Impact des mutations de la tyrosine phosphatase SHP2 dans la réponse à l'hormone de croissance : conséquences physiopathologiques dans le retard de croissance associé au syndrome de Noonan

Le syndrome de Noonan (SN) est une maladie génétique autosomique dominante relativement fréquente (1/2000 naissances), qui appartient à la famille des syndromes Neuro-Cardio-Facio-Cutanés (NCFC), et qui caractérisée par l'association d'une cardiopathie, d'une dysmorphie faciale et d'un retard de croissance. Dans 50% des cas, le SN est causé par des mutations sur le gène PTPN11, codant la tyrosine phosphatase SHP2. SHP2 joue un rôle essentiel dans le développement et l'homéostasie de multiples tissus et organes par sa capacité à réguler diverses voies de signalisation majeures, telles que les voies Ras/Mitogen-Activated Protein Kinase (MAPK), Phosphoinositide 3-Kinase (PI3K)/Akt et JAK2/STAT5. Si l'on sait que les mutations SN de SHP2 causent son hyperactivation, il reste à comprendre comment cette hyperactivation de SHP2 induit le développement des différents symptômes du SN et notamment le retard statural, dont l'origine reste mal comprise bien qu'il concerne 80% des patients SN. Or, des études cliniques ont révélé que les patients SN présentent des taux normaux d'hormone de croissance (Growth Hormone, GH) associés à des taux faibles d'Insulin-like-Growth-Factor-I (IGF-I), son médiateur biologique, un profil hormonal évocateur d'une résistance à GH. Ces travaux de thèse ont donc eu pour objectifs d'identifier le rôle de SHP2 en aval du récepteur de GH et de déterminer quelles étaient les conséquences physiopathologiques des mutations SN de SHP2 dans la réponse à GH. A l'aide de différents outils moléculaires et cellulaires et d'un modèle murin du SN (souris Ptpn11D61G/+), nous avons montré que SHP2 joue un rôle négatif dans la production d'IGF-I induite par GH, ce qui se traduit par des taux bas d'IGF-I chez des animaux SN en phase de croissance, en association avec un retard statural. D'un point de vue mécanistique, nous avons démontré, par différentes approches, que SHP2 joue un rôle positif dans l'activation de la voie Ras/MAPK induite par GH, sans affecter les autres voies de signalisation activées par le R-GH (JAK/STAT, PI3K/Akt). Nous avons, de plus, disséqué le mode d'action de SHP2 dans l'activation de la voie Ras/MAPK, en montrant que SHP2 joue un rôle positif dans l'activation de Ras en déphosphorylant la protéine adaptatrice Gab1 sur ses sites de recrutement pour un inhibiteur naturel de Ras, RASGAP. Enfin, nous avons pu faire le lien entre ces 2 processus dépendants de SHP2 sous induction par GH, en montrant que l'inhibition des MAPK induisait une augmentation de la production d'IGF-I induite par GH, in vitro et in vivo. De façon importante, cette restauration des taux d'IGF-I chez les souris SN s'accompagne d'une amélioration de leur croissance, suggérant que le ciblage de la voie Ras/MAPK puisse avoir un effet bénéfique sur le retard de croissance des sujets SN. A l'appui de cela, plusieurs études récentes ont montré un effet bénéfique d'un tel traitement sur d'autres traits phénotypiques (cardiopathies, dysmorphie).Noonan syndrome (NS) is a common congenital autosomal dominant disorder (estimated incidence 1/2000 live births), that belongs to the family of Neuro-Cardio-Facio-Cutaneous (NCFC) syndromes and is characterized by heart defects, facial dysmorphic features and growth retardation. Nearly half of the NS patients carry a mutation of the PTPN11 gene, encoding the tyrosine phosphatase SHP2, that plays a key role in development and homeostasis of multiple tissues and organs, through its ability to regulate various signaling pathways, including the Ras/Mitogen-Activated Protein Kinase (MAPK), Phosphoinositide 3-Kinase (PI3K)/Akt and JAK2/STAT5 module. It is known that NS-causing mutations induce a hyperactivation of the phosphatase. However, it remains to be established how this hyperactivation is responsible for the development of the different symptoms of the disease. This is notably the case for short stature, which origin is poorly understood although it concerns 80% of NS patients. Clinical studies have revealed that NS patients display normal level of growth hormone (GH) but low level of Insulin-like-Growth-Factor-I (IGF-I), its biological mediator, a hormonal profile that is evocative of GH resistance. This project thus aimed at identifying the role of SHP2 downstream of the GH receptor and at determining the pathophysiological outcomes of SN-causing SHP2 mutations in GH response. Using different molecular and cellular tools as well as a mouse model of NS (Ptpn11D61G/+ mice), we have shown that SHP2 plays a negative role in GH-induced IGF-I production, which results in low IGF-I levels in growing NS animals, in association with short stature. On a mechanistic point of view, we demonstrated through several approaches, that SHP2 plays a positive role in GH-evoked Ras/MAPK activation, without affecting other signaling pathways activated by the GH-R (JAK/STAT, PI3K/Akt). Moreover, we deciphered how SHP2 acts in the Ras/MAPK pathway, showing that it participates in Ras activation by dephosphorylating the adaptor Gab1 on its binding site for RASGAP, a natural inhibitor of Ras. Lastly, we could link these 2 SHP2-dependent processes under GH induction, showing that MAPK inhibition results in an increase in GH-induced IGF-I production in vitro and in vivo. Importantly, restoring IGF-I level in young NS animals results in an improvment of their growth parameters, suggesting that targeting that Ras/MAPK pathway could be beneficial on growth retardation in NS subjects. Supporting this view, recents reports have shown that such strategy could be effective to alleviate other NS features, notably cardiopathy and dysmorphic abnormalities

