CD146: a potential therapeutic target for systemic sclerosis

Systemic sclerosis (scleroderma, SSc) is a chronic disease of connective tissues, and is clinically characterized by persistent fibrosis in the skin as well as in a variety of organs (Katsumoto et al., 2011). The pathogenesis of SSc is complex and involves vasculopathy, autoimmunity and fibrosis. The hallmarks of late-stage SSc are the excessive secretion and accumulation of extracellular matrix (ECM) by aberrantly activated fibroblasts (myofibroblasts) in the skin and internal organs (Bhattacharyya et al., 2012). Similar to other fibrotic diseases, dysfunction of the affected organs is a common feature and results in high morbidity and significantly increased mortality (Akhmetshina et al., 2012). However, effective anti-fibrotic strategies for the treatment of SSc are not available to date, primarily due to an incomplete understanding of the precise mechanism governing skin fibroblast activation during SSc progression (Distler and Cozzio, 2016). Therefore, identification of novel therapeutic targets for fibrotic-targeted therapy of SSc is of paramount importance. CD146 was originally identified as a biomarker for metastatic melanoma (Lehmann et al., 1987). Mounting evidence suggests that CD146 plays an important role in the progression of many inflammatory diseases (Dagur and McCoy, 2015; Duan et al., 2013; Luo et al., 2017; Xing et al., 2014). Recent studies suggested that CD146/sCD146 represent a novel biomarker useful for assessing the disease activity of SSc (Ito et al., 2017; Kaspi et al., 2017). Consistently, we found that CD146 is up-regulated on dermal fibroblasts of BLM-induced mice and SSc patients (Fig. S1), and genetic deletion of CD146 attenuated dermal fibrosis, as indicated by decreased skin thickness, collagen content and myofibroblast accumulation (Fig. S2A–C). In addition, we observed decreased type I collagen and fibronectin expression (Fig. S2D–F), and fibroblasts accumulation (Fig. 2G–I) in the skin of CD146-deficient mice. Together, the data from both human and mice indicate that CD146 is a fibrosis-related gene and may be critical for the pathogenesis of dermal fibrosis. As fibroblast accumulation and excessive ECM secretion are the hallmarks of SSc and CD146 elimination reduced the number of skin fibroblasts and inhibited fibrosis, we explored the molecular mechanism behind fibroblast activation. Recently, canonical Wnt signal activation, characterized by increased β-catenin levels, has emerged as a key player in sustained and pathological activation of fibroblasts during the progression of fibrotic diseases (Bergmann and Distler, 2016). Several studies suggest that CD146 expression is potentially linked to Wnt/β-catenin activation in various types of cells (Liu et al., 2012; Tung and Lee, 2017). Therefore, CD146 might facilitate SSc development by mediating Wnt/ β-catenin-induced fibrosis. First, we tested whether CD146 expression was associated with Wnt/β-catenin activation in skin fibroblasts, and found that BLM-induced CD146 mice exhibited a decreased number of nuclear β-catenin fibroblasts (Fig. S3A), decreased expression levels of β-catenin target genes, including axin 2, cyclin D1 and c-Myc (Fig. S3B) in lesion skin relative to those in CD146 mice. These results imply that CD146 expression is tightly linked to Wnt/β-catenin activation in skin fibroblasts during tissue fibrosis. To investigate the molecular mechanism whereby the absence of CD146 results in reduced fibrogenesis and impaired Wnt/ β-catenin activation in skin fibroblasts, we next examined the role of CD146 in Wnt/β-catenin-induced fibroblast activation using primary human skin fibroblasts. Since Wnt1 is the major canonical Wnt ligand specifically up-regulated in fibrotic skin and promotes SSc, we used Wnt1 as a stimulator to trigger fibroblast proliferation and ECM production, mimicking skin fibroblast activation in vivo. Results showed that knockdown of CD146 eliminated Wnt1-induced proliferation (Fig. 1A) and the expression of fibrotic genes (α-SMA and type I collagen) (Fig. 1B and 1C), while rescuing CD146 expression restored these functions, demonstrating that CD146 is essential for Wnt1-induced fibroblast proliferation and ECM production. Then, we examined the role of CD146 in β-catenin activation and the expression of its downstream effectors. We found that Wnt1-induced GSK3β phosphorylation and βcatenin accumulation was impaired by CD146 knockdown and was rescued after restoration of CD146 expression (Fig. 1D). The requirement of CD146 in Wnt/β-catenin activation was further supported by the results of β-catenin/TCF transcription activity assay (Fig. 1E). To establish whether CD146 is required for the transcription of downstream β-


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For transfection, scrambled non-targeting control siRNA was used as a negative control. Lipofectamine 2000-mediated transfection was employed according to the manufacturer's instructions. To downregulate CD146 expression, 50 nM siRNA targeting CD146 was transfected into human skin fibroblasts. The co-transfection of CD146-siRNA (50 nM) and CD146 expression plasmid (Flag-CD146 plasmid, 2 µg) was performed to restore CD146 expression. The empty vector (Flag-empty) was used as an internal control.

