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.

Human Skin Aging and the Anti-Aging Properties of Retinol.

The skin is the most-extensive and -abundant tissue in the human body. Like many organs, as we age, human skin experiences gradual atrophy in both the epidermis and dermis. This can be primarily attributed to the diminishing population of epidermal stem cells and the reduction in collagen, which is the primary structural protein in the human body. The alterations occurring in the epidermis and dermis due to the aging process result in disruptions to the structure and functionality of the skin. This creates a microenvironment conducive to age-related skin conditions such as a compromised skin barrier, slowed wound healing, and the onset of skin cancer. This review emphasizes the recent molecular discoveries related to skin aging and evaluates preventive approaches, such as the use of topical retinoids. Topical retinoids have demonstrated promise in enhancing skin texture, diminishing fine lines, and augmenting the thickness of both the epidermal and dermal layers.

Molecular insights of human skin epidermal and dermal aging.

Human skin is the most widespread and abundant type of tissue in the human body. With the passage of time, most of our organs, including a substantial part of the skin, tend to undergo a gradual thinning or decrease in size. As we age, there is a gradual and progressive reduction in the thickness of both the epidermis and dermis layers of our skin. This is primarily attributed to the decline of epidermal stem cells and the loss of dermal collagen, which is the most abundant protein in the human body. Age-related alterations of the epidermis and dermis impair skin structure/function and create a tissue microenvironment that promotes age-related skin diseases, such as impaired skin barrier, delayed wound healing, and skin cancer development. This review will examine the current body of literature pertaining to our knowledge of skin epidermal and dermal aging.

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.

Skin aging from the perspective of dermal fibroblasts: the interplay between the adaptation to the extracellular matrix microenvironment and cell autonomous processes.

This article summarizes important molecular mechanisms that drive aging in human skin from the perspective of dermal fibroblasts. The dermis comprises the bulk of the skin and is largely composed of a collagen-rich extracellular matrix (ECM). The dermal ECM provides mechanical strength, resiliency, and an environment that supports the functions of ibroblasts and other types of dermal cells. Fibroblasts produce the dermal ECM and maintain its homeostasis. Fibroblasts attach to the ECM and this attachment controls their morphology and function. During aging, the ECM undergoes gradual degradation that is nitiated by matrix metalloproteinases (MMPs). This degradation alters mechanical forces within the dermal ECM and disrupts he interactions between fibroblasts and the ECM thereby generating an aged fibroblast phenotype. This aged fibroblast phenotype is characterized by collapsed morphology, altered mechanosignaling, induction of CCN1, and activation of transcription factor AP-1, with consequent upregulation of target genes including MMPs and pro-inflammatory mediators. The TGF-beta pathway coordinately regulates ECM production and turnover. Altered mechanical forces, due to ECM fragmentation, down-regulate the type II TGF-beta receptor, thereby reducing ECM production and further increasing ECM breakdown. Thus, dermal aging involves a feed-forward process that reinforces the aged dermal fibroblast phenotype and promotes age-related dermal ECM deterioration. As discussed in the article, the expression of the aged dermal fibroblast phenotype involves both adaptive and cell-autonomous mechanisms.

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.

Reduced expression of Collagen 17A1 in naturally aged, photoaged, and UV-irradiated human skin in vivo: Potential links to epidermal aging.

Collagen 17A1 (COL17A1) is a transmembrane structural component of the hemidesmosome that mediate adhesion of keratinocytes to the underlying membrane. Recent work in mouse showed that COL17A1 deficiency leads to premature skin aging. Although the role COL17A1 in skin aging is becoming recognized in mouse models, its connection to human skin natural aging/photoaging/ultraviolet (UV)-irradiated human skin has received little attention. To determine COL17A1 expression in naturally aged and photoaged as well as acutely UV irradiated human skin, skin samples were obtained from: (1) young (N = 10, 26.7±1.3 years) and aged (N = 10, 84.0 ± 1.7 years) sun-protected buttock skin; (2) photoaged extensor forearm and subject matched sun-protected underarm skin (N = 6, 56.0 ± 3.4 years); (3) solar-simulated UV-irradiated buttock skin (N = 6, 51.2 ± 3.6 years). COL17A1 levels were determined by immunohistology and RT-PCR, and the potential role of COL17A1 in epidermal aging was investigated by immunostaining of the marker for interfollicular epidermal stem cells and keratinocytes proliferation. We found that COL17A1 is specifically expressed in interfollicular epidermal stem cell niches, and that significantly reduced in naturally aged, photoaged, and acute UV-irradiated human skin in vivo. COL17A1 is identified as keratinocyte-specific collagen, and UV irradiation significantly downregulates COL17A1 expression in keratinocytes. Reduced expression of COL17A1 is positively correlated with impaired regeneration of keratinocytes and reduced dermal-epidermal junction as well as thin epidermis in aged human skin (epidermal aging). We also confirmed that keratinocyte-specific integrin ß4 (ITGB4), which interacts with COL17A1, is reduced in aged human skin. Mechanistically, we found that matrix metalloproteinases (MMPs) are responsible for UV-mediated COL17A1 degradation in both in vitro keratinocytes and in vivo mouse skin. These data suggest the possible links between reduced expression of COL17A1 and epidermal aging in human skin.

