S100A13, a member of the S100 gene family of Ca 2؉ -binding proteins has been previously characterized as a component of a brain-derived heparin-binding multiprotein aggregate/complex containing fibroblast growth factor 1 (FGF1). We report that while expression of S100A13 in NIH 3T3 cells results in the constitutive release of S100A13 into the extracellular compartment at 37°C, co-expression of S100A13 with FGF1 represses the constitutive release of S100A13 and enables NIH 3T3 cells to release S100A13 in response to temperature stress. S100A13 release in response to stress occurs with kinetics similar to that observed for the stress-induced release of FGF1, but S100A13 expression is able to reverse the sensitivity of FGF1 release to inhibitors of transcription and translation. The release of FGF1 and S100A13 in response to heat shock results in the solubility of FGF1 at 100% (w/v) ammonium sulfate saturation, and the expression of a S100A13 deletion mutant lacking its novel basic residue-rich domain acts as a dominant negative effector of FGF1 release in vitro. Surprisingly, the expression of S100A13 also results in the stress-induced release of a Cys-free FGF1 mutant, which is normally not released from NIH 3T3 cells in response to heat shock. These data suggest that S100A13 may be a component of the pathway for the release of the signal peptide-less polypeptide, FGF1, and may involve a role for S100A13 in the formation of a noncovalent FGF1 homodimer. FGF11 and FGF2 are the prototype members of a large family of heparin-binding growth factor genes that regulate numerous biological processes such as neurogenesis, mesoderm formation, and angiogenesis (1, 2). FGF1 and FGF2 lack a classical signal peptide sequence that provides access to the conventional endoplasmic reticulum (ER)-Golgi secretion pathway, a characteristic that led to the hypothesis that the release of these polypeptides may proceed through novel release/export pathways (2). Our laboratory previously demonstrated that FGF1, but not FGF2, is released as a latent homodimer by a transcription-and translation-dependent mechanism in response to a variety of cellular stresses including heat shock (3), hypoxia (4), and serum starvation (5). Conversely, the disruption of communication between the ER and Golgi apparatus by brefeldin A does not prevent the release of FGF1 from NIH 3T3 cells, confirming that FGF1 release may occur through a nonconventional pathway (6).FGF1 is released in vitro as a reducing agent-and denaturant-sensitive complex, which contains the p40 extravesicular domain of the Ca 2ϩ -binding protein, p65 synaptotagmin (Syt)1 (7). The release of FGF1 in response to stress is dependent on Syt1 expression, since the expression of either a deletion mutant lacking 95 amino acids from the extravesicular portion of Syt1 or an antisense-Syt1 gene is able to repress FGF1 release in NIH 3T3 cells (7,8). In addition, FGF1 purified from ovine brain as a high molecular weight aggregate exists as a component of a noncovalent heparin-binding complex wit...

Non-classical protein release independent of the ER-Golgi pathway has been reported for an increasing number of proteins lacking an N-terminal signal sequence. The export of FGF1 and IL-1α, two pro-angiogenic polypeptides, provides two such examples. In both cases, export is based on the Cu 2+ -dependent formation of multiprotein complexes containing the S100A13 protein and might involve translocation of the protein across the membrane as a 'molten globule'. FGF1 and IL-1α are involved in pathological processes such as restenosis and tumor formation. Inhibition of their export by Cu 2+ chelators is thus an effective strategy for treatment of several diseases.

The Jagged/Notch signaling pathways control cell fate determination and differentiation, and their dysfunction is associated with human pathologies involving cardiovascular abnormalities. To determine the presence of these genes during vascular response to injury, we analyzed expression of Jagged1, Jagged2, and Notch1 through 4 after balloon catheter denudation of the rat carotid artery. Although low levels of Jagged1, Jagged2, and constitutive expression of Notch1 were seen in uninjured endothelium, expression of all was significantly increased in injured vascular cells. High Jagged1 expression was restricted to the regenerating endothelial wound edge, whereas Notch transcripts were abundant in endothelial and smooth muscle cells. To understand the basis for Jagged/Notch control of cellular phenotype, we studied an in vitro model of NIH3T3 cells transfected with a secreted form of the extracellular domain of Jagged1. We report that the soluble Jagged1 protein caused decreased cell-matrix adhesion and cell migration defects. Cadherin-mediated intercellular junctions as well as focal adhesions were modified in soluble Jagged1 transfectants, demonstrating that cell-cell contacts and adhesion plaques may be targets of Jagged/Notch activity. We suggest that Jagged regulation of cell-cell and cell-matrix interactions may contribute to the control of cell migration in situations of tissue remodeling in vivo.

