Michael L. Wilson, Ph.D. - US grants

The general objectives of this proposal are 1. to biochemically characterize the types of protein kinase and phosphoproteins in human prostatic fluid and 2. to identify and age- or disease-related changes in these enzymes and phosphoproteins. Preliminary experiments indicate that the pattern of phosvitin and histone protein kinase activities in prostatic fluid change with prostate cancer. Therefore, other types of protein kinase (e.g., Ca++ dependent, tyrosine-specific, etc.) which may be in prostatic fluid may be susceptible to change. The presence of other types of protein kinase will be examined and characterized by use of specific substrates. activators, and inhibitors. Since cancer of the prostate appears to reflect specific alterations in the pattern of activities of different types of protein kinase, the activities of individual isoenzyme forms of these kinases may be responsible for these overall changes. If specific isoenzymes are responsive to change due to cancer, isoenzyme measurements would make the use of protein kinase activities more sensitive to detect the pathological change. The study of protein kinase isoenzymes will utilize electrophoretic separation methods in combination with the assay of individual types of protein kinase activities right in the electrophoretic gel. Other separation techniques will be explored to determine whether a simple preliminary step(s) befire electrophoresis and kinase assay might increase the sensitivity of measurement of some forms of protein kinase. Some examples are: separation of soluble and membrane fractions by ultracentrifugation, affinity chromatography, ion-exchange chromatography, etc. Prostatic fluid samples from men with chronic prostatitis, benign prostatic hyperplasia, prostatic cancer, or no known prostate disease will be studied. Comparisons of the findings among these groups will establish the relationship of various changes in protein kinase activities to particular prostatic diseases, and determine the specificity of such changes to prostatic cancer. In addition, since protein phosphorylation is an improtant mechanism for regulating biological functions, these basic studies should provide important information on protein kinases themselves, and prostatic secretory function, the prostatic acinar cell milieu, and male reproductive function.

DESCRIPTION (adapted from the application) This R-21 application is submitted in response to the RFA (DK00-015), "Cell Specific Delineation of Prostate and Genitourinary Development." Although it is known that androgen influence on gene expression of cells in the mesenchyme are critical for differentiation of the prostatic epithelium, the molecular processes and cell interactions that lead to the tissue organization and the full cellular complexity of the mature gland are poorly understood. Our long-term goal is to understand the role of proteases in regulating development, function, and pathogenesis of diseases of the prostate gland. The objective of this application is to study cell surface proteolytic enzymes critical to prostate development because of their controlling roles in cell growth and cell-cell and cell-extracellular matrix interactions. The central hypothesis of this proposal is that there is a temporal dependent and cell type specific expression of metalloproteases of the matrix metalloprotease (MMP) and metalloprotease disintegrin (ADAM) families, and of other kinds of protease, controlling individual steps during prostate development. The rationale for this proposed research is that once we know the time in development of a cell type expressing a controlling protease, we can determine the regulation of the protease expression in normal. prostate development and potentially in neoplastic growth. In addition, these cell surface proteases can be tagged by antibodies or specific inhibitors for characterization and purification of individual cell types. The central hypothesis will be tested and objectives of the proposal accomplished by pursuing two specific aims: 1) determine the stage of prostate-development for expression of cell surface proteases, and 2) determine the cell types expressing individual cell surface proteases at specific stages of prostate development. The proposed work addresses several needs detailed in the RFA. It systematically assesses gene expression in specific cell types in the developing and adult mouse prostate. These are protease genes that are expected to be important in prostate development because they function at the cell surface in growth factor processing and modification of plasma membrane and extracellular matrix proteins that are critical in cell growth and cell-cell and cell-extracellular matrix interactions. Such outcomes will be significant because it is expected that this new knowledge will suggest novel protease targets important in prostatic neoplasia and the cell surface protease about can be used to develop methods (antibodies and/or immobilized inhibitors) to isolate the identified cell types for culture and development of cell lines.

