Inceptor counteracts insulin signalling in ß-cells to control glycaemia.

Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic ß-cells causes overt diabetes in mice; thus, therapies that sensitize ß-cells to insulin may protect patients with diabetes against ß-cell failure1-3. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse ß-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate receptor domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir-/-) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir-/- mice showed an increase in the activation of INSR-IGF1R in Iir-/- pancreatic tissue, resulting in an increase in the proliferation and mass of ß-cells. Similarly, inducible ß-cell-specific Iir-/- knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR-IGF1R and increased proliferation of ß-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR-IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR-IGF1R in ß-cells. Together, our findings show that inceptor shields insulin-producing ß-cells from constitutive pathway activation, and identify inceptor as a potential molecular target for INSR-IGF1R sensitization and diabetes therapy.

Reversed Phase-Liquid Chromatography for Recombinant AAV Genome Integrity Assessment.

After decades of research, gene therapy products have reached market maturity in recent years. Recombinant adeno-associated viruses (rAAVs) are one of the most promising gene delivery vehicles and are currently under intense scientific investigation. These next-generation medicines remain very challenging when it comes to designing appropriate analytical techniques for quality control. One critical quality attribute is the integrity of ssDNA incorporated in these vectors. The genome is the active compound driving rAAV therapy and therefore requires proper assessment and quality control. Current techniques for rAAV genome characterization include next-generation sequencing, quantitative polymerase chain reaction, analytical ultracentrifugation (AUC), and capillary gel electrophoresis (CGE), yet each of them presents their limitations or lack of user-friendliness. In this work, we demonstrate for the first time the potential of ion pairing-reverse phase-liquid chromatography (IP-RP-LC) to characterize the integrity of rAAV genomes. The obtained results were supported by two orthogonal techniques, AUC and CGE. IP-RP-LC can be performed above DNA melting temperatures, avoiding the detection of secondary DNA isoforms, and does not require the use of dyes due to UV detection. We demonstrate that this technique is suitable for batch comparability, different rAAV serotypes (AAV2 and AAV8), internal vs external (inside vs outside the capsid) DNA analysis, and contaminated samples. Overall, it is exceptionally user-friendly, needs limited sample preparation, has high reproducibility, and permits fractionation for further peak characterization. All of these factors add significant value of IP-RP-LC to the analytical toolbox of rAAV genome assessment.

Understanding pancreas development for ß-cell repair and replacement therapies.

The lack or dysfunction of insulin-producing ß-cells is the cause of all forms of diabetes. In vitro generation of ß-cells from pluripotent stem cells for cell-replacement therapy or triggering endogenous mechanisms of ß-cell repair have great potential in the field of regenerative medicine. Both approaches rely on a thorough understanding of ß-cell development and homeostasis. Here, we briefly summarize the current knowledge of ß-cell differentiation during pancreas development in the mouse. Furthermore, we describe how this knowledge is translated to instruct differentiation of both mouse and human pluripotent stem cells towards the ß-cell lineage. Finally, we shortly summarize the current efforts to identify stem or progenitor cells in the adult pancreatic organ and to harness the endogenous regenerative potential. Understanding development and regeneration of ß-cells already led to identification of molecular targets for therapy and informed on pathomechanisms of diabetes. In the future this knowledge might [corrected] lead to ß-cell repair and replacement therapies.

The global gene expression profile of the secondary transition during pancreatic development.

