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.

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.

Cysteine-rich protein 61 (CCN1) domain-specific stimulation of matrix metalloproteinase-1 expression through αVß3 integrin in human skin fibroblasts.

Human skin largely comprises collagenous extracellular matrix. The hallmark of skin aging is fragmentation of collagen fibrils. Matrix metalloproteinases (MMPs) are largely responsible for collagen degradation. MMP-1, principally derived from dermal fibroblasts, is the major protease capable of initiating degradation of native fibrillar collagens. Presently, we report that CCN1, a secreted and extracellular matrix-associated protein, is elevated in aged human skin dermal fibroblasts in vivo and stimulates MMP-1 expression through functional interaction with αVß3 integrin in human dermal fibroblasts. CCN1 contains four conserved structural domains. Our results indicate that the three N-terminal domains (IGFBP, VWC, and TSP1), but not the C-terminal CT domain, are required for CCN1 to stimulate MMP-1 expression. This stimulation is dependent on interaction between the active structural domains and αVß3 integrin. The interaction of VWC domain with integrin αVß3 is necessary and requires functional cooperation with adjacent IGFBP and TSP1 domains to stimulate MMP-1 expression. Finally, induction of MMP-1 expression in dermal fibroblasts by CCN1 N-terminal domains resulted in fragmentation of type I collagen fibrils in a three-dimensional collagen lattice model. These data suggest that domain-specific interactions of CCN1 with αVß3 integrin contribute to human skin aging by stimulating MMP-1-mediated collagen fibril fragmentation.

CCN1 secretion and cleavage regulate the lung epithelial cell functions after cigarette smoke.

Despite extensive research, the pathogenesis of cigarette smoking (CS)-associated emphysema remains incompletely understood, thereby impeding development of novel therapeutics, diagnostics, and biomarkers. Here, we report a novel paradigm potentially involved in the development of epithelial death and tissue loss in CS-associated emphysema. After prolonged exposure of CS, CCN1 cleavage was detected both in vitro and in vivo. Full-length CCN1 (flCCN1) was secreted in an exosome-shuttled manner, and secreted plasmin converted flCCN1 to cleaved CCN1 (cCCN1) in extracellular matrix. Interestingly, exosome-shuttled flCCN1 facilitated the interleukin (IL)-8 and vascular endothelial growth factor (VEGF) release in response to cigarette smoke extract (CSE). Therefore, flCCN1 potentially promoted CS-induced inflammation via IL-8-mediated neutrophil recruitment and also maintained the lung homeostasis via VEGF secretion. Interestingly, cCCN1 abolished these functions. Furthermore, cCCN1 promoted protease and matrix metalloproteinase (MMP)-1 production after CSE. These effects were mainly mediated by the COOH-terminal fragments of CCN1 after cleavage. Both the decrease of VEGF and the elevation of MMPs favor the development of emphysema. cCCN1, therefore, likely contributes to the epithelial cell damage after CS. Additionally, CSE and cCCN1 both stimulated integrin-α7 expressions in lung epithelial cells. The integrin-α7 appeared to be the binding receptors of cCCN1 and, subsequently, mediated its cellular function by promoting MMP1. Consistent with our observation on the functional roles of cCCN1 in vitro, elevated cCCN1 level was found in the bronchoalveolar lavage fluid from mice with emphysematous changes after 6 mo CS exposure. Taken together, we hypothesize that cCCN1 promoted the epithelial cell death and tissue loss after prolonged CS exposure.

Targeting fatty acid synthase in preclinical models of TNBC brain metastases synergizes with SN-38 and impairs invasion.

Fatty acid synthesis (FAS) has been shown to play a key role in the survival of brain-metastatic (BM) breast cancer. We demonstrate that the fatty acid synthase inhibitor TVB-2640 synergizes with the topoisomerase inhibitor SN-38 in triple-negative breast cancer (TNBC) BM cell lines, upregulates FAS and downregulates cell cycle progression gene expression, and slows the motility of TNBC BM cell lines. The combination of SN-38 and TVB-2640 warrants further consideration as a potential therapeutic option in TNBC BMs.

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.

Cysteine-rich protein 61 (CCN1) mediates replicative senescence-associated aberrant collagen homeostasis in human skin fibroblasts.

