Elevated YAP and its downstream targets CCN1 and CCN2 in basal cell carcinoma: impact on keratinocyte proliferation and stromal cell activation.

Yes-associated protein (YAP) is a transcriptional co-activator of hippo signaling pathway, which plays an important role in organ size control and tumorigenesis. Here we report that YAP and its downstream transcriptional targets CCN1 and CCN2 are markedly elevated in keratinocytes in human skin basal cell carcinoma tumor islands. In human keratinocytes, knockdown of YAP significantly reduced expression of CCN1 and CCN2, and repressed proliferation and survival. This inhibition of proliferation and survival was rescued by restoration of CCN1 expression, but not by CCN2 expression. In basal cell carcinoma stroma, CCN2-regulated genes type I collagen, fibronectin, and α-smooth muscle actin were highly expressed. Furthermore, atomic force microscopy revealed increased tissue stiffness in basal cell carcinoma stroma compared to normal dermis. These data provide evidence that up-regulation of YAP in basal cell carcinoma impacts both aberrant keratinocyte proliferation, via CCN1, and tumor stroma cell activation and stroma remodeling, via CCN2. Targeting YAP and/or CCN1 and CCN2 may provide clinical benefit in basal cell carcinoma.

Ultraviolet irradiation represses TGF-ß type II receptor transcription through a 38-bp sequence in the proximal promoter in human skin fibroblasts.

Transforming growth factor-ß (TGF-ß) is a major regulator of collagen gene expression in human skin fibroblasts. Cellular responses to TGF-ß are mediated primarily through its cell surface type I (TßRI) and type II (TßRII) receptors. Ultraviolet (UV) irradiation impairs TGF-ß signalling largely due to reduced TßRII gene expression, thereby decreasing type I procollagen synthesis, in human skin fibroblasts. UV irradiation does not alter either TßRII mRNA or protein stability, indicating that UV reduction in TßRII expression likely results from transcriptional or translational repression. To understand how UV irradiation regulates TßRII transcription, we used a series of TßRII promoter-luciferase 5'-deletion constructs (covering 2 kb of the TßRII proximal promoter) to determine transcriptional rate in response to UV irradiation. We identified a 137-bp region upstream of the transcriptional start site that exhibited high promoter activity and was repressed 60% by UV irradiation, whereas all other TßRII promoter reporter constructs exhibited either low promoter activities or no regulation by UV irradiation. Mutation of potential transcription factor binding sites within the promoter region revealed that an inverted CCAAT box (-81 bp from transcription start site) is required for promoter activity. Mutation of the CCAAT box completely abolished UV irradiation regulation of the TßRII promoter. Protein-binding assay, as determined by electrophoretic mobility-shift assays (EMSAs) using the inverted CCAAT box as probe (-100/-62), demonstrated significantly enhanced protein binding in response to UV irradiation. Super shift experiments indicated that nuclear factor Y (NFY) is able to binding to this sequence, but NFY binding was not altered in response to UV irradiation, indicating additional protein(s) are capable of binding this sequence in response to UV irradiation. Taken together, these data indicate that UV irradiation reduces TßRII expression, at least partially, through transcriptional repression. This repression is mediated by a 38-bp sequence in TßRII promoter, in human skin fibroblasts.

The effects of carbon pricing instruments on carbon emission reduction in China's refining industry: an evolutionary game between heterogeneous refineries.

Carbon emissions from the refining industry are receiving increasing national attention. In view of long-term sustainable development, a carbon pricing mechanism oriented to carbon emission reduction needs to be developed. Currently, the two most common carbon pricing instruments are emission trading system and carbon tax. Therefore, it is important to study the carbon emission problems in the refining industry under emission trading system or carbon tax. Based on the current situation of China's refining industry, this paper constructs an evolutionary game model for backward and advanced refineries to explore which instrument is more effectively applied in the refining industry and identify the effective factors that can promote carbon emission reduction in refineries. According to the numerical results, if the heterogeneity of enterprises is small, the government's implementation of an emission trading system is the most effective measure, while carbon tax can only ensure that the equilibrium strategy solution is (1,1) when the tax rate is high. If the heterogeneity is large, the carbon tax policy will not have any effect, indicating that government implementation of an emission trading system is more effective than the carbon tax. In addition, there is a positive relationship between carbon price, carbon tax, and refineries' agreement to carbon emission reduction. Finally, consumers' preference for low-carbon products, R&D investment level, and R&D spillover effect have nothing to do with carbon emission reduction. Only by reducing refinery heterogeneity and improving the R&D efficiency of backward refineries can all enterprises agree to carbon emission reduction.

