Search

The main laminin-binding integrins α3β1, α6β1 and α6β4 are co-expressed in the developing kidney collecting duct system. We previously showed that deleting the integrin α3 or α6 subunit in the ureteric bud, which gives rise to the kidney collecting system, caused either a mild or no branching morphogenesis phenotype, respectively. To determine whether these two integrin subunits cooperate in kidney collecting duct development, we deleted α3 and α6 in the developing ureteric bud. The collecting system of the double knockout phenocopied the α3 integrin conditional knockout. However, with age, the mice developed severe inflammation and fibrosis around the collecting ducts, resulting in kidney failure. Integrin α3α6-null collecting duct epithelial cells showed increased secretion of pro-inflammatory cytokines and displayed mesenchymal characteristics, causing loss of barrier function. These features resulted from increased nuclear factor kappa-B (NF-κB) activity, which regulated the Snail and Slug (also known as Snai1 and Snai2, respectively) transcription factors and their downstream targets. These data suggest that laminin-binding integrins play a key role in the maintenance of kidney tubule epithelial cell polarity and decrease pro-inflammatory cytokine secretion by regulating NF-κB-dependent signaling.

Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

Objective Enteroendocrine cells (EECs) of the gastro-intestinal tract sense gut luminal factors and release peptide hormones or serotonin (5-HT) to coordinate energy uptake and storage. Our goal is to decipher the gene regulatory networks controlling EECs specification from enteroendocrine progenitors. In this context, we studied the role of the transcription factor Rfx6 which had been identified as the cause of Mitchell–Riley syndrome, characterized by neonatal diabetes and congenital malabsorptive diarrhea. We previously reported that Rfx6 was essential for pancreatic beta cell development and function; however, the role of Rfx6 in EECs differentiation remained to be elucidated. Methods We examined the molecular, cellular, and metabolic consequences of constitutive and conditional deletion of Rfx6 in the embryonic and adult mouse intestine. We performed single cell and bulk RNA-Seq to characterize EECs diversity and identify Rfx6-regulated genes. Results Rfx6 is expressed in the gut endoderm; later, it is turned on in, and restricted to, enteroendocrine progenitors and persists in hormone-positive EECs. In the embryonic intestine, the constitutive lack of Rfx6 leads to gastric heterotopia, suggesting a role in the maintenance of intestinal identity. In the absence of intestinal Rfx6, EECs differentiation is severely impaired both in the embryo and adult. However, the number of serotonin-producing enterochromaffin cells and mucosal 5-HT content are increased. Concomitantly, Neurog3-positive enteroendocrine progenitors accumulate. Combined analysis of single-cell and bulk RNA-Seq data revealed that enteroendocrine progenitors differentiate in two main cell trajectories, the enterochromaffin (EC) cells and the Peptidergic Enteroendocrine (PE) cells, the differentiation programs of which are differentially regulated by Rfx6. Rfx6 operates upstream of Arx, Pax6 and Isl1 to trigger the differentiation of peptidergic EECs such as GIP-, GLP-1-, or CCK-secreting cells. On the contrary, Rfx6 represses Lmx1a and Tph1, two genes essential for serotonin biosynthesis. Finally, we identified transcriptional changes uncovering adaptive responses to the prolonged lack of enteroendocrine hormones and leading to malabsorption and lower food efficiency ratio in Rfx6-deficient mouse intestine. Conclusion These studies identify Rfx6 as an essential transcriptional regulator of EECs specification and shed light on the molecular mechanisms of intestinal failures in human RFX6-deficiencies such as Mitchell–Riley syndrome., Highlights • The lack of Rfx6 impairs the differentiation of peptide-producing enteroendocrine cells. • The number of 5-HT-expressing-cells is increased in Rfx6-deficient intestine. • Intestinal inactivation of Rfx6 leads to lipid malabsorption and decreased food efficiency.

