Mevastatin (MVS) has been previously shown to induce heme oxygenase (HO)-1 expression through Nox/ROS-dependent PDGFRα/PI3K/Akt/Nrf2/ARE axis in human pulmonary alveolar epithelial cells (HPAEpiCs). However, alternative signaling pathways might involve in MVS-induced HO-1 expression. We found that tumor necrosis factor α (TNFα) induced vascular cell adhesion protein 1 (VCAM-1) expression and NF-κB p65 phosphorylation which were attenuated by pretreatment with MVS via up-regulation of HO-1, determined by Western blot and real-time qPCR. TNFα-induced VCAM-1 expression was attenuated by an NF-κB inhibitor, Bay117082. The inhibitory effects of MVS were reversed by tin protoporphyrin (SnPP)IX (an inhibitor of HO-1 activity). In addition, pretreatment with the inhibitor of pan-Protein kinase C (PKC) (GF109203X), PKCα (Gö6983), Pyk2 (PF431396), p38α MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA), and transfection with their respective siRNAs abolished MVS-induced HO-1 expression in HPAEpiCs. c-Jun (one of AP-1 subunits) was activated by PKCα, Pyk2, p38α MAPK, and JNK1/2, which turned on the transcription of the homx1 gene. The interaction between c-Jun and HO-1 promoter was confirmed by a chromatin immunoprecipitation (ChIP) assay, which was attenuated by these pharmacological inhibitors. These results suggested that MVS induces AP-1/HO-1 expression via PKCα/Pyk2/p38α MAPK- or JNK1/2-dependent c-Jun activation, which further binds with AP-1-binding site on HO-1 promoter and suppresses the TNFα-mediated inflammatory responses in HPAEpiCs. Thus, upregulation of the AP-1/HO-1 system by MVS exerts a potentially therapeutic strategy to protect against pulmonary inflammation.

Recently, we found that 5,8-dihydroxy-4',7-dimethoxyflavone (DDF) upregulated the expression of heme oxygenase (HO)-1 via p38 mitogen-activated protein kinase/nuclear factor-erythroid factor 2-related factor 2 (MAPK/Nrf2) pathway in human cardiac fibroblasts (HCFs). However, the alternative processes by which DDF induces the upregulation of HO-1 expression are unknown. Activation of epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), and protein kinase C (PKC)α may initiate specificity protein (Sp)1 activity, which has been reported to induce expression of antioxidant molecules. Thus, we explored whether these components are engaged in DDF-induced HO-1 upregulation in HCFs. Western blotting, promoter-reporter analyses, and real-time polymerase chain reactions were adopted to measure HO-1 and vascular cell adhesion molecule (VCAM)-1 expressions in HCFs. Respective small interfering (si)RNAs and pharmacological inhibitors were employed to investigate the signaling components engaged in DDF-induced HO-1 upregulation. The chromatin immunoprecipitation assay was conducted to detect the binding interaction of Sp1 and antioxidant response elements (ARE) on the promoter of HO-1. An adhesion assay of THP-1 monocyte was undertaken to examine the functional effect of HO-1 on tumor necrosis factor (TNF)-α-induced VCAM-1 expression. DDF stimulated the EGFR/PKCα/PI3K/Akt pathway leading to activation of Sp1 in HCFs. The roles of these protein kinases in HO-1 induction were ensured by transfection with their respective siRNAs. Chromatin immunoprecipitation assays revealed the interaction between Sp1 and the binding site of proximal ARE on the HO-1 promoter, which was abolished by glutathione, AG1478, Gö6976, LY294002, or mithramycin A. HO-1 expression enhanced by DDF abolished the monocyte adherence to HCFs and VCAM-1 expression induced by TNF-α. Pretreatment with an inhibitor of HO-1: zinc protoporphyrin IX reversed these inhibitory effects of HO-1. We concluded that DDF-induced HO-1 expression was mediated via an EGFR/PKCα/PI3K/Akt-dependent Sp1 pathway and attenuated the responses of inflammation in HCFs.

Staphylococcus aureus (S. aureus) is a very common Gram-positive bacterium. It is widely distributed in air, soil, and water. S. aureus often causes septicemia and pneumonia in patients. In addition, it is considered to play a key role in mediating cell adhesion molecules upregulation. Resveratrol is a natural antioxidant with diverse biological effects, including the modulation of immune function, anti-inflammation, and cancer chemoprevention. In this study, we proved that S. aureus-upregulated vascular cell adhesion molecule-1 (VCAM-1) expression in human lung epithelial cells (HPAEpiCs) was inhibited by resveratrol. We also observed that resveratrol downregulated S. aureus-enhanced leukocyte count in bronchoalveolar lavage (BAL) fluid in mice. In HPAEpiCs, S. aureus stimulated c-Src, PDGFR, p38 MAPK, or JNK1/2 phosphorylation, which was inhibited by resveratrol. S. aureus induced the adhesion of THP-1 cells (a human monocytic cell line) to HPAEpiCs, which was also reduced by resveratrol. Finally, we found that S. aureus induced c-Src/PDGFR/p38 MAPK and JNK1/2-dependent c-Jun and ATF2 activation and in vivo binding of c-Jun and ATF2 to the VCAM-1 promoter, which were inhibited by resveratrol. Thus, resveratrol functions as a suppressor of S. aureus-induced inflammatory signaling, not only by inhibiting VCAM-1 expression but also by diminishing c-Src, PDGFR, JNK1/2, p38 MAPK, and AP-1 activation in HPAEpiCs.

