Ethylene modulates gene expression in cells of the marine sponge Suberites domuncula and reduces the degree of apoptosis.

Sponges (phylum Porifera) live in an aqueous milieu that contains dissolved organic carbon. This is degraded photochemically by ultraviolet radiation to alkenes, particularly to ethylene. This study demonstrates that sponge cells (here the demosponge Suberites domuncula has been used), which have assembled to primmorphs, react to 5 microM ethylene with a significant up-regulation of intracellular Ca(2+) concentration and with a reduction of starvation-induced apoptosis. In primmorphs from S. domuncula the expression of two genes is up-regulated after exposure to ethylene. The cDNA of the first gene (SDERR) isolated from S. domuncula encodes a potential ethylene-responsive protein, termed ERR_SUBDO; its putative M(r) is 32,704. Data bank search revealed that the sponge polypeptide shares high similarity (82% on amino acid level) with the corresponding plant molecule, the ethylene-inducible protein from Hevea brasiliensis. Until now no other metazoan ethylene-responsive proteins have been identified. The second gene, whose expression is up-regulated in response to ethylene is a Ca(2+)/calmodulin-dependent protein kinase II. Its cDNA, SDCCdPK, encodes a M(r) 54,863 putative kinase that shares 69% similarity with the corresponding enzyme from Drosophila melanogaster. The expression of both genes in primmorphs from S. domuncula is increased by approximately 5-fold after a 3-day incubation period with ethylene. It is concluded that also metazoan cells, with sponge cells as a model, may react to ethylene with an activation of cell metabolism including gene induction.     

Increased gene expression of a cytokine-related molecule and profilin after activation of Suberites domuncula cells with xenogeneic sponge molecule(s)

Porifera (sponges) constitute the lowest metazoan phylum. Experiments examined whether sponges can recognize self/nonself molecules. Cells from the marine sponge Suberites domuncula were incubated with membranes from either S. domuncula or another marine sponge, Geodia cydonium, as well as with recombinant alpha-integrin from G. cydonium. The cells responded immediately with a rise of intracellular Ca2+ ([Ca2+i]) if they were treated with membranes from G. cydonium but not after treatment by those from S. domuncula. This change of [Ca2+i] was also recorded with G. cydonium alpha-integrin. In parallel, the expression of two genes was strongly upregulated; one codes for a cytokine-related molecule, pre-B-cell colony-enhancing factor, and the other for profilin. These genes have previously been found to be highly expressed in human or echinoderm cells in the presence of xenogeneic proteins. Our data support the hypothesis that a primordial immune response system is present in sponges.     

Urokinase-type plasminogen activator receptor induces conformational changes in the integrin alphaMbeta2 headpiece and reorientation of its transmembrane domains

The glycosylphosphatidylinositol-linked urokinase-type plasminogen activator receptor (uPAR) interacts with the heterodimer cell adhesion molecules integrins to modulate cell adhesion and migration. Devoid of a cytoplasmic domain, uPAR triggers intracellular signaling via its associated molecules that contain cytoplasmic domains. Interestingly, uPAR changes the ectodomain conformation of one of its partner molecules, integrin alpha(5)beta(1), and elicits cytoplasmic signaling. The separation or reorientation of integrin transmembrane domains and cytoplasmic tails are required for integrin outside-in signaling. However, there is a lack of direct evidence showing these conformational changes of an integrin that interacts with uPAR. In this investigation we used reporter monoclonal antibodies and fluorescence resonance energy transfer analyses to show conformational changes in the alpha(M)beta(2) headpiece and reorientation of its transmembrane domains when alpha(M)beta(2) interacts with uPAR.     

Critical roles for the COOH-terminal NITY and RGT sequences of the integrin beta3 cytoplasmic domain in inside-out and outside-in signaling

Bidirectional signaling of integrin alphaIIbbeta3 requires the beta3 cytoplasmic domain. To determine the sequence in the beta3 cytoplasmic domain that is critical to integrin signaling, cell lines were established that coexpress the platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX, integrin alphaIIb, and mutants of beta3 with truncations at sites COOH terminal to T741, Y747, F754, and Y759. Truncation at Y759 did not affect integrin activation, as indicated by vWF-induced fibrinogen binding, but affected cell spreading and stable adhesion. Thus, the COOH-terminal RGT sequence of beta3 is important for outside-in signaling but not inside-out signaling. In contrast, truncation at F754, Y747, or T741 completely abolished integrin activation. A point mutation replacing Y759 with alanine also abolished integrin activation. Thus, the T755NITY759 sequence of beta3, containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling. In addition, we found that the calcium-dependent protease calpain preferentially cleaves at Y759 in a population of beta3 during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.     

