A Role for Estrogen Receptor-α and Estrogen Receptor-β in Collagen Biosynthesis in Mouse Skin  Margaret Markiewicz, Sergey Znoyko, Lukasz Stawski, Angela Ghatnekar, Gary Gilkeson, Maria Trojanowska  Journal of Investigative Dermatology  Volume 133, Issue 1, Pages 120-127 (January 2013) DOI: 10.1038/jid.2012.264 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Histopathological analyses of collagen in female mouse skin tissue. (a) Representative examples of skin sections stained with hematoxylin–eosin. (b) Skin samples were processed and stained with Masson's Trichrome as described in the Materials and Methods section. Collagen staining appears blue (original magnification × 100; bar=15.0μm). (c) Graphical presentation of skin thickness measured from the top of the granular layer to the junction between the dermis and the subcutaneous fat, using Sot Advanced Image Software. Values are presented as means±SD. *P<0.05. Journal of Investigative Dermatology 2013 133, 120-127DOI: (10.1038/jid.2012.264) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Absence of estrogen receptor (ER)α or ERβ differentially modulates collagen content in female skin tissue. (a) Hydroxyproline levels were measured as described in Materials and Methods section. The amount of hydroxyproline in each sample was calculated by comparison with a hydroxyproline standard curve and expressed as μg of hydroxyprolineml−1. Values are presented as means±SD. *P<0.05. (b, c) The acetic acid extraction of collagen was performed with the addition of pepsin (see Materials and Methods section). Arrows indicate collagen α1(I) and α2(I) subunits. Slower migrating bands represent cross-linked α-chain dimers (also termed β-components). The molecular weight marker for protein (kDa) is shown on the left. A graphical representation of collagen levels quantified using NIH Image densitometry software is shown on the right. Values are presented as means±SD. *P<0.05. Journal of Investigative Dermatology 2013 133, 120-127DOI: (10.1038/jid.2012.264) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Absence of estrogen receptor (ER)α and ERβ differentially modulates collagen synthesis in the skin and cultured fibroblasts. (a, b) Skin samples were taken with an 8-mm biopsy punch from the dorsal side of wild-type (WT), ERα−/−, or ERβ−/− mice. Acetic acid extraction of collagen was performed as described in Materials and Methods section. Arrows indicate collagen α1(I) and α2(I) subunits. β-Components represent cross-linked α-chain dimers. The molecular weight marker for proteins (kDa) is shown on the left. A graphical representation of collagen level quantified using NIH Image densitometry software is shown on the right. Values are presented as means±SD. *P<0.05. (c) Quantitative PCR (qPCR) analysis of fibril-forming collagens in skin isolated from control (black bars), ERα−/−, and ERβ−/− mice (white bars). Fold change shown in the graphs is normalized to mRNA expression by one of the control mice. Values are presented as means±SD. *P<0.05. (d) Dermal fibroblasts obtained from ERα−/− and ERβ−/− mice were treated for 24 hours with 17β-estradiol (10nM). Collagen a1 (1) mRNA levels were determined by qPCR. Values were normalized to control (arbitrarily set at 100) and presented as means±SD. *P<0.05. Representative western blot of collagen protein levels is shown (e). Journal of Investigative Dermatology 2013 133, 120-127DOI: (10.1038/jid.2012.264) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Absence of estrogen receptor (ER)α and ERβ modulates mRNA and protein expression levels of matrix metalloproteinases (MMPs) in female mouse skin. Quantitative real-time PCR analyses of gene expression of indicated MMPs in wild-type (WT; black bars), ERα−/− (a), and ERβ−/− (b; white bars) mice. Fold change shown in the graphs is normalized to mRNA expression by one of the control mice. Values are presented as means±SD. *P<0.05. (c, d) Representative western blots of MMP-8 and MMP-15 in skin samples from WT, ERα−/−, and ERβ−/− mice. Immunodetection of β-actin is shown as loading control. *P<0.05. Journal of Investigative Dermatology 2013 133, 120-127DOI: (10.1038/jid.2012.264) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Quantitative real-time PCR and protein analyses of small leucine proteoglycans in wild-type (WT), estrogen receptor (ER)α−/−, and ERβ−/− mice. (a, b) Quantitative PCR analysis of gene expression of lumican (Lum), fibromodulin (Fmod), and decorin (Dcn) in skin isolated from control (black bars), ERα−/−, and ERβ−/− mice (white bars). Fold change shown in the graphs is normalized to mRNA expression by one of the control mice. Values are presented as means±SD. *P<0.05. (c) Immunostaining was performed on paraffin-embedded, formalin-fixed skin tissue from control, ERα−/−, and ERβ−/− female mice (original magnification × 200; bar=50μm). To expose core proteins, sections were treated with appropriate enzymes (see Materials and Methods section). Journal of Investigative Dermatology 2013 133, 120-127DOI: (10.1038/jid.2012.264) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions