Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item DIFFERENTIAL ABILITIES OF THE CHICKEN PIT1 ISOFORMS TO REGULATE THE CHICKEN GROWTH HORMONE PROMOTER(2011) Mukherjee, Malini; Porter, Tom E; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Pit1, a pituitary-specific transcription factor, regulates differentiation of cells of the PIT1 lineage in the anterior pituitary. PIT1 also regulates the synthesis of peptide hormones from these cell types, including growth hormone (GH). A founding member of the POU-homeodomain family of transcription factors, PIT1 is characterized by a serine-threonine rich N-terminal transactivation domain and a C-terminal POU-domain. Alternative forms of PIT1, differing from each other in the N-terminal domain have been reported in several species, but the functional implication of having multiple isoforms is not known. Several Pit1 isoform mRNAs exist in chickens which have not been characterized. The main aim of this study was to determine which, if any, of the chicken PIT1 isoforms regulated the chicken Gh (cGh) promoter. PIT1β2, a novel isoform of chicken PIT1 was discovered, and known and novel isoforms (PIT1α, PIT1β1, PIT1β2 and PIT1γ) were characterized. A luciferase reporter construct containing 1775bp of the cGh promoter driving expression of firefly luciferase was used to determine the ability of the isoforms to regulate the target gene promoter activity in chicken LMH cells. We showed that three of the isoforms, PIT1α, PIT1β1 and PIT1β2, expressed from recombinant plasmids, regulated the cGh promoter, while PIT1γ did not. All the isoforms localized to the nucleus in both non-pituitary and pituitary cells. Results from gel-shift assays show that PIT1γ did not bind the proximal PIT1-binding site of the cGh promoter as well as the other isoforms, suggesting a possible mechanism behind the inactivity. Our result did not suggest a negative regulatory role for this isoform. In contrast, we found a functional advantage for having multiple isoforms. PIT1β1, the isoform that activated the promoter most strongly, when co-transfected with other activating isoforms, such as PIT1α and PIT1β2, induced significantly higher level of activation than one isoform alone. Whether this increased activation required, or was facilitated by, heterodimerization of two isoforms is not known. Nevertheless, identification of isoforms with specific functions will facilitate identification of their respective interacting partners, which are essential for GH gene expression.Item Ontogenic and glucocorticoid-regulated gene expression in the developing neuroendocrine system(2010) Ellestad, Laura Elizabeth; Porter, Tom E; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The neuroendocrine system is a critical regulator of vertebrate homeostasis that includes five hypothalamic-pituitary axes which develop during embryogenesis. Adrenal glucocorticoids play an important role in functional maturation of the anterior pituitary through initiation of growth hormone (GH) production. These studies were aimed at characterizing ontogenic and glucocorticoid-regulated changes in gene expression during neuroendocrine system development in the chick. First, to ascertain timing of initiation and establishment of each neuroendocrine axis, we measured mRNA levels of hypothalamic regulatory factors, their pituitary receptors, and pituitary hormones from embryonic day (e) 10 through post-hatch day (d) 7. We found that the adrenocorticotropic axis is the first to be established (e12), followed by establishment of the thyrotropic (e18), somatotropic (e20), lactotropic (d1), and gonadotropic (d5) axes. Next, we examined in detail mechanisms through which glucocorticoids initiate pituitary GH expression during embryogenesis. We determined that glucocorticoids elevate GH mRNA levels on e11 by increasing transcriptional activity of the GH gene rather than enhancing mRNA stability, and protein synthesis, histone deacetylase activity, ras signaling, and ERK1/2 signaling are required for this activation. Conversely, sustained activation of ERK1/2 and p38MAPK pathways reduced glucocorticoid stimulation of GH expression, indicating the requirement for ERK1/2 activity is transitory. Finally, we identified ras-dva as a novel Pit-1 and glucocorticoid-regulated gene in the chicken embryonic pituitary gland. Pituitary ras-dva mRNA levels increased between e10 and e18, decreased just prior to hatch, and remained low or undetectable post-hatch. Ras-dva expression was highly enriched within the pituitary gland on e18, and glucocorticoids rapidly induced ras-dva mRNA in cultured pituitary cells through a mechanism involving transcriptional activation. Potential regulatory elements within the 5'-flanking region of chicken ras-dva responsible for pituitary-specific expression were identified, as was a 2 kb fragment necessary for its glucocorticoid induction in embryonic pituitary cells. These results enhance our understanding of neuroendocrine system development and establishment during embryogenesis, reveal mechanisms underlying glucocorticoid initiation of GH expression in somatotrophs, and identify a new Pit-1 and glucocorticoid target gene that may play an important role in pituitary development.