β-adrenergic agonists,besides their cardiac and bronchial muscle relaxing effects, have been shown to possess anti-inflammatory functions. These functions are most notable during systemic inflammatory response syndrome when lipopolysaccharide (LPS) induces the production of proinflammatory cytokines and, at the same time, stimulates the release of catecholamines. A number of published studies demonstrated that β-adrenergic agonists suppress LPS-induced production of tumor necrosis factor (TNF)-α (24, 31) and, in some cases, interleukin (IL)-1β (11) and IL-6 (33). It is believed that accumulation of intracellular cAMP after activation of adenylyl cyclase by Gsα proteins that couple to β-adrenergic receptors is responsible for the suppressive effect of β-adrenergic agonists. Other agents that elevate intracellular cAMP, e.g., forskolin (30, 34), prostaglandin (PG) E2 (28), and theophylline (31), have anti-inflammatory properties similar to those of β-adrenergic agonists. Although these findings were made years ago, the mechanism by which cAMP negatively regulates cytokine production remains incompletely understood.
TNF-α and IL-1β are produced primarily by monocytes in response to LPS stimulation. Like other cytokine proteins, TNF-α and IL-1β production are regulated at transcriptional and posttranscriptional levels. It was reported that elevation of cAMP has no effect on IL-1β mRNA accumulation (31). mRNA obtained from cells treated with PGs, theophylline, cholera toxin, or dibutyryl cAMP was able to produce IL-1β when injected into frog oocytes similar to the production from untreated cells (10). Cells treated with cAMP-elevating agents have unchanged levels of cell-associated IL-1β but have reduced IL-1β secretion (34). Unlike IL-1β, the production of TNF-α in monocytes appears to be regulated primarily at the transcriptional level. Agents that increase intracellular cAMP level have been shown to reduce TNF-α messages (31) but have no effect on its stability (30). Transcription of the TNF-α gene is regulated by nuclear factor (NF)-κB. Therefore, factors that influence the translocation and activation of NF-κB proteins as well as the phosphorylation and degradation of the inhibitory protein for NF-κB (IκB) are targets of regulation for TNF-α gene expression.
NF-κB is a ubiquitously expressed and highly regulated dimeric transcription factor (2, 3). Although initially found constitutively activated in plasma cells (23), NF-κB remains dormant in most other cell types due to association with IκB proteins (1). A large number of extracellular stimuli, including LPS, TNF-α, IL-1β, phorbol ester, viral infection, ionizing irradiation, and selective agonists for G protein-coupled receptors, can induce rapid phosphorylation of IκB proteins (9), which subsequently degrade by a ubiquitin-mediated process (5). The released p50/p65 (Rel A) proteins then migrate to the nucleus, bind the κB sequence, and induce transcriptional activation. Protein kinases play an important role in NF-κB activation through phosphorylation of IκB-α and IκB-β, which is necessary for IκB degradation (9), as well as through phosphorylation of p65, which is required for the effective recruitment of coactivators such as proteins that bind the cAMP-responsive element-binding protein (CREB) (15, 16,37).
A paper published in this issue by Farmer and Pugin (6a) examined a collection of β-adrenergic agonists and antagonists for their effects on LPS-induced TNF-α and IL-8 production in the THP-1 human monocytic cell line. Results obtained from that study indicate a long-term (>1-h and up to 8-h) effect of cAMP-elevating agents in the inhibition of NF-κB activation. Treatment of the cells with isoproterenol did not have an immediate effect on nuclear translocation and DNA binding of NF-κB proteins as measured by electrophoresis mobility shift assay. However, in 3 h, a decrease in DNA binding by NF-κB proteins was observed. Isoproterenol did not affect the degradation of IκB-α, which occurred within 30 min after LPS stimulation. However, a significant increase in IκB-α protein level was observed 3 h after treatment with LPS in the presence of isoproterenol. This increase became more prominent 8 h after the cells were stimulated. PGE2, which also induces elevation of intracellular cAMP, produced an effect similar to that of isoproterenol. H-89, a potent inhibitor of cAMP-dependent protein kinase [protein kinase A (PKA)], blocked this effect of isoproterenol. These results suggest that elevated cAMP is responsible for the increased cellular IκB-α level.
The finding that isoproterenol used together with LPS increased the level of IκB-α raises the question of whether and how isoproterenol induces IκB-α production. Farmer and Pugin (6a) conducted two experiments that provided some clues. They found that isoproterenol alone did not induce IκB-α. Therefore, LPS supplies at least part of the signals. It has been known that the IκB-α gene contains a κB binding site. Therefore, signals that induce NF-κB activation may also stimulate IκB-α gene expression (4, 6, 29). This autoregulation mechanism contributes to the synthesis of IκB-α after its degradation. Farmer and Pugin (6a) then showed an increased half-life of IκB-α protein in isoproterenol-treated cells. These results when combined suggest that isoproterenol stabilizes newly synthesized IκB-α, although how the β-adrenergic agonist affects the half-life of IκB-α protein remains unclear.
