Abstract GPR4 is a proton-sensing G protein-coupled receptor highly expressed in vascular endothelial cells and has been shown to potentiate intestinal inflammation in murine colitis models. Herein, we evaluated the proinflammatory role of GPR4 in the development of colitis-associated colorectal cancer (CAC) using the dextran sulfate sodium (DSS) and azoxymethane (AOM) mouse models in wild-type and GPR4 knockout mice. We found GPR4 contributed to chronic intestinal inflammation and heightened DSS/AOM-induced intestinal tumor burden. Tumor blood vessel density was markedly reduced in mice deficient in GPR4 which correlated with increased tumor necrosis and reduced tumor cell proliferation. These data demonstrate GPR4 ablation alleviates intestinal inflammation and reduces tumor angiogenesis, development, and progression in the AOM/DSS mouse model. Author summary Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease, is a debilitating condition with chronic inflammation in the digestive tract. Patients with IBD are at higher risk of developing colitis-associated colorectal cancer (CAC), compared with the general population. The etiology of IBD is not well understood, but both genetic and environmental factors have been implicated. In this study, we investigated the role of the pH-sensing GPR4 receptor in colitis and CAC using the DSS and AOM induced mouse models. GPR4 knockout alleviated intestinal inflammation, reduced tumor angiogenesis, and impeded CAC development. Our data suggest that inhibition of GPR4 may be explored as a potential therapeutic approach for IBD treatment and CAC prevention.
Common minimally invasive methods for acquiring samples for flow cytometric immunophenotyping (FCI) include fine-needle aspiration (FNA) and needle core biopsy (NCB). FCI requires a sufficient quantity of viable cells for adequate evaluation.We collected patient data from our files of all FCI cases sampled via FNA or NCB from January 1, 2003 and June 1, 2012. Total Viable Cells (TVC) was calculated by multiplying the volume, viability, and concentration and then converted to logarithmic scale as "Log TVC." Statistical analysis was performed using SPSS.Five hundred seventy-one FCI cases at our institution were reviewed covering the period from 2003 to 2012 and 456 total cases were analyzed. One hundred sixteen cases were sampled by NCB and 340 were sampled by FNA. Comparing FNA to NCB subgroups demonstrated FNA to be superior in mean specimen viability, TVC, and cases with a final FCI interpretation. The cellularity of the sample (in Log TVC) correlates with the likelihood of achieving a FCI interpretation. The point where at least 50% of cases have a diagnostic FCI interpretation occurs between Log TVC of 5.0-5.25. However, FNA based cases had a higher proportion of samples with an indeterminate final diagnosis.FNA was found to be significantly superior to NCB in obtaining material for FCI. However, NCB resulted in fewer indeterminate final diagnoses due to benefit of histologic correlation. In our opinion, NCB for histology combined with dedicated FNA material for FCI may yield the best results for a minimally invasive approach to the diagnosis of hematologic neoplasms.
Abstract The τ2-region of steroid hormone receptors is a highly conserved region located at the extreme N-terminal end of the hormone-binding domain. A protein fragment encoding τ2 has been shown to function as an independent transcriptional activation domain; however, because this region is essential for hormone binding, it has been difficult to determine whether the τ2-region also contributes to the transactivation function of intact steroid receptors. In this study a series of amino acid substitutions were engineered at conserved positions in the τ2-region of the mouse glucocorticoid receptor (mGR, amino acids 533–562) to map specific amino acid residues that contribute to the hormone-binding function, transcriptional activation, or both. Substitution of alanine or glycine for some amino acids (mutations E546G, P547A, and D555A) reduced or eliminated hormone binding, but the transactivation function of the intact GR and/or the minimum τ2-fragment was unaffected for each of these mutants. Substitution of alanine for amino acid S561 reduced transactivation activity in the intact GR and the minimum τ2-fragment but had no effect on hormone binding. The single mutation L550A and the double amino acid substitution L541G+L542G affected both hormone binding and transactivation. The fact that the S561A and L550A substitutions each caused a loss of transactivation activity in the minimum τ2-fragment and the full-length GR indicated that the τ2-region does contribute to the overall transactivation function of the full-length GR. Overall, the N-terminal portion of the τ2-region (mGR 541–547) was primarily involved in hormone binding, whereas the C-terminal portion of theτ 2-region (mGR 548–561) was primarily involved in transactivation.