Pancreas organogenesis is a highly dynamic process where neighboring tissue interactions lead to dynamic changes in gene regulatory networks that orchestrate endocrine, exocrine, and ductal lineage formation. To understand the spatio-temporal regulatory logic we have used the Forkhead transcription factor Foxa2-Venus fusion (FVF) knock-in reporter mouse to separate the FVF(+) pancreatic epithelium from the FVF(−) surrounding tissue (mesenchyme, neurons, blood, and blood vessels) to perform a genome-wide mRNA expression profiling at embryonic days (E) 12.5-15.5. Annotating genes and molecular processes suggest that FVF marks endoderm-derived multipotent epithelial progenitors at several lineage restriction steps, when the bulk of endocrine, exocrine and ductal cells are formed during the secondary transition. In the pancreatic epithelial compartment, we identified most known endocrine and exocrine lineage determining factors and diabetes-associated genes, but also unknown genes with spatio-temporal regulated pancreatic expression. In the non-endoderm-derived compartment, we identified many well-described regulatory genes that are not yet functionally annotated in pancreas development, emphasizing that neighboring tissue interactions are still ill defined. Pancreatic expression of over 635 genes was analyzed with them RNA in situ hybridization Genepaint public database. This validated the quality of the profiling data set and identified hundreds of genes with spatially restricted expression patterns in the pancreas. Some of these genes are also targeted by pancreatic transcription factors and show active chromatin marks in human islets of Langerhans. Thus, with the highest spatio-temporal resolution of a global gene expression profile during the secondary transition, our study enables to shed light on neighboring tissue interactions, developmental timing and diabetes gene regulation.

The membrane-targeted alkylphosphocholine erufosine interferes with survival signals from the extracellular matrix.

Integrin-dependent adhesion of tumor cells to extracellular matrix proteins provides anchorage-dependent protection from cell death. In the present investigation we aimed to understand whether and how the paradigmatic membrane-targeted synthetic phospholipid analog erufosine is relevant for tumor cell adhesion to extracellular matrix proteins, cell survival and migration. The antineoplastic action of erufosine was analyzed with glioblastoma and prostate cancer cells adhering to fibronectin or collagen I using proliferation, adhesion and migration assays. The composition of adhesion contacts containing activated ß1 integrins was studied using immunofluorescence. The importance of ß1 integrins for the observed effects was analyzed in fibroblasts proficient or deficient in ß1 integrin expression. Adhesion to collagen I and fibronectin increased the death threshold in serum-deprived tumor cells. Moreover, ß1 integrin-deficient cells were more sensitive to erufosine-treatment compared to ß1 integrin proficient cells suggesting a role of ß1 integrins for matrix-mediated death resistance. Most importantly, erufosine disturbed the maturation of the cell adhesion complexes containing paxillin, activated ß1 integrins and phosphorylated FAK, leading to a reduction of survival signals and inhibition of tumor cell adhesion and migration. These findings suggest that membrane-targeted synthetic phospholipids analogs may be of value for counteracting matrix-mediated treatment resistance in combined treatment approaches with radiotherapy or chemotherapy.

ß1- and αv-class integrins cooperate to regulate myosin II during rigidity sensing of fibronectin-based microenvironments.

How different integrins that bind to the same type of extracellular matrix protein mediate specific functions is unclear. We report the functional analysis of ß1- and αv-class integrins expressed in pan-integrin-null fibroblasts seeded on fibronectin. Reconstitution with ß1-class integrins promotes myosin-II-independent formation of small peripheral adhesions and cell protrusions, whereas expression of αv-class integrins induces the formation of large focal adhesions. Co-expression of both integrin classes leads to full myosin activation and traction-force development on stiff fibronectin-coated substrates, with αv-class integrins accumulating in adhesion areas exposed to high traction forces. Quantitative proteomics linked αv-class integrins to a GEF-H1-RhoA pathway coupled to the formin mDia1 but not myosin II, and α5ß1 integrins to a RhoA-Rock-myosin II pathway. Our study assigns specific functions to distinct fibronectin-binding integrins, demonstrating that α5ß1integrins accomplish force generation, whereas αv-class integrins mediate the structural adaptations to forces, which cooperatively enable cells to sense the rigidity of fibronectin-based microenvironments.

ß1A integrin is a master regulator of invadosome organization and function.

