SIRT1 modulates expression of matrix metalloproteinases in human dermal fibroblasts

Background SIRT1, an NAD⁺‐dependent histone/protein deacetylase, controls a broad range of cellular functions. Objectives We examined if SIRT1 is involved in the regulation of matrix metalloproteinase (MMP) expression in human dermal fibroblasts. Methods We studied the effect of inhibition of SIRT1 by specific inhibitor and small interfering RNA (siRNA) on MMP‐1 and MMP‐3 expression in human dermal fibroblasts. Results Treatment with a potent and selective inhibitor of SIRT1, EX‐527, increased the basal expression levels of MMP‐1 and MMP‐3 proteins. Knockdown of endogenous SIRT1 by siRNA led to increased expression of MMP‐1 and MMP‐3 at both mRNA and protein levels. SIRT1 knockdown also upregulated MMP protein induction caused by an inflammatory cytokine, interleukin (IL)‐1β. Moreover, treatment with a SIRT1 activator, resveratrol, significantly suppressed IL‐1β‐mediated induction of MMP‐1, which was attenuated by pretreatment with EX‐527. Finally, MMP‐1 promoter activity was increased by EX‐527 in cells treated with or without IL‐1β. Conclusions Our findings suggest that SIRT1 exerts a negative regulatory role in the production of MMP‐1 and MMP‐3 in human dermal fibroblasts.     

Interleukin-1beta and macrophage migration inhibitory factor (MIF) in dermal fibroblasts mediate UVA-induced matrix metalloproteinase-1 expression

BACKGROUND: Exposure to solar UV radiation is the main environmental factor that causes premature aging of the skin. Matrix metalloproteinases (MMP)-1 is a member of the MMP family and degrades types I and III collagens, which are the major structural components of the dermis. OBJECTIVE: We evaluated the involvement IL-1beta and macrophage migration inhibitory factor (MIF) in MMP-1 expression under ultraviolet A (UVA) irradiation. METHODS: IL-1beta and MIF in MMP-1 expression in cultured human dermal fibroblasts and the UVA effects on MMPs production using IL-1alpha/beta-deficient mice were analyzed. Furthermore, fibroblasts derived from MIF-deficient mice were used to analyze the effect of IL-1beta-induced MMPs production. RESULTS: IL-1beta-enhanced MIF expression and induced MMP-1 in cultured human dermal fibroblasts. IL-1beta-induced MMP-1 expression is inhibited by neutralizing anti-MIF antibody. Dermal fibroblasts of IL-1alpha/beta-deficient mice produced significantly decreased levels of MMPs compared to wild-type mice after UVA irradiation. Furthermore, fibroblasts of MIF-deficient mice were much less sensitive to IL-1beta-induced MMPs production. On the contrary, IL-1beta produced significantly decreased levels of MMPs in MIF-deficient mice fibroblasts. The up-regulation of MMP-1 mRNA by IL-1beta stimulation was found to be inhibited by a p38 inhibitor and a JNK inhibitor. In contrast, the MEK inhibitor and inhibitor were found to have little effect on expression of MMP-1 mRNA. CONCLUSIONS: IL-1beta is involved in the up-regulation of UVA-induced MMP-1 in dermal fibroblasts, and IL-1beta and MIF cytokine network induce MMP-1 and contribute to the loss of interstitial collagen in skin photoaging.     

Matrix metalloproteinase-19 expression in dermal wounds and by fibroblasts in culture

Here, we have examined the expression of matrix metalloproteinase-19 (MMP-19) in human cutaneous wounds and by human skin fibroblasts in culture. Expression of MMP-19 was detected by immunohistochemistry in fibroblasts, capillary endothelial cells, and macrophages in the dermal layer of large granulating wounds, as well as in chronic venous and decubitus ulcers. MMP-19 mRNA expression and pro-MMP-19 production by dermal fibroblasts in culture was potently enhanced by tumor necrosis factor-alpha (TNF-alpha). Induction of MMP-19 expression by TNF-alpha was prevented partially by blocking the activation of extracellular signal-regulated kinase (ERK)-1/2 by PD98059 and p38 activity by SB203580. Activation of ERK1/2 by adenovirus-mediated delivery of constitutively active MAPK/ERK kinase 1 resulted in the induction of MMP-19 expression. Activation of p38 alone by adenovirally delivered constitutively active MAPK kinase 3b (MKK3b) and MKK6b also enhanced MMP-19 production, and the most potent induction of MMP-19 expression was noted when ERK1/2 was activated in combination with p38. Activation of c-Jun N-terminal kinase (NK). Abundant pro-MMP-19 production by fibroblasts was associated with proteolytic processing of secreted pro-MMP-19. These results suggest a role of MMP-19 in cutaneous wound repair and identify three distinct signaling pathways, which coordinately mediate induction of MMP-19 expression in fibroblasts: mitogen-activated ERK1/2 pathway and stress-activated JNK and p38 pathways, of which control proteolytic activity of dermal fibroblasts.     