Dermal fibroblasts produce a collagen-rich extracellular matrix, which confers mechanical strength and resiliency to human skin. During aging, collagen production is reduced and collagen fragmentation is increased, which is initiated by matrix metalloproteinase-1 (MMP-1). This aberrant collagen homeostasis results in net collagen deficiency, which impairs the structural integrity and function of skin. Cysteine-rich protein 61 (CCN1), a member of the CCN family, negatively regulates collagen homeostasis, in primary human skin dermal fibroblasts. As replicative senescence is a form of cellular aging, we have utilized replicative senescent dermal fibroblasts to further investigate the connection between elevated CCN1 and aberrant collagen homeostasis. CCN1 mRNA and protein levels were significantly elevated in replicative senescent dermal fibroblasts. Replicative senescent dermal fibroblasts also expressed significantly reduced levels of type I procollagen and increased levels of MMP-1. Knockdown of elevated CCN1 in senescent dermal fibroblasts partially normalized both type I procollagen and MMP-1 expression. These data further support a key role of CCN1 in regulation of collagen homeostasis. Elevated expression of CCN1 substantially increased collagen lattice contraction and fragmentation caused by replicative senescent dermal fibroblasts. Atomic force microscopy (AFM) further revealed collagen fibril fragmentation and disorganization were largely prevented by knockdown of CCN1 in replicative senescent dermal fibroblasts, suggesting CCN1 mediates MMP-1-induced alterations of collagen fibrils by replicative senescent dermal fibroblasts. Given the ability of CCN1 to regulate both production and degradation of type I collagen, it is likely that elevated-CCN1 functions as an important mediator of collagen loss, which is observed in aged human skin.

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.

Retinoids suppress cysteine-rich protein 61 (CCN1), a negative regulator of collagen homeostasis, in skin equivalent cultures and aged human skin in vivo.

Alterations in connective tissue collagen are prominent features of both chronologically aged and photoaged (ageing because of sun exposure) human skin. These age-related abnormalities are mediated in part by cysteine-rich protein 61 (CCN1). CCN1 is elevated in the dermis of both chronologically aged and photoaged human skin in vivo and promotes aberrant collagen homeostasis by down-regulating type I collagen, the major structural protein in skin, and promoting collagen degradation. Vitamin A and its metabolites have been shown to improve chronologically aged and photoaged skin by promoting deposition of new collagen and preventing its degradation. Here, we investigated regulation of CCN1 expression by retinoids in skin equivalent cultures and chronologically aged and photoaged human skin in vivo. In skin equivalent cultures, all-trans retinoic acid (RA), the major bioactive form of vitamin A in skin, significantly increased type I procollagen and reduced collagenase (matrix metalloproteinases-1, MMP-1). Addition of recombinant human CCN1 to skin equivalent cultures significantly reduced type I procollagen and increased MMP-1. Importantly, RA significantly reduced CCN1 expression in skin equivalent cultures. Topical treatment with retinol (vitamin A, 0.4%) for 7days significantly reduced CCN1 mRNA and protein expression in both chronologically aged (80+years) and photoaged human skin in vivo, compared to vehicle-treated skin. These data indicate that the mechanism by which retinoids improve aged skin, through increased collagen production, involves down-regulation of CCN1.

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.

Targeted RNA sequencing of adrenal zones using immunohistochemistry-guided capture of formalin-fixed paraffin-embedded tissue.

Adequate access to fresh or frozen normal adrenal tissue has been a primary limitation to the enhanced characterization of the adrenal zones via RNA sequencing (RNAseq). Herein, we describe the application of targeted RNAseq to formalin-fixed paraffin-embedded (FFPE) normal adrenal gland specimens. Immunohistochemistry (IHC) was used to visualize and guide the capture of the adrenocortical zones and medulla. Following IHC-based tissue capture and isolation of RNA, high-throughput targeted RNAseq highlighted clear transcriptomic differences and identified differentially expressed genes among the adrenal zones. Our data demonstrate the ability to capture FFPE adrenal zone tissue for targeted transcriptomic analyses. Future comparison of normal adrenal zones will improve our understanding of transcriptomic patterns and help identify potential novel pathways controlling zone-specific steroid production.

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.

Matrix-degrading metalloproteinases in photoaging.

UV radiation from the sun impacts skin health adversely through complex, multiple molecular pathways. Premature skin aging (photoaging) is among the most widely appreciated harmful effects of chronic exposure to solar UV radiation. Extensive damage to the dermal connective tissue is a hallmark of photoaged skin. Disruption of the normal architecture of skin connective tissue impairs skin function and causes it to look aged. UV irradiation induces expression of certain members of the matrix metalloproteinase (MMP) family, which degrade collagen and other extracellular matrix proteins that comprise the dermal connective tissue. Although the critical role of MMPs in photoaging is undeniable, important questions remain. This article summarizes our current understanding of the role of MMPs in the photoaging process and presents new data that (1) describe the expression and regulation by UV irradiation of all members of the MMP family in human skin in vivo and (2) quantify the relative contributions of epidermis and dermis to the expression of UV irradiation-induced MMPs in human skin in vivo.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 20-24; doi:10.1038/jidsymp.2009.8.

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.

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.