Age-related elevation of HGF is driven by the reduction of fibroblast size in a YAP/TAZ/CCN2 axis-dependent manner.

BACKGROUND: Aged human skin is primarily attributable to the loss of collagen. Hepatocyte growth factor (HGF) acts as an anti-fibrotic factor by suppression of collagen production. In aged human skin, HGF is elevated in dermal fibroblasts and thus contributes to dermal aging (thin dermis) by suppression of collagen production. OBJECTIVE: We aimed to investigate the underlying mechanisms of age-related elevation of HGF expression. METHODS: Collagen fibrils in the aged skin dermis are fragmented and disorganized, which impairs collagen-fibroblast interaction, resulting in reduced fibroblast spreading and size. To explore the connection between reduced dermal fibroblast size and age-related elevation of HGF expression, we manipulate dermal fibroblast size, and cell-size dependent regulation of HGF was investigated by laser capture microdissection, immunostaining, capillary electrophoresis immunoassay, and quantitative RT-PCR. RESULTS: We found that reduced fibroblast size is responsible for age-related elevation of HGF expression. Further investigation indicated that cell size-dependent upregulation of HGF expression was mediated by impeded YAP/TAZ nuclear translocation and their target gene, CCN2. Conversely, restoration of dermal fibroblast size rapidly reversed cell-size-dependent upregulation of HGF in a YAP/TAZ-dependent manner. Finally, we confirmed that elevated HGF expression is accompanied by the reduced expression of YAP/TAZ and CCN2 in the aged human skin in vivo. CONCLUSION: Age-related elevation of HGF is driven by the reduction of fibroblast size in a YAP/TAZ/CCN2 axis-dependent manner. These data reveal a novel mechanism by which reduction of fibroblast size upregulates HGF expression, which in turn contributes to loss of collagen, a prominent feature of aged human skin.

Dermal Fibroblast CCN1 Expression in Mice Recapitulates Human Skin Dermal Aging.

The aging process deleteriously alters the structure and function of dermal collagen. These alterations result in thinning, fragility, wrinkles, laxity, impaired wound healing, and a microenvironment conducive to cancer. However, the key factors responsible for these changes have not been fully elucidated, and relevant models for the study of skin aging progression are lacking. CCN1, a secreted extracellular matrix‒associated matricellular protein, is elevated in dermal fibroblasts in aged human skin. Toward constructing a mouse model to study the key factors involved in skin-aging progression, we demonstrate that transgenic mice, with selective expression of CCN1 in dermal fibroblasts (COL1A2-CCN1), display accelerated skin dermal aging. The aged phenotype in COL1A2-CCN1 mice resembles aged human dermis: the skin is wrinkled and the dermis is thin and composed of loose, disorganized, and fragmented collagen fibrils. These dermal alterations reflect reduced production of collagen due to impaired TGFß signaling and increased expression of matrix metalloproteinases driving the induction of c-Jun/activator protein-1. Importantly, similar mechanisms drive human dermal aging. Taken together, the data demonstrate that elevated expression of CCN1 by dermal fibroblasts functions as a key mediator of dermal aging. The COL1A2-CCN1 mouse model provides a novel tool for understanding and studying the mechanisms of skin aging and age-related skin disorders.

Alterations in extracellular matrix composition during aging and photoaging of the skin.

