Developmental Expression and Cellular Origin of the Laminin α2, α4, and α5 Chains in the Intestine

AbstractLaminins are extracellular matrix glycoproteins that are involved in various cellular functions, including adhesion, proliferation, and differentiation. In this study, we examine the expression patterns and the cellular origins of the laminin α2, α4, and α5 chains in the developing mouse intestine and inin vitromouse/chick or chick/mouse interspecies hybrid intestines.In situhybridization and Northern blot analysis revealed that mRNA levels for all three laminin α chains are highest in the fetal intestine undergoing intense morphogenetic movements. Laminin α4 mRNA and polypeptide are associated with mesenchyme-derived cell populations such as endothelium and smooth muscle. In contrast, laminin α2 and α5 chains participate in the structural organization of the subepithelial basement membrane and, in the mature intestine, show a complementary pattern of expression. All three laminin α chains occur in the smooth muscle basement membrane, with a differential expression of laminin α5 chain in the circular and longitudinal smooth muscle layers. The cellular origin of laminin α2 and α5 chains found in the subepithelial cell basement membrane was studied by immunocytochemical analysis of mouse/chick or chick/mouse interspecies hybrid intestines at various stages of development using mouse-specific antibodies. Laminin α2 was found to be deposited into the basement membrane exclusively by mesenchymal cells, while the laminin α5 chain was deposited by both epithelial and mesenchymal cells in an apparently developmentally regulated pattern. We conclude that (1) multiple laminin α chains are expressed in the intestine, implying specific roles for individual laminin isoforms during intestinal development, and (2) reciprocal epithelial/mesenchymal interactions are essential for the formation of a structured subepithelial basement membrane

Laminin α5 guides tissue patterning and organogenesis

Laminins (LM) are extracellular matrix molecules that contribute to and are required for the formation of basement membranes. They participate in the modulation of epithelial/mesenchymal interactions and are implicated in organogenesis and maintenance of organ homeostasis. Among the LM molecules, the LM α5 chain (LMα5) is one of the most widely distributed LM in the developing and mature organism. Its presence in some basement membranes during embryogenesis is absolutely required for maintenance of basement membrane integrity and thus for proper organogenesis. LMα5 also regulates the expression of genes important for major biological processes, in part by repressing or activating signaling pathways, depending upon the physiological context

Abnormal Wnt and PI3Kinase Signaling in the Malformed Intestine of lama5 Deficient Mice

Laminins are major constituents of basement membranes and are essential for tissue homeostasis. Laminin-511 is highly expressed in the intestine and its absence causes severe malformation of the intestine and embryonic lethality. To understand the mechanistic role of laminin-511 in tissue homeostasis, we used RNA profiling of embryonic intestinal tissue of lama5 knockout mice and identified a lama5 specific gene expression signature. By combining cell culture experiments with mediated knockdown approaches, we provide a mechanistic link between laminin α5 gene deficiency and the physiological phenotype. We show that laminin α5 plays a crucial role in both epithelial and mesenchymal cell behavior by inhibiting Wnt and activating PI3K signaling. We conclude that conflicting signals are elicited in the absence of lama5, which alter cell adhesion, migration as well as epithelial and muscle differentiation. Conversely, adhesion to laminin-511 may serve as a potent regulator of known interconnected PI3K/Akt and Wnt signaling pathways. Thus deregulated adhesion to laminin-511 may be instrumental in diseases such as human pathologies of the gut where laminin-511 is abnormally expressed as it is shown here

