**5. Furin**

Furin, also known as PACE, is a 94 kDa, type I transmembrane, Ca2+-dependant serine pro‐ tease. It is a member of the proprotein convertase (PC) family which is related to the bacterial subtisilin enzyme [23, 97-99]. The PC family consists of seven distinct members (furin and PC1- PC7) that vary in regards to their tissue and subcellular distribution as well as enzymatic and biochemical properties [23, 24, 97, 100]. Furin, PACE 4, PC5/6 and PC7/8 are widely expressed in the epidermis whereas PC2 and PC1/3 are limited to neuroendocrine tissues and PC4 is restricted to the testis [23, 24, 98]. The PC enzymes recognize basic motifs, cleaving after paired basic residues (PC2 and PC1/3); or after a canonical Rx (R/K) R (Arg-x-(Arg/Lys)-Arg) motif (furin and PACE4) [24, 97, 98, 100-102]. Both PC7 and furin share cleave similar substrates and theselectivityofwhichdependsontheircellularlocalization.Astheircytosolicdomainsregulate intracellulartraffickingitis likelythatthe cellularlocalizationofPC7differs tothatforfurin[85].

Structurally furin and other PCs consist of a signal peptide followed by pro, catalytic, middle, and cytoplasmic domains, respectively [24]. The signal peptide directs the translocation of the peptide chain to the endoplasmic reticulum and the secretory pathway [82, 97, 103]. The proregion is cleaved in the endoplasmic reticulum, where it then associates with the catalytic domain and helps to guide the protein through this region to the Golgi apparatus where it becomes catalytically active [97, 103]. The trans-membrane region anchors the enzyme in the membrane of the trans Golgi network (TGN) or on the cell membrane. The cytosolic tail contains the information necessary for furin's sorting to various intracellular compartments [82, 97, 103]. In the epidermis, furin can exist either as: (a) a mature 97 kDa membrane bound enzyme or (b) a smaller 75 kDa form that lacks the transmembrane domain [97]. This suggests that post-translational cleavage at the C-terminus occurs within in the cell [97, 98, 103]. Furin 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 and viral infection [23, 101, 103-106].


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

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

Furin, also known as PACE, is a 94 kDa, type I transmembrane, Ca2+-dependant serine pro‐ tease. It is a member of the proprotein convertase (PC) family which is related to the bacterial subtisilin enzyme [23, 97-99]. The PC family consists of seven distinct members (furin and PC1- PC7) that vary in regards to their tissue and subcellular distribution as well as enzymatic and biochemical properties [23, 24, 97, 100]. Furin, PACE 4, PC5/6 and PC7/8 are widely expressed in the epidermis whereas PC2 and PC1/3 are limited to neuroendocrine tissues and PC4 is restricted to the testis [23, 24, 98]. The PC enzymes recognize basic motifs, cleaving after paired basic residues (PC2 and PC1/3); or after a canonical Rx (R/K) R (Arg-x-(Arg/Lys)-Arg) motif (furin and PACE4) [24, 97, 98, 100-102]. Both PC7 and furin share cleave similar substrates and theselectivityofwhichdependsontheircellularlocalization.Astheircytosolicdomainsregulate intracellulartraffickingitis likelythatthe cellularlocalizationofPC7differs tothatforfurin[85]. Structurally furin and other PCs consist of a signal peptide followed by pro, catalytic, middle, and cytoplasmic domains, respectively [24]. The signal peptide directs the translocation of the peptide chain to the endoplasmic reticulum and the secretory pathway [82, 97, 103]. The proregion is cleaved in the endoplasmic reticulum, where it then associates with the catalytic domain and helps to guide the protein through this region to the Golgi apparatus where it becomes catalytically active [97, 103]. The trans-membrane region anchors the enzyme in the membrane of the trans Golgi network (TGN) or on the cell membrane. The cytosolic tail contains the information necessary for furin's sorting to various intracellular compartments [82, 97, 103]. In the epidermis, furin can exist either as: (a) a mature 97 kDa membrane bound enzyme or (b) a smaller 75 kDa form that lacks the transmembrane domain [97]. This suggests that post-translational cleavage at the C-terminus occurs within in the cell [97, 98, 103]. Furin

may indirectly play a role in this process.

**5. Furin**

278 Highlights in Skin Cancer

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 potential in the treatment of a variety of diseases.

