Loss of alcohol dehydrogenase 1B in cancer-associated fibroblasts: contribution to the increase of tumor-promoting IL-6 in colon cancer.

BACKGROUND: Increases in IL-6 by cancer-associated fibroblasts (CAFs) contribute to colon cancer progression, but the mechanisms involved in the increase of this tumor-promoting cytokine are unknown. The aim of this study was to identify novel targets involved in the dysregulation of IL-6 expression by CAFs in colon cancer. METHODS: Colonic normal (N), hyperplastic, tubular adenoma, adenocarcinoma tissues, and tissue-derived myo-/fibroblasts (MFs) were used in these studies. RESULTS: Transcriptomic analysis demonstrated a striking decrease in alcohol dehydrogenase 1B (ADH1B) expression, a gene potentially involved in IL-6 dysregulation in CAFs. ADH1B expression was downregulated in approximately 50% of studied tubular adenomas and all T1-4 colon tumors, but not in hyperplastic polyps. ADH1B metabolizes alcohols, including retinol (RO), and is involved in the generation of all-trans retinoic acid (atRA). LPS-induced IL-6 production was inhibited by either RO or its byproduct atRA in N-MFs, but only atRA was effective in CAFs. Silencing ADH1B in N-MFs significantly upregulated LPS-induced IL-6 similar to those observed in CAFs and lead to the loss of RO inhibitory effect on inducible IL-6 expression. CONCLUSION: Our data identify ADH1B as a novel potential mesenchymal tumor suppressor, which plays a critical role in ADH1B/retinoid-mediated regulation of tumor-promoting IL-6.

Traumatic brain injury induces long-lasting changes in immune and regenerative signaling.

There are no existing treatments for the long-term degenerative effects of traumatic brain injury (TBI). This is due, in part, to our limited understanding of chronic TBI and uncertainty about which proposed mechanisms for long-term neurodegeneration are amenable to treatment with existing or novel drugs. Here, we used microarray and pathway analyses to interrogate TBI-induced gene expression in the rat hippocampus and cortex at several acute, subchronic and chronic intervals (24 hours, 2 weeks, 1, 2, 3, 6 and 12 months) after parasagittal fluid percussion injury. We used Ingenuity pathway analysis (IPA) and Gene Ontology enrichment analysis to identify significantly expressed genes and prominent cell signaling pathways that are dysregulated weeks to months after TBI and potentially amenable to therapeutic modulation. We noted long-term, coordinated changes in expression of genes belonging to canonical pathways associated with the innate immune response (i.e., NF-κB signaling, NFAT signaling, Complement System, Acute Phase Response, Toll-like receptor signaling, and Neuroinflammatory signaling). Bioinformatic analysis suggested that dysregulation of these immune mediators-many are key hub genes-would compromise multiple cell signaling pathways essential for homeostatic brain function, particularly those involved in cell survival and neuroplasticity. Importantly, the temporal profile of beneficial and maladaptive immunoregulatory genes in the weeks to months after the initial TBI suggests wider therapeutic windows than previously indicated.

Early Inhibition of Fatty Acid Synthesis Reduces Generation of Memory Precursor Effector T Cells in Chronic Infection.

Understanding the mechanisms of CD4 memory T cell (Tmem) differentiation in malaria is critical for vaccine development. However, the metabolic regulation of CD4 Tmem differentiation is not clear, particularly in persistent infections. In this study, we investigated the role of fatty acid synthesis (FAS) in Tmem development in Plasmodium chabaudi chronic mouse malaria infection. We show that T cell-specific deletion and early pharmaceutical inhibition of acetyl CoA carboxylase 1, the rate limiting step of FAS, inhibit generation of early memory precursor effector T cells (MPEC). To compare the role of FAS during early differentiation or survival of Tmem in chronic infection, a specific inhibitor of acetyl CoA carboxylase 1, 5-(tetradecyloxy)-2-furoic acid, was administered at different times postinfection. Strikingly, the number of Tmem was only reduced when FAS was inhibited during T cell priming and not during the Tmem survival phase. FAS inhibition during priming increased effector T cell (Teff) proliferation and strongly decreased peak parasitemia, which is consistent with improved Teff function. Conversely, MPEC were decreased, in a T cell-intrinsic manner, upon early FAS inhibition in chronic, but not acute, infection. Early cure of infection also increased mitochondrial volume in Tmem compared with Teff, supporting previous reports in acute infection. We demonstrate that the MPEC-specific effect was due to the higher fatty acid content and synthesis in MPEC compared with terminally differentiated Teff. In conclusion, FAS in CD4 T cells regulates the early divergence of Tmem from Teff in chronic infection.

