Long-term growth arrest of PUVA-treated fibroblasts in G2/M in the absence of p16(INK4a) p21(CIP1) or p53

Premature aging of the skin is a prominent side-effect of psoralen photoactivation, a therapy used for different skin disorders. Recently, we demonstrated that treatment of fibroblasts with 8-methoxypsoralen and ultraviolet A irradiation resulted in growth arrest with morphological and functional changes reminiscent of replicative senescence. To further elucidate the underlying molecular mechanisms, we analysed the cell-cycle phases of the growth-arrested fibroblasts. After PUVA treatment, fibroblasts arrested in G2/M, in contrast to spontaneously senesced fibroblasts arresting in a cell-cycle phase with many features similar to G1. To address the role of the cell-cycle controlling genes p16(INK4a), p21(CIP1) and p53, we analysed the expression of these genes. p16(INK4a), p21(CIP1) and p53 protein levels increased substantially with different time kinetics in growth-arrested fibroblasts. Because p16(INK4a), p21(CIP1) and p53 are involved in replicative senescence, we applied the PUVA regimen to fibroblasts deficient in either of these genes. p16(INK4a), p21(CIP1) and p53 null mutant fibroblast strains underwent growth arrest with a senescent phenotype similar to wild-type human fibroblasts. Based on these results, we propose that redundant or alternate pathways are involved in the response of dermal fibroblasts to PUVA treatment resulting in a phenocopy of replicative senescence in vitro.     

Isolation and identification of psoralen plus ultraviolet A (PUVA)-induced genes in human dermal fibroblasts by polymerase chain reaction-based subtractive hybridization

Premature aging of the skin is a prominent side-effect of psoralen photoactivation, a therapy used for a variety of skin disorders. Recently, we demonstrated that treatment of human dermal fibroblasts with 8-methoxypsoralen and ultraviolet A irradiation resulted in a permanent growth arrest with a switch of mitotic to postmitotic fibroblasts. Furthermore, an upregulation of matrix-degrading metalloproteinases and a high level of de novo expression of the senescence-associated beta-galactosidase was detected in the PUVA-treated postmitotic fibroblasts. The molecular basis for this PUVA-induced change in the functional and morphologic phenotype of fibroblasts resembling or mimicking replicative senescence is, however, unknown. Herein after, we have used a polymerase chain reaction-based subtractive hybridization protocol to identify human genes that are induced by PUVA treatment. Application of polymerase chain reaction-Select resulted in the cloning of four PUVA genes. Sequence analysis and homology searches identified three cDNA clones of known genes related to cell cycle regulation (p21waf1/cip1), stress response (ferritin H) and connective tissue metabolism (tissue inhibitor of metalloproteinases-3), whereas one cDNA clone represented a novel gene (no. 478). Northern blot analyses were performed to confirm a PUVA-dependent increase in specific mRNA levels in human dermal fibroblasts in vitro. This report on the identification of growth arrest related genes in PUVA-treated fibroblasts may stimulate further research addressing the causal role of these known and novel genes in extrinsic and intrinsic aging processes on a molecular and cellular level.     

皮肤细胞DNA光损伤后反应机制研究进展

紫外线可引起皮肤细胞DNA损伤,诱发皮肤肿瘤和光老化.DNA损伤类型包括:DNA单链断裂、链间交联、核苷酸碱基修饰等.细胞拥有DNA损伤后反应机制以维持基因组稳定,如细胞周期监控点、DNA修复和细胞早衰等.细胞周期监控系统检测到染色体结构异常时将引起细胞周期停滞并启动修复进程;而在检测到无法修复的损伤时则会诱使细胞衰老.因此皮肤光老化也是一种预防肿瘤发生的防御机制,ATR-Chk1信号通路在其中起着关键的调控作用.

Abstract：

Ultraviolet radiation can induce DNA damage in skin cells, cause skin tumors and accelerate skin photoaging. UV-induced DNA damage in skin cells includes DNA single strand breaks, DNA interstrand cross-links and nucleotide base modifications. Skin cells could exert DNA damage responses, such as cell cycle checkpoints, DNA repair and premature senescence to prevent genomic instability. When cell cycle checkpoint systems sense the abnormal chromosomal DNA structures, they execute cell cycle arrest and DNA repair process will be initiated. Cellular senescence is also executed by checkpoint responses when unrepairble and extensive chromosomal abnormalities are detected. So skin photoaging is thought to be one of the major mechanisms against skin carcinogenesis. ATR-Chk1 signaling pathway plays a key role in the regulation of DNA damage response process.     

Reduced expression of the adhesion protein tensin1 in cultured human dermal fibroblasts affects collagen gel contraction

Abstract:  As revealed by immunohistochemistry and RT-QPCR, the focal adhesion protein tensin1 is expressed in cultured human dermal fibroblasts and reduced by 60% after transfection with tensin1 siRNA. Tensin1 silenced fibroblast exhibited a strongly reduced capacity to contract collagen gels. Aged fibroblasts, generated with the Hayflick replicative senescence model, exhibit as siRNA silences fibroblasts, a reduced tensin1 expression and an impaired gel contraction capacity. Based on these results, we speculate that in human dermal fibroblasts, tensin1 plays an important role in cell–matrix interaction and that a reduced expression might contribute to the dermal alterations observed during skin ageing.     

Phenotypic and functional changes in dermal primary fibroblasts isolated from intrinsically aged human skin

Dermal fibroblasts play a key role in maintaining skin homoeostasis by synthesizing and degrading extracellular matrix components. During ageing, they are subjected to changes, such as the loss of type I collagen expression and an increased synthesis of metalloproteinase I, leading to fragmentation of collagen fibrils with consequent reduction of the mechanical tension and defects of skin wound healing. Most information about fibroblast ageing was obtained from experiments performed on replicative‐senescent dermal fibroblasts in vitro. However, the senescence status of fibroblasts isolated from intrinsically aged skins and its consequences on functionality need to be deeper investigated. Herein, we studied age‐related phenotypic and functional alteration of fibroblasts from ‘young’ (<35 years) and ‘old’ (>50 years) donors. Our results brought evidence of the senescent status of ‘old’ fibroblasts by senescence associated β‐galactosidase (SA‐βgal) positive staining and p16 expression. A PCR array focusing on senescence highlighted a subset of downregulated genes including cell cycle progression and ECM genes in ‘old’ fibroblasts as well as a subset of upregulated genes involved in senescence features. In ‘old’ fibroblasts, we measured a downregulation of proliferative and contractile capacities of migratory potential under PDGF stimulation and activation into myofibroblasts under TGFβ. Old fibroblasts were also more sensitive to oxidative stress than ‘young’ ones. Of interest, downregulation of p16 expression partially reversed the senescent phenotype of ‘old’ fibroblasts but failed to restore their functional properties. In conclusion, our data brought evidence of phenotypic and functional differences between fibroblasts from young and intrinsically aged skin that may contribute to the alterations observed with ageing.     

Knockdown of paraoxonase 1 expression influences the ageing of human dermal microvascular endothelial cells

Skin is one of the most commonly studied tissues for microcirculation research owing to its close correlation of cutaneous vascular function, ageing and age‐related cardiovascular events. To elucidate proteins that determine this correlation between endothelial cell function and ageing in the vascular environment of the skin, we performed a proteomic analysis of plasma samples from six donors in their 20s (young) and six donors in their 60s (old). Among identified proteins, paraoxonase 1 (PON1) was selected in this study. To elucidate the role of PON1 on skin ageing and determine how it controls cellular senescence, the characteristics of PON1 in human dermal microvascular endothelial cells (HDMECs) were determined. When the expression of endogenous PON1 was knocked‐down by small interfering RNA (siRNA) targeting PON1, HDMECs showed characteristic features of cellular senescence such as increases in senescence‐associated β‐galactosidase stained cells and enlarged and flattened cell morphology. At 48 h post‐transfection, the protein expression of p16 in PON1 siRNA‐treated HDMECs was higher than that in scrambled siRNA‐treated HDMECs. In addition, the expressions of moesin and rho GTP dissociation inhibitor, additional age‐related candidate biomarkers, were decreased by PON1 knock‐down in HDMECs. In conclusion, these results suggest that PON1 functions as an ageing‐related protein and plays an important role in the cellular senescence of HDMECs.     