Fibroblasts support migration of monocyte‐derived dendritic cells by secretion of PGE2 and MMP‐1

The outcome of a cutaneous immune response is critically dependent upon the ability of dendritic cells (DC) to migrate from skin to the draining lymph nodes – a process that is influenced by the cutaneous tissue microenvironment. Here, the role of fibroblasts – a major component of the dermal microenvironment – on the migratory capacity of monocyte‐derived DC (MoDC) was investigated in a 3D collagen I matrix. Indeed, dermal fibroblasts supported the migration of pre‐activated MoDC through a 3D collagen I matrix. Activation of human MoDC resulted in the release of TNFα and IL‐1β that in turn stimulated MMP‐1 (human collagenase) and PGE₂ secretion by human dermal fibroblasts. Transmigration assays confirmed the importance of fibroblast‐derived MMP‐1 and PGE₂ for the migration of MoDC through a 3D collagen I matrix. Finally, in mice initiation of inflammation by induction of an irritant contact dermatitis or a psoriasis‐like skin inflammation, the expression of the PGE₂ generating cox‐2 and the mouse collagen I degrading enzyme matrix metalloproteinases (MMP)‐13 was strongly up‐regulated. Our study indicates that MoDC are able to instruct dermal fibroblasts resulting in enhanced migratory capability of MoDC, thus highlighting the role of a crosstalk of DC with their stromal microenvironment for the control of cutaneous immune responses.     

Akt inhibition up‐regulates MMP1 through a CCN2‐dependent pathway in human dermal fibroblasts

Please cite this article as: Akt inhibition up‐regulates MMP1 through a CCN2‐dependent pathway in human dermal fibroblasts. Experimental Dermatology 2010. Abstract: Akt is a key signalling molecule that was found to be down‐regulated in chronic wounds. Akt blockade has dual antifibrotic effects in human dermal fibroblasts, by up‐regulating matrix metalloproteinase 1 (MMP1) and down‐regulating collagen gene expression (J Invest Dermatol 2008: 128: 1906). The aim of this study was to gain additional insights into the mechanism of MMP1 up‐regulation following Akt blockade. As previous studies showed that CCN2 can be a positive regulator of MMP1, we examined the effects of Akt inhibition on CCN2 expression. Akt blockade using a specific pharmacological inhibitor and Akt siRNA resulted in a significant up‐regulation of CCN2, which correlated with the increase in MMP1. The MMP1 up‐regulation following Akt blockade was partially suppressed by CCN2 siRNA, suggesting that CCN2 is contributing to this effect. Additional experiments showed that CCN2 induces phosphorylation of ERK1/2, Ets1 and c‐Jun. Consistent with the stimulatory role of ERK1/2/Ets1 in the expression of MMP1, the ERK1/2 inhibitor UO126 prevented the phosphorylation of ERK1/2 and Ets1 and completely abrogated the induction of MMP1 after CCN2 overexpression, while having no effect on c‐Jun activation. Taken together these results establish CCN2 as a key regulator of MMP1 induction via activation of the ERK1/2/Ets1 pathway. Down‐regulation of Akt signalling leads to inappropriate activation of the CCN2/MMP1 pathway that may contribute to the pathogenesis of chronic wounds. Coordinate expression of CCN2, Akt and MMP1 could be important for normal wound healing to occur. Thus, targeting these specific proteins may represent a promising approach to the therapy of dysregulated wound healing.     

Pentoxifylline inhibits certain constitutive and tumor necrosis factor-alpha-induced activities of human normal dermal fibroblasts.