Transcriptomics of Environmental Enrichment Reveals a Role for Retinoic Acid Signaling in Addiction.

There exists much variability in susceptibility/resilience to addiction in humans. The environmental enrichment paradigm is a rat model of resilience to addiction-like behavior, and understanding the molecular mechanisms underlying this protective phenotype may lead to novel targets for pharmacotherapeutics to treat cocaine addiction. We investigated the differential regulation of transcript levels using RNA sequencing of the rat nucleus accumbens after environmental enrichment/isolation and cocaine/saline self-administration. Ingenuity Pathways Analysis and Gene Set Enrichment Analysis of 14,309 transcripts demonstrated that many biofunctions and pathways were differentially regulated. New functional pathways were also identified for cocaine modulation (e.g., Rho GTPase signaling) and environmental enrichment (e.g., signaling of EIF2, mTOR, ephrin). However, one novel pathway stood out above the others, the retinoic acid (RA) signaling pathway. The RA signaling pathway was identified as one likely mediator of the protective enrichment addiction phenotype, an interesting result given that nine RA signaling-related genes are expressed selectively and at high levels in the nucleus accumbens shell (NAcSh). Subsequent knockdown of Cyp26b1 (an RA degradation enzyme) in the NAcSh of rats confirmed this role by increasing cocaine self-administration as well as cocaine seeking. These results provide a comprehensive account of enrichment effects on the transcriptome and identify RA signaling as a contributing factor for cocaine addiction.

Convergent transcriptomics and proteomics of environmental enrichment and cocaine identifies novel therapeutic strategies for addiction.

Transcriptomic and proteomic approaches have separately proven effective at identifying novel mechanisms affecting addiction-related behavior; however, it is difficult to prioritize the many promising leads from each approach. A convergent secondary analysis of proteomic and transcriptomic results can glean additional information to help prioritize promising leads. The current study is a secondary analysis of the convergence of recently published separate transcriptomic and proteomic analyses of nucleus accumbens (NAc) tissue from rats subjected to environmental enrichment vs. isolation and cocaine self-administration vs. saline. Multiple bioinformatics approaches (e.g. Gene Ontology (GO) analysis, Ingenuity Pathway Analysis (IPA), and Gene Set Enrichment Analysis (GSEA)) were used to interrogate these rich data sets. Although there was little correspondence between mRNA vs. protein at the individual target level, good correspondence was found at the level of gene/protein sets, particularly for the environmental enrichment manipulation. These data identify gene sets where there is a positive relationship between changes in mRNA and protein (e.g. glycolysis, ATP synthesis, translation elongation factor activity, etc.) and gene sets where there is an inverse relationship (e.g. ribosomes, Rho GTPase signaling, protein ubiquitination, etc.). Overall environmental enrichment produced better correspondence than cocaine self-administration. The individual targets contributing to mRNA and protein effects were largely not overlapping. As a whole, these results confirm that robust transcriptomic and proteomic data sets can provide similar results at the gene/protein set level even when there is little correspondence at the individual target level and little overlap in the targets contributing to the effects.

Regulation of limited N-terminal proteolysis of APE1 in tumor via acetylation and its role in cell proliferation.

Mammalian apurinic/apyrimidinic (AP) endonuclease 1 (APE1), a ubiquitous and multifunctional protein, plays an essential role in the repair of both endogenous and drug-induced DNA damages in the genome. Unlike its E.coli counterpart Xth, mammalian APE1 has a unique N-terminal domain and possesses both DNA damage repair and transcriptional regulatory functions. Although the overexpression of APE1 in diverse cancer types and the association of APE1 expression with chemotherapy resistance and poor prognosis are well documented, the cellular and molecular mechanisms that alter APE1 functions during tumorigenesis are largely unknown. Here, we show the presence of full-length APE1 and N-terminal truncated isoforms of APE1 in tumor tissue samples of various cancer types. However, primary tumor tissue has higher levels of acetylated APE1 (AcAPE1) as well as full-length APE1 compared to adjacent non-tumor tissue. We found that APE1 is proteolytically cleaved by an unknown serine protease at its N-terminus following residue lysine (Lys) Lys6 and/or Lys7 and after Lys27 and Lys31 or Lys32. Acetylation of these Lys residues in APE1 prevents this proteolysis. The N-terminal domain of APE1 and its acetylation are required for modulation of the expression of hundreds of genes. Importantly, we found that AcAPE1 is essential for sustained cell proliferation. Together, our study demonstrates that increased acetylation levels of APE1 in tumor cells inhibit the limited N-terminal proteolysis of APE1 and thereby maintain the functions of APE1 to promote tumor cells' sustained proliferation and survival.

