Stressed-Induced Nonclassical Export of Growth Factors and Cytokines
Unlike other secreted proteins, the potent pro-angiogenic factor FGF1 and pro-inflammatory cytokine IL1alpha lack a signal peptide for classical secretion, and are released through Golgi-independent pathways. Cell stress induces the export of FGF1 and IL1 alpha. At stress, FGF1 and IL1alpha translocate to the vicinity of cell membrane (Fig 1), where they co-localize with cortical actin cytoskeleton to be further exported to the extracellular milieu.
Fig 1. Heat shock-induced peripheral localization of FGF1 (green).
We found that the export of FGF1 relies on copper-dependent formation of a complex containing several secreted signal-peptide-less proteins: S100A13, sphingosine kinase 1 (SphK1), 40 kDa form of synaptotagmin 1 (p40 Syt1) and, apparently, annexin II (Anx II). A similar complex is required for stress-induced export of IL1alpha. The export of FGF1 release complex depends on transmembrane translocation of acidic phospholipids (Fig 2).
Fig 2. Stress-induced FGF1 export.
Our current aims are to understand: 1.how the release complex assembles; 2. what is the role of actin cytoskeleton in the export of FGF1 and IL1α; 3.what is the molecular mechanism of transmembrane translocation of the release complex. In addition, to study the regulation of FGF1 export in vivo, we produced transgenic mice with FGF1 expression in endothelial cells and macrophages.
Regulation of Nonclassical Protein Export by Notch Signaling
FGF, IL1 and Notch are important regulators of angiogenesis and cancer growth. We found that the inhibition of Notch signaling results in strong spontaneous release of FGF1 (Figure 3) and IL1, and the increase of their expression.
Fig 3. Inhibition of Notch signaling by a dn form of transcription factor MAML induces spontaneous FGF1 export.
Fibroblasts with inhibited Notch pathway acquire transformed phenotype (Fig 4), which is dependent on FGF signaling.
Fig 4. Fibroblasts where Notch signaling is inhibited by a dn form of transcription factor CBF1 form tumors in chicken chorioallanthoic membrane (A) and produce highly angiogenic tumors in nude mice (B, PECAM staining).
Our aim is to understand the transcriptional regulation of nonclassical protein export by Notch signaling (Fig 5).
Fig 5. Regulation of FGF1 expression and release by Notch signaling.
Restricted Proliferation of FGF-Stimulated Cells
We found that several cell types of mesenchymal origin respond to FGF presented as a single mitogen by a strictly limited proliferation. They undergo one cell cycle and then get blocked in the non-proliferating state characterized by dramatic accumulation of cell cycle inhibitors (Figure 6) and active FGFR/Erk signaling.
Fig 6. Unlike serum-stimulated cells, FGF1 treated fibroblasts express large amounts of cell cycle inhibitor p21 (green).
We hypothesize that the restricted character of FGF-induced proliferative response prevents hyperplasia in damaged tissue, where FGF is released under stress conditions. We are currently studying the molecular mechanisms, which limit the growth of FGF-stimulated cells.
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