Glucocorticoid-induced changes in gene expression in embryonic anterior pituitary cells.

Within the anterior pituitary gland, glucocorticoids such as corticosterone (CORT) provide negative feedback to inhibit adrenocorticotropic hormone secretion and act to regulate production of other hormones including growth hormone (GH). The ontogeny of GH production during chicken embryonic and rat fetal development is controlled by glucocorticoids. The present study was conducted to characterize effects of glucocorticoids on gene expression within embryonic pituitary cells and to identify genes that are rapidly and directly regulated by glucocorticoids. Chicken embryonic pituitary cells were cultured with CORT for 1.5, 3, 6, 12, and 24 h in the absence and presence of cycloheximide (CHX) to inhibit protein synthesis. RNA was analyzed with custom microarrays containing 14,053 chicken cDNAs, and results for selected genes were confirmed by quantitative reverse transcription real-time PCR (qRT-PCR). Levels of GH mRNA were maximally induced by 6 h of CORT treatment, and this response was blocked by CHX. Expression of 396 genes was affected by CORT, and of these, mRNA levels for 46 genes were induced or repressed within 6 h. Pathway analysis of genes regulated by CORT in the absence of CHX revealed networks of genes associated with endocrine system development and cellular development. Eleven genes that were induced within 6 h in the absence and presence of CHX were identified, and eight were confirmed by qRT-PCR. The expression profiles and canonical pathways defined in this study will be useful for future analyses of glucocorticoid action and regulation of pituitary function.

Engaging Ly-6A/Sca-1 triggers lipid raft-dependent and -independent responses in CD4+ T-cell lines.

INTRODUCTION: The lymphocyte antigen 6 (Ly-6) supergene family encodes proteins of 12-14 kda in molecular mass that are either secreted or anchored to the plasma membrane through a glycosyl-phosphatidylinisotol (GPI) lipid anchor at the carboxy-terminus. The lipidated GPI-anchor allows localization of Ly-6 proteins to the 10-100 nm cholesterol-rich nano-domains on the membrane, also known as lipid rafts. Ly-6A/Sca-1, a member of Ly-6 gene family is known to transduce signals despite the absence of transmembrane and cytoplasmic domains. It is hypothesized that the localization of Ly-6A/Sca-1 with in lipid rafts allows this protein to transduce signals to the cell interior. METHODS AND RESULTS: In this study, we found that cross-linking mouse Ly-6A/Sca-1 protein with a monoclonal antibody results in functionally distinct responses that occur simultaneously. Ly-6A/Sca-1 triggered a cell stimulatory response as gauged by cytokine production with a concurrent inhibitory response as indicated by growth inhibition and apoptosis. While production of interleukin 2 (IL-2) cytokine by CD4+ T cell line in response to cross-linking Ly-6A/Sca-1 was dependent on the integrity of lipid rafts, the observed cell death occurred independently of it. Growth inhibited CD4+ T cells showed up-regulated expression of the inhibitory cell cycle protein p27kip but not of p53. In addition, Ly-6A/Sca-1 induced translocation of cytochrome C to the cytoplasm along with activated caspase 3 and caspase 9, thereby suggesting an intrinsic apoptotic cell death mechanism. CONCLUSIONS: We conclude that opposing responses with differential dependence on the integrity of lipid rafts are triggered by engaging Ly-6A/Sca-1 protein on the membrane of transformed CD4+ T cells.

Gene expression profiling during cellular differentiation in the embryonic pituitary gland using cDNA microarrays.

The anterior pituitary is comprised of five major hormone-secreting cell types that differentiate during embryonic development in a temporally distinct manner. Microarrays containing 5,128 unique cDNAs expressed in the chicken neuroendocrine system were produced and used to identify genes with potential involvement in the onset of thyroid-stimulating hormone beta-subunit (TSHbeta), growth hormone (GH), and prolactin (PRL) mRNA during embryonic development. We identified 352 cDNAs that were differentially expressed (P < or = 0.05) on embryonic day 10 (e10), e12, e14, or e17, the period of thyrotroph, somatotroph, and lactotroph differentiation. Self-organizing maps were used to identify genes that may function to initiate hormone gene transcription. Consistent with cellular ontogeny, TSHbeta mRNA increased steadily between e10 and e17, GH mRNA increased between e12 and e17, and PRL mRNA did not increase until e17. Expression of 141 genes increased in a manner similar to TSHbeta mRNA, and 64 genes decreased between e10 and e17. Although genes with these expression profiles are likely involved in development of the pituitary gland as a whole, some of these could be specifically associated with thyrotroph differentiation. Similarly, the expression profiles of 69 and 61 genes indicate a potential involvement in the induction of GH and PRL mRNA, respectively. Quantitative real-time RT-PCR was used to confirm microarray results for 31 genes. This is the first study to evaluate changes in anterior pituitary gene expression during embryonic development of any species using microarrays, and numerous transcription factors and signaling molecules not previously implicated in pituitary development were identified.