EuroDia: a beta-cell gene expression resource.

Type 2 diabetes mellitus (T2DM) is a major disease affecting nearly 280 million people worldwide. Whilst the pathophysiological mechanisms leading to disease are poorly understood, dysfunction of the insulin-producing pancreatic beta-cells is key event for disease development. Monitoring the gene expression profiles of pancreatic beta-cells under several genetic or chemical perturbations has shed light on genes and pathways involved in T2DM. The EuroDia database has been established to build a unique collection of gene expression measurements performed on beta-cells of three organisms, namely human, mouse and rat. The Gene Expression Data Analysis Interface (GEDAI) has been developed to support this database. The quality of each dataset is assessed by a series of quality control procedures to detect putative hybridization outliers. The system integrates a web interface to several standard analysis functions from R/Bioconductor to identify differentially expressed genes and pathways. It also allows the combination of multiple experiments performed on different array platforms of the same technology. The design of this system enables each user to rapidly design a custom analysis pipeline and thus produce their own list of genes and pathways. Raw and normalized data can be downloaded for each experiment. The flexible engine of this database (GEDAI) is currently used to handle gene expression data from several laboratory-run projects dealing with different organisms and platforms. Database URL: http://eurodia.vital-it.ch.

A corneal dystrophy associated with transforming growth factor beta-induced Gly623Asp mutation an amyloidogenic phenotype.

PURPOSE: To present the light and electron microscopic findings of a unique corneal dystrophy never before described in a German family carrying the Gly623Asp Mutation of the TGFBI gene with late clinical onset. DESIGN: Experimental study. PARTICIPANTS: Four affected and 6 nonaffected family members. METHODS: Slit-lamp examination, photographic documentation, and isolation of genomic DNA from peripheral blood leucocytes obtained from each family member examined. Exons 3, 4, 5, and 11 to 14 of the TGFBI gene were amplified and sequenced in these family members. Five corneal buttons of 3 affected siblings were excised at the time of penetrating keratoplasty. Light and electron microscopic examination were performed including immunohistochemistry with antibodies against keratoepithelin (KE) 2 and 15. MAIN OUTCOME MEASURES: Clinical and histologic characteristics of corneal opacification in affected patients and presence of coding region changes in the TGFBI gene. RESULTS: The specimens showed destructive changes in Bowman's layer and the adjacent stroma. Patchy Congo red-positive amyloid deposits were found within the epithelium in 1 cornea, in Bowman's layer and in the anterior stroma of all specimens also showing KE2, but not KE15, immunostaining. Electron microscopy revealed deposits mainly located in the anterior stroma and Bowman's layer and in small amounts in the basal area of some epithelial cells. The destroyed areas were strongly Alcian blue-positive, the Masson Trichrome stain proved mainly negative for the deposits. All affected but none of the unaffected family members had a heterozygous missense mutation in exon 14 of the TGFBI gene (G-->A transition at nucleotide 1915) replacing glycin by aspartic acid amino acid (Gly623Asp) at position 623 of the KE protein. CONCLUSIONS: In contrast with the patient carrying the Gly623Asp mutation of the TGFBI gene described by Afshari et al, our cases presented with Salzmann's nodular degeneration-like clinical features and their specimens contained KE2-positive amyloid. The reason for this now "meeting the expectation histologic phenotype" is unclear. The histologic findings emphasize that this is a unique corneal dystrophy, which shares no clinical characteristics with Reis-Bücklers' dystrophy and should be treated as a distinct entity. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any materials discussed in this article.

Contribution of TIR domain-containing adapter inducing IFN-beta-mediated IL-18 release to LPS-induced liver injury in mice.

BACKGROUND/AIMS: After treatment with heat-killed Propionibacterium acnes mice show dense hepatic granuloma formation. Such mice develop liver injury in an interleukin (IL)-18-dependent manner after challenge with a sublethal dose LPS. As previously shown, LPS-stimulated Kupffer cells secrete IL-18 depending on caspase-1 and Toll-like receptor (TLR)-4 but independently of its signal adaptor myeloid differentiation factor 88 (MyD88), suggesting importance of another signal adaptor TIR domain-containing adapter inducing IFN-beta (TRIF). Nalp3 inflammasome reportedly controls caspase-1 activation. Here we investigated the roles of MyD88 and TRIF in P. acnes-induced hepatic granuloma formation and LPS-induced caspase-1 activation for IL-18 release. METHODS: Mice were sequentially treated with P. acnes and LPS, and their serum IL-18 levels and liver injuries were determined by ELISA and ALT/AST measurement, respectively. Active caspase-1 in LPS-stimulated Kupffer cells was determined by Western blotting. RESULTS: Macrophage-ablated mice lacked P. acnes-induced hepatic granuloma formation and LPS-induced serum IL-18 elevation and liver injury. Myd88(-/-) Kupffer cells, but not Trif(-/-) cells, exhibited normal caspase-1 activation upon TLR4 engagement in vitro. Myd88(-/-) mice failed to develop hepatic granulomas after P. acnes treatment and liver injury induced by LPS challenge. In contrast, Trif(-/-) mice normally formed the hepatic granulomas, but could not release IL-18 or develop the liver injury. Nalp3(-/-) mice showed the same phenotypes of Trif(-/-) mice. CONCLUSIONS: Propionibacterium acnes treatment MyD88-dependently induced hepatic granuloma formation. Subsequent LPS TRIF-dependently activated caspase-1 via Nalp3 inflammasome and induced IL-18 release, eventually leading to the liver injury.