Age-related changes in dermal collagen physical properties in human skin.

Collagen is the major structural protein in the skin. Fragmentation and disorganization of the collagen fibrils are the hallmarks of the aged human skin dermis. These age-related alterations of collagen fibrils impair skin structural integrity and make the tissue microenvironment more prone to skin disorders. As the biological function of collagen lies predominantly in its physical properties, we applied atomic force microscopy (AFM) and nanoindentation to evaluate the physical properties (surface roughness, stiffness, and hardness) of dermal collagen in young (25±5 years, N = 6) and aged (75±6 years, N = 6) healthy sun-protected hip skin. We observed that in the aged dermis, the surface of collagen fibrils was rougher, and fiber bundles were stiffer and harder, compared to young dermal collagen. Mechanistically, the age-related elevation of matrix metalloproteinase-1 (MMP-1) and advanced glycation end products (AGEs) are responsible for rougher and stiffer/harder dermal collagen, respectively. Analyzing the physical properties of dermal collagen as a function of age revealed that alterations of the physical properties of collagen fibrils changed with age (22-89 years, N = 18). We also observed that the reticular dermis is rougher and mechanically stiffer and harder compared to the papillary dermis in human skin. These data extend the current understanding of collagen beyond biological entities to include biophysical properties.

CCN1 is predominantly elevated in human skin dermis by solar-simulated ultraviolet irradiation and accumulated in dermal extracellular matrix.

Skin primarily comprises a collagen-rich extracellular matrix (ECM) that provides structural and functional support to the skin. Aging causes progressive loss and fragmentation of dermal collagen fibrils, leading to thin and weakened skin (Dermal aging). We previously reported that CCN1 is elevated in naturally aged human skin, photoaged human skin, and acute UV-irradiated human skin dermal fibroblasts in vivo. Elevated CCN1 alters the expression of numerous secreted proteins that have deleterious effects on the dermal microenvironment, impairing the structural integrity and function of the skin. Here we show that CCN1 is predominantly elevated in the human skin dermis by UV irradiation and accumulated in the dermal extracellular matrix. Laser capture microdissection indicated that CCN1 is predominantly induced in the dermis, not in the epidermis, by acute UV irradiation in human skin in vivo. Interestingly, while UV-induced CCN1 in the dermal fibroblasts and in the medium is transient, secreted CCN1 accumulates in the ECM. We explored the functionality of the matrix-bound CCN1 by culturing dermal fibroblasts on an acellular matrix plate that was enriched with a high concentration of CCN1. We observed that matrix-bound CCN1 activates integrin outside-in signaling resulting in the activation of FAK and its downstream target paxillin and ERK, as well as elevated MMP-1 and inhibition of collagen, in human dermal fibroblasts. These data suggest that accumulation of CCN1 in the dermal ECM is expected to progressively promote the aging of the dermis and thereby negatively impact the function of the dermis.

Matrix Metalloproteinase-1 Expression in Fibroblasts Accelerates Dermal Aging and Promotes Papilloma Development in Mouse Skin.

