Signal transduction by the cloned glucagon-like peptide-1 receptor: comparison with signaling by the endogenous receptors of beta cell lines.

Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that potentiates glucose-induced insulin secretion by pancreatic beta cells. The mechanisms of interaction between GLP-1 and glucose signaling pathways are not well understood. Here we studied the coupling of the cloned GLP-1 receptor, expressed in fibroblasts or in COS cells, to intracellular second messengers and compared this signaling with that of the endogenous receptor expressed in insulinoma cell lines. Binding of GLP-1 to the cloned receptor stimulated formation of cAMP with the same dose dependence and similar kinetics, compared with the endogenous receptor of insulinoma cells. Compared with forskolin-induced cAMP accumulation, that induced by GLP-1 proceeded with the same initial kinetics but rapidly reached a plateau, suggesting fast desensitization of the receptor. Coupling to the phospholipase C pathway was assessed by measuring inositol phosphate production and variations in the intracellular calcium concentration. No GLP-1-induced production of inositol phosphates could be measured in the different cell types studied. A rise in the intracellular calcium concentration was nevertheless observed in transfected COS cells but was much smaller than that observed in response to norepinephrine in cells also expressing the alpha 1B-adrenergic receptor. Importantly, no such increase in the intracellular calcium concentration could be observed in transfected fibroblasts or insulinoma cells, which, however, responded well to thrombin or carbachol, respectively. Together, our data show that interaction between GLP-1 and glucose signaling pathways in beta cells may be mediated uniquely by an increase in the intracellular cAMP concentration, with the consequent activation of protein kinase A and phosphorylation of elements of the glucose-sensing apparatus or of the insulin granule exocytic machinery.

Vascular Integrins: Therapeutic and Imaging Targets of Tumor Angiogenesis

Cells, including endothelial cells, continuously sense their surrounding environment and rapidly adapt to changes in order to assure tissues and organs homeostasis. The extracellular matrix (ECM) provides a physical scaffold for cell positioning and represents an instructive interface allowing cells to communicate over short distances. Cell surface receptors of the integrin family emerged through evolution as essential mediators and integrators of ECM-dependent communication. In preclinical studies, pharmacological inhibition of vascular integrins suppressed angiogenesis and inhibited tumor progression. alpha(V)beta(3) and alpha(V)beta(5) were the first integrins targeted to suppress tumor angiogenesis. Subsequently, additional integrins, in particular alpha(1)beta(1), alpha(2)beta(1), alpha(5)beta(1), and alpha(6)beta(4), emerged as potential therapeutic targets. Integrin inhibitors are currently tested in clinical trials for their safety and antiangiogenic/antitumor activity. In this chapter, we review the role of integrins in angiogenesis and present recent advances in the use of integrin antagonists as potential therapeutics in cancer and discuss future perspectives.

Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus.

We have recently reported that Notch 1, a member of the Notch multigene family, is essential for the development of murine T cells. Using a mouse model in which Notch 1 is inactivated in bone marrow (BM) precursors we have shown that B cells instead of T cells are found in the thymus of BM chimeras. However, it is not clear whether these B cells develop by default from a common lymphoid precursor due to the absence of Notch 1 signaling, or whether they arise as a result of perturbed migration of BM-derived B cells and/or altered homeostasis of normal resident thymic B cells. In this report we show that Notch 1-deficient thymic B cells resemble BM B cells in phenotype and turnover kinetics and are located predominantly in the medulla and corticomedullary junction. Peripheral blood lymphocyte analysis shows no evidence of recirculating Notch1(-/)- BM B cells. Furthermore, lack of T cell development is not due to a failure of Notch1(-/)- precursors to home to the thymus, as even after intrathymic reconstitution with BM cells, B cells instead of T cells develop from Notch 1-deficient precursors. Taken together, these results provide evidence for de novo ectopic B cell development in the thymus, and support the hypothesis that in the absence of Notch 1 common lymphoid precursors adopt the default cell fate and develop into B cells instead.

ERK1/2 controls Na,K-ATPase activity and transepithelial sodium transport in the principal cell of the cortical collecting duct of the mouse kidney.

