Elevated cysteine-rich 61 mediates aberrant collagen homeostasis in chronologically aged and photoaged human skin

Abstract

Alterations of human skin connective tissue structure and function are prominent features of chronological aging and solar UV irradiation-induced premature aging (photoaging). These skin connective tissue abnormalities result, in part, from reduced synthesis and elevated degradation of type I collagen, the major structural protein in skin. Here, we report that cysteine-rich 61 (CYR61/CCN1), a novel mediator of collagen homeostasis, is predominantly expressed in human skin connective tissue and is significantly elevated in fibroblasts in chronologically aged (80+ years) and photoaged human skin in vivo. In cultured human skin fibroblasts, elevated CYR61 expression substantially reduces type I procollagen and concurrently increases matrix metalloproteinase-1 (MMP-1), which initiates fibrillar collagen degradation. Elevated CYR61 caused down-regulation of transforming growth factor-beta type II receptor mRNA and protein levels, thereby impairing the transforming growth factor-beta pathway, which reduced type I procollagen and raised MMP-1 expression. Furthermore, elevated CYR61 induced transcription factor activator protein-1 (AP-1), which functions to stimulate MMP-1 expression. Thus, elevated expression of CYR61 in human skin fibroblasts acts through multiple pathways to cause alterations of collagen homeostasis similar to those pathways observed in aged human skin in vivo. These data identify CYR61 as a pivotal regulator of collagen production and degradation in aged and photoaged human skin.

Figure 1-6922

CYR61 is predominantly expressed in dermal cells and colocalizes with fibroblast proteins type I procollagen and HSP47 in human skin in vivo. Skin samples were obtained from healthy adult subjects (32 to 55 years). A: Immunofluorescence staining of CYR61 protein reveals predominant dermal localization. Isotype control antibody immunofluorescence staining is shown in the left panel. Data are representative of five subjects. B: CYR61 mRNA is predominantly expressed in dermis of human skin. Epidermis and dermis were captured by LCM, and CYR61 and 36B4 (internal normalization control) mRNA levels were quantified by real-time RT-PCR. Data are means ± SEM, n = 5, *P < 0.05. CYR61 protein colocalizes with type I procollagen (C) and HSP47 (D) in dermal fibroblasts in human skin in vivo. Colocalization of CYR61 and type I procollagen (COL-1) or HSP47 is indicated by orange-yellow in the merged immunofluorescence images. Data are representative of three subjects.

Figure 2-6922

CYR61 is increased in the dermis of sun-protected chronologically aged human skin in vivo. A: Skin samples were obtained from chronologically aged (80+ years) and young (20 to 30 years) individuals. Dermis was captured by LCM, and CYR61 and 36B4 (internal normalization control) mRNA levels were quantified by real-time RT-PCR. Data are means ± SEM, n = 6, *P < 0.05. B: Dermis was separated from epidermis by dissection. Dermal CYR61 and β-actin (internal normalization control) protein levels were determined by Western analysis. Inset shows representative Western blot. Data are means ± SEM, n = 8, *P < 0.05.

Figure 3-6922

CYR61 is increased in the dermis of photoaged human skin in vivo. A: Skin samples were obtained from severely photoaged forearm and subject-matched, sun-protected hip skin. Dermis was captured by LCM, and CYR61 and 36B4 (internal normalization control) mRNA levels were quantified by real-time RT-PCR. Data are means ± SEM, n = 4, *P < 0.05. B: Dermis was separated from epidermis by dissection. Dermal CYR61 and β-actin protein levels were determined by Western analysis. Inset shows representative Western blot. Data are means ± SEM, n = 6, *P < 0.05.

Figure 4-6922

Overexpression of CYR61 down-regulates type I procollagen and up-regulates MMP-1 in human skin fibroblasts. Fibroblasts were transfected with empty control vector (CTRL) or CYR61 expression vector. Total RNA and whole cell proteins were prepared 48 hours after transfection. mRNA levels were quantified by real-time RT-PCR. 36B4 (housekeeping gene) mRNA was used as internal control for normalization of data. Protein levels were quantified by Western analysis. β-actin protein was used as internal control for normalization of data. Insets show representative Western blot. Overexpression of CYR61 mRNA (A) and protein (B) in human skin fibroblasts. Data are expressed as means ± SEM, n = 3, *P < 0.05. CYR61 overexpression down-regulates type I procollagen (COL-1) mRNA (C) and protein (D) in human skin fibroblasts. Data are means ± SEM, n = 3, *P < 0.05. CYR61 overexpression increases MMP-1 mRNA (E) and protein (F) in human skin fibroblasts. Data are means ± SEM, n = 3, *P < 0.05.

Figure 5-6922

Elevated CYR61 down-regulates TGF-β type II receptor and impairs TGF-β pathway in human skin fibroblasts. Human skin fibroblasts were treated with the TGF-β type I receptor kinase inhibitor SB431542 (10 μmol/L). Total RNA and whole-cell protein extracts were prepared 24 hours after treatment. Type I procollagen (COL-1), MMP-1, and 36B4 (internal normalization control) mRNA levels were quantified by real-time RT-PCR. Type I procollagen (COL-1), MMP-1, and β-actin proteins were quantified by Western analysis. Insets show representative Western blot. A–D: Inhibition of the TGF-β type I receptor kinase reduces type I procollagen mRNA (A) and protein (B) and increases MMP-1 mRNA (C) and protein (D) in human dermal fibroblasts. Data are means ± SEM, n = 3, *P < 0.05. E and F: Elevated CYR61 down-regulates TGF-β type II receptor (TβRII) mRNA (E) and protein (F). Fibroblasts were transfected with empty control vector (CTRL) or CYR61 expression vector. Total RNA and cellular protein were prepared 48 hours after transfection. TGF-β type II receptor and 36B4 (internal normalization control) mRNA levels were quantified by real-time RT-PCR. TGF-β type II receptor and β-actin (internal normalization control) protein levels were quantified by Western analysis. Inset shows representative Western blot. Data are means ± SEM, n = 3, *P < 0.05. G: Elevated CYR61 inhibits TGF-β responsiveness. TGF-β reporter (p3TP-Luc) was cotransfected with empty vector (−) or CYR61 (+) expression vector. Fibroblasts were treated with TGF-β1 (10 ng/ml) 32 hours after transfection for 16 hours. Cell lysates were prepared 48 hours after transfection. Reporter activity was determined by luciferase assay. Data are means ± SEM, n = 3, *P < 0.05.

Figure 6-6922

TGF-β type II receptor mRNA expression is reduced in the dermis of sun-protected chronologically aged, and photodamaged human skin in vivo. A: Skin samples were obtained from chronologically aged (80+ years) and young (20 to 30 years) individuals. Dermis was captured by LCM, and TβRII and 36B4 (internal normalization control) mRNA levels were quantified by real-time RT-PCR. Data are means ± SEM, n = 5, *P < 0.05. B: Skin samples were obtained from severely photoaged forearm and subject-matched, sun-protected hip skin. Dermis was captured by LCM, and CYR61 and 36B4 (internal normalization control) mRNA levels were quantified by real-time RT-PCR. Data are means ± SEM, n = 5, *P < 0.05.

Figure 7-6922

Elevated CYR61 activates AP-1 in human skin fibroblasts. AP-1 reporter construct (pAP-1-TA-Luc) was cotransfected with empty vector (CTRL) or CYR61 expression vector. Cell lysates were prepared 48 hours after transfection. Reporter activity was determined by luciferase assay. Data are means ± SEM, n = 3, *P < 0.05.