Integrin alpha3beta1: A novel regulator of epithelial-to-endothelial cell crosstalk during physiological and pathological tissue remodeling processes

Integrin alpha3beta1, a major cell surface receptor, has been shown to play important roles in regulating keratinocyte migration, one important aspect of wound healing. However its role in regulating other important processes associated with wound healing, such as angiogenesis are unknown. In Chapter 1, we demonstrate that mice with an epidermis-specific knockout of the integrin subunit alpha3 show markedly reduced blood vessel density during wound healing compared to control mice indicating that alpha3beta1 expression in keratinocytes is critical for the regulation of wound angiogenesis. Additionally, we show that endothelial cell migration is decreased in response to conditioned medium from alpha3 deficient keratinocytes compared to alpha3 expressing keratinocytes. These data indicate there is an alpha3beta1-dependent crosstalk from keratinocytes to endothelial cells which is important for regulating wound angiogenesis. In addition we demonstrate that alpha3beta1 dependent regulation of Mitogen Regulated Protein/Proliferin 3 (MRP3), a pro-angiogenic factor, is an important mediator of this crosstalk. Indeed, absence of alpha3beta1 from the epidermis leads to reduced expression of MRP3 in hair follicles adjacent to wounded tissue, and in the migrating epidermis. Furthermore, stable knockdown of MRP3 using shRNA in alpha3beta1 expressing keratinocytes decreases the rate of endothelial cell migration in response to conditioned medium, implicating MRP3 as an important mediator of keratinocyte to endothelial cell crosstalk. These findings identify a novel role for alpha3beta1 in promoting wound angiogenesis through a mechanism of crosstalk from epidermal to endothelial cells, and they implicate MRP3 in this integrin-dependent crosstalk. Such a mechanism represents a novel paradigm for integrin-mediated regulation of wound angiogenesis that extends beyond traditional roles for integrins in cell adhesion and migration.;Integrins are also known to have important roles in promoting tumor growth and progression. Integrin alpha3beta1 is expressed at high levels in breast cancer cells where it is thought to promote invasion and metastasis; however, its roles in regulating malignant tumor cell behavior remain unclear. In Chapter 2, we used short-hairpin RNA (shRNA) to down-regulate alpha3beta1 in the human breast cancer cell line, MDA-MB-231. Stable suppression of alpha3beta1 led to decreased tumorigenicity in vivo, reduced invasiveness in vitro, and decreased production of factors that stimulate endothelial cell migration. Real-time PCR arrays of breast cancer-associated genes revealed that suppression of alpha3beta1 caused a dramatic reduction in the cyclooxygenase-2 ( COX-2) gene, which is frequently over-expressed in breast cancers and has been exploited as a therapeutic target. Decreased COX-2 was accompanied by reduced production of prostaglandin E2 (PGE2), a major prostanoid produced downstream of COX-2 and an important effector of COX-2 signaling. shRNA-mediated suppression of COX-2 showed that it was required for both tumor cell invasion and crosstalk to endothelial cells. Furthermore, treatment with PGE2 restored these functions in alpha3beta1-deficient MDA-MB-231 cells. These findings identify a novel role for alpha3beta1 in regulating two properties of tumor cells that facilitate cancer progression: invasive potential and ability to stimulate endothelial cells. They also identify COX-2 as an effector of alpha3beta1 in tumor cells, thereby revealing a novel pathway that might serve as a potential therapeutic target to inhibit breast cancer.;Previous studies from our lab have identified integrin alpha3beta1 as an important regulator of MMP-9 mRNA stability, however the mechanisms by which this regulation occurs remain unclear. In Chapter 3, we demonstrate that pharmacological inhibition of p38 and SFKs leads to reduced MMP-9 mRNA expression, suggesting that these two pathways may be involved in mediating MMP-9 mRNA stability. In addition, our preliminary data suggest that alpha3beta1 may regulate MMP-9 mRNA stability by promoting the generation of alternative 3'UTRs which contain or lack AREs thereby regulating its stability. Indeed, we were able to isolate a short variant of MMP-9 mRNA which lacks the four AREs from the WT keratinocytes. However this variant was not detected in the alpha3-keratinocytes under the same conditions, suggesting that alpha3beta1 may regulate alternative polyadenylation of MMP-9. These data may represent a novel regulatory role for integrins in gene regulation and may indicate a unique mechanism of how integrin alpha3beta1 may regulate MMP-9 expression during tumorigenesis. (Abstract shortened by UMI.)...