NADPH oxidase is internalized by clathrin-coated pits and localizes to a Rab27A/B GTPase-regulated secretory compartment in activated macrophages

Here, we report that activation of different types of tissue macrophages, including microglia, by lipopolysaccharide (LPS) or GM-CSF stimulation correlates with the quantitative redistribution of NADPH oxidase (cyt b(558)) from the plasma membrane to an intracellular stimulus-responsive storage compartment. Cryo-immunogold labeling of gp91(phox) and CeCl(3) cytochemistry showed the presence of gp91(phox) and oxidant production in numerous small (<100 nm) vesicles. Cell homogenization and sucrose gradient centrifugation in combination with transferrin-HRP/DAB ablation showed that more than half of cyt b(558) is present in fractions devoid of endosomal markers, which is supported by morphological evidence to show that the cyt b(558)-containing compartment is distinct from endosomes or biosynthetic organelles. Streptolysin-O-mediated guanosine 5′-3-O-(thio)triphosphate loading of Ra2 microglia caused exocytosis of a major complement of cyt b(558) under conditions where lysosomes or endosomes were not mobilized. We establish phagocytic particles and soluble mediators ATP, TNFα, and CD40L as physiological inducers of cyt b(558) exocytosis to the cell surface, and by shRNA knockdown, we identify Rab27A/B as positive or negative regulators of vesicular mobilization to the phagosome or the cell surface, respectively. Exocytosis was followed by clathrin-dependent internalization of cyt b(558), which could be blocked by a dominant negative mutant of the clathrin-coated pit-associated protein Eps15. Re-internalized cyt b(558) did not reach lysosomes but associated with recycling endosomes and undefined vesicular elements. In conclusion, cyt b(558) depends on clathrin for internalization, and in activated macrophages NADPH oxidase occupies a Rab27A/B-regulated secretory compartment, which allows rapid agonist-induced redistribution of superoxide production in the cell

NAD(P)H oxidase activity of Nox4 in chondrocytes is both inducible and involved in collagenase expression.

International audienceReactive oxygen species (ROS) are regulators of redox-sensitive cell signaling pathways. In osteoarthritis, human interleukin-1beta is implicated in cartilage destruction through an ROS-dependent matrix metalloproteinase production. To determine the molecular source of ROS production in the human IL-1beta (hIL-1beta)-sensitive chondrocyte immortalized cell line C-20/A4, transfected cells were constructed that overexpress NAD(P)H oxidases. First, RT-PCR analysis showed that the C-20/A4 cell line expressed Nox2, Nox4, p22( phox ), and p67( phox ), but not p47( phox ). It was found that ROS production by C-20/A4 chondrocytes does not depend on PMA and ionomycin activation. This indicates that Nox2 was not involved in the production of ROS. In C- 20/A4 cells that overexpress Nox4, hIL-1beta stimulated ROS production three times more than the normal production of C-20/A4 cells. Moreover, there was a fourfold increase in the production of collagenase (MMP-1) by chondrocytes that overexpress Nox4. Interestingly, MMP-1 production in cells that overexpress Nox2 was not sensitive to hIL-1beta. These data suggest that under hIL-1beta stimulation, C-20/A4 chondrocytes produce MMP-1 through a Nox4-mediated, ROS-dependent pathway