IL-13-induced STAT3-dependent signaling networks regulate esophageal epithelial proliferation in eosinophilic esophagitis.

BACKGROUND: Basal zone hyperplasia (BZH) and dilated intercellular spaces (DISs) are thought to contribute to the clinical manifestations of eosinophilic esophagitis (EoE); however, the molecular pathways that drive BZH remain largely unexplored. OBJECTIVE: We sought to define the role of IL-13-induced transcriptional programs in esophageal epithelial proliferation in EoE. METHODS: We performed RNA sequencing, bioinformatics, Western blot, reverse transcriptase quantitative PCR, and histologic analyses on esophageal biopsies from healthy control and patients with EoE, primary esophageal cells derived from patients with EoE, and IL-13-stimulated esophageal epithelial keratinocytes grown at the air-liquid interface (EPC2-ALI). Genetic (shRNA) and pharmacologic (proteolysis-targeting chimera degrader) approaches and in vivo model of IL-13-induced esophageal epithelial remodeling (Krt5-rtTA x tetO-IL-13Tg) were used to define the role of signal transducer and activator of transcription 3 (STAT3) and STAT6 and secreted frizzled-related protein 1 (SFRP1) in esophageal epithelial proliferation. RESULTS: RNA-sequencing analysis of esophageal biopsies (healthy control vs EoE) and EPC2-ALI revealed 82 common differentially expressed genes that were enriched for putative STAT3 target genes. In vitro and in vivo analyses revealed a link between IL-13-induced STAT3 and STAT6 phosphorylation, SFRP1 mRNA expression, and esophageal epithelial proliferation. In vitro studies showed that IL-13-induced esophageal epithelial proliferation was STAT3-dependent and regulated by the STAT3 target SFRP1. SFRP1 mRNA is increased in esophageal biopsies from patients with active EoE compared with healthy controls or patients in remission and identifies an esophageal suprabasal epithelial cell subpopulation that uniquely expressed the core EoE proinflammatory transcriptome genes (CCL26, ALOX15, CAPN14, ANO1, and TNFAIP6). CONCLUSIONS: These studies identify SFRP1 as a key regulator of IL-13-induced and STAT3-dependent esophageal proliferation and BZH in EoE and link SFRP1+ esophageal epithelial cells with the proinflammatory and epithelial remodeling response in EoE.

Eosinophilic esophagitis: Immune mechanisms and therapeutic targets.

Eosinophilic esophagitis (EoE) is an emerging chronic inflammatory disease of the oesophagus and is clinically characterized by upper gastrointestinal (GI) symptoms including dysphagia and esophageal food impaction. Histopathologic manifestations, which include intraepithelial eosinophilic inflammation and alterations of the esophageal squamous epithelium, such as basal zone hyperplasia (BZH) and dilated intercellular spaces (DIS), are thought to contribute to esophageal dysfunction and disease symptoms. Corroborative clinical and discovery science-based studies have established that EoE is characterized by an underlying allergic inflammatory response, in part, related to the IL-13/CCL26/eosinophil axis driving dysregulation of several key epithelial barrier and proliferative regulatory genes including kallikrein (KLK) serine proteases, calpain 14 (CAPN14) and anoctamin 1 (ANO1). The contribution of these inflammatory and proliferative processes to the clinical and histological manifestations of disease are not fully elucidated. Herein, we discuss the immune molecules and cells that are thought to underlie the clinical and pathologic manifestations of EoE and the emerging therapeutics targeting these processes for the treatment of EoE.

Bilirubin Prevents Atherosclerotic Lesion Formation in Low-Density Lipoprotein Receptor-Deficient Mice by Inhibiting Endothelial VCAM-1 and ICAM-1 Signaling.