In this report, we demonstrate that, in contrast to most previously characterized nuclear receptors, hERR1 and hERR2 (human estrogen receptor-related protein 1 and -2) are constitutive activators of the classic estrogen response element (ERE) as well as the palindromic thyroid hormone response element (TREpal) but not the glucocorticoid response element (GRE). This intrinsically activated state of hERR1 and hERR2 resides in the ligand-binding domains of the two genes and is transferable to a heterologous receptor. In addition, we show that members of the p160 family of nuclear receptor coactivators, ACTR (activator of thyroid and retinoic acid receptors), GRIP1 (glucocorticoid receptor interacting protein 1), and SRC-1 (steroid receptor coactivator 1), potentiate the transcriptional activity by hERR1 and hERR2 in mammalian cells, and that both orphan receptors bind the coactivators in a ligand-independent manner. Together, these results suggest that hERR1 and hERR2 activate gene transcription through a mechanism different from most of the previously characterized steroid hormone receptors.
Orphan nuclear receptors share sequence homology with members of the nuclear receptor superfamily, but ligands are unknown or unnecessary. A novel orphan receptor, estrogen receptor-related protein 3 (ERR3), was identified by yeast two-hybrid screening, using the transcriptional coactivator glucocorticoid receptor interacting protein 1 (GRIP1) as bait. The putative full-length mouse ERR3 contains 458 amino acids and is closely related to two known orphan receptors ERR1 and ERR2. All the ERR family members share an almost identical DNA-binding domain, which has 68% amino acid identity with that of estrogen receptor. ERR3 bound specifically to an estrogen response element and activated reporter genes controlled by estrogen response elements, both in yeast and in mammalian cells, in the absence of any added ligand. A conserved AF-2 activation domain located in the hormone-binding domain of ERR3 was primarily responsible for transcriptional activation. The ERR3 AF-2 domain bound GRIP1 in a ligand-independent manner both in vitro and in vivo, through the LXXLL motifs of GRIP1, and GRIP1 functioned as a transcriptional coactivator for ERR3 in both yeast and mammalian cells. Expression of ERR3 in adult mouse was restricted; highest expression was observed in heart, kidney, and brain. In the mouse embryo no expression was observed at day 7, and highest expression occurred around the 11-15 day stages. Although ERR3 is much more closely related to ERR2 than to ERR1, the expression pattern for ERR3 was similar to that of ERR1 and distinct from that for ERR2, suggesting a unique role for ERR3 in development.
1022 δ-Catenin is an armadillo domain-containing p120ctn family protein, and its distribution is restricted to the brain. However, our recent study showed that δ-catenin expression is significantly increased in a number of cancer types outside the brain. These include cancerous tissues of breast, esophagus, ovary, as well as human prostatic adenocarcinomas, although the nature of δ-catenin upregulation in prostate cancer was unclear. In this study, we systematically analyzed the δ-catenin gene (CTNND2) transcription and translation patterns in human prostate cancer cell lines and primary human prostatic adenocarcinomas. δ-Catenin showed consistent upregulation at both transcriptional and translational levels when compared to that of controls. Analyses of δ-catenin CpG islands in the promoter region in benign and prostate cancer specimens revealed no significant differences in methylation status. Real-time PCR analyses found no evidence of gene amplification in δ-catenin. However, we have observed an increased incidence of mutations in the promoter region of δ-catenin gene when compared to that of benign specimens as well as to that of peripheral blood samples of normal control subjects. At the protein level, there was no evidence of mutations in the entire coding region of δ-catenin. These studies support the hypothesis that increased gene transcription activity is an important primary cause of δ-catenin upregulation in human prostate cancer. This study was supported in part by NIH CA111891 and Department of Defense W81XWH-05-1-0028.
Members of the 160-kDa nuclear receptor coactivator family (p160 coactivators) bind to the conserved AF-2 activation function found in the hormone binding domains of nuclear receptors (NR) and are potent transcriptional coactivators for NRs. Here we report that the C-terminal region of p160 coactivators glucocorticoid receptor interacting protein 1 (GRIP1), steroid receptor coactivator 1 (SRC-1a), and SRC-1e binds the N-terminal AF-1 activation function of the androgen receptor (AR), and p160 coactivators can thereby enhance transcriptional activation by AR. While they all interact efficiently with AR AF-1, these same coactivators have vastly different binding strengths with and coactivator effects on AR AF-2. p160 activation domain AD1, which binds secondary coactivators CREB binding protein (CBP) and p300, was previously implicated as the principal domain for transmitting the activating signal to the transcription machinery. We identified a new highly conserved motif in the AD1 region which is important for CBP/p300 binding. Deletion of AD1 only partially reduced p160 coactivator function, due to signaling through AD2, another activation domain located at the C-terminal end of p160 coactivators. C-terminal coactivator fragments lacking AD1 but containing AD2 and the AR AF-1 binding site served as efficient coactivators for full-length AR and AR AF-1. The two signal input domains (one that binds NR AF-2 domains and one that binds AF-1 domains of some but not all NRs) and the two signal output domains (AD1 and AD2) of p160 coactivators played different relative roles for two different NRs: AR and thyroid hormone receptor.
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