**3. UPS in immune and inflammatory response**

A role for UPS in the pathogenesis of human diseases was first suggested some two decades ago. With the broad spectrum of protein substrates and the complex enzymatic machinery involved in targeting them and practically all intracellular processes being controlled by the UPS, it is not surprising that the proteasome pathway is involved in the pathogenesis of malignant, autoimmune, and neurodegenerative diseases.

The UPS plays significant role in immune and inflammatory processes. It has been shown that UPS takes part in the antigen processing in antigen presenting cells, regulates the transmission of signals from T-cell antigen receptors and the co-stimulatory CD28 molecule and is involved in activation of transcription factor-ĸB (NF-κB). NF-κB is the key regulator of the activity of genes of many inflammatory cytokines, chemokines and cell adhesion molecules [Sorokin, 2009]. The function of UPS in the activation of NF-kB is the most important and will be discussed here in details.

NF-kB is a family of dimeric transcription factors. The NF-kB family consists of five members: p50, p52, p65/RelA, c-rel, and RelB [Neumann & M. Neumann, 2007]. p50 and p52 are formed as a result of processing from precursors p105 and p100, respectively. The processing of p105 can be performed both by the Ub-dependent pathway by the 26S proteasome [Coux & Goldberg, 1998] and by the ATP-/Ub-independent pathway by the 20S proteasome [Moorthy, 2006]. NF-ĸB activation promotes the expression of variety of target genes involved in the immune response, reparation reactions, and apoptosis. These include the pro-inammatory cytokines IL-1β and TNF-α, extracellular matrix metalloproteinase (MMPs), prostaglandins and nitric oxide. IL-1β and TNF-α, in particular, have been shown to play pivotal roles in the pathogenesis of RA both in preclinical [Han et al., 1998] and clinical studies using biological agents such as etanercept and iniximab [Carteron, 2000; Cunnane, 2001].

The UPS activate NF-κB in two stages. At first, the proteasome performs ubiquitin dependent processing of phosphorylated precursors p105 and p100 with the formation of active subunits of transcription factors p50 (NF-κB1) and p52 (NF-κB2). NF-κB is composed of p50 and p65 subunits, and in non-stimulated cells it is retained in the cytoplasm in a latent form associated with inhibitory protein IĸB. Following exposure of the cell to a variety of extracellular stimuli such as cytokines, viral and bacterial products and stress, IκB is phosphorylated, poly-ubiquitinated (which is recognized by the 19S regulatory subunit of Proteasome) and is finally rapidly degraded by the 26S proteasome. The released active heterodimer is translocated into the nucleus where it activates the transcription of corresponding genes [Van Waes et al., 2007] (Fig 5).

Proteasome Targeted Therapies in Rheumatoid Arthritis 139

NF-κB complexes and both the NF-κB classical oligonucleotide decoy and antisense oligonucleotide specic to p65, and they produced a concentration dependent decrease in IL-1-stimulated PGE2 production [Handel, 1995]. Additionally, NF-κB activator, IL-18 can indirectly stimulate osteoclast formation through up-regulation of RANKL production from T cells in RA synovitis [Dai et al., 2004]. Blocking of IKKβ *in vitro* with a dominant negative adenoviral construct was shown to inhibit the induction of IL-6, IL-8, and intercellular adhesion molecule-1 (ICAM-1) after stimulation with IL-1 or TNF-α

The signicance of NF-κB in inammatory joint disease has been validated by numbers of arthritis models such as carrageenan-induced paw edema, collagen-induced arthritis and adjuvant-induced arthritis [Min et al, 2009, Campo et al, 2011, Ahmed et al., 2010]. In animal models of arthritis the activation of NF-κB appears to precede the onset of disease, and the blockade of NF-κB decreases arthritis severity [Tsao et al., 1997; Ahmed et al., 2010]. Intraarticular gene transfer of IKKβ-wild type into the joints of normal rats resulted in signicant paw swelling and accompanied synovial inammation. Increased IKK activity was detectable in the IKKβ-wt-injected ankle joints which was coincident with enhanced NF-κB-DNA-binding activity. Intra-articular gene transfer of IKKβ-dominant negative signicantly ameliorated the severity of adjuvant arthritis, accompanied by a signicant decrease in NFκB DNA expression in the joints of adenoviral IKKb-dominant negative-treated animals

Progressive destruction of bone and articular cartilage plays a pivotal role in the pathogenesis of RA. During joint inflammation, the inflamed synovium forms a pannus tissue, which grows into the bone and causes destruction, initially as marginal erosions at the site of synovial proliferation where bone is unprotected by hyaline cartilage. Subsequent bone destruction leads to sublaxation and deformity. Cytokines such as TNF-α, IL-1β and IL-17 stimulate the activation of bone destroying osteoaclasts, and the production of destructive proteases - matrix metalloproteinases (MMPs). MMPs have been suggested to be involved in the pathogenesis of RA and OA through their ability to degrade proteoglycans

NF-κB is essential for osteoclast formation and survival through the receptor activator of the nuclear factor kappa-B ligand (RANKL) pathway [Soysa et al., 2009]. Abnormal activation of NF-κB signalling in osteoclasts has been observed in osteolytic conditions, including arthritis, Paget's disease of bone, and periodontitis [Xu et al., 2009]. Inhibition or deletion of RANKL prevents bone destruction [Zwerina et al., 2004]. Further, it is demonstrated that inhibition of IκB-kinase complex can suppress RANKL stimulated NF-κB activation and osteoclastogenesis both *in vitro* and *in vivo*. Additionally, this peptide signicantly reduced the severity of collagen-induced arthritis in mice by reducing levels of TNF-α and IL-1β, and thereby abrogating joint swelling and reducing destruction of bone and cartilage [Jim et al., 2004]. Elevated levels of MMP-1 (collagenase-1) in the synovial fluid and serum of RA patients [Green et al., 2003; Yamanaka et al., 2000] and MMP-3 (stromelysin) in the synovial fluid from RA patients has been determined [Hembry et al., 1995]. Interestingly, it has been reported that NF-κB regulates synthesis of MMPs including MMP-I and MMP-3 [Thurberg

[Aupperle et al., 2001].

[Tak et al., 2001].

et al., 1998].

**3.1.1 NF-κB in RA joint destruction** 

[Flannery et al., 1992; Humbry et al., 1995].

Fig. 5. Activation of Nuclear factor-ĸB by the proteasome system

NF-κB promotes transcription of genes which encode cytokines (TNF-α, IL-6, IL-1), stress response factors (Cyclooxygenase-2, NO), cell cycle regulators, and anti-apoptotic proteins (IAP-1, Bcl-2 family) [Delhalle et al., 2004]. The pathological activation of NF-κB is a cause of many inflammatory diseases including RA and has been an important target for therapeutic drug research in recent years [Elliott et al., 2003].
