SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes.

BACKGROUND: Exposure to heat stress after UVB irradiation induces a reduction of apoptosis, resulting in survival of DNA damaged human keratinocytes. This heat-mediated evasion of apoptosis appears to be mediated by activation of SIRT1 and inactivation of p53 signalling. In this study, we assessed the role of SIRT1 in the inactivation of p53 signalling and impairment of DNA damage response in UVB plus heat exposed keratinocytes. RESULTS: Activation of SIRT1 after multiple UVB plus heat exposures resulted in increased p53 deacetylation at K382, which is known to affect its binding to specific target genes. Accordingly, we noted decreased apoptosis and down regulation of the p53 targeted pro-apoptotic gene BAX and the DNA repair genes ERCC1 and XPC after UVB plus heat treatments. In addition, UVB plus heat induced increased expression of the cell survival gene Survivin and the proliferation marker Ki67. Notably, keratinocytes exposed to UVB plus heat in the presence of the SIRT1 inhibitor, Ex-527, showed a similar phenotype to those exposed to UV alone; i.e. an increase in p53 acetylation, increased apoptosis and low levels of Survivin. CONCLUSION: This study demonstrate that heat-induced SIRT1 activation mediates survival of DNA damaged keratinocytes through deacetylation of p53 after exposure to UVB plus heat.

Heat-mediated reduction of apoptosis in UVB-damaged keratinocytes in vitro and in human skin ex vivo.

BACKGROUND: UV radiation induces significant DNA damage in keratinocytes and is a known risk factor for skin carcinogenesis. However, it has been reported previously that repeated and simultaneous exposure to UV and heat stress increases the rate of cutaneous tumour formation in mice. Since constant exposure to high temperatures and UV are often experienced in the environment, the effects of exposure to UV and heat needs to be clearly addressed in human epidermal cells. METHODS: In this study, we determined the effects of repeated UVB exposure 1 kJ/m(2) followed by heat (39 °C) to human keratinocytes. Normal human ex vivo skin models and primary keratinocytes (NHEK) were exposed once a day to UVB and/or heat stress for four consecutive days. Cells were then assessed for changes in proliferation, apoptosis and gene expression at 2 days post-exposure, to determine the cumulative and persistent effects of UV and/or heat in skin keratinocytes. RESULTS: Using ex vivo skin models and primary keratinocytes in vitro, we showed that UVB plus heat treated keratinocytes exhibit persistent DNA damage, as observed with UVB alone. However, we found that apoptosis was significantly reduced in UVB plus heat treated samples. Immunohistochemical and whole genome transcription analysis showed that multiple UVB plus heat exposures induced inactivation of the p53-mediated stress response. Furthermore, we demonstrated that repeated exposure to UV plus heat induced SIRT1 expression and a decrease in acetylated p53 in keratinocytes, which is consistent with the significant downregulation of p53-regulated pro-apoptotic and DNA damage repair genes in these cells. CONCLUSION: Our results suggest that UVB-induced p53-mediated cell cycle arrest and apoptosis are reduced in the presence of heat stress, leading to increased survival of DNA damaged cells. Thus, exposure to UVB and heat stress may act synergistically to allow survival of damaged cells, which could have implications for initiation skin carcinogenesis.

The elastin peptide (VGVAPG)3 induces the 3D reorganisation of PML-NBs and SC35 speckles architecture, and accelerates proliferation of fibroblasts and melanoma cells.

During melanoma tumour growth, cancerous cells are exposed to the immediate surrounding the micro- and macro environment, which is largely modified through the degradation of the extracellular matrix by fibroblast-derived metalloproteinases. Among the degradation products, (VGVAPG)3, an elastin peptide is known to stimulate the proliferation of both fibroblasts and cancerous cells by binding to the elastin-binding receptor and activating the MEK/ERK signal transduction pathway. As this process strongly modifies mRNA synthesis, we investigated its effect on the relative three-dimensional organisation of the major partners of the mRNA splicing machinery: promyelocytic nuclear bodies (PML-NBs ) and splicing component 35 speckles (SC35) of normal fibroblasts and melanoma SK-MEL-28 cells. SC35 and PML-NBs proteins were immunolabeled and imaged by confocal microscopy within these cells cultured with (VGVAPG)3. Three-dimensional reconstruction was performed to elucidate the organisation of PML-NBs and SC35 speckles and their spatial relationship. In G0 cells, SC35 speckles were sequestered in PML-NBs. Shortly after (VGVAPG)3 stimulation, the three-dimensional organisation of PML-NBs and SC35 speckles changed markedly. In particular, SC35 speckles gradually enlarged and adopted a heterogeneous organisation, intermingled with PML-NBs. Conversely, inhibition of the elastin-binding protein or MEK/ERK pathway induced a remarkable early sequestration of condensed SC35 speckles in PML-NBs, the hallmark of splicing inhibition. The 3D architecture of speckles/PML-NBs highlights the modulation in their spatial relationship, the multiple roles of PML-NBs in activation, inhibition and sequestration, and provides the first demonstration of the dependence of PML-NBs and SC35 speckles on the elastin peptide for these functions.

