FOXS1 is increased in liver fibrosis and regulates TGFß responsiveness and proliferation pathways in human hepatic stellate cells.

Liver fibrosis commences with liver injury stimulating transforming growth factor beta (TGFß) activation of hepatic stellate cells (HSCs), causing scarring and irreversible damage. TGFß induces expression of the transcription factor Forkhead box S1 (FOXS1) in hepatocytes and may have a role in the pathogenesis of hepatocellular carcinoma (HCC). To date, no studies have determined how it affects HSCs. We analyzed human livers with cirrhosis, HCC, and a murine fibrosis model and found that FOXS1 expression is significantly higher in fibrotic livers but not in HCC. Next, we treated human LX2 HSC cells with TGFß to activate fibrotic pathways, and FOXS1 mRNA was significantly increased. To study TGFß-FOXS1 signaling, we developed human LX2 FOXS1 CRISPR KO and scrambled control HSCs. To determine differentially expressed gene transcripts controlled by TGFß-FOXS1, we performed RNA-seq in the FOXS1 KO and control cells and over 400 gene responses were attenuated in the FOXS1 KO HSCs with TGFß-activation. To validate the RNA-seq findings, we used our state-of-the-art PamGene PamStation kinase activity technology that measures hundreds of signaling pathways nonselectively in real time. Using our RNA-seq data, kinase activity data, and descriptive measurements, we found that FOXS1 controls pathways mediating TGFß responsiveness, protein translation, and proliferation. Our study is the first to identify that FOXS1 may serve as a biomarker for liver fibrosis and HSC activation, which may help with early detection of hepatic fibrosis or treatment options for end-stage liver disease.

Assessment of Opioid-Induced Immunomodulation in Experimental and Clinical Sepsis.

Opioids remain a standard supportive therapy in patients admitted to the ICU with sepsis. However, as preclinical models indicate an association between opioid exposure and immunosuppression, the use of this class of drugs warrants investigation. The objective of this study was to investigate whether opioid exposure causes immunosuppression in patients with sepsis, and to use a murine sepsis model to determine the effects of opioid exposure on secondary infection. HYPOTHESIS: We hypothesized opioid exposure would be associated with immunosuppression in patients with sepsis and secondary infection in a murine sepsis model. METHODS AND MODELS: This was a two-phase preclinical and clinical study. The clinical phase included a subgroup of patients with sepsis from an existing randomized controlled trial while the preclinical phase used a murine model of sepsis with C57BL/6 mice. In the clinical phase, a post hoc analysis was performed in subjects receiving fentanyl versus no opioid receipt. In the preclinical phase, a murine cecal slurry-induced sepsis model followed by secondary infection was used. Mice were randomized to fentanyl versus no fentanyl concomitantly. RESULTS: In clinical sepsis, a significant decrease in interleukin-23 (IL-23) level in patients with fentanyl exposure was observed and lower IL-23 was associated with mortality (p < 0.001). Other measured cytokines showed no significant differences. Concomitant fentanyl exposure during murine sepsis was associated with a significantly higher bacterial burden (p < 0.001) after secondary infection; however, immune cell counts and plasma cytokine levels were largely unaffected by fentanyl. INTERPRETATION AND CONCLUSIONS: Minimal alterations in cytokines were seen with opioid exposure during clinical sepsis. In a preclinical model, opioid exposure during sepsis was associated with ineffective bacterial clearance upon secondary infection. Further studies are warranted to evaluate the immunomodulatory role of opioids and their implications, especially in the post-sepsis period.

Mechanisms of γδ T cell accumulation in visceral adipose tissue with aging.

