**3. Tumor necrosis factor α**

UVB radiation can increase cyclo-oxygenase (COX)-2 expression and activity in keratinocytes [20, 28, 42]. High levels of COX-2 activity have been observed in human epithelial skin cancers [43]. Nonsteroidal anti-inflammatory drugs can inhibit COX-2 activity and subsequent PGE formation in the skin, and have been used in the treatment of actinic keratosis (AK) [44], BCC, SCC and melanoma [45]. This suggests a role for COX-2 in the formation of skin cancers, and

Upregulation of TNFα is a key early response observed in keratinocytes exposed to UVB radiation [8, 38, 47] and represents an important component of the inflammatory cascade in skin. The expression of TNFα mRNA was enhanced a few hours post-UVB irradiation in both keratinocytes and dermal fibroblasts [38, 47]. IL-1α was shown to stimulate TNFα expression in UVB-irradiated keratinocytes [47] and melanocytes [48]. While Bashir *et al.* [38] observed that TNFα expression in keratinocytes was only induced by UVB irradiation, others have shown that UVA can also induce expression in these cells [49, 50]. This increase in TNFα released by the cells is due to elevated gene transcription [38, 49]. The IL-1α formed in the skin, can in turn, induce mast cells to express inflammatory cytokines (e.g. TNFα and IL-1α), as well as prostaglandins which can enhance the inflammation caused by direct UV exposure on the epidermis [15, 20, 28, 51]. Histamine released from the mast cells can induce vasodilation of the surrounding blood vessels, which assists leucocytes in undergoing diapedesis and entering this region [20, 51]. UVB radiation can induce the synthesis and release of IL-6 and IL-8 from irradiated keratinocytes and fibroblasts [33, 36, 37, 51]. IL-8 assists in the homing of leucocytes, primarily neutrophils, from surrounding blood vessels into the inflamed region, while IL-6 can trigger the activation of monocytes and other infiltrating leucocytes to secrete cytokines and chemokines [51]. Figure 1 shows the complex interaction that occurs between different

TNFα can induce the expression of adhesion molecules and chemokines in surrounding epithelial cells, resulting in the recruitment of inflammatory leucocytes from surrounding blood vessels via diapedesis [15, 20, 51, 52]. These inflammatory cells in turn can express additional cytokines that form a positive feedback loop that further upregulates TNFα as well as downstream TNF − induced chemokines, cytokines, and other pro-inflammatory media‐ tors in irradiated skin [8, 38, 53]. The effects elicited by these infiltrating inflammatory cells occur some hours following exposure to UV irradiation, thereby prolonging the inflammatory response. UVB radiation also induces inducible nitric oxide synthase (iNOS) activity in dermal

TNFα plays a pro-inflammatory role in the skin due to; (a) the direct effects of UV radiation and (b) the indirect effects of inflammatory cells that chemotax to the skin. UV- and inflam‐ matory cell-derived cytokines further enhance TNFα gene transcription in human skin cells [38], which can again increase its production by epidermal cells. In contrast, clustering and internalization of the TNF receptors may lessen the cell's response to TNFα, which may account for why the upregulation of TNFα mRNA is not sustained over time in culture [20]. For further information on the complex interplay of cytokines, chemokines and other media‐

tors in UV-induced inflammation please refer to the following reviews [15, 20, 41, 42].

high levels of activity have been observed in many of these tumours [46].

274 Highlights in Skin Cancer

bioactive molecules in the skin following exposure to UV radiation.

endothelial cells, through a TNFα-dependent pathway [38, 54].

TNFα, is a member of the TNF ligand superfamily, and is a type II transmembrane glycoprotein of 234 amino acids possessing an extracellular carboxy-terminus and a cytoplasmic amino group [53, 55, 56]. It can exist in one of two forms; a 26 kDa membrane-bound form (mTNFα) and a 17 kDa soluble form (sTNFα). sTNFα is cleaved from its membrane bound precursor between Ala76 - Val77 by the action of the metalloprotease TACE [22, 55].

Numerous cells produce TNFα, including macrophages, leucocytes, dendritic cells, keratino‐ cytes, melanocytes and fibroblasts [8, 47, 57, 58]. It plays a role in apoptosis, cellular prolifera‐ tion, differentiation, inflammation, tumorigenesis, viral replication, immune response to extracellular stimuli, as well as in local andsystemic inflammation [21, 53, 55-57, 59].Most ofthe cellular actions described for TNFα correspond to its secreted, mature soluble form. There is increasing evidence that mTNFα is also biologically active [58]. Both forms of TNFα can specifically bind to one of two receptors: TNF-R1 (CD120a receptor), a 55 kDa protein; TNF-R2 (CD120b receptor), a 75 kDa protein [57]. The receptors are both transmembrane glycopro‐ teins,anddisplayahighdegreeof structuralhomologyandareexpressedonmost celltypes [60]. TNF-R1 is expressed on a wide range of cell types and its signalling mediates cytotoxicity, cell proliferation, antiviral activity and many of the proinflammatory actions of TNFα [58, 61]. TNF-R2 is expressed on a limited range of cells, including leucocytes, endothelial cells, Langerhans cells (LC) and epithelial cells but its actions are less clear [58, 61]. Membrane-bound TNF-R1 and TNF-R2 can be cleaved by TACE to release the soluble forms of these receptors and this process is activated by IL-10 [58]. The soluble forms of TNF-R may act as (a) an antagonist to the surface receptors by competing for sTNFα or (b) an agonist by stabilizing the TNF trimer; therefore maintaining saturating concentrations in extracellular fluids [58, 62].

also known as ADAM 17 [22, 75-77]. ADAM proteases belong to the adamalysin/reprolysin subfamilyofthemetzincinsuperfamily, andcontainaZn2+-dependent catalyticdomain[75, 77].

