Richard I. Weiner - US grants

The major objectives of this research are to characterize dopamine, adrenergic and opiate receptors involved in neuroendocrine regulation of LH and prolactin at both the level of the anterior pituitary and median eminence. Dopamine receptors in the anterior pituitary will be solubilized and purified. Antibodies will be generaged to dopamine receptor in rabbits and by the monoclonal antibody techniques. The antibodies as well as derivatized dopaminergic antagonist linked to ferritin will be used to label dopamine receptor in the anterior pituitary and hypothalamus at the light and electron microscopic levels. Modulation, distribution, aggregation and internalization of anterior pituitary dopamine receptors will also be studied. Stable hybrid cell lines will be established which secrete prolactin and express cyclase or non-cyclase dependent dopamine receptors or beta-adrenergic recptors. Dispersed anterior pituitary cells will be maintained in long term cultures on extracellular matrix. These cultures will be used to study the role of dopamine in regulating responsiveness of mammotrophs to the suppression of prolactin release. Responsiveness will be correlated with dopamine receptor number and affinity in whol cell radioligand assays.

Development of estradiol: (E2) induced prolactinomas involves cell division of lactotrophs, morphological changes in folliculi- stellate cells (FSC) and development of a direct arterial blood supply. We hypothesize that these changes are caused by direct actions of E2 at various target sites as well as the indirect actions of E2 to inhibit hypothalamic dopaminergic (DA) regulation. We will test this hypothesis in two strains of rats, Fisher 344 rats that are extremely sensitive to the tumorogenic action of E2 and Sprague-Dawley rats that are relatively insensitive. E2 will be administered alone or in combination with the DA antagonist trifluoperazine or agonist bromoergocryptine (CB154). FSC contain large amounts of basic fibroblast growth factor (bFGF) and show dramatic morphological changes following treatment of Fisher 344 rats with E2. We will determine whether during tumor formation there is an increase in bFGF production by measuring bFGF content by RIA and mRNA by northern blot hybridization or bFGF activity by measuring the number and affinity of FGF receptors. The site of bFGF production will be determined by immunocytochemistry. We will determine if FCS also produces large amounts of the proteolytic enzymes, plasminogen activator and type IV collagenase during tumor formation. These enzymes are an important component of tissue remodeling necessary for tumor growth and possibly a mechanism for the release of bFGF sequestered in basement membrane. Thirdly, we will determine if the expression of proto-oncogenes related to key regulatory processes in the AP is increased during various stages of tumor formation. mRNA levels will be determined for int-2 and hst which have considerable homology with FGF as well as erb-B, sis, Ha-ras, N-ras, Ki-ras, fos and myc. Cellular sites of oncogene expression will be determined by immunocytochemistry. Lastly, we have recently shown that the 16K fragment of prolactin (PRL) inhibits growth of endothelial cells. We will determine if 16K PRL production and receptors change during tumor formation. We will also test whether 16K PRL can inhibit growth of E2 produced prolactinomas in Fisher 344 rats, GH3 tumors in Wistar Furth rats and 7315a tumors in female Buffalo rats. These studies will increase our understanding of the mechanisms and cellular sites of action of E2 in tumor formation. Studies with 16K PRL could lead to the development of a new class of angiolytic drugs for treatment of cancer.

Development of estradiol: (E2) induced prolactinomas involves cell division of lactotrophs, morphological changes in folliculi- stellate cells (FSC) and development of a direct arterial blood supply. We hypothesize that these changes are caused by direct actions of E2 at various target sites as well as the indirect actions of E2 to inhibit hypothalamic dopaminergic (DA) regulation. We will test this hypothesis in two strains of rats, Fisher 344 rats that are extremely sensitive to the tumorogenic action of E2 and Sprague-Dawley rats that are relatively insensitive. E2 will be administered alone or in combination with the DA antagonist trifluoperazine or agonist bromoergocryptine (CB154). FSC contain large amounts of basic fibroblast growth factor (bFGF) and show dramatic morphological changes following treatment of Fisher 344 rats with E2. We will determine whether during tumor formation there is an increase in bFGF production by measuring bFGF content by RIA and mRNA by northern blot hybridization or bFGF activity by measuring the number and affinity of FGF receptors. The site of bFGF production will be determined by immunocytochemistry. We will determine if FCS also produces large amounts of the proteolytic enzymes, plasminogen activator and type IV collagenase during tumor formation. These enzymes are an important component of tissue remodeling necessary for tumor growth and possibly a mechanism for the release of bFGF sequestered in basement membrane. Thirdly, we will determine if the expression of proto-oncogenes related to key regulatory processes in the AP is increased during various stages of tumor formation. mRNA levels will be determined for int-2 and hst which have considerable homology with FGF as well as erb-B, sis, Ha-ras, N-ras, Ki-ras, fos and myc. Cellular sites of oncogene expression will be determined by immunocytochemistry. Lastly, we have recently shown that the 16K fragment of prolactin (PRL) inhibits growth of endothelial cells. We will determine if 16K PRL production and receptors change during tumor formation. We will also test whether 16K PRL can inhibit growth of E2 produced prolactinomas in Fisher 344 rats, GH3 tumors in Wistar Furth rats and 7315a tumors in female Buffalo rats. These studies will increase our understanding of the mechanisms and cellular sites of action of E2 in tumor formation. Studies with 16K PRL could lead to the development of a new class of angiolytic drugs for treatment of cancer.

