3. CELL DEATH IN SKIN PATHOLOGY

3.1. Sunburn

Although CE formation does not occur through classical apoptosis, all components required for apoptosis are present in keratinocytes. One of the major environmen-

tal insults to which our skin is exposed is ultraviolet radiation (UVR), a potent agent that causes DNA damage and apoptotic sunburn cells. UV-induced apoptosis is a complex cellular process that involves many players, which are discussed next (Figure 28-3).

UVB (290–320 nm) can be directly absorbed by DNA, resulting in the formation of photo lesions such as

cyclobutane-pyrimidine dimers (CPDs) and pyrimidine (6–4) pyrimidone. UVA radiation (>320 nm) penetrates deeply into the skin, and the genotoxic effects are mainly mediated by accumulation of reactive oxygen species (ROS) generated by activation of photosensitizers (riboflavin, porphyrins, quinones). ROS production is also excessive in UVB-irradiated skin and contributes to apoptosis, as witnessed by the protective effect of antioxidants. Most of the generated photoproducts are removed by the cellular DNA excision repair. If not, permanent mutations can lead to cancer development. Upon excessive DNA damage, the cell will undergo apoptosis, resulting in the formation of so-called sunburn cells, characterized by the presence of photo lesions, pyknotic nuclei, and eosinophilic cytoplasm.

An important player in UVB-induced apoptosis is the transcription factor p53. p53 is involved in a plethora of cellular functions, such as cell cycle arrest and activation of apoptosis, and is generally considered a tumor suppressor. By activating apoptosis, p53 protects the organism against accumulation of damaged cells that may develop into cancerous cells. The choice between cellular responses depends on the type of cell and stress and action of p53 co-activators, such as p300, CREBbinding protein (CBP), and P300/CBP-associated factor (PCAF). As mentioned previously, UVB can induce severe DNA damage. Upon DNA damage, MDM2, a p53-binding ubiquitinating enzyme that targets p53 for degradation, is inactivated, and as a consequence, p53 becomes stabilized. The important role of p53 in UVinduced apoptosis is demonstrated by the significant reduction in sunburn cells in p53 knockout mice after UVB irradiation. p53 is able to induce apoptosis through transcription-dependent or -independent mechanisms and can occur through the extrinsic and intrinsic mitochondrial apoptotic pathway. Cytoplasmic p53 can bind directly to several antiapoptotic members of the Bcl- 2 superfamily, such as Bcl-2 and Bcl-XL, thereby neutralizing their antiapoptotic activity and acting itself as a proapoptotic protein. Nuclear p53 can transactivate proapoptotic Bcl-2 family members, such as Bax, p53upregulated modulator of apoptosis (PUMA), Bik/Nbk, and Noxa. In Noxa-deficient animals, UV-induced keratinocyte apoptosis is suppressed, whereas this is not the case in PUMA-deficient mice. This is consistent with the observation that the UV-induced Noxa levels are far higher than the PUMA levels. Mouse embryonal fibroblasts deficient for both caspase-3 and -7 are also resistant to UVB-induced cell death. This may imply the existence of a positive feedback loop between pre- (e.g., Noxa) and postmitochondrial (caspases) signaling molecules required for UVB-induced cell death.

Among the transcriptional targets of p53, there are several genes involved in ROS generation, whereas other p53-dependent genes have an antioxidant function, suggesting a role for p53 in controlling ROS levels in homeostatic and pathogenic conditions. Interestingly, p53 also protects the skin by augmenting skin tanning upon UV irradiation. It was shown that p53 regulates induction of pro-opiomelanocortin–derived bioactive peptides such as α-melanocyte-stimulating hormone, adrenocorticotropic hormone, and β-endorphin, which are key players in melanin production and secretion.

The DNA repair-promoting transcription factor E2F1, which is upregulated after DNA damage, is also involved in UV-induced apoptosis. In several cellular systems, the proapoptotic potential of E2F1 has been demonstrated. However, in vivo data suggest that the function of E2F1 in skin is mainly antiapoptotic. Its in vivo antiapoptotic potential is clear from mice over-expressing E2F1 in the skin. In accordance, E2F1-deficient mice exhibit a higher abundance of sunburn cells upon UVR. E2F1−/– p53−/– double-knockout mice exhibit the elevated UVB-induced apoptosis, which is seen in E2F1 single-knockout animals. This implies that E2F1 can function in a p53-independent way. Chronic exposure to UVR can induce inactivating mutations in the p53 gene and thereby hamper its potency to induce apoptosis, making cells more prone to malignant transformation.

