TNFa plays a central role in inflammation; it induces the expression of other proinflammatory molecules, chemotactic cytokines and adhesion factors[9]. In vivo and in vitro studies have shown that high levels of TNFa lead to exacerbation of the inflammatory response[10]. This, together with its potent immunomodulator activities, has been suggested as important to the pathogenesis of diseases such as asthma and Atherosclerosis[11]. In this study, we evaluated a potential mechanism of TNFa on atherogenic effects. As shown in Figure 1A and B, TNFa at 5 ng/ml, 10 ng/ml significantly inhibited both apoA-I- and HDL-mediated cholesterol efflux from human macrophages. Next, we determined the mRNA and protein levels of ABCA1, ABCG1, LXRa in order to elucidate the molecular mechanisms underlying the inhibited cholesterol efflux in human macrophages due to TNFa.
A previous study noted that ATP binding cassette transporters (ABCA1, ABCG1) and LXRa are the best characterized cellular transporters/receptor involved in macrophage RCT[12]. The initial comparison of macrophage-specific RCT between ABCA1-deficient and wild-type mice showed direct evidence that ABCA1 deficiency reduces RCT from macrophages to feces in vivo[13]. ABCG1 is another ABC transporter that is able to load more cholesterol onto mature HDLs from the peripheral tissues and is important in allowing macrophages to efflux arterial wall cholesterol, eventually preventing atherosclerotic vascular disease[14].
Our results showed that TNFa at 5 ng/ml, 10 ng/ml significantly decreased the mRNA expression levels of ABCA1, ABCG1 and LXRa compared with the control group (Figure 2), resulting in significantly down-regulated protein expression levels in human macrophages (Figure 3A, B). This indicates that TNFa inhibit reverse cholesterol transport in human macrophages via ABCA1 and ABCG1 -mediated pathways.
Interestingly, we also found that the mRNA (Figure 2) and protein (Figure 3A, C) expression levels of pattern-recognition scavenger receptors CD-36 and SR-A, were significantly up-regulated in human macrophages treated with TNFa at 5 ng/ml, 10 ng/ml. SR-A belongs to scavenger receptor A family, and has been implicated in atherosclerosis[15]. CD36 is a member of the class B scavenger receptor family and its activation has been implicated in foam cell formation. It appears that SR-A and CD36 account for greater than 90% of the lipid accumulation in macrophages exposed to oxidized LDL[16]. So it suggested that TNFa can promote cholesterol uptake by enhancing the expression of CD-36 and SR-A in human macrophages.
Protein kinase C (PKC) comprises a family of serine/threonine kinases that are involved in the regulation of many cellular responses, including proliferation, differentiation, stress responses, and lipid metabolism[17]. PKC isozymes have been classified in three subfamilies: conventional (cPKCs α, βI, βII and γ), novel (nPKCs δ, ϵ, θ and η) and atypical (aPKCs λ, ι, μ, ζ). Conventional PKCs are regulated by diacylglycerol (DAG), hosphatidylserine and calcium, whereas novel PKCs are calcium-independent, but regulated by DAG and phosphatidylserine, and atypical PKC isoforms are regulated by phosphatidylserine, but calcium, DAG- and TPA-independent[18]. Previous study showed that PKC activation mediates production of granulocyte/macrophage colony-stimulating factor (GM-CSF), which plays a priming role in Ox-LDL-induced macrophage proliferation[19]. Protein kinase C-β and –δ mediate cholesterol accumulation in PMA-activated macrophages and another report shows that stably overexpressed a dominant-negative of PKC-α inhibits LPS-induced iNOS expression in RAW 264.7 macrophages[20, 21]. HIV-1 Tat protein induces TNF-α and IL-10 production via a PKC-βII and -δ isozymes dependent way in human macrophages[22]. 6-Gingerol inhibits ROS and iNOS in lipopolysaccharide-stimulated mouse macrophages,through the suppression of PKC-α and NF-κB pathways,[23].
PKC-θ mediates the critical T cell receptor (TCR) signals selectively required for T cell activation in vivo[24]. PKC-θ plays a crucial role in the activation of various transcription factors such as NF-kB, AP-1 and nuclear factor of activated T cells (NFAT)[25, 26]. Mature PKC-θ−/− T cells failed to proliferate and produce interleukin 2 (IL-2) upon TCR stimulation due to defective activation of NF-kB and AP1[27]. Mice deficient in other isoforms of PKC do not have a defect in T cell activation, thereby reinforcing the importance of PKC-θ in T cell activation[28]. Considering the multiple functions of PKC-θ in T cell activation, it would be interesting to examine whether PKC-θ plays a role in the effect of TNFa on ABCA1, ABCG1, LXRa, SR-A, CD-36 expression in human macrophages.
To confirm whether PKC-θ is involved in the TNFa-induced down-regulation the expression of ABCA1, ABCG1, LXRa and up-regulation the expression of CD-36, SR-A in human macrophages, we utilized PKC-θ-siRNA to inhibit PKC-θ. The results show that PKC-θ-siRNA significantly attenuated the effect of TNFa on ABCA1, ABCG1, LXRa, SR-A, CD-36 expression compared with the negative control (Figure 4A, B, C). This phenomenon confirmed that PKC-θ is involved in the effect of TNFa on ABCA1, ABCG1, LXRa, SR-A, CD-36 expression in human macrophages. These data suggest that PKC-θ activity is involved in the effect of TNFa on ABCA1, ABCG1, LXRa, SR-A, CD-36 expression in human macrophages, and the isotype-selective inhibitor to PKC-θ may suppress atherosclerotic progression.