The heparin-binding activity of secreted modular calcium-binding protein 1 (SMOC-1) modulates its cell adhesion properties.

Secreted modular calcium-binding proteins 1 and 2 (SMOC-1 and SMOC-1) are extracellular calcium- binding proteins belonging to the BM-40 family of proteins. In this work we have identified a highly basic region in the extracellular calcium-binding (EC) domain of the SMOC-1 similar to other known glycosaminoglycan-binding motifs. Size-exclusion chromatography shows that full length SMOC-1 as well as its C-terminal EC domain alone bind heparin and heparan sulfate, but not the related chondroitin sulfate or dermatan sulfate glycosaminoglycans. Intrinsic tryptophan fluorescence measurements were used to quantify the binding of heparin to full length SMOC-1 and the EC domain alone. The calculated equilibrium dissociation constants were in the lower micromolar range. The binding site consists of two antiparallel alpha helices and mutagenesis experiments have shown that heparin-binding residues in both helices must be replaced in order to abolish heparin binding. Furthermore, we show that the SMOC-1 EC domain, like the SMOC-2 EC domain, supports the adhesion of epithelial HaCaT cells. Heparin-binding impaired mutants failed to support S1EC-mediated cell adhesion and together with the observation that S1EC in complex with soluble heparin attenuated cell adhesion we conclude that a functional and accessible S1EC heparin-binding site mediates adhesion of epithelial cells to SMOC-1.

An indirect basal ganglia pathway in anuran amphibians?

The mammalian subthalamic nucleus (STN) is a glutamatergic cell group within the indirect pathway of the basal ganglia. It receives input from the external globus pallidus (GP) and in turn projects to the internal GP and the substantia nigra pars reticulata (SNr). While the direct pathway from striatum to SNr is well established in anurans, it is unknown whether they possess an indirect pathway including a STN homologue. The subthalamic region comprises the dorsocaudal suprachiasmatic nucleus (dcSC), the posterior entopeduncular nucleus (EP), and the ventral part of the ventral thalamus (vVM/VL). In the fire-bellied toad Bombina orientalis we investigated whether one of these areas match the criteria established for the mammalian STN. We delineated the SNr in the midbrain tegmentum by labeling the striatonigral terminal field by means of GABA-, substance P-, and enkephalin immunohistochemistry and striatal tracer injections. Subsequently, we used double fluorescence tracing with injections into the SNr and GP to stain different parts of the indirect pathway. Confocal laser scan analysis revealed that dcSC, EP, and vVM/VL contain retrogradely labeled neurons projecting to the SNr, contacted by anterogradely labeled terminals arising in the GP. Immunohistochemical stainings with antibodies against glutamate and the glutamate transporters EAAC1 and vGluT2 demonstrated that the investigated nuclei contain glutamatergic neurons. Our results suggest that all regions in the subthalamic region fulfill our morphological criteria, except the connection back to the GP. An indirect basal ganglia pathway seems to be present in anuran amphibians, although we cannot exclusively delineate an STN homologue.

From mechanotransduction to extracellular matrix gene expression in fibroblasts.

Tissue mechanics provide an important context for tissue growth, maintenance and function. On the level of organs, external mechanical forces largely influence the control of tissue homeostasis by endo- and paracrine factors. On the cellular level, it is well known that most normal cell types depend on physical interactions with their extracellular matrix in order to respond efficiently to growth factors. Fibroblasts and other adherent cells sense changes in physical parameters in their extracellular matrix environment, transduce mechanical into chemical information, and integrate these signals with growth factor derived stimuli to achieve specific changes in gene expression. For connective tissue cells, production of the extracellular matrix is a prominent response to changes in mechanical load. We will review the evidence that integrin-containing cell-matrix adhesion contacts are essential for force transmission from the extracellular matrix to the cytoskeleton, and describe novel experiments indicating that mechanotransduction in fibroblasts depends on focal adhesion adaptor proteins that might function as molecular springs. We will stress the importance of the contractile actin cytoskeleton in balancing external with internal forces, and describe new results linking force-controlled actin dynamics directly to the expression of specific genes, among them the extracellular matrix protein tenascin-C. As assembly lines for diverse signaling pathways, matrix adhesion contacts are now recognized as the major sites of crosstalk between mechanical and chemical stimuli, with important consequences for cell growth and differentiation.

