Chronic inflammatory diseases, such as inflammatory bowel disease, chronic obstructive pulmonary disease, rheumatoid arthritis (RA), psoriasis and psoriatic arthritis, are affecting a large segment of the population. In addition, cancer and even metabolic diseases, such as type 2 diabetes or atherosclerosis, are believed to have an inflammatory component . It is thought that in several of these diseases chemotactic/chemoattractant proteins and cytokines are released at the side of injury or infection, which then attracts innate and adaptive immune cells. The cytokine milieu together with the immune cells triggers a cascade of events, called the inflammatory process. Interestingly, many cytokine genes are regulated cooperatively by a transcription factor complex consisting of AP-1 and nuclear factor of activated T cells (NFAT). NFAT-dependent gene regulation has been demonstrated for IL-2, IL-3, granulocyte–macrophage colony-stimulating factor, IL-4, IL-5, IL-13, IFNγ, TNFα, CD40L, FasL, CD5, Igκ, CD25 and the chemokines IL-8 and MIP1α. Importantly, for the majority of these genes, the induction with AP-1 appears essential.
The innate immune system employs cellular components such as macrophages or dendritic cells and humoral components of the complement system to respond to infectious agents. The activation of Toll-like receptors is an important starting point for the activation of innate immunity. Once activated, Toll-like receptors lead among other events to the differentiation of macrophages and to the production of several cytokines such as TNFα, IL-1, IL-6 or IL-12. The signaling of Toll-like receptors leading to cytokine production is integrated by adapter molecules such as MyD88 and TRAF6 that eventually activate NF-κB and AP-1 .
Allergic asthma, RA and psoriasis are thought to be inflammatory diseases mediated by activated T cells. AP-1 has been shown to be involved in the differentiation of naïve T cells into T helper 1 cells and T helper 2 cells, which is a hallmark of the T cell-dependent immune response. JunB positively regulates IL-4 expression and accumulates in T helper 2 cells during differentiation . In agreement, loss of JunB in polarized T helper 2 cells in vitro is followed by deregulated expression of T-helper-2-specific cytokines and by expression of IFNγ and T-bet, which are known as key regulators of T helper 1 cells . The molecular mechanisms by which Jun and JunB regulate T helper 2 cytokine expression has been identified recently. The turnover of Jun and JunB is regulated by ubiquitin-dependent proteolysis after targeting for degradation by the E3-ligase Itch in a JNK-dependent pathway . In contrast, ectopic overexpression of JunD suppresses T cell proliferation and activation due to reduced expression of IL-4, CD25 and CD69 . Together, these data implicate Jun proteins as important players in T cell-mediated diseases that are characterized by an imbalanced ratio of T helper 1 effector cells and T helper 2 effector cells.
Glucocorticoids are very effective in controlling inflammation and are used for the treatment of autoimmune diseases such as RA. Expression of several cytokines such as IL-1, IL-2 or IFNγ is activated by AP-1 and other transcription factors, but is repressed by the glucocorticoid receptor (GR). Recent data suggest that the GR prevents the interaction between DNA-bound AP-1 complexes and transcriptional coactivators. Irrespective of the exact mechanism, the ability of the GR to repress the proinflammatory transcription factors AP-1 and NF-κB seems the most important function of the GR. This has been demonstrated with genetically modified GRdim/dim mice, whose GR is unable to bind to GR-responsive DNA elements but is still capable of transrepressing AP-1 and NF-κB .
Functions of activator protein 1 in the pathogenesis of inflammatory bone diseases
Bone is a highly dynamic organ that is continuously remodeled by osteoclasts and osteoblasts. Any disturbance in the balance between these cells causes a pathogenic change in bone mass. This could either be a loss of bone mass as observed in postmenopausal osteoporosis or a gain of bone mass as observed in osteopetrosis. Evidence from a variety of mouse models suggests that the AP-1 transcription factor is directly or indirectly implicated in the development of several bone diseases . AP-1 influences the pathogenic outcome of bone diseases not only via differentiation of bone cells but also via inflammatory processes. We shall focus on two types of inflammatory diseases, RA and psoriatic arthritis, and shall discuss the potential role of the AP-1 transcription factor.
Rheumatoid arthritis and activator protein 1
RA is considered an autoimmune disorder where the immune system preferentially attacks the joints. Extraarticular tissues such as skin, blood vessels, the heart, the lungs and muscles, however, can also be affected in a systemic manner. Besides aging, several risk factors have been identified, such as gender, environmental conditions and genetic predisposition. In addition, a strong genetic association between the major histocompatibility complex antigen DR4 and the prevalence for RA has been observed .
Histopathologically, RA is characterized by synovial inflammation, cartilage destruction and erosion of subchondral bone, eventually leading to a substantial loss of joint mobility. Activated T cells are considered the major inflammatory component that affects the severity of RA ; however, others see cells of the monocyte/macrophage lineage or synovial fibroblasts as the main culprits . The cellular mechanism by which T cells promote joint destruction in RA has been unravelled using different animal models. For example, collagen-induced arthritis has been widely used as an animal model for RA. The disease is induced by immunization of mice or rats with type II collagen and an adjuvant.