γδ T cells are resident in visceral adipose tissue (VAT) where they show an age-associated increase in numbers and contribute to local and systemic chronic inflammation. However, regulation of this population and mechanisms for the age-dependent accumulation are not known. In this study, we identified a progressive trend of γδ T cell accumulation in VAT over the lifespan in mice and explored physiological mechanisms contributing to accumulation. Using isochronic parabiotic pairs of wild-type (WT) and T cell receptor delta knockout (TCRδ KO) mice at young and old age, we confirmed that VAT γδ T cells are predominately a tissue-resident population which is sustained in aging. Migration of peripheral γδ T cells into VAT was observed at less than 10%, with a decreasing trend by aging, suggesting a minor contribution of recruitment to γδ T cell accumulation with aging. Since tissue-resident T cell numbers are tightly regulated by a balance between proliferation and programmed cell death, we further explored these processes. Using in vivo EdU incorporation and the proliferation marker Ki67, we found that the absolute number of proliferating γδ T cells in VAT is significantly higher in the aged compared to young and middle-aged mice, despite a decline in the proportion of proliferating to non-proliferating cells by age. Analysis of apoptosis via caspase 3/7 activation revealed that VAT γδ T cells show reduced apoptosis starting at middle age and continuing into old age. Further, induction of apoptosis using pharmacological inhibitors of Bcl2 family proteins revealed that VAT γδ T cells at middle age are uniquely protected from apoptosis via a mechanism independent of traditional anti-apoptotic Bcl2-family proteins. Collectively, these data indicate that protection from apoptosis at middle age increases survival of tissue-resident γδ T cells resulting in an increased number of proliferative cells from middle age onward, and leading to the age-associated accumulation of γδ T cells in VAT. These findings are important to better understand how adipose tissue dysfunction and related changes in the immune profile contribute to inflammaging among the elderly.

PAI-1 as a critical factor in the resolution of sepsis and acute kidney injury in old age.

Elevated plasma levels of plasminogen activator inhibitor type 1 (PAI-1) are documented in patients with sepsis and levels positively correlate with disease severity and mortality. Our prior work demonstrated that PAI-1 in plasma is positively associated with acute kidney injury (AKI) in septic patients and mice. The objective of this study was to determine if PAI-1 is causally related to AKI and worse sepsis outcomes using a clinically-relevant and age-appropriate murine model of sepsis. Sepsis was induced by cecal slurry (CS)-injection to wild-type (WT, C57BL/6) and PAI-1 knockout (KO) mice at young (5-9 months) and old (18-22 months) age. Survival was monitored for at least 10 days or mice were euthanized for tissue collection at 24 or 48 h post-insult. Contrary to our expectation, PAI-1 KO mice at old age were significantly more sensitive to CS-induced sepsis compared to WT mice (24% vs. 65% survival, p = 0.0037). In comparison, loss of PAI-1 at young age had negligible effects on sepsis survival (86% vs. 88% survival, p = 0.8106) highlighting the importance of age as a biological variable. Injury to the kidney was the most apparent pathological consequence and occurred earlier in aged PAI-1 KO mice. Coagulation markers were unaffected by loss of PAI-1, suggesting thrombosis-independent mechanisms for PAI-1-mediated protection. In summary, although high PAI-1 levels are clinically associated with worse sepsis outcomes, loss of PAI-1 rendered mice more susceptible to kidney injury and death in a CS-induced model of sepsis using aged mice. These results implicate PAI-1 as a critical factor in the resolution of sepsis in old age.

Accumulation of γδ T cells in visceral fat with aging promotes chronic inflammation.

Adipose tissue dysfunction is strongly linked to the development of chronic inflammation and cardiometabolic disorders in aging. While much attention has been given to the role of resident adipose tissue immune cells in the disruption of homeostasis in obesity, age-specific effects remain understudied. Here, we identified and characterized a population of γδ T cells, which show unique age-dependent accumulation in the visceral adipose tissue (VAT) of both mice and humans. Diet-induced obesity likewise increased γδ T cell numbers; however, the effect was greater in the aged where the increase was independent of fat mass. γδ T cells in VAT express a tissue-resident memory T cell phenotype (CD44hiCD62LlowCD69+) and are predominantly IL-17A-producing cells. Transcriptome analyses of immunomagnetically purified γδ T cells identified significant age-associated differences in expression of genes related to inflammation, immune cell composition, and adipocyte differentiation, suggesting age-dependent qualitative changes in addition to the quantitative increase. Genetic deficiency of γδ T cells in old age improved the metabolic phenotype, characterized by increased respiratory exchange ratio, and lowered levels of IL-6 both systemically and locally in VAT. Decreased IL-6 was predominantly due to reduced production by non-immune stromal cells, primarily preadipocytes, and adipose-derived stem cells. Collectively, these findings suggest that an age-dependent increase of tissue-resident γδ T cells in VAT contributes to local and systemic chronic inflammation and metabolic dysfunction in aging.

