AEONES O SEPHIROTHS, EL ARBOL DE LA VIDA

Photoaging is a major contributor to skin ageing (Friedman, 2005), and its effects are more prominent in fair skin individuals compared to people with darker skin, since melanin acts as a natural protective barrier against UV radiation. In fact, darker skin individuals contain a higher proportion of the dark pigment eumelanin, which is much more efficient in blocking UV than the lighter pigment, pheomelanin. Conversely, fairer skin is much more susceptible to the damaging effects of UV due to a low amount of epidermal eumelanin (Vincensi et al., 1998). As well as the pigmentation status of the skin, the severity of photoageing also depends on the cumulative dose of UV exposure experienced during life (Kammeyer and Luiten, 2015). Solar UV radiation is divided into three categories according to their wavelength, which include UVA (320-400 nm), UVB (290-320nm) and UVC (200-290 nm). Sunlight is primarily composed of UVA (90-95%) and UVB (5-10%), whereas UVC is completely filtered by the atmosphere, and does not actually reach the earth (Amaro-Ortiz et al., 2014). UVA plays a significant role in photoageing since it penetrates through the epidermis and into the dermis, where it can damage ECM components. On the other hand, UVB does not penetrate deeply into the skin, with the majority of the energy being absorbed in the epidermis, and thus it is largely responsible for the development of sunburn (D’Orazio et al., 2013). Nevertheless, both UVA and UVB can cause damage to epidermal and dermal components and contribute to photoageing.

Although the majority of UVB is absorbed in the stratum corneum, some also reaches viable epidermal cells, where it causes DNA damage through the formation of cyclobutane-pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts ((6-4)-PP) (Goodsell, 2001). UV radiation also leads to ROS generation, which in turn can damage DNA, giving rise to oxidative DNA lesions such as 8-hydroxy-2’-deoxyguanine (8-OHdG) (Meyskens et al., 2001; Kunisada et al., 2005). If this damage is not correctly repaired by the nuclear excision repair (NER) or base excision repair (BER) mechanism, keratinocytes can arrest in the cell cycle and become senescent (Lewis et al., 2008). Accumulation of damage can also lead to keratinocyte apoptosis (Qin et al., 2002), whereas other mutations, such as those in the p53 gene, can lead to malignant transformation and tumour initiation (Ziegler et al., 1994). Moreover, UVB is absorbed by aromatic amino acids in epidermal proteins, resulting in protein modifications which can be affect its function. Modified proteins can also form aggregates which are detrimental for cellular function if not efficiently degraded by the ubiquitin-proteasome system (Pattison and Davies, 2006).

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Studies have shown that ROS produced in response to UVA irradiation stimulates the synthesis of MMPs via the activation of MAPKs in the epidermis and upper dermis (Klotz et al., 1999; Maziere et al., 2001). Briefly, activated MAPKs phosphorylate the transcription factor c-Jun, which is then translocated into the nucleus where it interacts with c-Fos, and increases the expression of the transcription factor AP-1 (Rittie and Fisher, 2002). AP-1 stimulates the transcription of different ECM-degrading enzymes such as MMP-1, -3, and -9, which together have the ability to completely degrade fibrillary collagen and elastin in the skin (Fisher et al., 1996; Benbow and Brinckerhoff, 1997). Indeed, UV irradiation of human skin in vivo has been shown to induce expression of MMP-1, -3, and -9 with a concomitant increase in collagen degradation (Fisher et al., 1996; Fisher et al., 1997). Moreover, repeated UV exposure has been shown to trigger deposition of abnormal elastin in the dermis (Lavker et al., 1995). AP-1 also represses expression of collagen precursors (procollagen I and III), thus significantly impairing collagen synthesis (Chung et al., 1996). Imbalance in collagen homeostasis by UV exposure is also mediated through downregulation of the TGF-β pathway, which plays a role in stimulating production of procollagen I and III, and in reducing transcription on MMP-1 (also known as collagenase) in skin fibroblasts (Massague, 1998). It has been shown that UV irradiation downregulates expression of TGF-β type II receptor in human skin in vivo (Quan et al., 2004), induces expression of Smad-7, an inhibitor of TGF-β signalling (Quan et al., 2005), and also reduces the levels of connective tissue growth factor (CCN2), which mediates the stimulatory effects of TGF-β on procollagen I synthesis (Quan et al., 2002). This results in diminished TGF- β signalling, and impaired collagen homeostasis. Furthermore, it has been reported that UV exposure induces the expression of pro-inflammatory cytokines such as IL-1β in fibroblasts and keratinocytes in vitro, resulting in increased expression of MMP-1, which is one of the enzymes responsible for collagen degradation (Wlaschek et al., 1994; Wan et al., 2001). Another study proposed that UVB-induced wrinkling was mediated by keratinocyte IL-1α secretion, which can then diffuse into the dermis and stimulate the expression of elastase in skin fibroblasts. Increased elastase activity then leads to deterioration of elastic fibres in the dermis and contributes to wrinkle formation (Imokawa, 2009). Overall, UV irradiation activates intracellular signalling pathways that favour collagen and elastin degradation over production, contributing to the ECM deficit that is characteristic of aged skin.

