Zhaoping Qin

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.

Elevated Matrix Metalloproteinases and Collagen Fragmentation in Photodamaged Human Skin: Impact of Altered Extracellular Matrix Microenvironment on Dermal Fibroblast Function

Exposure of the human skin to acute solar ultraviolet (UV) irradiation induces several members of the matrix metalloproteinase family (MMPs), which degrade collagen fibrils and other components of the dermal extracellular matrix (ECM) (Fisher et al., 1996; Yaar and Gilchrest, 2007). Elevation of MMPs due to chronic sun exposure can promote accumulation of dermal ECM fragmentation, which contributes to impaired skin function and aged appearance of chronically sun-exposed skin (Fisher et al., 1997). Histological and ultrastructural studies have revealed that major alterations in dermal extracellular matrix, most notably the accumulation of amorphous elastin-containing material in the upper dermis, and disorganized collagen fibrils, which constitute the bulk (90% dry weight) of skin connective tissue.

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.

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

Abstract:  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 7 days 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.

CCN1 contributes to skin connective tissue aging by inducing age-associated secretory phenotype in human skin dermal fibroblasts.

Dermal connective tissue collagen is the major structural protein in skin. Fibroblasts within the dermis are largely responsible for collagen production and turnover. We have previously reported that dermal fibroblasts, in aged human skin in vivo, express elevated levels of CCN1, and that CCN1 negatively regulates collagen homeostasis by suppressing collagen synthesis and increasing collagen degradation (Quan et al. Am J Pathol 169:482–90, 2006, J Invest Dermatol 130:1697–706, 2010). In further investigations of CCN1 actions, we find that CCN1 alters collagen homeostasis by promoting expression of specific secreted proteins, which include matrix metalloproteinases and proinflammatory cytokines. We also find that CCN1-induced secretory proteins are elevated in aged human skin in vivo. We propose that CCN1 induces an “Age-Associated Secretory Phenotype”, in dermal fibroblasts, which mediates collagen reduction and fragmentation in aged human skin.

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

The dermal compartment of human skin is largely composed of dense collagen‐rich fibrils, which provide structural and mechanical support. Skin dermal fibroblasts, the major collagen‐producing cells, are interact with collagen fibrils to maintain cell spreading and mechanical force for function. A characteristic feature of aged human skin is fragmentation of collagen fibrils, which is initiated by matrix metalloproteinase 1 (MMP‐1). Fragmentation impairs fibroblast attachment and thereby reduces spreading. Here, we investigated the relationship among fibroblast spreading, mechanical force, MMP‐1 expression, and collagen fibril fragmentation. Reduced fibroblast spreading due to cytoskeletal disruption was associated with reduced cellular mechanical force, as determined by atomic force microscopy. These reductions substantially induced MMP‐1 expression, which led to collagen fibril fragmentation and disorganization in three‐dimensional collagen lattices. Constraining fibroblast size by culturing on slides coated with collagen micropatterns also significantly induced MMP‐1 expression. Reduced spreading/mechanical force induced transcription factor c‐Jun and its binding to a canonical AP‐1 binding site in the MMP‐1 proximal promoter. Blocking c‐Jun function with dominant negative mutant c‐Jun significantly reduced induction of MMP‐1 expression in response to reduced spreading/mechanical force. Furthermore, restoration of fibroblast spreading/mechanical force led to decline of c‐Jun and MMP‐1 levels and eliminated collagen fibril fragmentation and disorganization. These data reveal a novel mechanism by which alteration of fibroblast shape/mechanical force regulates c‐Jun/AP‐1‐dependent expression of MMP‐1 and consequent collagen fibril fragmentation. This mechanism provides a foundation for understanding the cellular and molecular basis of age‐related collagen fragmentation in human skin.

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) 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. J. Cell. Biochem. 113: 3011–3018, 2012.

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.

Elevated cysteine-rich protein 61 (CCN1) promotes skin aging via upregulation of IL-1β in chronically sun-exposed human skin

Chronic exposure of human skin to solar ultraviolet (UV) irradiation causes premature skin aging, which is characterized by reduced type I collagen production and increased fragmentation of the dermal collagenous extracellular matrix. This imbalance of collagen homeostasis is mediated, in part, by elevated expression of the matricellular protein cysteine-rich protein 61 (CCN1), in dermal fibroblasts, the primary collagen producing cell type in human skin. Here, we report that the actions of CCN1 are mediated by induction of interleukin 1β (IL-1β). CCN1 and IL-1β are strikingly induced by acute UV irradiation, and constitutively elevated in sun-exposed prematurely aged human skin. Elevated CCN1 rapidly induces IL-1β, inhibits type I collagen production, and upregulates matrix metalloproteinase-1, which degrades collagen fibrils. Blockade of IL-1β actions by IL-1 receptor antagonist largely prevents the deleterious effects of CCN1 on collagen homeostasis. Furthermore, knockdown of CCN1 significantly reduces induction of IL-1β by UV irradiation, and thereby partially prevents collagen loss. These data demonstrate that elevated CCN1promotes inflammaging and collagen loss via induction of IL-1β and thereby contributes to the pathophysiology of premature aging in chronically sun-exposed human skin.