The HMG box transcription factor HBP1: a cell cycle inhibitor at the crossroads of cancer signaling pathways

HMG box protein 1 (HBP1) is a transcription factor and a potent cell cycle inhibitor in normal and cancer cells. HBP1 activates or represses the expression of different cell cycle genes (such as CDKN2A, CDKN1A, and CCND1) through direct DNA binding, cofactor recruitment, chromatin remodeling, or neutralization of other transcription factors. Among these are LEF1, TCF4, and MYC in the WNT/beta-catenin pathway. HBP1 also contributes to oncogenic RAS-induced senescence and terminal cell differentiation. Collectively, these activities suggest a tumor suppressor function. However, HBP1 is not listed among frequently mutated cancer driver genes. Nevertheless, HBP1 expression is lower in several tumor types relative to matched normal tissues. Several micro-RNAs, such as miR-155, miR-17-92, and miR-29a, dampen HBP1 expression in cancer cells of various origins. The phosphatidylinositol-3 kinase (PI3K)/AKT pathway also inhibits HBP1 transcription by preventing FOXO binding to the HBP1 promoter. In addition, AKT directly phosphorylates HBP1, thereby inhibiting its transcriptional activity. Taken together, these findings place HBP1 at the center of a network of micro-RNAs and oncoproteins that control cell proliferation. In this review, we discuss our current understanding of HBP1 function in human physiology and diseases

Impact des mutations de la tyrosine phosphatase SHP2 dans la réponse à l'hormone de croissance (conséquences physiopathologiques dans le retard de croissance associé au syndrome de Noonan)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

HBP1 phosphorylation by AKT regulates its transcriptional activity and glioblastoma cell proliferation.

