Tianyuan He

Solar Ultraviolet Irradiation Reduces Collagen in Photoaged Human Skin by Blocking Transforming Growth Factor-β Type II Receptor/Smad Signaling

Ultraviolet (UV) irradiation from the sun reduces production of type I procollagen (COLI), the major structural protein in human skin. This reduction is a key feature of the pathophysiology of premature skin aging (photoaging). Photoaging is the most common form of skin damage and is associated with skin carcinoma. TGF-beta/Smad pathway is the major regulator of type I procollagen synthesis in human skin. We have previously reported that UV irradiation impairs transforming growth factor-beta (TGF-beta)/Smad signaling in mink lung epithelial cells. We have investigated the mechanism of UV irradiation impairment of the TGF-beta/Smad pathway and the impact of this impairment on type I procollagen production in human skin fibroblasts, the major collagen-producing cells in skin. We report here that UV irradiation impairs TGF-beta/Smad pathway in human skin by down-regulation of TGF-beta type II receptor (TbetaRII). This loss of TbetaRII occurs within 8 hours after UV irradiation and precedes down-regulation of type I procollagen expression in human skin in vivo. In human skin fibroblasts, UV-induced TbetaRII down-regulation is mediated by transcriptional repression and results in 90% reduction of specific, cell-surface binding of TGF-beta. This loss of TbetaRII prevents downstream activation of Smad2/3 by TGF-beta, thereby reducing expression of type I procollagen. Preventing loss of TbetaRII by overexpression protects against UV inhibition of type I procollagen gene expression in human skin fibroblasts. UV-induced down-regulation of TbetaRII, with attendant reduction of type I procollagen production, is a critical molecular mechanism in the pathophysiology of photoaging.

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.

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.

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-1β (∼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.

Ultraviolet Irradiation Induces CYR61/CCN1, a Mediator of Collagen Homeostasis, through Activation of Transcription Factor AP-1 in Human Skin Fibroblasts

UV irradiation from the sun elevates the production of collagen-degrading matrix metalloproteinases (MMPs) and reduces the production of new collagen. This imbalance of collagen homeostasis impairs the structure and function of the dermal collagenous extracellular matrix (ECM), thereby promoting premature skin aging (photoaging). We report here that aberrant dermal collagen homeostasis in UV-irradiated human skin is mediated in part by a CCN-family member, cysteine-rich protein-61 (CYR61/CCN1). CYR61 is significantly elevated in acutely UV-irradiated human skin in vivo, and UV-irradiated human skin fibroblasts. Knockdown of CYR61 significantly attenuates UV irradiation-induced inhibition of type-I procollagen and upregulation of MMP-1. Determination of CYR61 mRNA and protein indicates that the primary mechanism of CYR61 induction by UV irradiation is transcriptional. Analysis of CYR61 proximal promoter showed that a sequence conforming to the consensus binding site for transcription factor activator protein-1 (AP-1) is required for promoter activity. UV irradiation increased the binding of AP-1-family members c-Jun and c-Fos to this AP-1 site. Furthermore, functional blockade of c-Jun or knockdown of c-Jun significantly reduced the UV irradiation-induced activation of CYR61 promoter and CYR61 gene expression. These data show that CYR61 is transcriptionally regulated by UV irradiation through transcription factor AP-1, and mediates altered collagen homeostasis that occurs in response to UV irradiation in human skin fibroblasts.

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.

Oxidative exposure impairs TGF-β 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-β (TGF-β) pathway, through reduction of type II TGF-β receptor (TβRII) 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-β. Restoration of TβRII and SMAD3 together, but not separately, reinstates TGF-β signaling and increases CTGF/CCN2 and type I collagen levels. Treatment with the anti-oxidant N-acetylcysteine reduces ROS elevation and normalizes TGF-β 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-β 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‐β 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.

Reduction of fibroblast size/mechanical force down‐regulates TGF‐β 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‐β type II receptor (TβRII) and thus impairs TGF‐β/Smad signaling pathway. Both TβRII mRNA and protein were decreased, resulting in 90% loss of TGF‐β binding to fibroblasts. Down‐regulation of TβRII 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 TβRII significantly increased TGF‐β induction of Smad3 phosphorylation and stimulated expression of ECM components. Reduced expression of TβRII 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 TβRII 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.

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.