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UVA/B induced apoptosis in human melanocytes involves translocation of cathepsins and Bcl-2 family members
Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology . Linköping University, Faculty of Health Sciences.
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2006 (English)In: Journal of Investigative Dermatology, ISSN 0022-202X, Vol. 126, no 5, 1119-1127 p.Article in journal (Refereed) Published
Abstract [en]

We demonstrate UVA/B to induce apoptosis in human melanocytes through the mitochondrial pathway, displaying cytochrome c release, caspase-3 activation, and fragmentation of nuclei. The outcome of a death signal depends on the balance between positive and negative apoptotic regulators, such as members of the Bcl-2 protein family. Apoptotic melanocytes, containing fragmented nucleus, show translocation of the proapoptotic proteins Bax and Bid from the cytosol to punctate mitochondrial-like structures. Bcl-2, generally thought to be attached only to membranes, was in melanocytes localized in the cytosol as well. In the fraction of surviving melanocytes, that is, cells with morphologically unchanged nucleus, the antiapoptotic proteins Bcl-2 and Bcl-XL were translocated to mitochondria following UVA/B. The lysosomal proteases, cathepsin B and D, which may act as proapoptotic mediators, were released from lysosomes to the cytosol after UVA/B exposure. Proapoptotic action of the cytosolic cathepsins was confirmed by microinjection of cathepsin B, which induced nuclear fragmentation. Bax translocation and apoptosis were markedly reduced in melanocytes after pretreatment with either cysteine or aspartic cathepsin inhibitors. No initial caspase-8 activity was detected, excluding involvement of the death receptor pathway. Altogether, our results emphasize translocation of Bcl-2 family proteins to have central regulatory functions of UV-induced apoptosis in melanocytes and suggest cathepsins to be proapoptotic mediators operating upstream of Bax.

Place, publisher, year, edition, pages
2006. Vol. 126, no 5, 1119-1127 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-14400DOI: 10.1038/sj.jid.5700124OAI: oai:DiVA.org:liu-14400DiVA: diva2:23438
Available from: 2008-11-13 Created: 2008-11-13 Last updated: 2017-08-30
In thesis
1. Regulation of UV induced apoptosis in human melanocytes
Open this publication in new window or tab >>Regulation of UV induced apoptosis in human melanocytes
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Malignant melanoma arises from the pigment producing melanocytes in epidermis and is the most aggressive type of skin cancer. The incidence of malignant melanoma is increasing faster than any other type of cancer in white population worldwide, with a doubling rate every 10-20 years. So far, the only identified external risk factor for malignant melanoma is UV exposure. Elimination of photodamaged cells by apoptosis (programmed cell death) is essential to prevent tumor formation. Melanocytes are considered relatively resistant to apoptosis, however, the regulation of apoptosis in melanocytes is still unknown.

The aim of this thesis was to investigate the apoptotic process following ultraviolet (UV) irradiation in primary cultures of human melanocytes. Focus was on regulation of mitochondrial stability by Bcl-2 family proteins and the possible participation of lysosomal proteases, cathepsins. UV irradiation activated the mitochondrial pathway of apoptosis, leading to cytochrome c release, caspase activation, and nuclear fragmentation. No change in protein expression of Bax and Bcl-2 was observed in response to UV. Instead, translocation of the Bcl-2 family proteins from cytosol to mitochondia was important in the regulation of survival and death of melanocytes. The findings further demonstrated permeabilization of the lysosomal membrane to occur early in the apoptotic process, resulting in cathepsin release into the cytosol. The cathepsins were potent pro-apoptotic mediators and triggered apoptosis upstream of Bax translocation and mitochondrial membrane permeabilization. In response to both heat and UV irradiation, there was a marked increase in expression of stress-induced heat shock protein 70 (Hsp70), which inhibited apoptosis by binding lysosomal and mitochondrial membranes and counteracting the release of cathepsins and cytochrome c. Furthermore, UV irradiation activated c-jun N-terminal kinase (JNK), which triggered apoptosis upstream of cathepsins release from the lysosomes. In addition, JNK mediated apoptosis through phosphorylation of pro-apoptotic Bim, which was released from anti-apoptotic Mcl-1, by UV induced Mcl-1 depletion.