BACKGROUND: Numerous epidemiological studies support an inverse association between serum bilirubin levels and the incidence of cardiovascular disease; however, the mechanism(s) by which bilirubin may protect against atherosclerosis is undefined. The goals of the present investigations were to assess the ability of bilirubin to prevent atherosclerotic plaque formation in low-density lipoprotein receptor-deficient (Ldlr-/- ) mice and elucidate the molecular processes underlying this effect. METHODS AND RESULTS: Bilirubin, at physiological concentrations (≤20 µmol/L), dose-dependently inhibits THP-1 monocyte migration across tumor necrosis factor α-activated human umbilical vein endothelial cell monolayers without altering leukocyte binding or cytokine production. A potent antioxidant, bilirubin effectively blocks the generation of cellular reactive oxygen species induced by the cross-linking of endothelial vascular cell adhesion molecule 1 (VCAM-1) or intercellular adhesion molecule 1 (ICAM-1). These findings were validated by treating cells with blocking antibodies or with specific inhibitors of VCAM-1 and ICAM-1 signaling. When administered to Ldlr-/- mice on a Western diet, bilirubin (30 mg/kg intraperitoneally) prevents atherosclerotic plaque formation, but does not alter circulating cholesterol or chemokine levels. Aortic roots from bilirubin-treated animals exhibit reduced lipid and collagen deposition, decreased infiltration of monocytes and lymphocytes, fewer smooth muscle cells, and diminished levels of chlorotyrosine and nitrotyrosine, without changes in VCAM-1 or ICAM-1 expression. CONCLUSIONS: Bilirubin suppresses atherosclerotic plaque formation in Ldlr-/- mice by disrupting endothelial VCAM-1- and ICAM-1-mediated leukocyte migration through the scavenging of reactive oxygen species signaling intermediaries. These findings suggest a potential mechanism for the apparent cardioprotective effects of bilirubin.

Bilirubin prevents acute DSS-induced colitis by inhibiting leukocyte infiltration and suppressing upregulation of inducible nitric oxide synthase.

Bilirubin is thought to exert anti-inflammatory effects by inhibiting vascular cell adhesion molecule-1 (VCAM-1)-dependent leukocyte migration and by suppressing the expression of inducible nitric oxide synthase (iNOS). As VCAM-1 and iNOS are important mediators of tissue injury in the dextran sodium sulfate (DSS) murine model of inflammatory colitis, we examined whether bilirubin prevents colonic injury in DSS-treated mice. Male C57BL/6 mice were administered 2.5% DSS in the drinking water for 7 days, while simultaneously receiving intraperitoneal injections of bilirubin (30 mg/kg) or potassium phosphate vehicle. Disease activity was monitored, peripheral blood counts and serum nitrate levels were determined, and intestinal specimens were analyzed for histological injury, leukocyte infiltration, and iNOS expression. The effect of bilirubin on IL-5 production by HSB-2 cells and on Jurkat cell transendothelial migration also was determined. DSS-treated mice that simultaneously received bilirubin lost less body weight, had lower serum nitrate levels, and exhibited reduced disease severity than vehicle-treated animals. Concordantly, histopathological analyses revealed that bilirubin-treated mice manifested significantly less colonic injury, including reduced infiltration of eosinophils, lymphocytes, and monocytes, and diminished iNOS expression. Bilirubin administration also was associated with decreased eosinophil and monocyte infiltration into the small intestine, with a corresponding increase in peripheral blood eosinophilia. Bilirubin prevented Jurkat migration but did not alter IL-5 production. In conclusion, bilirubin prevents DSS-induced colitis by inhibiting the migration of leukocytes across the vascular endothelium and by suppressing iNOS expression.

Bilirubin inhibits the up-regulation of inducible nitric oxide synthase by scavenging reactive oxygen species generated by the toll-like receptor 4-dependent activation of NADPH oxidase.

It has been previously shown that bilirubin prevents the up-regulation of inducible nitric oxide synthase (iNOS) in response to LPS. The present study examines whether this effect is exerted through modulation of Toll-Like Receptor-4 (TLR4) signaling. LPS-stimulated iNOS and NADPH oxidase (Nox) activity in RAW 264.7 murine macrophages was assessed by measuring cellular nitrate and superoxide ( [Formula: see text] ) production, respectively. The generation of both nitrate and [Formula: see text] in response to LPS was suppressed by TLR4 inhibitors, indicating that activation of iNOS and Nox is TLR4-dependent. While treatment with superoxide dismutase (SOD) and bilirubin effectively abolished LPS-mediated [Formula: see text] production, hydrogen peroxide and nitrate release were inhibited by bilirubin and PEG-catalase, but not SOD, supporting that iNOS activation is primarily dependent upon intracellular H2O2. LPS treatment increased nuclear translocation of the redox-sensitive transcription factor Hypoxia Inducible Factor-1α (HIF-1α), an effect that was abolished by bilirubin. Cells transfected with murine iNOS reporter constructs in which the HIF-1α-specific hypoxia response element was disrupted exhibited a blunted response to LPS, supporting that HIF-1α mediates Nox-dependent iNOS expression. Bilirubin, but not SOD, blocked the cellular production of interferon-ß, while interleukin-6 production remained unaffected. These data support that bilirubin inhibits the TLR4-mediated up-regulation of iNOS by preventing activation of HIF-1α through scavenging of Nox-derived reactive oxygen species. Bilirubin also suppresses interferon-ß release via a ROS-independent mechanism. These findings characterize potential mechanisms for the anti-inflammatory effects of bilirubin.