Fragmentation, disorganization, and depletion of the collagen-rich dermal extracellular matrix are hallmarks of aged human skin. These deleterious alterations are thought to critically mediate many of the prominent clinical attributes of aged skin, including thinning, fragility, impaired wound healing, and a propensity for carcinoma. Matrix metalloproteinase-1 (MMP1) initiates the cleavage of collagen fibrils and is significantly increased in dermal fibroblasts in aged human skin. To investigate the role of elevated MMP1 in skin aging, we generated a conditional bitransgenic mouse (type I collagen alpha chain 2; human MMP1 [Col1a2;hMMP1]) that expresses full-length, catalytically active hMMP1 in dermal fibroblasts. hMMP1 expression is activated by a tamoxifen-inducible Cre recombinase that is driven by the Col1a2 promoter and upstream enhancer. Tamoxifen induced hMMP1 expression and activity throughout the dermis Col1a2:hMMP1 mice. At 6 months of age, Col1a2;hMMP1 mice displayed loss and fragmentation of dermal collagen fibrils, which was accompanied by many of the features of aged human skin, such as contracted fibroblast morphology, reduced collagen production, increased expression of multiple endogenous MMPs, and proinflammatory mediators. Interestingly, Col1a2;hMMP1 mice displayed substantially increased susceptibility to skin papilloma development. These data demonstrate that fibroblast expression of hMMP1 is a critical mediator of dermal aging and creates a dermal microenvironment that promotes keratinocyte tumor development.

Age-Related Downregulation of CCN2 Is Regulated by Cell Size in a YAP/TAZ-Dependent Manner in Human Dermal Fibroblasts: Impact on Dermal Aging.

CCN2, a member of the CCN family of matricellular proteins, is a key mediator and biomarker of tissue fibrosis. We previously reported that CCN2 is significantly reduced in aged human dermis, which contributes to dermal aging through the downregulation of collagen production, the major structural protein in the skin. In this study, we investigated the underlying mechanisms of the age-related downregulation of CCN2 in human skin dermal fibroblasts. Dermal fibroblasts isolation and laser-capture microdissection‒coupled RT-PCR from human skin confirmed that age-related reduction of CCN2 expression is regulated by epigenetics. Mechanistic investigation revealed that age-related reduction of CCN2 is regulated by impaired dermal fibroblast spreading/cell size, which is a prominent feature of aged dermal fibroblasts in vivo. Gain-of-function and loss-of-function analysis confirmed that age-related downregulation of CCN2 is regulated by YAP/TAZ in response to reduced cell size. We further confirmed that restoration of dermal fibroblast size rapidly reversed the downregulation of CCN2 in a YAP/TAZ-dependent manner. Finally, we confirmed that reduced YAP/TAZ nuclear staining is accompanied by loss of CCN2 in aged human skin in vivo. Our data reveal a mechanism by which age-related reduction in fibroblast spreading/size drives YAP/TAZ-dependent downregulation of CCN2 expression, which in turn contributes to loss of collagen in aged human skin.

Collagen fragmentation promotes oxidative stress and elevates matrix metalloproteinase-1 in fibroblasts in aged human skin.

Aged human skin is fragile because of fragmentation and loss of type I collagen fibrils, which confer strength and resiliency. We report here that dermal fibroblasts express increased levels of collagen-degrading matrix metalloproteinases-1 (MMP-1) in aged (>80 years old) compared with young (21 to 30 years old) human skin in vivo. Transcription factor AP-1 and alpha2beta1 integrin, which are key regulators of MMP-1 expression, are also elevated in fibroblasts in aged human skin in vivo. MMP-1 treatment of young skin in organ culture causes fragmentation of collagen fibrils and reduces fibroblast stretch, consistent with reduced mechanical tension, as observed in aged human skin. Limited fragmentation of three-dimensional collagen lattices with exogenous MMP-1 also reduces fibroblast stretch and mechanical tension. Furthermore, fibroblasts cultured in fragmented collagen lattices express elevated levels of MMP-1, AP-1, and alpha2beta1 integrin. Importantly, culture in fragmented collagen raises intracellular oxidant levels and treatment with antioxidant MitoQ(10) significantly reduces MMP-1 expression. These data identify positive feedback regulation that couples age-dependent MMP-1-catalyzed collagen fragmentation and oxidative stress. We propose that this self perpetuating cycle promotes human skin aging. These data extend the current understanding of the oxidative theory of aging beyond a cellular-centric view to include extracellular matrix and the critical role that connective tissue microenvironment plays in the biology of aging.

Limethason reduces airway inflammation in a murine model of ovalbumin-induced chronic asthma without causing side effects.

