Functional Roles of p38 Mitogen-Activated Protein Kinase in Macrophage-Mediated Inflammatory Responses

Inflammation is a natural host defensive process that is largely regulated by macrophages during the innate immune response. Mitogen-activated protein kinases (MAPKs) are proline-directed serine and threonine protein kinases that regulate many physiological and pathophysiological cell responses. p38 MAPKs are key MAPKs involved in the production of inflammatory mediators, including tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2). p38 MAPK signaling plays an essential role in regulating cellular processes, especially inflammation. In this paper, we summarize the characteristics of p38 signaling in macrophage-mediated inflammation. In addition, we discuss the potential of using inhibitors targeting p38 expression in macrophages to treat inflammatory diseases.


Introduction
Inflammatory response is a basic protective immune process of the organism and is accompanied by symptoms such as redness, heat, swelling, and pain associated with damage to tissues or organs [1]. This is one of the mechanisms by which our body defends us from pathogens such as parasites, bacteria, viruses, and other harmful microorganisms. Diseases induced by chronic inflammation, including gastritis, colitis, dermatitis, rheumatoid arthritis, pulmonary diseases, and type II diabetes, damage millions of people's health every year. Of concern is the increase in prevalence of these chronic inflammatory diseases. Furthermore, there is growing evidence that inflammation is a critical initiation factor inducing a variety of other major diseases such as cancer, atherosclerosis, Alzheimer's disease, cardiovascular disease, neurological disorders, and pulmonary diseases [2][3][4][5][6][7]. Therefore, a better understanding of inflammation is clinically significant and could improve treatment strategies.
Macrophages within tissues play an essential role in the initiation, development, and resolution of inflammation [8][9][10][11]. Macrophages are white blood cells that are differentiated from monocytes. Their roles are to clean up damaged cells and pathogens by phagocytosis and to activate immune cells, such as neutrophils, dendritic cells, macrophages, and monocytes, in response to pathogens and diseases. They can be activated or deactivated during inflammatory processes depending on the signaling molecules produced. Stimulation signals include lipopolysaccharide (LPS), cytokines (interleukin-1 (IL-1) and tumor necrosis factor-(TNF-)), 2 Mediators of Inflammation Cytokine production (IL-1 , TNF-, and IL-6); regulation of enzymes (iNOS, COX2); involvement of cell proliferation and differentiation; induction of cardiomyocyte apoptosis. [21,27,73] p38 (39) Ubiquitous Endothelial cells, T cells Regulation of cell differentiation; induction of cardiomyocyte hypertrophy. [21,27,73] p38 (43) Skeletal muscle Not detected Muscle differentiation. [25,27,73] p38 (40) Lung, kidney, testis, pancreas, and small intestine T cells, endothelial cells, and macrophages Developmentally regulated; involvement of cell differentiation. [26,27,73] other chemical mediators, and extracellular matrix proteins. A variety of membrane receptors are expressed on the surfaces of macrophages, including pattern recognition receptors (PRRs) such as dectin-1 and Toll-like receptors (TLRs) [12,13]. These receptors recognize activation signals and subsequently activate downstream protein kinases, eventually resulting in the stimulation of transcription factors including activator protein-1 (AP-1), nuclear factor-kappa B (NF-B), and cAMP response element-binding protein (CREB). Various intracellular proteins can initiate inflammation. p38 proteins are a class of mitogen-activated protein kinases (MAPKs) that are major players during inflammatory responses, especially in macrophages. p38, also called RK or cytokinin-specific binding protein (CSBP), was identified in 1994 and is the mammalian ortholog of the yeast Hog1p MAP kinase [14]. p38 was isolated as a 38 kDa protein that is rapidly phosphorylated at a tyrosine residue in response to LPS stimulation, and the p38 gene was cloned through binding of the p38 protein with pyridinyl imidazole derivatives [15]. p38 expression is upregulated in response to inflammatory and stress stimuli, such as cytokines, ultraviolet irradiation, osmotic shock, and heat shock, and is involved in autophagy, apoptosis, and cell differentiation [16][17][18][19][20]. Accumulating evidence suggests that p38 plays an important role in arthritis and inflammation of the liver, kidney, brain, and lung and that it acts as a critical player in inflammatory diseases mediated by macrophages [21][22][23].
In this paper, we summarize the characteristics of p38 and highlight the physiological significance of p38 activation in macrophage-mediated inflammatory responses. Moreover, we discuss the possibility of using plant extracts, natural products, and chemicals that target p38 as therapeutic drug candidates for the treatment of inflammatory diseases.  (Table 1). Genes encoding p38 and p38 show 74% sequence homology, whereas and are more distant relatives, with approximately 62% sequence identity [24][25][26]. Genes encoding p38 and p38 are ubiquitously expressed within tissues, and especially highly expressed in heart and brain. However, p38 and p38 show tissue-specific expression patterns; p38 is highly expressed in skeletal muscle, whereas p38 expression is concentrated in the kidneys, lungs, pancreas, testis, and small intestine [27]. In addition, p38 expression can be induced during muscle differentiation, and its expression can also be developmentally regulated. Moreover, we demonstrated very high expression of the active form of p38 in inflammatory diseases, such as gastritis, colitis, arthritis, and hepatitis [28,29] (unpublished data). p38 and p38 are abundantly expressed in macrophages, whereas p38 is undetectable. p38 and p38 are also expressed in endothelial cells, neutrophils, and CD4+ T cells, whereas p38 is abundant in endothelial cells. These findings indicate that, even though the four p38 family members share sequence homology, their expression is celland tissue dependent and their functions may therefore be different.

