Repeated exposure of human fibroblasts to UVR induces secretion of stem cell factor and senescence

Background Some of chronic hyperpigmentary diseases, such as melasma, induced by multiple factors including chronic sunlight exposure, can recur even after chemical epidermal removal. Dermal factors may be involved in the pathogenesis of melasma. Changes in dermal fibroblasts resulting from chronic sun exposure might cause melanocytes to synthesize melanin in the epidermis. Objective This study aimed at determining the effects of repetitive ultraviolet (UV) radiation on cultured fibroblasts and the secretion of melanogenic factors. Methods Cultured human fibroblasts were exposed to ultraviolet A (UVA) or ultraviolet B (UVB) for five consecutive days. After each irradiation, the supernatant medium was isolated from each dish and measured for levels of stem cell factor (SCF) and hepatocyte growth factor using an ELISA kit assay. To assess the effect of the keratinocyte-derived factors on fibroblast-secretion of SCF and hepatocyte growth factor, we added supernatants of the UV-irradiated keratinocytes to the non-irradiated fibroblasts. Finally, the irradiated fibroblasts were stained with senescence associated-β-galactosidase to assess their senescent change. Results Fibroblasts irradiated with UVA or UVB for five consecutive days, secreted SCF at levels that increased with repeated UVA or UVB exposure. Conditioned culture medium from UV-irradiated keratinocytes also induced SCF release from fibroblasts, depending on the number of UV exposures. UVA- or UVB-irradiated fibroblasts stained positive for senescence associated-β-galactosidase, and the staining intensity increased with repeated exposure. Conclusion These results suggest that fibroblast senescence and increased SCF secretion after repeated UV irradiation may be related to the pathogenesis of recurring hyperpigmentation disorders induced by chronic sun exposure.     

Hepatocyte growth factor establishes autocrine and paracrine feedback loops for the protection of skin cells after UV irradiation

Hepatocyte growth factor (HGF) is a multifunctional cytokine, which, among various other activities, acts as a growth factor for melanocytes and has recently been implicated in the pathogenesis of malignant melanoma. In the skin, the main source for HGF is dermal fibroblasts (FB). Here, we have investigated the regulation of HGF production and secretion by cytokines derived from UV-irradiated keratinocytes (KC) and by direct UV irradiation. We demonstrate that supernatants of ultraviolet (UV)B-irradiated KC strongly induce HGF production in FB, and that this effect was mediated primarily by IL-1alpha. Direct irradiation of FB with UVB had no effect on HGF expression. In contrast, irradiation with UVA1 strongly upregulated HGF mRNA production and secretion of the functional protein. Addition of neutralizing anti-HGF antibodies after UVA1 irradiation, as well as transfection of FB with HGF small-interfering RNA (siRNA); which completely abrogated HGF secretion led to a dramatic rise of FB apoptosis demonstrating that autocrine HGF efficiently protected FB from UVA1-induced apoptosis. Our data suggest that upregulation of HGF plays a role in skin homeostasis after UV irradiation. However, a negative side effect of UV-induced HGF secretion by dermal FB might represent a decisive factor for induction and/or progression of melanoma.     

Attachment and chemotaxis of melanocytes after ultraviolet irradiation in vitro

Because ultraviolet (UV) radiation is able to influence the spatial distribution of melanocytes in melanocytic naevi in vivo, we investigated the influence of UV radiation on the ability of melanocytes to adhere to the extracellular matrix proteins fibronectin, laminin and collagen type IV in vitro. In addition, chemotaxis of melanocytes was studied using both fibronectin and the supernatants from irradiated, as well as non-irradiated, keratinocytes and fibroblasts as attractants. Melanocyte attachment to fibronectin was significantly increased 48 h after a single UV irradiation at 30 mJ/cm2 in comparison with that of non-irradiated melanocytes, whereas attachment to laminin and collagen type IV showed only minor changes after UV exposure. The UV-induced increase in attachment to fibronectin was suppressed by preincubation with antibodies against alpha5beta1 or alphavbeta3 integrin. Both immunohistochemistry and flow cytometric analysis showed an increase in alpha5beta1 integrin expression on melanocytes after UV exposure. The chemotaxis of melanocytes to fibronectin was not influenced by UV exposure. A decreasing migration rate of melanocytes towards the supernatants of UVA-irradiated fibroblasts was observed with increasing UVA doses. The chemotactic effects of conditioned medium of keratinocytes towards melanocytes was not influenced either by UVB or by UVA. The results indicate that UV radiation may alter the ability of melanocytes to adhere to certain substrates by modification of integrin expression. Because fibronectin, as the major target protein of UV-altered attachment, is located in the dermis, the UV-induced morphological changes in melanocytic lesions, with an increase in suprabasally located melanocytes within the epidermis, may be due to other changes in the adhesive properties of melanocytes.     