Airway inflammation is the major pathological feature of asthma. Thus, the current therapeutic strategy for asthma is to control inflammation. Limethason, an anti-inflammation drug, is widely used in rheumatoid arthritis treatment. The aim of the present study was to detect the anti-inflammatory effect and side effects of limethason on airways that were sensitized with ovalbumin in a murine model of chronic asthma. In the present study, BALB/c mice were sensitized with ovalbumin. Airway hyperresponsiveness was estimated, and hematoxylin and eosin staining, Periodic acid-Schiff staining and bronchoalveolar lavage were used to detect the effect on chronic asthma. Limethason effectively reduced airway hyperresponsiveness, and inhibited inflammatory cell infiltration and mucus secretion. Bronchoalveolar lavage fluid analysis revealed that limethason suppressed levels of airway eosinophils. In the period of treatment, limethason exhibited no influence on morphology of the femoral head, bone mineral content or bone mineral density, which were detected by histological studies and dual-energy X-ray absorptiometry. The index of liver, spleen, kidney, gastrocnemius and brown adipose tissue also demonstrated that limethason had no adverse effects on organs and tissues. The present study revealed that limethason could effectively reduce inflammation in an asthma mouse model without side effects. Therefore, limethason may have therapeutic potential for treating chronic asthma clinically.

Reduction of fibroblast size/mechanical force down-regulates TGF-ß type II receptor: implications for human skin aging.

The structural integrity of human skin is largely dependent on the quality of the dermal extracellular matrix (ECM), which is produced, organized, and maintained by dermal fibroblasts. Normally, fibroblasts attach to the ECM and thereby achieve stretched, elongated morphology. A prominent characteristic of dermal fibroblasts in aged skin is reduced size, with decreased elongation and a more rounded, collapsed morphology. Here, we show that reduced size of fibroblasts in mechanically unrestrained three-dimensional collagen lattices coincides with reduced mechanical force, measured by atomic force microscopy. Reduced size/mechanical force specifically down-regulates TGF-ß type II receptor (TßRII) and thus impairs TGF-ß/Smad signaling pathway. Both TßRII mRNA and protein were decreased, resulting in 90% loss of TGF-ß binding to fibroblasts. Down-regulation of TßRII was associated with significantly decreased phosphorylation, DNA-binding, and transcriptional activity of its key downstream effector Smad3 and reduced expression of Smad3-regulated essential ECM components type I collagen, fibronectin, and connective tissue growth factor (CTGF/CCN2). Restoration of TßRII significantly increased TGF-ß induction of Smad3 phosphorylation and stimulated expression of ECM components. Reduced expression of TßRII and ECM components in response to reduced fibroblast size/mechanical force was fully reversed by restoring size/mechanical force. Reduced fibroblast size was associated with reduced expression of TßRII and diminished ECM production, in aged human skin. Taken together, these data reveal a novel mechanism that provides a molecular basis for loss of dermal ECM, with concomitant increased fragility, which is a prominent feature of human skin aging.

Computationally analyzing the possible biological function of YJL103C--an ORF potentially involved in the regulation of energy process in yeast.

Although the complete genomes of a number of organisms have been sequenced, the biological functions of many genes are still not known. Because experimentally studying the functions of those genes one by one requires tremendous time, it is vital to use published resources like microarray gene expression data for computational analysis of gene functions. One example is YJL103C, a yeast gene of unknown function in the Saccharomyces Genome Database (SGD). It is possible to quickly infer its biological function by computational analysis. In this study, we present an efficient model to explore the biological function of a novel gene using microarray data. We showed that the expression pattern of YJL103C is most similar to the genes in the energy group and respiratory chain subgroup. We further found that YJL103C contains a HAP2,3,4 box in its promoter region and a cytochrome C heme-binding signature in its protein sequence. Our findings define a potential role for YJL103C in the regulation of energy metabolism, specifically in the process of oxidative phosphorylation. Similar bioinformatics methods can be applied to infer the biological functions of other novel genes in organisms for which microarray data are available. In this work, we selected a single gene of unknown function as a case study. By focusing on the power of computer analysis and bioinformatics on the available microarray data, we have determined the likely biological function of YJL103C. Our study provides a method by which to explore the potential function of other genes currently annotated as having an unknown function in any organism for which global gene expression data are available.

Connective tissue growth factor: expression in human skin in vivo and inhibition by ultraviolet irradiation.