Pentoxifylline (PFN), analog of theobromine, which phenotypically and functionally alters various cell types including dermal fibroblasts, has been reported to inhibit tumor necrosis factor-alpha (TNF alpha) activation of neutrophils. We investigated the ability of PFN to alter constitutive and TNF alpha-induced biosynthetic activities of human normal dermal fibroblasts. The sixteenfold increase over constitutive intracellular 2'-5' oligo-adenylate synthetase (2'-5' A synthetase) activity induced by TNF alpha (400 U/ml) failed to occur when PFN (1 mg/ml) was added prior to cytokine treatment. This loss of biologic activity paralleled a reduction in 2'-5' A synthetase proteins and 2'-5' A synthetase-specific m-RNA. PFN failed to inhibit constitutive or TNF alpha-induced IL-6 hybridoma proliferative activity, IL-6 protein, or IL-6-specific m-RNA levels. The presence of PFN (1 mg/ml) in fibroblast cultures reduced constitutive synthesis of collagen and glycosaminoglycan (GAG) by 87% and 45%, respectively, and blocked induction of their synthesis by TNF alpha (10(4) U/ml). Total non-collagenous protein synthesis was not inhibited following PFN treatment (1 mg/ml). PFN did not inhibit TNF alpha induction of only those biosynthetic activities also susceptible to PFN in the constitutive state, with PFN failing to reduce constitutive collagenolytic activity but reducing TNF alpha-induced enhanced collagenolytic activity by 26% and collagenase m-RNA by 51%. Furthermore, PFN did inhibit, by 98%, TNF alpha-dependent murine and human fibroblast cytotoxicity. The selective nature of PFN inhibition of certain TNF alpha activities, the failure of PFN (1 mg/ml) to alter constitutive and TNF alpha-induced levels of type 1 and 2 TNF alpha receptor m-RNA, and the finding that PFN-treated fibroblasts express a similar number of receptors, of similar molecular weight and high affinity for TNF alpha as control, untreated cells, suggest that inhibitory activities of PFN are mediated at a locus other than receptors for TNF alpha.     

Inhibition of ATP-binding cassette transporter downregulates interleukin-1beta-mediated autocrine activation of human dermal fibroblasts.

Fibroblasts constitute an important source of cytokines during inflammatory processes in the skin. Interleukin-1 is a potent, pleiotropic cytokine that is induced in activated human dermal fibroblasts. Interleukin-1 further induces many inflammatory mediators, including the chemokine interleukin-8. As fibroblasts express both interleukin-1 and the interleukin-1 receptor complex, the cellular response may be enhanced by autocrine activation. Interleukin-1alpha and interleukin-1beta lack a signal peptide and are translocated at the plasma membrane using an alternative secretory pathway, which may involve ATP-binding cassette transporter proteins. We hypothesize that inhibition of this pathway prevents secretion of interleukin-1, thereby downregulating interleukin-1-dependent autocrine induction of interleukin-8. We used the ATP-binding cassette 1 transporter inhibitor glybenclamide, which has been previously shown to block interleukin-1beta secretion in human monocytes. Using enzyme-linked immunosorbent assay, we assessed the effect of glybenclamide on interleukin-8 production in human dermal fibroblasts. In interleukin-1beta-transfected human dermal fibroblasts, interleukin-8 was induced through an autocrine activity of interleukin-1beta. Glybenclamide disabled this activation loop and significantly reduced interleukin-8. In human dermal fibroblasts that were stimulated with tumor necrosis factor alpha to reach high interleukin-1 expression levels, glybenclamide similarly suppressed interleukin-8. In contrast, glybenclamide did not affect interleukin-8 production in cells stimulated with interleukin-1 only. Glybenclamide did not affect caspase-1 in fibroblasts, which was expressed as an inactive precursor form, irrespective of treatments with tumor necrosis factor alpha and/or glybenclamide. Using overexpressing, interleukin-1-transfected COS-1 cells, inhibition of interleukin-1alpha and interleukin-1beta secretion was directly demonstrated on Western blots. These results are consistent with glybenclamide preventing externalization of interleukin-1 and subsequent autocrine induction of interleukin-8 in human dermal fibroblasts. Acting through such a mechanism, ATP-binding cassette transporter inhibitors may downregulate inflammation locally.     