Azelaic acid reduced senescence‐like phenotype in photo‐irradiated human dermal fibroblasts: possible implication of PPARγ

Azelaic acid (AzA) has been used for the treatment for inflammatory skin diseases, such as acne and rosacea. Interestingly, an improvement in skin texture has been observed after long‐time treatment with AzA. We previously unrevealed that anti‐inflammatory activity of AzA involves a specific activation of PPARγ, a nuclear receptor that plays a relevant role in inflammation and even in ageing processes. As rosacea has been considered as a photo‐aggravated disease, we investigated the ability of AzA to counteract stress‐induced premature cell senescence (SIPS). We employed a SIPS model based on single exposure of human dermal fibroblasts (HDFs) to UVA and 8‐methoxypsoralen (PUVA), previously reported to activate a senescence‐like phenotype, including long‐term growth arrest, flattened morphology and increased synthesis of matrix metalloproteinases (MMPs) and senescence‐associated β‐galactosidase (SA‐β‐gal). We found that PUVA‐treated HDFs grown in the presence of AzA maintained their morphology and reduced MMP‐1 release and SA‐β‐galactosidase‐positive cells. Moreover, AzA induced a reduction in ROS generation, an up‐modulation of antioxidant enzymes and a decrease in cell membrane lipid damages in PUVA‐treated HDFs. Further evidences of AzA anti‐senescence effect were repression of p53 and p21, increase in type I pro‐collagen and abrogation of the enhanced expression of growth factors, such as HGF and SCF. Interestingly, PUVA‐SIPS showed a decreased activation of PPARγ and AzA counteracted this effect, suggesting that AzA effect involves PPARγ modulation. All together these data showed that AzA interferes with PUVA‐induced senescence‐like phenotype and its ability to activate PPAR‐γ provides relevant insights into the anti‐senescence mechanism.     

Epidermal growth factor receptor pathway mitigates UVA-induced G2/M arrest in keratinocyte cells

UVA irradiation contributes largely to photocarcinogenesis. In the process of keratinocyte transformation, the activation of EGFR by UV is now considered as a critical event. However, the mechanism that links the EGFR pathway and photocarcinogenesis is not totally understood. In this study, we report that the EGFR/Akt pathway mitigated G2/M arrest in human HaCaT keratinocytes and normal human keratinocytes treated with low doses of UVA irradiation. EGFR-mediated Akt activation resulted in increased level of checkpoint 1 kinase (Chk1) inhibitory phosphorylation (Ser280). In contrast, EGFR/Akt pathway inhibition resulted in the abrogation of Ser280 Chk1 phosphorylation, increased level of Chk1 stimulatory phosphorylation (Ser345), and restoration of G2/M arrest. Altogether, these results suggest that, after UVA exposure, the EGFR/Akt pathway subverts the G2/M checkpoint. This effect may have serious implications in photocarcinogenesis by allowing damaged cells to transit through the cell cycle.     

Oxytocin alleviates cellular senescence through oxytocin receptor-mediated extracellular signal-regulated kinase/Nrf2 signalling

Intrinsic skin aging is skin aging affected by natural factors within the body, such as hormones, rather than by external factors like UV light or smoking. Skin ageing causes the appearance of wrinkles, skin sagging and reduced elasticity. Like elsewhere in the body, skin ageing involves an increase in the proportion of cells undergoing cellular senescence (a type of deterioration due to age). Senescent skin cells called dermal fibroblasts have several characteristics that are different to cells that are not senescent, including having what is known as a senescence-associated secretory phenotype (SASP). Oxytocin (OT) is a neuropeptide hormone that has many beneficial effects in the body, including protection against age-related disorders. However, less is known about the role of OT in intrinsic skin aging. The authors looked into the role of oxytocin in prevention against senescence in skin cells called normal human dermal fibroblasts (NHDFs), taken from female donors of different ages. They found that OT suppressed SASP-induced senescence in NHDFs derived from young but not old female donors. The authors were also able to learn more about the processes within the body by which OT suppresses SASP and why it affects younger women differently to older women. Their results demonstrate that oxytocin may be effective in the prevention of skin aging.     