Dopamine receptors in human adipocytes: expression and functions.

INTRODUCTION: Dopamine (DA) binds to five receptors (DAR), classified by their ability to increase (D1R-like) or decrease (D2R-like) cAMP. In humans, most DA circulates as dopamine sulfate (DA-S), which can be de-conjugated to bioactive DA by arylsulfatase A (ARSA). The objective was to examine expression of DAR and ARSA in human adipose tissue and determine whether DA regulates prolactin (PRL) and adipokine expression and release. METHODS: DAR were analyzed by RT-PCR and Western blotting in explants, primary adipocytes and two human adipocyte cell lines, LS14 and SW872. ARSA expression and activity were determined by qPCR and enzymatic assay. PRL expression and release were determined by luciferase reporter and Nb2 bioassay. Analysis of cAMP, cGMP, leptin, adiponectin and interleukin 6 (IL-6) was done by ELISA. Activation of MAPK and PI3 kinase/Akt was determined by Western blotting. RESULTS: DAR are variably expressed at the mRNA and protein levels in adipose tissue and adipocytes during adipogenesis. ARSA activity in adipocyte increases after differentiation. DA at nM concentrations suppresses cAMP, stimulates cGMP, and activates MAPK in adipocytes. Acting via D2R-like receptors, DA and DA-S inhibit PRL gene expression and release. Acting via D1R/D5R receptors, DA suppresses leptin and stimulates adiponectin and IL-6 release. CONCLUSIONS: This is the first report that human adipocytes express functional DAR and ARSA, suggesting a regulatory role for peripheral DA in adipose functions. We speculate that the propensity of some DAR-activating antipsychotics to increase weight and alter metabolic homeostasis is due, in part, to their direct action on adipose tissue.

Lactogens and estrogens in breast cancer chemoresistance.

Tumor resistance to chemotherapy in advanced breast cancer is a major impediment to treatment success. Resistance can be induced by the drugs themselves or result from the action of internal factors. The role of hormones in chemoresistance has received little attention. This article focuses on two classes of hormones: lactogens and estrogens. Lactogens include prolactin, growth hormone and placental lactogen, all of which can activate the prolactin receptor. Estrogens include endogenous steroids and nonsteroidal compounds from the environment termed endocrine disruptors, all of which can activate 'classical' estrogen receptors (ERα and ERß), as well as other types of receptors. Both lactogens and estrogens antagonize cytotoxicity of multiple chemotherapeutic agents through complementary mechanisms. The implications of chemoresistance by these hormones to patients with breast cancer, and the potential benefits of developing combinatorial anti-lactogen/anti-estrogen treatment regimens, are discussed.

Histone deacetylase 9 is a negative regulator of adipogenic differentiation.

Differentiation of preadipocytes into mature adipocytes capable of efficiently storing lipids is an important regulatory mechanism in obesity. Here, we examined the involvement of histone deacetylases (HDACs) and histone acetyltransferases (HATs) in the regulation of adipogenesis. We find that among the various members of the HDAC and HAT families, only HDAC9 exhibited dramatic down-regulation preceding adipogenic differentiation. Preadipocytes from HDAC9 gene knock-out mice exhibited accelerated adipogenic differentiation, whereas HDAC9 overexpression in 3T3-L1 preadipocytes suppressed adipogenic differentiation, demonstrating its direct role as a negative regulator of adipogenesis. HDAC9 expression was higher in visceral as compared with subcutaneous preadipocytes, negatively correlating with their potential to undergo adipogenic differentiation in vitro. HDAC9 localized in the nucleus, and its negative regulation of adipogenesis segregates with the N-terminal nuclear targeting domain, whereas the C-terminal deacetylase domain is dispensable for this function. HDAC9 co-precipitates with USF1 and is recruited with USF1 at the E-box region of the C/EBPα gene promoter in preadipocytes. Upon induction of adipogenic differentiation, HDAC9 is down-regulated, leading to its dissociation from the USF1 complex, whereas p300 HAT is up-regulated to allow its association with USF1 and accumulation at the E-box site of the C/EBPα promoter in differentiated adipocytes. This reciprocal regulation of HDAC9 and p300 HAT in the USF1 complex is associated with increased C/EBPα expression, a master regulator of adipogenic differentiation. These findings provide new insights into mechanisms of adipogenic differentiation and document a critical regulatory role for HDAC9 in adipogenic differentiation through a deacetylase-independent mechanism.

