La résistance aux anti-aromatases (AAs) constitue un obstacle thérapeutique majeur dans le traitement des cancers du sein RE+. Les objectifs de ce travail étaient : (i) de caractériser les événements moléculaires associés à la résistance acquise aux AAs ; (ii) d’identifier de manière globale de nouveaux profils de miRNAs spécifiquement associés à la résistance aux AAs. Notre étude a mis en évidence le rôle central de la voie Akt/mTOR dans la résistance acquise et de novo aux AAs dans des modèles cellulaires, mais également dans des échantillons de patientes ayant récidivé sous anastrozole. La combinaison d’un AA avec le MK-2206, inhibiteur d’Akt ou avec la rapamycine, inhibiteur de mTOR, augmente la sensibilité à l’AA dans les cellules contrôles et est suffisante pour surmonter la résistance et restaurer la sensibilité à l'hormonothérapie dans les cellules résistantes. Notre travail propose également un modèle de résistance acquise aux AAs basé sur la sélection de cellules « cancer-initiating-like » dotées de propriétés d'auto-renouvellement, d’une résistance intrinsèque aux AAs et d’une sensibilité au MK-2206. Notre étude à grande échelle des miRNAs a identifié la voie Akt/mTOR comme une des cibles privilégiées de ces miRNAs. Nous avons identifié et validé trois miRNAs dérégulés capables de moduler le statut d’activation de la voie Akt/mTOR, qui représentent des cibles potentielles. En conclusion, notre projet a mis en évidence de nouvelles voies de signalisations ciblées par ces miRNAs et de nouveaux évènements moléculaires, qui représentent des candidats potentiels dans la résistance aux AAs / Resistance to aromatase inhibitors (AIs) remains a major drawback in the treatment of ER+ breast cancers. Our objectives were (i) to characterize molecular events associated with acquired AI resistance (ii) to capture a global view of the miRNA expression profiles associated with AI resistance. Our results showed the major role of the Akt/mTOR pathway in both de novo and acquired resistance to AI in cellular models and also in breast tumors of patients who relapsed under anastrozole. Combining AI with the Akt inhibitor MK-2206 or with the mTOR inhibitor rapamycin increased sensitivity to this AI in the control cells and was sufficient to overcome resistance and restore sensitivity to endocrine therapy in the resistant cells. Our findings propose a model of AI-acquired resistance based on the selection of cancer-initiating-like cells possessing self-renewing properties, intrinsic resistance to AI and sensitivity to MK-2206. Our large-scale study identified the Akt/mTOR pathway as one of the main targets of the deregulated miRNAs. We identified and validated three miRNAs able to modulate the Akt/mTOR activation status, suggesting these miRNAs as potential targets. To conclude, our project identified new miRNA-targeted signaling pathways and new molecular events, representing strong candidates in the mediation of AI resistance

Les β2-agonistes sont couramment utilisés pour prévenir et réduire les symptômes de l'asthme et de la bronchoconstriction induite par l'exercice. Mais, pris en quantités supérieures aux doses thérapeutiques, les β2-agonistes ont un effet anabolisant qui a été clairement démontré in vivo. Un certain nombre d’acteurs sont mis en jeu dans la réponse biologique du tissu musculaire aux β2-agonistes. L’un de ces acteurs est la voie de signalisation PI3K/Akt/mTOR, voie d’initiation de la traduction, ayant un rôle majeur dans la synthèse protéique. Dans ce contexte, notre première étude avait pour objectif de déterminer la cinétique des événements moléculaires responsables de l’hypertrophie du muscle squelettique de rat après administration de formotérol pendant 1 jour (J1), 3 jours (J3) et 10 jours (J10). Nous avons montré que l’administration de formotérol induisait une hypertrophie musculaire à J3 et J10 associée à l’activation transitoire de la voie de signalisation PI3K/Akt/mTOR (J1 et J3), et à une diminution de l’expression de l’E3 ubiquitine ligase MAFbx/Atrogin-1 (J3). La voie autophagie lysosome ne semblait pas être affectée. Ainsi, l’ensemble de ces résultats suggère que l’activation de la voie PI3K/Akt/mTOR est associée à la voie ubiquitine-protéasome mais pas à la voie autophagie-lysosome. La régulation transitoire de la voie PI3K/Akt/mTOR suggère que d’autres voies de signalisation sont impliquées dans l’hypertrophie musculaire induite par le formotérol. Le 007-AM, analogue de l’AMPc, a été décrit comme pouvant stimuler la voie de signalisation PI3K/Akt/mTOR via l’activation de la protéine Epac, suggérant que le 007-AM puisse constituer une molécule de substitution à l’utilisation des β2-agonistes. Notre seconde étude avait pour but de déterminer si le 007-AM avait une action anabolisante sur le tissu musculaire, mais également de déterminer si la 007-AM était une molécule stable permettant d’envisager son usage dans un cadre pharmacologique. L’administration de 007-AM