Our study firstly finds that in alveolar macrophage (AM) of chronic bronchitis (CB) rats the expression of cyclooxygenase-2 (COX-2) is enhanced significantly, whereas the activities of PGE2 and transforming growth factor-β (TGF-β) shared the similar tendency. Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPK) signal transduction is involved in increased COX-2 expression, while ERK MAPK signal transduction participates in TGF-β expression in AM of CB rats. This study has also demonstrated that ERK and p38 MAPK signal transduction exert synergistic effect on COX-2 expression in AM of CB rats.
Prostaglandins (PGEs) are arachidonic acid metabolites that influence inflammatory responses, bone development, wound healing, hemostasis, reproductive function, cardiovascular disease, atherosclerosis, cancer and renal homeostasis.(13) Prostanoids play an important modulatory role in the immune response through complex interactions with leukocytes and parenchymal cells in the inflamed organ. Prostaglandin E2 (PGE2), the classic model of a proinflammatory lipid mediator, also has anti-inflammatory effects depending upon the inflammatory stimulus, the predominant prostanoid produced, prostaglandin E synthase and the profile of prostanoid receptor for cyclooxygenase, which transforms it into eicosanoids. There are two cyclooxygenase isozymes that have been identified in humans bearing: cyclooxygenase-1 (COX-1) and COX-2. COX-1 is the enzyme responsible for basal, constitutive PG synthesis and expressed in the majority of cells. PG synthetized by COX-1 has anti-inflammatory effect. COX-1 deficient mice exhibited significantly increased lung inflammation and airway hyper-responsiveness, which is correlated with abrogation of PGE2 biosynthesis.(16) COX-2 gene expression is minimally or not present in most tissues. It is important in various inflammatory environments. It is inducible and can be up-regulated by various stimuli including tumor promoters, lipopolysaccharide, interleukin (IL)-1, platelet-derived growth factor, v-Src transformation and serum.(17–19) COX-2, which is expressed abundantly in inflammation, is an enzyme that regulates proinflammatory prostaglandin synthesis. Induction of COX-2 expressions promotes inflammation with PGEs. The molecular mechanisms by which this up-regulation is mediated remain unclear. The inhibition of COX-2 expression and its activity is a component of treatments for inflammatory diseases and tumor growth. Unfortunately, controversy has surrounded selective COX-2 inhibitors recently, owing to an apparent increase in the risk of cardiovascular disease and stroke. Several investigators have postulated that these procoagulant effects may result, in whole or part, from the fact that selective COX-2 inhibitors reduce endothelial production of prostacyclin (antithrombotic) while permitting platelet production of thromboxane A2 (prothrombotic), thus disrupting the homeostatic mechanisms of platelet regulation.
Prostaglandin E synthases (PGEs) have been of increasing interest since the controversy surrounding selective COX-2 inhibitors emerged. Microsomal prostaglandin E synthase-1 (mPGES-1), one of PGEs, is inducible, is increased in RA and OA cells, and is correlated with PGE2 production. In unstimulated cells, COX-2 and mPGES-1 are not expressed. Inflammatory stimulation results in calcium influx which leads to the translocation of cPLA2α from the cytosol to the nuclear membrane where it enzymatically hydrolyzes membrane phospho-lipids to release arachidonic acid. Inflammatory stimuli also induce the transcription and protein expression of both COX-2 and mPGES-1 at the nuclear membrane and endoplasmic reticulum.(20) COX-2 transforms arachidonic acid to PGG2 which is subsequently converted to PGH2. mPGES-1 may then act on PGH2 to generate PGE2. Theoretically, pharmacologic blockade of mPGES-1 (and perhaps other PGEs) could decrease proinflammatory PGE2 production while sparing other, non-E prostanoids including prostacyclin and thromboxane A2. Since no specific pharmacologic inhibitors of mPGES-1 or other PGEs are currently available, however, the benefits of such a strategy remain theoretical. Further investigation into the basic biology, regulation, and role of PGEs may illuminate the processes of inflammation, as well as the potential utility of clinically targeting these important enzymes. PGE2 synthase inhibition has the potential to be a safer anti-inflammatory strategy than either nonselective, or COX-2-selective COX inhibition. Expression of PGEs in AM of CB rats and effect of MAPK signal transduction pathway on mPGES-1 and other PGEs expression need to be further investigated.