The widely expressed extracellular matrix protein SMOC-2 promotes keratinocyte attachment and migration.

SMOC-2 is a recently discovered member of the BM-40/SPARC/osteonectin family of extracellular multidomain proteins of so far unknown function. While we have shown earlier that the homologous protein SMOC-1 is associated with basement membranes, in this study we demonstrate that, in the mouse, SMOC-2 could be detected in a large number of non-basement membrane localizations, often showing a diffuse tissue distribution. A more distinct expression pattern was seen in skin where SMOC-2 is mainly present in the basal layers of the epidermis. Functionally, recombinant SMOC-2 stimulated attachment of primary epidermal cells as well as several epidermal-derived cell lines but had no effect on the attachment of non-epidermal cells. Inhibition experiments using blocking antibodies against individual integrin subunits allowed the identification of alphavbeta6 and alphavbeta1 integrins as important cellular receptors for SMOC-2. Cell attachment as well as the formation of focal adhesions could be attributed to the extracellular calcium-binding domain. The calcium-binding domain also stimulated migration, but not proliferation of keratinocyte-like HaCaT cells. We conclude that SMOC-2, like other members of the BM40/SPARC family, acts as a regulator of cell-matrix interactions.

Tenascin-C induction by cyclic strain requires integrin-linked kinase.

Induction of tenascin-C mRNA by cyclic strain in fibroblasts depends on RhoA and Rho dependent kinase (ROCK). Here we show that integrin-linked kinase (ILK) is required upstream of this pathway. In ILK-deficient fibroblasts, RhoA was not activated and tenascin-C mRNA remained low after cyclic strain; tenascin-C expression was unaffected by ROCK inhibition. In ILK wild-type but not ILK-/- fibroblasts, cyclic strain-induced reorganization of actin stress fibers and focal adhesions, as well as nuclear translocation of MAL, a transcriptional co-activator that links actin assembly to gene expression. These findings support a role for RhoA in ILK-mediated mechanotransduction. Rescue of ILK -/- fibroblasts by expression of wild-type ILK restored these responses to cyclic strain. Mechanosensation is not entirely abolished in ILK -/- fibroblasts, since cyclic strain activated Erk-1/2 and PKB/Akt, and induced c-fos mRNA in these cells. Conversely, lysophosphatidic acid stimulated RhoA and induced both c-fos and tenascin-C mRNA in ILK -/- cells. Thus, the signaling pathways controlling tenascin-C expression are functional in the absence of ILK, but are not triggered by cyclic strain. Our results indicate that ILK is selectively required for the induction of specific genes by mechanical stimulation via RhoA-mediated pathways.

Is there evidence for social rhythm instability in people at risk for affective disorders?

Social rhythm disruptions are thought to be related to the etiology of affective symptoms. 'Hypomanic personality' and 'rigidity' are hypothesized to be risk factors for affective disorders. We examined whether people scoring high on such scales would demonstrate instability of social rhythms and sleep. In a short-term prospective diary study with one group factor, the following three groups were selected from a non-university student sample: 'bipolar risk' (scoring high on the 'Hypomanic Personality Scale'; n=56); 'Unipolar risk' (scoring high on the 'Rigidity Scale'; n=37); and a control group (scoring low on both scales; n=48). The participants completed ratings of their activities and sleep for 28 days. People at risk for bipolar disorders showed a lower regularity of daily activities than controls. Their sleeping pattern was not characterized by fewer but by more variable hours of sleep. The unipolar risk group did not differ from the control group at all. Despite some limitations, there is partial evidence for social rhythm and sleep irregularities in people putatively at risk for bipolar disorders. Further research is, however, needed to replicate and extend these results.