Background: The inflammasome is a multimolecular complex that regulates the processing of the pro-inflammatory cytokine interleukin-1β.Results: Inhibitors of deubiquitinase (DUB) enzymes inhibited the release of interleukin-1β.Conclusion: DUBs regulate assembly of the inflammasome.Significance: DUBs may represent new anti-inflammatory drug targets for the treatment of inflammatory disease.

Fibroblast growth factor (FGF) 1 is known to be released in response to stress conditions as a component of a multiprotein aggregate containing the p40 extravescicular domain of p65 synaptotagmin (Syt) 1 and S100A13. Since FGF1 is a Cu 2؉ -binding protein and Cu 2؉is known to induce its dimerization, we evaluated the capacity of recombinant FGF1, p40 Syt1, and S100A13 to interact in a cell-free system and the role of Cu 2؉ in this interaction. We report that FGF1, p40 Syt1, and S100A13 are able to bind Cu 2؉ with similar affinity and to interact in the presence of Cu 2؉ to form a multiprotein aggregate which is resistant to low concentrations of SDS and sensitive to reducing conditions and ultracentrifugation. The formation of this aggregate in the presence of Cu 2؉ is dependent on the presence of S100A13 and is mediated by cysteine-independent interactions between S100A13 and either FGF1 or p40 Syt1. Interestingly, S100A13 is also able to interact in the presence of Cu 2؉with Cys-free FGF1 and this observation may account for the ability of S100A13 to export Cys-free FGF1 in response to stress. Lastly, tetrathiomolybdate, a Cu 2؉ chelator, significantly represses in a dose-dependent manner the heat shock-induced release of FGF1 and S100A13. These data suggest that S100A13 may be involved in the assembly of the multiprotein aggregate required for the release of FGF1 and that Cu 2؉ oxidation may be an essential post-translational intracellular modifier of this process. FGF11 and FGF2, the prototype members of a family of heparin-binding growth factors involved in the regulation of neurogenesis, mesoderm formation, and angiogenesis (1, 2), function as extracellular mitogens for diverse populations of target cells, yet they both lack the presence of a classical signal peptide sequence that enables their secretion through the endoplasmic reticulum-Golgi apparatus (1, 2). FGF1, but not FGF2 (3), is released from NIH 3T3 cells in response to heat shock (4), hypoxia (5), and serum starvation (6) in a transcription-and translation-dependent manner, as a biologically inactive homodimer with decreased affinity for heparin (4). Recent studies have suggested that FGF1 may be released as a multiprotein aggregate containing FGF1 and the p40 extravesicular domain of p65 synaptotagmin (Syt)1 (7, 8). These studies are consistent with the observation that FGF1 is present in neuronal tissue as a component of a non-covalent aggregate containing p40 Syt1 and S100A13 (9). It is also known that (i) both p40 Syt1 and S100A13 are constitutively released at 37°C from NIH 3T3 (8, 10), (ii) the expression of FGF1 is able to repress the constitutive release of S100A13 but not p40 Syt1 at 37°C (8, 10), and (iii) FGF1 and S100A13 NIH 3T3 cell cotransfectants are able to release FGF1 and S100A13 in response to temperature stress in a form which alters the solubility of FGF1 at 100% (w/v) ammonium sulfate (10). Moreover, S100A13 appears to play a role in the regulation of FGF1 release since (i) a deletion mutant of S100A13, lacking the carboxyl...