Akt blockade downregulates collagen and upregulates MMP1 in human dermal fibroblasts

Acutely transforming retrovirus AKT8 in rodent T-cell lymphoma (Akt) is a serine/threonine kinase that plays important roles in survival, cell-cycle progression, and cell proliferation, and has recently been implicated in collagen regulation. The aim of this study was to determine the role of Akt in collagen deposition by normal dermal fibroblasts, and to determine the sensitivity of cultured systemic sclerosis (SSc) fibroblasts to Akt inhibition. We show that blockade of Akt using pharmacological inhibitors, small interfering RNA (siRNA), and a dominant-negative Akt mutant led to inhibition of the basal type I collagen production. Furthermore, inhibition of Akt upregulated basal matrix metalloproteinase 1 (MMP1) production and reversed the inhibitory effect of transforming growth factor-beta (TGF-beta) on MMP1 gene expression. In addition, SSc fibroblasts were more sensitive to Akt inhibition, with respect to collagen and MMP1 production. These findings suggest that in human dermal fibroblasts, Akt has dual profibrotic effects, increasing collagen synthesis and decreasing its degradation via downregulation of MMP1. Akt could directly contribute to elevated collagen in SSc fibroblasts and it may represent an attractive target for therapy of SSc fibrosis.     

Iron sensitizes keratinocytes and fibroblasts to UVA‐mediated matrix metalloproteinase‐1 through TNF‐α and ERK activation

Abstract: Oestrogen deficiency is regarded as the main causative factor in postmenopausal skin ageing and photoageing. While women after menopause experience low levels of oestrogen because of cease of ovarian function, they are also exposed to high levels of iron as a result of cessation of menstruation. In this study, we investigated whether this increase in iron presents a risk to the postmenopausal skin. Because of the lack of appropriate animal models to closely mimic the low oestrogen and high iron conditions, we tested the hypothesis in a high iron and low oestrogen culture model. Here, we showed that primary human dermal fibroblasts exposed to iron did not affect the baseline levels of matrix metalloproteinase‐1 (MMP‐1) activity. However, the iron‐exposed fibroblasts were sensitized to UVA exposure, which resulted in a synergistic increase in MMP‐1. UVA activated the three members of MAPK family: ERKs, p38, and JNKs. Additional activation of ERKs by iron contributed to the synergistic increases. Primary normal human epidermal keratinocytes (NHEK) did not respond to iron or UVA exposure as measured by MMP‐1, but produced tumor necrosis factor‐alpha (TNF‐α) in the media, which then stimulated MMP‐1 in fibroblasts. Our results indicate that iron and UVA increase MMP‐1 activity in dermal fibroblasts not only directly through ERK activation but also by an indirect paracrine loop through TNF‐α released by NHEK. We conclude that in addition to oestrogen deficiency, increased iron as a result of menopause could be a novel risk factor by sensitizing postmenopausal skin to solar irradiation.     

Induction of manganese superoxide dismutase in human dermal fibroblasts: a UV-B-mediated paracrine mechanism with the release of epidermal interleukin 1 alpha,interleukin 1 beta,and tumor necrosis factor alpha

BACKGROUND: Reactive oxygen species generated in the skin by UV irradiation promote photoaging and photocarcinogenesis. The manganese (Mn) superoxide dismutase (SOD) is a primary antioxidant enzyme that crucially contributes to the homeostasis of oxygen radicals within the mitochondria, and thus critically participates in the control of senescence and tumor generation. OBJECTIVE: To determine whether repetitive UV-B exposure, as practiced for light hardening during phototherapy for various photodermatoses, can enhance the adaptive antioxidant response by up-regulating MnSOD activity in either the epidermal or the dermal skin compartment. DESIGN: In vitro experiments to determine MnSOD activity levels in cultured human dermal fibroblasts and epidermal cells (HaCaT cells and primary keratinocytes) at different times after direct UV-B exposure or after incubation of human dermal fibroblasts with supernatants from UV-B-irradiated epidermal cells. SETTING: Photobiological research laboratory in a university dermatology department. INTERVENTION: Irradiation of cultured human dermal fibroblasts and epidermal cells with UV-B. MAIN OUTCOME MEASURES: Manganese SOD messenger RNA and activity levels in cultured irradiated or mock-treated skin cells. RESULTS: No increase in MnSOD activity could be detected in fibroblasts or epidermal cells until 24 hours after UV-B irradiation. However, fibroblasts incubated with supernatants from UV-B-irradiated epidermal cells showed a marked increase in specific MnSOD messenger RNA and activity. Removal of interleukin 1alpha, interleukin 1beta, and tumor necrosis factor alpha from the supernatants led to a significant reduction of MnSOD mRNA in fibroblasts. CONCLUSION: Irradiation of the epidermal cells with UV-B induced a release of soluble factors that amplified MnSOD activity in fibroblasts via a paracrine mechanism.     

