Age-associated reduction of cell spreading induces mitochondrial DNA common deletion by oxidative stress in human skin dermal fibroblasts: implication for human skin connective tissue aging

Abstract Background Reduced cell spreading is a prominent feature of aged dermal fibroblasts in human skin in vivo. Mitochondrial DNA (mtDNA) common deletion has been reported to play a role in the human aging process, however the relationship between age-related reduced cell spreading and mtDNA common deletion has not yet been reported. Results To examine mtDNA common deletion in the dermis of aged human skin, the epidermis was removed from full-thickness human skin samples using cryostat. mtDNA common deletion was significantly elevated in the dermis of both naturally aged and photoaged human skin in vivo. To examine the relationship between age-related reduced cell spreading and mtDNA common deletion, we modulated the shape of dermal fibroblasts by disrupting the actin cytoskeleton. Reduced cell spreading was associated with a higher level of mtDNA common deletion and was also accompanied by elevated levels of endogenous reactive oxygen species (ROS). Boosting cellular antioxidant capacity by using antioxidants was found to be protective against mtDNA common deletion associated with reduced cell spreading. Conclusion mtDNA common deletion is highly prevalent in the dermis of both naturally aged and photoaged human skin in vivo. mtDNA common deletion in response to reduced cell spreading is mediated, at least in part, by elevated oxidative stress in human dermal fibroblasts. These data extend current understanding of the mitochondrial theory of aging by identifying the connection between mtDNA common deletion and age-related reduction of cell spreading.http://deepblue.lib.umich.edu/bitstream/2027.42/112063/1/12929_2015_Article_167.pd

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

Transforming growth factor‐β ( TGF ‐β) is a major regulator of collagen gene expression in human skin fibroblasts. Cellular responses to TGF ‐β are mediated primarily through its cell surface type I (TβRI) and type II (TβRII) receptors. Ultraviolet ( UV ) irradiation impairs TGF ‐β signalling largely due to reduced TβRII gene expression, thereby decreasing type I procollagen synthesis, in human skin fibroblasts. UV irradiation does not alter either TβRII m RNA or protein stability, indicating that UV reduction in TβRII expression likely results from transcriptional or translational repression. To understand how UV irradiation regulates TβRII transcription, we used a series of TβRII promoter‐luciferase 5′‐deletion constructs (covering 2 kb of the TβRII 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 TβRII 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 TβRII 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 TβRII expression, at least partially, through transcriptional repression. This repression is mediated by a 38‐bp sequence in TβRII promoter, in human skin fibroblasts.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108702/1/exd12389.pd

CYR61/CCN1: A Novel Mediator of Epidermal Hyperplasia and Inflammation in Psoriasis?

The complex pathogenesis of psoriasis is still not fully understood. The study by Sun et al. (2015) suggests that CYR61 (now named CCN1), a secreted matricellular protein, has a role in the pathogenesis of psoriasis, and thus targeting CCN1 represents a potential therapeutic strategy in its treatment

Age‐associated reduction of cellular spreading/mechanical force up‐regulates matrix metalloproteinase‐1 expression and collagen fibril fragmentation via c‐Jun/ AP ‐1 in human dermal fibroblasts

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109644/1/acel12265.pd

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.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150500/1/exd13728_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150500/2/exd13728.pd

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

Abstract 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-β/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-β/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-β1-induced Smad3 phosphorylation and Smad3 transcriptional activity, thereby inhibiting the expression of TGF-β target genes. This reduction by reduced levels of YAP/TAZ results from induction of inhibitory Smad7, which inhibits Smad3 phosphorylation and activity by TGF-β1. 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-β/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-β signaling, in human dermal fibroblasts. Reduction of YAP/TAZ levels leads to activation of AP-1 activity, which induces Smad7. Smad7 suppresses the TGF-β pathway.https://deepblue.lib.umich.edu/bitstream/2027.42/143193/1/12964_2018_Article_232.pd

Matrix protein CCN1 induced by bacterial DNA and CpG ODN limits lung inflammation and contributes to innate immune homeostasis

Summary To defend against pulmonary infections, lung epithelial cells are equipped with complex innate immunity closely linked to inflammation. Dysregulated innate immunity / inflammation leads to self-perpetuating lung injury. The CpG motif in bacterial DNA is one of the factors involved in bacterial infection-associated inflammation. Bacterial DNA and synthetic CpG oligonucleotide (ODN) induced CCN1 secretion from lung epithelial cells, functioning as a potential “braking” signal to prevent uncontrolled inflammatory responses. CpG ODN-induced ER stress resulted in Src-Y527 phosphorylation (pY527) and Src/CCN1 vWF domain dissociation. Src-Y527 activated caveolin-1 (cav-1) phosphorylation at Y14 and then modulated CCN1 secretion via pCav-1 interaction with CCN1 IGFbp domain. Functionally, secreted CCN1 promoted anti-inflammatory cytokine IL-10 release from epithelial cells via integrin αVβ6 PKC, and this subsequently suppressed TNF-α, MIP-2 secretion and neutrophil infiltration in the lungs. Collectively, bacterial DNA/CpG ODN-stimulated CCN1 secretion via BiP/GRP78-Src(Y527)-JNK-Cav-1(Y14) pathway and CpG-induced CCN1 conferred anti-inflammatory roles. Our studies suggested a novel paradigm by which the lung epithelium maintains innate immune homeostasis after bacterial infection

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 (ECM) associated matricellular protein, is elevated in dermal fibroblasts in aged human skin. Towards constructing a mouse model to study 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 TGF-β signaling and increased expression of matrix metalloproteinases, driven up induction of c-Jun/AP-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 mechanisms of skin aging and age-related skin disorders

Expression of CCN family of genes in human skin in vivo and alterations by solar-simulated ultraviolet irradiation

The CCN family of proteins is involved in diverse biological functions such as cell growth, adhesion, migration, angiogenesis, and regulation of extracellular matrix. We have investigated expression of CCN family genes and alternations induced by solar-simulated ultraviolet irradiation in human skin in vivo. Transcripts of all six CCN genes were expressed in human skin in vivo. CCN5 was most abundantly expressed followed by CCN2>CCN3>CCN1>CCN4>CCN6. Solar-simulated ultraviolet irradiation increased mRNA expression of CCN1 and CCN2. In contrast, mRNA levels of CCN3, CCN4, CCN5, and CCN6, were reduced. Knowledge gained from this study provides the foundation to explore the functional roles of CCN gene products in cutaneous biology and responses to solar ultraviolet irradiation