The HMG-box protein 1 (HBP1) is a transcriptional regulator and a potential tumor suppressor that controls cell proliferation, differentiation and oncogene-mediated senescence. In a previous study, we showed that AKT activation through the PI3K/AKT/FOXO pathway represses HBP1 expression at the transcriptional level in human fibroblasts as well as in cancer cell lines. In the present study, we investigated whether AKT could also regulate HBP1 directly. First, AKT1 phosphorylated recombinant human HBP1 in vitro on three conserved sites, Ser380, Thr484 and Ser509. In living cells, we confirmed the phosphorylation of HBP1 on residues 380 and 509 using phospho-specific antibodies. HBP1 phosphorylation was induced by growth factors, such as EGF or IGF-1, which activated AKT. Conversely, it was blocked by treatment of cells with an AKT inhibitor (MK-2206) or by AKT knockdown. Next, we observed that HBP1 transcriptional activity was strongly modified by mutating its phosphorylation sites. The regulation of target genes such as DNMT1, P47phox, p16INK4A and cyclin D1 was also affected. HBP1 had previously been shown to limit glioma cell growth. Accordingly, HBP1 silencing by small-hairpin RNA increased human glioblastoma cell proliferation. Conversely, HBP1 overexpression decreased cell growth and foci formation. This effect was amplified by mutations that prevented phosphorylation by AKT, and blunted by mutations that mimicked phosphorylation. In conclusion, our results suggest that HBP1 phosphorylation by AKT blocks its functions as transcriptional regulator and tumor suppressor

The expression of the tumor suppressor HBP1 is down-regulated by growthfactors via the PI3K – PKB – FOXO pathway

Growth factors inactivate the Forkhead-box O (FOXO) transcription factors through phosphatidylinositol-3 kinase (PI3K) and protein kinase B (PKB). By comparing microarray data from multiple model systems, we identified the HMG-box protein 1 (HBP1) as a novel downstream target of this pathway. HBP1 mRNA was down-regulated by PDGF, FGF, PI3K and PKB, while it was up-regulated by FOXO factors. This observation was confirmed in human and murine fibroblasts as well as in cell lines derived from leukemia, breast adenocarcinoma and colon carcinoma. Bioinformatics analysis led to the identification of a conserved consensus FOXO-binding site in the HBP1 promoter. By luciferase activity assay and chromatin immunoprecipitation, we demonstrated that FOXO bound to this site and regulated the HBP1 promoter activity in a PI3K-dependent manner. Silencing of HBP1 by small-hairpin RNA (shRNA) increased human fibroblasts proliferation in response to growth factors, suggesting that HBP1 limits cell growth. Finally, by analyzing a transcriptomics data set from The Cancer Genome Atlas, we observed that HBP1 expression was lower in breast tumors that had lost FOXO expression. In conclusion, HBP1 is a novel target of the PI3K - FOXO pathway and controls cell proliferation in response to growth factor

The gut microbiota metabolite indole alleviates liver inflammation in mice

The gut microbiota regulates key hepatic functions, notably through the production of bacterial metabolites that are transported via the portal circulation. We evaluated the effects of metabolites produced by the gut microbiota from aromatic amino acids (phenylacetate, benzoate, p-cresol, and indole) on liver inflammation induced by bacterial endotoxin. Precision-cut liver slices prepared from control mice, Kupffer cell (KC)-depleted mice, and obese mice ( ob/ ob) were treated with or without LPS and bacterial metabolites. We observed beneficial effects of indole that dose-dependently reduced the LPS-induced up-regulation of proinflammatory mediators at both mRNA and protein levels in precision-cut liver slices prepared from control or ob/ ob mice. KC depletion partly prevented the antiinflammatory effects of indole, notably through a reduction of nucleotide-binding domain and leucine-rich repeat containing (NLR) family pyrin domain-containing 3 (NLRP3) pathway activation. In vivo, the oral administration of indole before an LPS injection reduced the expression of key proteins of the NF-κB pathway and downstream proinflammatory gene up-regulation. Indole also prevented LPS-induced alterations of cholesterol metabolism through a transcriptional regulation associated with increased 4β-hydroxycholesterol hepatic levels. In summary, indole appears as a bacterial metabolite produced from tryptophan that is able to counteract the detrimental effects of LPS in the liver. Indole could be a new target to develop innovative strategies to decrease hepatic inflammation.-Beaumont, M., Neyrinck, A. M., Olivares, M., Rodriguez, J., de Rocca Serra, A., Roumain, M., Bindels, L. B., Cani, P. D., Evenepoel, P., Muccioli, G. G., Demoulin, J.-B., Delzenne, N. M. The gut microbiota metabolite indole alleviates liver inflammation in mice.status: publishe

The gut microbiota metabolite indole alleviates liver inflammation in mice.

