Osteopontin in Systemic Sclerosis and Its Role in Dermal Fibrosis Minghua Wu, Daniel J. Schneider, Maureen D. Mayes, Shervin Assassi, Frank C. Arnett, Filemon K. Tan, Michael R. Blackburn, Sandeep K. Agarwal  Journal of Investigative Dermatology  Volume 132, Issue 6, Pages 1605-1614 (June 2012) DOI: 10.1038/jid.2012.32 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Osteopontin (OPN) expression in systemic sclerosis (SSc) patients and fibrotic mouse dermis. (a) Plasma levels of OPN are increased in SSc patients overall and in limited and diffuse SSc compared with controls. Data are presented as mean±SEM. (b) Plasma levels of OPN are increased in SSc-associated antibody (Ab; anti-centromere (ACA), anti-topoisomerase I (ATA), and anti-RNA polymerase III (ARA)) subsets of patients compared with controls. (c–h) Immunohistochemical (IHC) analyses with anti-OPN antibody demonstrate increased OPN immunoreactivity in the skin from SSc patients (f–h) compared with controls (c–e). Bar=200μm (c, f), 50μm (d, e), and 20μm (e, h). (i) Increased numbers of fibroblasts (spindle-shaped cells) and inflammatory cells (round cells) with OPN immunoreactivity were observed in control (open bars) compared with SSc skin (black bars). (j) Quantitative real-time reverse transcriptase–PCR analysis of total RNA from lesional skin of wild-type (WT) mice that received daily injection of bleomycin (bleo) or phosphate-buffered saline (PBS) for 28 days demonstrates increased expression of OPN after bleo injection. Data are presented as mean±SEM of duplicated determinations from 10 mice per group. *P<0.05. (k) IHC analysis of bleo-injected mouse skin demonstrated increased OPN expression compared with PBS-injected skin. Bar=200μm in panels 1 and 3, and 50μm in panel 4. **P<0.01; ***P<0.001. Journal of Investigative Dermatology 2012 132, 1605-1614DOI: (10.1038/jid.2012.32) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Osteopontin (OPN)−/− mice have reduced skin fibrosis in the bleomycin (bleo)-induced dermal fibrosis model. OPN−/− and wild-type mice received daily s.c. injections of phosphate-buffered saline (PBS) or bleo for 28 days, and lesional skin was examined. (a) Hematoxylin and eosin (H&E) stain and Masson's Trichrome (MT). Bar=200μm. (b) Quantification of dermal thickness. The results represent the mean±SEM, 15 mice/group. Open bars, PBS; closed bars, bleo. *P<0.001. (c) Soluble collagen was quantified by Sircol colorimetric assay. Data are presented as mean±SEM, from 15 mice/group. Open bars, PBS; closed bars, bleo. *P<0.05. (d) Total RNA from lesional skin was analyzed for Col1a1 mRNA by qRT-PCR. Data are presented as mean±SEM of duplicate determinations from 6 mice/group, *P<0.05. Journal of Investigative Dermatology 2012 132, 1605-1614DOI: (10.1038/jid.2012.32) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Osteopontin (OPN)−/− mice have decreased dermal inflammation. (a) Immunohistochemistry with anti-Mac-3 antibody was performed on lesional skin from mice injected with phosphate-buffered saline (PBS) or bleomycin (bleo). The number of Mac-3-positive macrophages was quantified in six high-powered fields (HPs) per mouse (four mice per group, **P<0.01). Inflammatory cytokine mRNA levels were decreased in OPN−/− mice injected with bleo compared with wild-type (WT) mice ((b) CCL-2, (c) IL-6; six mice per group, *P<0.05). Data are presented as mean±SEM. Open bars, PBS; closed bars, bleo. Journal of Investigative Dermatology 2012 132, 1605-1614DOI: (10.1038/jid.2012.32) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Osteopontin (OPN)−/− mice have decreased transforming growth factor (TGF)-β and downstream pathways. (a) Immunohistochemistry (IHC) with anti-smooth muscle actin (SMA) antibody was performed on lesional skin from mice injected with phosphate-buffered saline (PBS) or bleomycin (bleo). IHC with anti-SMA antibody demonstrated decreased myofibroblasts in OPN−/− mice. Data are presented as mean±SEM of triplicate determinations in at least six microscopic fields, four mice/group. Open bars, PBS; closed bars, bleo. *P=0.0076. Total mRNA from lesional skin of OPN−/− mice injected with bleo demonstrated decreased levels of (b) SMA, (c) TGF-β, (d) CCN2, and (e) PAI-1. Data are presented as mean±SEM of duplicate determinations from six mice/group, *P<0.05. PPIA, peptidylprolyl isomerase A. Journal of Investigative Dermatology 2012 132, 1605-1614DOI: (10.1038/jid.2012.32) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Osteopontin (OPN)−/− mice have decreased phosphorylated SMAD2 (pSMAD2) in vivo but not in vitro. Immunohistochemistry with anti-pSMAD2 antibody (a, b) or phosphorylated extracellular signal–regulated kinase (pERK) antibody (c, d) was performed on lesional skin from mice injected with phosphate-buffered saline or bleomycin (bleo). OPN−/− mice had decreased numbers of (a) pSMAD2 inflammatory cells and (b) fibroblasts after bleo injection. OPN−/− mice also had decreased numbers of (c) pERK inflammatory cells and (d) fibroblasts after bleo injection. Data are presented as mean±SEM in at least six microscopic fields, four mice per group. (e) In contrast, in vitro stimulation of dermal fibroblasts with transforming growth factor-β (TGF-β) resulted in similar levels of pSMAD2 in wild-type (WT) and OPN−/− cells. (f) OPN−/− dermal fibroblasts had less migratory capacity in the scratch-wound assay compared with WT dermal fibroblasts, which was partially restored by 5μgml−1 of OPN (representative of n=3 WT and 3 OPN−/−). Bone marrow–derived macrophages from OPN−/− mice produced less (g) tumor necrosis factor-α (TNF-α) and (h) TGF-β relative to WT macrophages (WT n=4, OPN−/−n=4). Data are presented as mean±SEM. Cont, control; KO, knockout; LPS, lipopolysaccharide. Journal of Investigative Dermatology 2012 132, 1605-1614DOI: (10.1038/jid.2012.32) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions