SUMMARY The tumor stroma is believed to contribute to some of the most malignant characteristics of epithelial tumors. However, signaling between stromal and tumor cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumors. This was associated with the massive remodeling of the extra-cellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumors ameliorated disruption of the tumor microenvironment and was sufficient to decrease tumor growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumor stroma of breast cancer patients. These findings identify the Pten-Ets2 axis as a critical stroma-specific signaling pathway that suppresses mammary epithelial tumors.

Osteoporosis results from an imbalance in skeletal remodeling that favors bone resorption over bone formation. Bone matrix is degraded by osteoclasts, which differentiate from myeloid precursors in response to the cytokine RANKL. To gain insight into the transcriptional regulation of bone resorption during growth and disease, we generated a conditional knockout of the transcription factor nuclear factor of activated T cells c1 (Nfatc1). Deletion of Nfatc1 in young mice resulted in osteopetrosis and inhibition of osteoclastogenesis in vivo and in vitro. Transcriptional profiling revealed NFATc1 as a master regulator of the osteoclast transcriptome, promoting the expression of numerous genes needed for bone resorption. In addition, NFATc1 directly repressed osteoclast progenitor expression of osteoprotegerin, a decoy receptor for RANKL previously thought to be an osteoblast-derived inhibitor of bone resorption. "Cherubism mice", which carry a gain-of-function mutation in SH3-domain binding protein 2 (Sh3bp2), develop osteoporosis and widespread inflammation dependent on the proinflammatory cytokine, TNF-α. Interestingly, deletion of Nfatc1 protected cherubism mice from systemic bone loss but did not inhibit inflammation. Taken together, our study demonstrates that NFATc1 is required for remodeling of the growing and adult skeleton and suggests that NFATc1 may be an effective therapeutic target for osteoporosis associated with inflammatory states.

Regulatory T cells (Treg) are critical to the maintenance of immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses. Treg specifically express the transcription factor Foxp3, which mediates the coordinate activation of genes such as CTLA-4 and GITR along with repression of T effector cytokines such as interleukin-2 and interferon-γ. Despite progress in understanding mechanisms of Foxp3-dependent gene activation, the molecular mechanism of Foxp3-dependent gene repression remains largely unknown. Herein we report the identification of Eos, a zinc-finger transcription factor of the Ikaros family, as a critical mediator of Foxp3-dependent gene silencing in Treg. Eos interacts directly with Foxp3 and is necessary for gene silencing without affecting expression of Foxp3 activated genes. We further demonstrate that Eos and its corepressor C-terminal binding protein 1 (CtBP1) are necessary for histone modifications and ultimately promoter methylation involved in selective gene silencing in Treg. Knockdown of Eos in Treg abrogates their ability to suppress immune responses in vitro and in vivo and endows them with partial effector function. This transcriptional control of Treg function through association between Foxp3 and Eos/co-repressor can potentially be exploited for immune-based therapies.

The methylation status of the CpG island located within the ribosomal RNA (rRNA) promoter in human hepatocellular carcinomas and pair-matched liver tissues was analyzed by bisulfite genomic sequencing. Significant hypomethylation of methyl-CpGs in the rRNA promoter was observed in the tumor samples compared with matching normal tissues, which was consistent with the relatively high level of rRNA synthesis in rapidly proliferating tumors. To study the effect of CpG methylation on RNA polymerase I (pol I)-transcribed rRNA genes, we constructed pHrD-IRES-Luc (human rRNA promoter-luciferase reporter). In this plasmid, Kozak sequence of the pGL3-basic vector was replaced by the internal ribosome entry site (IRES) of encephalomyocarditis viral genome to optimize pol I-driven reporter gene expression. Transfection of this plasmid into HepG2 (human) cells revealed reduced pol I-driven luciferase activity with an increase in methylation density at the promoter. Markedly reduced luciferase activity in Hepa (mouse) cells compared with HepG2 (human) cells showed that pHrD-IRES-Luc is transcribed by pol I. Site-specific methylation of human rRNA promoter demonstrated that methylation of CpG at the complementary strands located in the promoter (؊9, ؊102, ؊347 with respect to the ؉1 site) inhibited luciferase activity, whereas symmetrical methylation of a CpG in the transcribed region (؉152) did not affect the promoter activity. Immunofluorescence studies showed that the methyl-CpG-binding proteins, MBD1, MBD2, MBD3, and MeCP2, are localized both in the nuclei and nucleoli of HepG2 cells. Transient overexpression of MBD2 suppressed luciferase activity specifically from the methylated rRNA promoter, whereas MBD1 and MBD3 inhibited rRNA promoter activity irrespective of the methylation status. Chromatin immunoprecipitation analysis confirmed predominant association of MBD2 with the endogenous methylated rRNA promoter, which suggests a selective role for MBD2 in the methylationmediated inhibition of ribosomal RNA gene expression.