Stimulation of protein (collagen) synthesis in sponge cells by a cardiac myotrophin-related molecule from Suberites domuncula.

The body wall of sponges (Porifera), the lowest metazoan phylum, is formed by two epithelial cell layers of exopinacocytes and endopinacocytes, both of which are associated with collagen fibrils. Here we show that a myotrophin-like polypeptide from the sponge Suberites domuncula causes the expression of collagen in cells from the same sponge in vitro. The cDNA of the sponge myotrophin was isolated; the potential open reading frame of 360 nt encodes a 120 aa long protein (Mr of 12,837). The sequence SUBDOMYOL shares high similarity with the known metazoan myotrophin sequences. The expression of SUBDOMYOL is low in single cells but high after formation of primmorph aggregates as well as in intact animals. Recombinant myotrophin was found to stimulate protein synthesis by fivefold, as analyzed by incorporation studies using [3H] lysine. In addition, it is shown that after incubation of single cells with myotrophin, the primmorphs show an unusual elongated, oval-shaped appearance. It is demonstrated that in the presence of recombinant myotrophin, the cells up-regulate the expression of the collagen gene. The cDNA for S. domuncula collagen was isolated; the deduced aa sequence shows that the collagenous internal domain is rather short, with only 24 G-x-y collagen triplets. We conclude that the sponge myotrophin causes in homologous cells the same/similar effect as the cardiac myotrophin in mammalian cells, where it is involved in initiation of cardial ventricular hypertrophy. We assume that an understanding of sponge molecular cell biology will also contribute to a further elucidation of human diseases, here of the cardiovascular system.     

Calcium signaling through the beta 2-cytoplasmic domain of LFA-1 requires intracellular elements of the T cell receptor complex

The beta(2) integrin LFA-1 is an important cell-cell adhesion receptor of the immune system. Evidence suggests that the molecule also participates in signaling and co-stimulatory function. We show here that clustering of the intracellular domain of the beta(2) chain but not of the alpha(L)- or beta(1)-cytoplasmic domains, respectively, triggers intracellular Ca(2+) mobilization in Jurkat cells. A beta(2)-specific NPXF motif, located in the C-terminal portion of the beta(2) tail, is required for Ca(2+) signaling, and we show that this motif is important for the induction of allo-specific target cell lysis by cytotoxic T cells in vitro. Significantly, the Ca(2+)-signaling capacity of the beta(2) integrin is abrogated in T cells that do not express the T cell receptor but may be reconstituted by co-expression of the T cell receptor-zeta chain. Our data suggest a specific function of the cytoplasmic domain of the beta(2) integrin chain in T cell signaling.     

Specification of the direction of adhesive signaling by the integrin beta cytoplasmic domain

Integrin adhesion receptors can signal in two directions: first, they can regulate cellular behaviors by modulating cellular signaling enzymes ("outside-in signaling"); second, cells can regulate the affinity of integrins ("inside-out signaling") by such pathways. Integrin beta cytoplasmic domains (tails) mediate both types of signaling, and Src family kinases (SFKs) and talin, which bind to beta tails, are important for integrin signaling. Here, we utilized "homology scanning" mutagenesis to identify beta tail mutants selectively defective in c-Src binding and found that amino acid exchanges affecting a combination of an Arg and Thr residue in the integrin beta3 tail control the binding specificity for SFKs but have no effect on talin binding. Using beta tail mutants at these residues, we found that SFK binding to integrin beta tails is dispensable for inside-out signaling but is obligatory for cell spreading, a marker of outside-in signaling. Conversely, we found that point mutations that disrupt talin binding abolish integrin activation, but they do not inhibit SFK binding to the beta3 tail or the initiation of outside-in signaling once the integrins are in a high affinity form. Thus, we show that inside-out and outside-in integrin signaling are mediated by distinct and separable interactions of the integrin beta tails. Furthermore, based on our results, it is possible to discern the relative contributions of the direction of integrin signaling on biological functions in cell culture and, ultimately, in vivo.     