Emerging role of dermal compartment in skin pigmentation: comprehensive review

The variations in human skin colour mainly occur due to differences in the distribution of melanin pigment throughout the body, synthesized by epidermal melanocytes which are further taken up by keratinocytes present in epidermis. Recently, it has been discovered that besides these cells, dermis derived fibroblast factors also play a prominent role in regulating skin pigmentation. There exists a signal crosstalk between epidermal melanocytes, keratinocytes and dermal fibroblasts and any impairment in these signalling pathways may give rise to pigmentary disorders. Vitiligo is a hypopigmentary disorder and alteration in the expression level of several fibroblast-specific factors has been reported in the lesional skin of vitiligo patients. In such patients, there is decrease in the expression levels of factors such as basic fibroblast growth factor, stem cell factor (SCF) and keratinocyte growth factor (KGF) along with a steep increase in the expression levels of Dickkopf 1. Patients affected with hyperpigmentary disorder like melasma exhibit a marked increase in SCF and KGF expression levels leading to increase in melanin production and those affected with solar lentigo experience upregulation in the expression levels of SCF, KGF and HGF (hepatocyte growth factor). Hence, we conclude that new therapeutic strategies can be adopted to cure these pigmentary disorders by targeting factors involved in crosstalk signalling between epidermal melanocytes, keratinocytes and dermal fibroblasts.     

Less keratinocyte-derived factors related to more keratinocyte apoptosis in depigmented than normally pigmented suction-blistered epidermis may cause passive melanocyte death in vitiligo

Stem cell factor (SCF) of keratinocyte origin regulates melanocyte growth and survival. Deprivation of survival factors causes the apoptosis of melanocytes. Vitiligo often develops following physical trauma, even if this is minor. The exact mechanism of the Koebner phenomenon in vitiligo is unclear. Apoptosis of keratinocytes, which occurs more in depigmented suction-blistered epidermis than in the normally pigmented counterpart, could reduce levels of keratinocyte-derived factors such as SCF and basic fibroblast growth factor (bFGF). Levels of SCF expression were examined in the depigmented and normally pigmented paired epidermis of 19 patients with vitiligo, and bFGF expression in six patients. The expression of SCF (p<0.001) and bFGF was usually reduced in the depigmented compared with the normally pigmented epidermis. Apoptosis of cultured normal human keratinocytes, which was induced by staurosporine, resulted in a concentration-dependent decrease in levels of SCF mRNA and protein. Normal human melanocytes proliferated more in medium containing SCF or keratinocyte (XB-2) feeder than in medium with neither. Deprivation of SCF or keratinocyte feeder in the culture medium induced a marked decrease in melanocytes as a result of apoptosis. Therefore, lower expression of keratinocyte-derived factors, including SCF, in vitiliginous keratinocytes, which could result from keratinocyte apoptosis, might be responsible for passive melanocyte death and may explain the Koebner phenomenon.     

Association study between keratinocyte-derived growth factor gene polymorphisms and susceptibility to vitiligo vulgaris in a Taiwanese population: potential involvement of stem cell factor

