The gene encoding AIB1, an estrogen receptor coactivator, is amplified in a subset of human breast cancers. Here we show that overexpression of AIB1 in transgenic mice (AIB1-tg) leads to mammary hypertrophy, hyperplasia, abnormal postweaning involution, and the development of malignant mammary tumors. Tumors are also increased in other organs, including the pituitary and uterus. AIB1 overexpression increases mammary IGF-I mRNA and serum IGF-I protein levels. In addition, IGF-I receptor and downstream signaling molecules are activated in primary mammary epithelial cells and mammary tumor cells derived from AIB1-tg mice. Knockdown of AIB1 expression in cultured AIB1-tg mammary tumor cells leads to reduced IGF-I mRNA levels and increased apoptosis, suggesting that an autocrine IGF-I loop underlies the mechanism of AIB1-induced oncogenesis.

Severe dietary restriction, catabolic states and even short-term caloric deprivation impair fertility in mammals. Likewise, obesity is associated with infertile conditions such as polycystic ovary syndrome. The reproductive status of lower organisms such as Caenorhabditis elegans is also modulated by availability of nutrients. Thus, fertility requires the integration of reproductive and metabolic signals. Here we show that deletion of insulin receptor substrate-2 (IRS-2), a component of the insulin/insulin-like growth factor-1 signalling cascade, causes female infertility. Mice lacking IRS-2 have small, anovulatory ovaries with reduced numbers of follicles. Plasma concentrations of luteinizing hormone, prolactin and sex steroids are low in these animals. Pituitaries are decreased in size and contain reduced numbers of gonadotrophs. Females lacking IRS-2 have increased food intake and obesity, despite elevated levels of leptin. Our findings indicate that insulin, together with leptin and other neuropeptides, may modulate hypothalamic control of appetite and reproductive endocrinology. Coupled with findings on the role of insulin-signalling pathways in the regulation of fertility, metabolism and longevity in C. elegans and Drosophila, we have identified an evolutionarily conserved mechanism in mammals that regulates both reproduction and energy homeostasis.

Growth factor modulation of estrogen receptor (ER) activity plays an important role in both normal estrogen physiology and the pathogenesis of breast cancer. Growth factors are known to stimulate the ligand-independent activity of ER through the activation of mitogen-activated protein kinase (MAPK) and the direct phosphorylation of ER. We found that the transcriptional activity of AIB1, a ligand-dependent ER coactivator and a gene amplified preferentially in ER-positive breast cancers, is enhanced by MAPK phosphorylation. We demonstrate that AIB1 is a phosphoprotein in vivo and can be phosphorylated in vitro by MAPK. Finally, we observed that MAPK activation of AIB1 stimulates the recruitment of p300 and associated histone acetyltransferase activity. These results suggest that the ability of growth factors to modulate estrogen action may be mediated through MAPK activation of the nuclear receptor coactivator AIB1.The estrogen receptor (ER) is a member of the class I family of nuclear receptors (NRs) (for a review, see reference 24). It contains three major functional domains: an N-terminal activation domain (AF-1), a DNA-binding domain (DBD) highly conserved among other NRs, and a C-terminal hormone-binding domain which contains a second activation domain (AF-2) (19, 38). Although AF-1 and AF-2 contribute synergistically to the transcription of targeted genes, they have different mechanisms of activation. AF-1 activity is highly dependent on phosphorylation of serine 118 by mitogen-activated protein kinase (MAPK) (18). In contrast, the more potent AF-2 is activated by the binding of estrogenic ligands (3).ER-mediated gene transcription is regulated at yet another level depending on the ligand; ER interacts with corepressors or coactivators that inhibit or enhance its activity on target genes. In the absence of ligand, ER is sequestered in the nucleus by interaction with heat shock proteins (29, 33). When activated by agonist ligand binding, ER exerts its action by promoting chromatin remodeling and stimulating the basal transcriptional machinery through interaction with a variety of coactivators (1, 14-16, 23, 27, 28, 34). One of the best-characterized groups of NR coactivators is the p160 family. When bound to agonists such as estradiol, ER AF-2 engages signature motifs (LXXLL) in the center of the p160 molecule (11). Recent studies have shown that the AF-1 domain of ER also interacts with p160 coactivators (40, 42), though the interaction seems to occur at a different site found in the C terminus of the p160 molecule. The p160 coactivators also contain two activation domains, AD1 and AD2, which are localized in the C terminus (6,7,41) and bind the secondary coactivators p300/CREB binding protein (CBP) and coactivator-associated arginine methyltransferase 1 (CARM1), respectively. Thus, AD1 and AD2 act as signal output domains in the process of transcriptional activation (22). Mutations in the AD1 region greatly reduced or eliminated the ability of p160 proteins to bind CBP or p300 and to serve as coactivators f...

