in the ability of the RU486-bound receptor to remodel chromatin and associate with chromatin-remodeling proteins. Similarly, the observation that the RU486-bound receptor does not fully activate transcription is consistent with its inability to recruit receptor coactivator proteins.

Differential regulation of glucocorticoid receptor transcriptional activation via AF-1-associated proteins.

The hormone-activated glucocorticoid receptor (GR), through its N- and C-terminal transcriptional activation functions AF-1 and AF-2, controls the transcription of target genes presumably through interaction(s) with transcriptional regulatory factors. Utilizing a modified yeast two-hybrid approach, we have identified the tumor susceptibility gene 101 (TSG101) and the vitamin D receptor-interacting protein 150 (DRIP150) as proteins that interact specifically with a functional GR AF-1 surface. In yeast and mammalian cells, TSG101 represses whereas DRIP150 enhances GR AF-1-mediated transactivation. Thus, GR AF-1 is capable of recruiting both positive and negative regulatory factors that differentially regulate GR transcriptional enhancement. In addition, we show that another member of the DRIP complex, DRIP205, interacts with the GR ligand binding domain in a hormone-dependent manner and facilitates GR transactivation in concert with DRIP150. These results suggest that DRIP150 and DRIP205 functionally link GR AF-1 and AF-2, and represent important mediators of GR transcriptional enhancement.

Potentiation of human estrogen receptor alpha transcriptional activation through phosphorylation of serines 104 and 106 by the cyclin A-CDK2 complex.

Both estradiol binding and phosphorylation regulate transcriptional activation by the human estrogen receptor alpha (ER). We have previously shown that activation of the cyclin A-CDK2 complex by overexpression of cyclin A leads to enhanced ER-dependent transcriptional activation and that the cyclin A-CDK2 complex phosphorylates the ER N-terminal activation function-1 (AF-1) between residues 82 and 121. Within ER AF-1, serines 104, 106, and 118 represent potential CDK phosphorylation sites, and in this current study, we ascertain their importance in mediating cyclin A-CDK2-dependent enhancement of ER transcriptional activity. Cyclin A overexpression does not enhance transcriptional activation by an ER derivative bearing serine-to-alanine changes at residues 104, 106, and 118. Likewise, the cyclin A-CDK2 complex does not phosphorylate this triple-mutated derivative in vitro. Individual serine-to-alanine mutations at residues 104 and 106, but not 118, decrease ER-dependent transcriptional enhancement in response to cyclin A. The same relationship holds for ER phosphorylation by cyclin A-CDK2 in vitro. Finally, enhancement of ER transcriptional activation by cyclin A is evident in the absence and presence of estradiol, as well as in the presence of tamoxifen, suggesting that the effect of the cyclin A-CDK2 on ER transcriptional activation is AF-2-independent. These results indicate that the enhancement of ER transcriptional activation by the cyclin A-CDK2 complex is mediated via the AF-1 domain by phosphorylation of serines 104 and 106. We propose that these residues control ER AF-1 activity in response to signals that affect cyclin A-CDK2 function.

Distinct glucocorticoid receptor transcriptional regulatory surfaces mediate the cytotoxic and cytostatic effects of glucocorticoids.