Hemidesmosome integrity protects the colon against colitis and colorectal cancer

OBJECTIVE: Epidemiological and clinical data indicate that patients suffering from IBD with long-standing colitis display a higher risk to develop colorectal high-grade dysplasia. Whereas carcinoma invasion and metastasis rely on basement membrane (BM) disruption, experimental evidence is lacking regarding the potential contribution of epithelial cell/BM anchorage on inflammation onset and subsequent neoplastic transformation of inflammatory lesions. Herein, we analyse the role of the alpha6beta4 integrin receptor found in hemidesmosomes that attach intestinal epithelial cells (IECs) to the laminin-containing BM. DESIGN: We developed new mouse models inducing IEC-specific ablation of alpha6 integrin either during development (alpha6DeltaIEC) or in adults (alpha6DeltaIEC-TAM). RESULTS: Strikingly, all alpha6DeltaIEC mutant mice spontaneously developed long-standing colitis, which degenerated overtime into infiltrating adenocarcinoma. The sequence of events leading to disease onset entails hemidesmosome disruption, BM detachment, IL-18 overproduction by IECs, hyperplasia and enhanced intestinal permeability. Likewise, IEC-specific ablation of alpha6 integrin induced in adult mice (alpha6DeltaIEC-TAM) resulted in fully penetrant colitis and tumour progression. Whereas broad-spectrum antibiotic treatment lowered tissue pathology and IL-1beta secretion from infiltrating myeloid cells, it failed to reduce Th1 and Th17 response. Interestingly, while the initial intestinal inflammation occurred independently of the adaptive immune system, tumourigenesis required B and T lymphocyte activation. CONCLUSIONS: We provide for the first time evidence that loss of IECs/BM interactions triggered by hemidesmosome disruption initiates the development of inflammatory lesions that progress into high-grade dysplasia and carcinoma. Colorectal neoplasia in our mouse models resemble that seen in patients with IBD, making them highly attractive for discovering more efficient therapies.PMC559510

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis

La laminine-511, acteur de l inflammation et de la progression tumorale dans l intestin?

La laminine-511 (a5b1y1), élément de la matrice extracellulaire, fait partie des molécules médiatrices des interactions épithélio-mésenchymateuses qui contrôlent le développement intestinal puis le renouvellement cellulaire dans l organe mature. Une rupture de l homéostasie de l ensemble de ces mécanismes est centrale dans les maladies inflammatoires chroniques de l intestin (MICI) qui, dans une certaine proportion, peuvent dégénérer en cancer colorectal. Ce travail de thèse a permis de démontrer que la chaîne a5 de laminine, caractéristique de la laminine-511, pouvait d une part réguler l expression de gènes cibles importants dans les fonctions biologiques majeures des cellules intestinales et d autre part était capable de réprimer ou d activer deux voies de signalisation, Wnt et PI3K/Akt, suivant le contexte physiologique. Dans le contexte pathologique particulier des MICI, j ai pu définir la composition en laminines de la lame basale dans des segments pathologiques issus d exérèses chirurgicales de côlons de patients atteints de maladie de Crohn ou de rectocolite hémorragique. De façon très intéressante, des glandes atypiques y ont été trouvées, délimitées par une lame basale enrichie en chaînes a1 et a5 de laminines et caractérisées par une accumulation nucléaire de la protéine p53, suppresseur de tumeur. J ai pu définir, par des expériences in vitro, une chronologie d activation montrant que la protéine p53 était capable de stimuler l expression endogène de la chaîne a1 de laminine. J ai également pu modéliser les résultats obtenus chez l humain par des inductions chimiques d inflammation et de tumorigenèse associée grâce à des modèles de souris transgéniques sur-exprimant soit la chaîne a1 soit la chaîne a5 dans l épithélium intestinal, modèles établis par l équipe. Les dérégulations des laminines qui ont été observées peuvent jouer un rôle important dans la rupture de l épithélium, l inflammation et les mécanismes de réparation qui leur sont inhérents ou dans la progression des MICI en cancer.The laminin-511 (a5b1y1), key element of the extracellular matrix, mediates the epithelialmesenchymal interactions controlling the intestinal development and the cell renewal in the mature organ. A deregulation of the mechanisms implied in the homeostasis is obvious in inflammatory bowel diseases (IBD). IBD can in some cases degenerate into colorectal cancer. My present work demonstrates that the a5 chain, characteristic of the laminin-511 molecule, regulates the expression of target genes important for major biological functions of the intestinal cells as well as is able to repress or activate two signaling pathways, Wnt and PI3K/Akt, according to the physiological context. In the particular context of IBD, I could define the laminin composition of the basement membrane in pathological samples from surgical resection of colon from patients with Crohn s disease or Ulcerative colitis. Interestingly, atypical glands are also found in these samples. These glands are encircled with a basement membrane enriched in laminin a1 and a5 chains and are characterized by a nuclear accumulation of p53, a tumor suppressor. By in vitro experiments, I have defined a chronology of activation showing that the p53 protein is able to stimulate the endogenous expression of the laminin a1 chain. I could mimic the results obtained in humans by triggering inflammation and inflammation-associated tumorigenesis chemically to transgenic murine models overexpressing either the laminin a1 or a5 chain, models that were previously established in the team. The laminin-observed deregulations could play an important role in the epithelium rupture, the inflammation and the inherent repair mechanisms, as well as in the progression of IBD into cancer.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Recherche du rôle de la chaîne a5 de laminine dans l intestin normal et pathologique

