Application of the differential display RT-PCR strategy for the identification of inflammation-related mouse genes

The inflammatory response elicited by various stimuli such as microbial products or cytokines is determined by differences in the pattern of cellular gene expression. We have used the differential display RT-PCR (DDRT-PCR) strategy to identify mRNAs that are differentially expressed in various murine cell types stimulated with pro-inflammatory cytokines, microbial products or anti-inflammatory drugs. Mouse embryonic fibroblasts (MEFs) were treated with IFNs, TNF, or sodium salicylate. Also, peritoneal macrophages from C3H/Hej mice were stimulated with T. cruzi-derived GPI-mucin and/or IFN-g. After DDRT-PCR, various cDNA fragments that were differentially represented on the sequencing gel were recovered, cloned and sequenced. Here, we describe a summary of several experiments and show that, when 16 of a total of 28 recovered fragments were tested for differential expression, 5 (31%) were found to represent mRNAs whose steady-state levels are indeed modulated by the original stimuli. Some of the identified cDNAs encode for known proteins that were not previously associated with the inflammatory process triggered by the original stimuli. Other cDNA fragments (8 of 21 sequences, or 38%) showed no significant homology with known sequences and represent new mouse genes whose characterization might contribute to our understanding of inflammation. In conclusion, DDRT-PCR has proven to be a potent technology that will allow us to identify genes that are differentially expressed when cells are subjected to changes in culture conditions or isolated from different organs.

Les cibles transcriptionnelles du polycomb Rae28 lors du développement de l'oeil : l'hypothèse du locus Ink4a/Arf

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Role of receptor and non-receptor protein tyrosine kinases in vasoactive peptide-induced signaling

L'endothéline-1 (ET-1) et l'angiotensine II (Ang II) jouent un rôle important dans le maintien de la pression artérielle et l'homéostasie vasculaire. Une activité accrue de ces peptides vasoactifs est présumée contribuer au développement de pathologies vasculaires, telles que l'hypertension, l'athérosclérose, l'hypertrophie et la resténose. Ceci est causé par une activation excessive de plusieurs voies de signalisation hypertrophiques et prolifératives, qui incluent des membres de la famille des Mitogen Activated Protein Kinases (MAPK), ainsi que la famille phosphatidylinositol 3-kinase (PI3-K) / protéine kinase B (PKB). Bien que l'activation de ces voies de signalisation soit bien élucidée, les éléments en amont responsables de l'activation des MAPK et de la PKB, induite par l'ET-1 et Ang II, demeurent mal compris. Durant les dernières années, le concept de la transactivation de récepteurs et/ou non-récepteurs protéines tyrosine kinases (PTK) dans le déclenchement des événements de signalisation induits par les peptides vasoactifs a gagné beaucoup de reconnaissance. Nous avons récemment démontré que la PTK Insulin-like Growth Factor type-1 Receptor (IGF-1R) joue un rôle dans la transduction des signaux induits par l‟H2O2, menant à la phosphorylation de la PKB. Étant donné que les peptides vasoactifs génèrent des espèces réactives d'oxygène, telles que l‟H2O2 lors de leur signalisation, nous avons examiné le rôle de d‟IGF-1R dans la phosphorylation de la PKB et les réponses hypertrophiques dans les cellules muscle lisse vasculaires (CMLV) induites par l'ET-1 et Ang II. AG-1024, un inhibiteur spécifique de l'IGF-1R, a atténué la phosphorylation de la PKB induite à la fois par l'ET-1 et Ang II. Le traitement des CMLVs avec l‟ET-1 et Ang II a également induit une phosphorylation des résidus tyrosine dans les sites d'autophosphorylation d'IGF-1R, celle-ci a été bloquée par l‟AG-1024. En outre, l‟ET-1 et l‟Ang II on tous les deux provoqué la phosphorylation de c-Src, une PTK non-récepteur, bloqué par PP-2, inhibiteur spécifique de la famille Src. La PP-2 a également inhibé la phosphorylation de PKB et d‟IGF-1R induite par l‟ET-1 et l‟Ang II. De plus, la synthèse de protéines ainsi que d‟ADN, marqueurs de la prolifération cellulaire et de l'hypertrophie, ont également été atténuée par l‟AG-1024 et le PP-2. Bien que ce travail démontre le rôle de c-Src dans la phosphorylation PKB induite par l'ET-1 et Ang II, son rôle dans l'activation des MAPK induit par l'ET-1 dans les CMLVs reste controversé. Par conséquent, nous avons examiné l'implication de c-Src dans l'activation de ERK 1/2, JNK et p38MAPK, par l'ET-1 et Ang II, ainsi que leur capacité à régulariser l'expression du facteur de transcription Early growth transcription factor-1 « Egr-1 ». ET-1 et Ang II ont induit la phosphorylation de ERK 1/2, JNK et p38 MAPK, et ont amplifié l'expression d'Egr-1 dans les CMLVs. Cette augmentation de la phosphorylation des MAPK a été diminuée par la PP-2, qui a aussi atténué l'expression d'Egr-1 induite par l'ET-1 et l'Ang II. Une preuve supplémentaire du rôle de c-Src dans ce processus a été obtenue en utilisant des fibroblastes embryonnaires de souris déficientes en c-Src (Src -/- MEF). L'expression d'Egr-1, ainsi que l'activation des trois MAPKs par l'ET-1 ont été atténuées dans les cellules Src -/- par rapport au MEF exprimant des taux normaux Src. En résumé, ces données suggèrent que l'IGF-1R et c-Src PTK jouent un rôle essentiel dans la régulation de la phosphorylation de PKB et des MAPK dans l‟expression d'Egr-1, ainsi que dans les réponses hypertrophiques et prolifératives induites par l'ET-1 et Ang II dans les CMLVs.