Dynamic bookmarking of primary response genes by p300 and RNA polymerase II complexes.

Profiling the dynamic interaction of p300 with proximal promoters of human T cells identified a class of genes that rapidly coassemble p300 and RNA polymerase II (pol II) following mitogen stimulation. Several of these p300 targets are immediate early genes, including FOS, implicating a prominent role for p300 in the control of primary genetic responses. The recruitment of p300 and pol II rapidly transitions to the assembly of several elongation factors, including the positive transcriptional elongation factor (P-TEFb), the bromodomain-containing protein (BRD4), and the elongin-like eleven nineteen lysine-rich leukemia protein (ELL). However, transcription at many of these rapidly induced genes is transient, wherein swift departure of P-TEFb, BRD4, and ELL coincides with termination of transcriptional elongation. Unexpectedly, both p300 and pol II remain accumulated or "bookmarked" at the proximal promoter long after transcription has terminated, demarking a clear mechanistic separation between the recruitment and elongation phases of transcription in vivo. The bookmarked pol II is depleted of both serine-2 and serine-5 phosphorylation of its C-terminal domain and remains proximally positioned at the promoter for hours. Surprisingly, these p300/pol II bookmarked genes can be readily reactivated, and elongation factors can be reassembled by subsequent addition of nonmitogenic agents that, alone, have minimal effects on transcription in the absence of prior preconditioning by mitogen stimulation. These findings suggest that p300 is likely to play an important role in biological processes in which transcriptional bookmarking or preconditioning influences cellular growth and development through the dynamic priming of genes for response to rechallenge by secondary stimuli.

Proinflammatory phenotype of perivascular adipocytes: influence of high-fat feeding.

Adipose tissue depots originate from distinct precursor cells, are functionally diverse, and modulate disease processes in a depot-specific manner. However, the functional properties of perivascular adipocytes, and their influence on disease of the blood vessel wall, remain to be determined. We show that human coronary perivascular adipocytes exhibit a reduced state of adipocytic differentiation as compared with adipocytes derived from subcutaneous and visceral (perirenal) adipose depots. Secretion of antiinflammatory adiponectin is markedly reduced, whereas that of proinflammatory cytokines interleukin-6, interleukin-8, and monocyte chemoattractant protein-1, is markedly increased in perivascular adipocytes. These depot-specific differences in adipocyte function are demonstrable in both freshly isolated adipose tissues and in vitro-differentiated adipocytes. Murine aortic arch perivascular adipose tissues likewise express lower levels of adipocyte-associated genes as compared with subcutaneous and visceral adipose tissues. Moreover, 2 weeks of high-fat feeding caused further reductions in adipocyte-associated gene expression, while upregulating proinflammatory gene expression, in perivascular adipose tissues. These changes were observed in the absence of macrophage recruitment to the perivascular adipose depot. We conclude that perivascular adipocytes exhibit reduced differentiation and a heightened proinflammatory state, properties that are intrinsic to the adipocytes residing in this depot. Dysfunction of perivascular adipose tissue induced by fat feeding suggests that this unique adipose depot is capable of linking metabolic signals to inflammation in the blood vessel wall.

Transcriptional networks inferred from molecular signatures of breast cancer.