pendant 7 jours chez des souris n’engendrait pas d’hypertrophie musculaire. En revanche, in vitro sur cellules C2C12, le 007-AM activait la voie de signalisation PI3K/Akt/mTOR comme en témoignait l’augmentation de la phosphorylation des protéines rpS6 et 4E-BP1. Nos résultats montraient également que le 007-AM était instable dans le plasma alors que son produit de dégradation, le 007 était plus stable. Pris ensembles, ces résultats suggèrent qu’un traitement de 7 jours au 007-AM n’est pas suffisant pour induire une hypertrophie musculaire et que l’absence d’hypertrophie musculaire pourrait provenir de l’instabilité du 007-AM dans le plasma. Toutefois, des études supplémentaires seront nécessaires pour confirmer ces résultats / Β2-agonists are traditionally used to prevent and reduce asthma symptoms and bronchoconstriction induced by exercise. Nevertheless, when administrated in vivo, at relatively high, far away from therapeutic doses, β2-agonists induce anabolic effects. Numerous actors are involved in biological response of the skeletal muscle, induced by β2-agonists. PI3K/Akt/mTOR signaling pathway, which initiates translation, is one of these actors. In this context, our first study aimed at determined the kinetic of molecular events responsible for skeletal muscle hypertrophy after 1 day (D1), 3 days (D3) and 10 days (D10) of formoterol administration. We have shown that formoterol administration induced skeletal muscle hypertrophy at D3 and D10 associated with a transient activation of PI3K/Akt/mTOR signaling pathway (D1 and D3), and, with a decrease in E3 ubiquitin ligase MAFbx/atrogin-1 expression (D3). The autophagy-lysosome pathway seems not to be regulated by formoterol administration. Taken together, these results suggest that PI3K/Akt/mTOR activation is temporally associated with the regulation of ubiquitin-proteasome but not the autophagy-lysosome pathway. The transient nature of the regulation of PI3K/Akt/mTOR signaling pathway also indicates that other unidentified pathways are probably activated to sustain the increase in skeletal muscle mass. Recently, 007-AM synthetic molecule has been described to stimulate PI3K/Akt/mTOR signaling pathway through Epac protein activation, suggesting that 007-AM could be an alternative to the use of β2-agonists. The purpose of our second study was to determine whether 007-AM had an anabolic action on skeletal muscle and if 007-AM was stable allowing considering its use in pharmacology. 007-AM administration for 7 days to mice does not lead to muscle hypertrophy. Nonetheless, in vitro on C2C12 cells, 007-AM activated PI3K/Akt/mTOR signaling pathway by increasing phosphorylation of rpS6 and 4E-BP1. Our results showed that contrary to 007, 007-AM was instable in plasma. Altogether, these results suggest that a 7-day 007-AM treatment is not sufficient to induce skeletal muscle hypertrophy. This lack of hypertrophy could be due to 007-AM instability in plasma. However, supplemental studies are needed to confirm these results

The influenza A virus non-structural (NS1) protein is multifunctional, and during virus-infection NS1 interacts with several factors in order to manipulate host-cell processes. This study reports that NS1 binds directly to p85β, a regulatory subunit of phosphoinositide 3-kinase (PI3K), but not to the related p85α. Expression of NS1 was sufficient to activate PI3K and cause the phosphorylation of a downstream mediator of PI3K signalling, Akt. However, in virus-infected MDCK cells, the kinetics of Akt phosphorylation did not correlate with NS1 expression, and suggested that negative regulation of this signalling pathway occurs subsequent to ~8h post-infection. Mapping studies showed that the NS1:p85β interaction is primarily mediated by the NS1 C-terminal domain and the p85β inter-SH2 (Src homology 2) domain. Additionally, the highly conserved tyrosine at residue 89 (Y89) of NS1 was found to be important for binding and activating PI3K in a phosphorylation-independent manner. The inter-SH2 domain of p85β is a coiled-coil structure that acts as a scaffold for the p110 catalytic subunit of PI3K. As NS1 does not displace p110 from the inter-SH2 domain, a model is proposed whereby NS1 forms an active heterotrimeric complex with PI3K, and disrupts the ability of p85β to control p110 function. Biological studies revealed that a mutant influenza A virus (Udorn/72) expressing NS1 with phenylalanine substituted for tyrosine-89 (Y89F) exhibited a small-plaque phenotype, and grew more slowly in MDCK cells than wild-type virus. Unexpectedly, another mutant influenza A virus strain (WSN/33) expressing NS1-Y89F was not attenuated in MDCK cells, yet appeared to be less pathogenic than wild-type in vivo. Overall, these data indicate a role for NS1-mediated PI3K activation in efficient influenza A virus replication. The potential application of this work to the design of novel anti-influenza drugs and vaccine production is discussed.