Apatite-Polymer Composites for the Controlled Dual Delivery of BMP-2 and BMP-6 for Bone Tissue Engineering

The release of growth factors from tissue engineering scaffolds provides signals that influence the migration, differentiation, and proliferation of cells. The incorporation of a drug delivery platform that is capable of tunable release will give tissue engineers greater versatility in the direction of tissue regeneration. We have prepared a novel composite of two biomaterials with proven track records - apatite and poly(lactic-co-glycolic acid) (PLGA) – as a drug delivery platform with promising controlled release properties. These composites have been tested in the delivery of a model protein, bovine serum albumin (BSA), as well as therapeutic proteins, recombinant human bone morphogenetic protein-2 (rhBMP-2) and rhBMP-6. The controlled release strategy is based on the use of a polymer with acidic degradation products to control the dissolution of the basic apatitic component, resulting in protein release. Therefore, any parameter that affects either polymer degradation or apatite dissolution can be used to control protein release. We have modified the protein release profile systematically by varying the polymer molecular weight, polymer hydrophobicity, apatite loading, apatite particle size, and other material and processing parameters. Biologically active rhBMP-2 was released from these composite microparticles over 100 days, in contrast to conventional collagen sponge carriers, which were depleted in approximately 2 weeks. The released rhBMP-2 was able to induce elevated alkaline phosphatase and osteocalcin expression in pluripotent murine embryonic fibroblasts. To augment tissue engineering scaffolds with tunable and sustained protein release capabilities, these composite microparticles can be dispersed in the scaffolds in different combinations to obtain a superposition of the release profiles. We have loaded rhBMP-2 into composite microparticles with a fast release profile, and rhBMP-6 into slow-releasing composite microparticles. An equi-mixture of these two sets of composite particles was then injected into a collagen sponge, allowing for dual release of the proteins from the collagenous scaffold. The ability of these BMP-loaded scaffolds to induce osteoblastic differentiation in vitro and ectopic bone formation in a rat model is being investigated. We anticipate that these apatite-polymer composite microparticles can be extended to the delivery of other signalling molecules, and can be incorporated into other types of tissue engineering scaffolds.