Glucocorticoids act through the glucocorticoid receptor (GR), which can function as a transcriptional activator or repressor, to elicit cytostatic and cytotoxic effects in a variety of cells. The molecular mechanisms regulating these events and the target genes affected by the activated receptor remain largely undefined. Using cultured human osteosarcoma cells as a model for the GR antiproliferative effect, we demonstrate that in U20S cells, GR activation leads to irreversible growth inhibition, apoptosis, and repression of Bcl2. This cytotoxic effect is mediated by GR's transcriptional repression function, since transactivation-deficient mutants and ligands still bring about apoptosis and Bcl2 down-regulation. In contrast, the antiproliferative effect of GR in SAOS2 cells is reversible, does not result in apoptosis or repression of Bcl2, and is a function of the receptor's ability to stimulate transcription. Thus, the cytotoxic versus cytostatic outcome of glucocorticoid treatment is cell context dependent. Interestingly, the cytostatic effect of glucocorticoids in SAOS2 cells involves multiple GR activation surfaces. GR mutants and ligands that disrupt individual transcriptional activation functions (activation function 1 [AF-1] and AF-2) or receptor dimerization fail to fully inhibit cellular proliferation and, remarkably, discriminate between the targets of GR's cytostatic action, the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). Induction of p21(Cip1) is agonist dependent and requires AF-2 but not AF-1 or GR dimerization. In contrast, induction of p27(Kip1) is agonist independent, does not require AF-2 or AF-1, but depends on GR dimerization. Our findings indicate that multiple GR transcriptional regulatory mechanisms that employ distinct receptor surfaces are used to evoke either the cytostatic or cytotoxic response to glucocorticoids.

Occupational chlorophenol exposure and non-Hodgkin's lymphoma.

Occupational exposure to chlorophenols is suspected to increase non-Hodgkin's lymphoma (NHL) risk. This association was examined using data on 995 NHL cases and 1783 controls from the Selected Cancers Study, a population-based case-control study of men aged 32 to 60 years from eight population-based cancer registries conducted from 1984 to 1988. Potential chlorophenol exposure was characterized by an industrial hygienist using intensity estimates and confidence ratings, based upon review of verbatim job histories. Cases with substantial chlorophenol exposure had a significantly greater number of years of chlorophenol exposure (median years: cases, 4.0; controls, 2.0; P = 0.046); however, in conditional logistic regression models, the odds ratio for more than 8 years of substantial exposure was 1.51 (95% CI, 0.88 to 2.59). Overall, the findings do not provide strong support for an association with NHL risk. Chlorophenol exposure in this study is not based upon measured values and, therefore, may fail to characterize actual chlorophenol exposures accurately. Because of the large presence of machinists in the potentially chlorophenol-exposed group, these results may be underestimated by exposure misclassification if these subjects were not exposed to chlorophenolic biocides. However, these results are consistent with other findings, which suggest that chlorophenol exposure is not likely to be a strong risk factor for NHL.

Role for Hsp90-associated cochaperone p23 in estrogen receptor signal transduction.

The mechanism of signal transduction by the estrogen receptor (ER) is complex and not fully understood. In addition to the ER, a number of accessory proteins are apparently required to efficiently transduce the steroid hormone signal. In the absence of estradiol, the ER, like other steroid receptors, is complexed with Hsp90 and other molecular chaperone components, including an immunophilin, and p23. This Hsp90-based chaperone complex is thought to repress the ER's transcriptional regulatory activities while maintaining the receptor in a conformation that is competent for high-affinity steroid binding. However, a role for p23 in ER signal transduction has not been demonstrated. Using a mutant ER (G400V) with decreased hormone binding capacity as a substrate in a dosage suppression screen in yeast cells (Saccharomyces cerevisiae), we identified the yeast homologue of the human p23 protein (yhp23) as a positive regulator of ER function. Overexpression of yhp23 in yeast cells increases ER transcriptional activation by increasing estradiol binding in vivo. Importantly, the magnitude of the effect of yhp23 on ER transcriptional activation is inversely proportional to the concentration of both ER and estradiol in the cell. Under conditions of high ER expression, ER transcriptional activity is largely independent of yhp23, whereas at low levels of ER expression, ER transcriptional activation is primarily dependent on yhp23. The same relationship holds for estradiol levels. We further demonstrate that yhp23 colocalizes with the ER in vivo. Using a yhp23-green fluorescent protein fusion protein, we observed a redistribution of yhp23 from the cytoplasm to the nucleus upon coexpression with ER. This nuclear localization of yhp23 was reversed by the addition of estradiol, a finding consistent with yhp23's proposed role as part of the aporeceptor complex. Expression of human p23 in yeast partially complements the loss of yhp23 function with respect to ER signaling. Finally, ectopic expression of human p23 in MCF-7 breast cancer cells increases both hormone-dependent and hormone-independent transcriptional activation by the ER. Together, these results strongly suggest that p23 plays an important role in ER signal transduction.

