Ren G, Kim JY, Smas CM. Identification of RIFL, a novel adipocyte-enriched insulin target gene with a role in lipid metabolism. Am J Physiol Endocrinol Metab 303: E334 -E351, 2012. First published May 8, 2012; doi:10.1152/ajpendo.00084.2012.-To identify new genes that are important in fat metabolism, we utilized the Lexicon-Genentech knockout database of genes encoding transmembrane and secreted factors and whole murine genome transcriptional profiling data that we generated for 3T3-L1 in vitro adipogenesis. Cross-referencing null models evidencing metabolic phenotypes with genes induced in adipogenesis led to identification of a new gene, which we named RIFL (refeeding induced fat and liver). RIFL-null mice have serum triglyceride levels approximately one-third of wild type. RIFL transcript is induced Ͼ100-fold during 3T3-L1 adipogenesis and is also increased markedly during adipogenesis of murine and human primary preadipocytes. siRNA-mediated knockdown of RIFL during 3T3-L1 adipogenesis results in an ϳ35% decrease in adipocyte triglyceride content. Murine RIFL transcript is highly enriched in white and brown adipose tissue and liver. Fractionation of WAT reveals that RIFL transcript is exclusive to adipocytes with a lack of expression in stromal-vascular cells. Nutritional and hormonal studies are consistent with a prolipogenic function for RIFL. There is evidence of an approximately eightfold increase in RIFL transcript level in WAT in ob/ob mice compared with wild-type mice. RIFL transcript level in WAT and liver is increased ϳ80-and 12-fold, respectively, following refeeding of fasted mice. Treatment of 3T3-L1 adipocytes with insulin increases RIFL transcript Յ35-fold, whereas agents that stimulate lipolysis downregulate RIFL. Interestingly, the 198-amino acid RIFL protein is predicted to be secreted and shows ϳ30% overall conservation with the NH 2-terminal half of angiopoietin-like 3, a liver-secreted protein that impacts lipid metabolism. In summary, our data suggest that RIFL is an important new regulator of lipid metabolism.triglyceride; adipogenesis; differentiation WHITE ADIPOCYTES OF WHITE ADIPOSE TISSUE (WAT) are of major importance in lipid metabolism because they are involved in storage and release of triacylglycerol (10,30,31,42,58,81). Adipogenesis is the conversion of fibroblast-like preadipocytes to rounded lipid-filled mature adipocytes (5,35,47,48). Peroxisome proliferator-activated receptor-␥ (PPAR␥), a nuclear hormone receptor family member, functions as a master transcriptional regulator of adipogenesis (29,33,64,72). Multiple additional transcriptional and other signaling mechanisms positively or negatively regulate the process of adipogenesis (15,35,53,69). Because adipocytes are highly specialized for lipid metabolism, genes that are markedly upregulated during adipogenesis are strong candidates as new regulators of lipid metabolism. First established as an in vitro model of adipogenic conversion in the 1970s, the 3T3-L1 cell line has remained the dominant in vitro model of adipogene...

MicroRNAs (miRNAs) are 21-24-nucleotide non-coding RNAs found in diverse organisms. Although hundreds of miRNAs have been cloned or predicted, only very few miRNAs have been functionally characterized. Embryo implantation is a crucial step in mammalian reproduction. Many genes have been shown to be significantly changed in mouse uterus during embryo implantation. However, miRNA expression profiles in the mouse uterus between implantation sites and inter-implantation sites are still unknown. In this study, miRNA microarray was used to examine differential expression of miRNAs in the mouse uterus between implantation sites and inter-implantation sites. Compared with inter-implantation sites, there were 8 up-regulated miRNAs at implantation sites, which were confirmed by both Northern blot and in situ hybridization. miR-21 was highly expressed in the subluminal stromal cells at implantation sites on day 5 of pregnancy. Because miR-21 was not detected in mouse uterus during pseudopregnancy and under delayed implantation, miR-21 expression at implantation sites was regulated by active blastocysts. Furthermore, we showed that Reck was the target gene of miR-21. Our data suggest that miR-21 may play a key role during embryo implantation.

