Protein folding does not prevent the nonclassical export of FGF1 and S100A13.

Newly synthesized proteins are usually exported through the endoplasmic reticulum (ER) and Golgi due to the presence in their primary sequence of a hydrophobic signal peptide that is recognized by the ER translocation system. However, some secreted proteins lack a signal peptide and are exported independently of ER-Golgi. Fibroblast growth factor (FGF)1 is included in this group of polypeptides, as well as S100A13 that is a small calcium-binding protein critical for FGF1 export. Classically secreted proteins are transported into ER in their unfolded states. To determine the role of protein tertiary structure in FGF1 export through the cell membrane, we produced the chimeras of FGF1 and S100A13 with dihydrofolate reductase (DHFR). The specific DHFR inhibitor, aminopterin, prevents its unfolding. We found that aminopterin did not inhibit the release of FGF1:DHFR and S100A13:DHFR. Thus, FGF1 and S100A13 can be exported in folded conformation.

Opposite effects of Notch-1 and Notch-2 on mesothelioma cell survival under hypoxia are exerted through the Akt pathway.

Malignant mesothelioma (MM) is a cancer of the lining of the lungs, heart, and intestine and is known to respond poorly to chemotherapy. Here we show that malignant mesothelial cells have an elevated Notch signaling pathway compared with normal human mesothelial cells. We studied the role of Notch in MM under normoxic and hypoxic conditions, the latter condition best recapitulating the MM microenvironment. Genetic and chemical modulation of the Notch pathway indicated that MM cells are dependent on Notch signaling. More specifically, this signaling was Notch-1 dependent as the result of its negative transcriptional regulation on phosphatase and tensin homologue (PTEN), which led to activation of the prosurvival phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. Our study also provides evidence that whereas Notch-1 is elevated in the malignant setting, Notch-2 is diminished. This differential expression of the two Notch isoforms benefits cancer cell survival because reexpression of Notch-2 was toxic to MM cells. The mechanism of Notch-2 toxicity to MM cells countered that of Notch-1, as it was the result of positive transcriptional regulation of PTEN and inhibition of the PI3K/Akt/mTOR signaling pathway. These results provide new insight into the role of Notch in MM and suggest that Notch pathway inhibitors may be useful in the treatment of this deadly disease.

Novel cross-talk between three cardiovascular regulators: thrombin cleavage fragment of Jagged1 induces fibroblast growth factor 1 expression and release.

Angiogenesis is controlled by several regulatory mechanisms, including the Notch and fibroblast growth factor (FGF) signaling pathways. FGF1, a prototype member of FGF family, lacks a signal peptide and is released through an endoplasmic reticulum-Golgi-independent mechanism. A soluble extracellular domain of the Notch ligand Jagged1 (sJ1) inhibits Notch signaling and induces FGF1 release. Thrombin, a key protease of the blood coagulation cascade and a potent inducer of angiogenesis, stimulates rapid FGF1 release through a mechanism dependent on the major thrombin receptor protease-activated receptor (PAR) 1. This study demonstrates that thrombin cleaves Jagged1 in its extracellular domain. The sJ1 form produced as a result of thrombin cleavage inhibits Notch-mediated CBF1/Suppressor of Hairless [(Su(H)]/Lag-1-dependent transcription and induces FGF1 expression and release. The overexpression of Jagged1 in PAR1 null cells results in a rapid thrombin-induced export of FGF1. These data demonstrate the existence of novel cross-talk between thrombin, FGF, and Notch signaling pathways, which play important roles in vascular formation and remodeling.

Oxygen concentration determines the biological effects of NOTCH-1 signaling in adenocarcinoma of the lung.

NOTCH signaling is an evolutionarily conserved signaling pathway that regulates cell fate during development and postnatal life. It has been increasingly linked to carcinogenesis, although its role in cancer seems to be highly context and tissue specific. Although NOTCH signaling is required for lung development, little is known about its role in lung cancer. In this study, we show that NOTCH signaling, as measured by the gamma-secretase cleavage product N(IC)-1, is active in both normal human and lung tumor samples; however, downstream NOTCH readouts (i.e., HES-1 and HES-5) are elevated in lung tumors. Levels of NOTCH signaling components in primary human lung cells reflect observations in tissue samples, yet lung tumor cell lines showed little NOTCH signaling. Because oxygen concentrations are important in normal lung physiology and lung tumors are hypoxic, the effect of low oxygen on these lung tumor cell lines was evaluated. We found that hypoxia dramatically elevates NOTCH signaling (especially NOTCH-1) in lung tumor cell lines and concomitantly sensitizes them to inhibition via small-molecule gamma-secretase inhibitors or NOTCH-1 RNA interference. gamma-Secretase inhibitor-induced apoptosis of lung tumor cells grown under hypoxic conditions could be rescued by reintroduction of active NOTCH-1. Our data strengthen the role of NOTCH in lung cancer and as a therapeutic target for the treatment of lung and other hypoxic tumor types.