Phosphorylation and inhibition of rat glucocorticoid receptor transcriptional activation by glycogen synthase kinase-3 (GSK-3). Species-specific differences between human and rat glucocorticoid receptor signaling as revealed through GSK-3 phosphorylation.

Transcriptional activation by the glucocorticoid receptor (GR) is regulated by both glucocorticoid binding and phosphorylation. The rat GR N-terminal transcriptional regulatory domain contains four major phosphorylation sites: threonine 171 (Thr171), serine 224 (Ser224), serine 232 (Ser232), and serine 246 (Ser246). We have previously demonstrated that Ser224 and Ser232 are phosphorylated by cyclin-dependent kinases, while Ser246 is phosphorylated by the c-Jun N-terminal kinase. We report here that the remaining GR phosphorylation site, Thr171, is a target for glycogen synthase kinase-3 (GSK-3) in vitro and in cultured mammalian cells. Increasing GSK-3 activity through its overexpression in cultured cells inhibits GR transcriptional enhancement, an effect dependent upon Thr171. Correspondingly, overexpression of a constitutively active form of the GSK-3 inhibitor, protein kinase B/Akt, increases GR transcriptional enhancement. Overexpression of GSK-3 had no effect on GR-mediated transcriptional repression of AP1-dependent gene expression. Importantly, transcriptional activation by the human GR (hGR), which contains an alanine (Ala150) at the position equivalent to Thr171 in rat GR, is not affected by GSK-3 overexpression. Introduction of a threonine residue at this position (A150T) establishes GSK-3-mediated inhibition of hGR transcriptional activation. These findings demonstrate species-specific differences in GR signaling, as revealed through GSK-3 phosphorylation, which suggests that GR function in rodents may not fully recapitulate receptor action in humans and that hGR is capable of adopting the GSK-3 signaling pathway through a somatic mutation.

Antagonism of glucocorticoid receptor transcriptional activation by the c-Jun N-terminal kinase.

The mitogen-activated protein kinases ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38 phosphorylate and activate transcription factors that promote proliferative and inflammatory responses, whereas glucocorticoid receptor (GR) activation inhibits cell growth and inflammation. We demonstrate that JNK and ERK but not p38 phosphorylate GR in vitro primarily at Ser-246. Selective activation of either ERK or JNK in vivo inhibits GR-mediated transcriptional activation, which depends on receptor phosphorylation at Ser-246 by JNK but not ERK. Thus, JNK inhibits GR transcriptional activation by direct receptor phosphorylation, whereas ERK does so indirectly. We propose that phosphorylation of GR by JNK or of a GR cofactor by ERK provides mechanisms to ensure the rapid inhibition of GR-dependent gene expression when it conflicts with mitogenic or proinflammatory signals.

Regulation of estrogen receptor transcriptional enhancement by the cyclin A/Cdk2 complex.

We have found that ectopic expression of cyclin A increases hormone-dependent and hormone-independent transcriptional activation by the estrogen receptor in vivo in a number of cell lines, including HeLa cells, U-2 OS osteosarcoma cells and Hs 578Bst breast epithelial cells. This effect can be further enhanced in HeLa cells by the concurrent expression of the cyclin-dependent kinase activator, cyclin H, and cdk7, and abolished by expression of the cdk inhibitor, p27(KIP1), or by the expression of a dominant negative catalytically inactive cdk2 mutant. ER is phosphorylated between amino acids 82 and 121 in vitro by the cyclin A/cdk2 complex and incorporation of phosphate into ER is stimulated by ectopic expression of cyclin A in vivo. Together, these results strongly suggest a direct role for the cyclin A/cdk2 complex in phosphorylating ER and regulating its transcriptional activity.