The gut microbiota regulates key hepatic functions, notably through the production of bacterial metabolites that are transported via the portal circulation. We evaluated the effects of metabolites produced by the gut microbiota from aromatic amino acids (phenylacetate, benzoate, p-cresol, and indole) on liver inflammation induced by bacterial endotoxin. Precision-cut liver slices prepared from control mice, Kupffer cell (KC)-depleted mice, and obese mice ( ob/ ob) were treated with or without LPS and bacterial metabolites. We observed beneficial effects of indole that dose-dependently reduced the LPS-induced up-regulation of proinflammatory mediators at both mRNA and protein levels in precision-cut liver slices prepared from control or ob/ ob mice. KC depletion partly prevented the antiinflammatory effects of indole, notably through a reduction of nucleotide-binding domain and leucine-rich repeat containing (NLR) family pyrin domain-containing 3 (NLRP3) pathway activation. In vivo, the oral administration of indole before an LPS injection reduced the expression of key proteins of the NF-κB pathway and downstream proinflammatory gene up-regulation. Indole also prevented LPS-induced alterations of cholesterol metabolism through a transcriptional regulation associated with increased 4β-hydroxycholesterol hepatic levels. In summary, indole appears as a bacterial metabolite produced from tryptophan that is able to counteract the detrimental effects of LPS in the liver. Indole could be a new target to develop innovative strategies to decrease hepatic inflammation.-Beaumont, M., Neyrinck, A. M., Olivares, M., Rodriguez, J., de Rocca Serra, A., Roumain, M., Bindels, L. B., Cani, P. D., Evenepoel, P., Muccioli, G. G., Demoulin, J.-B., Delzenne, N. M. The gut microbiota metabolite indole alleviates liver inflammation in mice

PDGFRB gain-of-function mutations in sporadic infantile myofibromatosis.

Infantile myofibromatosis is one of the most prevalent soft tissue tumors of infancy and childhood. Multifocal nodules with visceral lesions are associated with a poor prognosis. A few familial cases have been linked to mutations in various genes including PDGFRB. In this study, we sequenced PDGFRB, which encodes a receptor tyrosine kinase, in 16 cases of myofibromatosis or solitary myofibroma. Mutations in the coding sequence of PDGFRB were identified in 6 out of 8 patients with the sporadic multicentric form of the disease and in 1 out of 8 patients with isolated myofibroma. Two patients had the same mutation in multiple separated lesions. By contrast, a third patient had three different PDGFRB mutations in the three nodules analyzed. Mutations were located in the transmembrane, juxtamembrane and kinase domains of the receptor. We showed that these mutations activated receptor signaling in the absence of ligand and transformed fibroblasts. In one case, a weakly-activating germline variant was associated with a stronger somatic mutation, suggesting a two-hit model for familial myofibromatosis. Furthermore, the mutant receptors were sensitive to the tyrosine kinase inhibitor imatinib, except D850V, which was inhibited by dasatinib and ponatinib, suggesting a targeted therapy for severe myofibromatosis. In conclusion, we identified gain-of-function PDGFRB mutations in the majority of multifocal infantile myofibromatosis cases, shedding light on the mechanism of disease development, which is reminiscent of multifocal venous malformations induced by TIE2 mutations. Our results provide a genetic test to facilitate diagnosis, and preclinical data for development of molecular therapies

LEOPARD syndrome-associated SHP2 mutation confers leanness and protection from diet-induced obesity

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