In the majority of aggressive tumorigenic prostate cancer cells, the transcription factor Egr1 is overexpressed. We provide new insights of Egr1 involvement in proliferation and survival of TRAMP C2 prostate cancer cells by the identification of several new target genes controlling growth, cell cycle progression, and apoptosis such as cyclin D2, P19ink4d, and Fas. Egr1 regulation of these genes, identified by Affymetrix microarray, was confirmed by real-time PCR, immunoblot, and chromatin immunoprecipitation assays. Furthermore we also showed that Egr1 is responsible for cyclin D2 overexpression in tumorigenic DU145 human prostate cells. The regulation of these genes by Egr1 was demonstrated using Egr1 antisense oligonucleotides that further implicated Egr1 in resistance to apoptotic signals. One mechanism was illustrated by the ability of Egr1 to inhibit CD95 (Fas/Apo) expression, leading to insensitivity to FasL. The results provide a mechanistic basis for the oncogenic role of Egr1 in TRAMP C2 prostate cancer cells.

The early growth response 1 (Egr1) gene is a transcription factor that acts as both a tumor suppressor and a tumor promoter. Egr1-null mouse embryo fibroblasts bypass replicative senescence and exhibit a loss of DNA damage response and an apparent immortal growth, suggesting loss of p53 functions. Stringent expression analysis revealed 266 transcripts with >2-fold differential expression in Egr1-null mouse embryo fibroblasts, including 143 known genes. Of the 143 genes, program-assisted searching revealed 66 informative genes linked to Egr1. All 66 genes could be placed on a single regulatory network consisting of three branch points of known Egr1 target genes: TGFb1, IL6, and IGFI. Moreover, 19 additional genes that are known targets of p53 were identified, indicating that p53 is a fourth branch point. Electrophoretic mobility shift assay as well as chromatin immunoprecipitation confirmed that p53 is a direct target of Egr1. Because deficient p53 expression causes tumors in mice, we tested the role of Egr1 in a two-step skin carcinogenesis study (144 mice) that revealed a uniformly accelerated development of skin tumors in Egr1-null mice (P < 0.005). These studies reveal a new role for Egr1 as an in vivo tumor suppressor. (Cancer Res 2005; 65(12): 5133-43)

Aberrant accumulation of lipids in the liver ("fatty liver" or hepatic steatosis) represents a hallmark of the metabolic syndrome and is tightly associated with obesity, type II diabetes, starvation, or glucocorticoid (GC) therapy. While fatty liver has been connected with numerous abnormalities of liver function, the molecular mechanisms of fatty liver development remain largely enigmatic. Here we show that liver-specific disruption of glucocorticoid receptor (GR) action improves the steatotic phenotype in fatty liver mouse models and leads to the induction of transcriptional repressor hairy enhancer of split 1 (Hes1) gene expression. The GR directly interferes with Hes1 promoter activity, triggering the recruitment of histone deacetylase (HDAC) activities to the Hes1 gene. Genetic restoration of hepatic Hes1 levels in steatotic animals normalizes hepatic triglyceride (TG) levels. As glucocorticoid action is increased during starvation, myotonic dystrophy, and Cushing's syndrome, the inhibition of Hes1 through the GR might explain the fatty liver phenotype in these subjects.

Transcription factor early growth response-1 (Egr-1) is a crucial regulator of cell growth, differentiation and survival. Several observations suggest that Egr-1 is growth promoting in prostate cancer cells and that blocking its function may impede cancer progression. To test this hypothesis, we developed phosphorothioate antisense oligonucleotides that efficiently inhibit Egr-1 expression without altering the expression of other family members Egr-2, Egr-3 and Egr-4. In TRAMP mouse-derived prostate cancer cell lines, our optimal antisense oligonucleotide decreased the expression of the Egr-1 target gene transforming growth factor-b1 whereas a control oligonucleotide had no effect, indicating that the antisense blocked Egr-1 function as a transcription factor. The antisense oligonucleotide deregulated cell cycle progression and decreased proliferation of the three TRAMP cell lines by an average of 5473%. Both colony formation and growth in soft agar were inhibited by the antisense oligonucleotide. When TRAMP mice were treated systemically for 10 weeks, the incidence of palpable tumors at 32 weeks of age in untreated mice or mice injected with the control scramble oligonucleotide was 87%, whereas incidence of tumors in antisense-Egr-1-treated mice was significantly reduced to 37% (P ¼ 0.026). Thus, Egr-1 plays a functional role in the transformed phenotype and may represent a valid target for prostate cancer therapy.