Notch, a transmembrane receptor member of the homeotic epidermal growth factor-like family of proteins, participates in cell-to-cell signaling to control cell fate during development. Activated Notch-1 constructs lacking the extracellular region prevent differentiation of several mammalian cells in vitro. This effect, however, bypasses the normal mechanisms of cell-to-cell interactions in which Notch-1 participates. We investigated the role of Notch-1 in the hormone-induced adipocyte differentiation of 3T3-L1 fibroblasts, a paradigmatic model of adipogenesis that requires cell-to-cell contact. Unlike other differentiation models, Notch-1 expression and function were necessary conditions for adipogenesis. Impaired Notch-1 expression by antisense Notch-1 constructs prevented adipocyte differentiation. Strategies aimed at blocking putative Notch/ligand interactions also blocked adipogenesis, implicating Notch as a critical molecule in cell-to-cell signaling necessary for differentiation. Inhibition of Notch-1 expression or function decreased the expression of peroxisomal proliferatoractivated receptors ␦ and ␥, transcription factors that control adipocyte differentiation and that are up-regulated at cell confluence. These results implicate Notch in the commitment of 3T3-L1 cells to undergo adipogenesis by controlling the expression of the principal regulators of this process.

In 3T3-L1 fibroblasts, Ras proteins mediate both insulin-induced differentiation to adipocytes and its activation of cytosolic serine/threonine kinases, including Raf-1 kinase, mitogen-activated protein kinase (MAPK), and Rsk. Here, we report that insulin-and Ras-induced activation of MAPK is not required for the differentiation process and in fact antagonizes it. The treatment of 3T3-L1 preadipocytes with MEK-specific inhibitor PD98059 blocked insulin-and Ras-induced MAPK activation but had no effect on or slightly enhanced adipocytic differentiation. Tumor necrosis factor alpha (TNF-␣), an inhibitor of insulin-stimulated adipogenesis, activated MAPK in 3T3-L1 cells. PD98059 treatment blocked MAPK activation by TNF-␣ and reversed the blockade of adipogenesis mediated by low (1 ng/ml) TNF-␣ concentrations. 3T3-L1 transfectants containing hyperactivated MEK1 or overexpressed MAPK displayed impaired adipocytic differentiation. PD98059 treatment also reversed the blockade of differentiation in MEK1 transfectants. These results indicate that MAPK does not promote but can contribute to inhibition of the process of adipocytic differentiation of 3T3-L1 cells.Adipocytic differentiation is a complex process regulated by many hormones, growth factors, and cytokines. Whereas some of those signals stimulate differentiation to adipocytes, others inhibit this event (13,20), presumably based on the ability to ultimately alter gene expression. Preadipocytic cell lines, such as 3T3-L1, undergo differentiation after continuous exposure to pharmacological doses of insulin or physiologic doses of insulin-like growth factor 1 and are additionally induced by glucocorticoids and fatty acids (27,37,41,43). In contrast, tumor necrosis factor alpha (TNF-␣), a cytokine secreted by macrophages and adipose tissue, inhibits adipocytic differentiation (44, 45). However, the mechanisms that mediate the actions of insulin and other ligands on differentiation have yet to be clearly defined.We demonstrated previously that overexpression of Ras induces adipocytic differentiation of 3T3-L1 cells (3, 34), implicating Ras proteins as obligatory signaling intermediates in insulin-stimulated differentiation pathways. Our prior studies also showed that Ras proteins are necessary and sufficient for insulin activation of the cytosolic kinases Raf-1, mitogen-activated protein kinase (MAPK), and Rsk (35, 36); furthermore, Raf-1 participates downstream of Ras in the signaling cascade resulting in adipocytic differentiation. However, in contrast to its role in proliferating cells (39, 51, 54), Raf-1 kinase activation in differentiating 3T3-L1 cells is completely dissociated from activation of MAPK by insulin (35,36), suggesting that at least two separate signals emerge from Ras after insulin stimulation, regulating either growth or differentiation depending on the state of the cell or the presence of other environmental factors. Dissociation between Raf-1 kinase and MAPK has also previously been described for other cell types (15,39,(55)(56)(57).To further de...