Particulate matter (PM), a major air pollutant, may be associated with adverse cardiovascular effects. Reactive oxygen species- (ROS-) dependent proinflammatory cytokine production, such as interleukin-6 (IL-6), is a possible underlying mechanism. Carbon monoxide- (CO-) releasing molecule-2 (CORM-2) which liberates exogenous CO can exert many beneficial effects, particularly anti-inflammation and antioxidant effects. The purpose of this study was to explore the protective effects and underpinning mechanisms of CORM-2 on PM-induced aorta inflammation. Here, human aortic vascular smooth muscle cells (HASMCs) were utilized as in vitro models for the assessment of signaling pathways behind CORM-2 activities against PM-induced inflammatory responses, including Toll-like receptors (TLRs), NADPH oxidase, ROS, nuclear factor-kappa B (NF-κB), and IL-6. The modulation of monocyte adherence and HASMC migration, that are two critical cellular events of inflammatory process, along with their regulators, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinase-2 (MMP-2) and MMP-9, in response to PM by CORM-2, were further evaluated. Finally, mice experiments under different conditions were conducted for the in vivo evaluation of CORM-2 benefits on the expression of inflammatory molecules including IL-6, ICAM-1, VCAM-1, MMP-2, and MMP-9. Our results found that PM could induce aorta inflammation in vitro and in vivo, as evidenced by the increase of IL-6 expression that was regulated by the TLR2 and TLR4/NADPH oxidase/ROS/NF-κB signaling pathway, thereby promoting ICAM-1- and VCAM-1-dependent monocyte adhesion and MMP-2- and MMP-9-dependent HASMC migration. Importantly, our experimental models demonstrated that CORM-2-liberated CO effectively inhibited the whole identified PM-induced inflammatory cascade in HASMCs and tissues. In conclusion, CORM-2 treatment may elicit multiple beneficial effects on inflammatory responses of aorta due to PM exposure, thereby providing therapeutic value in the context of inflammatory diseases of the cardiovascular system.

Oxidative stresses are believed to play an important role in the induction of both cell adhesion molecules and pro-inflammatory cytokines, a key event in a variety of inflammatory processes. The enzyme heme oxygenase-1 (HO-1) functions as an antioxidant and serves to protect against tissue injury. In this study, we report that HO-1 was induced in cultured human tracheal smooth muscle cells after either treatment with a potent inducer of HO-1 activity, cobalt protoporphyrin IX, or infection with a recombinant adenovirus that carries the human HO-1 gene. Overexpression of HO-1 protected against tumor necrosis factor (TNF)-alpha-mediated airway inflammation via the down-regulation of oxidative stress, adhesion molecules, and interleukin-6 in both cultured human tracheal smooth muscle cells and the airways of mice. In addition, HO-1 overexpression inhibited TNF-alpha-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression, adherence of THP-1 cells, generation of interleukin-6, p47(phox) translocation, and nuclear factor-kappaB activation. HO-1 overexpression also attenuated TNF-alpha-induced oxidative stress, which was abrogated in the presence of both the HO-1 inhibitor, zinc protoporphyrin IX, as well as a carbon monoxide scavenger. In addition, HO-1 overexpression reduced the formation of a TNFR1/c-Src/p47(phox) complex. These results suggest that HO-1 functions as a suppressor of TNF-alpha signaling, not only by inhibiting the expression of adhesion molecules and generation of interleukin-6, but also by diminishing intracellular reactive oxygen species production and nuclear factor-kappaB activation in both cultured human tracheal smooth muscle cells and the airways of mice.

In bacteria-induced glomerulonephritis, Toll-like receptor 4 (TLR4) activation by lipopolysaccharide (LPS, a key component of the outer membranes of Gram-negative bacteria) can increase oxidative stress and the expression of vascular cell adhesion molecule-1 (VCAM-1), which recruits leukocytes to the glomerular mesangium. However, the mechanisms underlying VCAM-1 expression induced by LPS are still unclear in human renal mesangial cells (HRMCs).

Histone acetylation regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) plays a critical role in the expression of inflammatory genes, such as vascular cell adhesion molecule-1 (VCAM-1). Oxidative processes have been shown to induce VCAM-1 expression. Here, we investigated the mechanisms underlying IL-1beta-induced VCAM-1 expression in human tracheal smooth muscle cells (HTSMCs). Our results showed that IL-1beta enhanced HTSMCs-monocyte adhesion through up-regulation of VCAM-1, which was inhibited by pretreatment with selective inhibitors of PKCalpha (Gö6976), c-Src (PP1), NADPH oxidase [diphenylene iodonium (DPI) and apocynin (APO)], intracellular calcium chelator (BAPTA/AM), PI-PLC (U73122), CaM (calmidazolium chloride), CaM kinase II (KN62), p300 (garcinol), NF-kappaB (Bay11-7082), HDAC (trichostatin A), and ROS scavenger [N-acetyl-L-cysteine (NAC)] or transfection with siRNAs of MyD88, PKCalpha, Src, p47(phox), p300, and HDAC4. Moreover, IL-1beta stimulated NF-kappaB and CaMKII phosphorylation through MyD88-dependent PI-PLC/PKCalpha/c-Src/ROS and PI-PLC/Ca2+/CaM pathways, respectively. Activation of NF-kappaB and CaMKII may eventually lead to the acetylation of histone residues and phosphorylation of histone deacetylases. These findings suggested that IL-1beta induced VCAM-1 expression via these multiple signaling pathways in HTSMCs. Blockade of these pathways may reduce monocyte adhesion via VCAM-1 suppression and attenuation of the inflammatory responses in airway diseases.