Sphingosine kinase 1 is a critical component of the copper-dependent FGF1 export pathway.

Sphingosine kinase 1 catalyzes the formation of sphingosine-1-phosphate, a lipid mediator involved in the regulation of angiogenesis. Sphingosine kinase 1 is constitutively released from cells, even though it lacks a classical signal peptide sequence. Because copper-dependent non-classical stress-induced release of FGF1 also regulates angiogenesis, we questioned whether sphingosine kinase 1 is involved in the FGF1 release pathway. We report that (i) the coexpression of sphingosine kinase 1 with FGF1 inhibited the release of sphingosine kinase 1 at 37 degrees C; (ii) sphingosine kinase 1 was released at 42 degrees C in complex with FGF1; (iii) sphingosine kinase 1 null cells failed to release FGF1 at stress; (iv) sphingosine kinase 1 is a high affinity copper-binding protein which formed a complex with FGF1 in a cell-free system, and (v) sphingosine kinase 1 over expression rescued the release of FGF1 from inhibition by the copper chelator, tetrathiomolybdate. We propose that sphingosine kinase 1 is a component of the copper-dependent FGF1 release pathway.

Thrombin induces rapid PAR1-mediated non-classical FGF1 release.

Thrombin induces cell proliferation and migration during vascular injury. We report that thrombin rapidly stimulated expression and release of the pro-angiogenic polypeptide fibroblast growth factor 1 (FGF1). Thrombin failed to induce FGF1 release from protease-activated receptor 1 (PAR1) null fibroblasts, indicating that this effect was dependent on PAR1. Similarly to thrombin, FGF1 expression and release were induced by TRAP, a specific oligopeptide agonist of PAR1. These results identify a novel aspect of the crosstalk between FGF and thrombin signaling pathways which both play important roles in tissue repair and angiogenesis.

The non-classical export routes: FGF1 and IL-1alpha point the way.

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-1alpha, two pro-angiogenic polypeptides, provides two such examples. In both cases, export is based on the Cu2+-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-1alpha are involved in pathological processes such as restenosis and tumor formation. Inhibition of their export by Cu2+ chelators is thus an effective strategy for treatment of several diseases.

S100A13 mediates the copper-dependent stress-induced release of IL-1alpha from both human U937 and murine NIH 3T3 cells.

Copper is involved in the promotion of angiogenic and inflammatory events in vivo and, although recent clinical data has demonstrated the potential of Cu2+ chelators for the treatment of cancer in man, the mechanism for this activity remains unknown. We have previously demonstrated that the signal peptide-less angiogenic polypeptide, FGF1, uses intracellular Cu2+ to facilitate the formation of a multiprotein aggregate that enables the release of FGF1 in response to stress and that the expression of the precursor form but not the mature form of IL-1alpha represses the stress-induced export of FGF1 from NIH 3T3 cells. We report here that IL-1alpha is a Cu2+-binding protein and human U937 cells, like NIH 3T3 cells, release IL-1alpha in response to temperature stress in a Cu2+-dependent manner. We also report that the stress-induced export of IL-1alpha involves the intracellular association with the Cu2+-binding protein, S100A13. In addition, the expression of a S100A13 mutant lacking a sequence novel to this gene product functions as a dominant-negative repressor of IL-1alpha release, whereas the expression of wild-type S100A13 functions to eliminate the requirement for stress-induced transcription. Lastly, we present biophysical evidence that IL-1alpha may be endowed with molten globule character, which may facilitate its release through the plasma membrane. Because Cu2+ chelation also represses the release of FGF1, the ability of Cu2+ chelators to potentially serve as effective clinical anti-cancer agents may be related to their ability to limit the export of these proinflammatory and angiogenic signal peptide-less polypeptides into the extracellular compartment.