The transcription factor grainyhead-like 2 regulates the molecular composition of the epithelial apical junctional complex.

Differentiation of epithelial cells and morphogenesis of epithelial tubes or layers is closely linked with the establishment and remodeling of the apical junctional complex, which includes adherens junctions and tight junctions. Little is known about the transcriptional control of apical junctional complex components. Here, we show that the transcription factor grainyhead-like 2 (Grhl2), an epithelium-specific mammalian homolog of Drosophila Grainyhead, is essential for adequate expression of the adherens junction gene E-cadherin and the tight junction gene claudin 4 (Cldn4) in several types of epithelia, including gut endoderm, surface ectoderm and otic epithelium. We have generated Grhl2 mutant mice to demonstrate defective molecular composition of the apical junctional complex in these compartments that coincides with the occurrence of anterior and posterior neural tube defects. Mechanistically, we show that Grhl2 specifically associates with cis-regulatory elements localized at the Cldn4 core promoter and within intron 2 of the E-cadherin gene. Cldn4 promoter activity in epithelial cells is crucially dependent on the availability of Grhl2 and on the integrity of the Grhl2-associated cis-regulatory element. At the E-cadherin locus, the intronic Grhl2-associated cis-regulatory region contacts the promoter via chromatin looping, while loss of Grhl2 leads to a specific decrease of activating histone marks at the E-cadherin promoter. Together, our data provide evidence that Grhl2 acts as a target gene-associated transcriptional activator of apical junctional complex components and, thereby, crucially participates in epithelial differentiation.

A stomatin-domain protein essential for touch sensation in the mouse.

Touch and mechanical pain are first detected at our largest sensory surface, the skin. The cell bodies of sensory neurons that detect such stimuli are located in the dorsal root ganglia, and subtypes of these neurons are specialized to detect specific modalities of mechanical stimuli. Molecules have been identified that are necessary for mechanosensation in invertebrates but so far not in mammals. In Caenorhabditis elegans, mec-2 is one of several genes identified in a screen for touch insensitivity and encodes an integral membrane protein with a stomatin homology domain. Here we show that about 35% of skin mechanoreceptors do not respond to mechanical stimuli in mice with a mutation in stomatin-like protein 3 (SLP3, also called Stoml3), a mammalian mec-2 homologue that is expressed in sensory neurons. In addition, mechanosensitive ion channels found in many sensory neurons do not function without SLP3. Tactile-driven behaviours are also impaired in SLP3 mutant mice, including touch-evoked pain caused by neuropathic injury. SLP3 is therefore indispensable for the function of a subset of cutaneous mechanoreceptors, and our data support the idea that this protein is an essential subunit of a mammalian mechanotransducer.

Nociceptive tuning by stem cell factor/c-Kit signaling.

The molecular mechanisms regulating the sensitivity of sensory circuits to environmental stimuli are poorly understood. We demonstrate here a central role for stem cell factor (SCF) and its receptor, c-Kit, in tuning the responsiveness of sensory neurons to natural stimuli. Mice lacking SCF/c-Kit signaling displayed profound thermal hypoalgesia, attributable to a marked elevation in the thermal threshold and reduction in spiking rate of heat-sensitive nociceptors. Acute activation of c-Kit by its ligand, SCF, resulted in a reduced thermal threshold and potentiation of heat-activated currents in isolated small-diameter neurons and thermal hyperalgesia in mice. SCF-induced thermal hyperalgesia required the TRP family cation channel TRPV1. Lack of c-Kit signaling during development resulted in hypersensitivity of discrete mechanoreceptive neuronal subtypes. Thus, c-Kit can now be grouped with a small family of receptor tyrosine kinases, including c-Ret and TrkA, that control the transduction properties of sensory neurons.

M line-deficient titin causes cardiac lethality through impaired maturation of the sarcomere.