Summary Integrins, which bind laminin, a major component of the mammary basement membrane, are strongly expressed in basal stem cell-enriched populations, but their role in controlling mammary stem cell function remains unclear. We found that stem cell activity, as evaluated in transplantation and mammosphere assays, was reduced in mammary basal cells depleted of laminin receptors containing α3- and α6-integrin subunits. This was accompanied by low MDM2 levels, p53 stabilization, and diminished proliferative capacity. Importantly, disruption of p53 function restored the clonogenicity of α3/α6-integrin-depleted mammary basal stem cells, while inhibition of RHO or myosin II, leading to decreased p53 activity, rescued the mammosphere formation. These data suggest that α3/α6-integrin-mediated adhesion plays an essential role in controlling the proliferative potential of mammary basal stem/progenitor cells through myosin II-mediated regulation of p53 and indicate that laminins might be important components of the mammary stem cell niche., Highlights • α3- and α6-integrins are required for mammary basal stem cell function • p53 is activated in mammary basal cells depleted of α3- and α6-integrins • RHO and myosin II mediate p53 activation in α3- and α6-integrin-depleted cells, Faraldo and colleagues report that major cellular receptors for laminins, α3-and α6-subunit-containing integrins play an important role in the control of the proliferative potential of mammary basal stem/progenitor cells regulating the p53 activity through an RHOA/myosin II-dependent mechanism. These findings indicate that laminins might be essential components of the mammary stem cell niche.

In a recently published letter,1 Dr Beaulieu questions our recent publication suggesting that α6 integrin acts as a tumour suppressor gene in colorectal cancer (CRC),2 given the apparent antagonistic role of α6A and α6B integrin isoforms in the intestine. We thank him for raising this important issue and welcome the opportunity of clarifying the topic. We showed that mice failing to express the α6 integrin (both α6A and α6B isoforms) specifically in intestinal epithelial cells (α6ΔIEC) spontaneously develop fully penetrant colitis and adenocarcinoma afterwards.2 These mice display epithelium detachment from the basement membrane, followed …

Laminins are a major constituent of the basement membranes of the kidney collecting system. Integrins, transmembrane receptors formed by non-covalently bound alpha and beta subunits, serve as laminin receptors, but their role in development and homeostasis of the kidney collecting system is poorly defined. Integrin alpha3beta1, one of the major laminin receptors, plays a minor role in kidney collecting system development, while the role of alpha6 containing integrins (alpha6beta1 and alpha6beta4), the other major laminin receptors, is unknown. Patients with mutations in alpha6 containing integrins not only develop epidermolysis bullosa, but also have abnormalities in the kidney collecting system. In this study, we show that selectively deleting the alpha6 or beta4 integrin subunits at the initiation of ureteric bud development in mice does not affect morphogenesis. However, the collecting system becomes dilated and dysmorphic as the mice age. The collecting system in both null genotypes was also highly susceptible to unilateral ureteric obstruction injury with evidence of excessive tubule dilatation and epithelial cell apoptosis. Mechanistically, integrin alpha6-null collecting duct cells are unable to withstand high mechanical force when adhered to laminin. Thus, we conclude that alpha6 integrins are important for maintaining the integrity of the kidney collecting system by enhancing tight adhesion of the epithelial cells to the basement membrane. These data give a mechanistic explanation for the association between kidney collecting system abnormalities in patients and epidermolysis bullosa.

International audience; Instability in the composition of gut bacterial communities (dysbiosis) has been linked to common human intestinal disorders, such as Crohn's disease and colorectal cancer. Here, we show that dysbiosis caused by Nod2 deficiency gives rise to a reversible, communicable risk of colitis and colitis-associated carcinogenesis in mice. Loss of either Nod2 or RIP2 resulted in a proinflammatory microenvironment that enhanced epithelial dysplasia following chemically induced injury. The condition could be improved by treatment with antibiotics or an anti-interleukin-6 receptor-neutralizing antibody. Genotype-dependent disease risk was communicable via maternally transmitted microbiota in both Nod2-deficient and WT hosts. Furthermore, reciprocal microbiota transplantation reduced disease risk in Nod2-deficient mice and led to long-term changes in intestinal microbial communities. Conversely, disease risk was enhanced in WT hosts that were recolonized with dysbiotic fecal microbiota from Nod2-deficient mice. Thus, we demonstrated that licensing of dysbiotic microbiota is a critical component of disease risk. Our results demonstrate that NOD2 has an unexpected role in shaping a protective assembly of gut bacterial communities and suggest that manipulation of dysbiosis is a potential therapeutic approach in the treatment of human intestinal disorders.

