Zhengzhi Li

Crosstalk between the α2β1 integrin and c-met/HGF-R regulates innate immunity

Data from several investigators suggest that the alpha2beta1 integrin, a receptor for collagens, laminins, decorin, E-cadherin, matrix metalloproteinase-1, endorepellin, and several viruses, is required for innate immunity and regulation of autoimmune/allergic disorders. We demonstrated that the innate immune response to Listeria monocytogenes required alpha2beta1 integrin expression by peritoneal mast cells (PMCs). Ligation of the alpha2beta1 integrin by C1q contained in immune complexes comprised of Listeria and antibody was required for PMC activation in vitro and in vivo. However, ligation of the alpha2beta1 integrin alone was insufficient to activate cytokine secretion, suggesting that one or more additional signals emanating from a coreceptor were required for PMC activation. Here, we demonstrate that C1q, but neither other complement proteins nor FcRgamma, is required for early innate immune response to Listeria. The binding of Listerias Internalin B (InlB) to hepatocyte growth factor receptor (HGF-R)/c-met provides the costimulatory function required for PMC activation. Either HGF or Listeria InlB bound to c-met and either C1q or type I collagen bound to alpha2beta1 integrin stimulates PMC activation. These findings suggest that crosstalk between c-met and the alpha2beta1 integrin may contribute to mast-cell activation in autoimmune and inflammatory disorders.

Loss of the α2β1 integrin alters human papilloma virus-induced squamous carcinoma progression in vivo and in vitro.

Expression of the α2β1 integrin, a receptor for collagens and laminin, is altered during tumor progression. Recent studies have linked polymorphisms in the α2 integrin gene with oral, squamous cell carcinoma (SCC). To determine the α2β1 integrins role in SCC progression, we crossed α2-null mice with K14-HPV16 transgenic animals. Pathological progression to invasive carcinoma was evaluated in HPV-positive, α2-null (HPV/KO) and HPV-positive, wild-type (HPV/WT) animals. α2β1 integrin expression stimulated progression from hyperplasia and papillomatosis to dysplasia with concomitant dermal mast cell infiltration. Moreover, lymph node metastasis was decreased by 31.3% in HPV/KO, compared to HPV/WT, animals. To evaluate the integrin-specific impact on the malignant epithelium versus the microenvironment, we developed primary tumor cell lines. Although transition from dysplasia to carcinoma was unaltered during spontaneous tumor development, isolated primary HPV/KO SCC cell lines demonstrated decreased migration and invasion, compared to HPV/WT cells. When HPV/WT and HPV/KO SCC cells were orthotopically injected into WT or KO hosts, tumor α2β1 integrin expression resulted in decreased tumor latency, regardless of host integrin status. HPV/WT SCC lines failed to demonstrate a proliferative advantage in vitro, however, the HPV/WT tumors demonstrated increased growth compared to HPV/KO SCC lines in vivo. Although contributions of the integrin to the microenvironment cannot be excluded, our studies indicate that α2β1 integrin expression by HPV-transformed keratinocytes modulates SCC growth and progression.

Selective, α2β1 integrin-dependent secretion of il-6 by connective tissue mast cells.

Mast cells, critical mediators of inflammation and anaphylaxis, are poised as one of the first lines of defense against external assault. Mast cells release several classes of preformed and de novo synthesized mediators. Cross-linking of the high-affinity FcΕRI results in degranulation and the release of preformed, proinflammatory mediators including histamine and serotonin. We previously demonstrated that mast cell activation by Listeria monocytogenes requires the α2β1 integrin for rapid IL-6 secretion both in vivo and in vitro. However, the mechanism of IL-6 release is unknown. Here, we demonstrate the Listeria- and α2β1 integrin-mediated mast cell release of preformed IL-6 without the concomitant release of histamine or β-hexosaminidase. α2β1 integrin-dependent mast cell activation and IL-6 release is calcium independent. In contrast, IgE cross-linking-mediated degranulation is calcium dependent and does not result in IL-6 release, demonstrating that distinct stimuli result in the release of specific mediator pools. These studies demonstrate that IL-6 is presynthesized and stored in connective tissue mast cells and can be released from mast cells in response to distinct, α2β1 integrin-dependent stimulation, providing the host with a specific innate immune response without stimulating an allergic reaction.

Mitigation of oxygen-induced retinopathy in α2β1 integrin-deficient mice.

PURPOSE The α2β1 integrin plays an important but complex role in angiogenesis and vasculopathies. Published GWAS studies established a correlation between genetic polymorphisms of the α2β1 integrin gene and incidence of diabetic retinopathy. Recent studies indicated that α2-null mice demonstrate superior vascularization in both the wound and diabetic microenvironments. The goal of this study was to determine whether the vasculoprotective effects of α2-integrin deficiency extended to the retina, using the oxygen-induced retinopathy (OIR) model for retinopathy of prematurity (ROP). METHODS In the OIR model, wild-type (WT) and α2-null mice were exposed to 75% oxygen for 5 days (postnatal day [P] 7 to P12) and subsequently returned to room air for 6 days (P12-P18). Retinas were collected at postnatal day 7, day 13, and day 18 and examined via hematoxylin and eosin and Lectin staining. Retinas were analyzed for retinal vascular area, neovascularization, VEGF expression, and Müller cell activation. Primary Müller cell cultures from WT and α2-null mice were isolated and analyzed for hypoxia-induced VEGF-A expression. RESULTS In the retina, the α2β1 integrin was minimally expressed in endothelial cells and strongly expressed in activated Müller cells. Isolated α2-null primary Müller cells demonstrated decreased hypoxia-induced VEGF-A expression. In the OIR model, α2-null mice displayed reduced hyperoxia-induced vaso-attenuation, reduced pathological retinal neovascularization, and decreased VEGF expression as compared to WT counterparts. CONCLUSIONS Our data suggest that the α2β1 integrin contributes to the pathogenesis of retinopathy. We describe a newly identified role for α2β1 integrin in mediating hypoxia-induced Müller cell VEGF-A production.