Titin, the largest protein known to date, has been linked to sarcomere assembly and function through its elastic adaptor and signaling domains. Titin's M-line region contains a unique kinase domain that has been proposed to regulate sarcomere assembly via its substrate titin cap (T-cap). In this study, we use a titin M line-deficient mouse to show that the initial assembly of the sarcomere does not depend on titin's M-line region or the phosphorylation of T-cap by the titin kinase. Rather, titin's M-line region is required to form a continuous titin filament and to provide mechanical stability of the embryonic sarcomere. Even without titin integrating into the M band, sarcomeres show proper spacing and alignment of Z discs and M bands but fail to grow laterally and ultimately disassemble. The comparison of disassembly in the developing and mature knockout sarcomere suggests diverse functions for titin's M line in embryonic development and the adult heart that not only involve the differential expression of titin isoforms but also of titin-binding proteins.

Selective inflammatory pain insensitivity in the African naked mole-rat (Heterocephalus glaber).

In all mammals, tissue inflammation leads to pain and behavioral sensitization to thermal and mechanical stimuli called hyperalgesia. We studied pain mechanisms in the African naked mole-rat, an unusual rodent species that lacks pain-related neuropeptides (e.g., substance P) in cutaneous sensory fibers. Naked mole-rats show a unique and remarkable lack of pain-related behaviors to two potent algogens, acid and capsaicin. Furthermore, when exposed to inflammatory insults or known mediators, naked mole-rats do not display thermal hyperalgesia. In contrast, naked mole-rats do display nocifensive behaviors in the formalin test and show mechanical hyperalgesia after inflammation. Using electrophysiology, we showed that primary afferent nociceptors in naked mole-rats are insensitive to acid stimuli, consistent with the animal's lack of acid-induced behavior. Acid transduction by sensory neurons is observed in birds, amphibians, and fish, which suggests that this tranduction mechanism has been selectively disabled in the naked mole-rat in the course of its evolution. In contrast, nociceptors do respond vigorously to capsaicin, and we also show that sensory neurons express a transient receptor potential vanilloid channel-1 ion channel that is capsaicin sensitive. Nevertheless, the activation of capsaicin-sensitive sensory neurons in naked mole-rats does not produce pain-related behavior. We show that capsaicin-sensitive nociceptors in the naked mole-rat are functionally connected to superficial dorsal horn neurons as in mice. However, the same nociceptors are also functionally connected to deep dorsal horn neurons, a connectivity that is rare in mice. The pain biology of the naked mole-rat is unique among mammals, thus the study of pain mechanisms in this unusual species can provide major insights into what constitutes "normal" mammalian nociception.

Beyond the sarcomere: CSRP3 mutations cause hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a frequent genetic cardiac disease and the most common cause of sudden cardiac death in young individuals. Most of the currently known HCM disease genes encode sarcomeric proteins. Previous studies have shown an association between CSRP3 missense mutations and either dilated cardiomyopathy (DCM) or HCM, but all these studies were unable to provide comprehensive genetic evidence for a causative role of CSRP3 mutations. We used linkage analysis and identified a CSRP3 missense mutation in a large German family affected by HCM. We confirmed CSRP3 as an HCM disease gene. Furthermore, CSRP3 missense mutations segregating with HCM were identified in four other families. We used a newly designed monoclonal antibody to show that muscle LIM protein (MLP), the protein encoded by CSRP3, is mainly a cytosolic component of cardiomyocytes and not tightly anchored to sarcomeric structures. Our functional data from both in vitro and in vivo analyses suggest that at least one of MLP's mutated forms seems to be destabilized in the heart of HCM patients harbouring a CSRP3 missense mutation. We also present evidence for mild skeletal muscle disease in affected persons. Our results support the view that HCM is not exclusively a sarcomeric disease and also suggest that impaired mechano-sensory stress signalling might be involved in the pathogenesis of HCM.

Impaired synapse function during postnatal development in the absence of CALEB, an EGF-like protein processed by neuronal activity.

In an attempt to characterize the molecular components by which electric activity influences the development of synapses, we searched for cell surface proteins modulated by calcium influx and glutamate receptor activity. Here, we report that neuronal depolarization facilitates the conversion of CALEB, which results in a truncated transmembrane form with an exposed EGF domain. To characterize the role of CALEB in synapse development, synaptic features were investigated in slices of the colliculus superior from CALEB-deficient mice. In the absence of CALEB, the number of synapses and their morphological characteristics remained unchanged. However, in CALEB-deficient mice, synapses displayed higher paired-pulse ratios, less depression during prolonged repetitive activation, a lower rate of spontaneous postsynaptic currents, and a lower release probability at early but not mature postnatal stages. Our findings indicate that CALEB provides a molecular basis for maintaining normal release probability at early developmental stages.