Connective tissue growth factor, which is induced by transforming growth factor beta, has been reported to mediate the stimulatory actions of transforming growth factor beta on type I procollagen synthesis. Connective tissue growth factor is expressed in fibrotic disease such as scleroderma, where it is believed to promote abnormal deposition of collagen. Connective tissue growth factor expression has not been described in normal human skin or cultured skin cells, however. We report here that connective tissue growth factor mRNA is constitutively expressed in normal human skin. In situ hybridization demonstrated that connective tissue growth factor mRNA was expressed in keratinocytes throughout the epidermis and in dermal cells. Quantitative real-time reverse transcription polymerase chain reaction revealed that the level of connective tissue growth factor mRNA in the epidermis and dermis of normal human skin was comparable to the level of housekeeping gene 36B4. Ultraviolet irradiation (2 minimal erythema dose, UVB/A2 source) reduced connective tissue growth factor mRNA expression throughout the epidermis and dermis in normal human skin in vivo. Connective tissue growth factor mRNA was reduced (30%) within 4 h post ultraviolet irradiation, and remained reduced (50%) 8-24 h post ultraviolet. Connective tissue growth factor mRNA and protein were also constitutively highly expressed in normal cultured human skin keratinocytes and fibroblasts. Ultraviolet irradiation of cultured normal human skin fibroblasts resulted in a time-dependent inhibition of connective tissue growth factor mRNA expression. At 24 h post ultraviolet, connective tissue growth factor mRNA expression was reduced 80%. Transforming growth factor beta1 rapidly induced connective tissue growth factor mRNA levels (5-fold within 4 h) in skin fibroblasts, but not keratinocytes, and this induction was attenuated 80% by ultraviolet irradiation. Electrophoretic mobility shift assays demonstrated that ultraviolet irradiation reduced protein binding to the transforming growth factor beta/Smad responsiveness elements in the connective tissue growth factor gene promoter, in human skin in vivo and human skin fibroblasts. Constitutive expression of connective tissue growth factor in normal human skin suggests that it is a physiologic regulator of procollagen synthesis. Ultraviolet reduction of connective tissue growth factor expression may contribute to reduced procollagen synthesis observed in ultraviolet-irradiated normal human skin and human skin fibroblasts.

Ultraviolet irradiation alters transforming growth factor beta/smad pathway in human skin in vivo.

Solar ultraviolet irradiation damages human skin and causes premature skin aging and skin cancer. As transforming growth factor beta plays an important role in regulating cell growth and extracellular matrix synthesis, we investigated expression of transforming growth factor beta isoforms, transforming growth factor beta receptors, and transforming growth factor beta regulated Smad transcription factors following irradiation with an ultraviolet B source and solar-simulated ultraviolet irradiation of human skin in vivo. Full-thickness, sun-protected adult human skin expressed transforming growth factor beta1, beta2, and beta3 transcripts in a ratio of 1:5:3, as determined by quantitative real-time reverse transcription polymerase chain reaction. Northern analysis demonstrated that the ultraviolet irradiation (2 minimal erythema dose) caused moderate (2-3-fold) gradual increases of transforming growth factor beta1 and beta3 mRNA expression during 3 d post exposure. In contrast, expression of transforming growth factor beta2 mRNA, the predominant form of transforming growth factor beta in human skin, decreased within 4 h after ultraviolet irradiation. In situ hybridization revealed transforming growth factor beta1, beta2, and beta3 mRNA expression in cells throughout the epidermis and the dermis in nonirradiated skin. Following ultraviolet or solar-simulated ultraviolet irradiation, transforming growth factor beta1 and beta3 mRNA were increased and transforming growth factor beta2 mRNA was reduced throughout the epidermis and dermis. No significant changes were observed in transforming growth factor beta type I receptor mRNA expression after ultraviolet irradiation. In contrast, transforming growth factor beta type II receptor mRNA expression was reduced 60% within 4 h following ultraviolet exposure in human skin in vivo. Transforming growth factor beta type II receptor mRNA levels remained reduced for 8 h and recovered by 24 h post ultraviolet. In situ hybridization revealed that ultraviolet or solar-simulated ultraviolet irradiation caused loss of transforming growth factor beta type II receptor mRNA in basal and suprabasal cells in the epidermis and dermal cells. In addition, no significant changes were observed in Smad2, Smad3, and Smad4 expression after ultraviolet irradiation. In contrast, ultraviolet and solar-simulated ultraviolet irradiation rapidly induced gene expression of Smad7, which antagonizes the actions of the transforming growth factor beta/Smad pathway. Smad7 mRNA induction occurred throughout the epidermis and dermal cells as determined by in situ hybridization. Ultraviolet irradiation also caused reduced DNA binding of Smad3/4 in human skin in vivo. Reduced Smad3/4 DNA binding was observed within 4 h following irradiation. Taken together, these results demonstrate that ultraviolet and solar-simulated ultraviolet irradiation alter the transforming growth factor beta/Smad pathway in human skin in vivo. Ultraviolet induction of Smad7 and reduction of transforming growth factor beta2 and transforming growth factor beta type II receptor should diminish transforming growth factor beta signaling, and probably contribute to the decrease of transforming growth factor beta regulated type I and type III procollagen gene expression observed in ultraviolet and solar-simulated ultraviolet irradiated human skin in vivo.

