Ultraviolet (UV) irradiation from the sun adversely impacts skin health through complex, multiple molecular pathways. Premature skin aging (photoaging) is among the most widely appreciated harmful effects of chronic exposure to solar UV irradiation. 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 (ECM) proteins that comprise the dermal connective tissue. Although the critical role of MMPs in photoaging process is undeniable, important questions remain. This article summarizes our current understanding regarding the role of MMPs in the photoaging process and presents new data that 1) describe 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 the epidermis and dermis to expression of UV irradiation-induced MMPs in human skin in vivo.

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 ␣2␤1 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 ␣2␤1 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. Skin connective tissue (dermis) provides structural support for the skin's vasculature, appendages, and epidermis, which are vital to the function of skin. Structural integrity and function of the dermis are primarily dependent on its extracellular matrix, which is primarily composed of type I collagen fibrils. Type I collagen is the most abundant structural protein in skin, 1 and fragmented collagen fibrils are prominent, characteristic features of aged human skin in vivo.2-4 This fragmentation seriously impairs both the mechanical properties of skin, and the functions of cells that reside within the dermis. Clinically, this impairment manifests as delayed wound healing, reduced vascularization, propensity to bruise, and thin skin. Failure of normal functional interactions among dermal cells and their extracellular matrix microenvironment underlie these age-dependent phenotypic alterations. 6Damage to the collagenous extracellular matrix of the dermis can be observed at both the histological and ultrastructural level. 5,[7][8][9] In young dermis, intact, tightly packed, well-organized, long collagen fibrils are abundant. In contrast, in aged dermis, collagen fibrils are fragmented, disorganized, and sparse, resulting in the appearance of amorphous open space. Quantitative biochemical analysis reveals that the amount of fragmented collagen is 4.3-fold greater in aged (Ͼ80 years old) compared with young (21 to 30 years old) human dermis in vivo.

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.

Human skin is largely composed of a collagen-rich connective tissue, which provides structural and functional support. The collagen-rich connective tissue is produced, organized, and maintained by dermal fibroblasts. During aging, dermal collagen fibrils undergo progressive loss and fragmentation, leading to thin and structurally weakened skin. Age-related alterations of collagen fibrils impairs skin structure and function and creates a tissue microenvironment that promotes age-related skin diseases, such as delayed wound healing and skin cancer development. This mini-review describes cellular mechanisms that give rise to self-perpetuating, collagen fibril fragmentation that creates an age-associated dermal microenvironment, which contributes to decline of human skin function.

IntroductionSolar ultraviolet (UV) irradiation damages human skin and causes premature skin aging (photoaging) characterized by thickening, rough texture, coarse wrinkles, and mottled pigmentation (1). Histologic and ultrastructural studies have revealed that the major alterations in photoaged skin are localized in the connective tissue (dermis), which is composed predominantly of type I and type III collagen, elastin, proteoglycans, and fibronectin. Damage induced by UV irradiation is manifested primarily as the disorganization of collagen fibrils (2) that constitute the bulk (90% dry weight) of skin connective tissue and accumulation of abnormal, amorphous, elastin-containing material (3). Since collagen fibrils and elastin are responsible for the strength and resiliency of skin (4), their disarrangement with photoaging causes skin to appear aged.Biochemical evidence of connective tissue alterations in photoaged human skin includes reduced levels of types I and III collagen precursors (5, 6) and cross-links (7), increased ratio of type III to type I collagen (6), and increased levels of elastin (8). Additionally, wrinkle reduction in photodamaged human and mouse skin, after treatment with topical all-trans retinoic acid, correlates with increased dermal procollagen synthesis (9-11).Fibroblasts that reside within skin connective tissue synthesize and secrete type I and type III procollagens. Type I procollagen typically is composed of two α1 chains and one α2 chain, although homotrimers of α1 chains have been described in normal skin and certain diseases (12, 13). Type III procollagen is composed of three identical α1 chains (distinct from type I α1 chains). Type I and III procollagens contain globular amino-and carboxy-terminal domains, which make these proteins soluble. After secretion of type I and type III procollagen, their amino-and carboxy-terminal domains are cleaved by specific proteases (14, 15), resulting in formation of mature collagen, which spontaneously assembles into thin collagen fibrils. Because type I and type III procollagens and their partially processed forms are precursor molecules of mature collagen, their levels generally reflect the level of collagen biosynthesis (16,17).Mature type I collagen in skin undergoes continuous turnover, which is required for optimal connective-tissue function (18). Collagen turnover is regulated by both its rate of synthesis and its rate of breakdown. In mammals, breakdown of collagen fibrils is dependent on the action of one of three known collagenases, MMP-1, MMP-8, or MMP-13, which initiates collagen cleavage at one specific site. Imbalance in collagen syn- The aged appearance of skin following repeated exposure to solar ultraviolet (UV) irradiation stems largely from damage to cutaneous connective tissue, which is composed primarily of type I and type III collagens. We report here that a single exposure to UV irradiation causes significant loss of procollagen synthesis in human skin. Expression of type I and type III procollagens is substantially reduce...

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-β (TGF-β)/Smad pathway, is highly expressed in numerous fibrotic disorders, where 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-β/Smad/CTGF axis is significantly reduced in dermal fibroblasts, the major collagen-producing cells, in aged (80+ years) human skin in vivo. In primary human skin fibroblasts, neutralization of endogenous TGF-β or knockdown of CTGF substantially reduced 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-β 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-β and CTGF, which act through interdependent yet distinct mechanisms. Down regulation of the TGF-β/Smad/CTGF axis likely mediates reduced type I procollagen expression, in aged human skin in vivo.

The dermal extracellular matrix (ECM) provides strength and resiliency to skin. The ECM consists mostly of type I collagen fibrils, which are produced by fibroblasts. Binding of fibroblasts to collagen fibrils generates mechanical forces, which regulate cellular morphology and function. With aging, collagen fragmentation reduces fibroblast-ECM binding and mechanical forces, resulting in fibroblast shrinkage and reduced function including collagen production. Here, we report that these age-related alterations are largely reversed by enhancing structural support of the ECM. Injection of dermal filler, cross-linked hyaluronic acid, into the skin of persons over seventy years-old stimulates fibroblasts to produce type I collagen. This stimulation is associated with localized increased of mechanical forces, indicated by fibroblast elongation/spreading, and mediated by up-regulation of type II TGF-β receptor and connective tissue growth factor. Interestingly, enhanced mechanical support of the ECM also stimulates fibroblast proliferation, expands vasculature, and increases epidermal thickness. Consistent with our observations in human skin, injection of filler into dermal equivalent cultures causes elongation of fibroblasts, coupled with type I collagen synthesis, which is dependent on the TGF-β signaling pathway. Thus, fibroblasts in aged human skin retain their capacity for functional activation, which is restored by enhancing structural support of the ECM.

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