Analysis of the TGFß-induced program in primary airway epithelial cells shows essential role of NF-κB/RelA signaling network in type II epithelial mesenchymal transition.

BACKGROUND: The airway epithelial cell plays a central role in coordinating the pulmonary response to injury and inflammation. Here, transforming growth factor-ß (TGFß) activates gene expression programs to induce stem cell-like properties, inhibit expression of differentiated epithelial adhesion proteins and express mesenchymal contractile proteins. This process is known as epithelial mesenchymal transition (EMT); although much is known about the role of EMT in cellular metastasis in an oncogene-transformed cell, less is known about Type II EMT, that occurring in normal epithelial cells. In this study, we applied next generation sequencing (RNA-Seq) in primary human airway epithelial cells to understand the gene program controlling Type II EMT and how cytokine-induced inflammation modifies it. RESULTS: Generalized linear modeling was performed on a two-factor RNA-Seq experiment of 6 treatments of telomerase immortalized human small airway epithelial cells (3 replicates). Using a stringent cut-off, we identified 3,478 differentially expressed genes (DEGs) in response to EMT. Unbiased transcription factor enrichment analysis identified three clusters of EMT regulators, one including SMADs/TP63 and another NF-κB/RelA. Surprisingly, we also observed 527 of the EMT DEGs were also regulated by the TNF-NF-κB/RelA pathway. This Type II EMT program was compared to Type III EMT in TGFß stimulated A549 alveolar lung cancer cells, revealing significant functional differences. Moreover, we observe that Type II EMT modifies the outcome of the TNF program, reducing IFN signaling and enhancing integrin signaling. We confirmed experimentally that TGFß-induced the NF-κB/RelA pathway by observing a 2-fold change in NF-κB/RelA nuclear translocation. A small molecule IKK inhibitor blocked TGFß-induced core transcription factor (SNAIL1, ZEB1 and Twist1) and mesenchymal gene (FN1 and VIM) expression. CONCLUSIONS: These data indicate that NF-κB/RelA controls a SMAD-independent gene network whose regulation is required for initiation of Type II EMT. Type II EMT dramatically affects the induction and kinetics of TNF-dependent gene networks.

Caspase-1/ASC inflammasome-mediated activation of IL-1ß-ROS-NF-κB pathway for control of Trypanosoma cruzi replication and survival is dispensable in NLRP3-/- macrophages.

In this study, we have utilized wild-type (WT), ASC-/-, and NLRP3-/- macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1ß production in mφs in comparison to LPS-treated controls. When WT and ASC-/- macrophages were treated with inhibitors of caspase-1, IL-1ß, or NADPH oxidase, we found that IL-1ß production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1ß regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3-/- macrophages, despite an inability to elicit IL-1ß activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3-/- macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1ß-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1ß/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1ß/NFκB activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages.

Environmental enrichment alters protein expression as well as the proteomic response to cocaine in rat nucleus accumbens.

Prior research demonstrated that environmental enrichment creates individual differences in behavior leading to a protective addiction phenotype in rats. Understanding the mechanisms underlying this phenotype will guide selection of targets for much-needed novel pharmacotherapeutics. The current study investigates differences in proteome expression in the nucleus accumbens of enriched and isolated rats and the proteomic response to cocaine self-administration using a liquid chromatography mass spectrometry (LCMS) technique to quantify 1917 proteins. Results of complementary Ingenuity Pathways Analyses (IPA) and gene set enrichment analyses (GSEA), both performed using protein quantitative data, demonstrate that cocaine increases vesicular transporters for dopamine and glutamate as well as increasing proteins in the RhoA pathway. Further, cocaine regulates proteins related to ERK, CREB and AKT signaling. Environmental enrichment altered expression of a large number of proteins implicated in a diverse number of neuronal functions (e.g., energy production, mRNA splicing, and ubiquitination), molecular cascades (e.g., protein kinases), psychiatric disorders (e.g., mood disorders), and neurodegenerative diseases (e.g., Huntington's and Alzheimer's diseases). Upregulation of energy metabolism components in EC rats was verified using RNA sequencing. Most of the biological functions and pathways listed above were also identified in the Cocaine X Enrichment interaction analysis, providing clear evidence that enriched and isolated rats respond quite differently to cocaine exposure. The overall impression of the current results is that enriched saline-administering rats have a unique proteomic complement compared to enriched cocaine-administering rats as well as saline and cocaine-taking isolated rats. These results identify possible mechanisms of the protective phenotype and provide fertile soil for developing novel pharmacotherapeutics. Proteomics data are available via ProteomeXchange with identifier PXD000990.

