No abstract

The insulin-like growth factor binding proteins (IGFBPs) have IGF-independent differential effects on cell function. We investigated whether they can affect integrin-receptor-mediated cell attachment to different extracellular matrix (ECM) components in Hs578T cells.Cell attachment to a general ECM gel was unaffected by IGFBP-1 and -6 but was significantly increased by IGFBP-4 and -5 and decreased by IGFBP-2 and -3. Similar results were obtained for attachment to laminin or collagen type IV. Attachment to fibronectin, however, was increased by IGFBP-3 and decreased by IGFBP-5. The actions of IGFBP-3 and -5 on cell attachment to ECM were lost in the presence of a soluble Arg-Gly-Asp (RGD)-containing fibronectin fragment. Thrombospondin reversed the actions of IGFBP-3 on cell attachment, but IGFBP-5 still increased cell attachment.On plastic, neither IGFBP-3 nor -5 alone affected cell viability; although ceramide-induced apoptosis was enhanced by IGFBP-3 but reduced by IGFBP-5. The presence of RGD reversed the action of IGFBP-5 on cell death but attenuated that of IGFBP-3. With cells grown on fibronectin, the action of IGFBP-3 was reversed, and it conferred cell survival, whereas the survival effect of IGFBP-5 was lost.In summary we have demonstrated that IGFBP-3 and -5 both have intrinsic effects on cell survival. In each case the presence of fibronectin or fibronectin fragments determines whether susceptibility to apoptosis is increased or decreased. These effects on cell survival are paralleled by acute effects on integrin receptor function; IGFBP-3 and -5 were able to either enhance or inhibit cell attachment in the presence of fibronectin. Cell survival is tightly controlled by cues from the ECM and from growth factors, particularly the IGFs. Our findings indicate that, in addition to being crucial modulators of IGF actions, the IGFBPs have direct actions on cell attachment and survival that are specific and dependent upon the matrix components present.

Matrix metalloproteinase-7 (MMP-7) is localized to epithelial cells and is up-regulated in many cancers and in inflammation. We now report that MMP-7 targets a key mesenchymal cell type, the myofibroblast. Recombinant MMP-7 stimulated the proliferation and migration of human colonic myofibroblasts. These responses were partly attributable to activation of other MMPs, notably MMP-3 and MMP-8, and to stimulation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways. Using a proteomic approach, we identified insulin-like growth factor binding protein-5 (IGFBP-5) as a previously unsuspected target of MMP-7 produced by colonic myofibroblasts. We present evidence that the MMP-7 cleavage of IGFBP-5 liberates IGF-II that functions as an autocrine myofibroblast growth factor. Thus, MMP-7 may act as a signal from epithelial cells for local recruitment of myofibroblasts and stimulation of their proliferation. Similar effects of MMP-7 produced in epithelial tumors might account for the expansion of stroma through activation of myofibroblasts. (Cancer Res 2005; 65(16): 7363-9)

IGF-binding protein (IGFBP)-3 is generally considered to have actions that counterbalance those of IGFs and is therefore being developed as a cancer treatment. In breast tumors, however, high levels are associated with aggressive tumors and poor prognosis. Consistent with this we have demonstrated that although IGFBP-3 and a non-IGF-binding fragment (serine phosphorylation domain peptide) reduced attachment and enhanced apoptosis of Hs578T breast cancer cells cultured on collagen or laminin, it promoted their attachment and survival on fibronectin, which is abundant in the matrix of aggressive tumors. We have now examined the factors that determine whether IGFBP-3 has positive or negative actions on breast epithelial cells. IGFBP-3 also promoted survival of Hs578T cells in the presence of an antibody to the beta1-integrin subunit or when cholesterol-stabilized complexes were disrupted. These actions were blocked by IGF-I or a MAPK inhibitor. Serine phosphorylation domain peptide had similar actions on MCF-7 cells that were again reversed on fibronectin or with disruption of cholesterol-stabilized complexes and blocked by the beta1-integrin antibody. In contrast, IGFBP-3 promoted growth and survival for nonmalignant MCF-10A cells, but these effects were again reversed on fibronectin and blocked by the beta1 antibody or a MAPK inhibitor or by disruption of cholesterol-stabilized complexes. On Hs578T cells, IGFBP-3 bound to caveolin-1 and beta1-integrins, enhancing their aggregation, the recruitment of focal adhesion kinase, and the activation of MAPK. In summary, with three breast epithelial cell lines, IGFBP-3 had positive or negative effects on growth and survival dependent upon the status of cholesterol-stabilized integrin receptor complexes.