FTO influences adipogenesis by regulating mitotic clonal expansion

The fat mass and obesity-associated (FTO) gene plays a pivotal role in regulating body weight and fat mass; however, the underlying mechanisms are poorly understood. Here we show that primary adipocytes and mouse embryonic fibroblasts (MEFs) derived from FTO overexpression (FTO-4) mice exhibit increased potential for adipogenic differentiation, while MEFs derived from FTO knockout (FTO-KO) mice show reduced adipogenesis. As predicted from these findings, fat pads from FTO-4 mice fed a high-fat diet show more numerous adipocytes. FTO influences adipogenesis by regulating events early in adipogenesis, during the process of mitotic clonal expansion. The effect of FTO on adipogenesis appears to be mediated via enhanced expression of the pro-adipogenic short isoform of RUNX1T1, which enhanced adipocyte proliferation, and is increased in FTO-4 MEFs and reduced in FTO-KO MEFs. Our findings provide novel mechanistic insight into how upregulation of FTO leads to obesity.

Caratteristiche biologiche e potenziale applicativo delle cellule staminali derivate da membrane fetali

La ricerca sulle cellule staminali apre nuove prospettive per approcci di terapia cellulare. Molta attenzione è concentrata sulle cellule staminali isolate da membrane fetali, per la facilità di recupero del materiale di partenza, le limitate implicazioni etiche e le caratteristiche delle popolazioni di cellule staminali residenti. In particolare a livello dell’epitelio amniotico si concentra una popolazione di cellule (hAECs) con interessanti caratteristiche di staminalità, pluripotenza e immunomodulazione. Restano però una serie di limiti prima di arrivare ad un’applicazione clinica: l’uso di siero di origine animale nei terreni di coltura e le limitate conoscenze legate alla reazione immunitaria in vivo. La prima parte di questo lavoro è focalizzata sulle caratteristiche delle hAECs coltivate in un terreno privo di siero, in confronto a un terreno di coltura classico. Lo studio è concentrato sull’analisi delle caratteristiche biologiche, immunomodulatorie e differenziative delle hAECs. L’interesse verso le caratteristiche immunomodulatorie è legato alla possibilità che l’uso di un terreno serum free riduca il rischio di rigetto dopo trapianto in vivo. La maggior parte degli studi in vivo con cellule isolate da membrane fetali sono stati realizzati con cellule di derivazione umana in trapianti xenogenici, ma poco si sa circa la sopravvivenza di queste cellule in trapianti allogenici, come nel caso di trapianti di cellule di derivazione murina in modelli di topo. La seconda parte dello studio è focalizzata sulla caratterizzazione delle cellule derivate da membrane fetali di topo (mFMSC). Le caratteristiche biologiche, differenziative e immunomodulatorie in vitro e in vivo delle mFMSC sono state confrontate con i fibroblasti embrionali di topo. In particolare è stata analizzata la risposta immunitaria a trapianti di mFMSC nel sistema nervoso centrale (CNS) in modelli murini immunocompetenti.

The role of NPxY domains in LRP1 receptor maturation and function

Analyses of low density lipoprotein receptor-related protein 1 (LRP1) mutant mouse embryonic fibroblasts (MEFs) generated from LRP1 knock-in mice revealed that inefficient maturation and premature proteasomal degradation of immature LRP1 is causing early embryonic lethality in NPxY1 and NPxY1+2 mutant mice. In MEFs, NPxY2 mutant LRP1 showed efficient maturation but, as expected, decreased endocytosis. The single proximal NPxY1 and the double mutant NPxY1+2 were unable to reach the cell surface as an endocytic receptor due to premature degradation. In conclusion, the proximal NPxY1 motif is essential for early sorting steps in the biosynthesis of mature LRP1.rnThe viable NPxY2 mouse was used to provide genetic evidence for LRP1-mediated amyloid-β (Aβ) transport across the blood-brain barrier (BBB). Here, we show that primary mouse brain capillary endothelial cells (pMBCECs) express functionally active LRP1. Moreover, demonstrate that LRP1 mediates [125I]-Aβ1-40 transcytosis across pMBCECs in both directions, whereas no role for LRP1-mediated Aβ degradation was detected. Aβ transport across pMBCECs generated from NPxY2 knock-in mice revealed a reduced Aβ clearance in both directions compared to WT derived pMBCECs. Finally, we conclude that LRP1 is a bona-fide receptor involved in bidirectional transcytosis of Aβ across the BBB.rn