Epigenetic modifications such as histone methylation play an important role in human cancer metastasis. Enhancer of zeste homolog 2 (EZH2), which encodes the histone methyltransferase component of the polycomb repressive complex 2 (PRC2), is overexpressed widely in breast and prostate cancers and epigenetically silences tumor suppressor genes. Expression levels of the novel tumor and metastasis suppressor Raf-1 kinase inhibitor protein (RKIP) have been shown to correlate negatively with those of EZH2 in breast and prostate cell lines as well as in clinical cancer tissues. Here, we show that the RKIP/EZH2 ratio significantly decreases with the severity of disease and is negatively associated with relapse-free survival in breast cancer. Using a combination of loss-and gain-of-function approaches, we found that EZH2 negatively regulated RKIP transcription through repressionassociated histone modifications. Direct recruitment of EZH2 and suppressor of zeste 12 (Suz12) to the proximal E-boxes of the RKIP promoter was accompanied by H3-K27-me3 and H3-K9-me3 modifications. The repressing activity of EZH2 on RKIP expression was dependent on histone deacetylase promoter recruitment and was negatively regulated upstream by miR-101. Together, our findings indicate that EZH2 accelerates cancer cell invasion, in part, via RKIP inhibition. These data also implicate EZH2 in the regulation of RKIP transcription, suggesting a potential mechanism by which EZH2 promotes tumor progression and metastasis.

Channels formed by the canonical transient receptor potential (TRPC) subfamily of proteins are Ca 21 -permeable, nonselective cation channels with various functions. Through a phospholipase C (PLC)-dependent mechanism TRPC6, a member of TRPC subfamily, can be activated by receptor tyrosine kinases (RTK) or G protein-coupled receptors (GPCR), which are implicated in cell proliferation and human malignancies. Here, we report that TRPC6 has a critical role in human gastric cancer development. Expression of TRPC6 was greatly upregulated in human gastric cancer epithelial cells compared with that in normal gastric epithelial cells. Treatment of AGS or MKN45 cells, human gastric cancer cell lines, with SKF96365, an agent known to inhibit TRPC channels, arrested cell cycle in G2/M phase and suppressed cell growth. Importantly, expressing a dominant negative mutant of TRPC6 (DNC6) in these cells also arrested cell cycle in G2/M phase and inhibited cell growth. Transient receptor potential (TRP) channels are a large family of nonselective cation channels and initially characterized in the photoreceptor of Drosophila as a light sensor. 5,6 Canonical transient receptor potential (TRPC) channels, which share the highest homology to Drosophila TRP, are one subfamily of TRP channels in mammalian cells.7,8 TRPC channels include 7 members, TRPC1-7, which are mainly permeable to Ca 21 . The role of TRPC channels in cell proliferation has been revealed in artery smooth muscle cells 9 and in several types of cancer cells. 10,11We thus asked whether TRPC6 channels contribute to cell cycle progression of gastric cancer cells and to gastric cancer development. In this study, we found that TRPC6 expression was upregulated in human gastric cancer samples and blockade of TRPC6 induced G2/M phase arrest and growth retardation in gastric cancer cells. Furthermore, blockade of TRPC6 channels inhibited development of xenografted gastric tumor. These results support the idea that Ca 21 elevation regulated by TRPC6 was critical for G2/M phase transition in gastric cancer cells and contributed to the development of human gastric cancer. Material and methods Materials and cell cultureAntibody for TRPC6 was purchased from Alomone (Israel), antibodies for Cdc2 and pTyr15 Cdc2 from Cell Signaling (Danvers, MA), and for SKF96365 from Calbiochem (Darmstadt, Germany). Other reagents were from Sangon (Shanghai, China), unless otherwise noted. The AGS and MKN45 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum in a humidified cell incubator with an atmosphere of 5% CO 2 at 37°C. Adenoviral vectorsThe GFP, DNC6 (human) and WTC6 (mouse) were constructed into an adenoviral vector with the CMV promoter. The adenovirus contained an internal ribosome entry site (IRES)-GFP tag. The AGS and MKN45 cells were infected at multiplicity of infection (MOI) 5 10 and HEK293 cells at MOI 5 2. Clinical specimens, immunohistology and in situ hybridizationA total of 25 primary gastric cancer samples a...

