Heart failure represents an important cause of death in Western Countries. The pathophysiology of heart failure is mainly associated with abnormalities in intracellular calcium control. We previously showed that Egr-1 negatively regulates expression of sodium-calcium exchanger (NCX) in vivo and in vitro. Here we tested the hypothesis that Egr-1 regulates expression of calcium storage proteins in the sarco-endoplasmic reticulum (SER), calsequestrin (CSQ) and/or ER, calreticulin (CRT) directly or indirectly via Egr-1:NFAT (nuclear factor of activated T-cells) formation. Secondarily, we hypothesized that this will reduce calcium mobilization. We found that undifferentiated 1293F cells, overexpressing Egr-1, have reduced CSQ compared to control H9c2 cells. We demonstrated that Egr-1 negatively regulates CSQ but not CRT expression. The Egr-1 mediated decrease in CSQ is linked to decreased calcium availability. Repression is by a novel NAB-independent (NGFI-A binding protein) activity localized to a.a. region 1-307. We conclude that Egr-1-mediated reductions in calcium storage protein expression alter calcium availability for cardiac contraction/relaxation.

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains xv, 156 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 112-153).

All cells and organisms consume energy for survival. A robust system has evolved in vertebrates to serve as an energy reservoir. In particular, specialized cells, adipocytes, are responsible for the dynamic storage of energy by accumulating and releasing fatty acids. Fluctuating energy demands require adipose tissue to adjust in size, however complications can arise in both extremes giving rise to systemic diseases, such as obesity and diabetes mellitus (T2D). In mammals, leptin production in adipocytes is up-regulated by feeding and insulin to provide long-term post-prandial satiety. Although this regulatory connection is central to all physiological effects of leptin, the molecular mechanism remains unknown for leptin production. Here, we show that the transcription factor Egr1 is rapidly but transiently induced by insulin in adipose cells both in vitro and in vivo in a mTORC1-dependent fashion. Induction of Egr1 was immediately followed by an increase in leptin transcription. Chromatin immunoprecipitation and luciferase assays demonstrate that Egr1 directly binds to and activates the leptin promoter. Interestingly, the lipid droplet protein Fat specific protein 27 (FSP27) may work as a co-factor for Egr1 in regulating leptin expression. By using siRNA-mediated knock out of Egr1 along with its over-expression in adipocytes, we demonstrate that Egr1 is both necessary and sufficient for the stimulatory effect of insulin on leptin transcription. Knockout of the mTORC1-regulated translation repressor 4EBP1/2 increases leptin transcription both in vitro and in vivo. Adipose specific doxycycline-inducible constitutively active Rheb transgenic mouse lines contained higher circulating leptin and transcription of leptin following doxycycline treatment and were able to maintain elevated leptin levels following a 16 hour fast. Thus, insulin and nutrients, such as amino acids and glucose, activate leptin expression via the mTORC1-Egr1 regulatory axis.

No description available.

Angiopoietin-1 (Ang-1), the main ligand for the endothelial cell (EC)-selective Tie-2 receptors, promotes survival, proliferation, migration and differentiation of these cells. Despite its importance in various aspects of vascular biology, the mechanisms of action of the Ang-1/Tie-2 receptor pathway have not been fully explored. / To identify the downstream modulators of Ang-1, we evaluated changes in the transcriptome of human umbilical vein endothelial cells (HUVECs) treated with Ang-1 protein for four hours by employing the oligonucleotide rnicroarray technology. Eighty-six genes were significantly upregulated by this treatment and forty-nine genes were significantly downregulated. These genes are involved in the regulation of cell cycle, proliferation, apoptosis, transcription and differentiation. Furthermore, we found that the Erk1/2, PI3-Kinase and mTOR pathways are implicated in promoting gene expression in HUVECs in response to Ang-1. Analysis of the microarray data employing the Ingenuity Pathways analysis software to place the regulated genes in the context of biological networks revealed several highly connected nodes including the chemokine Interleukin-8 (IL-8) and the transcription factor Early growth response-1 (Egr-1). Due to the importance of these genes in promoting angiogenesis, we decided to evaluate their roles in Ang-1/Tie-2 receptor signaling and biological effects. / Ang-1 induced IL-8 expression in a time- and dose-dependent manner in ECs through both transcriptional and post-transcriptional mechanisms. To study the functional role of Ang-1-induced IL-8, we generated HUVECs that overexpress Ang-1. In these cells, neutralizing IL-8 significantly reduced EC proliferation and migration. IL-8 promoter activity experiments and gel shift assays revealed the involvement of the transcription factor AP-1 in Ang-1-induced IL-8. Ang-1 stimulated the phosphorylation of c-Jun through activation of Erk1/2, JNK and PI-3 kinase pathways. Similarly, Ang-1 provoked the expression and DNA binding of Egr-1 in HUVECs. Employing siRNA and DNAzyme to specifically knock-down Egr-1, we found that Ang-1-induced Egr-1 also promotes EC proliferation and migration. / We conclude that Ang-1 provokes a coordinated response intended to promote EC survival, proliferation, and angiogenesis and to inhibit EC apoptosis. Ang-1 induces EC proliferation and migration in part through the secretion of the soluble mediator Interleukin-8 and through induction of the transcription factor Egr-1.

No description available.

Thesis (M.S.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).