Mitogen-activated and cyclin-dependent protein kinases selectively and differentially modulate transcriptional enhancement by the glucocorticoid receptor.

Cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) phosphorylate the rat glucocorticoid receptor in vitro at distinct sites that together correspond to the major phosphorylated receptor residues observed in vivo; MAPK phosphorylates receptor residues threonine 171 and serine 246, whereas multiple CDK complexes modify serines 224 and 232. Mutations in these kinases have opposite effects on receptor transcriptional activity in vivo. Receptor-dependent transcriptional enhancement is reduced in yeast strains deficient in the catalytic (p34CDC28) or certain regulatory (cyclin) subunits of CDK complexes and is increased in a strain devoid of the mammalian MAPK homologs FUS3 and KSS1. These findings indicate that the glucocorticoid receptor is a target for multiple kinases in vivo, which either positively or negatively regulate receptor transcriptional enhancement. The control of receptor transcriptional activity via phosphorylation provides an increased array of regulatory inputs that, in addition to steroid hormones, can influence receptor function.

Glucocorticoid receptor-mediated cell cycle arrest is achieved through distinct cell-specific transcriptional regulatory mechanisms.

Glucocorticoids inhibit proliferation of many cell types, but the events leading from the activated glucocorticoid receptor (GR) to growth arrest are not understood. Ectopic expression and activation of GR in human osteosarcoma cell lines U2OS and SAOS2, which lack endogenous receptors, result in a G1 cell cycle arrest. GR activation in U2OS cells represses expression of the cyclin-dependent kinases (CDKs) CDK4 and CDK6 as well as their regulatory partner, cyclin D3, leading to hypophosphorylation of the retinoblastoma protein (Rb). We also demonstrate a ligand-dependent reduction in the expression of E2F-1 and c-Myc, transcription factors involved in the G1-to-S-phase transition. Mitogen-activated protein kinase, CDK2, cyclin E, and the CDK inhibitors (CDIs) p27 and p21 are unaffected by receptor activation in U2OS cells. The receptor's N-terminal transcriptional activation domain is not required for growth arrest in U2OS cells. In Rb-deficient SAOS2 cells, however, the expression of p27 and p21 is induced upon receptor activation. Remarkably, in SAOS2 cells that express a GR deletion derivative lacking the N-terminal transcriptional activation domain, induction of CDI expression is abolished and the cells fail to undergo ligand-dependent cell cycle arrest. Similarly, murine S49 lymphoma cells, which, like SAOS2 cells, lack Rb, require the N-terminal activation domain for growth arrest and induce CDI expression upon GR activation. These cell-type-specific differences in receptor domains and cellular targets linking GR activation to cell cycle machinery suggest two distinct regulatory mechanisms of GR-mediated cell cycle arrest: one involving transcriptional repression of G1 cyclins and CDKs and the other involving enhanced transcription of CDIs by the activated receptor.

GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors.

