Impact of PRG4 and HA treatments on ACTA2 expression, α-SMA immunostaining, and stress fiber formation in osteoarthritic fibroblast-like synoviocytes (OA FLS) and the role of CD44 in mediating the effect of rhPRG4 in TGF-β stimulated OA FLS
OA FLS (Cell Applications, USA) were isolated from synovial tissues from de-identified patients undergoing knee replacement surgery (n=4; 2 males and 2 females; 62-69 years old). Cells were received in their second passage and were cultured as previously described . OA FLS were used between the 3rd and 6th passages to avoid alterations in gene expression pattern and cell proliferation rate . OA FLS (300,000 cells/well) in serum-free DMEM media were treated with recombinant human TGF-b1 (R&D Systems, USA) (1ng/mL) ± native human synovial PRG4 (apparent mol mass 280 kDa as a monomer; 100mg/mL)  or high molecular weight hyaluronic acid (HA; ~1,200 kDa; R&D Systems; 100mg/mL) for 24 hours. RNA isolation, cDNA synthesis and quantitative PCR were performed as previously described . The cycle threshold (Ct) value of α-SMA (ACTA2) was normalized to the Ct value of GAPDH in the same sample, and the relative expression was calculated using the 2-ΔΔCt method . All primers and probes utilized in our study are commercially available (Thermo Fisher Scientific, USA).
Assessment of a-SMA content in OA FLS was conducted using immunofluorescence and determination of corrected total cell fluorescence (CTCF) using a Nikon E600 fluorescence microscope. OA FLS (200,000 cells/well) were cultured on collagen type I-coated 22 mm glass coverslips for 48 hours in DMEM medium + 10% fetal bovine serum (FBS). Cells were treated with TGF-b1 (1ng/mL) ± PRG4 or HA (100mg/mL for both treatments) for 48 hours in serum-free DMEM medium. Subsequently, cells were fixed in 10% neutral buffered formalin for 15 min and washed twice with phosphate buffered saline (PBS). Cells were permeabilized using 0.01% Triton X-100 in PBS and blocked using 2% bovine serum albumin (BSA; Sigma Aldrich, USA) in PBS for 2 hours at room temperature. Probing for α-SMA was performed using FITC-conjugated anti-α-SMA antibody (1:100 dilution; Abcam, USA) and counterstained using Alexa Fluor 594-conjugated anti-α-tubulin antibody (1:100 dilution: Abcam) overnight at 4oC. Following washing with PBS, cells were mounted on DAPI mounting shield (Abcam) and CTCF was quantified using 4 different fields per slide and mean CTCF was calculated. The presence of stress fibers in OA FLS was also evaluated.
Recombinant human PRG4 (rhPRG4; apparent mol mass 240 kDa) is an endotoxin-free full- length product produced by CHO-M cells (Lubris, Framingham, USA) . Rhodamine labeling of rhPRG4 was performed using the Pierce NHS-Rhodamine Antibody Labeling Kit (Thermo Fisher Scientific). OA FLS (200,000 cells/well) were cultured on collagen type-I coated cover slips and incubated with rhodamine-rhPRG4 (25mg/mL) ± anti-CD44 or isotype control (IC) antibodies (2mg/mL for both antibodies; Abcam) for 30 min. Cells were pre-incubated with the antibodies for 1-hour prior to rhPRG4 addition. Subsequently, cells were washed twice with PBS and mounted on DAPI mounting media and CTCF was quantified as described above. In another set of experiments, OA FLS (300,000 cells/well) were treated with TGF-b1 (1ng/mL) ± rhPRG4 (100mg/mL) ± anti-CD44 or IC antibodies (2mg/mL for both antibodies) for 24 hours followed by determination of ACTA2 expression as described above.
CD44-dependent uptake of rhPRG4, Smad3 phosphorylation and regulation of TGF-b/Smad pathway activation in TGF-b stimulated HEK Blue-TGF-b cells
HEK Blue-TGF-b is an engineered reporter cell line produced by transfecting human embryonic kidney (HEK) cells with TGF-b receptor 1 (TGF-b R1), Smad3 and Smad4 genes (Invivogen, USA). TGF-b1-treatment results in phosphorylation of Smad3 (pSmad3) and Smad4 (pSmad4), translocation to the nucleus and expression of secreted alkaline phosphatase (SEAP). The activity of SEAP can be detected colorimetrically in media supernatants using a specific substrate (Quanti Blue) at 620-655 nm (Invivogen). HEK-TGF-b cells (500,000) were plated onto sterile chamber slides and incubated with rhodamine-rhPRG4 (25mg/mL) for 1 hour ± anti CD44 or IC antibodies (2mg/mL for both antibodies). Cells were pre-incubated with the antibodies for 1-hour prior to rhPRG4 addition. Subsequently, cells were washed with PBS and intracellular CTCF was determined as described above. In another set of experiments, rhodamine labeled rhPRG4 (25mg/mL) was incubated with HEK-TGF-b cells and CD44 was probed using FITC-conjugated anti-CD44 antibody (Abcam) and CD44 (green) and rhPRG4 (red) co-localization was examined using a confocal microscope.