Ultraviolet-B-induced G1 arrest is mediated by downregulation of cyclin-dependent kinase 4 in transformed keratinocytes lacking functional p53

In order to identify potential novel targets for ultraviolet-B-induced skin tumorigenesis, we assessed the effect of ultraviolet-B exposure on cell cycle progression of transformed keratinocytes with mutant p53. We show that ultraviolet-B exposure of human epidermoid carcinoma A431 cells results in G1 cell cycle arrest in both asynchronously growing and synchronized cells. A significant increase in G1 cell population was observed following exposure to doses of ultraviolet-B as low as 10 mJ per cm2. When irradiated with ultraviolet-B, cells synchronized in G1 with mimosine did not exit G1. G1 cell cycle arrest was associated with a decrease in the hyperphosphorylated forms of retinoblastoma protein that was detectable within 4 h and gradually disappeared by 12 h. We also observed a decrease in cyclins D1, D2, and D3, and cyclin-dependent kinase 4 proteins, and a concomitant decrease in cyclin-dependent kinase 4/cyclin D1 associated kinase activity, whereas ultraviolet-B exposure had no effect on cyclin-dependent kinase 2 and cyclin-dependent kinase 6 levels during this time period. Incubation of cells with proteasome inhibitors MG-115 and MG-132 prevented the decrease in cyclin D1, D2, and D3, and cyclin-dependent kinase 4 protein. Taken together, our results suggest that ultraviolet-B-induced cell cycle arrest in A431 cells is mediated by cyclin-dependent kinase 4 downregulation. This identifies a novel pathway for G1 cell cycle arrest in transformed keratinocytes following ultraviolet-B irradiation.     

Papillary fibroblasts differentiate into reticular fibroblasts after prolonged in vitro culture

The dermis can be divided into two morphologically different layers: the papillary and reticular dermis. Fibroblasts isolated from these layers behave differently when cultured in vitro. During skin ageing, the papillary dermis decreases in volume. Based on the functional differences in vitro, it is hypothesized that the loss of papillary fibroblasts contributes to skin ageing. In this study, we aimed to mimic certain aspects of skin ageing by using high‐passage cultures of reticular and papillary fibroblasts and investigated the effect of these cells on skin morphogenesis in reconstructed human skin equivalents. Skin equivalents generated with reticular fibroblasts showed a reduced terminal differentiation and fewer proliferating basal keratinocytes. Aged in vitro papillary fibroblasts had increased expression of biomarkers specific to reticular fibroblasts. The phenotype and morphology of skin equivalents generated with high‐passage papillary fibroblasts resembled that of reticular fibroblasts. This demonstrates that papillary fibroblasts can differentiate into reticular fibroblasts in vitro. Therefore, we hypothesize that papillary fibroblasts represent an undifferentiated phenotype, while reticular fibroblasts represent a more differentiated population. The differentiation process could be a new target for anti‐skin‐ageing strategies.     

Inhibition of cyclin‐dependent kinase activity exacerbates H2O2‐induced DNA damage in Kindler syndrome keratinocytes

Kindler syndrome (KS) is an autosomal recessive skin disorder characterized by skin blistering and photosensitivity. KS is caused by loss of function mutations in FERMT1, which encodes Kindlin‐1. Kindlin‐1 is a FERM domain containing adaptor protein that is found predominantly at cell‐extracellular matrix adhesions where it binds to integrin β subunits and is required for efficient integrin activation. Using keratinocytes derived from a patient with KS, into which wild‐type Kindlin‐1 (Kin1WT) has been expressed, we show that Kindlin‐1 binds to cyclin‐dependent kinase (CDK)1 and CDK2. CDK1 and CDK2 are key regulators of cell cycle progression, however, cell cycle analysis showed only small differences between the KS and KS‐Kin1WT keratinocytes. In contrast, G2/M cell cycle arrest in response to oxidative stress induced by hydrogen peroxide (H₂O₂) was enhanced in KS keratinocytes but not KS‐Kin1WT cells, following inhibition of CDK activity. Furthermore, KS keratinocytes were more sensitive to DNA damage in response to H₂O₂ and this was exacerbated by treatment with the CDK inhibitor roscovitine. Thus, in Kindlin‐1 deficient keratinocytes, CDK activity can further regulate oxidative damage induced cell cycle arrest and DNA damage. This provides further insight into the key pathways that control sensitivity to oxidative stress in KS patients.     