The proposed program is designed to furnish training for two graduate level predoctoral and four postdoctoral fellows per year in reproductive biology. Trainees are expected to go on to academic investigative careers in basic science or clinical departments. The training program has been operative for the past 15 years during which time 74 postdoctoral trainees (48 M.D., 8 M.D./Ph.D.,18/Ph.D.) and 22 graduate students have received training. Trainees are selected by a five member Steering Committee following formal application to the Reproductive Endocrinology Center and, in the case of predoctoral students, to the Biomedical Sciences Graduate Program. Graduate students will pursue a course of study leading to the Ph.D. degree in Biomedical Sciences. Postdoctoral training will be offered to Ph.D. candidates in related disciplines, and also to clinically trained scientists with M.D. degrees and prior specialty training in Obstetrics and Gynecology, Pediatrics or Internal Medicine. All entering fellows will be guided by a mentoring team to determine appropriate course work, and to initiate and follow research progress. M.D. fellows with no prior graduate education will spend the first year of a 3 year fellowship attending graduate level courses and performing research. All trainees will participate in the weekly Reproductive Biology Seminar Program and Research Workshop and will take the Molecular and Cellular Reproductive Biology Course. Research training will be provided by the 16 members of the Reproductive Endocrinology Center who hold faculty positions at UCSF School of Medicine and Dentistry in both basic science and clinical departments. Current research programs address important issues in reproductive biology including: sex determination, development of the reproductive tract, biology of the ovary and testes, endocrine regulation of normal and abnormal reproductive function and genetics of infertility. The Reproductive Endocrinology Center (REC) laboratories and offices consist of approximately 13,000 square feet on two floors of the Health Sciences Instruction and Research Building. In addition a primate facility, clinical research unit and transgenic barrier facility are available for studies. Histology, hormone measurement, protein separation and sequencing, and molecular biology cores also are available for training.

reproduction; radioimmunoassay; peptide hormone; biomedical facility; endocrinology; chorionic gonadotropin; cyclic GMP; thyrotropin; prolactin; follicle stimulating hormone; adrenocorticotropic hormone; transferrin; desoxycorticosterone; second messengers; steroid hormone; prostaglandin F; iodination; luteinizing hormone; somatotropin; cyclic AMP; radiotracer; chromatography; computer data analysis; enzyme linked immunosorbent assay; monoclonal antibody;

biomedical facility; genetically modified animals; animal colony; growth factor receptors; receptor expression; genetic regulatory element; early embryonic stage; laboratory mouse; gene expression; embryo /fetus culture; microinjections; hybrid cells;

DESCRIPTION (adapted from the investigator's abstract): The objective of this proposal is to elucidate the signaling pathways in GnRH neurons which constitute the clock for pulsatile GnRH release and that mediate the action of neurotransmitters. The investigator proposes to determine the relationship between intracellular and extracellular cAMP levels and GnRH secretion; to determine whether GT1 cells express cAMP gated cation channels and how they are regulated; to determine which isoforms of adenyl cyclase and phosphodiesterase are expressed in GT1 cells and how they are regulated, particularly isoforms that are regulated by PKA; and, to determine cAMP and GnRH secretion dynamics in GT1 cells stably expressing either the constitutively-active PDE-4D isoform or a dominant-negative PKA regulatory subunit. Finally, if effects are observed in the biochemical studies, the effector molecules will be targeted to GnRH neurons using transgenic approaches in order to determine their physiological significance.

DESCRIPTION: Aberrant neovascularization of the retina is a major cause of adult blindness associated with diabetic retinopathy. The 16 kDa fragment of human prolactin (16K hPRL) is a potent and specific antiangiogenic factor that inhibits neovascularization of the retina in the oxygen-induced retinopathy (OIR) model. In a variety of endothelial cell models, 16K hPRL inhibits VEGF- and bFGF-induced endothelial cell proliferation and activates apoptosis. We will determine the signaling pathways responsible for the antiangiogenic action of the 16K hPRL in the retina in vitro in bovine adrenal capillary endothelial cells (BAEC) and human retinal endothelial cells (HREC), and in vivo in the mouse OIR model. The inhibition of VEGF-induced endothelial cell proliferation by 16K hPRL is mediated via inhibition of Ras activation. We hypothesize that 16K hPRL inhibits Ras activation by stimulating the association of Ras with two Ras inhibitory proteins, Ras-GAP and Sprouty2 (Spry). In vitro we will study the protein-protein interactions between Ras-GFP and Ras-GAP-RFP and Spry2-RFP in living BAEC and HREC by measuring fluorescence resonance energy transfer (FRET) intensity in cellular compartments. Cells will be treated with VEGF and VEGF + 16K hPRL and signaling interactions measured in real-time. We hypothesize that the organization of signaling molecules occurs within calveolae. The calveolae will be identified by caveolin-1 (Cav-1-CFP) and the co-localization and FRET of signaling molecules measured in calveolae with the GFP and RFP donor/acceptor pair. Immunoblotting of proteins purified from cell compartments will be used to confirm results. We hypothesize that the 16K hPRL-induced inhibition of retinal neovascularization in the OIR model is mediated by the induction of apoptosis and inhibition of MAPK signaling. We will determine if intravitreal injection of an adenovirus (Ad) expressing 16K hPRL (16K-Ad) or a NulI-Ad into one eye activates apoptosis in areas of neovascularization measured by the TUNEL assay and a caspase-3 assay. We will utilize quantitative fluorescence microscopy to measure MAPK activation in areas of neovascularization in retinal whole mounts from the OIR model. MAPK activation will be assessed with a recently established assay that measures co-localization and FRET intensity of Ras-GFP and Raf-I-RFP. These studies will provide the basis for developing potent antiangiogenic factors for the treatment of abnormal neovascualization of the retina.