Self-assembly of sensory neurons into ganglia-like microtissues

Unraveling intra- and inter-cellular signaling networks managing cell-fate control, coordinating complex differentiation regulatory circuits and shaping tissues and organs in living systems remain major challenges in the post-genomic era. Resting on the laurels of past-century monolayer culture technologies, the cell culture community has only recently begun to appreciate the potential of three-dimensional mammalian cell culture systems to reveal the full scope of mechanisms orchestrating the tissue-like cell quorum in space and time. Capitalizing on gravity-enforced self-assembly of monodispersed primary embryonic mouse cells in hanging drops, we designed and characterized a three-dimensional cell culture model for ganglion-like structures. Within 24h, a mixture of mouse embryonic fibroblasts (MEF) and cells, derived from the dorsal root ganglion (DRG) (sensory neurons and Schwann cells) grown in hanging drops, assembled to coherent spherical microtissues characterized by a MEF feeder core and a peripheral layer of DRG-derived cells. In a time-dependent manner, sensory neurons formed a polar ganglion-like cap structure, which coordinated guided axonal outgrowth and innervation of the distal pole of the MEF feeder spheroid. Schwann cells, present in embryonic DRG isolates, tended to align along axonal structures and myelinate them in an in vivo-like manner. Whenever cultivation exceeded 10 days, DRG:MEF-based microtissues disintegrated due to an as yet unknown mechanism. Using a transgenic MEF feeder spheroid, engineered for gaseous acetaldehyde-inducible interferon-beta (ifn-beta) production by cotransduction of retro-/ lenti-viral particles, a short 6-h ifn-beta induction was sufficient to rescue the integrity of DRG:MEF spheroids and enable long-term cultivation of these microtissues. In hanging drops, such microtissues fused to higher-order macrotissue-like structures, which may pave the way for sophisticated bottom-up tissue engineering strategies. DRG:MEF-based artificial micro- and macrotissue design demonstrated accurate key morphological aspects of ganglions and exemplified the potential of self-assembled scaffold-free multicellular micro-/macrotissues to provide new insight into organogenesis.

MODELING SPORADIC TUMOR FORMATION DRIVEN BY TELOMERE DYSFUNCTION IN THE GASTROINTESTINAL TRACT

Colorectal cancer is a complex disease that is thought to arise when cells accumulate mutations that allow for uncontrolled growth. There are several recognized mechanisms for generating such mutations in sporadic colon cancer; one of which is chromosomal instability (CIN). One hypothesized driver of CIN in cancer is the improper repair of dysfunctional telomeres. Telomeres comprise the linear ends of chromosomes and play a dual role in cancer. Its length is maintained by the ribonucleoprotein, telomerase, which is not a normally expressed in somatic cells and as cells divide, telomeres continuously shorten. Critically shortened telomeres are considered dysfunctional as they are recognized as sites of DNA damage and cells respond by entering into replicative senescence or apoptosis, a process that is p53-dependent and the mechanism for telomere-induced tumor suppression. Loss of this checkpoint and improper repair of dysfunctional telomeres can initiate a cycle of fusion, bridge and breakage that can lead to chromosomal changes and genomic instability, a process that can lead to transformation of normal cells to cancer cells. Mouse models of telomere dysfunction are currently based on knocking out the telomerase protein or RNA component; however, the naturally long telomeres of mice require multiple generational crosses of telomerase null mice to achieve critically short telomeres. Shelterin is a complex of six core proteins that bind to telomeres specifically. Pot1a is a highly conserved member of this complex that specifically binds to the telomeric single-stranded 3’ G-rich overhang. Previous work in our lab has shown that Pot1a is essential for chromosomal end protection as deletion of Pot1a in murine embryonic fibroblasts (MEFs) leads to open telomere ends that initiate a DNA damage response mediated by ATR, resulting in p53-dependent cellular senescence. Loss of Pot1a in the background of p53 deficiency results in increased aberrant homologous recombination at telomeres and elevated genomic instability, which allows Pot1a-/-, p53-/- MEFs to form tumors when injected into SCID mice. These phenotypes are similar to those seen in cells with critically shortened telomeres. In this work, we created a mouse model of telomere ysfunction in the gastrointestinal tract through the conditional deletion of Pot1a that recapitulates the microscopic features seen in severe telomere attrition. Combined intestinal loss of Pot1a and p53 lead to formation of invasive adenocarcinomas in the small and large intestines. The tumors formed with long latency, low multiplicity and had complex genomes due to chromosomal instability, features similar to those seen in sporadic human colorectal cancers. Taken together, we have developed a novel mouse model of intestinal tumorigenesis based on genomic instability driven by telomere dysfunction.