SSc patients and controls
Skin biopsies were obtained from SSc patients and unrelated healthy participants at PUMCH. All SSc patients met the American College of Rheumatology (ACR) criteria for SSc (LeRoy and Medsger, 2001). Patients had been diagnosed with diffuse cutaneous SSc, with the disease duration being less than 5 years in all participants recruited from PUMCH. Healthy participants showed no personal or family history of autoimmune diseases. Skin biopsies were obtained from affected forearm skin tissues, or from a similar location from healthy participants.

Mice, bleomycin (BLM) administration, and antibody treatment
All mice were housed and cared for in a pathogen-free facility at PUMCH. Wild-type (WT) (C57BL/6) mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Both CD146 +/+ (WT) mice and CD146 -/mice were from a C57BL/6 background. CD146 -/mice were generated using a Cre/loxP recombination system by crossing EIIa-cre mice (The Jackson Laboratory, Bar Harbor, ME, USA) with CD146 floxed/floxed mice. All genotypes were confirmed by 3 PCR analysis.

Histological assessment and immunohistochemistry (IHC)
On the day after the final injection, mice were sacrificed, and skin sections were taken.
Sections (5-µm thick) of formalin-fixed paraffin-embedded skin biopsies were stained with hematoxylin and eosin (H&E). To evaluate levels of skin fibrosis in mice treated with BLM, the thickness of the dermis, defined as the distance from the epidermal-dermal junction to the dermal-adipose layer junction, was measured at six randomly selected sites/microscopic fields in each animal (Wu et al., 2012). To analyze the accumulated collagen content in the lesioned skin, sections were stained with Masson's trichrome. Immunohistochemistry was performed on formalin-fixed paraffin-embedded skin biopsies using α-smooth muscle actin (α-SMA).

Collagen measurement
Collagen contents of skin tissues were quantified following the instructions of the 4 QuickZyme Total Collagen Assay (QuickZyme Biosciences, Leiden, Netherlands).
Skin samples from a 6-mm biopsy punch were used (Wu et al., 2014).

RNA isolation and real-time (RT) PCR
Tissue total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified with a High Pure RNA Tissue Kit (Roche, Basel, Switzerland). RNA (2 µg) was reverse-transcribed into cDNA. RT-PCR was carried out using SYBR Green Immunoprecipitated proteins were detected by western blot.

In vitro Fc pull-down assay
Fc or Fc-CD146 (0.15 µg) was mixed with Myc-Wnt1 in PBS for 2 h. Then, 25 µl of protein G-agarose beads were added and incubated for 1 h. Proteins bound to the beads were then boiled in sample loading buffer and subjected to immunoblotting.
For antibody inhibition assay, 0.15 µg His-CD146 or His-CD146 D4-5 was mixed with Myc-Wnt1 in PBS for 2 h in the presence or absence of anti-CD146 mAbs.
Anti-Myc or anti-His tagged antibody was used for precipitating the protein complex.
Precipitates were then boiled in sample loading buffer and subjected to immunoblotting.

Cell proliferation assays
Wnt1-induced fibroblast proliferation was performed using a CCK-8 Cell Counting Kit. Briefly, the same number of fibroblasts was seeded in each well of a 96-well plate. Anti-CD146 mAb AA98 or AA1 (50 µg/ml) was added 1 h before stimulation with Wnt1 supernatant. Then, 48 h after stimulation, the relative cell number was determined by measuring the optical density (OD) of CCK-8 at 450 nm.

Luciferase reporter assays
Fibroblasts used in the luciferase reporter assay were seeded in 48-well plates and transfected in triplicate with plasmids or siRNAs together with Super TOP-FLASH (Addgene, Cambridge, MA, USA). pRL-TK was used as an internal control, and luciferase activity was determined as described.

Statistical analysis
All experiments were performed in triplicate, and all summary data are expressed as mean ± SEM. A one-or two-way ANOVA test was used to compare differences between groups in the various experiments. SPSS 11.0 for Windows was used to perform the analyses. Differences with p-values < 0.05 were considered to be statistically significant.