Chronic bronchitis is chronic obstructive disease of the airways in which inflammation is followed by fibrosis with extracellular matrix (ECM) deposition on the airways wall. Fibroblast proliferation and collagen synthesis are known to be regulated by a complex interaction between stimulatory and inhibitory mediators. Growth factors such as TGF-β and glycoproteins of the ECM, such as fibronectin (FN), as stimulatory mediators, have been implicated in airway remodeling and pulmonary fibrosis.(21) TGF-β is a growth factor involved in fibrosis, since it induces the deposition of ECM by simultaneously stimulating cells to increase the synthesis of most ECM protein, decreasing the production of ECM degrading proteases and increasing the production of protease inhibitors (TIMPs). The TGF-β family can be generated by a number of different cell types, including macrophages.(22) TGF-β is considered to be a major fibrogenic cytokine and has been implicated in the generation of pulmonary fibrosis in humans. In rats with pulmonary fibrosis induced by the administration of bleomycin, total TGF-β content was several times higher than in normal rats, and increased production of TGF-β preceded the synthesis of ECM components.(23) There is quite a large volume of studies that indicate that COX metabolites might be a key mediator of the inhibitory signals on fibroblasts. As inhibitory mediator, PGE2 has been shown to decrease fibroblast proliferation and reduce collagen levels by inhibiting its synthesis and promoting its degradation.(24–26)
Our study shows that in chronic bronchitis, airway wall fibrosis has been demonstrated and AMs are an important source of biologically active TGF-β. AMs isolated from chronic bronchitis rats release greater amount of TGF-β, PGE2 and COX-2 than those from normal rats. The elevated levels of TGF-β and PGE2 released by AM from chronic bronchitis rats may be of great importance in the regulation of fibrosis and inflammation. In the early and later phase of chronic bronchitis, PGE2 may exert pro or anti-inflammation and anti-fibrosis effects, respectively, while in the later phase of chronic bronchitis, TGF-β exerts fibrotic response.
Recent papers have linked COX-2 and TGF-β expression with activation of the MAPK pathway in macrophages.(27–29) MAPKs are activated by a range of extracellularstimuli(growthfactors,cytokines,hormones, oxidants, toxins, physical stress) and regulate a variety of cellular responses including apoptosis, immune activation, as well as inflammation. MAPK pathways involve highly conserved cascades, and these processes are salient to several aspects of immune response.(30) MAPKs phosphorylate selected intracellular proteins, including transcription factors that regulate gene expression. The best described MAPK members are the ERK-1 and -2, the c-Jun-N-terminal kinases (JNKs) and the p38 MAPKs. p38 and JNK MAPKs are referred to as stress kinases and are activated by a host of stimuli, including heat shock, TNF-α, UV irradiation, and cytochalasin D; ERK1/2 is activated in response to growth stimuli and differentiation signals, including growth factors, cytokines, and viral infection.(31) Several researches have indicated the relationship between the MAPK signal transduction pathways and COX-2, PGE2, TGF-β expression in lipopolysaccharides (LPS) induced normal macrophage.(3,32,33) However, the MAPK signal transduction pathways that trigger the induction of inflammatory mediators COX-2 and growth factor TGF-β in LPS induced macrophages of CB rats have not yet been identified.
To analyze the exact MAPK signal transduction pathway about COX-2 expression in AM of CB rats, three special inhibitors of p38, ERK and JNK MAPK signal transduction pathways (SB202190, PD98059 and Curcumin) were used in this study. We determined that MAPK activation was related to COX-2 expression and PGE2, TGF-β production in AM of CB rats via RT-PCR, western blotting, radioimmunoassay and ELISA . It was founded that SB203580 (10 μM) and PD98059 (30 μM) could significantly inhibit COX-2 mRNA and protein expression and PGE2 activity in AM, which indicated that p38 and ERK MAPK signal transduction pathway participated in COX-2 expression and further affected PGE2 activity. Furthermore, ERK and p38 MAPK signal transduction pathway had synergistic effect on COX-2 protein expression. TGF-β activity from AM of model group was suppressed by PD98059 (30 uM), which showed that LPS selectively up-regulated TGF-β via ERK MAPK in AM of CB rats.
Our recent findings indicated that p38 and ERK plays important role in controlling COX-2 expression and PGE2 secretion, meanwhile, the TGF-β activity was regulated by ERK alone. However, it remains to be elucidated how AM recognizes LPS and secretes TGF-β, PGE2 and COX-2 in CB rats and their interaction.