Visceral fat-specific regulation of plasminogen activator inhibitor-1 in aged septic mice.

Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) are documented in patients with sepsis and levels positively correlate with disease severity and mortality. Our previous work demonstrated that visceral adipose tissues (VAT) are a major source of PAI-1, especially in the aged (murine endotoxemia), that circulating PAI-1 protein levels match the trajectory of PAI-1 transcript levels in VAT (clinical sepsis), and that PAI-1 in both VAT and plasma are positively associated with acute kidney injury (AKI) in septic patients. In the current study utilizing preclinical sepsis models, PAI-1 tissue distribution was examined and cellular sources, as well as mechanisms mediating PAI-1 induction in VAT, were identified. In aged mice with sepsis, PAI-1 gene expression was significantly higher in VAT than in other major organs. VAT PAI-1 gene expression correlated with PAI-1 protein levels in both VAT and plasma. Moreover, VAT and plasma levels of PAI-1 were positively associated with AKI markers, modeling our previous clinical data. Using explant cultures of VAT, we determined that PAI-1 is secreted robustly in response to recombinant transforming growth factor ß (TGFß) and tumor necrosis factor α (TNFα) treatment; however, neutralization was effective only for TNFα indicating that TGFß is not an endogenous modulator of PAI-1. Within VAT, TNFα was localized to neutrophils and macrophages. PAI-1 protein levels were fourfold higher in stromal vascular fraction (SVF) cells compared with mature adipocytes, and among SVF cells, both immune and nonimmune compartments expressed PAI-1 in a similar fashion. PAI-1 was localized predominantly to macrophages within the immune compartment and preadipocytes and endothelial cells within the nonimmune compartment. Collectively, these results indicate that induction and secretion of PAI-1 from VAT is facilitated by a complex interaction among immune and nonimmune cells. As circulating PAI-1 contributes to AKI in sepsis, understanding PAI-1 regulation in VAT could yield novel strategies for reducing systemic consequences of PAI-1 overproduction.

Exploring the Obesity Paradox in A Murine Model of Sepsis: Improved Survival Despite Increased Organ Injury in Obese Mice.

ABSTRACT: Despite the known deleterious effects of obesity, clinical data indicate that overweight or obese patients experience higher rates of sepsis survival compared to normal and underweight patients; a phenomenon called the obesity paradox. Results from preclinical sepsis studies have not been able to replicate these findings. The objective of this study was to test the existence of the obesity paradox in a murine model of cecal slurry (CS)-induced sepsis with insulin-resistant diet-induced obese mice. Male C57BL/6 mice were provided high-fat (HFD) or low-fat (LFD) diets for 20 weeks. HFD-fed mice experienced higher rates of survival compared to LFD-fed mice after septic challenge induced by CS injection (66% vs. 25%, P = 0.01, survival assessed for 14 days). Despite the survival advantage, HFD-fed mice had higher rates of positive bacterial cultures and increased markers of kidney injury. Circulating levels of IL-6, IL-1ß, TNFα, and IL-23 were equivalent 24 h after CS-injection; however, IL-17A was uniquely increased in HFD-fed mice. While LFD-fed mice maintained euglycemia, HFD-fed mice were hyperglycemic 6 and 12 h after CS-injection. Stable isotope resolved metabolomics analysis of liver tissue showed diverging pathways of glucose utilization during sepsis, with LFD-fed mice significantly upregulating glycolytic activity and HFD-fed mice decreasing glucose entry into the TCA cycle. This murine study corroborates clinical data that obesity confers a survival benefit in sepsis, albeit at the expense of more significant organ injury. The mechanisms promoting survival in the obese remain unknown; however, this model appears to be well-poised to begin answering this question. Differences in glucose utilization are a novel target to investigate this paradox.

