**4. TACE**

TNFα is cleaved from its proform by the action of the metalloprotease TACE [75]. This en‐ zyme is a member of the disintegrin and metalloprotease (ADAM) family of proteases, and is also known as ADAM 17 [22, 75-77]. ADAM proteases belong to the adamalysin/reprolysin subfamilyofthemetzincinsuperfamily, andcontainaZn2+-dependent catalyticdomain[75, 77].

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]. 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

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

TNFα is cleaved from its proform by the action of the metalloprotease TACE [75]. This en‐ zyme is a member of the disintegrin and metalloprotease (ADAM) family of proteases, and is

CHS, suggesting that TNFα plays a role in this process.

lupus erythematosus and cancer [21, 38, 46, 74].

**4. TACE**

276 Highlights in Skin Cancer

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

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

In UV-irradiated HaCaT cells, TACE was responsible for the increased cleavage of EGF family members [28, 94]. Inhibition of TACE by metalloprotease inhibitors reduced the release of these growth factors, resulting in an increase in apoptotic cell death [28, 94]. It appears that TACE mediates a EGF receptor/AKT signalling pathway in these cells that is activated as a result of its cleavage of EGF family members. In HaCaT cells exposed to UVA-radiation TACE mediated EGF receptor activation and cell cycle progression, which suggests that UVA, at non-lethal doses, has the potential to be a skin cancer promoter [28, 94]. TACE has also shown to be overexpressed in some tumours [21, 46, 56], as well in a large number of skin cancer cells lines compared to their non tumorous counterparts [28, 94]. It is also known that members of the EGF family are overexpressed in skin cancers [95], and this could be a mechanism by which skin cancer growth is stimulated by autogenic growth factors. The results of these recent studies suggest that inhibition of TACE following UV radiation may prevent the stimulation of surviving irradiated cells. This has the potential in reducing the incidence of skin cancer that may arise from prolonged sun exposure. It is not clear if the increase in TACE activity seen in UV-irradiated skin cells is due to increased numbers or a higher level of activity. Furin is known to activate TACE [83, 85, 96] as well as matrix metalloproteases (MMP) [97, 98] and may indirectly play a role in this process.

and other PC family members process inactive precursor proteins to their functional or mature form, and these include growth factor receptors, growth factors, hormones, plasma proteins, and MMPs [23, 24, 97, 98, 103] as seen in Table 2. PC family members play crucial roles in a variety of physiological processes and are involved in the pathology of diseases such as cancer

**Functional group Substrate References** Serum proteins Von Willebrand Factor [107]

Signalling peptides Endothelin-1 [103, 109] Growth factors TGFβ [103, 110]

Membrane proteins MT1-MMP [86, 113, 114]

Transmembrane receptors Notch1 Receptor [98, 116]

Extracellular matrix proteins N-Cadherin [113, 118]

Viral proteins Ebola virus glycoprotein [103, 120]

As a result of the role furin plays in many disease states, considerable effort has been directed at designing specific inhibitors that may have therapeutic applications. The first furin inhibi‐ tors that were synthesised where peptidyl chloromethyl ketones [124]. The next major furin inhibitor that was developed, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKRcmk, or CMK) was less cytotoxic and is cell permeable and has been used in many experimental studies [86, 106, 125]. It was recently shown to reduce the incidence of skin cancer in transgenic mice by inhibiting PACE4 as well as other PCs [126]. However a limitation of CMK's use is that it is not furin specific, and is also known to inhibit other proprotein convertases [86, 102, 127]. Zhu *et al*. [127] has recently developed an antibody-based single domain nanobody which is a furin specific inhibitor. Through the use of this and other furin-specific inhibitors, it will be possible to delineate the role furin plays in the processing of specific substrates within in the cell. This will help in development of specific inhibitors, which will have therapeutic

Furin and other PCs have been shown to be involved in the maturation of both TACE and MMP within skin cells. ProTACE is processed by both furin and PC7 to its mature form thereby increasing its proteolytic activity [83, 85]. The maturation of TACE occurs as it transits through

Bacterial toxins Anthrax toxin [122]

Coagulation factor IX [108]

The Role of Furin in the Development of Skin Cancer

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

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Vascular endothelial growth factor (VEGF) [111] β-Nerve growth factor [112]

Insulin growth factor 1 receptor [117]

Integrin α-chain subunits [119]

Papillomavirus minor capsid protein L2 [121]

*Clostridium septicum* alpha-toxin [103, 123]

TACE [77, 99, 115]

and viral infection [23, 101, 103-106].

**Table 2.** Some biological molecules cleaved by furin

potential in the treatment of a variety of diseases.