Two other members of the p53 family, p63 and p73, are capable of transactivating p53 target genes and have been implicated in the regulation of apoptosis. Mice deficient for p63 have no epidermis or other squamous epithelia and lack epithelial appendages. There is some debate concerning the mechanism by which p63 exerts its function in skin. It has been thought to mainly play a role in stem cell proliferation; others claim a role in epidermal lineage commitment. Studying p63 in skin differentiation is complicated by the fact that the p63 gene is transcribed into 10 isoforms, either containing (p63TA isoforms) or lacking the transactivation domain ( Np63 isoforms). A mouse model over-expressing Np63α specifically in the epidermis showed a reduction in UVBinduced skin apoptosis, probably as a result of competing with p53-dependent signaling. Interestingly, it has been demonstrated that in human keratinocytes deficient in p53 and p63, there is impaired repair of CPDs. In these cells there was a marked decrease observed in the expression of DDB2 and XPC, which are key DNA damage-recognition proteins. p73-deficient mice show, in contrast to p53-deficient mice, no increased spontaneous tumorigenesis, but exhibit neurologic defects; no skin abnormalities have been described.

Involvement of the intrinsic pathway upon UVBinduced keratinocyte apoptosis has been shown by inhibitor studies targeting caspase-9. In accordance with this, it has been shown that over-expression of Bcl-2 renders keratinocytes resistant to UVB-induced apoptosis. Expression of Apaf-1 is increased upon UV irradiation of keratinocytes. Caspase cleavage of PKCδ is required for UVB-induced apoptosis. The generated PKCδ catalytic fragment translocates to the mitochondria, targets antiapoptotic Mcl-1 for degradation, and triggers redistribution and activation of Bax. Besides apoptotic caspases, UVR can also activate inflammatory caspases, which are activated in inflammasome complexes. UV-irradiated human keratinocytes have been shown to secrete interleukin-1β, a caspase-1 substrate, in an inflammasome-dependent manner. This makes the skin an important component of innate immunity, which is logical, given the fact that keratinocytes are continuously exposed to environmental stressors. When mice deficient for caspase-1 are irradiated with UVB, the number of infiltrated neutrophils is strongly reduced as compared with that of wild-type mice. How UVB is able to induce assembly and activation of the inflammasome is currently unknown. In cultured keratinocytes, the activation of caspase-1 is dependent on an increase in cytoplasmic Ca2+ concentration. Interestingly, UV irradiation of human epidermal keratinocytes results in an increase in intracellular Ca2+ concentration.

The skin of caspase-14 deficient mice is highly sensitive to UVB irradiation, which is characterized by increased UVB-induced CPDs and consequent keratinocyte apoptosis. This is probably due to a reduced UV scavenging effect of the stratum corneum. It is of note that filaggrin degradation, which gives rise to NMFs and urocanic acid, a major absorber of UVB, is impaired in caspase-14 deficient mice. However, the exact mechanism by which caspase-14 protects against UVB-induced apoptosis in the skin remains to be determined. The implication of caspase-14 in UVB protection may be reflected in the fact that caspase-14 has thus far been found only in terrestrial mammals.

DRs are also involved in UV-induced apoptosis, and their triggering can occur in ligand-independent and ligand-dependent ways. Ligand-independent UVBinduced clustering of the DRs Fas and TNF-R1 has been described in human keratinocyte cultures. However, UVB irradiation can also induce upregulation of DRs and its ligands, such as Fas and FasL and TNF. In accordance with this, mice that are deficient for FasL or TNF-R1 show a reduction in the occurrence of sunburn cell formation.

3.2. Skin cancer

An imbalance toward too little apoptosis or too much cell survival in the epidermis can result in skin tumor formation. Epidermal tumors are divided into melanoma and nonmelanoma skin cancers (NMSCs). The majority of NMSCs (80%) are basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs), both arising from cutaneous keratinocytes. Apoptosis is a major cancer defense system in the skin, and generally, triggering or resensitizing of the apoptotic pathway is used in different anticancer treatments. Mutations in the p53 gene, induced by chemical or UVB exposure, are probably an early step during tumorigenesis in BCC and SCC. Mice deficient in p53 are very sensitive to UV-induced SCC formation, and skin-specific transgenes for the p53 regulator MDM2 inhibit UV induction of p53 and are more susceptible to chemical carcinogenesis.

In correlation with their elevated survival status, tumors often have an altered expression of antior proapoptotic proteins. Cancer treatment often aims to restore or invert this imbalance to make use of the apoptotic program to kill the tumor.

For instance, Fas levels are decreased in BCC and in melanomas, probably as a result of oncogenic Ras. Certain melanomas even have inactivating mutations in the death domain of Fas. Increase in Fas expression levels is accomplished by different anticancer drugs and cytokines. In addition, TRAIL-R activated cell death by antibodies or recombinant TRAIL appears promising as a cancer therapy. Interestingly, cancer cell lines, including melanomas, are sensitive to TRAIL killing, whereas nontransformed cells are not, therefore reducing the possible side effects of TRAIL therapy.