In this study, we investigated differential activation and the role of two mTOR complexes in cell migration of prostate cancer cells. Specific knock-down of endogenous RAPTOR and RICTOR by siRNA resulted in decreased cell migration in LNCaP, DU145, and PC3 cells indicating that both mTORC1 and mTORC2 are required for cell migration. EGF treatment induced the activation of both mTORC1 and mTORC2 as determined by complex-specific phosphorylation of mTOR protein. Specific knock-down or inhibition of Rac1 activity in PC3 cells blocked EGF-induced activation of mTORC2, but had no effect on mTORC1 activation. Furthermore, the over-expression of constitutively active Rac1 (Rac1Q61L) resulted in significant increase in cell migration and activation of mTORC2 in PC3 cells, but had no effect on mTORC1 activation. Constitutively active Rac1 (Rac1Q61L) in PC3 cells was localized in the plasma membrane and was found to be in a protein complex which contained mTOR and RICTOR proteins, but not RAPTOR. In conclusion, we suggested that EGF-induced activation of Rac1 causes the phosphorylation/activation of mTORC2 via RICTOR, specific regulator of mTORC2 activation in numerous cancer cells. The major role played by mTOR in a wide array of cancers has in the recent decades led to the development of numerous mTOR inhibitors. One of the drawback of these first generation mTOR inhibitors are that m TORC1 activity is inhibited but effect on mTORC2 activity require high dosages and prolonged exposure in different cancer cell types including HeLa, PC3, LNCaP, and A549. High dosage of rapamycin and its associated rapalogs required for mTORC2 inhibition is clinically unsuitable. Studies have shown that the dual mTORC1/C2 inhibitors trigger feedback loops causing metastasis and affect the cell viability of normal tissues in vitro and in vivo. There is a need for specific mTORC1 and mTORC2 inhibitor, which overcome the disadvantages of the previously developed mTOR inhibitors. The Rac1-RICTOR axis suggested in this study could be used as a potential target for the development of mTORC2 inhibitor and lead to a potential therapeutic treatment for aggressive prostate cancer.

Tumor suppressor genes play a role as a growth regulator and a gatekeeper of a cell. Their inactivation is often detected in malignant tumors. Identification of novel tumor suppressor gene candidates may help to further understand tumorigenesis and aid in the discovery of a new treatment leading toward cure of cancer. This PhD research project aimed to understand functional significance of a novel tumor suppressor gene candidate, myosin IC (MYO1C) and to identify potential interaction(s) of the MYO1C protein with key components of the signaling pathways involving in cancer development. In an experimental rat model for endometrial carcinoma (EC), detailed molecular genetic analysis of a candidate tumor suppressor region located distal to the tumor protein 53 (Tp53) suggested the myosin IC gene (Myo1c) as the best potential target for deletion of the genetic material. The question arising was whether and how MYO1C could function as a tumor suppressor gene. By using qPCR, Western blot or immunohistochemistry analyses, we examined MYO1C protein level in panels of well-stratified human colorectal cancer (CRC) and EC respectively. We found that MYO1C was significantly down-regulated in these cancer materials and that for the EC panel, the observed down-regulation of MYO1C correlated with tumor stage, where tumors at more advanced stages had less expression of MYO1C. In cell transfection experiments, we found that over-expression of MYO1C significantly decreased cell proliferation, and silencing MYO1C with siRNA increased cell viability. Additionally, knockdown of MYO1C impaired the ability of cells to migrate, spread and adhere to the surface. Recent published studies suggested a potential interplay between MYO1C and the phosphoinositide 3-kinase (PI3K)/AKT pathway. To examine this hypothesis, we analyzed the expression and/or activation of components of the PI3K/AKT and RAS/ERK signaling pathways in vivo in CRC samples, and in vitro in cells transfected with the MYO1C gene expression construct or MYO1C-targeted siRNA. To identify other potential pathways/ mechanisms through which MYO1C may exert its tumor suppressor activity, we additionally performed new sets of MYO1C-siRNA knockdown experiments. At different time points post transfection, we performed microarray global gene expression experiments followed by bioinformatics analysis of the data. Altogether, the results suggested an early PI3K/AKT response to altered MYO1C expression. We additionally identified several cancer-related genes/pathways with late response to MYO1C knockdown. All things considered, the identification of MYO1C-expression impact on cell proliferation, migration, and adhesion in combination with its interplay between several cancer-related genes and signaling pathways provide further evidence for the initial hypothesis of a tumor suppressor activity of MYO1C. / Cellular, Molecular and Functional Characterization of the Tumor Suppressor Candidate MYO1C

No description available.