Adipose-Derived Inflammatory and Coagulant Mediators in Patients With Sepsis.

ABSTRACT: Results from preclinical sepsis studies using rodents are often criticized as not being reproducible in humans. Using a murine model, we previously reported that visceral adipose tissues (VAT) are highly active during the acute inflammatory response, serving as a major source of inflammatory and coagulant mediators. The purpose of this study was to determine whether these findings are recapitulated in patients with sepsis and to evaluate their clinical significance. VAT and plasma were obtained from patients undergoing intra-abdominal operations with noninflammatory conditions (control), local inflammation, or sepsis. In mesenteric and epiploic VAT, gene expression of pro-inflammatory (TNFα, IL-6, IL-1α, IL-1ß) and pro-coagulant (PAI-1, PAI-2, TSP-1, TF) mediators was increased in sepsis compared with control and local inflammation groups. In the omentum, increased expression was limited to IL-1ß, PAI-1, and PAI-2, showing a depot-specific regulation. Histological analyses showed little correlation between cellular infiltration and gene expression, indicating a resident source of these mediators. Notably, a strong correlation between PAI-1 expression in VAT and circulating protein levels was observed, both being positively associated with markers of acute kidney injury (AKI). In another cohort of septic patients stratified by incidence of AKI, circulating PAI-1 levels were higher in those with versus without AKI, thus extending these findings beyond intra-abdominal cases. This study is the first to translate upregulation of VAT mediators in sepsis from mouse to human. Collectively, the data suggest that development of AKI in septic patients is associated with high plasma levels of PAI-1, likely derived from resident cells within VAT.

Correlation of Biomarker Expression in Colonic Mucosa with Disease Phenotype in Crohn's Disease and Ulcerative Colitis.

BACKGROUND: Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are characterized by chronic intestinal inflammation due to immunological, microbial, and environmental factors in genetically predisposed individuals. Advances in the diagnosis, prognosis, and treatment of IBD require the identification of robust biomarkers that can be used for molecular classification of diverse disease presentations. We previously identified five genes, RELA, TNFAIP3 (A20), PIGR, TNF, and IL8, whose mRNA levels in colonic mucosal biopsies could be used in a multivariate analysis to classify patients with CD based on disease behavior and responses to therapy. AIM: We compared expression of these five biomarkers in IBD patients classified as having CD or UC, and in healthy controls. RESULTS: Patients with CD were characterized as having decreased median expression of TNFAIP3, PIGR, and TNF in non-inflamed colonic mucosa as compared to healthy controls. By contrast, UC patients exhibited decreased expression of PIGR and elevated expression of IL8 in colonic mucosa compared to healthy controls. A multivariate analysis combining mRNA levels for all five genes resulted in segregation of individuals based on disease presentation (CD vs. UC) as well as severity, i.e., patients in remission versus those with acute colitis at the time of biopsy. CONCLUSION: We propose that this approach could be used as a model for molecular classification of IBD patients, which could further be enhanced by the inclusion of additional genes that are identified by functional studies, global gene expression analyses, and genome-wide association studies.

Secretory IgA is Concentrated in the Outer Layer of Colonic Mucus along with Gut Bacteria.

Antibodies of the secretory IgA (SIgA) class comprise the first line of antigen-specific immune defense, preventing access of commensal and pathogenic microorganisms and their secreted products into the body proper. In addition to preventing infection, SIgA shapes the composition of the gut microbiome. SIgA is transported across intestinal epithelial cells into gut secretions by the polymeric immunoglobulin receptor (pIgR). The epithelial surface is protected by a thick network of mucus, which is composed of a dense, sterile inner layer and a loose outer layer that is colonized by commensal bacteria. Immunofluorescence microscopy of mouse and human colon tissues demonstrated that the SIgA co-localizes with gut bacteria in the outer mucus layer. Using mice genetically deficient for pIgR and/or mucin-2 (Muc2, the major glycoprotein of intestinal mucus), we found that Muc2 but not SIgA was necessary for excluding gut bacteria from the inner mucus layer in the colon. Our findings support a model whereby SIgA is anchored in the outer layer of colonic mucus through combined interactions with mucin proteins and gut bacteria, thus providing immune protection against pathogens while maintaining a mutually beneficial relationship with commensals.