The Src family kinases Hck and Fgr are dispensable for inside-out, chemoattractant-induced signaling regulating beta 2 integrin affinity and valency in neutrophils, but are required for beta 2 integrin-mediated outside-in signaling involved in sustained adhesion

Neutrophil beta(2) integrins are activated by inside-out signaling regulating integrin affinity and valency; following ligand binding, beta(2) integrins trigger outside-in signals regulating cell functions. Addressing inside-out and outside-in signaling in hck(-/-)fgr(-/-) neutrophils, we found that Hck and Fgr do not regulate chemoattractant-induced activation of beta(2) integrin affinity. In fact, beta(2) integrin-mediated rapid adhesion, in static condition assays, and neutrophil adhesion to glass capillary tubes cocoated with ICAM-1, P-selectin, and a chemoattractant, under flow, were unaffected in hck(-/-)fgr(-/-) neutrophils. Additionally, examination of integrin affinity by soluble ICAM-1 binding assays and of beta(2) integrin clustering on the cell surface, showed that integrin activation did not require Hck and Fgr expression. However, after binding, hck(-/-)fgr(-/-) neutrophil spreading over beta(2) integrin ligands was reduced and they rapidly detached from the adhesive surface. Whether alterations in outside-in signaling affect sustained adhesion to the vascular endothelium in vivo was addressed by examining neutrophil adhesiveness to inflamed muscle venules. Intravital microscopy analysis allowed us to conclude that Hck and Fgr regulate neither the number of rolling cells nor rolling velocity in neutrophils. However, arrest of hck(-/-)fgr(-/-) neutrophils to >60 microm in diameter venules was reduced. Thus, Hck and Fgr play no role in chemoattractant-induced inside-out beta(2) integrin activation but regulate outside-in signaling-dependent sustained adhesion.     

Contribution of sponge genes to unravel the genome of the hypothetical ancestor of Metazoa (Urmetazoa)

Recently the term Urmetazoa, as the hypothetical metazoan ancestor, was introduced to highlight the finding that all metazoan phyla including the Porifera (sponges) are derived from one common ancestor. Sponges as the evolutionarily oldest, still extant phylum, are provided with a complex network of structural and functional molecules. Analyses of sponge genomes from Demospongiae (Suberites domuncula and Geodia cydonium), Calcarea (Sycon raphanus) and Hexactinellida (Aphrocallistes vastus) have contributed also to the reconstruction of the evolutionary position of Metazoa with respect to Fungi. Furthermore, these analyses have provided evidence that the characteristic evolutionary novelties of Metazoa, such as the extracellular matrix molecules, the cell surface receptors, the nervous signal transduction molecules as well as the immune molecule existing in Porifera, share high sequence and in some aspects also functional similarities to related polypeptides found in other metazoan phyla. During the transition to Metazoa new domains occurred; as one example, the formation of the death domain from the ankyrin is outlined. In parallel, domanial proteins have been formed, such as the receptor tyrosine kinases. The metazoan essentials have been defined by analyzing and comparing the sponge sequences with the related sequences from the metazoans Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster, the fungus Saccharomyces cerevisiae and the plant Arabidopsis thaliana. The data revealed that those sponge molecules grouped to cell adhesion cell recognition proteins are predominantly found in Protostomia and Deuterostomia while they are missing in Fungi and Viridiplantae. Moreover, evidence is presented allowing the conclusion that the sponge molecules are more closely related to the corresponding molecules from H. sapiens than to those of C. elegans or D. melanogaster. Especially surprising was the finding that the Demospongiae are provided with elements of adaptive immunity.     

Integrins and Src: dynamic duo of adhesion signaling

Src family protein tyrosine kinases (SFKs) play important roles downstream of integrin adhesion receptors, and they are necessary for the generation of "outside-in signals" that regulate cytoskeletal organization, cell motility and gene expression in response to cell adhesion. One relatively under-explored facet of this relationship is the possible physical interaction of integrins with SFKs. Recently, it has been established that beta3 integrins and c-Src can interact directly, and this pool of c-Src is activated by cell adhesion to initiate outside-in signaling in platelets, osteoclasts and cells of the vasculature. Here, the biochemical basis for and biological significance of this integrin-SFK interaction is summarized, and I propose a general mechanism for initiation of outside-in integrin signaling.     