Profile of dengue infection in Jamnagar city and district, west India.

Background and Methods India is one of the countries in the World Health Organization South-East Asia Region that regularly reports outbreaks of dengue fever (DF)/dengue hemorrhagic fever (DHF). As effective control and preventive programmes depend upon improved surveillance data, this study was carried out to report the seroprevalence of dengue virus infection in an area around Jamnagar city, Western India. [1] Methods The laboratory records of clinically suspected dengue patients from July 2008 to June 2011 were analysed retrospectively for the results of immunoglobulin M (IgM) anti-dengue antibodies, tested by dengue monoclonal antibody (IgM) capture enzyme-linked immunosorbent assay (MAC ELISA). Variations in disease incidence by sex, age group and season were assessed. Results A total of 903 serum samples were tested, of which 253 were positive. The majority were males (72%) and in the age group of 16-30 years. The incidence of dengue peaked in October and slowly tapered by December. Conclusion Dengue cases were higher during September to December, in the post-monsoon season. This observation is useful for planning special preventive strategies. The study draws attention to the susceptibility of the male, young adult age group.

A clinical microbiological study of corneal ulcer patients at western Gujarat, India.

Corneal ulcer is a major cause of blindness throughout the world. When the cornea is injured by foreign particles, there are chances of infection by the organism and development of ulcer. Bacterial infection in the cornea is invariably an alteration of the defense mechanism of the outer eye. It is essential to determine the local etiology within a given region when planning a corneal ulcer management strategy. Laboratory evaluation is necessary to establish the diagnosis and to guide the antibiotic therapy. One hundred corneal ulcer patients were studied by collecting their corneal scraping samples and processing at Clinical Microbiology department of Shree Meghaji Petharaj Shah Medical College, Jamnagar, Gujarat, India during a period of 17 months. All clinical microbiology laboratory procedures followed standard protocols described in the literature. 40 (40%) patients from the age group of 20-70 years had been confirmed as - any organism culture positive - within the corneal ulcer patient population. Fungi were isolated from 26 (26%) corneal ulcer patients. The bacterial etiology was confirmed in 14 (14%) corneal ulcer patients. The major risk factors for mycotic keratitis were vegetative injury (16, (62%)), followed by conjunctivitis (4, (15%)), and blunt trauma (3, (11%)). Pseudomonas aeruginosa was the most commonly isolated bacterium (6, (43%)), followed by Proteus spp. (4, (29%)). Corneal Infections due to bacteria and filamentous fungi are a frequent cause of corneal damage. Microbiological investigation is an essential tool in the diagnosis of these infections. The frequency of fungal keratitis has risen over the past 20 to 30 years. Prognosis of bacterial corneal infection has improved since the introduction of specific antibacterial therapy.

Pulmonary nocardiosis--a rare case of pulmonary nocardiosis in an immunocompetent patient.

Nocardiosis is localised or disseminated infection caused by soil dwelling aerobic actinomycetes, which habitually enter through the respiratory tract. There has been an increase in the incidence of nocardia species infections probably due to higher degree of clinical suspicion, aggressive diagnostic examinations, increased use of immunosuppressive treatments (chemotherapy agents and immunosuppressive agents) and the appearance of AIDS. Here we are presenting an atypical case of Nocardia asteroides in a 45-years-old immunocompetent female patient presenting with history of cough with scanty expectoration, haemoptysis and fever for 4 months. Clinical and radiological diagnostic consideration were tuberculosis and malignancy. Diagnosis of nocardiosis was confirmed microbiologically and the patient responded to cotrimoxazole.

Study of opportunistic intestinal parasitic infections in human immunodeficiency virus/acquired immunodeficiency syndrome patients.