A growing number of proteins devoid of signal peptides have been demonstrated to be released through the non-classical pathways independent of endoplasmic reticulum and Golgi. Among them are two potent proangiogenic cytokines FGF1 and IL1α. Stress-induced transmembrane translocation of these proteins requires the assembly of copper-dependent multiprotein release complexes. It involves the interaction of exported proteins with the acidic phospholipids of the inner leaflet of the cell membrane and membrane destabilization. Not only stress, but also thrombin treatment and inhibition of Notch signaling stimulate the export of FGF1. Non-classical release of FGF1 and IL1α presents a promising target for treatment of cardiovascular, oncologic, and inflammatory disorders. Keywords non-classical secretion; FGF1; IL1α VARIETY OF NON-CLASSICALLY RELEASED PROTEINSThe familiar textbook scheme of protein secretion starts with the cotranslational protein translocation into the endoplasmic reticulum (ER). This translocation requires a molecular exit visa, a hydrophobic signal peptide located usually at the N-terminus of a secretable protein [Blobel, 1995]. After the protein is translocated through the ER membrane, its folding, transport, sorting, and covalent modifications occur in the ER and Golgi. Finally, the protein is released from the cell as a result of the fusion of an exocytotic vesicle with the cell membrane.

We have previously demonstrated that the expression of the soluble extracellular domain of the transmembrane ligand for Notch receptors, Jagged 1 (sJ1), in NIH 3T3 cells results in the formation of a matrix-dependent chord-like phenotype, the loss of contact inhibition of growth, and an inhibition of pro-␣1(I) collagen expression. In an effort to define the mechanism by which sJ1 induces this phenotype, we report that sJ1 transfectants display biochemical and cytoskeletal alterations consistent with the activation of Src. Indeed, cotransfection of sJ1 transfectants with a dominant-negative mutant of Src resulted in the loss of matrix-dependent chord formation and correlated with the restoration of type I collagen expression and contact inhibition of growth. We also report that the sJ1-mediated induction of Src activity and related phenotypes, including chord formation, may result from the inhibition of endogenous Jagged 1-mediated Notch signaling since it was not possible to detect an sJ1-dependent induction of CSL-dependent transcription in these cells. Interestingly, NIH 3T3 cells transfected with dominant-negative (but not constitutively active) mutants of either Notch 1 or Notch 2 displayed a similar Src-related phenotype as the sJ1 transfectants. These data suggest that the ability of sJ1 to mediate chord formation is Src-dependent and requires the repression of endogenous Jagged 1-mediated Notch signaling, which is tolerant to the destabilization of the actin cytoskeleton, a mediator of cell migration.

The induction of an acute inflammatory response followed by the release of polypeptide cytokines and growth factors from peripheral blood monocytes has been implicated in mediating the response to vascular injury. Because the Cu 2؉ -binding proteins IL-1␣ and fibroblast growth factor 1 are exported into the extracellular compartment in a stress-dependent manner by using intracellular Cu 2؉ to facilitate the formation of S100A13 heterotetrameric complexes and these signal peptideless polypeptides have been implicated as regulators of vascular injury in vivo, we examined the ability of Cu 2؉ chelation to repress neointimal thickening in response to injury. We observed that the oral administration of the Cu 2؉ chelator tetrathiomolybdate was able to reduce neointimal thickening after balloon injury in the rat. Interestingly, although immunohistochemical analysis of control neointimal sections exhibited prominent staining for MAC1, IL-1␣, S100A13, and the acidic phospholipid phosphatidylserine, similar sections obtained from tetrathiomolybdate-treated animals did not. Further, adenoviral gene transfer of the IL-1 receptor antagonist during vascular injury also significantly reduced the area of neointimal thickening. Our data suggest that intracellular copper may be involved in mediating the response to injury in vivo by its ability to regulate the stress-induced release of IL-1␣ by using the nonclassical export mechanism employed by human peripheral blood mononuclear cells in vitro.phosphatidylserine ͉ interleukin 1 ͉ restenosis ͉ tetrathiomolybdate ͉ fibroblast growth factor