p38
Structure and Domains. p38 kinases have two domains: a 135 amino acid N-terminal domain and a 225 amino acid C-terminal domain. The main secondary structure of the N-terminal domain is -sheets, while the C-terminal domain has a -helical structure. The catalytic site is located in the region linking the two domains. The phosphorylation lip of p38 consists of 13 residues, Leu-171-Val-183, and the protein is activated by phosphorylation of a single threonine (Thr-180) and a single tyrosine residue (Tyr-182) in the lip [30]. Moreover, in Drosophila p38 MAPK, phosphorylation of tyrosine-186 was detected exclusively in the nucleus following osmotic stress [31]. p38 isoforms show various threedimensional structures with differences in the orientation of the N-and C-terminal domains, resulting in different sized ATP-binding pockets [  able to stimulate p38 kinases, for example, insulin signaling. Interestingly, with respect to inflammatory responses, a number of studies have reported p38 regulation in macrophages treated with LPS, endothelial cells stimulated with TNF-, U1 monocytic cells treated with IL-18, and human neutrophils activated with phorbol 12-myristate 13-acetate (PMA), LPS, TNF-, and fMLP [33,34]. It should also be noted that p38 activation in different cell types is dependent on the type of stimulus.
In addition, a number of studies have reported that distinct upstream kinases selectively activate p38 isoforms. p38 family kinases are all activated by MAP kinase kinases (MKKs). MKK6 activates all four p38 isoforms, while MKK3 can activate p38 , , and , but not p38 [35], and MKK4 activates p38 and [36]. This implies that p38 isoforms can be coactivated by the same upstream regulators and regulated specifically through different regulators.

p38
Deficiency. p38 deficiency affects placental development and erythropoietin expression and can result in embryonic lethality [37][38][39][40]. Tetraploid rescue of placental defects in p38 −/− embryos indicated that p38 was required for extraembryonic development, while it was not necessary for embryo development or adult mice survival. In accordance with the phenotype of p38 knockouts, knockout of two p38 activators, namely, MKK3 and MKK6, led to placental and vascular defects and induced embryonic lethality [41]. In contrast, p38 −/− mice were viable and exhibited no obvious health defects. Neither transcription of p38-dependent immediate-early genes, such as TNF-and IL-1 , nor T cell development was influenced by the loss of p38 [42,43]. Furthermore, mice harboring a T106M mutation in p38 resisted the drug inhibitory effect of collagen antibody-induced arthritis and LPS-induced TNF production, whereas the same mutation in p38 had the opposite effect [44], and p38 knockout mice responded normally to inflammatory stimuli. Single knockouts of either p38 or p38 , and even a double knockout, were viable [45]. However, reduced production of TNF-, IL-1 , and IL-10 in stimulated macrophages isolated from p38 / null mice has been observed, which indicates that p38 / are important regulatory components of the innate immune response [46]. Taken together, these findings suggest that p38 is the critical isoform in inflammatory responses but that other subtypes also play important roles.