Human skin is composed of the cell-rich epidermis, the extracellular matrix (ECM) rich dermis, and the hypodermis. Within the dermis, a dense network of ECM proteins provides structural support to the skin and regulates a wide variety of signaling pathways which govern cell proliferation and other critical processes. Both intrinsic aging, which occurs steadily over time, and extrinsic aging (photoaging), which occurs as a result of external insults such as solar radiation, cause alterations to the dermal ECM. In this study, we utilized both quantitative and global proteomics, alongside single harmonic generation (SHG) and two-photon autofluorescence (TPAF) imaging, to assess changes in dermal composition during intrinsic and extrinsic aging. We find that both intrinsic and extrinsic aging result in significant decreases in ECM-supporting proteoglycans and structural ECM integrity, evidenced by decreasing collagen abundance and increasing fibril fragmentation. Intrinsic aging also produces changes distinct from those produced by photoaging, including reductions in elastic fiber and crosslinking enzyme abundance. In contrast, photoaging is primarily defined by increases in elastic fiber-associated protein and pro-inflammatory proteases. Changes associated with photoaging are evident even in young (mid 20s) sun-exposed forearm skin, indicating that proteomic evidence of photoaging is present decades prior to clinical signs of photoaging. GO term enrichment revealed that both intrinsic aging and photoaging share common features of chronic inflammation. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD015982.

Physical properties of the photodamaged human skin dermis: Rougher collagen surface and stiffer/harder mechanical properties.

Fragmentation of collagen fibrils and aberrant elastic material (solar elastosis) in the dermal extracellular matrix (ECM) is among the most prominent features of photodamaged human skin. These alterations impair the structural integrity and create a dermal microenvironment prone to skin disorders. The objective of this study was to determine the physical properties (surface roughness, stiffness and hardness) of the dermal ECM in photodamaged and subject-matched sun-protected human skin. Skin samples were sectioned and analysed by histology, atomic force microscopy and nanoindentation. Dermal ECM collagen fibrils were more disorganized (ie, rougher surface), and the dermal ECM was stiffer and harder, in photodamaged forearm, compared to sun-protected underarm skin. Cleavage of collagen fibrils in sun-protected underarm dermis by recombinant human matrix metalloproteinase-1 resulted in rougher collagen fibril surface and reduced dermal stiffness and hardness. Degradation of elastotic material in photodamaged skin by treatment with purified neutrophil elastase reduced stiffness and hardness, without altering collagen fibril surface roughness. Additionally, expression of two members of the lysyl oxidase gene family, which insert cross-links that stiffen and harden collagen fibrils, was elevated in photodamaged forearm dermis. These data elucidate the contributions of fragmented collagen fibrils, solar elastosis and elevated collagen cross-linking to the physical properties of the dermal ECM in photodamaged human skin. This new knowledge extends current understanding of the impact of photodamage on the dermal ECM microenvironment.

Actin cytoskeleton assembly regulates collagen production via TGF-ß type II receptor in human skin fibroblasts.

The dermal compartment of skin is primarily composed of collagen-rich extracellular matrix (ECM), which is produced by dermal fibroblasts. In Young skin, fibroblasts attach to the ECM through integrins. During ageing, fragmentation of the dermal ECM limits fibroblast attachment. This reduced attachment is associated with decreased collagen production, a major cause of skin thinning and fragility, in the elderly. Fibroblast attachment promotes assembly of the cellular actin cytoskeleton, which generates mechanical forces needed for structural support. The mechanism(s) linking reduced assembly of the actin cytoskeleton to decreased collagen production remains unclear. Here, we report that disassembly of the actin cytoskeleton results in impairment of TGF-ß pathway, which controls collagen production, in dermal fibroblasts. Cytoskeleton disassembly rapidly down-regulates TGF-ß type II receptor (TßRII) levels. This down-regulation leads to reduced activation of downstream effectors Smad2/Smad3 and CCN2, resulting in decreased collagen production. These responses are fully reversible; restoration of actin cytoskeleton assembly up-regulates TßRII, Smad2/Smad3, CCN2 and collagen expression. Finally, actin cytoskeleton-dependent reduction of TßRII is mediated by induction of microRNA 21, a potent inhibitor of TßRII protein expression. Our findings reveal a novel mechanism that links actin cytoskeleton assembly and collagen expression in dermal fibroblasts. This mechanism likely contributes to loss of TßRII and collagen production, which are observed in aged human skin.

