Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated β1-integrin-dependent cell migration

uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of β1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.     

A collapsin response mediator protein 2 isoform controls myosin II-mediated cell migration and matrix assembly by trapping ROCK II

Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two binding domains but also trapped and inhibited the kinase. CRMP-2L protein levels profoundly affected haptotactic migration and the actin-myosin cytoskeleton of carcinoma cells as well as nontransformed epithelial cell migration in a ROCK activity-dependent manner. Moreover, the ectopic expression of CRMP-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions through the inhibition of ROCK II in nonneuronal cells.     

Proteomics of rat biological fluids--the tenth anniversary update

In addition to a remarkable sexual dimorphism of serum and urine proteomes, the rat is exceptional for the wide difference between the serum patterns during an acute phase reaction vs baseline conditions. This feature allows monitoring with high sensitivity onset and progression of any pathological state that involves an inflammatory component as well as assessing the outcome of any therapeutic intervention. Reference maps have been defined for the proteomes of serum, urine, cerebrospinal fluid and bronchoalveolar lavage fluid. For both serum and urine most of the proteomic investigations have dealt with toxicological testing, for BALF with allergic or irritative reactions, whereas with CSF the main aim was the characterization of rat models of neurological disorders. When surveying more than ten years of literature on rat biological fluid proteomics, it is puzzling to see how seldom a consistent analytical plan has been set up for the comparative investigation on two or more types of sample, whether to fully characterize a disease model or to evaluate pharmacological/toxicological effects of a drug. It is also regrettable that in several cases only a negligible part of the results is discussed at length whereas most data are not even made known to the scientific community.     

The structure of a complex glycosylphosphatidyl inositol-anchored glucoxylan from the kinetoplastid protozoan Leptomonas samueli.

The structure of a glycosylphosphatidyl inositol-anchored glucoxylan (GPI-glucoxylan) synthesized by the monogenetic trypanosomatid Leptomonas samueli has been determined. The glucoxylan is anchored to the membrane by phytoceramide and an oligosaccharide core, the structure of which is identical to glycoinositolphospholipids (GIPLs) expressed by this protozoan. The glucoxylan chain is linear, containing -->4Glcalpha1-->, -->4Xylbeta1--> and -->3Xylbeta1--> residues. A well defined sequence heterogeneity was analysed in terms of a series of overlapping trisaccharide substructures. A proportion of the chains are capped with a GlcAalpha1-->3Glcalpha1--> sequence. While an average GlcA-capped chain contained 10 Glc and 16 Xyl residues, uncapped chains have a higher molecular mass with an average of 30 Glc and 50 Xyl per chain. We propose a mode of biosynthesis based on the observed structural heterogeneity.     

The fraction of expanding to expanded leaves determines the biomass response of Populus to elevated CO2

We examined whether the effects of elevated CO₂ on growth of 1-year old Populus deltoides saplings was a function of the assimilation responses of particular leaf developmental stages. Saplings were grown for 100 days at ambient (approximately 350 ppm) and elevated (ambient + 200 ppm) CO₂ in forced-air greenhouses. Biomass, biomass distribution, growth rates, and leaf initiation and expansion rates were unaffected by elevated CO₂. Leaf nitrogen (N), the leaf C:N ratio, and leaf lignin concentrations were also unaffected. Carbon gain was significantly greater in expanding leaves of saplings grown at elevated compared to ambient CO₂. The Rubisco content in expanding leaves was not affected by CO₂ concentration. Carbon gain and Rubisco content were significantly lower in fully expanded leaves of saplings grown at elevated compared to ambient CO₂, indicating CO₂-induced down-regulation in fully expanded leaves. Elevated CO₂ likely had no overall effect on biomass accumulation due to the more rapid decline in carbon gain as leaves matured in saplings grown at elevated compared to ambient CO₂. This decline in carbon gain has been documented in other species and shown to be related to a balance between sink/source balance and acclimation. Our data suggest that variation in growth responses to elevated CO₂ can result from differences in leaf assimilation responses in expanding versus expanded leaves as they develop under elevated CO₂. Received: 28 September 1998 / Accepted: 23 June 1999     