Background  Vitiligo vulgaris is a depigmentary disorder resulting from the disappearance of functional melanocytes. Currently, the pathogenesis of this disorder remains obscure.
Objectives  Genetic analysis of patients with vitilgo may provide important clues for elucidating the complex pathomechanisms involved in the disease process. Because dysfunctional keratinocytes have recently been implicated in the pathogenesis of vitiligo vulgaris, we conducted a case–control association study to investigate this phenomenon.
Patients and methods  Fifty-one patients with vitiligo vulgaris and 118 healthy controls from Taiwan were recruited to investigate the association between relevant keratinocyte-related genes and the occurrence of vitiligo vulgaris. This study genotyped 11 single-nucleotide polymorphisms (SNPs) in five genes including stem cell factor ( SCF , also known as KITLG ), basic fibroblast growth factor ( bFGF , also known as NuDT6 ), endothelin-1 ( EDN1 ), hepatocyte growth factor ( HGF ) and stem cell growth factor ( SCGF , also known as CLEC11A ).
Results  Our results revealed that the A allele for SNP rs11104947 in the SCF gene and the T allele for SNP rs13866 in the SCGF gene were, respectively, associated with a 1·95- and a 2·14-fold risk of developing vitiligo vulgaris. A higher risk was also detected among subjects who carried the SCF rs995029/rs11104947 C/A haplotype (odds ratio = 2·45). Furthermore, the at-risk alleles for SCF rs11104947 (A allele) and for SCGF SNP rs13866 (T allele) were found to display a 7·92-fold increased gene–gene combined risk. No significant relationship between polymorphic frequency for genes bFGF , EDN1 as well as HGF and occurrence of vitiligo vulgaris was observed.
Conclusions  These novel genetic findings provide new insights in relation to the mechanisms that might be involved in the development of vitiligo vulgaris.     

Predominant effects of Polypodium leucotomos on membrane integrity,lipid peroxidation,and expression of elastin and matrixmetalloproteinase-1 in ultraviolet radiation exposed fibroblasts,and keratinocytes

BACKGROUND: Polypodium leucotomos has been reported to have antioxidant, anti-inflammatory and photoprotective properties. Exposure of skin to ultraviolet (UV) radiation can lead to deposition of excessive elastotic material, reduction in collagen, and increased expression of matrix metalloproteinases (MMPs). OBJECTIVE: The goal of this research was to determine the effects of P. leucotomos in the absence or presence of UVA or UVB radiation on membrane damage, lipid peroxidation, and expression of elastin and MMP-1 in fibroblasts and keratinocytes, respectively. METHODS: Fibroblasts and keratinocytes, respectively, were irradiated by a single exposure to UVA (0.6, 1.8 or 3.6 J) or UVB radiation (0.75, 2.5 or 7.5 mJ), and then incubated with, or without, P. leucotomos (0.01, 0.1 and 1%) and examined for membrane damage, lipid peroxidation, expression of elastin (protein levels) and MMP-1 (protein levels or MMP-1 promoter activity). RESULTS: UV radiation did not significantly alter membrane integrity, lipid peroxidation or MMP-1 expression, but increased elastin expression. P. leucotomos significantly improved membrane integrity, inhibited lipid peroxidation, increased elastin expression, and inhibited MMP-1 expression in both fibroblasts, and keratinocytes. The effects of P. leucotomos predominated in the presence of UVA or UVB in both fibroblasts and keratinocytes, respectively, with the exception of inhibition of MMP-1 protein levels in fibroblasts only in combination with UV radiation. CONCLUSION: Lower concentration of P. leucotomos (lower than 0.1%), may be beneficial in preventing photoaging by improving membrane integrity and inhibiting MMP-1, without increasing elastin expression. Higher concentration (greater than 0.1%) of P. leucotomos may reverse the loss of normal elastic fibers associated with intrinsic aging.     

Role of fibroblast‐derived growth factors in regulating hyperpigmentation of solar lentigo

Background Cutaneous pigmentation is regulated by a complex melanogenic network in which both keratinocytes and fibroblasts synthesize growth factors and cytokines. Solar lentigo (SL) is characterized by hyperpigmented lesions occurring on photodamaged skin areas. Despite the association of SL to ultraviolet (UV) exposure, the mechanisms underlying the development of these spots are not completely defined. Objectives To analyse the involvement of the fibroblast‐derived growth factors, hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and stem cell factor (SCF) in SL hyperpigmentation; to evaluate whether the photoageing process occurring in fibroblasts could be responsible for the altered expression of these cytokines; and to investigate a new possible role of KGF in regulating pigmentation through the specific induction of melanogenic cytokines by keratinocytes. Methods We performed immunohistochemical analysis of HGF, KGF and SCF on SL biopsies. We analysed the mRNA expression of these cytokines using an in vitro model of photoageing induced on fibroblasts. Finally, we evaluated the effects of KGF on the expression of melanogenic cytokines at the mRNA and protein levels on keratinocytes. Results We found positive staining for HGF, KGF and SCF in the upper dermis of SL lesions and a significant induction of the three cytokines in photoaged fibroblasts. We also demonstrated the contribution of KGF to pigmentation, showing its ability specifically to modulate the expression of SCF in keratinocytes. Conclusions Fibroblasts may be persistently activated by UV exposure to release melanogenic growth factors; this inducible cytokine network acts both directly and indirectly through keratinocytes and may contribute to the hyperpigmentation of SL.     

