Smooth muscle cell (SMC) degradation of the extracellular matrix and migration to the intima are fundamental processes in the vascular response to injury. NADPH oxidase-derived reactive oxygen species (ROS) are involved in development of vascular disease; however, the specific contribution of Nox1 and Nox4, the primary catalytic subunits of NADPH oxidase in SMC, is poorly understood. We hypothesized that Nox1-derived ROS mediate thrombin-dependent activation of matrix metalloproteinase 9 (MMP-9) and migration of SMCs. Studies were performed in SMCs cultured from the aorta of Nox1 null and littermate control mice. Thrombin (2 U/mL) increased superoxide levels in control SMCs, as measured by dihydroethidium, and this response was inhibited by the flavoenzyme inhibitor diphenylene iodonium (DPI, 10 mM). In contrast, thrombin failed to increase ROS in Nox1 null SMCs. Previous studies have identified Src and mitogen-activated protein kinases as key redox-dependent regulatory proteins in thrombin-stimulated responses. Five minutes following thrombin stimulation, both Src and ERK1/2 phosphorylation were significantly decreased in Nox1 null SMCs compared with normal SMCs, measured by densitometry of Western blots. In addition, in response to thrombin, epidermal growth factor receptor (EGFR) phosphorylation was reduced in Nox1 null VSMCs. Conditioned media was collected 24 hours after cells were treated with thrombin and MMP-9 activity measured by gelatin zymography. Thrombin increased MMP-9 more than twofold in control cells; however, thrombin failed to increase MMP-9 activity in Nox1 null cells. Using a wound-scratch assay, the number and distance of cells migrating into the injured area were markedly reduced in SMCs deficient in Nox1. In conclusion, the Nox1 subunit of NADPH oxidase is required by SMCs for thrombin-dependent activation of MMP-9 and cell migration. In addition, Nox1 generation of ROS participates in phosphorylation of Src and of ERK1/2. These findings suggest that Nox1 may play an important role in the pathogenesis of vascular disease.

Bacillus anthracis synthesizes two complex structures, a peptidoglycan cell wall and poly-γ-D-glutamic acid (PDGA) capsule, which require an accessible pool of D-glutamate. The mechanisms, however, underlying the establishment of accessible pools of D-glutamate for B. anthracis are poorly understood. B. anthracis harbors two genes, racE1 and racE2, which are each predicted to encode a glutamate racemase capable of converting L-glutamate to D-glutamate. However, the respective roles, if any, of RacE1 or RacE2 in catalyzing the racemization of D-glutamate to L-glutamate have not been investigated. The objective of this study was to compare the in vitro properties of the racE1 and racE2 gene products, with the explicit purpose of establishing whether either or both of these proteins have the capacity to catalyze the conversion of L-glutamate to D-glutamate. racE1 or racE2 were cloned from B. anthracis Sterne 7702 and expressed as recombinant proteins in Escherichia coli. Each protein was purified to homogeneity. We developed an assay based on circular dichroism for directly monitoring glutamate racemase activity. Both RacE1 and RacE2 exhibited detectable glutamate racemase activity. Characterization of both enzymes revealed similar pH profiles and a lack of dependency for various metal cofactors. However, the catalytic efficiency (kcat /KM) of RacE2 was twice that of RacE1. In addition, RacE2 exists in solution as a dimer, whereas RacE1 exists primarily as a monomer. RacE1 forms a higher-ordered complex in the presence of L-glutamate, whereas the quaternary structure of RacE2 is largely independent of substrate. Collectively, these data indicate that although both racE1 and racE2 encode proteins that catalyze the racemization of L-glutamate to D-glutamate in vitro, differences in the properties of these two enzymes suggest that these two enzymes may have distinct cellular roles.