La chaîne a5 de laminine, représentante majeure des lames basales, est médiatrice des interactions épithélio-mésenchymateuses indispensables à la morphogenèse et à l homéostasie de l intestin. Ce travail de thèse a révélé que les souris mutantes pour le gène Lama5 présentent une altération d expression de gènes de différenciation épithéliale et musculaire, de molécules d'adhésion et de 2 voies de signalisation, la voie de la PI3Kinase/Akt et la voie Wnt. Cette étude a permis de démontrer que la chaîne a5 est une molécule signal capable de réguler l expression de gènes cibles lui conférant un rôle dans plusieurs fonctions biologiques dans les cellules intestinales - prolifération, survie, migration et différenciation. La chaîne a5 favorise notamment la survie cellulaire en induisant l activation d'Akt via la PI3K. L invalidation génique d un des récepteurs de la chaîne a5, le Luthéran, a révélé l importance de la signalisation laminine a5/Luthéran pour l intégrité tissulaire du rein et du muscle intestinal. Cette signalisation est essentielle à une organisation normale des lames basales dans ces deux tissus. Enfin, j ai montré que la chaîne a5 de laminines est dérégulée dans deux pathologies intestinales : la maladie de Hirschsprung et le cancer colorectal. Les intestins atteints de maladie de Hirschsprung présentent une accumulation de chaîne a5 dans le muscle qui pourrait partiellement être à l origine de l aganglionose intestinale caractéristique de cette maladie. Par cette étude, nous avons mis en évidence une nouvelle caractéristique des cas d aganglionose qui est la présence anormale de cellules de Schwann au sein des plexus entériques et révélé une modification du profil d expression de plusieurs laminines qui pourra être utilisé comme marqueur diagnostique des cas d aganglionose. Ce travail a par ailleurs montré une dérégulation de la chaîne a5 dans les cancers colorectaux. La chaîne a5 est en effet trouvée sur-exprimée dans les zones tumorales encore bien différenciées où elle pourrait contribuer aux phases précoces de la tumorigenèse. En revanche, son expression chute dans les zones plus altérées permettant certainement l invasion tumorale. En conclusion, ce travail de thèse a montré que la chaîne a5 de laminine est une molécule signal du microenvironnement cellulaire qui influence le devenir des cellules intestinales en régulant de nombreux processus cellulaires essentiels au développement et à l homéostasie intestinale.The laminin a5 chain, a major component of the basement membrane, plays an essential role in intestinal morphogenesis and homeostasis by driving epithelial-mesenchymal interactions. The present work demonstrated, in intestines lacking the laminin a5 chain, an alteration of genes implied in epithelial and muscle differentiation, of some adhesion receptors and a deregulation of Wnt and PI3K/Akt signaling pathways. We identified the laminin a5 chain as a signaling molecule able to induce expression of specific genes that will trigger various cellular processes including proliferation, survival, migration and differentiation of intestinal cells. The a5 chain has notably an anti-apoptotic effect by inducing the PI3K-dependent activation of Akt. We also studied the role of Lutheran, an exclusive receptor for the a5 chain by gene invalidation in mice. We showed that the laminin a5 chain/Lutheran signaling is essential for renal and intestinal muscle integrity and is required for a normal organization of basement membranes. Finally, I have shown the deregulation of laminin a5 chain in two intestinal diseases: Hirschsprung disease and colorectal cancer. First, we found an accumulation of a5 chain in intestinal muscle of patients with Hirschsprung disease which could be implied in the pathogenesis. We identified the abnormal presence of Schwann cells and found modifications in the expression profile of several laminin chains in aganglionic plexuses that could be new markers of intestinal aganglionosis. Second, we hightlighted the deregulation of laminin a5 chain in colorectal tumors. Indeed, a5 chain is overexpressed in still differentiated tumor areas where it could participate in onset of tumorigenesis. With increasing grade of the tumors, the a5 chain expression becomes downregulated to permit tumoral cell invasion. In conclusion, this work identified the laminin a5 chain as a signal molecule of the cell microenvironment which determines intestinal cell fate during intestinal development and homeostasis.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Développement de la fonction digestive : régulation de la maturation des enzymes de la bordure en brosse intestinale

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