The collecting duct of normal kidney exhibits significant activity of the MEK1/2-ERK1/2 pathway as shown in vivo by immunostaining of phosphorylated active ERK1/2 (pERK1/2). The MEK1/2-ERK1/2 pathway controls many different ion transports both in proximal and distal nephron, raising the question of whether this pathway is involved in the basal and/or hormone-dependent transepithelial sodium reabsorption in the principal cell of the cortical collecting duct (CCD), a process mediated by the apical epithelial sodium channel and the basolateral sodium pump (Na,K-ATPase). To answer this question we used ex vivo microdissected CCDs from normal mouse kidney or in vitro cultured mpkCCDcl4 principal cells. Significant basal levels of pERK1/2 were observed ex vivo and in vitro. Aldosterone and vasopressin, known to up-regulate sodium reabsorption in CCDs, did not change ERK1/2 activity either ex vivo or in vitro. Basal and aldosterone- or vasopressin-stimulated sodium transport was down-regulated by the MEK1/2 inhibitor PD98059, in parallel with a decrease in pERK1/2 in vitro. The activity of Na,K-ATPase but not that of epithelial sodium channel was inhibited by MEK1/2 inhibitors in both unstimulated and aldosterone- or vasopressin-stimulated CCDs in vitro. Cell surface biotinylation showed that intrinsic activity rather than cell surface expression of Na,K-ATPase was controlled by pERK1/2. PD98059 also significantly inhibited the activity of Na,K-ATPase ex vivo. Our data demonstrate that the ERK1/2 pathway controls Na,K-ATPase activity and transepithelial sodium transport in the principal cell and indicate that basal constitutive activity of the ERK1/2 pathway is a critical component of this control.

Psoriasis triggered by toll-like receptor 7 agonist imiquimod in the presence of dermal plasmacytoid dendritic cell precursors.

BACKGROUND: It has been proposed that the innate immune system plays a central role in driving the autoimmune T-cell cascade leading to psoriasis; however, there is no direct evidence for this. OBSERVATIONS: We observed aggravation and spreading of a psoriatic plaque when treated topically with the toll-like receptor (TLR) 7 agonist imiquimod. The exacerbation of psoriasis was accompanied by a massive induction of lesional type I interferon activity, detected by MxA expression after imiquimod therapy. Since imiquimod induces large amounts of type I interferon production from TLR7-expressing plasmacytoid dendritic cell precursors (PDCs), the natural interferon-producing cells of the peripheral blood, we asked whether PDCs are present in psoriatic skin. We identified high numbers of PDCs in psoriatic skin lesions (up to 16% of the total dermal infiltrate) based on their coexpression of BDCA2 and CD123. By contrast, PDCs were present at very low levels in atopic dermatitis and not detected in normal human skin. CONCLUSIONS: This study shows that psoriasis can be driven by the innate immune system through TLR ligation. Furthermore, our finding that large numbers of PDCs infiltrate psoriatic skin suggests a role of lesional PDCs as type I interferon-producing targets for the TLR7 agonist imiquimod.

c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation.

The activity of adult stem cells is essential to replenish mature cells constantly lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. Here, we provide genetic evidence for an unexpected function of the c-Myc protein in the homeostasis of hematopoietic stem cells (HSCs). Conditional elimination of c-Myc activity in the bone marrow (BM) results in severe cytopenia and accumulation of HSCs in situ. Mutant HSCs self-renew and accumulate due to their failure to initiate normal stem cell differentiation. Impaired differentiation of c-Myc-deficient HSCs is linked to their localization in the differentiation preventative BM niche environment, and correlates with up-regulation of N-cadherin and a number of adhesion receptors, suggesting that release of HSCs from the stem cell niche requires c-Myc activity. Accordingly, enforced c-Myc expression in HSCs represses N-cadherin and integrins leading to loss of self-renewal activity at the expense of differentiation. Endogenous c-Myc is differentially expressed and induced upon differentiation of long-term HSCs. Collectively, our data indicate that c-Myc controls the balance between stem cell self-renewal and differentiation, presumably by regulating the interaction between HSCs and their niche.