Combined effect of the growth temperature and salinity of the medium on the accumulation of compatible solutes by Rhodothermus marinus and Rhodothermus obamensis

In this study we propose revised structures for the two major compatible solutes of Rhodothermus marinus. We have also examined the accumulation of compatible solutes by the type strains of the slightly halophilic and thermophilic species Rhodothermus marinus and Rhodothermus obamensis at several growth temperatures and salinities. The major solutes of R. marinus were identified as α-mannosylglycerate (α-MG) and α-mannosylglyceramide (α-MGA), whereas R. obamensis accumulated only α-mannosylglycerate. The total osmolyte content was higher during the early exponential phase and decreased abruptly as growth continued into the stationary phase. At low growth temperatures, R. marinus responded to water stress by accumulation of α-mannosylglycerate and its amide, in addition to low levels of trehalose, glutamate, and glucose. At the highest growth temperature, α-mannosylglycerate was the major compatible solute and α-mannosylglyceramide was not detected. When both compounds were present, an increase in the salinity of the growth medium favored the accumulation of α-mannosylglyceramide over α-mannosylglycerate. The absence of α-mannosylglyceramide in R. obamensis at all growth temperatures and salinities constituted the most pronounced difference in the profiles of compatible solute accumulation by the two strains. Trehalose was also a prominent solute in this organism. Both organisms accumulated higher levels of α-mannosylglycerate as the temperature was raised. The importance of the two compounds in the mechanisms of thermoadaptation and osmoadaptation is discussed. Received: February 10, 1998 / Accepted: January 11, 1999     

Themis2/ICB1 Is a Signaling Scaffold That Selectively Regulates Macrophage Toll-Like Receptor Signaling and Cytokine Production

Background

Thymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number {"type":"entrez-protein","attrs":{"text":"Q8BGW0","term_id":"81896053","term_text":"Q8BGW0"}}Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 ({"type":"entrez-protein","attrs":{"text":"Q91YX0","term_id":"73920022","term_text":"Q91YX0"}}Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages.

Methodology/Principal Findings

Here we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly I∶C). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-κB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase ({"type":"entrez-protein","attrs":{"text":"P25911","term_id":"2507208","term_text":"P25911"}}P25911), the Rho guanine nucleotide exchange factor, Vav ({"type":"entrez-protein","attrs":{"text":"P27870","term_id":"137483","term_text":"P27870"}}P27870), and the adaptor protein Grb2 ({"type":"entrez-protein","attrs":{"text":"Q60631","term_id":"2498425","term_text":"Q60631"}}Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo.

Conclusions/Significance

We hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses.     

The mammalian chitinase-like lectin, YKL-40, binds specifically to type I collagen and modulates the rate of type I collagen fibril formation

YKL-40 is expressed in arthritic cartilage and produced in large amounts by cultured chondrocytes, but its exact role is unclear, and the identities of its physiological ligands remain unknown. Purification of YKL-40 from resorbing bovine nasal cartilage and chondrocyte monolayers demonstrated the existence of three isoforms, a major and minor form from resorbing cartilage and a third species from chondrocytes. Affinity chromatography experiments with purified YKL-40 demonstrated specific binding of all three forms to collagen types I, II, and III, thus identifying collagens as potential YKL-40 ligands. Binding to immobilized type I collagen was inhibited by soluble native ligand, but not heat-denatured ligand, confirming a specific interaction. Binding of the chondrocyte-derived species to type I collagen was also demonstrated by surface plasmon resonance analysis, and the dissociation rate constant was calculated (3.42 x 10(-3) to 4.50 x 10(-3) s(-1)). The chondrocyte-derived species was found to prevent collagenolytic cleavage of type I collagen and to stimulate the rate of type I collagen fibril formation in a concentration-dependent manner. By contrast, the cartilage major form had an inhibitory effect on type I collagen fibrillogenesis. Digestion with N-glycosidase F, endoglycosidase H and lectin blotting did not reveal any difference in the carbohydrate component of these two YKL-40 species, indicating that this does not account for the opposing effects on fibril formation rate.