Calcipotriol counteracts betamethasone-induced decrease in extracellular matrix components related to skin atrophy.

The calcipotriol/betamethasone dipropionate fixed-combination gel is widely used for topical treatment of psoriasis vulgaris. It has been hypothesized that calcipotriol counteracts glucocorticoid-induced skin atrophy which is associated with changes in the extracellular matrix (ECM). To elucidate the combined effects of calcipotriol and betamethasone on key ECM components, a comparative study to the respective mono-treatments was carried out. The effect on collagen I synthesis, matrix metalloproteinase (MMP) secretion, and hyaluronic acid (HA) production was investigated in primary human fibroblast and keratinocyte cultures as well as in a human skin explant model. We show that calcipotriol counteracts betamethasone-induced suppression of collagen I synthesis. Similarly, calcipotriol and betamethasone have opposing effects on MMP expression in both fibroblasts and keratinocytes. Moreover, calcipotriol is able to restore betamethasone-impaired HA synthesis in keratinocytes and prevent betamethasone-induced epidermal thinning in minipigs upon treatment with the calcipotriol/betamethasone gel. In summary, our results show for the first time in primary human skin cultures that calcipotriol reduces early signs of betamethasone-induced skin atrophy by modulation of key ECM components. These results indicate that the calcipotriol component of the fixed-combination gel counteracts the atrophogenic effects of betamethasone on the skin.

Ultraviolet-B irradiation induces epidermal up-regulation of heparanase expression and activity.

Heparan sulfate (HS) glycosaminoglycans are abundant components of basement membranes and cell surfaces where they are present associated with specific core-proteins to form proteoglycans, mainly perlecan, glypicans and syndecans. They play many roles such as modulation of cell proliferation and differentiation, cell-matrix adhesion and assembly. It was previously shown that HS content decreases during skin aging. This decrease could be explained either by a decrease of HS synthesis or by an increased activity of its degrading enzyme, heparanase (Hpse-1). Since UV-B irradiation is one of the most important factor for skin photo-damage, we decided to study the effects of UV-B irradiation on heparanase expression and activity in human epidermal keratinocytes. Normal human keratinocytes and reconstructed epidermis were submitted to increasing doses of UV-B. HPSE1 mRNA levels were measured using real time PCR and heparanase enzymatic activity was quantified in human keratinocyte cultures using a microtiter-based assay. Expression and distribution of Hpse-1 were also studied in reconstructed epidermis by immunofluorescence. Both HPSE1 mRNA level and heparanase enzymatic activity were increased after UV-B irradiation of keratinocyte cultures in a time and dose-dependent manner. Protein expression of Hpse-1 was also up-regulated with increasing doses of UV-B in reconstructed epidermis. Increase of Hpse-1 expression and activity in the epidermis after UV-B irradiation could contribute to skin photo-aging.

Ultraviolet-B irradiation induces differential regulations of hyaluronidase expression and activity in normal human keratinocytes.

Skin aging is a complex process determined by genetic factors (intrinsic aging) and environmental factors (extrinsic aging). One of the most influential environmental factor is UV-B irradiation. Hyaluronic acid (HA) is an abundant component of skin extracellular matrix where it plays many roles such as hydration and architectural support. Downregulation of HA during photoaging was reported previously. Changes in expression and function of its degrading enzymes, the hyaluronidases (Hyals) might be involved in this decrease. In the present study, normal human keratinocytes were submitted to increasing doses of UV-B. The mRNA expression of HYAL1, HYAL2 and HYAL3 and the hyaluronidase enzymatic activity were quantified using real-time PCR and a microtiter-based assay, respectively. After UV-B irradiation, HYAL1 mRNA expression was upregulated whereas HYAL2 and HYAL3 mRNAs were downregulated and hyaluronidase enzymatic activity was increased in both cell layer and culture medium. In parallel, immunohistochemical studies performed on UV-B irradiated reconstructed epidermis confirmed that Hyal-1, Hyal-2 and Hyal-3 protein expression were differently regulated by UV-B. Taken together, our results demonstrate that UV-B irradiation induces differential regulations of hyaluronidase expression and enzymatic activity in human keratinocytes. These differential modulations of hyaluronidase expression and activity by UV-B could contribute to cutaneous photoaging.