Interferon gamma and lipopolysaccharide promote macrophage adherence to basement membrane glycoproteins

The ability of thioglycollate (TG)-elicited peritoneal macrophages, a population of recently recruited monocytes, to adhere to the basement membrane glycoproteins laminin and type IV collagen is not a constitutive function of these cells. Adherence can be induced, however, by treatment with IFN-gamma and LPS. In general, IFN-gamma is more potent than LPS in promoting this adherence. Maximal adherence, however, is observed when IFN-gamma (greater than or equal to 5 U/ml) is used together with LPS (2.0 ng/ml). These requirements parallel the conditions needed to obtain tumoricidal activation of TG-elicited macrophages. Adherence to laminin, in the presence of these stimuli, is transient, being maximal at 8 h after their addition and diminishing with longer periods of incubation. In contrast, adherence to type IV collagen does not appear to be transient and IFN-gamma and LPS induce a more prolonged association of macrophages with this substratum

Regulation of alpha 6 beta 1 integrin laminin receptor function by the cytoplasmic domain of the alpha 6 subunit

The alpha 6 beta 1 integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the alpha 6 subunit cytoplasmic domain in alpha 6 beta 1 integrin activation was examined. The use of P388D1 cells, an alpha 6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the alpha 6A or alpha 6B subunit cDNAs restores their activation responsive laminin adhesion (Shaw, L. S., M. Lotz, and A. M. Mercurio. 1993. J. Biol. Chem. 268:11401-11408). A truncated alpha 6 cDNA, alpha 6-delta CYT, was constructed in which the human cytoplasmic domain sequence was deleted after the GFFKR pentapeptide. Expression of this cDNA in P388D1 cells resulted in the surface expression of a chimeric alpha 6-delta CYT beta 1 integrin that was unable to mediate laminin adhesion or increase this adhesion in response to PMA under normal conditions, i.e., in medium that contained physiological concentrations of Ca++ and Mg++. The alpha 6A-delta CYT transfectants adhered to laminin, however, when Ca++/Mg++ was replaced with 150 microM Mn++. We also assessed the role of serine phosphorylation in the regulation of alpha 6A beta 1 integrin function by site-directed mutagenesis of the two serine residues present in the alpha 6A cytoplasmic domain because this domain is phosphorylated on serine residues in response to stimuli that activate the laminin receptor function of alpha 6 A beta 1. Point mutations were introduced in the alpha 6A cDNA that changed either serine residue #1064 (M1) or serine residue #1071 (M2) to alanine residues. In addition, a double mutant (M3) was constructed in which both serine residues were changed to alanine residues. P388D1 transfectants which expressed these serine mutations adhered to laminin in response to PMA to the same extent as cells transfected with wild-type alpha 6A cDNA. These findings provide evidence for a novel mode of integrin regulation that is distinct from that reported for other regulated integrins (O\u27Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. F. Plow, and M. H. Ginsberg. 1991. Science (Wash. DC). 254:845-847. Hibbs, M. L., H. Xu, S. A. Stacker, and T. A. Springer. 1991. Science (Wash. DC). 251:1611-1613), and they demonstrate that serine phosphorylation of the alpha 6A cytoplasmic domain is not involved in this regulation

Therapeutic Approaches to Aggressive Carcinomas Based on a Novel VEGF/Neuropilin Autocrine Pathway

Summary: Autocrine VEGF signaling in tumor cells contributes to de-differentiation and function of tumor initiating/stem cells. NRP2 is the nexus of a signaling pathway that promotes de-differentiation and sustains tumor initiating/stem sells. Anti-NRP2 therapy is worth pursuing, especially for high-grade cancers. Therapeutic Abs are available. This presentation was part of the retreat mini-symposium entitled: Biomarker Discovery and Targeted Therapeutics in Cancer

Protein Kinase C–Dependent Mobilization of the α6β4 Integrin from Hemidesmosomes and Its Association with Actin-Rich Cell Protrusions Drive the Chemotactic Migration of Carcinoma Cells

We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the α6β4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express α6β4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the α6β4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis (∼1 ng/ml), the α6β4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained α6β4 in association with F-actin. Importantly, we demonstrate that this mobilization of α6β4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-α and that it is associated with the phosphorylation of the β4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin–rich cell protrusions that mediate α6β4-dependent cell movement but also the disruption of α6β4-containing hemidesmosomes by protein kinase C

The activation dependent adhesion of macrophages to laminin involves cytoskeletal anchoring and phosphorylation of the alpha 6 beta 1 integrin