The guanine nucleotide releasing protein C3G was initially identi®ed as a Crk SH3-binding protein and recently shown to exhibit exchange activity on Rap1 proteins. Overexpression in NIH3T3 cells of a full-length C3G cDNA isolated from human placenta markedly reduced the focus forming activity of cotransfected, malignantly activated, ras oncogenes (5 ± 7-fold). C3G also had a reverting eect on sis-mediated transformation, decreasing the number of c-sis-induced foci by a factor of 5 ± 10-fold. The observed inhibitory eect of C3G on focus-forming activity of Ras and Sis was always higher than that observed with Rap1A, a known target of C3G. The inhibition of focus formation observed in the presence of C3G was not due to toxic eects on cell viability, since transfected C3G cells exhibited the same survival and growth rates as untransfected NIH3T3 cells or cells transfected with plasmid vector alone. Surprisingly, as opposed to Rap1A, which has no eect on Raf-1 oncogene-mediated transformation, C3G also reduced dramatically (6 ± 8-fold) the number of v-raf-induced foci in transfected NIH3T3 cells. The inhibitory eect on Raf-induced transformation suggests that C3G has other functional targets in addition to Rap1. A C3G mutant (C3G DCat) lacking the catalytic domain (CDC25-H) but retaining the rest of the N-terminal sequences, including the Crkbinding domain, exhibited similar ability than full length C3G to inhibit focus formation. In contrast, a C3G mutant (C3G Cat), containing the catalytic domain only but lacking the rest of the N-terminal sequences, did not have any inhibitory eect on transformation mediated by the oncogenes tested. The C3G-derived gene products overexpressed in our transfected cell lines localized to the cytoplasm and did not change the basal MAPK or JNK activity of those cell lines nor their ability to activate the kinases in response to agonists. Our results suggest that the N-terminal region of C3G, and not its catalytic domain, may be responsible for the inhibitory eects observed.

Development of diabetes generally re¯ects an inadequate mass of insulin-producing b-cells. b-cell proliferation and differentiation are regulated by a variety of growth factors and hormones, including insulin-like growth factor I (IGF-I). GRF1 is a Rasguanine nucleotide exchange factor known previously for its restricted expression in brain and its role in learning and memory. Here we demonstrate that GRF1 is also expressed in pancreatic islets. Interestingly, our GRF1-de®cient mice exhibit reduced body weight, hypoinsulinemia and glucose intolerance owing to a reduction of b-cells. Whereas insulin resistance is not detected in peripheral tissues, GRF1 knockout mice are leaner due to increased lipid catabolism. The reduction in circulating insulin does not re¯ect defective glucose sensing or insulin production but results from impaired b-cell proliferation and reduced neogenesis. IGF-I treatment of isolated islets from GRF1 knockouts fails to activate critical downstream signals such as Akt and Erk. The observed phenotype is similar to manifestations of preclinical type 2 diabetes. Thus, our observations demonstrate a novel and speci®c role for Ras-GRF1 pathways in the development and maintenance of normal b-cell number and function.

RasGRF1 is a Ras-guanine nucleotide exchange factor implicated in a variety of physiological processes including learning and memory and glucose homeostasis. To determine the role of RASGRF1 in aging, lifespan and metabolic parameters were analyzed in aged RasGrf1−/− mice. We observed that mice deficient for RasGrf1−/− display an increase in average and most importantly, in maximal lifespan (20% higher than controls). This was not due to the role of Ras in cancer because tumor-free survival was also enhanced in these animals. Aged RasGrf1−/− displayed better motor coordination than control mice. Protection against oxidative stress was similarly preserved in old RasGrf1−/−. IGF-I levels were lower in RasGrf1−/− than in controls. Furthermore, SIRT1 expression was increased in RasGrf1−/− animals. Consistent with this, the blood metabolomic profiles of RasGrf1-deficient mice resembled those observed in calorie-restricted animals. In addition, cardiac glucose consumption as determined PET was not altered by aging in the mutant model, indicating that RasGrf1-deficienct mice display delayed aging. Our observations link Ras signaling to lifespan and suggest that RasGrf1 is an evolutionary conserved gene which could be targeted for the development of therapies to delay age-related processes.