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Abstractβ-Carotene has shown antioxidant and antiinflammatory activities; however, its molecular mechanism has not been clearly defined. We examined in vitro and in vivo regulatory function of β-carotene on the production of nitric oxide (NO) and PGE2 as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, TNF-α, and IL-1β. β-Carotene inhibited the expression and production of these inflammatory mediators in both LPSstimulated RAW264.7 cells and primary macrophages in a dose-dependent fashion as well as in LPS-administrated mice. Furthermore, this compound suppressed NF-κB activation and iNOS promoter activity in RAW264.7 cells stimulated with LPS. β-Carotene blocked nuclear translocation of NF-κB p65 subunit, which correlated with its inhibitory effect on IκBα phosphorylation and degradation. This compound directly blocked the intracellular accumulation of reactive oxygen species in RAW264.7 cells stimulated with LPS as both the NADPH oxidase inhibitor diphenylene iodonium and antioxidant pyrrolidine dithiocarbamate did. The inhibition of NADPH oxidase also inhibited NO production, iNOS expression, and iNOS promoter activity. These results suggest that β-carotene possesses anti-inflammatory activity by functioning as a potential inhibitor for redox-based NF-κB activation, probably due to its antioxidant activity.

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Carbon monoxide (CO) plays a significant role in vascular functions. We here examined the molecular mechanism by which CO regulates HIF-1 (hypoxia-inducible transcription factor-1)-dependent expression of vascular endothelial growth factor (VEGF), which is an important angiogenic factor. We found that astrocytes stimulated with CORM-2 (CO-releasing molecule) promoted angiogenesis by increasing VEGF expression and secretion. CORM-2 also induced HO-1 (hemeoxygenase-1) expression and increased nuclear HIF-1␣ protein level, without altering its promoter activity and mRNA level. VEGF expression was inhibited by treatment with HIF-1␣ siRNA and a hemeoxygenase inhibitor, indicating that CO stimulates VEGF expression via up-regulation of HIF-1␣ protein level, which is partially associated with HO-1 induction. CORM-2 activated the translational regulatory proteins p70S6k and eIF-4E as well as phosphorylating their upstream signal mediators Akt and ERK. These translational signal events and HIF-1␣ protein level were suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K), MEK, and mTOR, suggesting that the PI3K/Akt/mTOR and MEK/ERK pathways are involved in a translational increase in HIF-1␣. In addition, CORM-2 also increased stability of the HIF-1␣ protein by suppressing its ubiquitination, without altering the proline hydroxylase-dependent HIF-1␣ degradation pathway. CORM-2 increased HIF-1␣/HSP90␣ interaction, which is responsible for HIF-1␣ stabilization, and HSP90-specific inhibitors decreased this interaction, HIF-1␣ protein level, and VEGF expression. Furthermore, HSP90␣ knockdown suppressed CORM-2-induced increases in HIF-1␣ and VEGF protein levels. These results suggest that CO stimulates VEGF production by increasing HIF-1␣ protein level via two distinct mechanisms, translational stimulation and protein stabilization of HIF-1␣.Carbon monoxide (CO) is a diffusible gas that has recently been found to play an important role in several biological processes, including angiogenesis and cytoprotection (1-4). CO is the product of the breakdown of heme by heme oxygenase (HO) 2 enzymes (5). There are two heme oxygenase functional isoforms: an inducible form, HO-1, and a constitutive form, HO-2. CO produced from heme by the catalytic reaction of HO induces the synthesis of angiogenic mediators, such as vascular endothelial growth factor (VEGF), IL-8, and stromal cell-derived factor-1, as well as decreasing the anti-angiogenic factors, namely soluble VEGF receptor-1 and soluble endoglin, resulting in the promotion of endothelial cell proliferation, migration, and anti-apoptotic responses (6 -9). In addition, glutamate-induced CO in astrocytes can act as a signal or regulatory molecule that contributes to the homeostatic regulation of vascular functions such as vasodilation (10, 11).HIF-1 (hypoxia-inducible factor) is a transcriptional complex involved in the regulation of crucial aspects of cellular functions, such as cell proliferation, survival, invasion, and glucose metabolism (12, 13). HIF-1 is composed of two s...

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