YAP/TAZ regulates TGF-ß/Smad3 signaling by induction of Smad7 via AP-1 in human skin dermal fibroblasts.

BACKGROUND: Transcription factors YAP and TAZ function as the primary mediators of the Hippo pathway. Yet, crosstalk of YAP and TAZ with other signaling pathways remains relatively unexplored. We have explored the impact of YAP and TAZ levels on the TGF-ß/Smad signaling pathway in human skin dermal fibroblasts. METHODS: YAP and TAZ levels in dermal fibroblasts were reduced in dermal fibroblasts by siRNA-mediated knockdown. The effects of YAP and TAZ reduction on TGF-ß/Smad signaling were examined by quantitative real-time PCR, Western analysis, and immunostaining. Luciferase reporter assays and electrophoretic mobility shift assays were conducted to investigate the transcription factor DNA-binding and transcriptional activities. RESULTS: Knockdown of both YAP and TAZ (YAP/TAZ), but not either separately, impaired TGF-ß1-induced Smad3 phosphorylation and Smad3 transcriptional activity, thereby inhibiting the expression of TGF-ß target genes. This reduction by reduced levels of YAP/TAZ results from induction of inhibitory Smad7, which inhibits Smad3 phosphorylation and activity by TGF-ß1. Conversely, prevention of Smad7 induction restores Smad3 phosphorylation and Smad3 transcriptional activity in fibroblasts that have reduced YAP/TAZ. In agreement with these findings, inhibition of YAP/TAZ transcriptional activity, similar to the reduction of YAP/TAZ levels, also significantly induced Smad7 and impaired TGF-ß/Smad signaling. Further investigations revealed that reduced levels of YAP/TAZ led to induction of activator protein-1 (AP-1) activity, Activated AP-1 bound to DNA sequences in the Smad7 gene promoter, and deletion of these AP-1 binding sequences substantially reduced Smad7 promoter reporter activity. CONCLUSION: YAP/TAZ functions in concert with transcription factor AP-1 and Smad7 to regulate TGF-ß signaling, in human dermal fibroblasts. Reduction of YAP/TAZ levels leads to activation of AP-1 activity, which induces Smad7. Smad7 suppresses the TGF-ß pathway.

Extracellular matrix regulation of fibroblast function: redefining our perspective on skin aging.

The dermal extracellular matrix (ECM) comprises the bulk of skin and confers strength and resiliency. In young skin, fibroblasts produce and adhere to the dermal ECM, which is composed primarily of type I collagen fibrils. Adherence allows fibroblasts to spread and exert mechanical force on the surrounding ECM. In this state, fibroblasts display a "youthful" phenotype characterized by maintenance of the composition and structural organization of the dermal ECM. During aging, fibroblast-ECM interactions become disrupted due to fragmentation of collagen fibrils. This disruption causes loss of fibroblast spreading and mechanical force, which inextricably lead to an "aged" phenotype; fibroblasts synthesize less ECM proteins and more matrix-degrading metalloproteinases. This imbalance of ECM homeostasis further drives collagen fibril fragmentation in a self-perpetuating cycle. This article summarizes age-related changes in the dermal ECM and the mechanisms by which these changes alter the interplay between fibroblasts and their extracellular matrix microenvironment that drive the aging process in human skin.

Ezrin regulates skin fibroblast size/mechanical properties and YAP-dependent proliferation.

Ezrin acts as a dynamic linkage between plasma membrane and cytoskeleton, and thus involved in many fundamental cellular functions. Yet, its potential role in human skin is virtually unknown. Here we investigate the role of Ezrin in primary skin fibroblasts, the major cells responsible extracellular matrix (ECM) production. We report that Ezrin play an important role in the maintenance of skin fibroblast size/mechanical properties and proliferation. siRNA-mediated Ezrin knockdown decreased fibroblast size and mechanical properties, and thus impaired the nuclear translocation of YAP, a protein commonly response to cell size and mechanical force. Functionally, depletion of Ezrin significantly inhibited YAP target gene expression and fibroblast proliferation. Conversely, restoration of YAP nuclear translocation by overexpression of constitutively active YAP reversed YAP target genes expression and rescued proliferation in Ezrin knockdown cells. These data reveal a novel role for Ezrin in maintenance of fibroblast size/mechanical force and regulating YAP-mediated proliferation.