The Role of Furin in the Development of Skin Cancer

http://dx.doi.org/10.5772/55569

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TACE was first purified, characterized and cloned in 1997 and is a multi-domain type I transmembrane protein of 824 amino acids in length [22, 76, 78]. While its amino acid sequence shows relatively low homology to other ADAM family members, its structure contains all the domain regions, which are characteristic for this family of metalloproteases [22, 76, 79]. Structurally TACE consists of a signal peptide followed by a pro, catalytic, disintegrin, cysteine-rich, transmembrane and cytoplasmic domain [55, 80]. The catalytic domain contains the zinc-binding consensus motif HEXGHXXGXXHD involved in coordinating Zn2+ with His residues and creating the active site of the enzyme [79, 81]. The cysteine-rich domain may play

TACE is synthesized as an inactive zymogen, which is subsequently proteolytically processed to the catalytically active form. In order for TACE to be activated its prodomain is removed at the furin cleavage site RVKR (Arg-Val-Lys-Arg) localized between the pro- and the catalytic domain, and is due to the action of a furin-type proprotein convertase [24, 77, 82-84]. In mammalian cells, proTACE is located in the endoplasmic reticulum and the proximal Golgi body whereas the mature form is located both intracellularly and on the cell membrane [83, 85]. TACE maturation is closely linked to the transport of proTACE through the medial Golgi, where upon exit, prodomain removal occurs before the enzyme reaches the cell's surface [77].

Apart from TNFα, TACE cleaves a wide range of molecules including transforming growth factor α (TGFα), amphiregulin, neuregulin, growth hormone receptor, TNF-R1, TNF-R2, Lselectin, amyloid precursor protein and IL-6R [77, 86-89]. TACE-knockout mice are far less efficient at processing TNFα on the cell membrane compared to wild type controls [75, 86]. This suggests that TACE is the main protease responsible for the processing of TNFα in the cell. Although some matrix metalloproteases (MMP) can cleave TNFα, the cleaved products are inactive due to hydrolysis occurring at different sites within the molecule [75, 81, 89].

Some metalloproteases are activated in epidermal cells following UV radiation [90-93]. Piva and co-workers found that there were a number of proteases whose activity was upregulated in UVC- or UVB-irradiated HeLa cells [91-93]. These enzymes included aminopeptidases and a "TGFαase" [91, 92]. On re-evaluation of their data, the TGFαase in questions is most likely TACE, because (a) the later enzyme is known to cleave TGFα among other growth factors [81, 88] and (b) the substrate used in these studies was a nonapeptide based on the N-terminal cleavage site of TGFα [90-93]. In cells undergoing UV-induced apoptosis, the level of cell surface protease activity (aminopeptidase and "TGFαase") was shown to be higher than that seen in viable or necrotic cells [91, 93]. The results of these studies were the first to show that TACE activity was elevated in cells exposed to UV radiation. Recently Skiba *et al*. [29] reported that UVA and UVB irradiation increased TACE mRNA levels in HaCaT cells, with higher induction induced by UVA. The expression patterns for both UVA- and UVB-irradiated cells in general appeared to be constant, although mRNA levels were significantly higher than

a role in enzyme maturation or substrate recognition [75, 76].

controls throughout the 48 h post-exposure period [29].

When TNFα is bound to the TNF-R1 receptor it plays a role in UVB-induced apoptosis in keratinocytes [54, 63]. Transgenic mice deficient for either TNF-R1 and/or TNF-R2 have been shown to be less susceptible to UVB-induced skin tumours than were wild type controls [64]. Through the use of TNF-R1 [65, 66] and TNF-R2 [65] gene-targeted mutant mice, it has been shown that TNF-R1 plays a decisive role in the host's defence against microorganisms, while TNF-R2 plays a role in the induction of tissue necrosis. Through the use of agonist and antago‐ nist antibodies, TNF-RI was shown to be the main mediator of TNFα action in the cell [67].

Dermal injection of TNFα resulted in the accumulation of dendritic cells in draining lymph nodes as well as in impairment of contact hypersensitivity (CHS) in the skin [60, 68]. This suggests that TNFα induces the migration of LC from the skin to the surrounding regional lymph nodes. Streilein and colleagues [69, 70] observed that UVB indirectly induced TNFα, which then caused morphologic and functional changes on LC resulting in the impairment of CHS, suggesting that TNFα plays a role in this process.

Studies using TNF-R1(-) mutant mice have shown that TNFα was not involved in UVBinduced immunosuppression [71]. UVB-induced immunosuppression is implicated in the pathogenesis of skin cancers, and is mediated in part by *cis*-urocanic acid (*cis*-UCA) [72, 73]. *trans*-Urocanic acid, a deamination product of histidine, is a major chromophore present at high concentrations in the stratum corneum [73]. Upon exposure to UV radiation, *trans*-UCA undergoes a photoisomerization to its *cis-*isomer until equilibrium is reached. In humans, this occurs after one minimal erythemal dose of UV radiation, which is the lowest dose that can induce a visibly perceptible erythema [72, 73]. *cis*-UCA does not exert its immunosuppressive effects via TNFα, but through other factors such as prostaglandin E2 [72]. Amerio *et al*. [71] showed that in TNF-R1 and TNF-R2 double knockout mice, TNFα played a minimal role in UVB-induced immunosuppression and therefore cannot be considered as a major mediator of *cis*-UCA-induced immunosuppression. While TNFα does not play a major role in UV-induced immunosuppression [60, 71] it does play a significant role in UV-induced inflammation [20] as well as in other inflammatory diseases such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus and cancer [21, 38, 46, 74].