Inflammatory responses represent a hallmark of numerous pathologies including sepsis, bacterial infection, insulin resistance, and malign obesity. Here we describe an unexpected coactivator function for the nuclear receptor interacting protein 140 (RIP140) for nuclear factor B (NFB), a master transcriptional regulator of inflammation in multiple tissues. Previous work has shown that IntroductionMetabolic diseases, such as insulin resistance, obesity, and atherosclerosis, have recently been recognized as low-grade, subacute inflammatory conditions, contributing to the development of type II diabetes and cardiovascular failure. 1 Similar to acute inflammation, all of these conditions are characterized by elevated levels of proinflammatory cytokines such as interleukin-1␤ (IL-1␤) and IL-6, and tumor necrosis factor ␣ (TNF␣). 2,3 Toward this end, levels of IL-6, IL-1␤, and TNF␣ are elevated in obese patients and mouse models of insulin resistance and obesity. [2][3][4] In this respect, ablation of the TNF␣ gene or of its receptor renders mice resistant to the development of insulin resistance and associated metabolic disorders. 5,6 A common polymorphism has been identified in the IL-6 receptor gene that is associated with energy intake and obesity in humans, 7 underlining the critical impact of cytokine signaling for metabolic diseases.The inflammatory response emerging in the presence of insulin resistance and obesity seems to reside predominantly in adipose tissue. 1,8 Indeed, transgenic overexpression of monocyte chemotactic protein 1 (MCP1) in adipose tissue results in enhanced macrophage infiltration, inflammation, and insulin resistance. 9,10 On the other hand, impairment of macrophage migration into adipose tissue by genetic knockout of the MCP1 receptor chemotactic cytokine receptor 2 (CCR2) has been found to substantially improve tissue inflammation and insulin sensitivity. 10,11 Importantly, macrophages have recently been shown to accumulate under obese conditions in adipose tissue of both mice and humans. 12,13 Resident macrophages in adipose tissue, therefore, seem to be in large part responsible for the cytokine release of this tissue and the systemic inflammation associated with obesity. 12,14 These studies highlighted the importance of adipose tissue as a key site for the interaction of metabolic cells with effectors of the immune system, specifically macrophages, to control systemic energy homeostasis and to trigger metabolic dysfunction under pathophysiologic conditions. 8 Consequently, not only adipose tissue quantity but also quality, as exemplified by its macrophage content and inflammatory status, seems to represent a critical determinant for the onset of insulin resistance and other components of the metabolic syndrome. 8,15 In this respect, the cytokine release from macrophages in response to external signals is largely determined by transcriptional mechanisms. A number of transcriptional regulators have been identified as critical checkpoints for the proinflammatory response of macrophages, ...

The proliferation of most primary cells in culture is limited by replicative senescence and crisis, p53-dependent events. However, the regulation of p53 itself has not been defined. We find that deletion of the early growth response 1 (EGR1) transcription factor leads to a striking phenotype, including complete bypass of senescence and apparent immortal growth consistent with loss of a suppressor gene. EGR1-null mouse embryo fibroblasts (MEFs) exhibit decreased expression of p53, p21 Cip1/Waf1 , and other p53 ''marker'' proteins. Precrisis WT but not EGR1-null cells exhibit irradiation-induced arrest. WT MEFs that emerge from crisis exhibit a mutated p53 (sequence confirmed), colony formation, and tumorigenicity. In contrast, high-passage EGR1-null MEFs retain the WT p53 sequence but with much reduced expression, remain untransformed, and grow continuously. An EGR1-expressing retrovirus restores p53 expression and sencescence to EGR1-null but not p53-null MEFs or postcrisis WT cells. Taken together, the results establish EGR1 as a major regulator of cell senescence and previously undescribed upstream ''gatekeeper'' of the p53 tumor suppressor pathway.early growth response 1 gene ͉ cancer ͉ retrovirus ͉ mouse embryo fibroblasts

Systemic bile acid (BA) homeostasis is a critical determinant of dietary fat digestion, enterohepatic function, and postprandial thermogenesis. However, major checkpoints for the dynamics and the molecular regulation of BA homeostasis remain unknown. Here we show that hypothalamic-pituitary-adrenal (HPA) axis impairment in humans and liver-specific deficiency of the glucocorticoid receptor (GR) in mice disrupts the normal changes in systemic BA distribution during the fasted-to-fed transition. Fasted mice with hepatocyte-specific GR knockdown had smaller gallbladder BA content and were more susceptible to developing cholesterol gallstones when fed a cholesterol-rich diet. Hepatic GR deficiency impaired liver BA uptake/transport via lower expression of the major hepatocyte basolateral BA transporter, Na(+)-taurocholate transport protein (Ntcp/Slc10a1), which affected dietary fat absorption and brown adipose tissue activation. Our results demonstrate a role of the HPA axis in the endocrine regulation of BA homeostasis through the liver GR control of enterohepatic BA recycling.

FOXO3 is a transcription factor involved in the regulation of multiple physiological processes including cell cycle arrest, apoptosis, oxidative stress-response and energy metabolism. Although much is known about its post-translational modification, the transcriptional regulation of FOXO3, as well as the cross-talk between transcription and post-translational events, is still poorly understood. In the present study, we show that FOXO3 is an immediate early glucocorticoid receptor (GR) target, whose transcription is even further enhanced by conditions that mimic metabolic stress. Induction of FOXO3 transcription by GR-binding steroids was reversed by concomitant treatment with the GR antagonist RU-486, but further enhanced by stimuli that activate the AMP-activated protein kinase (AMPK). Analysis of genomic DNA and chromatin immunoprecipitation, as well as luciferase reporter assays, revealed two functional glucocorticoid responsive elements within the FOXO3 promoter. Furthermore, we provide functional evidence for a phosphorylation switch that explains how glucocorticoids induce transcriptional activation of the gene but subsequently inactivate the corresponding protein by site-specific phosphorylation. Only when AMPK is stimulated, pre-existing FOXO3 becomes reverted toward an active form. Energy deprived conditions thus activate FOXO3 on two different levels, namely transcriptional and post-translational. In that way, FOXO3 acts as a metabolic stress sensor that coordinates expression of LKB1, the master upstream kinase involved in metabolic sensing, depending on the energy status of the cell. Additionally, we show that FOXO3 binds and activates its own promoter via a positive autoregulatory feedback loop. In conclusion, our data explain how catabolic glucocorticoid hormones and high intracellular AMP levels cooperate in inducing FOXO3 transcription and in activating the corresponding protein.