p38 Functions in Macrophage-Mediated Inflammatory Responses and Diseases
Macrophages are the first line of defense of organisms against pathogens. They represent a major cell population distributed in most tissues, and their numbers increase massively in inflammatory diseases. In particular, macrophages are critically involved in the pathogenesis of rheumatoid arthritis (RA) and produce a variety of proinflammatory cytokines and chemokines that contribute to cartilage and bone degradation. They are also the predominant cells in the synovial lining and sublining of patients with RA [83]. Macrophages also play a central role in the development of type 2 diabetic nephropathy. Macrophage accumulation in kidney, coronary arteries, nerves, and epiretinal membrane is regarded as one of major causing factors in terms of type 2 diabetic complications, including nephropathy, atherosclerosis, neuropathy, and retinopathy [84][85][86][87][88]. Components of the diabetic milieu, including high glucose, advanced glycation end products, and oxidized low-density lipoprotein, promote macrophage accumulation and activation within diabetic tissues [89].
Macrophage depletion studies have also demonstrated the crucial role of macrophages in the development of diabetic complications [89]. Moreover, macrophages play a pivotal role in the clearance of pulmonary pathogens. Alveolar macrophages (AM) constitute more than 90% of the cells present in bronchoalveolar lavage of naïve tissues [90]. AM can rapidly clear bacteria from airways and cellular debris, help to depress the immune characteristics of the airways, and aid in lung parenchyma modeling [90]. Furthermore, macrophages have significant roles in metabolic diseases, atherosclerosis, bowel disease, and liver fibrosis [91][92][93][94]. The fundamental roles of macrophages in inflammation highlight the need for macrophage-targeted studies and therapeutics. Accumulating evidence suggests that p38 plays an essential role in macrophage-mediated inflammation. p38 is involved in the expression of proinflammatory mediators in macrophages such as IL-1 , TNF-, PGE 2 , and IL-12 [95][96][97] as well as COX-2, IL-8, IL-6, IL-3, IL-2, and IL-1, all of which contain AU-rich elements (AREs) in their 3 untranslated regions to which p38 binds [98]. Moreover, p38 can regulate the production of endothelial vascular cell adhesion molecule-1 (VCAM-1), which participates in cell proliferation and differentiation of the immune response [99]. Furthermore, p38 is associated with various inflammatory diseases, including endotoxin-induced shock, collagen-induced arthritis, granuloma, diabetes, and acute lung inflammation [100][101][102][103], as well as joint diseases, including synovial inflammation, cartilage damage, and bone loss [104]. In contrast, p38 and also play important roles in regulation of TPAinduced skin inflammation and tumor development [105,106]. In addition, a large number of reports have suggested a close correlation between p38 and cell apoptosis, cell cycle progression, and differentiation [107][108][109][110].

Development of p38-Targeted Drugs as New Anti-Inflammatory Therapeutics
p38 MAPK signaling plays a significant role in the inflammatory response and other physiological processes. A better understanding of the functional and biological significance of p38 in inflammation has led to the development of p38 inhibitors. Currently, a number of p38 inhibitors have been developed such as AMG-548, SC-80036, SC-79659, and VXs ( Figure 2) [111]; however, few studies have reported their effects on macrophages.

Crude Plant Extracts.
Natural plant extracts that target p38 are promising therapeutics for the treatment of inflammatory diseases ( Table 2). For example, Scutellaria baicalensis extract attenuates MAPK phosphorylation, especially p38 activity, resulting in inhibition of inflammatory mediators such as COX-2, iNOS, L-1 , IL-12, IL-6, IL-2, PGE 2 , and TNF-in RAW 264.7 cells treated with LPS [118]. Phaseolus angularis ethanol extract suppressed the release of PGE 2 and NO in macrophages activated by LPS-, Poly(I:C)-, or pam3CSK through regulation of TAK1/p38 pathways and, moreover, it ameliorated gastritis induced by EtOH/HCl in mice, which implies a close relationship between p38 and gastritis [119]. Archidendron clypearia extract suppressed the production of PGE 2 in activated RAW264.7 and peritoneal macrophages, as well as gastritis lesions in mouse stomachs exposed to EtOH/HCl [28]. Unfortunately, p38 is not the only target of these extracts; they contain several other active ingredients and therefore are not good candidates for the development of p38-specific inhibitors. However, they are effective at treating inflammatory diseases because of their multiple targets and their ability to improve body's homeostatic defense responses [120][121][122][123]. Meanwhile, as reported previously [124], during covering years 1981-2006, of the 974 small molecule new chemical entities, 63% were naturally derived or semisynthetic derivatives of naturally occurring products, which indicate the importance of plant extract in the drug development [124]. In addition, we and other groups have found that various traditional plant extracts that target p38 kinase can reduce the symptoms of inflammatory diseases (unpublished data), such as gastritis, colitis, arthritis, and hepatitis [28,29]. Plant extract data are summarized in Table 2.

Plant-Derived Compounds.
Several compounds from natural products inhibit p38 activity and inflammatory responses (Table 3). Sugiol, an aditerpene that was isolated and purified from alcohol extracts of the bark of Calocedrus formosana, effectively decreased the production of intracellular reactive oxygen species (ROS), IL-1 , and TNF-in LPSstimulated macrophages through regulation of MAPKs [125].
Quercetin, a plant-derived flavonoid that is widely distributed in fruits and vegetables, strongly decreased the expression of the inflammatory cytokines iNOS and TNF-by targeting both MAPK (ERK and p38) and I B signaling pathways [126,127]. Sulfur-containing compounds from garlic inhibited the production of NO, PGE 2 , and proinflammatory cytokines such as TNF-, IL-1 , and IL-6 in macrophages by suppressing p38 transduction pathways [128]. A summary of natural products targeting p38 is provided in Table 3.
These studies indicate that natural products inhibiting p38 activity exhibit strong anti-inflammatory properties, and are therefore potential therapeutic drug candidates for inflammatory diseases. Moreover, studies of natural compounds, in addition to elucidating why these extracts have strong antiinflammatory effects, can also aid the design of novel p38 inhibitors to treat inflammatory diseases.