INTRODUCTION: Intestinal parasites predominantly coccidian parasites are a common cause for diarrhea in human immunodeficiency virus (HIV)-positive patients. MATERIALS AND METHODS: The study was conducted during January 2009-December 2010. A total of 1,088 stool samples from 544 seropositive HIV positive cases were examined microscopically for ova and cyst using wet mount preparations and stained smears. Out of 544 patients, 343 had prolonged diarrhea for more than 4 weeks, 57 had acute diarrhea of lesser than 7 days and 144 were asymptomatic cases who attended out-patient department; included in this study after taking consent from patients. Enteric pathogens were detected in 274 (50.36%) of the 544 patients. RESULTS AND CONCLUSIONS: The parasites identified were Cryptosporidium (135), Isospora belli (42), Cyclospora (12), Microsporidia (02), Entamoeba histolytica (49), Hookworm (34). Intestinal parasites in chronic diarrhea were significantly higher than the acute diarrhea (63.05% vs. 7.35%; P < 0.05). Parasitic pathogens were frequently associated with HIV-positive patients with diarrhea in Western India. Stools of all HIV-positive patients with diarrhea should thoroughly be investigated to identify etiologic agents for proper management. MATERIALS AND METHODS: The study was conducted during January 2009-December 2010. A total of 1,088 stool samples from 544 seropositive HIV positive cases were examined microscopically for ova and cyst using wet mount preparations and stained smears. Out of 544 patients, 343 had prolonged diarrhea for more than 4 weeks, 57 had acute diarrhea of lesser than 7 days and 144 were asymptomatic cases who attended out-patient department; included in this study after taking consent from patients. Enteric pathogens were detected in 274 (50.36%) of the 544 patients. RESULTS AND CONCLUSIONS: The parasites identified were Cryptosporidium (135), Isospora belli (42), Cyclospora (12), Microsporidia (02), Entamoeba histolytica (49), Hookworm (34). Intestinal parasites in chronic diarrhea were significantly higher than the acute diarrhea (63.05% vs. 7.35%; P > 0.05). Parasitic pathogens were frequently associated with HIV-positive patients with diarrhea in Western India. Stools of all HIV-positive patients with diarrhea should thoroughly be investigated to identify etiologic agents for proper management.

Innate immune tolerance and the role of kupffer cells in differential responses to interferon therapy among patients with HCV genotype 1 infection.

BACKGROUND & AIMS: In patients with hepatitis C virus (HCV) infection, interferon alfa (IFN-α) alters expression of IFN-stimulated genes (ISGs), but little is understood about factors that determine outcomes of therapy. We used a systems biology approach to evaluate the acute response of patients with chronic hepatitis C to IFN-α therapy. METHODS: We collected liver biopsy samples from 8 treatment-naïve patients with chronic HCV genotype 1 infection at baseline and 24 hours after treatment with IFN-α-2a (10 MU subcutaneously). Blood samples were collected before and up to 48 hours after administration of IFN-α-2a to measure HCV RNA levels and for gene expression analysis. Patients then received pegylated IFN-α-2a and ribavirin on day 5 of the study; therapy continued for up to 48 weeks. RESULTS: Based on the kinetics of HCV RNA during the first 12 weeks of therapy, 2 patients were rapid virologic responders, 4 were early virologic responders, and 2 did not respond to therapy (nonresponders). Nonresponders had high pretreatment levels of ISG expression in the liver but not in peripheral blood mononuclear cells. In responders, after administration of IFN-α, intrahepatic ISG expression increased significantly from baseline and was associated with a rapid phase 1 decrease in HCV. We identified distinct hepatic expression and tissue distribution patterns of ISGs that segregated with treatment outcome. Importantly, Kupffer cells were a local source of IFN that promoted basal expression of ISG in hepatocytes of nonresponders. This finding was validated in cultured THP1 human macrophages that expressed IFN-ß after exposure to viable HCV 2a. When Huh7 K2040 and Huh7 L2198S hepatoma cells were incubated with IFN-α-2a, expression of ISGs peaked by 4 hours and decreased by 72 hours, associated with an increase in level of HCV RNA. This indicates that constitutive exposure to IFN causes hepatoma cells to become tolerant of ISG function. CONCLUSIONS: In patients with chronic HCV infection, IFN production by Kupffer cells might promote innate immune tolerance, characterized by a lack of response to IFN therapy. Strategies to disrupt the virus-host interactions that induce innate immune tolerance should improve therapy.

