Therapeutic targeting of innate immunity in the failing heart.

Recent studies suggest that the heart possesses an intrinsic system that is intended to delimit tissue injury, as well as orchestrate homoeostatic responses within the heart. The extant literature suggests that this intrinsic stress response is mediated, at least in part, by a family of pattern recognition receptors that belong to the innate immune system, including CD14, the soluble pattern recognition receptor for lipopolysaccharide, and Toll-like receptors 2, 3, 4, 5, 6, 7, and 9. Although this intrinsic stress response system provides a short-term adaptive response to tissue injury, the beneficial effects of this phylogenetically ancient system may be lost if myocardial expression of these molecules either becomes sustained and/or excessive, in which case the salutary effects of activation of these pathways are contravened by the known deleterious effects of inflammatory signaling. Herein we present new information with regard to activation of innate immune gene expression in the failing human heart, as well as review the novel TLR antagonists that are being developed for other indications outside of heart failure. This review will discuss the interesting possibility that the TLR pathway may represent a new target for the development of novel heart failure therapeutics. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure."

Regulation of pancreas plasticity and malignant transformation by Akt signaling.

BACKGROUND & AIMS: Extensive evidence suggests that Akt signaling plays an important role in beta-cell mass and function, although its function in the regulation of the different pancreatic fates has not been adequately investigated. The goal of these studies was to assess the role of Akt signaling in the pancreatic differentiation programs. METHODS: For these experiments, we have generated a double reporter mouse model that provides activation of Akt signaling in a cell type-specific manner. This mouse model conditionally overexpresses a constitutively active form of Akt upon Cre-mediated recombination. Activation of Akt signaling in pancreatic progenitors and acinar and beta-cells was achieved by crossing this animal model to specific Cre-lines. RESULTS: We showed that overexpression of a constitutively active Akt in pancreatic and duodenal homeobox 1 (Pdx1) progenitors induced expansion of ductal structures expressing progenitor markers. This expansion resulted in part from increased proliferation of the ductal epithelium. Lineage-tracing experiments in mice with activation of Akt signaling in mature acinar and beta-cells suggested that acinar-to-ductal and beta-cell-to-acinar/ductal transdifferentiation also contributed to the expansion of the ductal compartment. In addition to the changes in cell plasticity, these studies demonstrated that chronic activation of Akt signaling in Pdx1 progenitors induced the development of premalignant lesions and malignant transformation in old mice. CONCLUSIONS: The current work unravels some of the molecular mechanisms of cellular plasticity and reprogramming, and demonstrates for the first time that activation of Akt signaling regulates the fate of differentiated pancreatic cells in vivo.

Modulation of subsets of cardiac B lymphocytes improves cardiac function after acute injury.

Despite the long-standing recognition that the immune response to acute myocardial injury contributes to adverse left ventricular (LV) remodeling, it has not been possible to effectively target this clinically. Using 2 different in vivo models of acute myocardial injury, we show that pirfenidone confers beneficial effects in the murine heart through an unexpected mechanism that depends on cardiac B lymphocytes. Naive hearts contained a large population of CD19+CD11b-CD23-CD21-IgD+IgMlo lymphocytes, and 2 smaller populations of CD19+CD11b+ B1a and B1b cells. In response to tissue injury, there was an increase in neutrophils, monocytes, macrophages, as well as an increase in CD19+ CD11b- B lymphocytes. Treatment with pirfenidone had no effect on the number of neutrophils, monocytes, or macrophages, but decreased CD19+CD11b- lymphocytes. B cell depletion abrogated the beneficial effects of pirfenidone. In vitro studies demonstrated that stimulation with lipopolysaccharide and extracts from necrotic cells activated CD19+ lymphocytes through a TIRAP-dependent pathway. Treatment with pirfenidone attenuated this activation of B cells. These findings reveal a previously unappreciated complexity of myocardial B lymphocytes within the inflammatory infiltrate triggered by cardiac injury and suggest that pirfenidone exerts beneficial effects in the heart through a unique mechanism that involves modulation of cardiac B lymphocytes.