Invadosomes are adhesion structures involved in tissue invasion that are characterized by an intense actin polymerization-depolymerization associated with ß1 and ß3 integrins and coupled to extracellular matrix (ECM) degradation activity. We induced the formation of invadosomes by expressing the constitutive active form of Src, SrcYF, in different cell types. Use of ECM surfaces micropatterned at the subcellular scale clearly showed that in mesenchymal cells, integrin signaling controls invadosome activity. Using ß1⁻/⁻ or ß3⁻/⁻ cells, it seemed that ß1A but not ß3 integrins are essential for initiation of invadosome formation. Protein kinase C activity was shown to regulate autoassembly of invadosomes into a ring-like metastructure (rosette), probably by phosphorylation of Ser785 on the ß1A tail. Moreover, our study clearly showed that ß1A links actin dynamics and ECM degradation in invadosomes. Finally, a new strategy based on fusion of the photosensitizer KillerRed to the ß1A cytoplasmic domain allowed specific and immediate loss of function of ß1A, resulting in disorganization and disassembly of invadosomes and formation of focal adhesions.

Beta1 integrin deficiency results in multiple abnormalities of the knee joint.

The lack of beta1 integrins on chondrocytes leads to severe chondrodysplasia associated with high mortality rate around birth. To assess the impact of beta1 integrin-mediated cell-matrix interactions on the function of adult knee joints, we conditionally deleted the beta1 integrin gene in early limb mesenchyme using the Prx1-cre transgene. Mutant mice developed short limbed dwarfism and had joint defects due to beta1 integrin deficiency in articular regions. The articular cartilage (AC) was structurally disorganized, accompanied by accelerated terminal differentiation, altered shape, and disrupted actin cytoskeleton of the chondrocytes. Defects in chondrocyte proliferation, cytokinesis, and survival resulted in hypocellularity. However, no significant differences in cartilage erosion, in the expression of matrix-degrading proteases, or in the exposure of aggrecan and collagen II cleavage neoepitopes were observed between control and mutant AC. We found no evidence for disturbed activation of MAPKs (ERK1/2, p38, and JNK) in vivo. Furthermore, fibronectin fragment-stimulated ERK activation and MMP-13 expression were indistinguishable in control and mutant femoral head explants. The mutant synovium was hyperplastic and frequently underwent chondrogenic differentiation. beta1-null synoviocytes showed increased proliferation and phospho-focal adhesion kinase expression. Taken together, deletion of beta1 integrins in the limb bud results in multiple abnormalities of the knee joints; however, it does not accelerate AC destruction, perturb cartilage metabolism, or influence intracellular MAPK signaling pathways.

Early detection of aging cartilage and osteoarthritis in mice and patient samples using atomic force microscopy.

The pathological changes in osteoarthritis--a degenerative joint disease prevalent among older people--start at the molecular scale and spread to the higher levels of the architecture of articular cartilage to cause progressive and irreversible structural and functional damage. At present, there are no treatments to cure or attenuate the degradation of cartilage. Early detection and the ability to monitor the progression of osteoarthritis are therefore important for developing effective therapies. Here, we show that indentation-type atomic force microscopy can monitor age-related morphological and biomechanical changes in the hips of normal and osteoarthritic mice. Early damage in the cartilage of osteoarthritic patients undergoing hip or knee replacements could similarly be detected using this method. Changes due to aging and osteoarthritis are clearly depicted at the nanometre scale well before morphological changes can be observed using current diagnostic methods. Indentation-type atomic force microscopy may potentially be developed into a minimally invasive arthroscopic tool to diagnose the early onset of osteoarthritis in situ.

Homing of mouse spermatogonial stem cells to germline niche depends on beta1-integrin.

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis. In a manner comparable to hematopoietic stem cell transplantation, SSCs colonize the niche of recipient testes and reinitiate spermatogenesis following microinjection into the seminiferous tubules. However, little is known about the homing mechanism of SSCs. Here we examined the role of adhesion molecules in SSC homing. SSCs isolated from mice carrying loxP-tagged beta1-integrin alleles were ablated for beta1-integrin expression by in vitro adenoviral cre transduction. The beta1-integrin mutant SSCs showed significantly reduced ability to recolonize recipient testes in vivo and to attach to laminin molecules in vitro. In contrast, genetic ablation of E-cadherin did not impair homing, and E-cadherin mutant SSCs completed normal spermatogenesis. In addition, the deletion of beta1-integrin on Sertoli cells reduced SSC homing. These results identify beta1-integrin as an essential adhesion receptor for SSC homing and its association with laminin is critical in multiple steps of SSC homing.