Mary M. Zutter

The α2 Integrin Subunit-Deficient Mouse: A Multifaceted Phenotype Including Defects of Branching Morphogenesis and Hemostasis

The alpha(2)beta(1) integrin is a collagen/laminin receptor expressed on platelets, endothelial cells, fibroblasts, and epithelial cells. To define the role of the alpha(2)beta(1) integrin in vivo, we created a genetically engineered mouse in which expression of the alpha(2)beta(1) integrin was completely eliminated. Mice deficient in the alpha(2)beta(1) integrin are viable, fertile, and develop normally with no excess lethality of homozygotes. Both alpha(2)beta(1)-integrin protein and alpha(2) mRNA were undetectable in the alpha(2)-null mice. Gross and histological evaluation of the heart, lungs, kidneys, gastrointestinal tract, pancreas, skin, and reproductive tracts revealed no abnormalities. However, quantitative analysis of mammary gland branching morphogenesis demonstrated that branching complexity is markedly diminished in the alpha(2)-deficient animals. Studies in the alpha(2)-deficient animals do not support the proposed roles for the alpha(2)beta(1) integrin on fibroblasts and keratinocytes in wound healing. When compared to platelets from wild-type littermates, platelets from alpha(2)-null mice failed to adhere to type I collagen under either static or shear-stress conditions. Although platelets from alpha(2)-deficient animals aggregated in response to collagen, they did so with prolonged lag time and lessened intensity. The alpha(2)beta(1) integrin-null mouse thus exhibits diverse, sometimes subtle, phenotypes consistent with the widespread pattern of alpha(2)beta(1) integrin expression.

Proteases, Extracellular Matrix, and Cancer: A Workshop of the Path B Study Section

The role of the extracellular matrix (ECM) in the tumor microenvironment is not limited to being a barrier against tumor invasion. The ECM is a reservoir of cell binding proteins and growth factors that affect tumor cell behavior. It is also substantially modified by proteases produced by tumor cells or stroma cells. As a result of the activity of these proteases, cell-cell and cell-ECM interactions are altered, new biologically active ECM molecules are generated, and the bioavailability and activity of many growth factors, growth factor receptors, and cytokines are modified. ECM-degrading proteases also play a critical role in angiogenesis, where they can act as positive as well as negative regulators of endothelial cell proliferation and vascular morphogenesis. This review article summarizes some of the most relevant findings made over the recent years that were discussed at a workshop organized by the Path B Study Section of the National Institutes of Health in October 2002.

The α 2 β 1 integrin is a metastasis suppressor in mouse models and human cancer

Integrins regulate cell-cell and cell-matrix adhesion and thereby play critical roles in tumor progression and metastasis. Although work in preclinical models suggests that β1 integrins may stimulate metastasis of a number of cancers, expression of the β1 subunit alone has not been shown to be a useful prognostic indicator in human cancer patients. Here we have demonstrated that the α2β1 integrin suppresses metastasis in a clinically relevant spontaneous mouse model of breast cancer. These data are consistent with previous studies indicating high expression of α2β1 integrin in normal breast epithelium and loss of α2β1 in poorly differentiated breast cancer. They are also consistent with our systematic analysis of microarray databases of human breast and prostate cancer, which revealed that decreased expression of the gene encoding α2 integrin, but not genes encoding α1, α3, or β1 integrin, was predictive of metastatic dissemination and decreased survival. The predictive value of α2 expression persisted within both good-risk and poor-risk cohorts defined by estrogen receptor and lymph node status. Thus, the α2β1 integrin functionally inhibits breast tumor metastasis, and α2 expression may serve as an important biomarker of metastatic potential and patient survival.

β1 Integrin Mediates Internalization of Mammalian Reovirus

ABSTRACT Reovirus infection is initiated by interactions between the attachment protein σ1 and cell surface carbohydrate and junctional adhesion molecule A (JAM-A). Expression of a JAM-A mutant lacking a cytoplasmic tail in nonpermissive cells conferred full susceptibility to reovirus infection, suggesting that cell surface molecules other than JAM-A mediate viral internalization following attachment. The presence of integrin-binding sequences in reovirus outer capsid protein λ2, which serves as the structural base for σ1, suggests that integrins mediate reovirus endocytosis. A β1 integrin-specific antibody, but not antibodies specific for other integrin subunits, inhibited reovirus infection of HeLa cells. Expression of a β1 integrin cDNA, along with a cDNA encoding JAM-A, in nonpermissive chicken embryo fibroblasts conferred susceptibility to reovirus infection. Infectivity of reovirus was significantly reduced in β1-deficient mouse embryonic stem cells in comparison to isogenic cells expressing β1. However, reovirus bound equivalently to cells that differed in levels of β1 expression, suggesting that β1 integrins are involved in a postattachment entry step. Concordantly, uptake of reovirus virions into β1-deficient cells was substantially diminished in comparison to viral uptake into β1-expressing cells. These data provide evidence that β1 integrin facilitates reovirus internalization and suggest that viral entry occurs by interactions of reovirus virions with independent attachment and entry receptors on the cell surface.