Cell-size-dependent upregulation of HGF expression in dermal fibroblasts: Impact on human skin connective tissue aging.

BACKGROUND: Aged human skin is primarily attributable to loss of collagen, the main structural component of skin. Hepatocyte growth factor (HGF) acts as an anti-fibrotic factor by suppression of collagen production. It is not known whether HGF is involved in age-related collagen deficit in human skin. OBJECTIVE: The objective of this study was to investigate the expression of HGF in human skin, and the underlying mechanisms of age-related elevation of HGF expression. METHODS: The expression of HGF in young (25±5years, six subjects) and aged (75±6years, six subjects) human skin was determined by laser capture microdissection (LCM) coupled real-time PCR and immunohistology. The underlying mechanisms of age-related elevation of HGF were investigated by reducing dermal fibroblast size, which is a prominent feature of aged skin fibroblast in vivo. RESULTS: HGF is predominantly expressed in human skin dermal fibroblasts, the major cells responsible for collagen production, and is significantly elevated in aged human skin in vivo. Mechanistically, reduced fibroblast size, which is a prominent feature of aged skin fibroblasts in vivo, is responsible for age-related elevation of HGF expression. Cell-size-dependent upregulation of HGF expression is driven by increased c-Jun and impaired TGF-ß signaling. Restoration of fibroblast size normalizes increased c-Jun expression and impaired TGF-ß signaling, and thus reversed the elevated HGF expression. Finally, we confirmed that application of retinoid (ROL), which has been shown to improve aged human skin, significantly reduced elevated HGF mRNA expression in aged human skin in vivo (78±4years, six subjects). CONCLUSION: These data reveal a novel mechanism by which reduction of fibroblast size upregulates HGF expression, which in turn contributes to loss of collagen, a prominent feature of aged skin.

Smad3-dependent CCN2 mediates fibronectin expression in human skin dermal fibroblasts.

The potential involvement of connective tissue growth factor (CCN2/CTGF) in extracellular matrix (ECM) production is recognized. However, the role CCN2 in fibronectin (FN) gene expression has remained incompletely understood and even controversial. Here we report that CCN2 is absolutely necessary for FN expression in primary human skin dermal fibroblasts, the major cells responsible for ECM production in skin. Gain- and loss-of-function approaches demonstrate that CCN2 is an essential component of FN expression in both basal and stimulation by TGF-ß signaling, the major regulator of FN expression. CCN2 is significantly induced by Smad3, a critical mediator of TGF-ß signaling. CCN2 acts as a downstream mediator of TGF-ß/Smad signaling and acting synergistically with TGF-ß to regulate FN gene expression. Finally, we observed that CCN2 and FN predominantly expressed in the dermis of normal human skin, stromal tissues of skin squamous cell carcinoma (SCC), and simultaneously induced in wounded human skin in vivo. These findings provide evidence that CCN2 is responsible for mediating the stimulatory effects of TGF-ß/Smad on FN gene expression, and attenuation of CCN2 expression may benefit to reduce fibrotic ECM microenvironment in disease skin.

PI3K/mTOR dual inhibitor BEZ235 and histone deacetylase inhibitor Trichostatin A synergistically exert anti-tumor activity in breast cancer.

Molecule-targeted therapy has achieved great progress in cancer therapy. Effective drug combinations are one way to enhance the therapeutic efficacy and combat resistance. Here, we determined the effect of the PI3K/mTOR dual inhibitor BEZ235 and the histone deacetylase inhibitor Trichostatin A (TSA) on human breast cancer. We demonstrated that the combination of BEZ235 and TSA results in significant synergistic growth inhibition of multiple breast cancer cell lines. Mechanistic studies revealed that the combined therapy induced apoptosis in a caspase-dependent manner, which might be related to the further depression of the PI3K/Akt/mTOR signalling pathway. Additionally, co-treatment with BEZ235 and TSA enhanced autophagic cell death by up-regulating the expression of LC3B-II and Beclin-1. The vivo tumour modelling studies revealed that BEZ235 combined with TSA blocked tumour growth without noticeable side effects. These data suggest that the combination of BEZ235 and TSA may be a new selective strategy, which may have significant clinical application in the treatment of breast cancer patients.