A molecular switch that controls cell spreading and retraction

Integrin-dependent cell spreading and retraction are required for cell adhesion, migration, and proliferation, and thus are important in thrombosis, wound repair, immunity, and cancer development. It remains unknown how integrin outside-in signaling induces and controls these two opposite processes. This study reveals that calpain cleavage of integrin beta(3) at Tyr(759) switches the functional outcome of integrin signaling from cell spreading to retraction. Expression of a calpain cleavage-resistant beta(3) mutant in Chinese hamster ovary cells causes defective clot retraction and RhoA-mediated retraction signaling but enhances cell spreading. Conversely, a calpain-cleaved form of beta(3) fails to mediate cell spreading, but inhibition of the RhoA signaling pathway corrects this defect. Importantly, the calpain-cleaved beta(3) fails to bind c-Src, which is required for integrin-induced cell spreading, and this requirement of beta(3)-associated c-Src results from its inhibition of RhoA-dependent contractile signals. Thus, calpain cleavage of beta(3) at Tyr(759) relieves c-Src-mediated RhoA inhibition, activating the RhoA pathway that confines cell spreading and causes cell retraction.     

A role for caveolin and the urokinase receptor in integrin-mediated adhesion and signaling

The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with beta1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with beta1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes beta1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, beta1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with beta1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal beta1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.     

Kindlin-3 mediates integrin αLβ2 outside-in signaling, and it interacts with scaffold protein receptor for activated-C kinase 1 (RACK1)

Integrins are heterodimeric type I membrane cell adhesion molecules that are involved in many biological processes. Integrins are bidirectional signal transducers because their cytoplasmic tails are docking sites for cytoskeletal and signaling molecules. Kindlins are cytoplasmic molecules that mediate inside-out signaling and activation of the integrins. The three kindlin paralogs in humans are kindlin-1, -2, and -3. Each of these contains a 4.1-ezrin-radixin-moesin (FERM) domain and a pleckstrin homology domain. Kindlin-3 is expressed in platelets, hematopoietic cells, and endothelial cells. Here we show that kindlin-3 is involved in integrin αLβ2 outside-in signaling. It also promotes micro-clustering of integrin αLβ2. We provide evidence that kindlin-3 interacts with the receptor for activated-C kinase 1 (RACK1), a scaffold protein that folds into a seven-blade propeller. This interaction involves the pleckstrin homology domain of kindlin-3 and blades 5-7 of RACK1. Using the SKW3 human T lymphoma cells, we show that integrin αLβ2 engagement by its ligand ICAM-1 promotes the association of kindlin-3 with RACK1. We also show that kindlin-3 co-localizes with RACK1 in polarized SKW3 cells and human T lymphoblasts. Our findings suggest that kindlin-3 plays an important role in integrin αLβ2 outside-in signaling.     

The alpha 3(IV)185-206 peptide from noncollagenous domain 1 of type IV collagen interacts with a novel binding site on the beta 3 subunit of integrin alpha Vbeta 3 and stimulates focal adhesion kinase and phosphatidylinositol 3-kinase phosphorylation

We have recently identified integrin alpha(v)beta(3) and the associated CD47/integrin-associated protein (IAP) together with three other proteins as the potential tumor cell receptors for the alpha(3) chain of basement membrane type IV collagen (Shahan, T.A., Ziaie, Z., Pasco, S., Fawzi, A., Bellon, G., Monboisse, J. C., and Kefalides, N. A. (1999) Cancer Res. 59, 4584-4590). Using different cell lines expressing alpha(v)beta(3), alpha(IIb)beta(3), and/or CD47 and a liquid phase receptor capture assay, we now provide direct evidence that the synthetic and biologically active alpha3(IV)185-206 peptide, derived from the alpha3(IV) chain, interacts with the beta(3) subunit of integrin alpha(v)beta(3), independently of CD47. Increased alpha3(IV) peptide binding was observed on transforming growth factor-beta(1)-stimulated HT-144 cells shown to up-regulate alpha(v)beta(3) independently of CD47. Also, incubation of HT-144 melanoma cells in suspension induced de novo exposure of ligand-induced binding site epitopes on the beta(3) subunit similar to those observed following Arg-Gly-Asp-Ser (RGDS) stimulation. However, RGDS did not prevent HT-144 cell attachment and spreading on the alpha3(IV) peptide, suggesting that the alpha3(IV) binding domain on the beta(3) subunit is distinct from the RGD recognition site. alpha3(IV) peptide binding to HT-144 cells in suspension stimulated time-dependent tyrosine phosphorylation, while the RGDS peptide did not. Two major phosphotyrosine proteins of 120-130 and 85 kDa were immunologically identified as focal adhesion kinase and phosphatidylinositol 3-kinase (PI3-kinase). A direct involvement of PI3-kinase in alpha3(IV)-dependent beta(3) integrin signaling could be documented, since pretreatment of HT-144 cells with wortmannin, a PI3-kinase inhibitor, reverted the known inhibitory effect of alpha3(IV) on HT-144 cell proliferation as well as membrane type 1-matrix metalloproteinase gene expression. These results provide evidence that the alpha3(IV)185-206 peptide, by directly interacting with the beta(3) subunit of alpha(v)beta(3), activates a signaling cascade involving focal adhesion kinase and PI3-kinase.     