Neuronal expression of SGK1 in aged human brain and its nuclear compartmentalization suggest a possible neuroprotective role. FOXO3a and NDRG1 data suggest augmented SGK1 activity (as reported for Akt) in severe AD. Increased intracellular SGK1 may complement enhanced Akt, with a distinct subcellular localization.

Chronic hypergastrinemia is associated with enterochromaffin-like (ECL) cell hyperplasia, which may progress to gastric carcinoid tumors. The latter consists of epithelial cells and stroma, and both compartments usually regress after normalization of hypergastrinemia. We previously showed that matrix metalloproteinase (MMP)-7 in gastric epithelial cells was upregulated by Helicobacter pylori and described MMP-7-dependent reciprocal signaling between the epithelium and a key stromal cell type, the myofibroblast. Here, we describe the regulation of gastric MMP-7 by gastrin and the potential significance for recruiting and maintaining myofibroblast populations. Biopsies of the gastric corpus and ECL cell carcinoid tumors were obtained from hypergastrinemic patients. Western blot analysis, ELISA, immunohistochemistry, and promoter-luciferase (luc) reporter assays were used to study MMP-7 expression. Gastric myofibroblasts were identified by alpha-smooth muscle actin (alpha-SMA) expression, and the effects of MMP-7 on myofibroblast proliferation were investigated. In hypergastrinemic patients, there was an increased abundance of MMP-7 and alpha-SMA in gastric corpus biopsies and ECL cell carcinoid tumors. In the latter, MMP-7 was localized to ECL cells but not stromal cells, which were nevertheless well represented. Gastrin stimulated MMP-7-luc expression in both AGS-G(R) and primary human gastric epithelial cells. Conditioned medium from gastrin-treated human gastric glands stimulated myofibroblast proliferation, which was inhibited by neutralizing antibodies to MMP-7. MMP-7 increased the proliferation of myofibroblasts via the MAPK and phosphatidylinositol 3-kinase (PI3K) pathways. In conclusion, stimulation of gastric MMP-7 by elevated plasma gastrin may activate epithelial-mesenchymal signaling pathways regulating myofibroblast function via MAPK and PI3K pathways and contribute to stromal deposition in ECL cell carcinoid tumors.

No abstract

We have demonstrated previously in Hs578T cells that insulin-like growth factor binding protein (IGFBP)-3 can significantly accentuate ceramide (C2)-induced apoptosis, but has no effect on cell death induced by integrin detachment [using an arginine-glycine-aspartic acid (RGD)-containing peptide]. In contrast we found that IGFBP-5 could inhibit apoptosis induced by either C2 or integrin detachment. It is now clear that the mitochondria not only provide the energy required for cell viability, but can also play an important role during the commitment phase to apoptosis. We used a mitochondrial respiratory chain inhibitor, antimycin A, at both apoptotic and nonapoptotic doses to further investigate the IGF-independent actions of IGFBP-3 and IGFBP-5 on C2 and RGD-induced apoptosis in the Hs578T cells. Hs578T cells had one of three treatments. 1: They were incubated with increasing doses of antimycin A for 24 h. 2: They were coincubated with an apoptotic dose of either C2 or RGD together with a nonapoptotic dose of antimycin A for 24 h. 3: They were incubated with a binding protein (100 ng/ml) for 24 h followed by coincubation of the binding protein with an apoptotic dose of antimycin A for a further 24 h. Cell viability was assessed by trypan blue dye exclusion and MTT assay, and apoptosis was confirmed and measured by morphologic assessment and flow cytometry. We found that antimycin A initiated apoptosis at 10 micromol/L and above. We also demonstrated that a nonapoptotic dose of antimycin A (0.1 micromol/L) significantly inhibited C2-induced apoptosis, whereas it significantly accentuated RGD-induced cell death. In addition, we found that cell death induced by antimycin A can be accentuated by IGFBP-3 but is not affected by IGFBP-5. These data indicate that IGFBP-3 can directly enhance apoptosis triggered via the mitochondria; either directly by a mitochondrial inhibitor or by C2 (which we demonstrate to act via effects on the mitochondria in this model). IGFBP-5, however, appears to confer survival effects via a distinct pathway not involving the mitochondria.