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.     

Differential regulation by IL-1beta and EGF of expression of three different hyaluronan synthases in oral mucosal epithelial cells and fibroblasts and dermal fibroblasts: quantitative analysis using real-time RT-PCR

Using "real-time RT-PCR", we assessed the expression of three different hyaluronan synthase genes, HAS1, HAS2, and HAS3, by measuring their mRNA amounts in cultured human oral mucosal epithelial (COME) cells, oral mucosal fibroblasts, and dermal fibroblasts, and investigated the effects of interleukin-1beta (IL-1beta) and epidermal growth factor (EGF). When COME cells were treated with IL-1beta or EGF, early and marked increases and subsequent rapid decreases were observed for all HAS genes and, moreover, actual changes in hyaluronan synthesis subsequently occurred. The effects of IL-1beta stimulation were concentration-dependent and the maximal response to the EGF stimulation was observed at a low concentration (0.1 ng per mL). When two different types of fibroblasts were treated with IL-1beta or EGF, increased expression with different degrees and rates of three different HAS genes and subsequent increased synthesis of hyaluronan were also observed. In addition, HAS1 gene expression was not detectable in the mucosal fibroblasts, while weak HAS3 gene expression was detected in the dermal fibroblasts. Taken together, it is likely that the regulation of the expression of the three different HAS genes is different between oral mucosa and skin, which may be of significance for elucidating some of the differences between these tissues in wound healing.     

Alterations in fibroblast alpha1beta1 integrin collagen receptor expression in keloids and hypertrophic scars

Keloids and hypertrophic scars are significant symptomatic clinical problems characterized by excess collagen. Although extensive research has focused on fibroblasts and collagen turnover in these aberrant scars, little work has been done on the expression of integrins (cell membrane structures that link cells to extracellular matrix) within these lesions. Integrin-mediated regulation of collagen synthesis has previously been observed in explanted fibroblasts from normal and fibrotic dermis, and integrin alpha1 knockout mice maintain increased collagen synthesis consistent with a role for alpha1beta1 in providing negative feedback on collagen synthesis. These findings suggested the need to evaluate integrin roles in keloids and hypertrophic scars. In this study we examined integrin expression in keloids (n = 11), hypertrophic scars (n = 5), radiation ulcers (n = 2), and normal skin specimens (n = 8). We used a novel approach to analysis by isolating dermal fibroblasts directly from tissue (without explant culture) and determining surface integrin expression by flow cytometry. We found that keloids and hypertrophic scars have marked alterations in fibroblast integrin expression and contain several distinct populations of fibroblasts. One of these populations expresses high levels of alpha1 integrin, and the proportion of these cells is higher in keloids (63% +/- 3.6% SEM) and hypertrophic scars (45% +/- 2.7% SEM) than in normal skin tissues (28% +/- 4.7% SEM). The different populations of fibroblasts defined by integrin expression merge, however, when the cells are serially cultured, suggesting that there may be aspects of the dermal microenvironment that maintain the integrin phenotypic heterogeneity in dermal fibroblasts.     

Keratinocyte-derived cytokines after photodynamic therapy and their paracrine induction of matrix metalloproteinases in fibroblasts

BACKGROUND: Recent studies have shown that collagen-degrading matrix metalloproteinase (MMP)-1 and MMP-3 are produced by fibroblasts in response to photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) and are considered to be involved in the antisclerotic effects of ALA-PDT observed in the treatment of localized scleroderma. OBJECTIVES: As the primary target of topical PDT is epidermal keratinocytes, we studied the indirect participation of keratinocytes in the production of MMPs and collagen by dermal fibroblasts. METHODS: Keratinocytes were treated with sublethal doses of ALA (100 micromol L(-1)) and red light. The conditioned media were collected 24 h after PDT and primary human fibroblasts were exposed to these media for 6-48 h. Further, a coculture model, keratinocytes seeded on to collagen type IV-coated transwells in the upper chamber and fibroblasts in the lower chamber, was used to study paracrine effects of keratinocytes after PDT. RESULTS: Keratinocyte supernatants after PDT showed a significant, up to 10-fold increase of interleukin (IL)-1alpha and a 2.5-fold increase of tumour necrosis factor-alpha as determined by enzyme-linked immunosorbent assay, while IL-6, MMP-1 and MMP-3 were not altered significantly. Fibroblasts treated with keratinocyte-conditioned media after PDT showed an induction of MMP-1 and MMP-3 protein levels up to threefold in both models used, suggesting that ALA-PDT modulates MMP-1 and MMP-3 production via indirect mechanisms. Collagen type I mRNA expression by fibroblasts was not altered significantly in either model. The addition of an IL-1 receptor antagonist to the keratinocyte-conditioned media completely inhibited the induction of MMP-1 and MMP-3 in stimulated fibroblasts, suggesting that IL-1 is mainly responsible for the observed paracrine effects. CONCLUSIONS: We present evidence that PDT can trigger MMP production in dermal fibroblasts not only directly as has been already shown, but also by an indirect paracrine loop mediated by soluble factors released by epidermal keratinocytes.     

