Class A scavenger receptors (SR-A) participate in multiple macrophage functions including macrophage adhesion to modified proteins. SR-A-mediated adhesion may therefore contribute to chronic inflammation by promoting macrophage accumulation at sites of protein modification. The mechanisms that couple SR-A binding to modified proteins with increased cell adhesion have not been defined. In this study, SR-A expressing HEK cells and SR-A+/+ or SR-A−/− macrophages were used to delineate the signaling pathways required for SR-A-mediated adhesion to modified protein. Inhibiting Gi/o activation, which decreases initial SR-A-mediated cell attachment, did not prevent the subsequent spreading of attached cells. In contrast, inhibition of Src kinases or PI3-kinase abolished SR-A-dependent cell spreading without affecting SR-A-mediated cell attachment. Consistent with these results, the Src kinase Lyn and PI3-kinase were sequentially activated during SR-A-mediated cell spreading. Furthermore, activation of both Lyn and PI3-kinase was required for enhancing paxillin phosphorylation. Activation of a Src kinase-PI3-kinase-Akt pathway was also observed in cells expressing a truncated SR-A protein that does not internalize indicating that SR-A-mediated activation of intracellular signaling cascades following adhesion to MDA-BSA is independent of receptor internalization. Thus SR-A binding to modified protein activates signaling cascades that have distinct roles in regulating initial cell attachment and subsequent cell spreading.

Objective-Thiol oxidative stress leads to macrophage dysfunction and cell injury, and has been implicated in the development of atherosclerotic lesions. We investigated if strengthening the glutathione-dependent antioxidant system in macrophages by overexpressing glutathione reductase (GR) decreases the severity of atherosclerosis. Methods and Results-Bone marrow cells infected with retroviral vectors expressing either enhanced green fluorescent protein (EGFP) or an EGFP-fusion protein of cytosolic GR (GR cyto -EGFP) or mitochondrial GR (GR mito -EGFP) were transplanted into low-density lipoprotein receptor-deficient mice. Five weeks after bone marrow transplantation, animals were challenged with a Western diet for 10 weeks. No differences in either plasma cholesterol and triglyceride levels or peritoneal macrophage content were observed. However, mice reconstituted with either GR cyto -EGFP or GR mito -EGFP-expressing bone marrow had lesion areas (PϽ0.009) that were 32% smaller than recipients of EGFP-expressing bone marrow. In cultured macrophages, adenovirus-mediated overexpression of GR cyto -EGFP or GR mito -EGFP protected cells from mitochondrial hyperpolarization induced by oxidized low-density lipoprotein. Conclusion-This study provides direct evidence that the glutathione-dependent antioxidant system in macrophages plays a critical role in atherogenesis, and suggests that thiol oxidative stress-induced mitochondrial dysfunction contributes to macrophage injury in atherosclerotic lesions.

The importance of cytoplasmic motifs in differentially regulating SR-A function was demonstrated by deleting the first 49 cytoplasmic aa (SR-A(Delta1-49)), which abolished SR-A-mediated ligand internalization without reducing cell adhesion. To identify additional cytoplasmic motifs within the first 49 aa that regulate SR-A function, the acidic residues in a conserved motif (EDAD) were changed to their amide derivatives (SR-A(QNAN)). The function and regulation of SR-A(QNAN) were compared with that of SR-A(Delta1-49) and SR-A in transfected HEK-293 cells. Blocking PI3K activation inhibited SR-A, but not SR-A(Delta1-49)- or SR-A(QNAN)-mediated cell adhesion. Although deleting (SR-A(Delta1-49)) or mutating (SR-A(QNAN)) the EDAD motif abolished the PI3K sensitivity of SR-A-mediated cell adhesion, these mutations did not affect ligand internalization or PI3K activation during cell adhesion. To define the mechanism by which PI3K regulates SR-A-mediated cell adhesion, the cellular localization of wild-type and mutant SR-A was examined. PI3K inhibition reduced surface localization of SR-A but not of SR-A(Delta1-49) or SR-A(QNAN). The regulation of SR-A surface localization by PI3K was confirmed in peritoneal macrophages, which endogenously express SR-A. Together, these results suggest a pathway in which SR-A binding to an immobilized ligand activates PI3K to recruit more receptor to the plasma membrane and enhances cell adhesion.

Cigarette smoke contains a high concentration of free radicals and induces oxidative stress in the lung and other tissues. Several transcription factors are known to be activated by oxidative stress, including nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and hypoxia-inducible factor (HIF). Studies were therefore undertaken to examine if cigarette smoke could activate these transcription factors, as well as other transcription factors that may be important in lung carcinogenesis. Female A/J mice were exposed to cigarette smoke for 2, 5, 10, 15, 20, 42, or 56 days (6 hr/day, 5 days/wk). Cigarette smoke did not increase NF-κB activation at any of these times, but NF-κB DNA binding activity was lower after 15 days and 56 days of smoke exposure. The DNA binding activity of AP-1 was lower after 10 days and 56 days but was not changed after 42 days of smoke exposure. The DNA binding activity of HIF was quantitatively increased after 42 days of smoke exposure but decreased after 56 days. Whether the activation of other transcription factors in the lung could be altered after exposure to cigarette smoke was subsequently examined. The DNA binding activities of FoxF2, myc-CF1, RORE, and p53 were examined after 10 days of smoke exposure. The DNA binding activities of FoxF2 and p53 were quantitatively increased, but those of myc-CF1 and RORE were unaffected. These studies show that cigarette smoke exposure leads to quantitative increases in DNA binding activities of FoxF2 and p53, while the activations of NF-κB, AP-1, and HIF are largely unaffected or reduced.

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