RA is also characterized by the overexpression of pro-inflammatory cytokines. In fact, a particularly important genetic model that was used to investigate the cellular interactions in RA is transgenic mice expressing human TNFα from a globin promoter (hTNFtg mice). The hTNFtg mice develop a RA-like disease that is characterized by inflammation of the joints, joint swelling and bone erosions. Breeding of hTNFtg mice with knockout mice lacking the AP-1 component Fos, and therefore devoid of osteoclasts, demonstrated the essential requirement for osteoclasts in RA. hTNFtg Fos-/- mice are completely protected from hTNFtg-induced bone erosion, although the severity of synovial inflammation as well as paw swelling and the reduction of grip strength were not ameliorated. Similar studies where osteoprotegerin was used to inhibit osteoclast differentiation suggest that activated cells present in the rheumatoid synovial membrane, such as T cells or fibroblasts, promote Fos-dependent differentiation of macrophage precursors into osteoclasts, thereby promoting bone resorption .
One key signaling molecule that was initially identified on activated T cells and as a regulator of T cell function is the receptor activator of NF-κB ligand (RANKL) – also called TRANCE, ODF, OPGL or TNFSF11 . Under pathogenic conditions such as RA, RANKL is also secreted by a variety of synovial cells including inflammatory T cells, thereby promoting extensive osteoclastogenesis and bone resorption . One potent negative regulator of RANKL is the decoy receptor osteoprotegerin, which competes with RANKL for binding to the receptor activator of NF-κB receptor on osteoclast precursors, thereby inhibiting RANKL-induced osteoclastogenesis . In RA, however, the ratio between RANKL and osteoprotegerin is shifted in favor of RANKL, resulting in a net increase of osteoclastogenesis. Based on this knowledge, a human anti-RANKL antibody called Denosumab has been developed and is currently being tested for treatment of postmenopausal osteoporosis as well as of local bone erosions in RA .
The most important transcription factor complexes that are activated by RANKL/TRAF signals are NF-κB and Fos/AP-1 . The inactivation of NF-κB or Fos causes severe osteopetrosis due to the lack of osteoclasts. Two key target genes of Fos in osteoclastogenesis have been identified recently.
The first gene, NFATc1, turned out to be a promoter of osteoclatogenesis, whereas the second gene, IFNβ, is an antagonist. NFATc1 is not solely a downstream target of Fos but also cooperates with Fos and Jun proteins to induce osteoclast-specific genes such as tartrate-resistant acid phosphatase or cathepsin K. Most importantly, ectopic expression of NFATc1 can rescue the osteoclast differentiation defect of Fos-deficient monocyte precursors, suggesting it is the most critical target gene of Fos in osteoclastogenesis . The other Fos target gene that is activated by RANKL is IFNβ. Surprisingly, IFNβ has been shown to reduce the expression of Fos in osteoclast precursors. This has led to a model where IFNβ provides a negative feedback loop that prevents extensive osteoclastogenic activity of Fos . The implication of NFATc1 and IFNβ in RA is very likely, since these proteins are key target genes of Fos. Further studies are required, however, before their potential use as therapeutic targets is taken into account.
AP-1 activity can also affect the severity of RA at a level different from osteoclastogenesis. In addition to ostecoclast-mediated bone erosion, several molecules are secreted by synovial fibroblasts that contribute to matrix degradation. Of particular importance are matrix metalloproteinases (MMPs) that are regulated by AP-1 and degrade collagen, fibronectin or other components of the extracellular matrix. The major MMPs that are implicated in RA are MMP-1, MMP-9, MMP-13 and MMP-14 (MT1-MMP) . These MMPs are expressed by activated osteoclasts or by synovial fibroblasts, or by both. The significance of AP-1-mediated MMP regulation in RA, however, has not yet been demonstrated in suitable mouse models.
Signals that lead to activation of Jun have been implicated in RA. In particular, JNK is highly activated in synovial fibroblasts of RA. The use of the JNK inhibitor SP600125 blocked accumulation of phospho-Jun in synovial fibroblasts, reduced the expression of the Jun target gene collagenase-3 and ameliorated bone erosion after collagen-induced arthritis in rats . JNK/Jun signaling should therefore also be considered a potential therapeutic target for RA.
In summary, AP-1 activity is induced in RA by inflammatory cytokines and has a complex impact on osteoclast differentiation and production of soluble mediators of bone erosion. It can be anticipated that several AP-1 components or signaling pathways leading to AP-1 activation may provide valuable drug targets for therapy of RA in the future. At present, however, therapies that target TNF-α, IL-1, IL-6, B cell and T cell costimulation are the most effective biological treatments .