Modulation of intercellular adhesion molecule-1 expression on human melanocytes and melanoma cells: evidence for a regulatory role of IL-6, IL-7, TNF beta, and UVB light.

Intercellular adhesion molecule-1 (ICAM-1) is involved in cell-cell interactions of leukocytes and parenchymal cells and thus plays an important role in immunologic and inflammatory reactions. The expression of ICAM-1 that is found on many different cells such as melanocytes and melanoma cells is induced by various cytokines, including interferon-gamma (IFN gamma), interleukin (IL)-1 and tumor necrosis factor alpha (TNF alpha). Because expression of ICAM-1 in melanoma was found to correlate with increased risk of metastasis, the regulation of ICAM-1 expression on human melanocytes and melanoma cells was investigated. Foreskin-derived melanocytes and melanoma cell lines (A375, G361) were incubated with different cytokines and ICAM-1 expression was evaluated by fluorescence-activated cell sorter. IFN gamma, IL-1, IL-7, TNF alpha, and TNF beta significantly upregulated ICAM-1 expression in a dose-dependent manner. Most interestingly, the cytokine IL-6, which does not influence adhesion-molecule expression on other cells, significantly upregulated melanocyte and melanoma cell ICAM-1 expression. This effect was dose dependent and could be blocked by an IL-6 antibody. Irradiation with ultraviolet (UVB) light did not influence constitutive ICAM-1 expression on melanoma cells and melanocytes, but suppressed cytokine-induced ICAM-1 expression when cells were harvested 16 h after irradiation. These findings were further confirmed by Northern blot analysis, showing a marked accumulation of ICAM-1 mRNA after cytokine treatment, which was reduced by irradiation with UVB light. However, when UVB-exposed melanoma cells were cultured for at least 48 h induction of ICAM-1 expression was observed. These data indicate that, similar to other cells, ICAM-1 expression on melanoma cells and melanocytes is regulated by cytokines and that UVB light affects ICAM-1 expression on melanocytic cells in a biphasic manner.     

Protection against TGF-β1-induced fibrosis effects of IL-10 on dermal fibroblasts and its potential therapeutics for the reduction of skin scarring

Scarring, tightly associated with fibrosis, is a significant symptomatic clinical problem. Interleukin 10 (IL-10) has been identified as a candidate scar-improving therapy based on preclinical studies. However, the molecular mechanism of IL-10 in scar improvement is still uncertain. In this study, human dermal fibroblasts stimulated with TGF-β1 were treated with IL-10 to analyze the mRNA and some of proteins’ expression levels of type I collagen (Col1), type III collagen (Col3), alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-1 (MMP1), MMP2, MMP8 and tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2 by real-time PCR and Western blot, to observe α-SMA-positive fibroblasts by immunocytochemistry. The contracture and improvement of fibroblast-populated collagen lattice (FPCL) and a murine model of wound healing were used to evaluate the scar-improving effects by histological staining. The results showed that IL-10 can significantly down-regulate the mRNA and protein expression levels of Col1, Col3, α-SMA, and up-regulate the mRNA expression levels of MMP1 and MMP8, and decrease α-SMA-positive fibroblasts. FPCL analysis showed that the IL-10 (20 ng/ml) can significantly inhibit the contracture, improve the architecture of FPCL. Wounds injected with IL-10 demonstrated that the appearance of scar was improved, the wound margin of scarring was narrow, and the deposition of collagens (Col1 and Col3) in regenerated tissue was relieved. These results provide direct evidences that IL-10 has the inhibitory effects on the excessive deposition of extracellular matrix components and fibroblast-to-myofibroblast transition, and show that IL-10 has the potential therapy in prevention and reduction of skin scarring.