Effects of progesterone treatment on expression of genes involved in uterine quiescence.

An important action of progesterone during pregnancy is to maintain the uterus in a quiescent state and thereby prevent preterm labor. The causes of preterm labor are not well understood, so progesterone action on the myometrium can provide clues about the processes that keep the uterus from contracting prematurely. Accordingly, we have carried out Affymetrix GeneChip analysis of progesterone effects on gene expression in immortalized human myometrial cells cultured from a patient near the end of pregnancy. Progesterone appears to inhibit uterine excitability by a number of mechanisms, including increased expression of calcium and voltage-operated K(+) channels, which dampens the electrical activity of the myometrial cell, downregulation of agents, and receptors involved in myometrial contraction, reduction in cell signal components that lead to increased intracellular Ca(2+) concentrations in response to contractile stimuli, and downregulation of proteins involved in the cross-linking of actin and myosin filaments to produce uterine contractions.

Aging and microRNA expression in human skeletal muscle: a microarray and bioinformatics analysis.

A common characteristic of aging is loss of skeletal muscle (sarcopenia), which can lead to falls and fractures. MicroRNAs (miRNAs) are novel posttranscriptional modulators of gene expression with potential roles as regulators of skeletal muscle mass and function. The purpose of this study was to profile miRNA expression patterns in aging human skeletal muscle with a miRNA array followed by in-depth functional and network analysis. Muscle biopsy samples from 36 men [young: 31 ± 2 (n = 19); older: 73 ± 3 (n = 17)] were 1) analyzed for expression of miRNAs with a miRNA array, 2) validated with TaqMan quantitative real-time PCR assays, and 3) identified (and later validated) for potential gene targets with the bioinformatics knowledge base software Ingenuity Pathways Analysis. Eighteen miRNAs were differentially expressed in older humans (P < 0.05 and >500 expression level). Let-7 family members Let-7b and Let-7e were significantly elevated and further validated in older subjects (P < 0.05). Functional and network analysis from Ingenuity determined that gene targets of the Let-7s were associated with molecular networks involved in cell cycle control such as cellular proliferation and differentiation. We confirmed with real-time PCR that mRNA expression of cell cycle regulators CDK6, CDC25A, and CDC34 were downregulated in older compared with young subjects (P < 0.05). In addition, PAX7 mRNA expression was lower in older subjects (P < 0.05). These data suggest that aging is characterized by a higher expression of Let-7 family members that may downregulate genes related to cellular proliferation. We propose that higher Let-7 expression may be an indicator of impaired cell cycle function possibly contributing to reduced muscle cell renewal and regeneration in older human muscle.

Requirement of TGFbeta signaling for SMO-mediated carcinogenesis.

Hedgehog (Hh) signaling, via the key signal transducer Smoothened (SMO) and Gli transcription factors, is essential for embryonic development and carcinogenesis. At present, the molecular mechanism of Hh signaling-mediated carcinogenesis is not completely understood. Using a mouse model (K14cre/R26SmoM2) of SMO-mediated basal cell carcinoma development, we identified TGFß2 as a major Hh-regulated gene. TGFß2 expression was high in the keratinocytes, with activated TGFß signaling (indicated by elevated expression of phosphorylated SMAD2/3) detected in both tumor and stroma. The significance of TGFß signaling for SMO function was demonstrated in two assays. Down-regulation of TGFß2 expression prevented Hh signaling-dependent osteoblast differentiation and motor neuron differentiation. Furthermore, inhibition of TGFß signaling by TGFß receptor I inhibitor SD208 significantly reduced tumor area in K14cre/R26SmoM2 mice. Tumor shrinkage in mice was associated with an increased number of lymphocytes, suggesting an immune suppression role of TGFß signaling. The relevance of our results to human cancer is reflected by the fact that human basal cell carcinomas, which almost always harbor activated Hh signaling, have activated TGFß signaling, as indicated by high levels of phosphorylated SMAD2 and SMAD3 in tumor and stroma. Together, our data indicate that TGFß signaling is critical for Hh signaling-mediated carcinogenesis.