Secretory antibodies in breast milk promote long-term intestinal homeostasis by regulating the gut microbiota and host gene expression.

Maintenance of intestinal homeostasis requires a healthy relationship between the commensal gut microbiota and the host immune system. Breast milk supplies the first source of antigen-specific immune protection in the gastrointestinal tract of suckling mammals, in the form of secretory IgA (SIgA). SIgA is transported across glandular and mucosal epithelial cells into external secretions by the polymeric Ig receptor (pIgR). Here, a breeding scheme with polymeric Ig receptor-sufficient and -deficient mice was used to study the effects of breast milk-derived SIgA on development of the gut microbiota and host intestinal immunity. Early exposure to maternal SIgA prevented the translocation of aerobic bacteria from the neonatal gut into draining lymph nodes, including the opportunistic pathogen Ochrobactrum anthropi. By the age of weaning, mice that received maternal SIgA in breast milk had a significantly different gut microbiota from mice that did not receive SIgA, and these differences were magnified when the mice reached adulthood. Early exposure to SIgA in breast milk resulted in a pattern of intestinal epithelial cell gene expression in adult mice that differed from that of mice that were not exposed to passive SIgA, including genes associated with intestinal inflammatory diseases in humans. Maternal SIgA was also found to ameliorate colonic damage caused by the epithelial-disrupting agent dextran sulfate sodium. These findings reveal unique mechanisms through which SIgA in breast milk may promote lifelong intestinal homeostasis, and provide additional evidence for the benefits of breastfeeding.

Multifactorial patterns of gene expression in colonic epithelial cells predict disease phenotypes in experimental colitis.

BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) is complex and the need to identify molecular biomarkers is critical. Epithelial cells play a central role in maintaining intestinal homeostasis. We previously identified five "signature" biomarkers in colonic epithelial cells (CEC) that are predictive of disease phenotype in Crohn's disease. Here we investigate the ability of CEC biomarkers to define the mechanism and severity of intestinal inflammation. METHODS: We analyzed the expression of RelA, A20, pIgR, tumor necrosis factor (TNF), and macrophage inflammatory protein (MIP)-2 in CEC of mice with dextran sodium sulfate (DSS) acute colitis or T-cell-mediated chronic colitis. Factor analysis was used to combine the five biomarkers into two multifactorial principal components (PCs). PC scores for individual mice were correlated with disease severity. RESULTS: For both colitis models, PC1 was strongly weighted toward RelA, A20, and pIgR, and PC2 was strongly weighted toward TNF and MIP-2, while the contributions of other biomarkers varied depending on the etiology of inflammation. Disease severity was correlated with elevated PC2 scores in DSS colitis and reduced PC1 scores in T-cell transfer colitis. Downregulation of pIgR was a common feature observed in both colitis models and was associated with altered cellular localization of pIgR and failure to transport IgA. CONCLUSIONS: A multifactorial analysis of epithelial gene expression may be more informative than examining single gene responses in IBD. These results provide insight into the homeostatic and proinflammatory functions of CEC in IBD pathogenesis and suggest that biomarker analysis could be useful for evaluating therapeutic options for IBD patients.

HIV protease inhibitors alter innate immune response signaling to double-stranded RNA in oral epithelial cells: implications for immune reconstitution inflammatory syndrome?

In this investigation, several HIV protease inhibitors altered the virally associated, double-stranded RNA (dsRNA)-stimulated, innate immune response. Lopinavir, the most potent inducer of interleukin (IL)-8 expression, also inhibited dsRNA-induced monocyte chemotactic protein 1 expression. Further analyses demonstrated that nuclear factor-κB is required for lopinavir's induction of IL-8. These findings demonstrate that protease inhibitors, such as lopinavir, differentially dysregulate innate immune signaling in a manner that could affect immune (reconstitution) inflammatory responses in oral epithelium.