This thesis illustrates that permeabilization of mitochondria and lysosomes and release of their constituents to the cytosol participates in UV induced apoptosis signaling in human melanocytes in vitro. The process is regulated by a complex network of pro- and anti-apoptotic proteins, exerting their effects through intracellular translocation and alteration of protein expression.

Place, publisher, year, edition, pages
Institutionen för biomedicin och kirurgi, 2007
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 997
Keyword
apoptosis, UV, melanocyte, lysosome, cathepsin, Bcl-2, Bax, Hsp70, JNK
National Category
Dermatology and Venereal Diseases
Identifiers
urn:nbn:se:liu:diva-8749 (URN)978-91-85831-97-5 (ISBN)
Public defence
2007-05-25, Berzeliussalen, Campus US, Linköpings Universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2007-05-14 Created: 2007-05-14 Last updated: 2017-08-30
2. UVA/B induced redox alterations and apoptosis in human melanocytes
Open this publication in new window or tab >>UVA/B induced redox alterations and apoptosis in human melanocytes
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Malignant melanoma is one of the most rapidly increasing cancers and accounts for about three-quarter of all skin cancer deaths worldwide. Despite compelling evidence that ultraviolet (UV) irradiation causes melanoma the knowledge how various wavelength spectra affect the balance between proliferation and apoptosis controlling the homeostasis of the melanocyte population is still limited. The aim of this thesis was to elucidate the regulation of UVA/B induced apoptotic signaling in human epidermal melanocytes in vitro in relation to redox alterations and antioxidant photoprotection.

UVA irradiation induced changes in plasma membrane stability, decreased cell proliferation and increased apoptosis. In comparison, melanocyte plasma membrane was markedly resistant to UVB irradiation although apoptosis was triggered. Thus, UVA irradiation should not be overlooked as an etiologic factor in melanoma development. Further, after irradiation with UVA/B we found alterations in redox state manifested by a reduction of intracellular GSH levels, translocation of nuclear factor-κB from the cytosol to the nucleus, an increase of γ-glutamylcysteine synthetase, the rate-limiting enzyme in GSH synthesis, and an increased apoptosis frequency. α-Tocopherol provided photoprotection through several modes of action affecting redox alterations and signaling, stabilizing the plasma membrane, and decreased proliferation and apoptosis rate, while β-carotene did not show the same protective capacity. Altogether, α-tocopherol might be a useful substance in protecting melanocytes from UV induced damage.

We demonstrate UVA/B irradiation to activate the intrinsic pathway of apoptosis in melanocytes where translocation of Bcl-2 family proteins to the mitochondria modulates the apoptosis signal. Interestingly, the anti-apoptotic Bcl-2 family proteins generally thought to be attached to membranes, were localized in the cytosol before UV irradiation and translocated to the mitochondria in the surviving population, which might be a critical event in preventing apoptotic cell death. Lysosomal cathepsins were released to the cytosol acting as pro-apoptotic mediators upstream of activation and translocation of Bax to the mitochondria. When melanocytes were exposed to UVA, p53 participated in apoptosis regulation through interaction with Bcl-2 family proteins, while UVB induced p53-transcriptional activity and apoptosis involving lysosomal membrane permeabilization. Thus, depending on the UV wavelength p53 mediated apoptosis in melanocytes by transcriptional dependent or independent activity. These results emphasize p53 as an important pro-apoptotic component in the regulation of apoptosis.

This thesis gives new insight in the harmful and various effects of different wavelengths within the UV spectrum on human melanocytes in vitro. Improved knowledge of the apoptosis regulatory systems in melanocytes might lead to a better understanding of the formation of pigment nevi and malignant melanoma and, in the future, provide better strategies to prevent and eliminate tumor development and progression.

Place, publisher, year, edition, pages
Institutionen för biomedicin och kirurgi, 2007. 58 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1004
Keyword
UV, melanocyte, apoptosis, glutathione, p53
National Category
Dermatology and Venereal Diseases
Identifiers
urn:nbn:se:liu:diva-8880 (URN)978-91-85831-84-5 (ISBN)
Public defence
2007-06-05, Berzeliussalen, Campus US, Ingång 65, Hälsouniversitetet, Linköpings universitet, SE-581 85 Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2007-05-21 Created: 2007-05-21 Last updated: 2017-08-30

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Bivik, CeciliaWäster, PetraKågedal, KatarinaRosdahl, IngerÖllinger, Karin

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