Pathway signature and cellular differentiation in clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer. The purpose of this study is to define a biological pathway signature and a cellular differentiation program in ccRCC. METHODOLOGY: We performed gene expression profiling of early-stage ccRCC and patient-matched normal renal tissue using Affymetrix HG-U133a and HG-U133b GeneChips combined with a comprehensive bioinformatic analyses, including pathway analysis. The results were validated by real time PCR and IHC on two independent sample sets. Cellular differentiation experiments were performed on ccRCC cell lines and their matched normal renal epithelial cells, in differentiation media, to determine their mesenchymal differentiation potential. PRINCIPAL FINDINGS: We identified a unique pathway signature with three major biological alterations-loss of normal renal function, down-regulated metabolism, and immune activation-which revealed an adipogenic gene expression signature linked to the hallmark lipid-laden clear cell morphology of ccRCC. Culturing normal renal and ccRCC cells in differentiation media showed that only ccRCC cells were induced to undergo adipogenic and, surprisingly, osteogenic differentiation. A gene expression signature consistent with epithelial mesenchymal transition (EMT) was identified for ccRCC. We revealed significant down-regulation of four developmental transcription factors (GATA3, TFCP2L1, TFAP2B, DMRT2) that are important for normal renal development. CONCLUSIONS: ccRCC is characterized by a lack of epithelial differentiation, mesenchymal/adipogenic transdifferentiation, and pluripotent mesenchymal stem cell-like differentiation capacity in vitro. We suggest that down-regulation of developmental transcription factors may mediate the aberrant differentiation in ccRCC. We propose a model in which normal renal epithelial cells undergo dedifferentiation, EMT, and adipogenic transdifferentiation, resulting in ccRCC. Because ccRCC cells grown in adipogenic media regain the characteristic ccRCC phenotype, we have identified a new in vitro ccRCC cell model more resembling ccRCC tumor morphology.

Progesterone receptor-induced gene expression in primary mouse granulosa cell cultures.

The progesterone receptor (PGR) is induced by luteinizing hormone (LH) in granulosa cells of preovulatory follicles, and the PGR-A isoform is essential for ovulation based on the phenotypes of Pgr isoform-specific knockout mice. Although several genes regulated by PGR-A in vivo have been identified, whether these genes are primary targets of PGR-A or if their expression also depends on other signaling molecules that are induced by the LH surge has not been resolved. Therefore, to identify genes that are either induced or repressed by PGR in the absence of LH-mediated signaling cascades, we infected primary cultures of mouse granulosa cells with either PGR-A or PGR-B adenoviral vectors without or with R-5020 as a PGR ligand. Total RNA was extracted from infected cells at 16 h and analyzed by Affymetrix Mouse 430 2.0 microarrays. PGR-A in the presence or absence of ligand significantly induced approximately 50 genes 2-fold or more (local pooled error test at P

Allergen challenge induces Ifng dependent GTPases in the lungs as part of a Th1 transcriptome response in a murine model of allergic asthma.

According to the current paradigm, allergic airway inflammation is mediated by Th2 cytokines and pro-inflammatory chemokines. Since allergic inflammation is self-limited, we hypothesized that allergen challenge simultaneously induces anti-inflammatory genes to counter-balance the effects of Th2 cytokines and chemokines. To identify these putative anti-inflammatory genes, we compared the gene expression profile in the lungs of ragweed-sensitized mice four hours after challenge with either PBS or ragweed extract (RWE) using a micro-array platform. Consistent with our hypothesis, RWE challenge concurrently upregulated Th1-associated early target genes of the Il12/Stat4 pathway, such as p47 and p65 GTPases (Iigp, Tgtp and Gbp1), Socs1, Cxcl9, Cxcl10 and Gadd45g with the Th2 genes Il4, Il5, Ccl2 and Ccl7. These Th1-associated genes remain upregulated longer than the Th2 genes. Augmentation of the local Th1 milieu by administration of Il12 or CpG prior to RWE challenge further upregulated these Th1 genes. Abolition of the Th1 response by disrupting the Ifng gene increased allergic airway inflammation and abrogated RWE challenge-induced upregulation of GTPases, Cxcl9, Cxcl10 and Socs1, but not Gadd45g. Our data demonstrate that allergen challenge induces two sets of Th1-associated genes in the lungs: 1) Ifng-dependent genes such as p47 and p65 GTPases, Socs1, Cxcl9 and Cxcl10 and 2) Ifng-independent Th1-inducing genes like Gadd45g. We propose that allergen-induced airway inflammation is regulated by simultaneous upregulation of Th1 and Th2 genes, and that persistent unopposed upregulation of Th1 genes resolves allergic inflammation.