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens.

Cell fusion reprogramming leads to a specific hepatic expression pattern during mouse bone marrow derived hepatocyte formation In Vivo

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-β1 (TGF-β1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation.

AMP-activated protein kinase plays an important evolutionary conserved role in the regulation of glucose metabolism in fish skeletal muscle cells

AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP:ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1α in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively) and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively). We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase) and mitochondrial biogenesis (PGC-1α) and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish.

A Novel Missense Mutation, I890T, in the Pore Region of Cardiac Sodium Channel Causes Brugada Syndrome

Brugada syndrome (BrS) is a life-threatening, inherited arrhythmogenic syndrome associated with autosomal dominant mutations in SCN5A, the gene encoding the cardiac Na₊ channel alpha subunit (Naᵥ1.5). The aim of this work was to characterize the functional alterations caused by a novel SCN5A mutation, I890T, and thus establish whether this mutation is associated with BrS. The mutation was identified by direct sequencing of SCN5A from the proband’s DNA. Wild-type (WT) or I890T Naᵥ1.5 channels were heterologously expressed in human embryonic kidney cells. Sodium currents were studied using standard whole cell patch-clamp protocols and immunodetection experiments were performed using an antibody against human Naᵥ1.5 channel. A marked decrease in current density was observed in cells expressing the I890T channel (from -52.0 ± 6.5 pA/pF, n=15 to 35.9 ± 3.4 pA/pF, n = 22, at -20 mV, WT and I890T, respectively). Moreover, a positive shift of the activation curve was identified (V½ =-32.0 ± 0.3 mV, n = 18, and -27.3 ± 0.3 mV, n = 22, WT and I890T, respectively). No changes between WT and I890T currents were observed in steady-state inactivation, time course of inactivation, slow inactivation or recovery from inactivation parameters. Cell surface protein biotinylation analyses confirmed that Nav1.5 channel membrane expression levels were similar in WT and I890T cells. In summary, our data reveal that the I890T mutation, located within the pore of Nav1.5, causes an evident loss-of-function of the channel. Thus, the BrS phenotype observed in the proband is most likely due to this mutation

Anchor or Accelerate – A Study on Cancer Cell Adhesion and Motility

Cell migration and adhesion to the extracellular matrix (ECM) are crucial in many biological and pathological processes such as morphogenesis, tissue repair, inflammatory responses, survival, and cancer. Cell-matrix adhesion is mediated by the integrin family of transmembrane receptors, which not only anchor cells to their surroundings, but also transmit bidirectional signalling at the cell surface and couple the ECM to the cytoskeleton. Another group of adhesion receptors are the syndecan proteoglycans, which engage the ECM and possess signalling activity in response to a variety of ligands. Cell migration is a complex process that requires spatial and temporal coordination of adhesion, cell contractility, intracellular traffic of integrins, and matrix turnover by matrix metalloproteinases (MMPs). Thus, integrins and syndecans, as well as MMPs, play essential roles in cancer cell migration and invasion. The understanding of the cooperation of syndecans and integrins was broadened in this thesis study. The results reveal that syndecan-1 functions in concert with 21 integrin in cell adhesion to collagen, whereas syndecan-4 is essential in 21 integrin-mediated matrix contraction. Finally, oncogenic K-Ras was shown to regulate 21 integrin, membrane-type 1 MMP, and syndecan-1 and -4 expression and their cooperation in cell invasion. Epithelial-mesenchymal transition (EMT) is fundamental during embryogenesis and organ development. Activation of EMT processes, including the upregulation of mesenchymal intermediate filament protein vimentin, has also been implicated in the acquisition of a malignant phenotype by epithelial cancer cells. Members of the protein kinase C (PKC) superfamily are involved in cell migration and various integrindependent cellular functions. One aim of this work was to shed light on the role of vimentin in the regulation of integrin traffic and cell motility. In addition, the mechanism by which vimentin participates in EMT was investigated. The results show that integrin recycling and motility are dependent on the PKC–mediated phosphorylation of vimentin. In addition, vimentin was found to be a positive regulator of EMT and regulate the expression of several migratory genes. Specifically, vimentin governs the expression of receptor tyrosine kinase Axl, which is implicated in tumour growth and metastasis. Taken together, the findings described in this thesis reveal novel aspects of the complex interplay between distinct cellular components: integrins, syndecans, and the vimentin cytoskeleton, which all contribute to the regulation of human cancer cell adhesion, migration, and invasion.

