Anthrax Toxin Receptor proteins function as receptors for anthrax toxin, however physiological activity remains unclear. To evaluate the biological role of Antxr2, we generated Antxr2−/− mice. Antxr2−/− mice were viable, however Antxr2 is required for parturition in young females and for preserving fertility in older female mice. Histological analysis of the uterus and cervix revealed aberrant deposition of extracellular matrix proteins such as type I collagen, type VI collagen and fibronectin. A marked disruption of both the circular and longitudinal myometrial cell layers was evident in Antxr2−/− mice. These changes progressed as the mice aged, resulting in a thickened, collagen dense, acellular stroma and the disappearance of normal uterine architecture. To investigate the molecular mechanism underlying the uterine fibrosis we performed immunoblotting for MMP2 using uterine lysates and zymography using conditioned medium from Antxr2−/− mouse embryonic fibroblasts and found reduced levels of activated MMP2 in both. This prompted us to investigate MT1-MMP status, as MMP2 processing is regulated by MT1-MMP. We found MT1-MMP activity, as measured by MMP2 processing and activation, was enhanced by expression of either ANTXR1 or ANTXR2. We identified an ANTXR2/MT1-MMP complex and demonstrated that MT1-MMP activity is dependent on ANTXR2 expression levels in cells. Thus, we have discovered that ANTXR1 and ANTXR2 function as positive regulators of MT1-MMP activity.
The genes encoding Anthrax Toxin Receptors (ANTXRs) were originally identified based on expression in endothelial cells suggesting a role in angiogenesis. The focus of this review is to discuss what has been learned about the physiological roles of these receptors through evaluation of the Antxr knockout mouse phenotypes. Mice mutant in Antxr genes have defects in extracellular matrix homeostasis. We discuss how knowledge of physiological ANTXR function relates to what is already known about anthrax intoxication.
Objective Previously we demonstrated anthrax toxin receptor 2 knockout (Antxr2−/−) mice are fertile but fail to deliver their pups at term. This parturition defect is associated with over-accumulation of extracellular matrix (ECM) proteins and decreased myometrial cell content in the uterus. Myometrial cell loss in Antxr2−/− uterine tissue prompted us to evaluate if ANTXR2 is essential for human uterine smooth muscle cell (HUSMC) viability and function. Study Design We subjected HUSMC to lentiviral-mediated knock down (R2KD) or retroviral-mediated over-expression (R2OE) of ANTXR2. Flow cytometry confirmed R2KD or R2OE in cell lines vs control (CTL). Cell behavior and function in CTL, R2KD and R2OE cells were evaluated for apoptosis via TUNEL assay, migration via Boyden chamber assay and with oxytocin-mediated collagen contraction assays. Matrix metalloproteinase (MMP) activity was evaluated using gelatin zymography. Cell lines and samples were run in duplicate. Student t-test was used for statistical analysis. Results ANTXR2 is expressed by HUSMC. HUMSC-R2KD cells exhibited increased apoptosis (p<0.05) and decreased migration (p<0.05) while HUSMC-R2OE cells exhibited no change in apoptosis (p=0.91) and increased migration (p=0.05) vs CTL. HUMSC-R2KD cells contracted significantly less than CTL while HUSMC-R2OE cells showed no difference in contractility vs CTL. MMP2 activity appeared slightly decreased in HUMSC-R2KD cells and increased in HUSMC-R2OE cells vs CTL. Conclusion ANTXR2 is expressed by HUSMC and appears important for normal HUSMC viability, migration and contractility. Further studies are needed to delineate if ANTXR2 is important for normal and abnormal labor patterns.
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