Dr. Jeffrey Pollard Lab

Female Sex Steroid Hormones and Growth Factors in Mammalian Development, Reproduction and Cancer

My laboratory has four major separate, but inter-related areas of research. These are focused on the interactions between sex steroid hormones, oncogenes and growth factors in normal reproductive processes and in cancers of the reproductive system. We also have a particular interest in the role of macrophages in development and cancer. We place a strong emphasis on using genetic approaches in mice to define mechanisms in vivo and are at the forefront of such technologies.

(1) Female sex steroid hormones action in the preparation of the uterus for implantation

Early pregnancy involves a complex interplay between the female sex steroid hormones, estradiol 17? (E2) and Progesterone (P4), that synchronizes the differentiation of the uterus with pre-implantation embryonic development such that successful blastocyst implantation is achieved. In the adult mouse uterus, E2 synthesized at pro-estus stimulates a synchronized wave of cell proliferation in the epithelium. P4 synthesized in response to copulation completely inhibits this E2-induced cell proliferation. Thus P4 is a unique anti-proliferative agent in vivo and as such is used to treat estrogen-responsive uterine cancers. The cell cycle is regulated by cyclins and their obligate partners, the cyclin dependent kinases (CDK). Various classes of these sequentially phosphorylate the retinoblastoma (pRB) class of proteins that regulate progression through the cell cycle dependent on their phosphorylation state. We have shown that E2 stimulates cyclin D1/CDK 4 translocation to the nucleus and the activity of cyclin E and A/CDK2 with the consequent phosphorylation of pRB. P4 completely prevents the cyclin D nuclear translocation and the activation of CDK2 and therefore an inhibition of pRB phosphorylation and cell cycle arrest. This constitutes a unique method of cell cycle regulation (1). We are using a variety of approaches to identify the mechanism of this inhibition of

cyclin D1 translocation and have recently found new mechanisms of P4 action. In addition, to inhibiting the epithelial cell proliferation, P4 also stimulates epithelial differentiation in preparation for implantation. We have used DNA microarrays along with subtractive cloning methods to identify new genes involved in this process. Several of these are novel and we have also demonstrated one that defines both a new lipid metabolic pathway in mammals and is also regulated by P4 through the PKC pathway (2). This demonstrates a novel pathway activated by P4. Future research is aimed at identifying the function of these genes through gene targeting and RNAi strategies.

Our research is also translational and we validate the data we obtain in mice in the human system. To this end we collaborate extensively with Dr. N. Santoro, the Director of the Reproductive Endocrinology Division of the Department of Obstetrics and Gynecology and Women’s Health. Clinical fellows work in my lab and through them we obtain important clinical material derived from Dr. Santoro research programs. We are currently studying cell cycle regulation in human uterine samples and determining if the same markers of implantation that we find in mice are also found in the human. This is important since E2 is the major risk factor

(2) Placental Immunity

Sex steroid hormones exert many of their actions through the intermediary of peptide growth factors. Of these, we have extensively studied the mononuclear phagocyte growth factor, colony stimulating factor-1 (CSF-1), whose uterine epithelial synthesis is regulated by E2 and P4 and whose receptor is expressed not only in macrophages but also upon trophoblast (the fetal cells of the placenta). Studies on this growth factor were dramatically enhanced by the identification of the natural recessive mutation, Csf1op, as being a null mutation in the CSF-1 gene. These mice have no CSF-1 and have severely depleted macrophage populations. They display many fertility defects but once pregnant they can carry litters to term with apparently normal placental function (3). We have shown that the trophoblast have taken over several CSF-1controlled macrophage functions through their expression of the CSF-1 receptor and have used them to regulate the maternal immune response to pathogens such as Listeria monocytogense that preferentially replicate at the maternal-fetal interface. This shows that the trophoblast becomes a part of the innate immune system during pregnancy (4). Our continuing research is to determine how pathogens are eradicated from the placenta while at the same time the allogeneic (foreign) fetus is spared (5).

(3) Macrophages in the brain regulate sex-steroid hormone feedback.

Mice homozygous for the CSF-1 null mutation have severe fertility defects. The major cause of this is the lack of sex-steroid regulated feedback in the hypothalamus. The mice also display nerophysiological defects that can in part, be ascribed to problems in GABAeric circuitry, a major regulator of the sex steroid hormone feedback system (6). We have shown that this is a developmental defect cause by a failure of CSF-1 signaling in the microglia, the macrophages of the brain. Using a mouse whose macrophages are marked with GFP, the MacGreen mouse, we are studying the development and function of microglia. In addition we are developing MRI methods to functionally map the defects in the mouse. This coupled with hypothalamic slice cultures will elucidate the important developmental roles of migroglial in the establishment of normal connectivity in the brain.