Cardiovascular disease is a leading cause of death in the United States. Toll-like receptor (TLR)-mediated pathways have been demonstrated to play a role in myocardial ischemia/reperfusion (I/R) injury. We and others have shown that PI3K/Akt signaling is involved in regulating cellular survival and protecting the myocardium from I/R induced injury. In this dissertation, we provide compelling evidence that miR-125b serves to “fine tune” TLR mediated NF-kB responses by repressing TNF-a and TRAF6 expression. We constructed lentiviral expressing miR-125b, delivered it into the myocardium. The data showed that delivery of lentivirus expressing miR-125b significantly reduces myocardial infarct size and improves cardiac function in I/R hearts. Mechanistic studies demonstrated that miR-125b negatively regulates TLR mediated NF-kB activation pathway by repressing TNF-a and TRAF6 expression in the myocardium. We also observed that transfection of the myocardium with lentivirus expressing miR-214 markedly attenuates I/R induced myocardial infarct size and cardiac dysfunction. We demonstrated that miR-214 activates PI3K/Akt signaling by targeting PTEN expression in the myocardium. We also investigated the role of TLR3 in neonatal heart repair and regeneration following myocardial infarction (MI). Wild type (WT) neonatal mice showed fully cardiac functional recovery and small infarct size, while TLR3 deficient mice exhibited impaired cardiac functional recovery and large infarct area after MI. Poly (I:C), a TLR3 ligand, administration significantly enhances glycolysis, YAP1 activation and the proliferation of WT neonatal cardiomyocytes. 2-deoxyglucose (2-DG), a glycolysis inhibitor treatment abolished cardiac functional recovery and YAP1 activation in neonatal mice after MI. In vitro either inhibition of glycolysis by 2-DG or inhibition of YAP1 activation prevents Poly (I:C) induced YAP1 activation and neonatal cardiomyocyte proliferation. Importantly, YAP1 activation increases miR-152 expression, leading to cardiomyocyte proliferation through suppression P27kip1 and DNMT1 expression. We conclude that microRNAs play an important role in TLR modulation induced protection against myocardial I/R injury by increasing the activation of PI3K/Akt signaling pathway, decreasing TLR/NF-kB mediated inflammatory response, and suppressing activation of apoptotic signaling following myocardial I/R injury. In addition, TLR3 is an essential for neonatal heart repair and regeneration after myocardial infarction. TLR3 modulation could be a novel strategy for heart regeneration and repair.