Keratinocytes suppress transforming growth factor-beta1 expression by fibroblasts in cultured skin substitutes

Transforming growth factor (TGF)-beta1 is a multipotent growth factor with an important role in tissue homeostasis. This growth factor regulates cell proliferation, adhesion, migration and differentiation, as well as extracellular matrix deposition. The temporal secretion and activation of latent TGF-beta1 is thus of major importance to physiological and pathological processes and in wound healing and tumour formation. Cultured skin substitutes, as used to treat extensive acute or chronic skin wounds, offer an attractive model to investigate cellular interactions in cytokine and growth factor expression and response in vitro. In the present investigation, expression of TGF-beta1 was analysed in keratinocyte, fibroblast and melanocyte monolayer cultures, as well as in the dermal vs. epidermal components of reconstituted human skin. Immunohistology, enzyme-linked immunosorbent assay (ELISA) and Northern blotting were used to demonstrate expression at the RNA and protein level. In the monolayer cultures, levels of TGF-beta1 synthesized by melanocytes were observed to be considerably elevated when compared with keratinocytes. Most TGF-beta1, however, was secreted by fibroblasts. The relative contribution of the epidermal and dermal components of the skin substitutes to overall TGF-beta1 levels was determined by comparing results obtained for either component in the presence and absence of fibroblasts and keratinocytes. From results obtained by ELISA it was apparent that TGF-beta1 levels generated predominantly by fibroblasts within the skin substitutes were greatly reduced over time in the presence of keratinocytes. Suppression of fibroblast TGF-beta1 expression in the presence of keratinocytes was also demonstrable at the RNA level by Northern blotting. Results obtained by immunohistochemistry suggest that most, if not all, of the growth factor was present in the latent form. It is therefore most likely that the observed effect results from a factor secreted by keratinocytes, which is capable of suppressing TGF-beta1 synthesis by fibroblasts. These results suggest that expression of TGF-beta1 by fibroblasts is downregulated by paracrine actions of keratinocytes in healing skin.     

Narrow-band ultraviolet-B stimulates proliferation and migration of cultured melanocytes

Narrow-band ultraviolet-B (UVB) radiation is an effective treatment for vitiligo vulgaris. However, the mechanisms of narrow-band UVB in inducing repigmentation of vitiligo lesions are not thoroughly clarified. The purpose of our study was to investigate the effects of narrow-band UVB irradiation on melanocyte proliferation and migration in vitro. Our results showed that the cell counts as well as [3H]thymidine uptake of melanocytes were significantly enhanced by narrow-band UVB-irradiated keratinocyte supernatants. In these supernatants, a significant increase in basic fibroblast growth factor (bFGF) and in endothelin-1 (ET-1) release was observed. bFGF is a natural mitogen for melanocytes, whereas ET-1 can stimulate DNA synthesis in melanocytes. This stimulatory effect of melanocyte proliferation by supernatants derived from narrow-band UVB-irradiated keratinocytes was significantly reduced by a selective endothelin-B (ET-B) receptor antagonist (BQ788), suggesting an essential role of ET-1 on melanocyte proliferation. Our results of time-lapse microphotography revealed a stimulatory effect of narrow-band UVB irradiation on melanocyte migration. Focal adhesion kinase (FAK) plays a pivotal role in cell migration. Phosphorylated FAK (p125(FAK)) expression on melanocyte was enhanced by narrow-band UVB irradiation. In this study, narrow-band UVB irradiation stimulated a significant increase in matrix metalloproteinase-2 (MMP-2) activity in melanocyte supernatants. Narrow-band UVB-irradiation-induced migration of melanocytes was significantly annihilated by the addition of p125(FAK) inhibitor (herbimycin-A) or MMP-2 inhibitor (GM6001). These results suggest that p125(FAK) and MMP-2 activity play important roles in narrow-band UVB-induced migration of melanocytes. Our results provide a theoretical basis for the effectiveness of narrow-band UVB irradiation in treating vitiligo.     