Stress-induced dilated cardiomyopathy in a knock-in mouse model mimicking human titin-based disease.

Mutations in a variety of myofibrillar genes cause dilated cardiomyopathy (DCM) in humans, usually with dominant inheritance and incomplete penetrance. Here, we sought to clarify the functional effects of the previously identified DCM-causing TTN 2-bp insertion mutation (c.43628insAT) and generated a titin knock-in mouse model mimicking the c.43628insAT allele. Mutant embryos homozygous for the Ttn knock-in mutation developed defects in sarcomere formation and consequently died before E9.5. Heterozygous mice were viable and demonstrated normal cardiac morphology, function and muscle mechanics. mRNA and protein expression studies on heterozygous hearts demonstrated elevated wild-type titin mRNA under resting conditions, suggesting that up-regulation of the wild-type titin allele compensates for the unstable mutated titin under these conditions. When chronically exposed to angiotensin II or isoproterenol, heterozygous mice developed marked left ventricular dilatation (p<0.05) with impaired fractional shortening (p<0.001) and diffuse myocardial fibrosis (11.95+/-2.8% vs. 3.7+/-1.1%). Thus, this model mimics typical features of human dilated cardiomyopathy and may further our understanding of how titin mutations perturb cardiac function and remodel the heart.

Estrogen and the inner ear: megalin knockout mice suffer progressive hearing loss.

Megalin, the largest member of the low-density lipoprotein receptor-related protein family, functions as an endocytic receptor for a variety of essential lipophilic metabolites, including the steroid hormone estrogen. In the cochlea, megalin is strongly expressed within the marginal cells of the stria vascularis, and previous studies demonstrated that beta-estrogen receptors are also expressed in megalin-expressing marginal cells. In the present study, we demonstrate that homozygous megalin mutant mice exhibit profound hearing loss at 3 months of age associated with features of presbycusis, enrichment of lipofuscin granules, and a reduced number of microvilli in marginal cells of the stria vascularis. FITC-labeled beta-estrogen is taken up into the strial marginal cells; however, in megalin-deficient mice the uptake of FITC-labeled beta-estrogen is reduced. This highlights beta-estrogen as a possible carrier-bound candidate ligand for megalin and supports the concept that estrogen may function via megalin within the inner ear. A crucial role of megalin in hearing should be considered and the megalin/estrogen interaction needs to be discussed in the context of early presbycusis in estrogen-deficient humans and mice.

Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation.

Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease. We have ablated the plakophilin 2 gene in mice. The resulting mutant mice exhibit lethal alterations in heart morphogenesis and stability at mid-gestation (E10.5-E11), characterized by reduced trabeculation, disarrayed cytoskeleton, ruptures of cardiac walls, and blood leakage into the pericardiac cavity. In the absence of plakophilin 2, the cytoskeletal linker protein desmoplakin dissociates from the plaques of the adhering junctions that connect the cardiomyocytes and forms granular aggregates in the cytoplasm. By contrast, embryonic epithelia show normal junctions. Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

Structural appearance of linker histone H1/siRNA complexes.

Efficient non-viral vectors for the in vivo siRNA transfer are still being searched for. Comparing the differences of the structural appearance of siRNA and pDNA one would assume differences in the assembling behaviour between these polyanions when using polycationic vectors such as nuclear proteins. The spontaneous assembly of nuclear proteins such as histone H1 (H1) with pDNA as polyanion which has intensively been investigated over the last decade, showed a particulate structure of the resulting complexes. For an efficient in vivo use small almost monomolecular structures are searched for. Using siRNA as the polyanion might enforce this structural prerequisite lacking unwanted aggregation processes, exploiting the molecular size of siRNA. We therefore investigated the structure of H1/siRNA complexes. Five commonly used methods characterizing the resulting assemblies such as retardation gels, static and dynamic light scattering, reduction of ethidium bromide fluorescence, analytical ultracentrifugation, and electron microscopy were used. From analytical ultracentrifugation we learned that under physiological salt conditions the siRNA-H1 binding was not cooperative, even though the gel analysis showed disproportionation which would be an indication for a cooperative binding mode. H1 formed very small and stable complexes with siRNA at a molar ratio of 1:1 and 1:2. In order to find out if the observed structural appearance of the H1/siRNA complexes is due to unspecific charge effects only or to special features of H1, polylysine was included in the study. Low molecular weight polylysine (K(16)) showed also non-cooperative binding with siRNA.