Abstract : Long-term survival of T lymphocytes in a quiescent state is essential to maintain their cell numbers in secondary lymphoid organs. Interaction of the T cell antigen receptor (TCR) with self-peptide/MHC synergizes with IL-7-induced anti-apoptotic signals to promote T cell survival. These extrinsic stimuli are also implicated in T cell metabolism and survival by regulating several signaling pathways including the phosphatidyl-inositol-3 kinase (PI3K)/Akt pathway. In mice and in rats, loss of functional GTPase of the immune associated nucleotide binding protein 5 (GIMAP5) causes peripheral T lymphopenia due to spontaneous death of T cells. The underlying mechanisms responsible for the pro-survival function of GIMAP5 in T lymphocytes remain largely unknown. Previous work from my laboratory has shown that T cells from GIMAP5-deficient rats show reduced influx of calcium (Ca[superscript 2+]) from the extracellular milieu following stimulation of the TCR complex. In this thesis, I characterized the mechanism by which GIMAP5 regulates Ca[superscript 2+] homeostasis, and elucidated the signaling pathways modulated by GIMAP5 to facilitate the survival of T cells. Firstly, I investigated if GIMAP5 prevents apoptotic death of T lymphocytes by affecting the Ca[superscript 2+] buffering capacity of mitochondria, which is required for sustained Ca[superscript 2+] influx via the plasma membrane channels. I observed that mitochondrial Ca[superscript 2+] accumulation following capacitative Ca[superscript 2+] entry is defective in T cells from Gimap5 deficient rats. Disruption of microtubules, but not the actin cytoskeleton, abrogated Ca[superscript 2+] sequestration by mitochondria in T cells from control but not Gimap5 deficient mice. Similarly, mice lacking functional GIMAP5 displayed defective T cell development and Ca[superscript 2+] influx. Furthermore, I observed that the proximal signaling events following TCR stimulation was reduced and was accompanied by defective proliferation in T cells from Gimap5 deficient mice. Additionally, IL-7-induced STAT5 phosphorylation was decreased in CD4[superscript +] T cells from Gimap5 deficient mice. I also showed that loss of functional Gimap5 results in increased basal activation of mammalian target of rapamycin (mTOR), independent of protein phosphatase 2A (PP2A) or AMP-activated protein kinase (AMPK). Instead, the constitutive activation the PI3K pathway contributed to the spontaneous high mTOR activation. Collectively, my observations suggest that the pro-survival function of GIMAP5 in T-lymphocytes may be linked to the regulation of diverse signaling pathways in a context dependent manner. GIMAP5 also facilitates microtubule-dependent mitochondrial buffering of Ca[superscript 2+] following capacitative entry. GIMAP5 is required to integrate the survival signals generated following activation through TCR and IL-7R. / Résumé : La survie à long terme des lymphocytes T en état de repos est essentielle pour maintenir leurs nombres dans les organes lymphoïdes secondaires. Le récepteur antigénique des cellules T (TCR) en contact avec les peptides du soi / CMH et en synergie avec l'IL-7 induit des signaux anti-apoptotiques pour favoriser la survie des cellules T. Ces stimuli extrinsèques sont également impliqués dans le métabolisme et la survie des cellules T grâce à la régulation de plusieurs voies de signalisation dont la voie phosphatidyl-inositol-3 kinase (PI3K) /AKT. Chez la souris et chez le rat, la perte de l’activité de GIMAP5 (GTPase of Immune Associated Nucleotide Binding Protein 5), provoque une lymphopénie T périphérique en raison de la mort spontanée des cellules T. Le mécanisme sous-jacent responsable de la fonction de survie de GIMAP5 dans les lymphocytes T reste largement inconnu. Nous avons observé que les cellules de rats déficients en GIMAP5, après stimulation par complexe TCR, montrent un afflux de calcium (Ca[indice supérieur 2+]) réduit provenant du milieu extracellulaire. Dans cette thèse, J’ai caractérisé le mécanisme d’action de GIMAP5 dans la régulation de l'homéostasie du Ca[indice supérieur 2+], ainsi que les voies de signalisation modulées par GIMAP5 pour faciliter la survie des cellules T. Tout d'abord, j’ai étudié si GIMAP5 empêche l’apoptose des lymphocytes T en affectant la capacité des mitochondries à réguler la concentration du Ca[indice supérieur 2+], ce qui est nécessaire pour soutenir l’influx de Ca[indice supérieur 2+]. J’ai trouvé que l’accumulation du Ca[indice supérieur 2+] mitochondrial après l’entrée capacitive de Ca[indice supérieur 2+] est défectueuse dans les cellules T de rat déficientes en Gimap5. La disruption des microtubules, mais pas du cytosquelette d'actine, abroge la séquestration du Ca[indice supérieur 2+] mitochondrial dans les cellules T primaires de rat, mais pas dans les cellules T déficientes en Gimap5. J’ai observé que les cellules T provenant de souris déficientes en Gimap5 démontrent une diminution de l’entrée de Ca[indice supérieur 2+]. De plus, la prolifération des cellules T déficientes en Gimap5 est diminuée suite à la stimulation du TCR. En outre, la phosphorylation de STAT5 induit par l'IL-7 est diminuée dans les cellules T CD4[indice supérieur +] de souris déficientes en Gimap5. Également, la perte de Gimap5 aboutit à une activation accrue de la cible mammalienne de la rapamycine (mTOR), indépendamment de la protéine phosphatase 2A (PP2A) ou de la protéine kinase activée par l'AMP (AMPK). Au lieu de cela, l'activation constitutive de la voie PI3K contribue à une forte activation spontanée de mTOR. Collectivement, la fonction de survie de GIMAP5 dans les lymphocytes T peut être liée à la régulation de différentes voies de signalisation. GIMAP5 facilite la fonction, microtubule dépendant, des mitochondries dans leurs actions de régulation du Ca[indice supérieur 2+] après l’entrée capacitive de Ca[indice supérieur 2+]. GIMAP5 est nécessaire pour intégrer les signaux de survie produits suite à l'activation du TCR et de l’IL-7R, qui pourrait être associée à la régulation de l'activité PI3K / AKT / mTOR.