Lethal weapons : novel approaches for receptor-targeted cancer cell elimination

The currently used forms of cancer therapy are associated with drug resistance and toxicity to healthy tissues. Thus, more efficient methods are needed for cancer-specific induction of growth arrest and programmed cell death, also known as apoptosis. Therapeutic forms of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are investigated in clinical trials due to the capability of TRAIL to trigger apoptosis specifically in cancer cells by activation of cell surface death receptors. Many tumors, however, have acquired resistance to TRAIL-induced apoptosis and sensitizing drugs for combinatorial treatments are, therefore, in high demand. This study demonstrates that lignans, natural polyphenols enriched in seeds and cereal, have a remarkable sensitizing effect on TRAIL-induced cell death at non-toxic lignan concentrations. In TRAIL-resistant and androgen-dependent prostate cancer cells we observe that lignans repress receptor tyrosine kinase (RTK) activity and downregulate cell survival signaling via the Akt pathway, which leads to increased TRAIL sensitivity. A structure-activity relationship analysis reveals that the γ-butyrolactone ring of the dibenzylbutyrolactone lignans is essential for the rapidly reversible TRAIL-sensitizing activity of these compounds. Furthermore, the lignan nortrachelogenin (NTG) is identified as the most efficient of the 27 tested lignans and norlignans in sensitization of androgen-deprived prostate cancer cells to TRAIL-induced apoptosis. While this combinatorial anticancer approach may leave normal cells unharmed, several efficient cancer drugs are too toxic, insoluble or unstable to be used in systemic therapy. To enable use of such drugs and to protect normal cells from cytotoxic effects, cancer-targeted drug delivery vehicles of nanometer scale have recently been generated. The newly developed nanoparticle system that we tested in vitro for cancer cell targeting combines the efficient drug-loading capacity of mesoporous silica to the versatile particle surface functionalization of hyperbranched poly(ethylene imine), PEI. The mesoporous hybrid silica nanoparticles (MSNs) were functionalized with folic acid to promote targeted internalization by folate receptor overexpressing cancer cells. The presented results demonstrate that the developed carrier system can be employed in vitro for cancer selective delivery of adsorbed or covalently conjugated molecules and furthermore, for selective induction of apoptotic cell death in folate receptor expressing cancer cells. The tested carrier system displays potential for simultaneous delivery of several anticancer agents specifically to cancer cells also in vivo.

Thymic atrophy in cattle poisoned with Solanum glaucophyllum

Solanum glaucophyllum (Sg) [= S. malacoxylon] is a calcinogenic plant inducing "Enzootic Calcinosis" in cattle. The 1,25-dihydroxyvitamin D3, its main toxic principle, regulates bone and calcium metabolism and also exerts immunomodulatory effects. Thymocyte precursors from bone marrow-derived progenitor cells differentiate into mature T-cells. Differentiation of most T lymphocytes is characterized not only by the variable expression of CD4/CD8 receptor molecules and increased surface density of the T cell antigen receptor, but also by changes in the glycosylation pattern of cell surface glycolipids or glycoproteins. Thymocytes exert a feedback influence on thymic non-lymphoid cells. Sg-induced modifications on cattle thymus T-lymphocytes and on non-lymphoid cells were analysed. Heifers were divided into 5 groups (control, intoxicated with Sg during 15, 30 or 60 days, and probably recovered group). Histochemical, immunohistochemical, lectinhistochemical and morphometric techniques were used to characterize different cell populations of the experimental heifers. Sg-poisoned heifers showed a progressive cortical atrophy that was characterized using the peanut agglutinin (PNA) lectin that recognizes immature thymocytes. These animals also increased the amount of non-lymphoid cells per unit area detected with the Picrosirius technique, WGA and DBA lectins, and pancytokeratin and S-100 antibodies. The thymus atrophy found in intoxicated animals resembled that of the physiological aging process. A reversal effect on these changes was observed after suppression of the intoxication. These findings suggest that Sg-intoxication induces either directly, through the 1,25-dihydroxyvitamin D3 itself, or indirectly through the hypercalcemia, the observed alteration of the thymus.