Immunostaining of pSmad3 was performed using an anti-Smad3 antibody that detects phosphorylated serine residues (1:1,000 dilution overnight at 4oC; Abcam). HEK-TGF-b cells were stimulated with TGF-b1 ± rhPRG4 (150mg/mL) for 1 hour. Subsequently, cells were fixed, washed with PBS and permeabilized as described above. Following probing with anti-pSmad3 antibody, cells were washed with PBS and incubated with goat anti-rabbit IgG (Alexa Fluor 488) (1:1,000 dilution for 1 hour at room temperature; Abcam). Cells were subsequently washed with PBS and the green immunofluorescence was quantified across all experimental groups. To investigate the impact of rhPRG4 on TGF-b/Smad pathway activation, HEK-TGF-b cells (100,000 cells/well) were cultured in sterile 96 well plates ± TGF-b1 (1ng/mL) ± rhPRG4 (50, 100 or 150mg/mL) for 24 hours in Quanti-Blue media and the 655 nm absorbance intensity was determined. Data are presented as fold absorbance intensities across experimental groups normalized to untreated controls.
TGF-b induction of stress fibers, focal adhesions (FAs) and cell migration in murine NIH3T3 fibroblasts, comparison of stress fibers and FAs in Prg4+/+ and Prg-/- synovial fibroblasts and role of rhPRG4 in regulating fibroblast migration
Murine fibroblasts (NIH3T3; ATCC; 20,000 cells/well) were cultured on collagen type I-coated 22-mm glass coverslips for 24 hours in DMEM supplemented with 10% bovine calf serum (BCS). NIH3T3 cells were starved in DMEM containing 1% BCS for 24 hours. Murine fibroblasts were treated with murine TGF-b1 (1ng/mL) ± rhPRG4 (200mg/mL) for 24 hours in serum-free DMEM. Subsequently, fibroblasts were fixed using neutral buffered formalin, washed with PBS, permeabilized using 0.1% triton X-100 and probed using an anti-a-SMA (marker of stress fibers; 1:1,000) or anti-vinculin (marker of FA complex; 1:1,000) overnight at 4oC. After washing with PBS, cells were incubated with goat anti rabbit IgG (Alexa Fluor®488) at 1:1,000 dilution for 1 hour at 4°C. Rhodamine-labeled phalloidin (cytoskeleton label; 1:1,000) was added with the secondary antibody. All antibodies were obtained from Abcam. Cells were washed and mounted with DAPI medium for 2 hours and viewed under a confocal microscope. A blinded investigator determined the number of stress fiber-positive fibroblasts. At least 100 cells over at least 5 different fields were evaluated, and the percentage of stress-fiber positive fibroblasts was calculated. Separately, mean fibroblast cell spread area was determined using the NIS-Elements imaging software (Nikon). The number and mean size of FAs per cell were determined using ImageJ software as previously described . Fibroblast migration was determined using a scratch assay. A 1,000 mL pipette tip was used to perform a uniform scratch in the confluent NIH3T3 fibroblast monolayer. TGF-b (1ng/mL) stimulation was performed for 48 hours ± rhPRG4 (200mg/mL). Subsequently, cells were washed and stained (Cell Biolabs) and imaged using an all-in-one fluorescence microscope (Keyence). A region of interest was defined and scratch widths were determined across multiple locations. Cell migration was expressed as percent wound closure across different experimental groups as previously described .
