Macrophage stimulating protein.
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Animals↗
Biomedical subjects
A Danilkovitch
Publications and source records attributed to A Danilkovitch.
Two independent signaling pathways mediate the antiapoptotic action of macrophage-stimulating protein on epithelial cells.
In addition to its effects on macrophage function, macrophage-stimulating protein (MSP) is a growth and motility factor for epithelial cells. The growth and survival of epithelial cells generally require two signals, one generated by interaction with extracellular matrix via integrins, the other initiated by a growth factor. Therefore we investigated the effect of MSP on epithelial cell survival. Survival of epithelial cells cultured overnight in serum-free medium was promoted by adhesion, which activated both the phosphatidylinositol 3'-kinase (PI3-K)/AKT and mitogen-activated protein kinase (MAPK) pathways, operating independently of one another. The number of apoptotic cells resulting from inhibition of either pathway alone was approximately doubled by simultaneous inhibition of both pathways. This shows that each pathway made a partial contribution to the prevention of apoptosis. In the presence of an inhibitor of either pathway, MSP increased the activity of the other pathway so that the single uninhibited pathway alone was sufficient to prevent apoptosis. In contrast to the results with adherent cells, although MSP also prevented apoptosis of cells in suspension (anoikis), its effect was mediated only by the PI3-K/AKT pathway. Despite activation of MAPK by MSP, anoikis was not prevented in suspended cells with a blocked PI3-K/AKT pathway. Thus, activation of MAPK alone is not sufficient to mediate MSP antiapoptotic effects. Cell adhesion generates an additional signal, which is essential for MSP to use MAPK in an antiapoptotic pathway. This may involve translocation of MSP-activated MAPK from the cytoplasm into the nucleus, which occurs only in adherent cells. Our results suggest that there is cross talk between cell matrix adhesion and growth factors in the regulation of cell survival via the MAPK pathway. Growth factors induce MAPK activation, and adhesion mediates MAPK translocation from the cytoplasm into the nucleus.
Animals↗
Interaction of macrophage-stimulating protein with its receptor. Residues critical for beta chain binding and evidence for independent alpha chain binding.
Macrophage-stimulating protein (MSP) and hepatocyte growth factor/scatter factor (HGF/SF) are plasminogen-related growth and motility factors that interact with cell-surface protein tyrosine kinase receptors. Each one is a heterodimeric protein comprising a disulfide-linked alpha chain and a serine protease-like beta chain. Despite structural similarities between MSP and HGF, the primary receptor binding site is located on the alpha chain of HGF/SF but on the beta chain of MSP. To obtain insight into the structural basis for MSP beta chain binding, beta chain structure was modeled from coordinates of an existing model of the HGF beta chain. The model revealed that the region corresponding to the S1 specificity pocket in trypsin is filled by the Asn(682)/Glu(648) interacting pair, leaving a shallow cavity for possible beta chain interaction with the receptor. Mutants in this region were created, and their binding characteristics were determined. A double mutation of Asn(682)/Glu(648) caused diminished binding of the beta chain to the MSP receptor, and a single mutation of neighboring Arg(683) completely abolished binding. Thus, this region of the molecule is critical for binding. We also found that at equimolar concentrations of free alpha and beta chains, alpha chain binding to receptor was detectable, at levels considerably lower than beta chain binding. The EC(50) values determined by quantitative enzyme-linked immunosorbent assay are 0.25 and 16.9 nM for beta and alpha chain, respectively. The data suggest that MSP has two independent binding sites with high and low affinities located in beta and alpha chain, respectively, and that the two sites together mediate receptor dimerization and subsequent activation.
Amino Acid Sequence↗
Macrophage stimulating protein-induced epithelial cell adhesion is mediated by a PI3-K-dependent, but FAK-independent mechanism.
Macrophage stimulating protein (MSP) is a growth and motility factor that mediates its activity via the RON/STK receptor tyrosine kinase. MSP promotes integrin-dependent epithelial cell migration, which suggests that MSP may regulate integrin receptor functions. Integrins are cell surface receptors for extracellular matrix. Epithelial cell adhesion and motility are mediated by integrins. We studied the enhancement by MSP of cell adhesion and the molecular mechanisms mediating this effect. MSP decreased the time required for adhesion of 293 and RE7 epithelial cells to substrates coated with collagen or fibronectin. Prevention of adhesion by an RGD-containing peptide showed that the cell-substrate interaction was mediated by integrins. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), blocked MSP-dependent adhesion, which shows that PI3-K is in the MSP-induced adhesion pathway. MSP also affected focal adhesion kinase (FAK) which is important for some types of cell adhesion and motility. Although MSP caused PI3-K-independent tyrosine phosphorylation and activation of FAK, experiments with dominant-negative FAK constructs showed that FAK does not mediate the effects of MSP on cell adhesion or motility. Thus PI3-K, but not FAK, mediates MSP-induced integrin-dependent adhesion of epithelial cells. Also, we found ligand-independent association between RON and beta1 integrin, which is additional evidence for a relationship between these two receptor systems.
Cell Adhesion↗
Kinases involved in MSP/RON signaling.
Macrophage stimulating protein (MSP) belongs to the plasminogen-related kringle domain family. In addition to stimulation of macrophages, MSP acts on other cell types including epithelial and hematopoietic cells. The MSP receptor is a transmembrane tyrosine kinase called RON in humans and STK in mice. MSP/receptor interaction induces activation of signal transduction pathways that mediate MSP biological activities. Cytoplasmic kinases are intracellular messengers occupying an important role in signal transduction. We have identified kinases that participate in RON signaling. In addition to previously identified involvement of phosphatidylinositol 3-kinase (PI3-K), JNK, and MAPK, we found that FAK, c-Src, and AKT are rapidly and transiently activated by MSP. FAK, MAPK, and c-Src are involved in MSP-induced cell proliferation. MAPK and c-Src are components of one signal transduction cascade, and MAPK is downstream of c-Src. FAK also regulates MSP-induced cell growth, but via a path different from c-Src/MAPK. AKT kinase is a component of a separate branch of the RON/PI3-K pathway that mediates the MSP anti-apoptotic effect on epithelial cells. PI3-K regulates MSP-induced adhesion and motility but via downstream components different from AKT. Thus, occupancy of the RON receptor by MSP activates distinct signal transduction pathways that mediate several cellular responses.
Animals↗
Anti-tumor activity of synthetic peptide fragments of the human interferon-alpha 2.
We have studied an influence of nine overlapping peptides from the region between amino acid residues 124 and 144 of the human interferon (FIN)-alpha 2 molecule on the growth of human lymphoid tumor cell lines in vitro. It was found that several, but not all, synthetic peptides inhibited proliferation of the same cell lines that IFN did. One of peptides, corresponding to the 124-138 amino acid residues of the IFN molecule (124-138) was most active. Using a human-mouse somatic hybrid cell line, we have shown that antiproliferative activity of the peptide 124-138 and IFN depended on the presence of human chromosome 21. Receptor binding studies also demonstrated that the peptide specifically interacted with membrane receptors on hybrid human-mouse cells carrying human chromosome 21, but not on parental mouse cells. Displacement experiments confirm that IFN and the peptide 124-138 compete for the same binding sites. Taken together, the data presented support a hypothesis that the C-terminal part of the IFN molecule contributes to antiproliferative activity possessed by IFN. Synthetic peptides studied in the present work may serve as a tool for studying tumor cell growth regulation by IFN and may be considered as potential nontoxic anti-tumor agents.
Animals↗