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 the Golgi compartment where the prodomain was removed by a furin-type proprotein convertase [77, 84, 85]. As increased amounts of mature TACE are detected in furin overexpressing cells, it appears that proTACE is a better substrate for furin than it is for PC7 [85]. A similar observation has been seen in cells overexpressing TACE [58, 83, 99] where furin was shown to be responsible for its cleavage [83, 99]. This finding was confirmed using cell permeable furin inhibitors CMK and PDX in Cos7 cells [83] and keratinocytes [98] where reduced levels of mature TACE were formed.

Furin mRNA, protein and enzyme activity has been observed in human epidermal keratino‐ cytes [29, 98, 111, 128, 129]. Skiba *et al*. [29] found that UVA and UVB radiation immediately increased furin mRNA levels in HaCaT cells. UVB irradiation induced higher levels of furin mRNA expression [29]. The time course for furin mRNA levels in cells irradiated with low dose of UVA or high dose of UVB was similar to that for TNFα, whereas maximal mRNA induction of both genes were detected 8 h post-irradiation [29]. Although UV irradiation does appear to have an effect on furin gene expression, no direct relationship was apparent between TACE and furin mRNA induction. A recent study has shown that following exposure to UVA and UVB, furin levels in HaCaT cells fell with respect to time [49, 129]. However, it was unknown whether this was due to the loss of the pro or mature form of the enzyme. Through its effect on stimulating MMPs, as well as activating TACE and the resultant effect this has on TNFα released by the cell, furin activity has an influence on the inflammation seen in the skin following exposure to UV radiation as seen in Figure 2.

Furin/PC processing of substrates has been shown to also contribute to tumour progression, aggressiveness, metastasis, and angiogenesis [23, 24, 104-106]. Tumour invasion and metasta‐ sis represent a multistep process that depends on the activity of many proteins [46, 101, 104, 130]. Proteolytic degradation of the ECM components is a central event of this process. Several classes of proteases, including MMPs, serine proteases and cysteine proteases have been implicated in the tumour cell invasive process [104, 130, 131]. Of these, MMPs appear to be primarily responsible for much of the ECM degradation observed during invasive processes [111, 130, 132-134]. They can contribute to tumour growth not only by degradation of the ECM but by the release of sequestered growth factors or the generation of bioactive fragments VEGF, bFGF or TGFβ, the suppression of tumour cell apoptosis and the destruction of immunemodulating chemokine gradients [131, 132, 135]. Furin also cleaves a number of MMPs from their proform, and activating them as a result [23, 86, 102, 105].

MMPs are also implicated in cell migration, proliferation, and tissue remodelling and thereby may also play a role in growth and development, angiogenesis, and atherosclerosis [138, 139].

**Figure 2.** The role furin plays in the maturation of TACE and MMPs in skin cells. Furin cleaves and activates TACE, which in turn can process TNFα from its proform. Keratinocytes secrete TNFα following exposure to UVB radiation, and this is enhanced if IL-1α is present. Furin also cleaves MMPs from their respective proforms, and the expression and activity of these proteases are elevated when the cells have been exposed to UVB radiation, and they are enhanced if either IL-1α (MMP-9) or TNFα (MMP-2) is present. The effect of UVB radiation on the expression of the enzymes and pTNFα in the cell is represented by dashed lines, if it is enhanced it is represented by (+), and if it is unknown (?)

The Role of Furin in the Development of Skin Cancer

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

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Structurally MMPs consist of a signal peptide followed by pro, catalytic, hemopexin and cytoplasmic domains, respectively [130]. MMPs cleave peptides and proteins, which have a myriad of functions that are independent of their proteolytic activity [140]. They have distinct but often overlapping substrate specificities, hence leading to the absence of distinct pheno‐

MMPs are generally expressed in very low amounts and their transcription is tightly regulated either positively or negatively by cytokines and growth factors such as IL-1, IL-4, IL-6, TGFβ, or TNFα [130, 135, 141, 142]. Some of these regulatory molecules can be proteolytically activated or inactivated by MMPs (via a feedback loop). MMPs are synthesized as latent proenzymes, which are converted into mature, catalytically active forms in the TGN by PCs [111, 139]. Activation of MMPs following secretion from the cell depends on disruption of the prodomain interaction with the catalytic site, which may occur either by conformational changes or proteolytic removal of the prodomain. With the exception of MMP-2, the mecha‐

types in most genetically-engineered mice with knockdown of specific MMPs [140].

nism for *in vivo* activation of secreted MMPs is not well understood [135].