ATM Signaling to TSC2: Mechanisms and Implications for Cancer Therapy

Ataxia telangiectasia mutated (ATM) is a critical component of the cellular response to DNA damage, where it acts as a damage sensor, and signals to a large network of proteins which execute the important tasks involved in responding to the damage, namely inducing cell cycle checkpoints, inducing DNA repair, modulating transcriptional responses, and regulating cell death pathways if the damage cannot be repaired faithfully. We have now discovered that an additional novel component of this ATM-dependent damage response involves induction of autophagy in response to oxidative stress. In contrast to DNA damage-induced ATM activation however, oxidative stress induced ATM, occurs in the cytoplasm, and does not require nuclear-to-cytoplasmic shuttling of ATM. Using several cell culture systems including MCF7 breast carcinoma cells, SKOV3 ovarian cancer cells, and various lineages of mouse embryonic fibroblasts, we showed that once activated by reactive oxygen species (ROS), ATM signals to mTORC1 to induce autophagy via the LKB1-AMPK-TSC2 pathway. Targeting dysregulation of mTORC1 in Atm-deficient mice, which succumb to lymphomagenesis within 3-4 months of age with daily administration of rapamycin, could significantly extend survival and cause regression of tumors, suggesting that pharmacologically targeting this pathway has therapeutic implications in cancer. We also identified a second contrasting pathway for DNA damage-induced mTORC1 repression which does not require AMPK activation, but does require ATM and TSC2. Several potential mechanisms including mTOR localization and p53-mediated pathways were ruled out however we identified that TSC2 may be an additional cytoplasmic direct ATM substrate that is engaged in response to DNA damage specifically. Lastly, a study was performed to examine whether autophagy induced by ovarian cancer therapeutics (focusing on cisplatin, since paclitaxel does not induce autophagy in the SKOV3 cell line model we used) plays a role in resistance to therapy since autophagy can play both pro-survival mechanisms or be a mechanism of cell death. Using a genetic approach to knock-down Atg5 expression with shRNA in SKOV3 ovarian carcinoma cells, we compared the cytotoxicity of cisplatin in vector or Atg5 knock-down cells, and demonstrated that autophagy does not play any significant role in the response to cisplatin in this cell line.

Functional studies on DNA double-strand break repair proteins (MmRad51, Ku80) in mice

RecA in Escherichia coli and it's homologue, ScRad51 in Saccharomyces cerevisiae, play important roles in recombinational repair. ScRad51 homologues have been discovered in a wide range of organisms including Schizosaccharomyces pombe, lily, chicken, mouse and human. To date there is no direct evidence to describe that mouse Rad51(MmRad51) is involved in DNA double-strand break repair. In order to elucidate the role of MmRad51 in vivo, it was mutated by the embryonic stem (ES) cell/gene targeting technology in mice. The mutant embryos arrested in development shortly after implantation. There was a decrease in cell proliferation followed by programmed cell death, and trophectoderm-derived cells were sensitive to $\gamma$-radiation. Severe chromosome loss was observed in most mitotically dividing cells. The mutant embryos lived longer and developed further in a p53 mutant background; however, double-mutant embryonic fibroblasts failed to proliferate in tissue culture, reflecting the embryos limited life span. Based on these data, MmRad51 repairs DNA damage induced by $\gamma$-radiation, is needed to maintain euplody, and plays an important role in proliferating cells.^ Ku is a heterodimer of 70 and 80 kDs subunit, which binds to DNA ends and other altered DNA structures such as hairpins, nicks, and gaps. In addition, Ku is required for DNA-PK activity through a direct association. Although the biochemical properties of Ku and DNA-PKcs have been characterized in cells, their physiological functions are not clear. In order to understand the function of Ku in vivo, we generated mice homozygous for a mutation of the Ku80 gene. Ku80-deficient mice, like scid mice, showed severe immunodeficiency due to a impairment of V(D)J recombination. Mutant mice were semiviable and runted, cells derived from mutant embryos displayed hypersensitivity to $\gamma$-radiation, a decreased growth rate, a slow entry into S phase, altered colony size distributions, and a short life span. Based on these results, mutant cells and mice appeared to prematurely age. ^