Integrins on the move

Cancer is a leading cause of death worldwide accounting for 13% of all deaths in 2005. The spread of cancer and formation of metastases is the major cause of mortality among cancer patients. The spread of cancer is based on the cancer cell’s ability to break away from the surrounding tissue and to migrate into new areas in the body. The ability of cells to bind its surroundings and to move is controlled by the mechanical cell surface adhesion receptors called the integrins. Integrins have a critical role in cell adhesion, cell motility and tissue homeostasis. By communicating with ECM, integrins transmit signals from the surrounding environment inside the cell and modulate the function of many important signalling pathways involved in cell survival, development, gene expression, proliferation, motility and cytoskeletal organization. During cell migration integrin-matrix adhesions are formed in front of the cell while rear-adhesions are released during migration. Integrins are endocytosed from the plasma-membrane into the cytoplasm and partly recycled back to new adhesion sites in a process called integrin trafficking. Also, the cell cytoskeleton and protrusions are important in cell migration. Finger-like actin protrusions called filopodia display an interesting cancer relevant cooperation with integrins that is required for cell migration. The expression and function of integrins changes markedly as cells acquire carcinogenic properties. Changed integrin function is partly responsible for detachment of tumor cells from neighbouring cells and for providing enhanced invasive capabilities for tumor cells to disseminate. Similarly, the formation of filopodia is increased in cancer. High myosin-10 expression is related to poor outcome in breast cancer and increased cell migration. The proper function of myosin-10 induced filopodia needs association with β1 integrins. The importance of integrin trafficking and filopodia formation is becoming increasingly more recognized in cancer. This thesis focusses on the role of integrins, integrin trafficking and myosin-10 induced filopodia cancer cell migration.

Vesicular Trafficking in Osteoclasts

Osteoclasts are multinucleated bone-degrading cells that undergo large changes in their polarisation and vesicular trafficking during the bone resorption cycle. Rab proteins are small GTPases that offer both temporal and spatial regulation to the transport between membranous organelles. Previously the presence and function of only few of the currently known 60 Rab proteins in osteoclasts have been reported. In this study, the expression of 26 Rab genes in bone-resorbing osteoclasts was demonstrated with gene-specific primer pairs. The further analysis of three Rab genes during human osteoclast differentiation revealed that Rab13 gene is highly induced during osteoclastogenesis. The presence of Rab13 protein in the secretory vesicles directed towards the ruffled border and in the endocytotic or transcytotic pathways in resorbing osteoclasts was excluded. The localisation of Rab13 suggests that that it is associated with a previously unknown vesicle population travelling between the trans-Golgi network and the basolateral membrane in bone resorbing osteoclasts. Rab proteins convey their functions by binding to specific effector proteins. We found a novel Rab13 interaction with endospanins-1 and -2 that are yet poorly characterised small transmembrane proteins. The Rab13 subfamily member Rab8 also bound to endospanins, while Rab10 and unrelated Rabs did not. Rab13 and endospanin-2 co-localised in perinuclear vesicles in transfected cells, demonstrating the interaction also in vivo. The inhibition of Rab13 did not interfere with the localisation of endospanin-2 nor did it affect the cell surface expression of growth hormone receptor, as has been previously described for endospanins. The physiological role of this novel protein-protein interaction thus remains to be clarified. The analysis of the transcytotic route in bone resorbing osteoclasts revealed that multiple vesicle populations arise from the ruffled border and transport the bone degradation products for exocytosis. These vesicles are directed to the functional secretory domain that is encircled by an actin-based molecular barrier. Furthermore, the transcytotic vesicles contain abundant Helix pomatia lectin binding sites and represent lipid raft concentrates. Finally, autophagosomal compartments may also be involved in the transcytosis.