Oxidant exposure induces cysteine-rich protein 61 (CCN1) via c-Jun/AP-1 to reduce collagen expression in human dermal fibroblasts.

Human skin is a primary target of oxidative stress from reactive oxygen species (ROS) generated from both extrinsic and intrinsic sources. Oxidative stress inhibits the production of collagen, the most abundant protein in skin, and thus contributes to connective tissue aging. Here we report that cysteine-rich protein 61 (CCN1), a negative regulator of collagen production, is markedly induced by ROS and mediates loss of type I collagen in human dermal fibroblasts. Conversely, antioxidant N-acetyl-L-cysteine significantly reduced CCN1 expression and prevented ROS-induced loss of type I collagen in both human dermal fibroblasts and human skin in vivo. ROS increased c-Jun, a critical member of transcription factor AP-1 complex, and increased c-Jun binding to the AP-1 site of the CCN1 promoter. Functional blocking of c-Jun significantly reduced CCN1 promoter and gene expression and thus prevented ROS-induced loss of type I collagen. Targeting the c-Jun/CCN1 axis may provide clinical benefit for connective tissue aging in human skin.

Oxidative exposure impairs TGF-ß pathway via reduction of type II receptor and SMAD3 in human skin fibroblasts.

Exposure to oxidants results in cellular alterations that are implicated in aging and age-associated diseases. Here, we report that brief, low-level oxidative exposure leads to long-term elevation of cellular reactive oxygen species (ROS) levels and oxidative damage in human skin fibroblasts. Elevated ROS impairs the transforming growth factor-ß (TGF-ß) pathway, through reduction of type II TGF-ß receptor (TßRII) and SMAD3 protein levels. This impairment results in reduced expression of connective tissue growth factor (CTGF/CCN2) and type I collagen, which are regulated by TGF-ß. Restoration of TßRII and SMAD3 together, but not separately, reinstates TGF-ß signaling and increases CTGF/CCN2 and type I collagen levels. Treatment with the anti-oxidant N-acetylcysteine reduces ROS elevation and normalizes TGF-ß signaling and target gene expression. These data reveal a novel linkage between limited oxidant exposure and altered cellular redox homeostasis that results in impairment of TGF-ß signaling. This linkage provides new insights regarding the mechanism by which aberrant redox homeostasis is coupled to decline of collagen production, a hallmark of human skin aging.

Expression of catalytically active matrix metalloproteinase-1 in dermal fibroblasts induces collagen fragmentation and functional alterations that resemble aged human skin.