Basic fibroblast growth factor promotes melanocyte migration via increased expression of p125(FAK) on melanocytes

Vitiligo is an acquired pigmentary disorder characterized by depigmentation of skin and hair. Melanocyte migration is an important event in re-pigmentation of vitiligo. We have demonstrated that narrow-band ultraviolet B (UVB) irradiation stimulated cultured keratinocytes to release a significant amount of basic fibroblast growth factor (bFGF). Furthermore, narrow-band UVB enhanced migration of melanocytes via increased expression of phosphorylated focal adhesion kinase (p125(FAK)) on melanocytes. The aim of this study was to investigate the effect of recombinant human bFGF (rhbFGF) on melanocyte migration. The relationship between the expression of p125(FAK) and melanocyte migration induced by rhbFGF was also studied. Our results demonstrated that rhbFGF significantly enhanced migration of melanocytes and p125(FAK) expression on melanocytes. Herbimycin A, a potent p125(FAK) inhibitor, effectively abolished rhbFGF-induced melanocyte migration. The combined results indicated that p125(FAK) plays an important role in the signal transduction pathway of melanocyte migration induced by bFGF.     

Modulation of endothelin-1 in normal human keratinocytes by UVA1/B radiations, prostaglandin E2 and peptidase inhibitors

In the skin, keratinocytes synthesize and secrete endothelin-(ET-1), a potent vasoconstrictor peptide which acts also as a growth factor for most skin cells. The aim of the study was to test the effects of UVA1 and the associations UVA1/B on the expression of ET-1 in normal human keratinocytes and to determine whether exogenously added prostaglandin E2 (PGE2) regulated ET-1 expression. As ET-1 is susceptible to degradation, we also evaluated whether ET-1 secretion was modulated by peptidase inhibitors. Our results showed that UVA1 (365 nm) did not modify the levels of preproET-1 mRNA and protein. Moreover, the associations UVA1+UVB or UVB+UVA1 down-regulated the overexpression of secreted ET-1 induced by UVB alone. PGE2 at 10(-5) M reduced the expression of ET-1 at the mRNA and protein levels but did not exert any significant modification at lower concentrations from 10(-10) to 10(6) M. Phosphoramidon, an endothelin converting enzyme (ECE) inhibitor, drastically decreased the amount of ET-1 accumulating in the culture medium in basal conditions or after UVB irradiation. Conversely, thiorphan, a specific inhibitor of neutral endopeptidase (NEP), rather increased the levels of ET-1 secretion mainly after UVB irradiation. Taken together, the results showed that normal human keratinocytes secrete and partly degrade ET-1 through ECE and NEP pathways and pointed out a differential regulation of ET-1 by UVB and UVA1 radiations without any noticeable role for PGE2.     

Solar ultraviolet radiation induces biological alterations in human skin in vitro: relevance of a well-balanced UVA/UVB protection

Cutaneous damages such as sunburn, pigmentation, and photoaging are known to be induced by acute as well as repetitive sun exposure. Not only for basic research, but also for the design of the most efficient photoprotection, it is crucial to understand and identify the early biological events occurring after ultraviolet (UV) exposure. Reconstructed human skin models provide excellent and reliable in vitro tools to study the UV-induced alterations of the different skin cell types, keratinocytes, fibroblasts, and melanocytes in a dose- and time-dependent manner. Using different in vitro human skin models, the effects of UV light (UVB and UVA) were investigated. UVB-induced damages are essentially epidermal, with the typical sunburn cells and DNA lesions, whereas UVA radiation-induced damages are mostly located within the dermal compartment. Pigmentation can also be obtained after solar simulated radiation exposure of pigmented reconstructed skin model. Those models are also highly adequate to assess the potential of sunscreens to protect the skin from UV-associated damage, sunburn reaction, photoaging, and pigmentation. The results showed that an effective photoprotection is provided by broad-spectrum sunscreens with a potent absorption in both UVB and UVA ranges.     