Induction of senescence pathways in Kindler syndrome primary keratinocytes

Background Individuals with Kindler syndrome (KS) have loss‐of‐function mutations in the FERMT1 gene that encodes the focal adhesion component kindlin‐1. The major clinical manifestation of KS is epidermal atrophy (premature skin ageing). This phenotypic feature is thought to be related to the decreased proliferation rate of KS keratinocytes; nevertheless, molecular mediators of such abnormal behaviour have not been fully elucidated. Objectives To investigate how kindlin‐1 deficiency affects the proliferative potential of primary human keratinocytes. Methods  We serially cultivated nine primary KS keratinocyte strains until senescence and determined their lifespan and colony‐forming efficiency (CFE) at each serial passage. The expression of molecular markers of stemness and cellular senescence were investigated by immunoblotting using cell extracts of primary keratinocyte cultures from patients with KS and healthy donors. In another set of experiments, kindlin‐1 downregulation in normal keratinocytes was obtained by small interfering RNA (siRNA) technology. Results We found that KS keratinocytes exhibited a precocious senescence and strongly reduced clonogenic potential. Moreover, KS cultures showed a strikingly increased percentage of aborted colonies (paraclones) already at early passages indicating an early depletion of stem cells. Immunoblotting analysis of KS keratinocyte extracts showed reduced levels of the stemness markers p63 and Bmi‐1, upregulation of p16 and scant amounts of hypophosphorylated Rb protein, which indicated cell cycle‐arrested status. Treatment of normal human primary keratinocytes with siRNA targeting kindlin‐1 proved that its deficiency was directly responsible for p63, Bmi‐1 and pRb downregulation and p16 induction. Conclusions Our data directly implicate kindlin‐1 in preventing premature senescence of keratinocytes.     

Deficiency of the parathyroid hormone‐related peptide nuclear localization and carboxyl terminal sequences leads to premature skin ageing partially mediated by the upregulation of p27

We previously reported that deficiency of the PTHrP nuclear localization sequence (NLS) and C‐terminus in PTHrP knockin (PTHrP KI) mice resulted in premature ageing of skin. P27, a cyclin‐dependent kinase inhibitor, was upregulated in PTHrP KI mice and acted as a downstream target of the PTHrP NLS to regulate the proliferation of vascular smooth muscle cells. To determine the effects of p27 deficiency on premature skin ageing of PTHrP KI mice, we compared the skin phenotypes of PTHrP KI mice to those of p27 knockout (p27^?/?) mice and to those of double homozygous p27‐deficient and PTHrP KI (p27^?/?PTHrP KI) mice at 2 weeks age. Compared with wild‐type littermates, PTHrP KI mice displayed thinner skin and decreased subcutaneous fat and collagen fibres, decreased skin cell proliferation and increased apoptosis, higher expression of p27, p19 and p53 and lower expression of cyclin E and CDK2, and increased reactive oxygen species levels and decreased antioxidant capacity. Deficiency of p27 in the PTHrP KI mice at least in part corrected the skin premature ageing phenotype resulting in thicker skin and increased subcutaneous fat and collagen. These alternations were associated with higher expression of CDK2 and cyclin E, lower expression of p19 and p53, and enhanced antioxidant capacity with increased skin cell proliferation and inhibition of apoptosis. Our results indicate that the NLS and C‐terminus of PTHrP play a critical role in preventing skin from premature ageing that is partially mediated by p27.     

Metformin alleviates ageing cellular phenotypes in Hutchinson–Gilford progeria syndrome dermal fibroblasts

Metformin is a popular antidiabetic biguanide, which has been considered as a candidate drug for cancer treatment and ageing prevention. Hutchinson–Gilford progeria syndrome (HGPS) is a devastating disease characterized by premature ageing and severe age‐associated complications leading to death. The effects of metformin on HGPS dermal fibroblasts remain largely undefined. In this study, we investigated whether metformin could exert a beneficial effect on nuclear abnormalities and delay senescence in fibroblasts derived from HGPS patients. Metformin treatment partially restored normal nuclear phenotypes, delayed senescence, activated the phosphorylation of AMP‐activated protein kinase and decreased reactive oxygen species formation in HGPS dermal fibroblasts. Interestingly, metformin reduced the number of phosphorylated histone variant H2AX‐positive DNA damage foci and suppressed progerin protein expression, compared to the control. Furthermore, metformin‐supplemented aged mice showed higher splenocyte proliferation and mRNA expression of the antioxidant enzyme, superoxide dismutase 2 than the control mice. Collectively, our results show that metformin treatment alleviates the nuclear defects and premature ageing phenotypes in HGPS fibroblasts. Thus, metformin can be considered a promising therapeutic approach for life extension in HGPS.     