Abstract 3900: Alpha2beta1 integrin regulation of endothelial notch signaling in the retina.

Angiogenesis expands the vascular network during normal development and in response to angiogenic stress. Dysregulation of this dynamic process contributes to tumor progression and to the pathogenesis of many diseases. Evidence suggests that the alpha2beta1 integrin, a collagen and laminin receptor, plays an important role in angiogenesis. In the wound-healing and tumor microenvironment, deletion of the alpha2beta1 integrin has been reported to increase neoangiogenesis. In contrast, small molecule inhibitor (SMI) targeting of the alpha2 integrin blocks sprouting angiogenesis. To reconcile these divergent findings and gain a fuller understanding of alpha2beta1 integrin9s role in angiogenesis we turned to the retina. The retinal model is uniquely suited for angiogenesis investigations as the retinal vasculature develops postnatally in a 2-dimensional plane in a well-characterized manner. Evaluation of the alpha2-null retina reveals a constellation of defects and delays in vascular development, including delayed vessel outgrowth, and increased vessel irregularity and decreased plexus density at the vascular front. Additionally we determined that alpha2 integrin-deletion has a protective effect in an oxygen-induced retinopathy model of retinopathy of prematurity (ROP) in mice by inhibiting both hyperoxia-induced vaso-obliteration and hypoxia-induced pathologic neovascularization. Confirming this result, our analysis of human microarray data shows, for the first time, that preterm infants with lower ITGA2 expression are less likely to suffer from ROP. This work clarifies the role of alpha2beta1 integrin in sprouting angiogenesis and raises the intriguing possibility of alpha2 integrin targeted therapies for prevention of ROP. These changes are reminiscent of changes observed in other models with dysregulated notch signaling. Recent studies reported that the alpha2beta1 integrin regulates sprouting angiogenesis by inducing DLL4 in ‘tip cells’. We show, for the first time, notch induced downregulation of alpha2beta1 integrin expression in ‘stalk cells’. Together these results suggest that the alpha2beta1 integrin coordinates endothelial notch signaling by stabilizing tip-stalk status. The apparent discrepancy between the effects of the alpha2 integrin inhibition and integrin-deletion may reflect differences between acute and chronic upregulation of notch signaling. We propose that synergistic use of notch and alpha2 integrin targeted therapies may provide enhanced anti-tumor angiogenesis. Citation Format: Aasakiran Madamanchi, Megan Capozzi, Ling Geng, Zhengzhi Li, Zhonghua Zhang, Richard Friedman, Kent Dickeson, John Penn, Mary Zutter. Alpha2beta1 integrin regulation of endothelial notch signaling in the retina. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3900. doi:10.1158/1538-7445.AM2013-3900

Abstract 415: Dynamic interplay of tumor and microenvironmental contributions of the α2β1 integrin in squamous cell carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The complex interplay between tumor cells and their surrounding microenvironment define cancer progression. The α2β1 integrin, expressed by subsets of epithelial, endothelial, fibroblast, and inflammatory cells, is a receptor for matrix and non-matrix ligands, including collagens, laminins, and immunomodulatory molecules C1q and collectins. Altered α2β1 integrin expression in cancer has been linked to tumor progression; recent studies have linked polymorphisms in the α2β1 integrin with oral, squamous cell carcinoma (SCC). We hypothesized that the α2β1 integrin functions in SCC initiation or progression by modulating interactions between the squamous epithelium and/or tumor microenvironment. To determine the role of α2β1 integrin expression on SCC progression, we crossed α2β1 integrin-null mice with K14-HPV transgenic animals, a well-established model of epithelial carcinogenesis. Progression from hyperplasia to dysplasia and invasive carcinoma was evaluated in HPV-positive, α2β1 integrin-null (HPV/KO) and HPV-positive, wild-type (HPV/WT) animals. Our data demonstrate that during SCC progression, expression of the α2β1 integrin stimulates progression from hyperplasia and papillomatosis to dysplasia. Decreased dysplasia in HPV/KO animals at early time points of tumor progression directly correlated with reduced dermal mast cell infiltration. Moreover, lymph node metastasis was decreased by 31.3% and associated with alterations in lymphatic vasculature within the tumor microenvironment of HPV/KO, compared to HPV/WT, animals. Since these studies utilized global α2β1 integrin knockout mice, we developed primary tumor cell lines to evaluate the integrin-specific impacts on the malignant epithelium versus the host microenvironment. Although transition from dysplasia to carcinoma was unaltered in our original tumor studies, isolated primary HPV/KO SCC cell lines had decreased invasion in a 3D transwell migration assay, compared to HPV/WT cells. When HPV/WT and HPV/KO SCC cell lines were orthotopically injected into either WT or KO hosts, α2β1 integrin-specific expression on tumor cells decreased tumor latency, regardless of host integrin status. HPV/WT SCC lines failed to demonstrate a proliferative advantage in vitro, however, the HPV/WT tumors demonstrated increased growth rates compared to HPV/KO SCC lines in vivo. Therefore, these latter data suggest that contributions of the tumor microenvironment cannot be excluded and are likely modulating SCC growth characteristics. Our studies indicate the α2β1 integrin plays diverse roles during tumor progression, acting on the tumor and tumor microenvironment. Although the α2β1 integrin plays no obvious role during normal development, exposure to physiological stresses, such as cancer, unmasks functions of the α2β1 integrin to reveal its importance in tissue biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 415. doi:10.1158/1538-7445.AM2011-415