Macrophages require activation with either PMA (Mercurio, A. M., and L. M. Shaw. 1988. J. Cell Biol. 107:1873-1880) or interferon-gamma (Shaw, L. M., and A. M. Mercurio. 1989. J. Exp. Med. 169:303-308) to adhere to a laminin substratum. In the present study, we identified an integrin laminin receptor on macrophages and characterized cellular changes that occur in response to PMA activation that facilitate laminin adhesion. A monoclonal antibody (GoH3) that recognizes the integrin alpha 6 subunit (Sonnenberg, A., H. Janssen, F. Hogervorst, J. Calafat, and J. Hilgers. 1987. J. Biol. Chem. 262:10376-10383) specifically inhibited adhesion to laminin-coated surfaces. This antibody precipitated an alpha 6 beta 1 heterodimer (Mr 130/110 kD) from 125I surface-labeled macrophages. The amount of radiolabeled receptor on the cell surface did not increase after PMA activation. Thus, the induction of laminin adhesion cannot be attributed to de novo or increased surface expression of alpha 6 beta 1. By initially removing the Triton X-100-soluble fraction of macrophages and then disrupting the remaining cytoskeletal framework, we observed that 75% of the alpha 6 beta 1 heterodimer on the cell surface is anchored to the cytoskeleton in macrophages that had adhered to a laminin substratum in response to PMA. Significant cytoskeletal anchoring of this receptor was not observed in macrophages that had adhered to fibronectin or tissue culture plastic, nor was it seen in nonadherent cells. PMA also induced phosphorylation of the cytoplasmic domain of the alpha 6 subunit, but not the beta 1 subunit. Phosphorylated alpha 6 was localized to the cytoskeletal fraction only in macrophages plated on a laminin substratum. In summary, our results support a mechanism for the regulation of macrophage adhesion to laminin that involves specific and dynamic matrix integrin-cytoskeletal interactions that may be facilitated by integrin phosphorylation

Endothelial cells assemble two distinct alpha6beta4-containing vimentin-associated structures: roles for ligand binding and the beta4 cytoplasmic tail

The alpha6beta4 laminin binding integrin functions in the assembly of type I hemidesmosomes, which are specialized cell-matrix adhesion sites found in stratified epithelial cells. Although endothelial cells do not express all the components of type I hemidesmosomes, endothelial cells can express the alpha6beta4 integrin. Because endothelial cells lose expression of alpha6beta4 in culture, we expressed recombinant alpha6beta4 in the dermal microvascular endothelial cell line, HMEC-1, to test whether endothelial cells can assemble adhesion structures containing alpha6beta4. Using immunofluorescence microscopy, we found that recombinant alpha6beta4 concentrates specifically in a novel fibrillar structure on the basal surface of endothelial cells in the absence of an exogenous laminin substrate. This localization is regulated by an intracellular mechanism, because the beta4 cytoplasmic domain is sufficient to direct a reporter domain (IL-2R) to the fibrillar structures independently of recombinant alpha6beta4. In addition, this IL-2R-beta4 chimera is sufficient to recruit the intermediate filament-associated protein HD1/plectin to these fibrillar structures and this also occurs in the absence of recombinant alpha6beta4. The fibrillar localization pattern, as well as the recruitment of HD1/plectin, requires the first and second fibronectin type III repeats and the connecting segment of the beta4 tail. In addition, when endothelial cells are provided a laminin 5-rich matrix, recombinant alpha6beta4 redistributes from the fibrillar structure to type I hemidesmosome-like structures. The beta4 cytoplasmic domain can also direct a reporter domain to these type I hemidesmosome-like structures; however, this process is dependent upon the expression of recombinant alpha6beta4 Biochemical analysis indicates that both the fibrillar and the type I hemidesmosome-like structures are associated with the vimentin intermediate filament cytoskeleton. Thus, the results illustrate that endothelial cells have the essential components necessary to assemble at least two distinct alpha6beta4-containing and vimentin-associated structures on their basal surface and that the alpha6beta4 cytoplasmic tail and the availability of specific alph6beta4 ligands regulate receptor localization to these structures

Release of cAMP gating by the alpha6beta4 integrin stimulates lamellae formation and the chemotactic migration of invasive carcinoma cells