Regulation of the polymeric immunoglobulin receptor in intestinal epithelial cells by Enterobacteriaceae: implications for mucosal homeostasis.

The commensal microbiota of the human colon profoundly impacts host gene expression and mucosal homeostasis. Secretory IgA antibodies, which influence the composition of the intestinal microbiota and provide immunity against pathogens, are transported across intestinal epithelial cells (IEC) by the polymeric immunoglobulin receptor (pIgR). To compare the effects of different colonic bacteria on pIgR expression, the human IEC line HT-29 was stimulated with various species representing the 4 major phyla of colonic bacteria. Only bacteria from the family Enterobacteriaceae (phylum Proteobacteria) induced expression of pIgR and other target genes of bacterial pattern recognition receptors. HT-29 cells responded to purified ligands for Toll-like receptor (TLR)4 but not TLR2. Expression of pIgR and transport of IgA were significantly reduced in colons of mice deficient in the TLR adaptor MyD88, consistent with a role for TLR signaling in the regulation of pIgR by colonic bacteria. Induction of pIgR expression in HT-29 cells required NF-kappaB signaling but not MAPK signaling, in contrast to the requirement for both NF-kappaB and MAPK signaling for induction of pro-inflammatory genes. These results suggest that commensal Enterobacteriaceae may promote intestinal homeostasis by enhancing pIgR expression in IEC.

Regulation of the polymeric Ig receptor by signaling through TLRs 3 and 4: linking innate and adaptive immune responses.

IgA Abs help to maintain homeostasis at mucosal surfaces by promoting defense mechanisms that protect against pathogens while suppressing inflammatory responses to commensal organisms and food Ags. The polymeric Ig receptor (pIgR) mediates transport of IgA across mucosal epithelial cells. We hypothesized that signaling through TLRs may up-regulate pIgR expression by intestinal epithelial cells and thus enhance IgA-mediated homeostasis. To test this hypothesis we treated the HT29 human intestinal epithelial cell line with dsRNA, a ligand for TLR3, or LPS, a ligand for TLR4. Both dsRNA and LPS up-regulated levels of pIgR mRNA and cell surface pIgR protein. By contrast, dsRNA but not LPS up-regulated expression of TLR3 and TLR4 mRNA. However, cell surface expression of both TLR3 and TLR4 was enhanced by treatment of HT29 cells with their respective ligands. Transfection of HT29 cells with wild-type and mutated promoter/enhancer plasmids suggested that TLR3 and TLR4 signal primarily through NF-kappaB to enhance transcription of pIgR mRNA. TLR3 signaling resulted in a more pronounced inflammatory response than did TLR4, as evidenced by up-regulation of the transcription factor IFN regulatory factor-1, chemokines IL-8 and RANTES, and the proinflammatory cytokine TNF. Signaling through LPS/TLR4 appears to up-regulate pIgR expression while minimizing proinflammatory responses, a mechanism that could promote IgA-mediated homeostasis in the presence of commensal Gram-negative bacteria.

Long-term exposure of the HT-29 human intestinal epithelial cell line to TNF causes sustained up-regulation of the polymeric Ig receptor and proinflammatory genes through transcriptional and posttranscriptional mechanisms.

Transport of IgA Abs across intestinal epithelial cells into gut secretions is mediated by the polymeric Ig receptor (pIgR). The cytokine TNF plays a central role in initiating and amplifying inflammatory reactions, and is implicated in the pathogenesis of inflammatory bowel diseases. Acute exposure of intestinal epithelial cell lines to TNF has been shown to up-regulate transcription of genes encoding pIgR and a number of proinflammatory factors, but the effects of chronic exposure to TNF have not been studied. We found that exposure of HT-29 human colon carcinoma cells to TNF for up to 20 days reduced the rate of cell proliferation, but did not cause gross morphological changes. Expression of mRNA encoding pIgR and several proinflammatory genes increased acutely, and then diminished but remained elevated above control levels throughout the experiment. Changes in gene expression were paralleled by increased expression of the transcription factors IFN regulatory factor-1 and the RelB subunit of NF-kappaB. HT-29 cells activated the endogenous TNF gene in response to TNF treatment, but the level of TNF production was insufficient to maintain pIgR and proinflammatory gene expression after withdrawal of exogenous TNF. Chronic exposure to TNF caused a marked increase in pIgR mRNA stability and a small but significant decrease in TNF mRNA stability, but no change in the half-lives of IL-8, c-Myc, and GAPDH. In summary, we observed different effects of acute vs chronic exposure to TNF on gene expression, and found evidence for transcriptional and posttranscriptional regulation of expression of the pIgR.