Global genomic approaches in cancer research have provided new and innovative strategies for the identification of signatures that differentiate various types of human cancers. Computational analysis of the promoter composition of the genes within these signatures may provide a powerful method for deducing the regulatory transcriptional networks that mediate their collective function. In this study we have systematically analyzed the promoter composition of gene classes derived from previously established genetic signatures that recently have been shown to reliably and reproducibly distinguish five molecular subtypes of breast cancer associated with distinct clinical outcomes. Inferences made from the trends of transcription factor binding site enrichment in the promoters of these gene groups led to the identification of regulatory pathways that implicate discrete transcriptional networks associated with specific molecular subtypes of breast cancer. One of these inferred pathways predicted a role for nuclear factor-kappaB in a novel feed-forward, self-amplifying, autoregulatory module regulated by the ERBB family of growth factor receptors. The existence of this pathway was verified in vivo by chromatin immunoprecipitation and shown to be deregulated in breast cancer cells overexpressing ERBB2. This analysis indicates that approaches of this type can provide unique insights into the differential regulatory molecular programs associated with breast cancer and will aid in identifying specific transcriptional networks and pathways as potential targets for tumor subtype-specific therapeutic intervention.

Functional profiling of uncommon VCAM1 promoter polymorphisms prevalent in African American populations.

Multiple variants of the vascular adhesion molecule-1 (VCAM1) promoter show increased nucleotide heterozygosity in the African American population. Using a novel transfection-based transcriptional pathway profiling method, we show that select uncommon variants are functionally hyperactive. Eight candidate VCAM1 promoter haplotypes comprising 13 previously identified SNPs were assessed for response to known mitogens. Activity was correlated with bioinformatic analysis of hyper- and hyporesponsive variants to identify the gain or loss of haplotype-specific transcription factor binding site (TFBS). Using this approach, a low frequency regulatory allele (c.-540A>G; dbSNP rs3783605:A>G), found in a hyperactive VCAM1 promoter haplotype, was shown to create a candidate binding site for ETS2 that was confirmed in vivo by chromatin immunoprecipitation. This report provides the first functional evaluation of VCAM1 promoter polymorphisms and establishes a hypothetical foundation for investigation of their role in the pathogenesis of VCAM1-associated diseases that disproportionately afflict African Americans, including thromboembolic diseases, asthma, and multiple myeloma.

A novel EWS-WT1 gene fusion product in desmoplastic small round cell tumor is a potent transactivator of the insulin-like growth factor-I receptor (IGF-IR) gene.

Desmoplastic small round cell tumor (DSRCT) is a primitive sarcoma characterized by a recurrent chromosomal translocation, t(11;22)(p13;q12), which fuses the 5' exons of the EWS gene to the 3' exons of the WT1 gene. EWS-WT1 chimeras are heterogeneous as a result of fusions of different regions of the EWS gene to the WT1 gene. We report here a rare and novel EWS-WT1 variant, EWS-WT1 5/10, in a 6-year-old boy diagnosed with DSRCT and analyze the potential transactivation effect of the fusion oncoprotein. The predicted product is comprised of the N-terminal transactivation domain of EWS and lacks any sequence derived from the WT1 gene product. Nonetheless, the truncated protein was able to stimulate expression of the insulin-like growth factor-I receptor gene, a potent antiapoptotic receptor tyrosine kinase with potentially important roles in DSRCT etiology. These findings raise the possibility that the oncogenic potential of EWS-WT1 fusions is not necessarily a consequence of the fusion protein product per se.

Transcriptional activation of the insulin-like growth factor I receptor gene by the Kruppel-like factor 6 (KLF6) tumor suppressor protein: potential interactions between KLF6 and p53.