Memo has a novel role in S1P signaling and crucial for vascular development

Memo is a conserved protein that was identified as an essential mediator of tumor cell motility induced by receptor tyrosine kinase activation. Here we show that Memo null mouse embryonic fibroblasts (MEFs) are impaired in PDGF-induced migration and this is due to a defect in sphingosine-1-phosphate (S1P) signaling. S1P is a bioactive phospholipid produced in response to multiple stimuli, which regulates many cellular processes. S1P is secreted to the extracellular milieu where it exerts its function by binding a family of G-protein coupled receptors (S1PRs), causing their activation in an autocrine or paracrine manner. The process, termed cell-autonomous S1PR signaling, plays a role in survival and migration. Indeed, PDGF uses cell-autonomous S1PR signaling to promote cell migration; we show here that this S1P pathway requires Memo. Using vascular endothelial cells (HUVECs) with Memo knock-down we show that their survival in conditions of serum-starvation is impaired. Furthermore, Memo loss in HUVECs causes a reduction of junctional VE-cadherin and an increase in sprout formation. Each of these phenotypes is rescued by S1P or S1P agonist addition, showing that Memo also plays an important role in cell-autonomous S1PR signaling in endothelial cells. We also produced conventional and endothelial cell-specific conditional Memo knock-out mouse strains and show that Memo is essential for embryonic development. Starting at E13.5 embryos of both strains display bleeding and other vascular problems, some of the phenotypes that have been described in mouse strains lacking S1PRs. The essential role of Memo in embryonic vascular development may be due in part to alterations in S1P signaling. Taken together our results show that Memo has a novel role in the S1P pathway and that Memo is needed to promote cell-autonomous S1PR activation.

Mammalian Snm1 participates in cellular responses to genotoxic and mitotic stress

Eukaryotic cells have evolved a complex network of metabolic processes and regulatory systems to help ensure that hereditary information is protected or restored when exposed to genotoxic agents. Two members of the Snm1 protein family have been characterized; scSNM1/PSO2, a yeast gene responsible for repair of DNA interstrand crosslinks, and hARTEMIS, a human gene that is mutated in radiosensitive severe combined immunodeficiency (RS-SCID). Here we report on another member of this protein family, hSNM1, and its response to DNA damage and mitotic stress. We have found that this protein colocalizes and physically associates with 53BP1, a crucial member of the mammalian response to DNA damage. In addition, hSnm1 interacts with several proteins involved in mitosis, and mSNM1 deficiency causes a mitotic checkpoint defect in mouse embryonic fibroblasts. ^

The role of coactivator associated arginine methyltransferase 1 (CARM1) in nuclear receptor mediated transcription