C/EBP and Cdx family factors regulate liver fatty acid binding protein transgene expression in the small intestinal epithelium.

A transgene constructed from the rat liver fatty acid binding protein gene (Fabp1) promoter is active in all murine small intestinal crypt and villus epithelial cells. Coincident Cdx and C/EBP transcription factor binding sites were identified spanning Fabp1 nucleotides -90 to -78. CDX-1, CDX-2, C/EBPalpha, and C/EBPbeta activated the Fabp1 transgene in CaCo-2 cells, and mutagenizing the -78 site prevented activation by these factors. CDX but not C/EBP factors bound to the site in vitro, although C/EBP factors competed with CDX factors for transgene activation. The -78 site adjoins an HNF-1 site, and CDX and C/EBP family factors cooperated with HNF-1alpha but not HNF-1beta to activate the transgene. Furthermore, CDX-1, CDX-2, C/EBPalpha, and C/EBPbeta bound to HNF-1alpha and HNF-1beta. The transgene with a mutagenized -78 site was silenced in vivo specifically in small intestinal crypt epithelial cells but remained active in villus cells. These results demonstrate functional interactions between HNF-1, C/EBP, and CDX family factors and suggest that these interactions may contribute to differential transcriptional regulation in the small intestinal crypt and villus compartments.

Mechanisms of mutual functional interactions between HNF-4alpha and HNF-1alpha revealed by mutations that cause maturity onset diabetes of the young.

Hepatic nuclear factor (HNF)-4alpha and HNF-1alpha are key endodermal transcriptional regulators that physically and functionally interact. HNF-4alpha and HNF-1alpha cooperatively activate genes with binding sites for both factors, whereas suppressive interactions occur at regulatory sequences with a binding site for only one factor. The liver fatty acid binding protein gene (Fabp1) has binding sites for both factors, and chromatin precipitation assays were utilized to demonstrate that HNF-4alpha increased HNF-1alpha Fabp1 promoter occupancy during cooperative transcriptional activation. The HNF4 P2 promoter contains a HNF-1 but not HNF-4 binding site, and HNF-4alpha suppressed HNF-1alpha HNF4 P2 activation and decreased promoter HNF-1alpha occupancy. The apolipoprotein C III (APOC3) promoter contains a HNF-4 but not HNF-1 binding site, and HNF-1alpha suppressed HNF-4alpha APOC3 activation and decreased HNF-4alpha promoter occupancy. Maturity onset diabetes of the young (MODY) as well as defects in hepatic lipid metabolism result from mutations in either HNF-4alpha or HNF-1alpha. We found that MODY missense mutant R127W HNF-4alpha retained wild-type individual Fabp1 activation and bound to HNF-1alpha better than wild-type HNF-4alpha, yet did not cooperate with HNF-1alpha or increase HNF-1alpha Fabp1 promoter occupancy. The R127W mutant was also defective in both suppressing HNF-1alpha activation of HNF4 P2 and decreasing HNF-1alpha promoter occupancy. The HNF-1alpha R131Q MODY mutant also retained wild-type Fabp1 activation and bound to HNF-4alpha as well as the wild type but was defective in both suppressing HNF-4alpha APOC3 activation and decreasing HNF-4alpha promoter occupancy. These results suggest HNF-1alpha-HNF-4alpha functional interactions are accomplished by regulating factor promoter occupancy and that defective factor-factor interactions may contribute to the MODY phenotype.

Cooperative interactions among intestinal GATA factors in activating the rat liver fatty acid binding protein gene.