The above findings appear to be in agreement with a previous report (28) that dibutyryl cAMP when added 1.5–3 h post-LPS stimulation was still able to reduce TNF-α production. Another study (32) demonstrated an early inhibitory effect of cAMP on TNF-α production that occurs shortly after treatment of the cells and peaks within 4 h after LPS stimulation. These studies suggest the presence of multiple mechanisms for NF-κB suppression by cAMP. Using THP-1 cells and human umbilical vein endothelial cells (HUVECs), Ollivier et al. (17) and Parry and Mackman (19) found that elevated cAMP inhibited NF-κB-mediated transcription of the tissue factor gene that contains a κB binding site. These studies were conducted within 1 h after LPS stimulation and demonstrated unaffected p65 translocation to the nucleus in cells treated with forskolin. In either LPS-stimulated THP-1 cells or TNF-α-stimulated HUVECs, a gel mobility shift assay showed unaltered NF-κB binding pattern, and phosphorylation of p65 was not noticeably changed in the presence or absence of forskolin (17). Using a GAL4-p65 chimeric reporter, these investigators subsequently found that in THP-1 cells, PKA-mediated phosphorylation of CREB leads to recruitment of CREB-binding protein (CBP), which is a limiting factor for effective transcription by NF-κB. Thus phosphorylated CREB competes with p65 for CBP, resulting in reduced NF-κB activation (19).
Gerritsen et al. (8), Sheppard et al. (26), and Wadgaonkar et al. (35) provided additional data that demonstrated the importance of CBP in NF-κB activation. Nuclear microinjection of antibodies against CBP or the CBP-associated factor p/CAF prevented NF-κB activation (27). Gerritsen et al. (8) also demonstrated a direct interaction of CBP and p300 (a protein related to CBP) with p65. These authors showed that inhibition of NF-κB activation by the adenovirus E1A 12S protein, which complexes with CBP and p300, could be reversed by overexpression of CBP or p300. That CBP is a limiting factor for NF-κB activation was also suggested by a study in which the glucocorticoid receptor was shown to compete with p65 for CBP and SRC-1, the steroid receptor coactivator-1 that is required for maximal NF-κB activity (26). Ultraviolet light-induced p53 activity represses p65-mediated transcription, apparently by competition for the limited CBP (35).
PKA is an immediate effector of elevated cytosolic cAMP. It is therefore reasonable to speculate that the NF-κB-suppressive effect of cAMP-elevating agents is mediated through PKA. Indeed, the potent PKA inhibitor H-89 has been shown to abrogate suppression of NF-κB by elevated cAMP (6a). Activation of PKA, however, does not always lead to the suppression of NF-κB. Zhong et al. (36) provided extensive experimental data demonstrating that NF-κB activation can be enhanced by increased PKA activity, an effect independent of cAMP. They showed association of the 42-kDa catalytic subunit of PKA with IκB proteins in a manner similar to that with the regulatory subunit of PKA. Signals (e.g., LPS) that induce degradation of IκB proteins release and therefore activate the catalytic subunit of PKA, which then phosphorylates the nearby p65 protein at Ser276. The p65 protein contains a number of potential phosphorylation sites for PKA. Phosphorylation of Ser276 by PKA presumably leads to a change in p65 conformation, exposing a binding site for CBP and p300 (36). Association of p65 with CBP/p300 creates a “transcriptional synergy” (15), thereby promoting NF-κB activation. This mechanism is supported by data published in another study (12) demonstrating a transient increase in NF-κB binding activity in splenocytes after stimulation with forskolin. However, PKA activity was sustained for 2 h, whereas κB binding activity peaked at 30 min and returned to basal level by 2 h.
An obvious question to the aforementioned effect of PKA is whether activation of PKA invariably phosphorylates p65 and therefore enhances NF-κB activation. Zhong et al. (36) showed that dibutyryl cAMP-induced PKA activation does not result in a significant increase in p65 phosphorylation (36). They suggested that efficient p65 phosphorylation occurs only when the catalytic subunit of PKA is activated as a result of IκB degradation, leading to phosphorylation of the closely associated p65. Indeed, an independent study demonstrated that treatment of HUVECs with forskolin did not increase p65 phosphorylation or alter TNF-α-induced p65 phosphorylation (17). Thus it appears that elevated cAMP induces PKA-dependent phosphorylation of CREB (19) but not of p65.
The above studies when combined suggest that cAMP imposes both short-term and long-term effects on NF-κB activation. In the short term (within 1 h after LPS stimulation), cAMP-stimulated activation of PKA results in phosphorylation of CREB, which then competes with p65 for recruitment of CBP and p300. The long-term effects include stabilization of IκB proteins and may involve other secondary functions of PKA. It was reported that elevated cAMP potentiates LPS-induced production of IL-10 (20, 32), a cytokine generally considered to be anti-inflammatory, and has recently been shown to inhibit IκB kinases and DNA binding by NF-κB (22). Finally, it is noted that although both the short-term and long-term NF-κB-inhibitory effects of cAMP appear to be mediated through PKA, activation of PKA does not always lead to suppression of NF-κB.
Inhibition of NF-κB activation provides an effective anti-inflammatory mechanism for β-adrenergic agonists, which act on heptahelical receptors coupled to Gsα proteins. It is interesting to note that many other G protein-coupled receptors transduce signals that lead to activation of NF-κB. These receptors bind a broad spectrum of agonists including thrombin (14,21), platelet-activating factor (13), endothelin (7), bradykinin (18), and lysophosphatidic acid (25). The actions of these agonists likely are mediated through other Gα and Gβγ proteins. Thus G protein-coupled heptahelical receptors possess the ability to regulate transcription events by selective activation of one or another class of Gα proteins.
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