A missense variant in desmoglein-2 predisposes to dilated cardiomyopathy.

Familial Dilated Cardiomyopathy (FDCM) is caused by mutations in genes encoding myocardial force transduction proteins. Desmoglein-2 (DSG2) and Desmocollin-2 (DSC2) provide cellular adhesion and force transduction by cell-to-cell anchorage. To test whether perturbations of DSG2 or DSC2 exhibit a pathogenic impact on DCM pathogenesis, we sequenced both genes in 73 patients with FDCM and assessed prevalence of missense variations in matched control cohorts. We detected two missense variations in DSG2 (V55M and V919G) which were absent in 360 control alleles. Surprisingly, both variants were previously reported in patients with arrhythmogenic right ventricular cardiomyopathy. Yet, in the present study only the DSG2-V55M variant showed segregation with DCM in a family pedigree. Subsequent, analysis of 538 patients with idiopathic DCM and 617 consecutive control individuals resulted in identification of thirteen DSG2-V55M carriers with DCM, whereas only three control subjects harbored the variant. DSG2 immunostaining revealed pale structures of the intercalated disc in myocardium of one unique homozygous DSG2-V55M carrier. Furthermore, myocardial desmosomal structures were significantly shortened when compared to DCM myocardium negative for DSG2-V55M. Thus, our study identified the DSG2-V55M polymorphism as a novel risk variant for DCM associated with shortened desmosomes of the cardiac intercalated disc.

Dysfunction of dysferlin-deficient hearts.

Mutations in the gene encoding dysferlin cause limb-girdle muscular dystrophy 2B (LGMD2B), a disorder that is believed to spare the heart. We observed dilated cardiomyopathy in two out of seven LGMD2B patients and cardiac abnormalities in three others. Cardiac biopsies showed that dysferlin was completely absent from the sarcolemma and appeared to be trapped within the cardiomyocytes. SJL/J mice (33-week-old) had diminished end-systolic pressure and reduced dP/dt; however, the hearts were histologically normal. Gene expression profiles of cardiac tissue were obtained and later confirmed by quantitative RT-PCR. Dysferlin-deficient and control mice had different gene expression patterns in terms of cardiomyocyte Z-disc and signal transduction proteins. CapZ, LIM-domain-binding protein 3 (LDB3, MLP), cypher (ZASP), desmin, and the cardiac ankyrin-repeated protein (CARP) were differentially expressed, compared to controls. Mechanical stress induced by the nonselective beta-adrenergic agonist isoproterenol (5 mg/kg body weight) given daily for 10 days resulted in reduced fractional shortening and increased cardiac fibrosis in SJL/J mice as compared to controls. Isoproterenol also caused metalloproteinase-2 upregulation in SJL/J mice. In A/J mice, the effect of isoproterenol injection was even more dramatic and lead to premature death as well as marked sarcolemmal injury as demonstrated by Evans blue dye penetration. Our data suggest that disturbances in dysferlin as well as Z-line proteins and transcription factors particularly under mechanical stress cause cardiomyopathy.

CY15, a malignant histiocytic tumor that is phenotypically similar to immature dendritic cells.

The origin and pathogenesis of histiocytic malignancies and the biology of the tumor cells are poorly understood. We have isolated a murine histiocytic tumor cell line (CY15) from a BALB/c IFNgamma(-/-) mouse and characterized it in terms of phenotype and function. The morphology, as judged by electron microscopy, and the surface marker phenotype suggests that CY15 cells are similar to immature dendritic cells (CD11c (low), MHC II (low), CD11b(+), B7.1(+), B7.2(+), and CD40(+)). The cells form tumors in BALB/c mice and metastasize to spleen, liver, lung, kidney, and to a lesser extend to lymph nodes and bone marrow, as judged by the growth of green fluorescent protein transfected tumor cells in mice. CY15 cells are capable of actively taking up antigen (FITC-ovalbumin) and can stimulate T lymphocytes in an allogenic mixed lymphocyte reaction but less effectively than their normal counterparts (immature dendritic cells). They respond to interleukin 4 (IL-4) with up-regulation of CD11c. If stimulated with IFNgamma the cells up-regulate MHC II, CD40 B7.1, and B7.2. Lipopolysaccharide induces the cells to up-regulate B7.1 and B7.2 and to secrete tumor necrosis factor alpha and IL-12. Based on these data, CY15 is a dendritic cell-like tumor cell line and may serve as a transplantable tumor model for histiocytosis in humans.

Reevaluation of the 22-1-1 antibody and its putative antigen, EBAG9/RCAS1, as a tumor marker.

BACKGROUND: Tumor-associated antigens are appreciated as diagnostic markers, but they have also prompted tremendous efforts to develop tumor-specific immunotherapy. A previously cloned tumor-associated antigen, EBAG9, was initially defined by reactivity with the monoclonal antibody 22-1-1. Functionally, the EBAG9-encoded gene-product was believed to induce apoptosis in activated immune cells. However, using a cell-biological approach we identified EBAG9 as a Golgi-resident modulator of O-linked glycan expression, the latter product was then recognized by the 22-1-1 antibody. Secondly, EBAG9 expression was found physiologically in all murine tissues examined. This raised the question if EBAG9 is tumor-specific and mediates apoptosis itself or through O-linked glycans generated, among them the cognate 22-1-1 antigen Tn. METHODS: We have used immunohistochemistry to detect the expression of 22-1-1 and EBAG9 in various tissues. Correlation between expression of both antigens in cell lines was analysed by immunoblot and flow cytometry. Apoptosis was studied by using flow cytometry and Caspase-Glo 3/7 assay kit. Cellular distribution of EBAG9 was analysed by electron and confocal microscopy. RESULTS: Here, we compared expression of the 22-1-1 and EBAG9-defined antigens in normal and neoplastic tissues in situ. In contrast to 22-1-1 staining, EBAG9 is a ubiquitously expressed antigen in all normal and cancerous tissues. Functional studies on the role of 22-1-1 reactive material did not support any evidence for apoptosis induction. Employing electron and confocal microscopy, a refined subcellular localization of EBAG9 at the Golgi was obtained. CONCLUSION: We suggest that the estrogen-inducible EBAG9 gene-product and the 22-1-1 defined antigen are structurally and functionally separate antigens.

Ultrastructural analysis of DNA complexes during transfection and intracellular transport.

We present a simple method based on transmission electron microscopy that allows investigation of the early steps of polyplex-mediated transfection without the use of labeled DNA. The ultrastructural analysis showed internalization of 0.2-1-micro m aggregates composed of 30-50-nm subunits. In addition, new details of the internalization process were revealed, suggesting an unspecific cell entry mechanism of large DNA aggregates.

Sortilin associates with Trk receptors to enhance anterograde transport and neurotrophin signaling.

Binding of target-derived neurotrophins to Trk receptors at nerve terminals is required to stimulate neuronal survival, differentiation, innervation and synaptic plasticity. The distance between the soma and nerve terminal is great, making efficient anterograde Trk transport critical for Trk synaptic translocation and signaling. The mechanism responsible for this trafficking remains poorly understood. Here we show that the sorting receptor sortilin interacts with TrkA, TrkB and TrkC and enables their anterograde axonal transport, thereby enhancing neurotrophin signaling. Cultured DRG neurons lacking sortilin showed blunted MAP kinase signaling and reduced neurite outgrowth upon stimulation with NGF. Moreover, deficiency for sortilin markedly aggravated TrkA, TrkB and TrkC phenotypes present in p75(NTR) knockouts, and resulted in increased embryonic lethality and sympathetic neuropathy in mice heterozygous for TrkA. Our findings demonstrate a role for sortilin as an anterograde trafficking receptor for Trk and a positive modulator of neurotrophin-induced neuronal survival.

The tumor-associated antigen EBAG9 negatively regulates the cytolytic capacity of mouse CD8+ T cells.

CTLs eliminate virus-infected and tumorigenic cells through exocytosis of cytotoxic agents from lytic granules. While insights into the intracellular mechanisms facilitating lytic granule release have been obtained through analysis of loss-of-function mutations in humans and mice, there is a paucity of information on negative regulators of secretory lysosome release at the molecular level. By generating and analyzing estrogen receptor-binding fragment-associated antigen 9-KO (Ebag9 KO) mice, we show here that loss of EBAG9 confers CTLs with enhanced cytolytic capacity in vitro and in vivo. Although loss of EBAG9 did not affect lymphocyte development, it led to an increase in CTL secretion of granzyme A, a marker of lytic granules. This resulted in increased cytotoxicity in vitro and an enhanced cytolytic primary and memory T cell response in vivo. We further found that EBAG9 interacts with the adaptor molecule gamma2-adaptin, suggesting EBAG9 is involved in endosomal-lysosomal biogenesis and membrane fusion. Indeed, granzyme B was sorted to secretory lysosomes more efficiently in EBAG9-deficient CTLs than it was in WT CTLs, a finding consistent with the observed enhanced kinetics of cathepsin D proteolytic processing. While EBAG9 deficiency did not disrupt the formation of the immunological synapse, lytic granules in Ebag9-/- CTLs were smaller than in WT CTLs. These data suggest that EBAG9 is a tunable inhibitor of CTL-mediated adaptive immune response functions.

Connective tissue growth factor overexpression in cardiomyocytes promotes cardiac hypertrophy and protection against pressure overload.

Connective tissue growth factor (CTGF) is a secreted protein that is strongly induced in human and experimental heart failure. CTGF is said to be profibrotic; however, the precise function of CTGF is unclear. We generated transgenic mice and rats with cardiomyocyte-specific CTGF overexpression (CTGF-TG). To investigate CTGF as a fibrosis inducer, we performed morphological and gene expression analyses of CTGF-TG mice and rat hearts under basal conditions and after stimulation with angiotensin II (Ang II) or isoproterenol, respectively. Surprisingly, cardiac tissues of both models did not show increased fibrosis or enhanced gene expression of fibrotic markers. In contrast to controls, Ang II treated CTGF-TG mice displayed preserved cardiac function. However, CTGF-TG mice developed age-dependent cardiac dysfunction at the age of 7 months. CTGF related heart failure was associated with Akt and JNK activation, but not with the induction of natriuretic peptides. Furthermore, cardiomyocytes from CTGF-TG mice showed unaffected cellular contractility and an increased Ca(2+) reuptake from sarcoplasmatic reticulum. In an ischemia/reperfusion model CTGF-TG hearts did not differ from controls.Our data suggest that CTGF itself does not induce cardiac fibrosis. Moreover, it is involved in hypertrophy induction and cellular remodeling depending on the cardiac stress stimulus. Our new transgenic animals are valuable models for reconsideration of CTGF's profibrotic function in the heart.

Sortilin-related receptor with A-type repeats (SORLA) affects the amyloid precursor protein-dependent stimulation of ERK signaling and adult neurogenesis.

Sortilin-related receptor with A-type repeats (SORLA) is a sorting receptor that impairs processing of amyloid precursor protein (APP) to soluble (s) APP and to the amyloid beta-peptide in cultured neurons and is poorly expressed in patients with Alzheimer disease (AD). Here, we evaluated the consequences of Sorla gene defects on brain anatomy and function using mouse models of receptor deficiency. In line with a protective role for SORLA in APP metabolism, lack of the receptor results in increased amyloidogenic processing of endogenous APP and in aggravated plaque deposition when introduced into PDAPP mice expressing mutant human APP. Surprisingly, increased levels of sAPP caused by receptor deficiency correlate with pro-found stimulation of neuronal ERK signaling and with enhanced neurogenesis, providing in vivo support for neurotrophic functions of sAPP. Our data document a role for SORLA not only in control of plaque burden but also in APP-dependent neuronal signaling and suggest a molecular explanation for increased neurogenesis observed in some AD patients.