The IGF system plays an important role in prostate cancer initiation and progression. Most of the biological actions of IGF-I and IGF-II are mediated by activation of the IGF-I receptor (IGF-IR). Evidence accumulated in recent years indicates that acquisition of the malignant phenotype is initially IGF-IR dependent, but progression toward metastatic stages is usually associated with a decrease in IGF-IR levels. The Kruppel-like factor 6 (KLF6) is a zinc finger-containing transcription factor that was shown to be mutated in a significant portion of prostate and other types of cancer. To examine the potential regulation of IGF-IR gene expression by KLF6, we measured KLF6 levels in prostate-derived cell lines displaying different levels of IGF-IR. The results of Western analysis showed that KLF6 levels were higher in nontumorigenic P69 cells expressing high IGF-IR levels than in metastatic M12 cells containing reduced IGF-IR levels. Transient coexpression of wild-type, but not mutated, KLF6 together with an IGF-IR promoter-luciferase reporter plasmid resulted in an approximately 3.4-fold stimulation of IGF-IR promoter activity. Furthermore, KLF6 expression induced a significant increment in endogenous IGF-IR levels. Deletion analysis of the IGF-IR promoter revealed that a cluster of four GC boxes located between nucleotides -399 and -331 mediates a significant portion of the transactivating effect of KLF6. KLF6, although unable to stimulate IGF-IR promoter activity in Sp1-null Drosophila-derived Schneider cells, significantly enhanced the effect of Sp1. To assess the potential interactions between KLF6 and p53 in the regulation of IGF-IR gene expression, transfections were performed in the colorectal cancer cell line HCT116(+/+), which expresses p53, and its HCT116(-/-) derivative, which lacks p53. KLF6 exhibited an enhanced activity in p53-containing, compared with p53-null, cells. In addition, we were able to detect a physical interaction between KLF6 and p53. In summary, we have identified the IGF-IR gene as a novel downstream target for transcription factor KLF6. The regulation of IGF-IR gene expression by KLF6 may have significant implications in terms of cancer initiation and/or progression.

WT1-p53 interactions in insulin-like growth factor-I receptor gene regulation.

The insulin-like growth factor-I receptor (IGF-IR) plays a critical role in transformation. The expression of the IGF-IR gene is negatively regulated by a number of transcription factors, including the WT1 and p53 tumor suppressors. Previous studies have suggested both physical and functional interactions between the WT1 and p53 proteins. The potential functional interactions between WT1 and p53 in control of IGF-IR promoter activity were addressed by transient coexpression of vectors encoding different isoforms of WT1, together with IGF-IR promoter-luciferase reporter constructs, in p53-null osteosarcoma-derived Saos-2 cells, wild-type p53-expressing kidney tumor-derived G401 cells, and mutant p53-expressing, rhabdomyosarcoma-derived RD cells. Similar studies were also performed to compare p53-expressing Balb/c-3T3 and clonally derived p53-null, (10)1 fibroblasts and the colorectal cancer cell line HCT116 +/+, which expresses a wild-type p53 gene, and its HCT116 -/- derivative, in which the p53 gene has been disrupted by homologous recombination. WT1 splice variants lacking a KTS insert between zinc fingers 3 and 4 suppressed IGF-IR promoter activity in the absence of p53 or in the presence of wild-type p53. WT1 variants that contain the KTS insert are impaired in their ability to bind to the IGF-IR promoter and are unable to suppress IGF-IR promoter. In the presence of mutant p53, WT1 cannot repress the IGF-IR promoter. Coimmunoprecipitation experiments showed that p53 and WT1 physically interact, whereas electrophoretic mobility shift assay studies revealed that p53 modulates the ability of WT1 to bind to the IGF-IR promoter. In summary, the transcriptional activity of WT1 proteins and their ability to function as tumor suppressors or oncogenes depends on the cellular status of p53.

Transcriptional regulation of IGF-I receptor gene expression by novel isoforms of the EWS-WT1 fusion protein.

The EWS family of genes is involved in numerous chromosomal translocations that are characteristic of a variety of sarcomas. A recently described member of this group is desmoplastic small round cell tumor (DSRCT), which is characterized by a recurrent t(11;22)(p13;q12) translocation that fuses the 5' exons of the EWS gene to the 3' exons of the WT1 gene. The originally described chimera comprises exons 1-7 of EWS and exons 8-10 of WT1. We have previously reported that the WT1 protein represses the expression of the IGF-I receptor gene, whereas the EWS(1-7)-WT1(8-10) fusion protein activates IGF-I receptor gene expression. It has recently become apparent that EWS-WT1 chimeras produced in DSCRT are heterogeneous as a result of fusions of different regions of the EWS gene to the WT1 gene. We have recently characterized additional EWS-WT1 translocations that involve the juxtaposition of EWS exons 7 or 8 to WT1 exon 8, and an EWS-WT1 chimera that lacks EWS exon 6. The chimeric transcription factors encoded by these various translocations differ in their DNA-binding characteristics and their ability to transactivate the IGF-I receptor promoter. These data suggest that the molecular pathology of DSRCT is more complex than previously appreciated, and that this diversity may provide the foundation for predictive genotype-phenotype correlations in the future.