A 5'-distal enhanceosome in the PDGF-A gene is activated in choriocarcinoma cells via ligand-independent binding of vitamin D receptor and constitutive jun kinase signaling.

Overexpression of platelet-derived growth factor A-chain (PDGF-A) is clearly linked to autocrine and paracrine stimulation of malignant growth in many human cancers. We have shown previously that PDGF-A overexpression in choriocarcinoma, hepatoma and lung carcinoma cell lines is driven by the activity of a 66 bp enhancer element (ACE66) located approximately 7 kb upstream of the PDGF-A transcription start site. In this study, the ACE66 element is shown to be activated in JEG-3 choriocarcinoma cells through synergistic interactions between consensus DNA motifs for binding of vitamin D receptor, AP1 and ELK1. Binding of the vitamin D/retinoid-X receptor (VDR/RXRalpha) heterodimer to the ACE66 element was reconstituted in vitro with recombinant VDR/RXRalpha and with JEG-3 nuclear extract, and was verified in living JEG-3 cells by chromatin immunoprecipitation analysis. Transcriptional activity of the ACE66 element, as well as occupancy of the element by VDR/RXRalpha, was shown to be independent of stimulation with the hormonal VDR ligand, 1,25-dihydroxyvitamin D3. The jun kinase pathway of mitogen-activated protein kinase (MAPK) signaling was shown to activate the ACE66 enhancer, most likely through activation of factors binding to the AP1 element. These results identify a novel mechanism of transcriptional enhancement involving ligand-independent activity of the VDR/RXR heterodimer and MAPK signaling pathways that appears to play an important role in the overexpression of PDGF in many different settings of human malignancy.

Upstream stimulatory factor but not c-Myc enhances transcription of the human polymeric immunoglobulin receptor gene.

Secretory antibodies protect mucosal surfaces from ingested, inhaled and sexually transmitted pathogens. The polymeric immunoglobulin receptor (pIgR) transports antibodies across mucosal epithelia into external secretions. We and others have identified a region of the human polymeric immunoglobulin receptor gene (locus PIGR) that is sufficient for basal transcriptional activity. An E-Box motif, which binds transcription factors of the basic helix-loop-helix/leucine zipper (bHLH/zip) family, was identified as a major regulatory element in the PIGR gene promoter. Transient transfection of PIGR promoter reporter plasmids in intestinal epithelial cell (IEC) lines suggested that the transcription factors upstream stimulatory factor (USF) and c-Myc may exert opposing effects on PIGR promoter activity. Mutations within and flanking the E-Box that favored USF binding enhanced promoter activity, while mutations that favored c-Myc binding reduced promoter activity. Ectopic expression of USF1 or USF2 enhanced PIGR promoter activity, while exogenous c-Myc did not. Electrophoretic mobility shift assays (EMSA) demonstrated that USF1 and USF2 bound to the E-Box motif as homo- and heterodimers. Chromatin immunoprecipitation (ChIP) demonstrated that USF proteins bind the PIGR promoter in vivo, which is enriched in acetylated histones. E-Box motifs are commonly observed in promoters of genes that are highly expressed in the human colon. Genes that are down-regulated in colorectal cancer, including PIGR, frequently have non-canonical E-Boxes, while genes that are up-regulated in colorectal cancer generally have canonical E-Boxes. The results of our experiments may shed light on the mechanisms of dysregulated expression of pIgR in inflammatory bowel disease and colorectal cancer, diseases associated with aberrant expression of c-Myc.