Enhanced expression of p16 in seborrhoeic keratosis; a lesion of accumulated senescent epidermal cells in G1 arrest

BACKGROUND: Seborrhoeic keratosis (SK) is a common skin disease associated with skin ageing and photoageing, but only limited studies have been performed on SK and the senescence of keratinocytes. OBJECTIVES: We sought to clarify the genetic basis of SK and the senescence of keratinocytes. METHODS: Expression of p16, cyclins A, D and E, p21, p53, retinoblastoma (Rb) gene product and telomerase-associated protein 1 (TP1) in SK was examined by immunohistochemistry. DNA fragmentation in SK was detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling method. We cultured keratinocytes from SK lesions and non-lesional epidermis and examined expression of p16, observed morphology of the cultured cells by light and electron microscopy and measured survival time. RESULTS: p16, a cyclin-dependent kinase inhibitor, was expressed in all cells from SK lesions, whereas normal keratinocytes expressed p16 only in the granular cells. Other factors such as cyclins A, D and E, p21, p53, Rb gene product, and TP1, were not expressed in SK cells. These results suggest that p16 expression is a marker of SK and that p16 has a role in the pathogenesis of SK. DNA fragmentation was not detected in four of five SK tissue samples; one of the SK tissue samples showed DNA fragmentation only in the superficial upper layer of an SK lesion, suggesting that apoptosis was inhibited in SK cells. In contrast, normal epidermis showed DNA fragmentation in the granular and squamous layers. Immunohistochemical examination of cultured SK cells also revealed the presence of p16. A greater number of SK cells survived after 3 weeks of culture in comparison with normal keratinocytes. Features of senescence, such as a balloon-like appearance after lengthy culture and increased amounts of tonofilaments in cytoplasm, were observed in SK cells in culture. CONCLUSIONS: These results suggest that SK is a benign neoplasm where keratinocytes in a senescent condition and G1 arrest are accumulated.     

Tiron protects against UVB-induced senescence-like characteristics in human dermal fibroblasts by the inhibition of superoxide anion production and glutathione depletion

Background/Objectives: Free radicals and reactive oxygen species (ROS), which are generated by UV irradiation, may induce an irreversible growth arrest similar to senescence. Tiron, 4,5-dihydroxy-1,3-benzene disulfonic acid, is a widely used antioxidant to rescue ROS-evoked cell death. The aim of the article was to explore the effects of tiron on skin photoaging and associated mechanisms. Methods: The effects of tiron on cell proliferation were determined using 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide. Senescent cells were determined by morphology and senescence-associated β-galactosidase activity analysis. Intracellular hydrogen peroxide, superoxide anion and glutathione concentration were analysed by a fluorescent probe. The concomitant changes of protein expression were analysed with Western blot. Results: Human dermal fibroblasts were induced to premature senescence by sub-cytotoxic doses of irradiated UVB. Strong senescence-associated β-galactosidase activity and increased intracellular superoxide anion were observed in human dermal fibroblasts irradiated by UVB. Tiron blocks UVB-induced glutathione depletion and increase of superoxide anion and protects against UVB-induced senescence-like characteristics in human dermal fibroblasts. Compared with normal fibroblasts, UVB-irradiated human dermal fibroblasts showed a higher ratio of active (hypophosphorylated) to inactive (phosphorylated) forms of Rb and p38, upregulation of p53 or p16 and c-Myc and insulin-like growth factor 1 (IGF-1) downregulation. After treatment with tiron, p53, p16 c-Myc and IGF-1 as well as phosphorylation Rb and p38 could partially recover. Conclusion: These results indicate that tiron protects against UVB-induced senescence-like characteristics in human dermal fibroblasts via the inhibition of production of superoxide anion and glutathione depletion, and modulation of related senescence proteins.