GATA-4, GATA-5, and GATA-6 are endodermal zinc-finger transcription factors that activate numerous enterocytic genes. GATA-4 and GATA-6 but not GATA-5 are present in adult murine small intestinal enterocytes, and we now report the simultaneous presence of all three GATA factors in murine small intestinal enterocytes before weaning age. An immunohistochemical survey detected enterocytic GATA-4 and GATA-6 at birth and 1 wk of age and GATA-5 at 1 wk but not birth. Interactions among GATA factors were explored utilizing a transgene constructed from the proximal promoter of the rat liver fatty acid binding protein gene (Fabp1). GATA-4 and GATA-5 but not GATA-6 activate the Fabp1 transgene through a cognate binding site at -128. A dose-response assay revealed a maximum in transgene activation by both factors, where additional factor did not further increase transgene activity. However, at saturated levels of GATA-4, additional transgene activation was achieved by adding GATA-5 expression construct, and vice versa. Similar cooperativity occurred with GATA-5 and GATA-6. Identical interactions were observed with a target transgene consisting of a single GATA site upstream of a minimal promoter. Furthermore, GATA-4 and GATA-5 or GATA-5 and GATA-6 bound to each other in solution. These results are consistent with tethering of one GATA factor to the Fabp1 promoter through interaction with a second GATA factor to produce increased target gene activation. Cooperative target gene activation was specific to an intestinal cell line and may represent a mechanism by which genes are activated in the small intestinal epithelium during the period before weaning.

Antisense transcripts at the EMX2 locus in human and mouse.

The homeodomain transcription factor EMX2 is critical for central nervous system and urogenital development. In addition, EMX2 maps to a region of allelic deletion corresponding to a putative endometrial tumor suppressor at 10q26. We now report another polyadenylated transcript that is transcribed on the strand opposite to EMX2 and overlaps with the EMX2 transcript. This transcript was designated EMX2OS (OS, opposite strand), and an orthologous transcript present at the murine Emx2 locus was designated Emx2os. Alternative splicing to generate transcripts with varying 5' sequences was detected in the human but not the mouse. Neither ortholog contains a significant open reading frame, nor is primary sequence conserved between the two species. The sense and antisense transcripts display coordinate expression in that EMX2 and EMX2OS are abundant in normal postmenopausal endometrium, reduced in premenopausal endometrium, and absent or reduced in a majority of primary endometrial tumors. EMX2, EMX2OS, Emx2, and Emx2os are abundant in the uterine endometrium, with sense and antisense transcripts exhibiting identical expression patterns. Conservation of functional human and murine EMX2 antisense genes, of overlap between the sense and the antisense transcripts, and of identical cellular expression patterns suggests a biological function for EMX2OS, presumably to regulate EMX2.

GATA-4, GATA-5, and GATA-6 activate the rat liver fatty acid binding protein gene in concert with HNF-1alpha.

Transcriptional regulation by GATA-4, GATA-5, and GATA-6 in intestine and liver was explored using a transgene constructed from the proximal promoter of the rat liver fatty acid binding protein gene (Fabpl). An immunohistochemical survey detected GATA-4 and GATA-6 in enterocytes, GATA-6 in hepatocytes, and GATA-5 in neither cell type in adult animals. In cell transfection assays, GATA-4 or GATA-5 but not GATA-6 activated the Fabpl transgene solely through the most proximal of three GATA binding sites in the Fabpl promoter. However, all three factors activated transgenes constructed from each Fabpl site upstream of a minimal viral promoter. GATA factors interact with hepatic nuclear factor (HNF)-1alpha, and the proximal Fabpl GATA site adjoins an HNF-1 site. GATA-4, GATA-5, or GATA-6 bounded to HNF-1alpha in solution, and all cooperated with HNF-1alpha to activate the Fabpl transgene. Mutagenizing all Fabpl GATA sites abrogated transgene activation by GATA factors, but GATA-4 activated the mutagenized transgene in the presence of HNF-1alpha. These in vitro results suggested GATA/HNF-1alpha interactions function in Fabpl regulation, and in vivo relevance was determined with subsequent experiments. In mice, the Fabpl transgene was active in enterocytes and hepatocytes, a transgene with mutagenized HNF-1 site was silent, and a transgene with mutagenized GATA sites had identical expression as the native transgene. Mice mosaic for biallelic Gata4 inactivation lost intestinal but not hepatic Fabpl expression in Gata4-deficient cells but not wild-type cells. These results demonstrate GATA-4 is critical for intestinal gene expression in vivo and suggest a specific GATA-4/HNF-1alpha physical and functional interaction in Fabpl activation.