Alpha beta lineage-committed thymocytes can be rescued by the gamma delta T cell receptor (TCR) in the absence of TCR beta chain.

Commitment of the alpha beta and gamma delta T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCR gamma delta-transgenic or TCR beta knockout mice, both of which are unable to generate TCR alpha beta-positive T cells, develop phenotypically alpha beta-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCR beta protein, the gamma delta TCR can promote the development of alpha beta-like thymocytes, which, however, do not expand significantly and do not mature into gamma delta T cells. These results show that commitment to the alpha beta lineage can be determined independently of the isotype of the TCR, and suggest that alpha beta versus gamma delta T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCR gamma and delta gene rearrangements on alpha beta T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.

Transcriptional regulation of CD4 gene expression by T cell factor-1/beta-catenin pathway.

By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/beta-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-xL. By both loss and gain of function studies, in this study we show additional function of TCF-1/beta-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4+ CD8+ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1(-/-) DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1(-/-) cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with beta-catenin. Furthermore, forced expression of a stabilized beta-catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized beta-catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and beta-catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/beta-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.

Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that can be activated by various xenobiotics and natural fatty acids. These transcription factors primarily regulate genes involved in lipid metabolism and also play a role in adipocyte differentiation. We present the expression patterns of the PPAR subtypes in the adult rat, determined by in situ hybridization using specific probes for PPAR-alpha, -beta and -gamma, and by immunohistochemistry using a polyclonal antibody that recognizes the three rat PPAR subtypes. In numerous cell types from either ectodermal, mesodermal, or endodermal origin, PPARs are coexpressed, with relative levels varying between them from one cell type to the other. PPAR-alpha is highly expressed in hepatocytes, cardiomyocytes, enterocytes, and the proximal tubule cells of kidney. PPAR-beta is expressed ubiquitously and often at higher levels than PPAR-alpha and -gamma. PPAR-gamma is expressed predominantly in adipose tissue and the immune system. Our results suggest new potential directions to investigate the functions of the different PPAR subtypes.

IL-2- and CD25-dependent immunoregulatory mechanisms in the homeostasis of T-cell subsets.

IL-2 plays a pivotal role in regulating the adaptive immune system by controlling the survival and proliferation of regulatory T (Treg) cells, which are required for the maintenance of immune tolerance. Moreover, IL-2 is implicated in the differentiation and homeostasis of effector T-cell subsets, including T(H)1, T(H)2, T(H)17, and memory CD8+ T cells. The IL-2 receptor is composed of 3 distinct subunits, namely the alpha (CD25), beta (CD122), and gamma (gammac) chains. Of crucial importance for the delivery of IL-2 signals to Treg cells is the expression of CD25, which, along with CD122 and gammac, confers high affinity binding to IL-2. Notably, recent findings suggest a novel role for CD25, whereby CD25 molecules on Treg cells and possibly other cells are capable of influencing T-cell homeostasis by means of IL-2 deprivation. This review explores these findings and integrates them into our current understanding of T-cell homeostasis.

RNA-driven cyclin-dependent kinase regulation: when CDK9/cyclin T subunits of P-TEFb meet their ribonucleoprotein partners.

The positive transcription elongation factor (P-TEFb) consists of CDK9, a cyclin-dependent kinase and its cyclin T partner. It is required for transcription of most class II genes. Its activity is regulated by non-coding RNAs. The 7SK cellular RNA turns the HEXIM cellular protein into a P-TEFb inhibitor that binds its cyclin T subunit. Thus, P-TEFb activity responds to variations in global cellular transcriptional activity and to physiological conditions linked to cell differentiation, proliferation or cardiac hypertrophy. In contrast, the Tat activation region RNA plays an activating role. This feature at the 5' end of the human immunodeficiency (HIV) viral transcript associates with the viral protein Tat that in turn binds cyclin T1 and recruits active P-TEFb to the HIV promoter. This results in enhanced P-TEFb activity, which is critical for an efficient production of viral transcripts. Although discovered recently, the regulation of P-TEFb becomes a paradigm for non-coding RNAs that regulate transcription factors. It is also a unique example of RNA-driven regulation of a cyclindependent kinase.

Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction. METHODS: Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting. RESULTS: MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

Dominant negative MyD88 proteins inhibit interleukin-1beta /interferon-gamma -mediated induction of nuclear factor kappa B-dependent nitrite production and apoptosis in beta cells.

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.

Transcriptional activation of the utrophin promoter B by a constitutively active Ets-transcription factor.

Duchenne muscular dystrophy is an X-linked genetic disease caused by the absence of functional dystrophin. Pharmacological upregulation of utrophin, the autosomal homologue of dystrophin, offers a potential therapeutic approach to treat Duchenne patients. Full-length utrophin mRNA is transcribed from two alternative promoters, called A and B. In contrast to the utrophin promoter A, little is known about the factors regulating the activity of the utrophin promoter B. Computer analysis of this second promoter revealed the presence of several conserved binding motives for Ets-transcription factors. Using electrotransfer of cDNA into mouse muscles, we demonstrate that a genetically modified beta-subunit of the Ets-transcription factor GA-binding protein potently activates a utrophin promoter B reporter construct in innervated muscle fibers in vivo. These results make the GA-binding protein and the signaling cascade regulating its activity in muscle cells, potential targets for the pharmacological modulation of utrophin expression in Duchenne patients.

Functional effects of Na+,K+-ATPase gene mutations linked to familial hemiplegic migraine.

Familial hemiplegic migraine type 2, an autosomal dominant form of migraine with aura, has been associated with four distinct mutations in the alpha2-subunit of the Na+,K+-ATPase. We have introduced these mutations in the alpha2-subunit of the human Na+,K+-ATPase and the corresponding mutations in the Bufo marinus alpha1-subunit and studied these mutants by expression in Xenopus oocyte. Metabolic labeling studies showed that the mutants were synthesized and associated with the beta-subunit, except for the alpha2HW887R mutant, which was poorly synthesized, and the alpha1BW890R, which was partially retained in the endoplasmic reticulum. [3H]ouabain binding showed the presence of the alpha2HR689Q and alpha2HM731T at the membrane, whereas the alpha2HL764P and alpha2HW887R could not be detected. Functional studies with the mutants of the B. marinus Na+,K+-ATPase showed a reduced or abolished electrogenic activity and a low K+ affinity for the alpha1BW890R mutant. Through different mechanisms, all these mutations result in a strong decrease of the functional expression of the Na+,K+-pump. The decreased activity in alpha2 isoform of the Na+,K+-pump expressed in astrocytes seems an essential component of hemiplegic migraine pathogenesis and may be responsible for the cortical spreading depression, which is one of the first events in migraine attacks.

Penicillin tolerance among Beta-hemolytic streptococci and production of the group carbohydrates, hemolysins, hyaluronidases and deoxyribonucleases

Penicillin tolerance among 67 strains of beta-hemolytic streptococci was examined by determining the ratio of the minimal bactericidal concentration to the minimal inhibitory concentration as 32 or greater. Tolerance was demonstrated in 15 group A strains and in 11,7, and 4 of groups B, C and G, respectively. Thereafter the effects of a subminimal inhibitory concentration (1/2MIC) of penicillin on the bacterial products of four tolerant and four nontolerant strains (two of each Lancefield group) were analyzed and compared. The antibiotic caused a marked increase in the expression of the group carbo-hydrates for strains of group B. Penicillin was found to reduce the cell-bound hemolysin activities of the four tolerant strains and to increase the activity of the other (free) form of nontolerant groups A, C and G hemolysins. Penicillin caused an increase in the extracellular hyaluronidase activities of one group A and groups B, C and G streptococci. With added antibiotic the production of deoxyribonuclease by tolerant groups A, C and G was greatly enhanced and that of the group B streptococcus was arrested.

Maintenance of hepatic nuclear factor 6 in postnatal islets impairs terminal differentiation and function of beta-cells.

The Onecut homeodomain transcription factor hepatic nuclear factor 6 (Hnf6) is necessary for proper development of islet beta-cells. Hnf6 is initially expressed throughout the pancreatic epithelium but is downregulated in endocrine cells at late gestation and is not expressed in postnatal islets. Transgenic mice in which Hnf6 expression is maintained in postnatal islets (pdx1(PB)Hnf6) show overt diabetes and impaired glucose-stimulated insulin secretion (GSIS) at weaning. We now define the mechanism whereby maintenance of Hnf6 expression postnatally leads to beta-cell dysfunction. We provide evidence that continued expression of Hnf6 impairs GSIS by altering insulin granule biosynthesis, resulting in a reduced response to secretagogues. Sustained expression of Hnf6 also results in downregulation of the beta-cell-specific transcription factor MafA and a decrease in total pancreatic insulin. These results suggest that downregulation of Hnf6 expression in beta-cells during development is essential to achieve a mature, glucose-responsive beta-cell.