NAG-2, a Novel Transmembrane-4 Superfamily (TM4SF) Protein That Complexes with Integrins and Other TM4SF Proteins

Transmembrane-4 superfamily (TM4SF) proteins form complexes with integrins and other cell-surface proteins. To further characterize the major proteins present in a typical TM4SF protein complex, we raised monoclonal antibodies against proteins co-immunoprecipitated with CD81 from MDA-MB-435 breast cancer cells. Only two types of cell-surface proteins were recognized by our 35 selected antibodies. These included an integrin (α6β1) and three different TM4SF proteins (CD9, CD63, and NAG-2). The protein NAG-2 (novelantigen-2) is a previously unknown 30-kDa cell-surface protein. Using an expression cloning protocol, cDNA encoding NAG-2 was isolated. When aligned with other TM4SF proteins, the deduced amino acid sequence of NAG-2 showed most identity (34%) to CD53. Flow cytometry, Northern blotting, and immunohistochemistry showed that NAG-2 is widely present in multiple tissues and cell types but is absent from brain, lymphoid cells, and platelets. Within various tissues, strongest staining was seen on fibroblasts, endothelial cells, follicular dendritic cells, and mesothelial cells. In nonstringent detergent, NAG-2 protein was co-immunoprecipitated with other TM4SF members (CD9 and CD81) and integrins (α3β1and α6β1). Also, two-color immunofluorescence showed that NAG-2 was co-localized with CD81 on the surface of spread HT1080 cells. These results confirm the presence of NAG-2 in specific TM4SF·TM4SF and TM4SF-integrin complexes.

α2 Integrin Subunit Cytoplasmic Domain-dependent Cellular Migration Requires p38 MAPK

The α2 integrin subunit cytoplasmic domain uniquely supported epidermal growth factor (EGF)-stimulated migration on type I collagen. p38 MAP kinase- and phosphatidylinositol 3-kinase-specific inhibitors, but not a MEK-specific inhibitor, eliminated EGF-stimulated and unstimulated α2-cytoplasmic domain-dependent migration. Following adhesion to collagenous matrices, cells expressing the full-length α2 integrin subunit, but not cells expressing a chimeric α2 integrin subunit in which the α2-cytoplasmic domain was replaced by the cytoplasmic domain of the α1-subunit, exhibited sustained and robust phosphorylation of p38 MAP kinase. Expression of dominant negative p38 MAP kinase inhibited α2-cytoplasmic domain-dependent, EGF-stimulated migration as well as unstimulated migration on collagen. Expression of constitutively active Rac1(Val-12) augmented p38 MAP kinase activation and α2-cytoplasmic domain-dependent migration. It also rescued the ability of cells expressing the α1-cytoplasmic domain to activate p38 MAPK and to migrate. These results suggest that the α2 integrin cytoplasmic domain uniquely stimulates the p38 MAP kinase pathway that is required for unstimulated and EGF-stimulated migration on type I collagen.

Diet-Induced Muscle Insulin Resistance Is Associated With Extracellular Matrix Remodeling and Interaction With Integrin α2β1 in Mice

OBJECTIVE The hypothesis that high-fat (HF) feeding causes skeletal muscle extracellular matrix (ECM) remodeling in C57BL/6J mice and that this remodeling contributes to diet-induced muscle insulin resistance (IR) through the collagen receptor integrin α2β1 was tested. RESEARCH DESIGN AND METHODS The association between IR and ECM remodeling was studied in mice fed chow or HF diet. Specific genetic and pharmacological murine models were used to study effects of HF feeding on ECM in the absence of IR. The role of ECM-integrin interaction in IR was studied using hyperinsulinemic-euglycemic clamps on integrin α2β1-null (itga2−/−), integrin α1β1-null (itga1−/−), and wild-type littermate mice fed chow or HF. Integrin α2β1 and integrin α1β1 signaling pathways have opposing actions. RESULTS HF-fed mice had IR and increased muscle collagen (Col) III and ColIV protein; the former was associated with increased transcript, whereas the latter was associated with reduced matrix metalloproteinase 9 activity. Rescue of muscle IR by genetic muscle-specific mitochondria-targeted catalase overexpression or by the phosphodiesterase 5a inhibitor, sildenafil, reversed HF feeding effects on ECM remodeling and increased muscle vascularity. Collagen remained elevated in HF-fed itga2−/− mice. Nevertheless, muscle insulin action and vascularity were increased. Muscle IR in HF-fed itga1−/− mice was unchanged. Insulin sensitivity in chow-fed itga1−/− and itga2−/− mice was not different from wild-type littermates. CONCLUSIONS ECM collagen expansion is tightly associated with muscle IR. Studies with itga2−/− mice provide mechanistic insight for this association by showing that the link between muscle IR and increased collagen can be uncoupled by the absence of collagen-integrin α2β1 interaction.