The alpha6beta4 integrin promotes carcinoma in-vasion by its activation of a phosphoinositide 3-OH (PI3-K) signaling pathway (Shaw, L.M., I. Rabinovitz, H.H.-F. Wang, A. Toker, and A.M. Mercurio. Cell. 91: 949-960). We demonstrate here using MDA-MB-435 breast carcinoma cells that alpha6beta4 stimulates chemotactic migration, a key component of invasion, but that it has no influence on haptotaxis. Stimulation of chemotaxis by alpha6beta4 expression was observed in response to either lysophosphatidic acid (LPA) or fibroblast conditioned medium. Moreover, the LPA-dependent formation of lamellae in these cells is dependent upon alpha6beta4 expression. Both lamellae formation and chemotactic migration are inhibited or gated by cAMP and our results reveal that a critical function of alpha6beta4 is to suppress the intracellular cAMP concentration by increasing the activity of a rolipram-sensitive, cAMP-specific phosphodiesterase (PDE). This PDE activity is essential for lamellae formation, chemotactic migration and invasion based on data obtained with PDE inhibitors. Although PI3-K and cAMP-specific PDE activities are both required to promote lamellae formation and chemotactic migration, our data indicate that they are components of distinct signaling pathways. The essence of our findings is that alpha6beta4 stimulates the chemotactic migration of carcinoma cells through its ability to influence key signaling events that underlie this critical component of carcinoma invasion

Id2 complexes with the SNAG domain of Snai1 inhibiting Snai1-mediated repression of integrin beta4

The epithelial-mesenchymal transition (EMT) is a fundamental process that underlies development and cancer. Although the EMT involves alterations in the expression of specific integrins that mediate stable adhesion to the basement membrane, such as alpha6beta4, the mechanisms involved are poorly understood. Here, we report that Snai1 inhibits beta4 transcription by increasing repressive histone modification (trimethylation of histone H3 at K27 [H3K27Me3]). Surprisingly, Snai1 is expressed and localized in the nucleus in epithelial cells, but it does not repress beta4. We resolved this paradox by discovering that Id2 complexes with the SNAG domain of Snai1 on the beta4 promoter and constrains the repressive function of Snai1. Disruption of the complex by depleting Id2 resulted in Snai1-mediated beta4 repression with a concomitant increase in H3K27Me3 modification on the beta4 promoter. These findings establish a novel function for Id2 in regulating Snai1 that has significant implications for the regulation of epithelial gene expression

Glycolipids of the mouse peritoneal macrophage. Alterations in amount and surface exposure of specific glycolipid species occur in response to inflammation and tumoricidal activation

We have characterized the major glycolipid constituents of the mouse peritoneal macrophage, and have demonstrated that alterations in the amount and in the accessibility of specific glycolipid species to galactose oxidase/NaB3H4 labeling, an indicator of glycolipid surface exposure, occur in response to inflammation and as a consequence of activation to a tumoricidal state. The key findings are: (a) Asialo GM1, a major neutral glycolipid constituent of all macrophage populations examined, is accessible to galactose oxidase/NaB3H4 labeling on the surface of TG-elicited and BCG-activated macrophages but not on resident macrophages; (b) GM1 is the predominant ganglioside constituent of the mouse macrophage. Resident macrophages contain two distinct GM1 species, as determined by cholera toxin binding, while TG-elicited and BCG-activated macrophages contain an additional GM1 species. Differences in the relative amounts of these GM1 species, as well as in their accessibility to galactose oxidase/NaB3H4 labeling, exist among the macrophage populations. These observations suggest that both a chemical and spatial reorganization of surface glycolipids occurs in response to inflammation and tumoricidal activation

ERbeta regulation of NF-kB activation in prostate cancer is mediated by HIF-1

We examined the regulation of NF-kappaB in prostate cancer by estrogen receptor beta (ERbeta) based on the inverse correlation between p65 and ERbeta expression that exists in prostate carcinomas and reports that ERbeta can inhibit NF-kappaB activation, although the mechanism is not known. We demonstrate that ERbeta functions as a gate-keeper for NF-kappaB p65 signaling by repressing its expression and nuclear translocation. ERbeta regulation of NF-kappaB signaling is mediated by HIF-1. Loss of ERbeta or hypoxia stabilizes HIF-1alpha, which we found to be a direct driver of IKKbeta transcription through a hypoxia response element present in the promoter of the IKKbeta gene. The increase of IKKbeta expression in ERbeta-ablated cells correlates with an increase in phospho-IkappaBalpha and concomitant p65 nuclear translocation. An inverse correlation between the expression of ERbeta and IKKbeta/p65 was also observed in the prostates of ERbeta knockout (BERKO) mice, Gleason grade 5 prostate tumors and analysis of prostate cancer databases. These findings provide a novel mechanism for how ERbeta prevents NF-kappaB activation and raise the exciting possibility that loss of ERbeta expression is linked to chronic inflammation in the prostate, which contributes to the development of high-grade prostate cancer