Arginine methylation has been implicated in the regulation of gene expression. The coactivator-associated arginine methyltransferase 1 (CARMI/PRMT4) binds the p160 family of steroid receptor coactivators (SRCs). This association enhances transcriptional activation by nuclear receptors. Here, we generated and characterized CARM1 knockout mice. Embryos with a targeted disruption of CARM1 are 35% smaller in size than the wild-type littermates and die perinatally. We also generated Carm1-/- and Carm1+/+ mouse embryonic fibroblasts and tested gene expression in response to estrogen. Estrogenresponsive gene expression was aberrant in Carm1-/- fibroblasts and embryos, thus emphasizing the role of arginine methylation as a transcription activation tag. We subsequently studied the role of CARM1 in estrogen signaling in viva in the mammary gland. Conditional knockout of CARM1 in mammary gland and Carml-1-embryonic mammary anlagen transplant experiments did not show any defects in growth and development of the glands. To further dissect the role of CARM1 in estrogen receptor mediated transactivation, we performed cDNA microarray and serial analysis of gene expression on Carm1-/- and Carm1+/+ embryos treated with the estrogen analog, DES. Our results indicate global changes in estrogen regulated genes as well as genes involved in lipid homeostasis. Marker genes for Peroxisome Proliferator Activated Receptor γ (PPARγ) activity, adipsin and aP2, are downregulated in the Carm1-/- embryos. Furthermore, OCT frozen sections of 18.5dpc embryos, processed simultaneously for oil red O staining to look for neutral fat, reveals greatly reduced brown fat accumulation in the Carm1-/- embryos in contrast to wild-type and gain-of-function Carm1 transgenic (ubiquitous) embryo. We used a well-established 3T3-L1 preadipocyte cell line to knockdown CARM1 by short hairpin RNA. 3T3-L1 cells with CARM1 knockdown showed greatly reduced potential to differentiate into mature lipid accumulating adipocytes upon administration of adipogenic stimuli. Ligand-dependent activation of reporter genes by the PPARγ receptor showed that PPRE-luciferase reporter activity was enhanced in the presence of CARM1, additionally, luciferase activity was reduced to background levels when enzyme dead CARM1 (CARM1-VLD) was used. Thus, in this study, we have identified novel pathways that use CARM1 as coactivator and showed that CARM1 functions as a key component of PPARγ receptor mediated gene expression. ^

MUTATIONS IN STAT3 ASSOCIATED WITH HUMAN HYPER IgE SYNDROME ENHANCE NFκB AND MAPK-MEDIATED GENE EXPRESSION

Hyper IgE syndrome (HIES) is a multisystem disorder resulting in bone and immune system abnormalities. It is associated with mutations in STAT3, which disrupt protein domains responsible for transcriptional function. Patients with HIES display osteoporosis and enhanced inflammatory cytokine production similar to hematopoietic Stat3-deficient mice. Since osteoclast and inflammatory cytokine genes are NFκB targets, these observations indicate a possible deregulation of NFκB signaling in both mice and humans with STAT3-deficiency. Here, we sought to examine the role of STAT3 in the regulation of NFκB-mediated gene expression through analysis of three HIES STAT3 point mutations in both hematopoietic and non- hematopoietic cells. We found that IL-6-induced tyrosine phosphorylation of STAT3 was partially or completely abrogated by HIES mutations in the transactivation domain (V713L) or SH2 domain (V637M), respectively, in both hematopoietic and non- hematopoietic cells. By contrast, IL-6-induced tyrosine phosphorylation of an HIES mutant in the STAT3 DNA-binding domain (R382W) was intact. The R382W and V713L mutants significantly reduced IL-6-dependent STAT3 transcriptional activity in reporter gene assays. Moreover, the R382W and V637M mutants significantly diminished IL-6-responsive expression of the endogenous STAT3 target gene, Socs3, as assessed by quantitative real-time PCR (qPCR) in the RAW macrophage cell line. These observations indicate the HIES mutants dominantly suppress the transcriptional activity of wild type STAT3, albeit to varying degrees. All three HIES mutants enhanced LPS-induced expression of the NFκB target genes IL6 (IL-6), Cxcl10 (IP- 10), and Tnf (TNFα) in RAW cells, as indicated by qPCR. Furthermore, overexpression of wild type STAT3 in Stat3-deficient murine embryonic fibroblasts significantlyreduced LPS-stimulated expression of IL6, Cxcl10, and IL12p35. In addition, in aprimary murine osteoclast differentiation assay, a STAT3-specific SH2 domain inhibitor led to significantly increased levels of osteoclast-specific gene expression. These results suggest that STAT3 serves as a negative regulator of NFκB-mediated gene expression, and furthermore imply that STAT3 mutations associated with HIES contribute to the osteopenia and inflammation observed in HIES patients.