Studies of human reproductive ecology have provided key insights into how evolution has shaped reproductive mechanisms (e.g., ovulation, gestation and lactation) to respond appropriately to environmental circumstances. For example, physiological mechanisms reduce ovulatory function in women already nursing or with low energy stores. Lactation, however, is largely unaffected by low energy stores as this process is given metabolic priority when maternal energy is partitioned. Though most such studies have focused on nutritionally stressed populations, recent research has demonstrated that overweight women face a higher rate of lactational dysfunction. Compared to normal weight women, overweight women establish lactation later postpartum and if established, stop breastfeeding sooner. This result is surprising from a reproductive ecology perspective because overweight women have ample energy stores needed for lactation and because of the metabolic priority generally afforded to lactation. Although studies consistently report an association between maternal obesity and late onset to and short duration of lactation, the causes of this relationship remain unknown. This project will focus on the central question: Why are overweight women at risk for lactational dysfunction? Because the process of establishing and maintaining milk production depends upon a finely tuned set of physiological, behavioral and social conditions, this research will utilize a bio-cultural approach. Pregnant women from Minneapolis-St. Paul will be recruited to participate in a matched-pair case-control study. Data will be collected using three methods: First, a prenatal survey will investigate how psychosocial and demographic factors influence breastfeeding to test the relationship between maternal obesity and these factors. Second, nursing episodes will be observed and participants will record attempts to breastfeed as well as factors influencing their decision to wean, in order to test the association of behavioral patterns and maternal obesity. Finally, serum samples will be drawn to test hormonal differences between obese and non-obese women. Statistical analyses will identify the relative contribution of the effects of maternal obesity, hormone concentrations, behavioral patterns and psychosocial factors on breastfeeding outcomes.

Breastfeeding provides numerous benefits for infants and their mothers. As obesity rates continue to rise globally, it becomes increasingly important to uncover the reasons why this cohort of women is at risk for lactational dysfunction. This project will lay the groundwork for knowledge that can be used by public health institutions to disseminate information on the special problems overweight women face when attempting to breastfeed. Elucidating the causes of lactational dysfunction in this population will help to explain lactational dysfunction in other environmental and cultural contexts and will make it easier to predict the proximate mechanisms leading to poor lactation performance. Finally, this project will advance knowledge regarding how modern human females manage trade-offs relating to reproductive strategies. It will address the puzzle of how a surplus of (stored) metabolic energy can interfere with the performance of an energetically demanding task essential for reproduction. In doing so, it will contribute to the understanding of how physiological pathways supporting milk production relate to pathways of fat storage and metabolism.

DESCRIPTION (provided by applicant): Tissue invasion and metastasis, one of the six purported capabilities acquired by human cancers, accounts for more than 90% of cancer deaths and represents an understudied but promising area for future therapeutic developments. The long-term goal of our program is to understand how malignant tumor cells acquire the invasive and metastatic phenotype. In the next five years, we plan to focus on the microenvironment of tumor cell surface and test the hypothesis that cell surface proteolysis regulates the invasive and metastatic properties of malignant tumors. Current evidence suggest that proteinases contribute to tumor invasion and metastasis by not only degrading the extracellular matrix as a barrier, but also functioning to regulate pathways controlling cell growth, migration and apoptosis through releasing latent growth factors or cleaving various receptors and their ligands for both activation and inactivation. Yet, efforts targeting tumor proteinases especially the MMPs have not achieved any clinical success so far. Several outstanding reviews have recently been published to address this apparent gap between "scientific success and clinical failure" for the MMP field. We would like to argue that one neglected area is proteolvsis on tumor cell surface. Our evidence both in vitro and in vivo suggests that the same proteinase behaves differently when it is tethered on cell surface or secreted. We hypothesize that the membrane-bound MMPs are more efficient for proteolysis and harder to inhibit than soluble ones, thus, enabling tumor invasion and metastasis. To test this idea, we designed three specific aims: 1) Characterize the invasive and metastatic phenotype conferred by MT1-MMP expressed on tumor cell surface both in vitro and in vivo;2) Determine the contributions of the hemopexin- and catalytic- domains of MT1-MMP towards the invasive and metastatic phenotype;and 3) Characterize the microenvironment on tumor cell surface that enables MT1-MMP to mediate invasion and metastasis. Accomplishment of these aims may empower the design of a new generation of MMP inhibitors targeting the tumor cell surface and provide model systems to test the efficacies of these potential therapeutics.

Previous studies have found that chimpanzees often give food-associated vocalizations when they find food, and that these vocalizations may provide other chimpanzees with information about the amount and quality of the food that has been discovered. This study will use a combination of experiments with captive chimpanzees and observations of wild chimpanzees to solve a long-standing puzzle:
given that chimpanzees often compete over food, why should they provide other chimpanzees with such information about food that they have found? This study will test the central hypothesis that chimpanzees benefit from giving these calls, not by sharing food, but by coordinating their travel with valued group members. Experiments with captive chimpanzees will test whether chimpanzees that hear recordings of food-associated vocalizations from other chimpanzees increase their own foraging effort. Observations of wild chimpanzees are expected to show that chimpanzees call more frequently when with valued members of their group, such as mates and allies, and that when chimpanzees hear such calls, they spend more time searching for food in a given spot. This research will result in a greater understanding of the role food-associated communication play in the social foraging decisions of chimpanzees. By approaching the puzzle of food-associated call production from a social decision-making perspective, these studies will transform our understanding of this calling behavior while simultaneously providing novel insight into the role communication plays in social decision-making processes.