The chemokine SDF-1alpha suppresses fibronectin-mediated in vitro lymphocytes adhesion

Stromal cell-derived factor (SDF-1) is a CXC chemokine that selectively activates the CXCR4 chemokine receptor. Fibronectin is an intracellular matrix component that binds integrin and mediates cell-matrix adhesion. Activation of the integrin receptor can occur in two ways: by ligand binding (outside-in signaling), and in response to intracellular events (inside-out signaling). In the current study we showed that SDF-1a inhibited adhesion of T lymphocyte Jurkat cells resulting from binding high concentrations of fibronectin as well as that of THP-1 monocytes. The effect of SDF-1a on fibronectin-mediated adhesion was partly reversed by the CXCR4 receptor antagonist T140. Our results suggest that an SDF-1/ CXCR4 signal pathway modulates fibronectin-mediated lymphocytes adhesion.     

Polarity factor 'Frizzled' in the demosponge Suberites domuncula: identification, expression and localization of the receptor in the epithelium/pinacoderm(1)

Until recently, it was assumed that polarity and axis formation have evolved only in metazoan phyla higher than Cnidaria. One key molecule involved in the signal transduction causing tissue polarity is Frizzled, a seven-transmembrane receptor that is activated by the Wnt family of secreted proteins. We report the isolation and characterization of a Frizzled gene from the demosponge Suberites domuncula (Sd-Fz). The deduced polypeptide comprises all characteristic domains known from Frizzled receptors of higher metazoans. In situ hybridization studies show that Sd-Fz is expressed in cells close to the surface of the sponges and in the pinacocytes of some canals. Northern blot analysis demonstrates its upregulation during the formation of three-dimensional sponge cell aggregates in culture. These data provide for the first time experimental evidence that already in the lowest metazoan phylum (Porifera) genes are present which are very likely involved in tissue polarity.     

RGDS and DGEA-induced [Ca2+]i signalling in human dermal fibroblasts

A pulse of short peptides, RGDS and DGEA in the millimolar range, immediately elicits in normal human fibroblasts a transient increase of intracellular Ca2+ ([Ca2+]i). In the present study, we show that this [Ca2+]i occurs in an increasing number of cells as a function of peptides concentration. It is specific of each peptide and inhibited at saturating concentration of the peptide in the culture medium. The [Ca2+]i transient depends on signalling pathways slightly different for DGEA and RGDS involving tyrosine kinase(s) and phosphatase(s), phospholipase C, production of inositol-trisphosphate and release of Ca2+ from the cellular stores. GFOGER, the classical collagen binding peptide of alpha1- alpha2- and alpha11-beta1 integrins, in triple helical or denatured form, does not produce any Ca2+ signal. The [Ca2+]i signalling induced by RGDS and DGEA is inhibited by antibodies against beta1 integrin subunit while that mediated by RGDS is also inhibited by antibodies against the alpha3 integrin. Delay in the acquisition of responsiveness is observed during cell adhesion and spreading on a coat of fibronectin for RGDS or collagen for DGEA or on a coat of the specific integrin-inhibiting antibodies but not by seeding cells on GFOGER or laminin-5. This delay is suppressed specifically by collagenase acting on the collagen coat or trypsin on the fibronectin coat. Our results suggest that free integrins and associated focal complexes generate a Ca2+ signal upon recognition of DGEA and RGDS by different cellular pathways.