RKIP was first identified as an inhibitor of the Raf-MEK-ERK signaling pathway. RKIP was also found to play an important role in the NF-ΚB pathway. Genetic and biochemical studies demonstrated that RKIP functioned as a scaffold protein facilitating the phosphorylation of IκB by upstream kinases. However, contrary to what one would expect of a scaffold protein, our results show that RKIP has an overall inhibitory effect on the NF-ΚB transcriptional activities. Since NF-ΚB target gene expression is subject to negative regulation involving the optimal induction of negative regulators, our data support a hypothesis that RKIP inhibits NF-ΚB activity via the auto-regulatory feedback loop by rapidly inducing the expression and synthesis of inhibitors of NF-ΚB activation.

Growing evidence demonstrates that long noncoding RNAs (lncRNAs) are involved in the progression of various cancers, including hepatocellular carcinoma (HCC). The role of nuclear-enriched abundant transcript 1 (NEAT1), an essential lncRNA for the formation of nuclear body paraspeckles, has not been fully explored in HCC. We aimed to determine the expression, roles and functional mechanisms of NEAT1 in the proliferation and invasion of HCC. Based on real-time polymerase chain reaction data, we suggest that NEAT1 is upregulated in HCC tissues compared with noncancerous liver tissues. The knockdown of NEAT1 altered global gene expression patterns and reduced HCC cell proliferation, invasion and migration. RNA immunoprecipitation and RNA pull-down assays confirmed that U2AF65 binds to NEAT1. Furthermore, the study indicated that NEAT1 regulated hnRNP A2 expression and that this regulation may be associated with the NEAT1–U2AF65 protein complex. Thus, the NEAT1-hnRNP A2 regulation mechanism promotes HCC pathogenesis and may provide a potential target for the prognosis and treatment of HCC.

Accumulating evidence suggests that Raf kinase inhibitor protein (RKIP), which negatively regulates multiple signaling cascades including the Raf and nuclear factor κB (NF-κB) pathways, functions as a metastasis suppressor. However, the basis for this activity is not clear. We investigated this question in a panel of breast cancer, colon cancer and melanoma cell lines. We found that RKIP negatively regulated the invasion of the different cancer cells through three-dimensional extracellular matrix barriers by controlling the expression of matrix metalloproteinases (MMPs), particularly, MMP-1 and MMP-2. Silencing of RKIP expression resulted in a highly invasive phenotype and dramatically increased levels of MMP-1 and MMP-2 expression, while overexpression of RKIP decreased cancer cell invasion in vitro and metastasis in vivo of murine tumor allografts. Knockdown of MMP-1 or MMP-2 in RKIP-knockdown cells reverted their invasiveness to normal. In contrast, when examining migration of the different cancer cells in a two-dimensional, barrier-less environment, we found that RKIP had either a positive regulatory activity or no activity, but in no case a negative one (as would be expected if RKIP suppressed metastasis at the level of cell migration itself). Therefore, RKIP’s function as a metastasis suppressor appears to arise from its ability to negatively regulate expression of specific MMPs, and thus invasion through barriers, and not from a direct effect on the raw capacity of cells to move. The NF-κB pathway, but not the Raf pathway, appeared to positively control the invasion of breast cancer cells. A regulatory loop involving an opposing relationship between RKIP and the NF-κB pathway may control the level of MMP expression and cell invasion.

How mammalian tissues maintain their architecture and tissue-specificity is poorly understood. Previously, we documented both the indispensable role of the extracellular matrix (ECM) protein, laminin-111 (LN1), in the formation of normal breast acini, and the phenotypic reversion of cancer cells to acini-like structures in 3-dimensional (3D) gels with inhibitors of oncogenic pathways. Here, we asked how laminin (LN) proteins integrate the signaling pathways necessary for morphogenesis. We report a surprising reciprocal circuitry comprising positive players: laminin-5 (LN5), nitric oxide (NO), p53, HOXD10 and three microRNAs (miRNAs) — that are involved in the formation of mammary acini in 3D. Significantly, cancer cells on either 2-dimensional (2D) or 3D and non-malignant cells on 2D plastic do not produce NO and upregulate negative players: NFκB, EIF5A2, SCA1 and MMP-9 — that disrupt the network. Introducing exogenous NO, LN5 or individual miRNAs to cancer cells reintegrates these pathways and induces phenotypic reversion in 3D. These findings uncover the essential elements of breast epithelial architecture, where the balance between positive- and negative-players leads to homeostasis.