Innate immune and inflammatory responses contribute to myocardial and cerebral ischemia/reperfusion (I/R) injury. Toll-like receptors (TLRs) play a critical role in the induction of innate immune and inflammatory responses via activation of nuclear factor kappa B (NF-κB). We have shown that activation of NF-κB contributes to myocardial and cerebral I/R injury. Indeed, inhibition of TLR4-mediated NF-κB activation significantly decreased myocardial and cerebral I/R injury via activation of PI3K/Akt signaling. PI3K/Akt signaling is an important pathway in regulating cellular survival and inflammatory responses. Therefore, an important question is how to differentially modulate PI3K/Akt signaling and TLR/NF-κB-mediated signaling pathway during I/R injury? We demonstrated that pretreatment of mice with Pam3CSK4, a specific TLR2 ligand, significantly decreased cerebral I/R injury and improved neuronal functional recovery. Importantly, therapeutic administration of Pam3CSK4 also markedly decreased cerebral I/R injury. The mechanisms involved suppression of NF-κB binding activity and activation of PI3K/Akt signaling. We also demonstrated that CpG-ODN, a specific TLR9 ligand, induced protection against cerebral I/R injury via activation of PI3K/Akt signaling. Our findings were consistent with our previous reports showing that administration of Pam3CSK4 or CpG-ODN protected against myocardial I/R injury via a PI3K/Akt-dependent mechanism. In addition, we demonstrated for the first time that TLR3 located in endosomes played a deleterious role in myocardial I/R injury via activation of NF-κB. To investigate how to activate PI3K/Akt signaling during I/R injury, we examined the role of microRNA (miRs) in regulating PI3K/Akt signaling during myocardial ischemic injury. We discovered that Pam3CSK4 or CpG-ODN treatment significantly increased the expression of miR-130a in the myocardium, while myocardial infarction markedly decreased the levels of miR-130a in the myocardium. The data indicated that miR-130a served a protective role in myocardial ischemic injury. Indeed, we demonstrated for the first time that increased expression of miR-130a significantly attenuated cardiac dysfunction and promoted angiogenesis after myocardial infarction. The mechanisms involved activation of PI3K/Akt signaling via targeting PTEN expression by microRNA-130a. This dissertation discovers novel mechanisms of cerebral and myocardial ischemic injury and provides solid basis for developing new approaches for the treatment and management of stroke and heart attack patients.

Insulin-like growth factor-1 (IGF-1), a known secretory product of intestinal subepithelial myofibroblasts (ISEMF), is essential for the intestinotrophic effects of glucagon-like peptide-2(GLP-2). I hypothesized that GLP-2 increases the production of IGF-1 by primary murine ISEMF in culture. Immunocytochemistry showed that the ISEMF stained appropriately for α smooth muscle actin and vimentin but not for desmin. The ISEMF also expressed GLP-2 receptor and IGF-1 mRNA transcripts. ISEMF treated with GLP-2 revealed a maximal increase in IGF-1 mRNA transcript levels at 10-8 M GLP-2 and 2hr. Interestingly, immunoblotting revealed an increase in P-AKT/T-AKT with GLP-2, but no changes in cAMP, P-ERK/T-ERK or calcium were detected. PI3K inhibition and kinase-dead AKT over-expression abrogated GLP-2-induction of IGF-1 mRNA, and ISEMF from GLP-2R null mice demonstrated reductions in IGF-1 mRNA and cellular IGF-1, but not in media IGF-1, vs. wild-type ISEMF. These findings suggest a possible mechanism by which GLP-2 increases intestinal growth in-vivo.