Nelson D. Horseman - US grants

Prolactin (PRL) and related hormones play major roles in parental care of offspring and the regulation of metabolism. In mammals this includes regulation of pregnancy and lactation. Prolactin not only controls the lactogenic differentiation of mammary gland, but also affects a variety of other target tissues. Many of those effects are important to support the growth, differentiation, and nutrition associated with pregnancy and nurturing. The general goal of this research program is understanding the molecular physiology of PRL action by studying the stimulation of calpactin expression in pigeon cropsac. This regulatory effect is a productive model system in which to examine simultaneously both the molecular mechanisms of PRL-induced gene expression and the function of genes which encode Ca2+- dependent membrane-binding proteins that are major cellular substrates of oncogene and growth-factor receptor tyrosine kinases. The PRL-regulated gene which we will be examining (calpactin) is inherently interesting as a component of basic cellular biology, as well as being a valuable endpoint from which to study PRL mechanisms. This is one positive aspect which distinguishes this system from others regulated by PRL (i.e. milk protein genes) which do not play any fundamental or general roles in cellular physiology. The aims of these experiments are focussed on three general topics. First a comprehensive analysis of the PRL-induced calpactin gene which we have cloned. These experiments will determine the unique structures which are associated with induction of this form of the calpactin genes. Hypotheses to be tested will include that the PRL-induced calpactin gene is spliced in a specific manner or that it is expressed from an alternative promoter site. Related mRNAs which are not PRL-regulated will be compared to determine special features of this gene's expression. Second, recombinations of segments of a human calpactin which are sites of regulation by tyrosine kinases with equivalent segments of the PRL-induced calpactin which is to tyrosine kinase-regulated will be made and tested. These experiments will test the hypothesis that productive phosphorylative regulation is determined by specific amino acid sequences downstream from the phosphorylation site in human calpactin. Thirdly, immunolocalization of calpactin protein and neutralization of calpactin mRNA expression with antisense oligonucleotides will be used to examine the function of this protein in PRL-induced proliferation and differentiation.

[unreadable] DESCRIPTION (provided by applicant): Using a combination of approaches, including gene-disrupted mice, expression profiling, and pharmacology in vitro and in vivo, we recently made the surprising discovery that the tryptophan hydroxylase (TPH) gene is an important regulator of mammary gland function. TPH catalyzes the conversion of L-tryptophan to 5-hydroxytryptophan, which is the rate-limiting substrate for serotonin (5-HT) synthesis. We showed that TPH is induced by milk stasis, and that 5-HT participates in an autocrine/paracrine feedback loop that inhibits lactation. These discoveries imply that biogenic monoamines (esp. 5-HT) are important mammary-derived signaling molecules. [unreadable] [unreadable] The central objective of our plan is to explore critical details of the mechanisms by which 5-HT acts within the mammary gland (aims 1-3). A subsidiary objective (aim 4) is to determine whether 5-HT is exported from the mammary gland into the maternal circulation and/or milk. [unreadable] [unreadable] The situation we now confront is that we know neither what specific aspects of mammary physiology and development are influenced by 5-HT, nor do we know the pharmacological profile (receptor types) for the 5-HT regulated functions in the mammary glands. To fill these knowledge gaps we will make use of the wealth of available serotonergic agents to examine mammary gene regulation, proliferation, and apoptosis in organotypic cultures and primary cultures of dissociated cells. [unreadable] [unreadable] The means to do in vivo studies of mammary TPH functions are very limited because of the confounding effects that pharmacological agents have on tissues other than the mammary gland (esp. the brain). To circumvent these problems we need genetic models in which to do the physiological and developmental studies, but useful models, such as TPH gene knockouts, have not been made by other labs. Consequently, we propose to make mice in which the TPH gene is disrupted in the mammary glands, and transgenic mice expressing the human serotonin transporter (hSERT) in the mammary glands. These models will allow us to examine the role of mammary-expressed TPH in development and physiology in animals that have interruptions in their autocrine/paracrine 5-HT exposure.