After binding to enhancer elements, transcription factors require transcriptional coactivator proteins to mediate their stimulation of transcription initiation. A search for possible coactivators for steroid hormone receptors resulted in identification of glucocorticoid receptor interacting protein 1 (GRIP1). The complete coding sequence for GRIP1, isolated from a mouse brain cDNA library, contains an open reading frame of 1,462 codons. GRIP1 is the probable ortholog of the subsequently identified human protein transcription intermediary factor 2 (TIF2) and is also partially homologous to steroid receptor coactivator 1 (SRC-1). The full-length GRIP1 interacted with the hormone binding domains (HBDs) of all five steroid receptors in a hormone-dependent manner and also with HBDs of class II nuclear receptors, including thyroid receptor alpha, vitamin D receptor, retinoic acid receptor alpha, and retinoid X receptor alpha. In contrast to agonists, glucocorticoid antagonists did not promote interaction between the glucocorticoid receptor and GRIP1. In yeast cells, GRIP1 dramatically enhanced the transcriptional activation function of proteins containing the HBDs of any of the above-named receptors fused to the GAL4 DNA binding domain and thus served as a transcriptional coactivator for them. This finding contrasts with previous reports of TIF2 and SRC-1, which in mammalian cells enhanced the transactivation activities of only a subset of the steroid and nuclear receptors that they physically interacted with. GRIP1 also enhanced the hormone-dependent transactivation activity of intact glucocorticoid receptor, estrogen receptor, and mineralocorticoid receptor. Experiments with glucocorticoid receptor truncation and point mutants indicated that GRIP1 interacted with and enhanced the activity of the C-terminal AF-2 but not the N-terminal AF-1 transactivation domain of the glucocorticoid receptor. These results demonstrate directly that AF-1 and AF-2 domains accomplish their transactivation activities through different mechanisms: AF-2 requires GRIP1 as a coactivator, but AF-1 does not.

Modular structure of glucocorticoid receptor domains is not equivalent to functional independence. Stability and activity of the steroid binding domain are controlled by sequences in separate domains.

A long-standing conundrum of glucocorticoid receptors has been why the steroid binding domain is active in hybrid proteins but not in isolation. For this reason, the precise boundaries of the steroid binding domain have not been defined. These questions have now been systematically examined with a variety of receptor deletion constructs. Plasmids encoding amino acids 537-673 and 537-795 of the rat receptor did not yield stable proteins, while the fusion of receptor or non-receptor sequences upstream of 537-673 afforded stable proteins that did not bind steroid. Wild type steroid binding affinity could be obtained, however, when proteins such as beta-galactosidase or dihydrofolate reductase were fused upstream of receptor amino acids 537-795. Studies of a series of dhfr/receptor constructs with deletions at the amino- and carboxyl-terminal ends of the receptor sequence localized the boundaries of the steroid binding domain to 550-795. The absence of steroid binding upon deletion of sequences in the carboxyl-terminal half of this domain was consistent with improperly folded receptor sequences. This conclusion was supported by analyses of the proteolysis and thermal stability of the mutant receptors. Thus, three independent regions appear to be required for the generation of the steroid binding form of receptors: 1) a protein sequence upstream of the steroid binding domain, which conveys stability to the steroid binding domain, 2) sequences of the carboxyl-terminal amino acids (674-795), which are required for the correct folding of the steroid binding domain, and 3) amino-terminal sequences (550-673), which may be sufficient for steroid binding after the entire steroid binding domain is properly folded. These results establish that the steroid binding domain of glucocorticoid receptors is not independently functional and illustrate the importance of both protein stability and protein folding when constructing mutant proteins.

Synergistic transcriptional activation of the tissue inhibitor of metalloproteinases-1 promoter via functional interaction of AP-1 and Ets-1 transcription factors.

The tissue inhibitor of metalloproteinases-1 (TIMP-1) is an inhibitor of the extracellular matrix-degrading metalloproteinases. We characterized response elements that control TIMP-1 gene expression. One contains a binding site that selectively binds c-Fos and c-Jun in vitro and confers a response to multiple AP-1 family members in vivo. Adjacent to this is a binding site for Ets domain proteins. Although c-Ets-1 alone did not activate transcription from this element, it enhanced transcription synergistically with AP-1 either in the context of the natural promoter or when the sequence was linked upstream of a heterologous promoter. Furthermore, a complex of c-Jun and c-Fos interacted with c-Ets-1 in vitro. These results suggest that AP-1 tethers c-Ets-1 to the TIMP-1 promoter via protein-protein interaction to achieve Ets-dependent transcriptional regulation. Collectively, our results indicate that TIMP-1 expression is controlled by several DNA response elements that respond to variations in the level and activity of AP-1 and Ets transcriptional regulatory proteins.