Aberrant expression of T-cell-associated antigens on B-cell non-Hodgkin lymphomas

We describe 9 well-characterized cases of B-cell non-Hodgkin lymphoma (NHL) that showed aberrant expression of T-cell-associated antigens by 2-color flow cytometry. Cases were as follows: chronic lymphocytic leukemia/small lymphocytic lymphoma, 4; follicle center cell lymphoma, 2; mantle cell lymphoma, 1; and diffuse large B-cell lymphoma, 2. CD2 was the most commonly expressed antigen (5 cases). CD8 and CD7 were identified in 2 cases each, including 1 case that expressed both CD7 and CD4. The disease course and response to treatment were compatible with the type and stage of lymphoma. No unusually aggressive behavior was noted in any case. A control group of 59 cases of benign lymph nodes analyzed during the same period showed no aberrant expression of T-cell-associated antigens; thus, such expression is not a feature of benign lymphoid proliferations. Study of these B-cell lymphomas may prove invaluable to study aberrant activation of silent or repressed T-cell differentiation genes. CD2-expressing B-cell NHLs may represent clonal expansion of CD2+ B lymphocytes that normally constitute a small fraction of peripheral B lymphocytes and should not be confused with composite B- and T-cell lymphomas. Unless aggressive behavior is noted consistently, no aggressive treatment is justified.

Integrin-Mediated Adhesion: Tipping the Balance between Chemosensitivity and Chemoresistance

The integrin family of extracellular matrix receptors plays an important role in normal development, epithelial morphogenesis, angiogenesis, and in tumor progression and metastasis. Integrins cooperate with growth factor receptors to control many cellular functions including proliferation and cell survival. Integrin-mediated adhesion regulates many of the cell cycle checkpoints including activation of cyclin D/cdk4/6 complexes, expression of cyclin D genes, and regulation of levels of cyclin-dependent kinase inhibitors. In addition, integrin-mediated cell adhesion regulates apoptosis by modulating the activity of both the mitochondrial pathway and the death receptor pathways. Therefore, integrin-mediated adhesion modulates the decision of life or death. A role for tumor-matrix interactions in the acquisition of drug resistance has been reported for many cancers including breast cancer. Recent evidence suggests that integrin-mediated adhesion to the ECM may undermine the response of tumors to chemotherapeutic agents. Integrins have been shown to be readily accessible drug targets and are therefore attractive potential targets for combined modality chemotherapy.

Collagen-induced release of interleukin 1 from human blood mononuclear cells. Potentiation by fibronectin binding to the alpha 5 beta 1 integrin.

PBMC express cell surface receptors for extracellular matrix components known as integrins. We have recently shown that ligand binding to one PBMC integrin, the collagen receptor alpha 2 beta 1, stimulates the secretion of interleukin 1 (IL-1). We have now investigated the role of fibronectin (Fn), an adherence protein that has binding sites for both PBMC and collagen, in the generation of the IL-1 response to collagen. In contrast to collagen, Fn did not stimulate IL-1 release but Fn-depleted serum decreased the release of IL-1 induced by collagen. A polyclonal antiserum directed against Fn also decreased the collagen-induced IL-1 secretion. The IL-1 response to collagen from cells incubated in Fn-depleted serum was restored by the addition of either purified Fn or the 120-kD cell-binding fragment of Fn, which contains the cell-binding site but not the collagen-binding domain. Smaller Arg-Gly-Asp (RGD) peptides failed to enhance the PBMC response to collagen but inhibited in a concentration-dependent fashion the potentiating effect Fn. As expected, a MAb against the alpha 2 beta 1 collagen receptor decreased collagen-induced IL-1 release. However collagen-induced IL-1 release was also inhibited by a MAb against the alpha 5 beta 1 Fn receptor. The effect of the two MAbs was not additive, suggesting that the occupancy of both receptors by ligands is required in order for collagen to induce an maximal response from PBMC. The mechanism by which Fn exerts its effect remains unknown. However, flow-cytometric analysis revealed that Fn does not alter expression of the alpha2beta1 receptor on PBMC. These data demonstrate a potentiating effect of Fn on the collagen-induced secretion of IL-1 from human PBMC and suggest that this effect is mediated via the integrin alpha5beta1. These findings indicate a complex interactive role for specific integrin receptors in the regulation of the mononuclear cell immune response.