(4) Regulation of Mammary gland development and cancer

The fourth project involves the role of stromal and epithelial elements in mammary gland development and cancer. Mammary development mostly occurs post-natally, initially under the influence of E2 and later in development, P4. The development begins with the formation of a terminal end bud (TEB), a multilaminate epithelial structure that grows out through the fatty stoma and that by bifurcation forms the rudimentary ductal tree. This then further arborises during the estrous cycle and particularly under the influence of P4 during pregnancy to give the milk-producing alveolar structures. Once lactation is finished then these alveolar structures regress to regenerate a virgin-like mammary gland. During development we have shown that the TEB recruits to itself large numbers of macrophages that persist until the TEB

reaches the end of the fat pad where they cease to develop. Using genetic means to remove macrophages from tissues, we have identified these cells as important regulators of ductal development in the mammary gland (7). Currently we are focused upon the mechanism of the macrophages in promoting mammary development using gene discovery methods and sophisticated multiphoton microscopy to visualize mammary development in vivo. We have also shown that the major regulator of cell death in the mammary gland is TGF?3 produced in response to milk stasis (8). We are using the power of genetics in the mammary gland system to determine the upstream and downstream regulators of this process.

In the breast cancer area we have also shown that the tumor recruit to itself, macrophages. These seem to act as they do during normal development to promote epithelial outgrowth into the stroma. Using genetics to remove these cells we have shown that they promote the progression and metastasis of cancer in this tissue (9). These tumor associated macrophages are therefore tumor promoter s and this is consistent with clinical data that shows an association of their density with poor prognosis (10). We have shown important interactions between these macrophages and tumor cells that stimulate their migration and invasion into the stroma to allow access into the vasculature. In addition these cells are involved in the vascularization of the tissue. Current work is aimed at elucidating the function of these macrophages at the molecular level and in developing therapies (11) against these cells to treat breast cancer patients.

Selected References

1. Tong, W., and J.W. Pollard. 1999. Progesterone inhibits estrogen-induced cyclin D1 and cdk4 nuclear translocation, cyclin E,A-cdk2 kinase activation and cell proliferation in uterine epithelial cells in mice. Mol. Cell. Biol. 19:2252-2264.

2. Chen, B., D. Zhang, and J.W. Pollard. 2003. Progesterone regulation of the mammalian ortholog of methylcitrate dehydratase (immune response gene 1) in the uterine epithelium during implantation through the protein kinase C pathway. Mol Endocrinol 17:2340-2354.

3. Pollard, J.W. 1997. Role of colony-stimulating factor-1 in reproduction and development. Molecular Reproduction and Development 46:54-61.

4. Guleria, I., and J.W. Pollard. 2000. The trophoblast is a component of the innate immune system during pregnancy. Nat Med 6:589-593.

5. Barber, E.M., and J.W. Pollard. 2003. The uterine NK cell population requires IL-15 but these cells are not required for pregnancy nor the resolution of a Listeria monocytogenes infection. J Immunol 171:37-46.

6. Cohen, P.E., L. Zhu, K. Nishimura, and J.W. Pollard. 2002. Colony-Stimulating Factor 1 Regulation of Neuroendocrine Pathways that Control Gonadal Function in Mice. Endocrinology 143:1413-1422.

7. Gouon-Evans, V., M.E. Rothenberg, and J.W. Pollard. 2000. Postnatal mammary gland development requires macrophages and eosinophils. Development 127:2269-2282.

8. Nguyen, A.V., and J.W. Pollard. 2000. Transforming growth factor beta3 induces cell death during the first stage of mammary gland involution. Development 127:3107-3118.

9. Lin, E.Y., V. Gouon-Evans, A.V. Nguyen, and J.W. Pollard. 2002. The macrophage growth factor, CSF-1, in mammary gland development and cancer. Journal of Mammary Gland Development and Neoplasia 7:147-162.

10. Pollard, J.W. 2004. Tumor educated macrophages promote tumor progression and metastasis. Nature Reveiws Cancer In Press.

11. Moadel, R.M., A.V. Nguyen, E.Y. Lin, P. Lu, J. Mani, M.D. Blaufox, J.W. Pollard, and E. Dadachova. 2003. Positron emission tomography agent 2-deoxy-2-[18F]fluoro-D-glucose has a therapeutic potential in breast cancer. Breast Cancer Res 5:R199-205.

Other recent references of interest.

12. Cohen, P.E., Pollard, J.W. 2001. Regulation of meiotic recombination and phosphase I progression in mammals. Bioessays 23:996-1009.

13. Kneitz, B., P.E. Cohen, E. Avdievich, L. Zhu, M.F. Kane, H. Hou, Jr., R.D. Kolodner, R. Kucherlapati, J.W. Pollard, and W. Edelmann. 2000. MutS homolog 4 localization to meiotic chromosomes is required for chromosome pairing during meiosis in male and female mice. Genes Dev 14:1085-1097.