Uterine Gab-1 Activation During Various Stages of the Estrous Cycle. To examine if there may be a physiological function for Gab-1 in uterine signaling, uteri were collected from adult ovary-intact, cycling mice. As demonstrated in Figure 1, activation of uterine Gab-1 (~115 kD), as identified through Gab-1 tyrosine phosphorylation, varied throughout the estrous cycle. Uterine Gab-1 activation was greatest during the late diestrus/early proestrus stages of the cycle. During this time serum P levels are declining and E2 levels are rising (7), implicating E2, P, or an appropriate ratio of the two hormones in the activation of uterine Gab-1. Protein levels of Gab-1 remained constant across the different stages of estrus.

Figure 1. Tyrosine phosphorylation of uterine Gab-1 (Gab-1 p-Tyr) varies throughout the mouse estrous cycle indicating that optimal Gab-1 stimulation may have a specific hormone requirement. D, diestrus; M, metestrus; P, proestrus.

Ovarian Steroid Hormone Activation of Uterine Gab-1. To determine a role for the ovarian steroid hormones in uterine Gab-1 activation, ovariectomized mice were treated with vehicle, E2, P, or E2 + P. Figure 2 shows that there was minimal Gab-1-pY in samples from vehicle-treated animals. Gab-1 phosphorylation was increased by all steroid hormone treatments according to the following hierarchy: E2 + P > E2 > P; treatment with the combination of E2 +

P resulted in the greatest level of uterine Gab-1-pY/activation. Hormone treatment did not alter Gab-1 protein levels.

The observation that each steroid hormone alone activated uterine Gab-1 to a differing degree may indicate that different cellular pools of Gab-1 are activated by the individual hormones. Alternatively, as Gab-1 has multiple tyrosine residues available for phosphorylation (reviewed in 3), the individual hormones may phosphorylate the same Gab-1 molecule but on tyrosine residues that are distinct for that hormone. For example, E and P may stimulate different RTKs that phosphorylate the same Gab-1 molecule on different tyrosine residues, and, therefore, the extent of phosphorylation of the Gab-1 molecule may vary with the nature of hormone treatment. Additionally, while E or P alone may be able to stimulate the production of a ligand necessary for RTK-stimulation of Gab-1, an appropriate ratio of the two hormones together may be necessary for optimal production of such a ligand.

Figure 2. Stimulation of uterine Gab-1-pY and signaling complexes by ovarian steroid hormones. A. Stimulation of Gab-1-pY (Gab-1-pY, top panel) and increase in the amount of the p85 subunit of PI3-kinase bound to Gab-1 (middle panel); bottom panel demonstrates the levels of Gab-1 protein. B. Stimulation of Gab-1-pY (top panel) and increase in the amount of SHP2 bound to Gab-1 (middle panel); bottom panel demonstrates the levels of Gab-1 protein.

In addition to activation of uterine Gab-1, ovarian steroid hormones also stimulated the formation of Gab-1 signaling complexes. Both E2 and E2 + P treatment resulted in an increase in the amount of the p85 subunit of PI3-kinase bound to Gab-1 (Figure 2A). Only E2 + P treatment stimulated formation of a complex between Gab-1 and SHP-2 (Figure 2B). The formation of Gab-1/p85 and Gab-1/SHP-2 signaling complexes in response to steroid hormones indicates that Gab-1 is actively participating in a signaling cascade in response to the ovarian steroids, and that the signaling cascades involve PI3-kinase and SHP2. This observation may be a further indication that multiple Gab-1-mediated signals are elicited in the uterus by different ovarian steroids or that a combination of the two steroids is obligatory for an optimal Gab-1 -mediated signal. The interaction between Gab-1 and p85 is necessary for activation of the PI3-kinase/Akt signaling pathway in response to many different stimuli, and the production of PIP3 by activated PI3-kinase may function to create a positive feedback loop between Gab-1 and PI3-kinase activation, since PIP3 can bind to Gab-1 to promote further PI3-kinase activation (reviewed in 3). Studies have shown that the Gab-1/SHP2 interaction is important for activation of SHP2 phosphatase activity, which can lead to activation of MAP kinase and subsequent MAP kinase-regulated cellular responses (reviewed in 3).

Other studies suggest that SHP-2 regulates the strength and duration of Gab-1-associated PI3-kinase activity by dephosphorylating the p85 binding sites of Gab-1 after EGF stimulation, preventing p85 from further binding to Gab-1 (8). Thus, another hypothesis is that E and P regulate competing signals, one that stimulates a Gab-1/p85 interaction and another that serves to promote the SHP-2 interaction and inhibit signaling from the Gab-1/p85 complex.

Growth Factor Activation of Gab-1. Levels of IGF-1 and several EGFR ligands are increased in the uterus by E2 and/or P (9, reviewed in 10, 11). To investigate whether EGF or IGF-1 stimulates uterine Gab-1-pY, ovariectomized adult mice were administered vehicle, EGF, or IGF-1. Figure 3 demonstrates that EGF, but not IGF-1, stimulated uterine Gab-1-pY. This suggests that in the uterus signaling through EGFR may contribute to the activation of Gab-1 and formation of Gab-1 signaling complexes in response to ovarian steroids. This particular experiment further indicates that IGF-1R signaling is not involved in uterine Gab-1 signaling after ovarian steroid hormone action. Both EGFR and IGF-1R were tyrosine phosphorylated after treatment with the respective ligands, demonstrating that both growth factors appropriately stimulated their cognate receptors (data not shown).

Figure 3. Growth factor-stimulated Gab-1-pY. EGF but not IGF-1 stimulated uterine Gab-1-pY(Gab-1 pY, top panel); Gab-1 protein levels were not altered by growth factor treatment (bottom panel).

Immunohistochemistry confirmed localization of Gab-1 in the uterine luminal and glandular epithelium (Figure 4); Gab-1 immunoreactivity, however, was not observed in the stroma or myometrium. When extracts of uterine luminal epithelial cells were analyzed, an increase in EGFR-pY and Gab-1-pY was observed in EGF-treated mice when compared to that of vehicle-treated controls (data not shown). This suggests that Gab-1 is a substrate for activated EGFR in the epithelium. In these and other experiments of the present study, Gab-1 was not detected in EGFR immunoprecipitates, nor was EGFR detected in Gab-1 immunoprecipitates.

The EGFR ligands EGF and TGF-a are increased in the uterus by E2 (10-12), and amphiregulin mRNA is increased in the uterus by P (9). A role in

E2-induced uterine cell proliferation has been suggested for EGF (13), while it has been hypothesized that TGF-a and amphiregulin function during implantation (9 and references therein). Furthermore, heparin binding-EGF (HB-EGF) is another EGFR ligand that is regulated in the uterus by the ovarian steroid hormones; treatment with E + P increases the level of HB-EGF in rodent uterine stromal cells (14). The number of different EGFR ligands that are stimulated in the uterus by ovarian steroids makes EGFR a good candidate for investigation of an RTK pathway that mediates hormone-stimulated Gab-1 signaling this organ. Indeed, in many other cell systems EGFR has been demonstrated to stimulate Gab-1 phosphorylation (3 and references therein). Thus, as a specific role for Gab-1 in the uterus remains undefined, EGFR continues to be a valid target for investigation in the activation of uterine Gab-1.

Figure 4. Immunolocalization of Gab-1 in the uterus. Gab-1 immunoreactivity is observed in the luminal and glandular epithelium.

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