Transforming growth factor-beta1 and ultraviolet A1 radiation increase production of vascular endothelial growth factor but not endothelin-1 in human dermal fibroblasts

BACKGROUND: Normal and dysregulated wound healing involves fibroblast activation and angiogenesis, in which polypeptide factors such as transforming growth factor (TGF)-beta, vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) play an important part. Ultraviolet (UV) A1 (365 nm) has recently received attention as a possible treatment for some dermal fibrotic disorders. OBJECTIVES: The aim of this study was to evaluate the effects of TGF-beta1 and UVA1 radiation, as well as that of cobalt chloride, reported to mimic hypoxia both in vivo and in vitro, on the expression of VEGF and ET-1 by cultured human dermal fibroblasts. METHODS: Levels of VEGF and ET-1 were measured by enzyme-linked immunosorbent assay and expression of neutral endopeptidase (NEP, CD10), known to degrade ET-1, was quantified by flow cytometric analysis after cell trypsinization. RESULTS: Our results showed that the cells released minor amounts of VEGF and ET-1. Both TGF-beta1 and UVA1 strongly increased VEGF secretion in a dose- and time-dependent manner, without significantly affecting ET-1 release. Irradiation of TGF-beta1-stimulated fibroblasts resulted in a synergistic effect on increasing levels of VEGF but not ET-1 after 48 h. Cobalt chloride stimulated the secretion of VEGF by fibroblasts; the effects of TGF-beta1 and cobalt were additive. However, no significant effect of cobalt chloride on ET-1 secretion was observed, suggesting that ET-1 production in fibroblasts is not oxygen-sensitive. The expression of NEP was not modified by TGF-beta1 or UVA1 radiation. Addition of a neutralizing anti-CD10 antibody to fibroblast cultures downregulated CD10 expression at the cell surface without changing ET-1 levels in cell supernatants after 24 or 48 h. This suggests that membrane-bound NEP has minimal or no activity against secreted ET-1. CONCLUSIONS: Taken together, these results underline the major role played by TGF-beta1 in increasing VEGF secretion by fibroblasts. This, as well as the documented effect of UVA1 on increasing VEGF production, may have implications for wound healing in vivo.     

Contrasting effects of an ultraviolet B and an ultraviolet A tanning lamp on interleukin-6, tumour necrosis factor-α and intercellular adhesion molecule-1 expression

BACKGROUND: Recent studies have demonstrated that a tanning lamp emitting predominantly ultraviolet (UV) A induces significant yields of the type of potentially mutagenic DNA damage that are associated with the onset of skin cancer (i.e. cyclobutane pyrimidine dimers). UV-induced immunosuppression is also an important event leading to skin cancer. OBJECTIVES: To the modulation of key immunological molecules following exposure to a broad-spectrum UVB lamp and a predominantly UVA-emitting tanning lamp using model in vitro systems. METHODS: We compared secretion and mRNA expression of interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha in normal human epidermal keratinocytes, and interferon (IFN)-gamma-induced intracellular adhesion molecule (ICAM)-1 in normal human fibroblasts irradiated in vitro with a broad-spectrum UVB lamp or with a Philips 'Performance' tanning lamp. RESULTS: With broad-spectrum UVB irradiation, upregulation of IL-6 and TNF-alpha mRNA was detected 6 h after irradiation, and a dose-dependent increase of cytokines in the supernatants of irradiated cells was found 24 h after irradiation. In contrast, there was no cytokine secretion and little evidence for mRNA upregulation following exposure to a tanning lamp. When cells were exposed first to broad-spectrum UVB, then the tanning lamp, UVB-induced cytokine secretion was inhibited, although mRNA levels were upregulated to a level close to that observed with UVB alone. By using a Schott WG 320 nm filter to attenuate the level of UVB relative to UVA emitted by the tanning lamp, the inhibition of cytokine secretion was shown to be associated with UVA exposure. Both UV sources inhibited IFN-gamma-induced ICAM-1 mRNA expression in a dose-dependent fashion. By using a Schott WG 335 nm filter, inhibition of ICAM-1 mRNA expression by the tanning lamp was shown to be associated with UVB exposure. CONCLUSIONS: These results suggest that UV sources emitting different levels of UVA and UVB have differential effects on the modulation of different immunoregulatory molecules, and indicate that there are potential interactions between these wavelengths.