Increased expression of matrix metalloproteinase-1 (MMP-1) and reduced production of type I collagen by dermal fibroblasts are prominent features of aged human skin. We have proposed that MMP-1-mediated collagen fibril fragmentation is a key driver of age-related decline of skin function. To investigate this hypothesis, we constructed, characterized, and expressed constitutively active MMP-1 mutant (MMP-1 V94G) in adult human skin in organ culture and fibroblasts in three-dimensional collagen lattice cultures. Expression of MMP-1 V94G in young skin in organ culture caused fragmentation and ultrastructural alterations of collagen fibrils similar to those observed in aged human skin in vivo. Expression of MMP-1 V94G in dermal fibroblasts cultured in three-dimensional collagen lattices caused substantial collagen fragmentation, which was markedly reduced by MMP-1 siRNA-mediated knockdown or MMP inhibitor MMI270. Importantly, fibroblasts cultured in MMP-1 V94G-fragmented collagen lattices displayed many alterations observed in fibroblasts in aged human skin, including reduced cytoplasmic area, disassembled actin cytoskeleton, impaired TGF-ß pathway, and reduced collagen production. These results support the concept that MMP-1-mediated fragmentation of dermal collagen fibrils alters the morphology and function of dermal fibroblasts and provide a foundation for understanding specific mechanisms that link collagen fibril fragmentation to age-related decline of fibroblast function.

Quercetin inhibits UV irradiation-induced inflammatory cytokine production in primary human keratinocytes by suppressing NF-κB pathway.

BACKGROUND: Topical flavonoids, such as quercetin, have been shown to reduce ultraviolet (UV) irradiation-mediated skin damage. However, the mechanisms and signaling pathways involved in this protective effect are not clear. UV irradiation leads to activation of two major signaling pathways, namely nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) pathways. Activation of NF-κB pathway by UV irradiation stimulates inflammatory cytokine expression, whereas activation of AP-1 pathway by UV irradiation promotes matrix metalloproteinase (MMP) production. Both pathways contribute to UV irradiation-induced skin damage, such as photoaging and skin tumor formation. OBJECTIVE: To elucidate the underlying mechanism, we examined the effect of quercetin on UV irradiation induced activation of NF-κB and AP-1 pathways. METHODS: Primary human keratinocytes, the major skin cell type subjected to physiological solar UV irradiation, were used to study the effects of quercetin on UV irradiation-induced signal transduction pathways. RESULTS: Quercetin decreased UV irradiation-induced NF-κB DNA-binding by 80%. Consequently, quercetin suppressed UV irradiation-induced expression of inflammatory cytokines IL-1ß (∼60%), IL-6 (∼80%), IL-8 (∼76%) and TNF-α (∼69%). In contrast, quercetin had no effect on UV irradiation activation of three MAP kinases, ERK, JNK, or p38. Accordingly, induction of AP-1 target genes such as MMP-1 and MMP-3 by UV irradiation was not suppressed by quercetin. CONCLUSION: Our data indicate that the ability of quercetin to block UV irradiation-induced skin inflammation is mediated, at least in part, by its inhibitory effect on NF-κB activation and inflammatory cytokine production.

Reduced expression of connective tissue growth factor (CTGF/CCN2) mediates collagen loss in chronologically aged human skin.

Reduced production of type I procollagen is a prominent feature of chronologically aged human skin. Connective tissue growth factor (CTGF/CCN2), a downstream target of the transforming growth factor-beta (TGF-beta)/Smad pathway, is highly expressed in numerous fibrotic disorders, in which it is believed to stimulate excessive collagen production. CTGF is constitutively expressed in normal human dermis in vivo, suggesting that CTGF is a physiological regulator of collagen expression. We report here that the TGF-beta/Smad/CTGF axis is significantly reduced in dermal fibroblasts, the major collagen-producing cells, in aged (> or = 80 years) human skin in vivo. In primary human skin fibroblasts, neutralization of endogenous TGF-beta or knockdown of CTGF substantially reduced the expression of type I procollagen mRNA, protein, and promoter activity. In contrast, overexpression of CTGF stimulated type I procollagen expression, and increased promoter activity. Inhibition of TGF-beta receptor kinase, knockdown of Smad4, or overexpression of inhibitory Smad7 abolished CTGF stimulation of type I procollagen expression. However, CTGF did not stimulate Smad3 phosphorylation or Smad3-dependent transcriptional activity. These data indicate that in human skin fibroblasts, type I procollagen expression is dependent on endogenous production of both TGF-beta and CTGF, which act through interdependent yet distinct mechanisms. Downregulation of the TGF-beta/Smad/CTGF axis likely mediates reduced type I procollagen expression in aged human skin in vivo.