UV irradiation has also been shown to cause mitochondrial dysfunction, likely through damaging mitochondrial DNA (mtDNA) as a result of increased in ROS generation. Indeed, mtDNA is highly susceptible to oxidative stress, and accumulates more mutations than genomic

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DNA due to a lack in repair mechanisms and histones (Yakes and Van Houten, 1997). Indeed, large-scale deletions in the mitochondrial genome have been associated with UV-induced photoageing of human skin (Berneburg et al., 1997; Birch-Machin et al., 1998). A study identified that a 4,977 base pair deletion in mtDNA, also known as “common deletion”, was more frequently found in sun-exposed skin compared with sun-protected sites of the same person, suggesting a role for mtDNA mutations in skin photoageing (Berneburg et al., 1997). Moreover, repeated exposure to sublethal doses of UVA irradiation induces these common deletions in the dermis of human skin in vivo and in skin fibroblasts in vitro, and causes mitochondrial dysfunction (Berneburg et al., 2005). Damage in mtDNA can also impair the oxidative phosphorylation process, which increases ROS generation, and in turn leads to further mutations in the mitochondrial genome (Wallace et al., 1998). In addition, inducing mitochondrial dysfunction by deletion of the mitochondrial antioxidant enzyme, Sod2, has been shown to trigger cellular senescence and lead to skin ageing phenotypes in mice in vivo (Velarde et al., 2012). Therefore, mitochondrial dysfunction that arises as a result of UV exposure might also play a role in human skin ageing.

Exposure of ex vivo human skin to suberythemal doses of UV radiation (i.e. doses that are not sufficient to provoke an erythemic response/reddening) has been shown to induce accumulation of p16 in basal epidermal cells (Pavey et al., 1999). Interestingly, p16 expression peaked at 24 hours post-irradiation, and reduced at 72 hours, with p16-positive cells moving from the innermost basal and suprabasal layers to the outermost spinous and granular layers with time, reflecting the turnover of keratinocytes in the epidermis (Pavey et al., 1999). Additionally, it was later demonstrated that this increase in p16 expression corresponded to a G2 cell-cycle arrest of keratinocytes and melanocytes (Pavey et al., 2001). As well as facilitating DNA damage repair, a G2 arrest was also suggested to contribute to melanin synthesis and increased melanisation of keratinocytes, which protects against further UV-induced damage (Pavey et al., 2001). Elevated p53 expression has also been demonstrated following UV radiation of human skin in vivo at doses that induce moderate erythema (Campbell et al., 1993). Therefore, given the damaging effects of UV radiation, it is likely that chronic UV exposure might contribute to senescent cell accumulation in the skin. Indeed, UVB radiation induces senescence in skin fibroblasts (Debacq-Chainiaux et al., 2005) and keratinocytes in vitro (Lewis et al., 2008), suggesting that senescence might occur as a protective response to ensure that UV-damaged cells harbouring extensive DNA mutations cannot replicate.

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