Tumors can sometimes escape from apoptosis because of increased levels of antiapoptotic proteins. First, higher levels of FLICE-like inhibitory protein (FLIP) allow melanomas to escape from DR-induced apoptosis by conventional T cells. Second, the inhibitor of apoptosis (IAP) family member survivin is not expressed in normal skin, but it is found in NMSCs. Third, tumor resistance to cytotoxic agents and radiotherapy is associated with increased expression of antiapoptotic Bcl-2 family members such as Bcl-XL and Bcl-2. In certain melanomas, low or absence of sensitivity to TRAIL-induced apoptosis can be overcome by knocking down FLIP, survivin, Bcl-2, or IAPs. One strategy to lower Bcl-2 levels in tumors is the use of antisense oligonucleotides, called oblimersen. However, this approach failed in clinical trials for melanoma (and other cancers) treatment. A promising alternative are

synthetic BH3-only peptides or small organic molecules that interact with the hydrophobic cleft of antiapoptotic Bcl-2 family members and that, as such, are able to induce apoptosis. An alternative therapy making use of IAP inhibitors is currently under development.

Instead of decreased sensitivity to apoptosis, overactivation of survival pathways can also be the basis of tumorigenesis in the skin. Transgenic mice that over-express Akt in basal keratinocytes, which leads both to increased proliferation and decreased apoptosis, develop spontaneous tumors and show increased sensitivity to tissue plasminogen activator (TPA)–induced carcinogenesis. Other survival pathways, such as the MAPK pathway, which controls cell growth and differentiation, are also important in skin tumorigenesis. However, these pathways can result in oncogenic or tumor-suppressor effects, depending on the cellular status. For example, the activator protein-1 transcription factor can promote or suppress skin tumor formation depending on its subunit composition.

As discussed previously (see Section 2, Cell Death in Skin Homeostasis), NF-κB signaling plays a crucial role in skin homeostasis. The role of NF-κB in skin tumorigenesis is diverse; both activation or inactivation can be advantageous for cancer development. The tumor-promoting role of NF-κB is demonstrated in familial cylindromatosis. This disease is caused by loss-of-function mutations in cylindromatosis (CYLD), a deubiquitinase responsible for dampening of NF-κB activation. Hence absence of CYLD will result in overactivation of NF-κB. Similar to human cylindromatosis, mice lacking CYLD are more sensitive to TPA-induced tumor formation, underscoring the importance of NF- κB activation in the development of certain types of skin cancer. In SCC, Ras activity is often increased as a result of mutations in the gene itself that render it constitutively active. Cell cycle arrest induced by oncogenic Ras can be overcome by blocking NF-κB, as shown by IkBα overexpression. So here NF-κB performs tumorsuppressor activity.

3.3. Necrolysis

Toxic epidermal necrolysis (TEN; Lyell’s syndrome) and Stevens-Johnson syndrome (SJS) are rare acute dermatological diseases defined by epidermal necrosis and mucosal erosions, with large areas of loss of contact between epidermis and dermis and massive keratinocyte apoptosis. In SJS and TEN, up to 10% and 30%, respectively, of skin surface can be detached. The main cause is a severe adverse drug reaction, or

in some cases, bacterial infection. The effector cells of the disease are thought to be drug-specific cytotoxic T cells, but the pathophysiologic mechanisms are largely unknown. So far, no specific treatment exists. However, FasL expression is increased in lesional skin keratinocytes of TEN patients. Interestingly, Fassensitive Jurkat cells undergo Fas-dependent apoptosis when incubated on skin cryosections of TEN patients. In addition, the therapeutic potential of intravenous administration of human intravenously collected immunoglobulins from healthy donors, also containing neutralizing anti-Fas antibodies, has been demonstrated in TEN patients. These results suggest an important role of the Fas signaling pathway in the development of TEN. SJS and TEN were long believed to belong to a spectrum of disorders that included erythema multiforme majus (EMM); however, now SJS and TEN are considered to be different diseases than EMM. In EMM, skin eruptions are mainly found on the extremities, whereas in TEN, blistering is widespread. In EMM, mucosal lesions may occur, but are mostly limited to the oral cavity, whereas in TEN, there are severe mucosal erosions. Finally, in TEN the mortality rate is much higher than in EMM.

3.4. Pemphigus

Pemphigus diseases are autoimmune cutaneous blistering disorders characterized by the presence of autoantibodies against structural proteins of the intracellular junctions.

Apoptotic keratinocytes are present in lesional tissue of pemphigus vulgaris (PV) patients. When PV immunoglobulin autoantibodies are added to keratinocytes in vitro, FasL is secreted, reduction of Bcl-2 is observed, and several apoptotic proteins are upregulated. Similar results were obtained after addition of an antibody against Fas Receptor to cultured keratinocytes. These data point to a possible involvement of the extrinsic apoptotic pathway in PV; however, it should be noted that this hypothesis has not been proven in vivo.

3.5. Eczema

The term eczematous dermatitis (eczema) comprises a group of inflammatory skin diseases, such as atopic dermatitis, and that are often characterized by the formation of vesicles associated with exudation. It has been shown that keratinocyte apoptosis is important for vesicle formation. The pathogenesis of eczema is T- cell mediated; secretion of interferon γ by these cells