International audience; Hemidesmosomes (HDs) are essential anchorage junctions which mediate the firm attachment of epithelia to the underlying basement membranes, of which one main component is the integrin α6β4. These specific junctions are also able to trigger signalling pathways, via the recruitment and interactions of signalling molecules with HD components such as the cytoplasmic tail of the β4 integrin or the plakin plectin. HDs must also assemble and disassemble depending on the tissue context for example during tissue remodelling. Alterations of HD components or their loss result in skin blistering disorders known as epidermolysis bullosa. Since mice lacking integrin α6 die at birth with severe skin blistering, we have produced a mouse line in which epidermal deletion of integrin α6 can be controlled by tamoxifen injection. We observed that the deletion was mosaic, but that hairless skin such as ears, tails and paws were affected and showed chronic inflammation associated with hyperproliferation, and expression of laminin-111. Interestingly, two cytokines, amphiregulin and epiregulin, previously found increased in integrin α6 deficient cultured keratinocytes, were also increased here in the affected skin. In detached areas, we validate clearly that the absence of integrin α6 leads to a delocalisation of plectin, and the complete disappearance of HD structures.

International audience; Muskelin is a member of the kelch-repeat superfamily of proteins, identified as an intracellular protein involved in cell spreading responses to thombospondin-1. Muskelin is expressed by many adult tissues and has an evolutionarily conserved, multidomain architecture consisting of an amino-terminal discoidin-like domain, a central alpha-helical region and six kelch-repeats that are predicted to form a beta-propeller structure. We previous demonstrated that muskelin molecules undergo head-to-tail association, however the physiological, post-translational regulation of muskelin is not well understood. Here, we have examined the expression of muskelin during mouse embryonic development and report widespread expression that includes muscle tissues, multiple epithelia and the brain. In cultured skeletal myoblasts and vascular smooth muscle cells, muskelin exists as a complex set of isoelectric variants. Five potential sites for phosphorylation by protein kinase C (PKC), are conserved between vertebrate and Drosophila muskelins, therefore we examined the hypothesis that muskelin is regulated post-translationally by PKC activity. We demonstrate that PKC activation or inhibition regulates the profile of endogenous muskelin isoelectric variants and that muskelin is a substrate for PKCalphain vitro. Wild-type GFP-muskelin and a panel of alanine point mutations were used to test the sensitivity of self-association to PKC activation. Mutation of two of the sites, S324 and T515, partially inhibited the ability of muskelin to self-associate in cells and inhibited responsiveness to activated PKC. Interestingly, both sites are predicted to lie in surface-exposed loops on the same side of the beta-propeller, implicating a common binding interface.

International audience; Radial glial cells have been shown to act as neuronal precursors in the developing cortex and to maintain their radial processes attached to the basement membrane (BM) during cell division. Here, we examined a potential role of direct signalling from the BM to radial glial cells in three mouse mutants where radial glia attachment to the BM is disrupted. This is the case if the nidogen-binding site of the laminin gamma1 chain is mutated, in the absence of alpha6 integrin or of perlecan, an essential BM component. Surprisingly, cortical radial glial cells lacking contact to the BM were not affected in their proliferation, interkinetic nuclear migration, orientation of cell division and neurogenesis. Only a small subset of precursors was located ectopically within the cortical parenchyma. Notably, however, neuronal subtype composition was severely disturbed at late developmental stages (E18) in the cortex of the laminin gamma1III4-/- mice. Thus, although BM attachment seems dispensable for precursor cells, an intact BM is required for adequate neuronal composition of the cerebral cortex.

International audience; Integrins are heterodimeric cell surface receptors that mediate developmental events by binding extracellular matrix ligands. Several lines of evidence suggest a role for integrins, specifically the alpha 6 subunit, in neuronal migration, neurite outgrowth, and axon guidance during olfactory development. Therefore, we undertook an analysis of the expression of the alpha 6 subunit in the olfactory system of the embryonic and early postnatal mouse to understand the role it may play during neural development. In addition, as a functional assay we examined the developmental effects of the loss of this subunit on olfactory development by analyzing an alpha 6 knockout (alpha 6-/-). Immunohistochemical analyses and confocal microscopy were used to examine alpha 6 expression in the CD-1 embryonic and early postnatal olfactory system and also to examine the organization of the olfactory system in the alpha 6-/- mouse. In CD-1 mice from E13 to E17, alpha 6 localizes in radial patterns extending from the core of the olfactory bulb to the nerve layer and colocalizes with RC2, an antibody specific for radial glia. By the day of birth (P0; approximately E19), expression is limited to the external plexiform layer and the olfactory nerve layer, where it colocalizes with laminin and p75. In the alpha 6-/- mouse, areas of ectopic granule cells were observed in the mitral cell layer of the olfactory bulb. These ectopias coincided with areas of disorganization of the radial glial processes and breaks in the mitral cell layer. These observations suggest a role for alpha 6 integrin in neural migration during olfactory development, likely secondary to organization of the radial glial scaffold.