Novel Inhibitors.
Pharmaceutical companies and researchers have worked hard to develop novel, safe, and specific p38 inhibitors. Based on the importance of p38 in inflammation, people have focused on inhibitors for this isoform rather than the other isoforms. ML3403, a SB203580 analogue, represses the expression of TNF-, IL-6, and IL-8. It can bind to both active and inactive forms of p38 kinase, which may reduce asthma-induced airway inflammation and remodeling [129]. AS1940477 has been shown to inhibit the production of proinflammatory cytokines such as TNF-, IL-1 , IL-6, PEG 2 , and MMP3 at very low concentrations. Moreover, it can reduce the enzyme activity of both p38 and but has no effect on 100 other kinases, including p38 and . It has been shown in rats experiment that low doses of this compound can also reduce the expression of LPSand Con A-stimulated proinflammatory cytokines, including TNF-and IL-6 [130]. Pamapimod strongly suppresses p38 and activity and therefore the expression of TNF-, IL-1 , and IL-6. It also shows high specific activity; when tested for binding to 350 kinases, it only bound to four other kinases in addition to p38. Furthermore, it can reduce clinical signs of inflammatory diseases, such as arthritis, bone loss, and renal diseases. Consistent with this, it inhibited Table 2: Plant extracts that inhibit the p38 signaling in macrophages.

Plant
Action target of p38 Reference

Archidendron clypearia
Suppression of PGE 2 production; amelioration of EtOH/HCl-induced gastritis [28] Scutellaria baicalensis Inhibition of iNOS, COX-2, PGE 2 , IL-1 , IL-2, IL-6, IL-12, and TNFexpression [118] Phaseolus angularis Suppression of the release of PGE 2 and NO; amelioration of EtOH/HCl-induced gastritis [119] Artemisia vestita Inhibition of TNF-release; beneficial for the treatment of endotoxin shock or sepsis [141] Boswellia   Percutaneous coronary intervention (PCI); neuropathic pain [139,140] TNF-production in RA synovial explants and reduced bone loss in murine collagen-induced arthritis. Meanwhile, it increased pain tolerance in a rat model of hyperalgesia [131]. Examples of other newly synthesized compounds are GSK-681323 to treat rheumatoid arthritis, SCIO-469 to treat multiple myeloma and dental pain, and RWJ67657 that was developed as an anti-inflammatory drug, all of which inhibit p38 activity [98]. In summary, most of these inhibitors were designed based on the structure of SB203580 but show more specific and stronger activity. They are therefore promising therapeutic agents for inflammatory diseases.

Inhibitors in Human Clinical
Trials. Based on the importance of p38 MAPK in disease development, inhibition of p38 was regarded as a promising therapeutic strategy to control inflammatory diseases. Right now, effectiveness of some p38 inhibitors is currently under evaluation in clinical trials to treat human diseases. For example, it has been reported that PH797804 and losmapimod were able to improve lung function parameters and to attenuate dyspnoea in patients with chronic obstructive pulmonary disease symptoms [132,133]. Also, losmapimod was reported to reduce vascular inflammation in the most inflamed regions in patients with atherosclerosis [134]. Clinical and histological improvements linked to the inhibition of TNF-level were clearly seen by p38 MAPK inhibitor adalimumab in lesional psoriatic skin [135]. Moreover, it was found that pamapimod can clearly alleviate various rheumatoid arthritis symptoms when coadministered with methotrexate [136]. Besides, there are still many other inhibitors which are ongoing clinical trials as summarized in Table 4 [137-140].

Summary and Perspectives
Inflammation has attracted great interest because of its significant role in several major diseases and the need to develop better ways to treat these diseases. Importantly, because inflammatory responses are largely mediated by macrophages, functional studies of macrophages in inflammation are crucial. Investigation of the roles of p38 MAPKs is particularly relevant as these are essential protein kinases in macrophage-mediated inflammatory responses. A number of studies have indicated that p38 plays a significant role in inflammatory diseases mediated by macrophages, and, as a consequence, several p38 inhibitors have been developed to treat inflammatory diseases. However, most of these inhibitors have shortcomings, such as low specificity, low efficacy, and high toxicity. As a result, new p38 inhibitors are urgently required. We are optimistic that novel and safe p38 inhibitors that possess strong anti-inflammatory properties will be developed in the near future to treat inflammatory diseases.

Conflict of Interests
The authors report no conflict of interests. The authors alone are responsible for the content and writing of the paper.

Authors' Contribution
Yanyan Yang, Seung Cheol Kim, and Tao Yu contributed equally to this work.