0 LA GESTION FINANCIERA

NAMPT is a ubiquitously-expressed protein, and has been reported to exert a variety of effects on a number of cell types. It is purported to have a cytokine-like function, and was initially identified by the sequence similarity in its coding region to that of other cytokines 167. Exogenous NAMPT can stimulate expression of cytokines, chemokines and matrix- degrading enzymes from synovial fibroblasts and monocytes 18, and it delays the apoptosis of neutrophils and macrophages 19,22. However, the mechanism by which NAMPT is released from cells is still a matter of debate 211,212, and no cell surface receptor for NAMPT has been identified. It was suggested that NAMPT can bind to the insulin receptor at a site distinct from that of insulin 31, although this has not been reconfirmed to date 26. The proposed role of NAMPT as a cytokine in inflammatory and metabolic diseases is discussed in this section.

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1.7.1 NAMPT expression and release

NAMPT is essential for development, as it has been shown that NAMPT gene knockouts are embryonic lethal in mice 26,31,174. NAMPT is ubiquitously expressed in the body, although it is particularly highly- expressed in the liver, adipose tissue and immune cells. It is well-validated that NAMPT expression can be upregulated in a number of immune cells in response to endotoxin and inflammatory cytokines 20. The expression of NAMPT in response to these stimuli, in a variety of cell types, is summarised in Table 1.3.

NAMPT is detected at relatively high levels in the circulation (~90 ng/mL), at a concentration around 9 times that of other common adipokines such as leptin, adiponectin and resistin 165; cytokines such as TNFα and IL-6 are generally observed to be around 10-20 pg/mL in the serum 214. NAMPT is reported to be significantly elevated during inflammation 18,32,165, obesity and diabetes 31,172, amongst other conditions. However, it is still not well understood how NAMPT is released from cells, and some argue that it is released solely through cell death and lysis 171,212. NAMPT does not contain a putative N-terminal signal sequence, commonly-seen in other secreted cytokines 167,176, but it has been suggested that NAMPT is actively-secreted by the 3T3-L1 adipocyte cell line during culture, in similar ways as other adipokines are secreted. 211. This secretion was independent of the endoplasmic reticulum (ER)-Golgi system, as it was not

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STIMULUS CELL TYPE REFERENCE

TNFα

Monocytes Dahl et al., 2007 185

Macrophages Iqbal & Zaidi, 2006 213 Neutrophils Jia et al., 2004 19 Amniotic epithelial cells Ognjanovic et al.,

2001 176 Lung microvascular endothelial

cells Ye et al., 2005

182

IL-1β

Neutrophils Jia et al., 2004 19 Amniotic epithelial cells Ognjanovic et al.,

2001 176 Lung microvascular endothelial

cells Ye et al., 2005

182

IL-6 Amniotic epithelial cells

Ognjanovic et al., 2001 176

Synovial fibroblasts Nowell et al., 2006 32

IL-8 Neutrophils Jia et al., 2004 19

GM-CSF Neutrophils Jia et al., 2004 19

Oncostatin-M Synovial Fibroblasts Nowell et al., 2006 32

Endotoxin/LPS

Neutrophils Jia et al., 2004 19 Amniotic epithelial cells Ognjanovic et al.,

2001 176 Lung microvascular endothelial

cells Ye et al., 2005

182

TABLE 1.3: Expression of NAMPT induced by inflammatory mediators in a number of cell types. Adapted from 23.

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blocked by addition of inhibitors of this pathway, and microvesicles secreted by these cells contained very little NAMPT. Thus, it was suggested that secretion occurs via a non-classical pathway, consisting of several discrete secretory machineries 211. One study has addressed the expression and release of NAMPT from neutrophils; it was shown that NAMPT mRNA transcripts were upregulated by exposure to LPS, TNFα and IL-1β, and conversely decreased by addition of pro-apoptotic stimuli (anti-CD95 antibody and heat-killed Candida albicans). NAMPT protein was also detected in the culture supernatant of LPS stimulated neutrophils 19_.

1.7.2 NAMPT signalling

It has been shown that exogenous NAMPT can activate expression of IL- 1β, IL-6 and TNFα in monocytes in a p38- and MEK-1-dependent manner; inhibition of p38 MAPK abrogates this NAMPT-induced cytokine expression 20. It is also reported that exogenous NAMPT can activate NF- κB signalling in endothelial cells; NAMPT-mediated expression of matrix metalloproteinase-2/9 (MMP-2/9) from these cells was abrogated by addition of an NF-κB inhibitor 215. In many of these studies of NAMPT cytokine-like activity, the role of NAMPT as an enzyme is not specifically addressed. Others, however, have attempted to reconcile these two supposed functions. For example, mutant recombinant NAMPT protein (with no enzyme function) was shown to stimulate expression of IL-8, IL-

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16 and CCR3 in human pulmonary epithelial cells but did not trigger NAD biosynthesis. It was identified that the transcription factor, AP-1, was required to mediate these effects, and MAPK and JNK inhibitors attenuated the activity 21. In other studies, the enzyme function of NAMPT did appear to be crucial for signalling. For example, in human vascular smooth muscle cells, NAMPT was shown to induce sustained activation of NF-κB and biphasic activation of ERK1/2, leading to enhanced iNOS production. The activation of these signalling pathways by NAMPT was abrogated by addition of the NAMPT enzyme inhibitor FK866, and was mimicked by addition of the NAMPT enzyme product nicotinamide mononucleotide (NMN) 216.

Exogenous NAMPT is also reported to have an anti-apoptotic effect on neutrophils and macrophages 19,22. This anti-apoptotic effect resulted in decreased activation of caspases-3 and -8, but not caspase-9, suggesting that NAMPT targets the extrinsic apoptotic pathway. It was shown that endogenous intracellular NAMPT was required for this process, but the role of NAMPT as an enzyme in this process was not discussed 19. In macrophages, NAMPT was shown to block ER-stress induced apoptosis via a two-step process that involved initial rapid induction of IL-6 secretion, followed by IL-6 mediated activation of STAT3. This effect was not altered by addition of the NAMPT enzyme inhibitor FK866, or by use of site-directed mutant NAMPT proteins 22.

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Current opinion still favours the dual-role hypothesis of NAMPT, activating cells like a cytokine in some circumstances and affecting cellular function via mediation of NAD biosynthesis and metabolism in others. It is clear that exogenous NAMPT can induce stimulatory effects on various cell types, but whether this is due to NAMPT induced expression of other cytokines and subsequent autocrine/paracrine activation of signalling pathways, is yet to be determined.