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The ras/Raf/Mek/Erk pathway plays a central role in coordinating endothelial cell activities during angiogenesis. Transcription factors Ets1 and Ets2 are targets of ras/Erk signaling pathways that have been implicated in endothelial cell function in vitro, but their precise role in vascular formation and function in vivo remains ill-defined. In this work, mutation of both Ets1 and Ets2 resulted in embryonic lethality at midgestation, with striking defects in vascular branching having been observed. The action of these factors was endothelial cell autonomous as demonstrated using Cre/loxP technology. Analysis of Ets1/Ets2 target genes in isolated embryonic endothelial cells demonstrated down-regulation of Mmp9, Bcl-X L , and cIAP2 in double mutants versus controls, and chromatin immunoprecipitation revealed that both Ets1 and Ets2 were loaded at target promoters. Consistent with these observations, endothelial cell apoptosis was significantly increased both in vivo and in vitro when both Ets1 and Ets2 were mutated. These results establish essential and overlapping functions for Ets1 and Ets2 in coordinating endothelial cell functions with survival during embryonic angiogenesis. (Blood. 2009;114:1123-1130) IntroductionAngiogenesis, the biologic process by which endothelial cells (ECs) form new blood vessels from an existing vascular network, is a critical process in normal vertebrate embryonic development, as well as in processes like wound healing and inflammation in adults. Angiogenesis is also an essential element in many pathologic conditions, including cancer. 1,2 Angiogenesis is regulated by a balance of both positive and negative signaling events mediated by growth factors and their receptors as well as by cell adhesion to the extracellular matrix. [1][2][3][4] These complex signaling and cell adhesion interactions alter the growth, migration, survival, and differentiation of ECs through modulation of the intracellular signaling pathways that control these processes. [1][2][3][4][5] Among these pathways, the ras/Raf/Mek/Erk pathway has been proposed to play a central role in coordinating these cellular activities during development and tumor angiogenesis. For example, gene knockouts of B-raf and Mek-1 point to their role in placental vascular formation during extraembryonic development, although their action in embryonic development is redundant. 6,7 Expression of dominant-negative Raf in the tumor vasculature in a transplantation model increases EC apoptosis and decreases tumor growth, 8 and sustained Erk activity is critical for EC migration and angiogenesis in the chick chorioallantoic membrane assay. 9 In cell culture studies, Erk signaling has been implicated in EC survival. [10][11][12] ECs are especially sensitive to apoptotic signals during angiogenesis, and the sustained activation of Erk signaling by the combination of growth factor receptors and integrin adhesion may be important in preventing cell death during this process. 9,10 The downstream targets of Erks that mediate these effects on ECs re...

The genes encoding the two isoenzymes of methyl coenzyme M reductase (MRI and MRII) in Methanobacterium thermoautotrophicum AH have been cloned and sequenced. The MRI-encoding mcr operon (mcrBDCGA) has been located immediately upstream from the mtr operon (mtrEDCBA) that encodes Ns-methyltetrahydromethanopterin:coenzyme M methyltransferase, the enzyme that catalyzes the step preceding the MRcatalyzed reaction in methanogenesis. The MRil-encoding mrt operon (mrtBDGA) (30-32), we chose to complete the sequence of the M. thermoautotrophicum AH mrt operon; therefore, for this project, we also needed to clone and sequence the M thermoautotrophicum AH mer operon. This mcr cloning fortuitously also resulted in cloning of the mtrEDCBA operon (the mtr operon [34]) that encodes the M thermoautotrophicum AH N5-methyltetrahydromethanopterin:CoM methyltransferase (methyl-H4MPT transferase), because the mtr operon was discovered immediately downstream from the mcr operon (see Fig. 1). Methyl H4MPT transferase (10,15,34) catalyzes the synthesis of the substrate (CH3-CoM) that is used by both MRI and MRII, and therefore analysis of the mtr transcripts was included in this study. This led to the discovery that transcription through the mcr transcription terminator and into the mtr operon appears to coordinate the synthesis of MRI and methyl H4MPT transferase.Sequencing of the mrt operon in Methanothermus fervidus was also recently completed (18). Surprisingly, both the M thermoautotrophicum AH and Methanothermus fervidus mrt operons contain only four genes, arranged mrtBDGA, in contrast to the mcrBDCGA organization of five genes found in all mcr operons (17,24,38

Abstract-The molecular events linking lipid accumulation in atherosclerotic plaques to complications such as aneurysm formation and plaque disruption are poorly understood. BALB/c-Apoe Ϫ/Ϫ mice bearing a null mutation in the Npc1 gene display prominent medial erosion and atherothrombosis, whereas their macrophages accumulate free cholesterol in late endosomes and show increased cathepsin K (Ctsk) expression. We now show increased cathepsin K immunostaining and increased cysteinyl proteinase activity using near infrared fluorescence imaging over proximal aortas of Apoemice. In mechanistic studies, cholesterol loading of macrophage plasma membranes (cyclodextrin-cholesterol) or endosomal system (AcLDLϩU18666A or Npc1 null mutation) activated Toll-like receptor (TLR) signaling, leading to sustained phosphorylation of p38 mitogen-activated protein kinase and induction of p38 targets, including Ctsk, S100a8, Mmp8, and Mmp14. Studies in macrophages from knockout mice showed major roles for TLR4, following plasma membrane cholesterol loading, and for TLR3, after late endosomal loading. TLR signaling via p38 led to phosphorylation and activation of the transcription factor Microphthalmia transcription factor, acting at E-box elements in the Ctsk promoter. These studies suggest that free cholesterol enrichment of either plasma or endosomal membranes in macrophages leads to activation of signaling via various TLRs, prolonged p38 mitogen-activated protein kinase activation, and induction of Mmps, Ctsk, and S100a8, potentially contributing to plaque complications.