The phenotype of Prg4-/- mice is characterized by synovial membrane hyperplasia, chondrocyte apoptosis, and cartilage surface fibrillation . The Prg4-/- mouse colony is maintained by Dr. Gregory Jay at Rhode Island Hospital (RIH) and is commercially available (stock no. 025737; JAX, USA). The IACUC committee at RIH approved all animal experiments and all experiments were performed according to all applicable guidelines and regulations. Synoviocyte isolation from Prg4-/- and Prg4+/+ synovial tissues was performed as previously described [38, 39]. Prg4-/- and Prg4+/+ synoviocytes were plated onto sterile chamber slides (Thermo Fisher Scientific) and allowed to adhere for 48 hours. Subsequently, cells were stained for a-SMA and vinculin as described above. The mean size of FAs per cell in Prg4-/- and Prg4+/+ synoviocytes was determined as described above. Prg4-/- and Prg4+/+ synoviocytes were seeded in 6 well plates and a scratch was performed in the confluent cell monolayer. Basal synovial fibroblast migration of both genotypes was quantified over 48 hours ± rhPRG4 (200mg/mL), anti-CD44 (2mg/mL) or IC (2mg/mL) antibodies and expressed as percent wound closure .
Generation of Active TGF-b in lipopolysaccharide-stimulated murine macrophage J774A and NIH3T3 fibroblast co-culture and impact of rhPRG4 treatment on fibroblast migration
Murine J774A macrophages (ATCC) were cultured in DMEM medium + 10% FBS. Macrophages (300,000 cells in DMEM medium + 10% FBS) were stimulated with lipopolysaccharide (LPS; Invivogen) (5mg/mL) for 24 hours. Subsequently, murine macrophages were washed five times with DMEM medium and transferred to the top chamber of a transwell co-culture system (0.4 mm pore size; Sigma Aldrich). Murine NIH3T3 cells were seeded in the lower chamber of the transwell system and a scratch was performed as described above. NIH3T3 migration was determined following a 48-hour incubation of fibroblasts with macrophages in the co-culture system as described above ± rhPRG4 (200mg/mL). Active and total TGF-b media levels were determined using an ELISA (R&D Systems).
Age-dependent expression of fibrotic markers in synovial tissues from Prg4GT/GT animals and the role of PRG4/CD44 interaction in modulating synovial fibrosis in vivo
The Prg4 gene-trap (Prg4GT) mouse colony is maintained by Dr. Gregory Jay at RIH and is commercially available (stock no. 025740; JAX) . Prg4GT animal is a genetically engineered PRG4-deficient mouse where the Prg4 expression can be restored via CRE-mediated recombination . The Prg4GT/GT mouse recapitulates the hallmark findings in Prg4-/- mouse, namely synovial tissue hyperplasia and cartilage surface fibrillations and recombination in 3-weeks old animals improved but did not completely normalize joint pathological findings . In our studies, recombination (Prg4GTR/GTR) occurred in 3-weeks old animals via intraperitoneal injection of tamoxifen (0.1 mg/g in 100mL corn oil vehicle) daily for 10 days. We compared gene expression and immunostaining of fibrotic markers: a-SMA, COL1A1 (collagen type-I) and PLOD2 in 2-months old Prg4GT/GT, 2-months old Prg4GTR/GTR, 9-months old Prg4GT/GT and 9-months old Prg4GTR/GTR animals. ACTA2, COL1A1 and PLOD2 expression levels in murine synovial tissues were performed as previously described . Tissues from each three consecutive mice were pooled and underwent RNA isolation, generating five pooled samples in each experimental group. Separately, animal joints (n=5 in each group) underwent decalcification and paraffin-embedded sectioning as previously described . Immunostaining of synovial tissues was performed using primary antibodies against a-SMA, COL1A1 or PLOD2 (1:1,000 dilutions performed overnight at 4oC) (All antibodies were purchased from Abcam). Subsequently, sections were washed and incubated with goat anti-rabbit IgG (Alexa Fluor 488) (1:1,000 dilution) for 1 hour and fluorescence intensities (expressed as lumens/mm2) were quantified using a fluorescence microscope.
To appreciate the significance of PRG4 and CD44 interaction in the context of fibrotic markers’ expression in the synovium, Prg4GT/GT animals were crossed with Cd44-/- mice (stock no. 005085; JAX)  to generate Cd44-/-&Prg4GT/GT animals. Recombination occurred in 3-weeks old animals to generate Cd44-/-&Prg4GTR/GTR animals as described in the last paragraph. Histological analyses of joints harvested from 2-months old animals (at least 4 animals per group) were performed as described above, using Cd44+/+&Prg4GT/GT animals from the same litters as controls. We probed synovial tissues for the following fibrotic markers: a-SMA and PLOD2 using specific primary antibodies (1:1,000 dilutions) followed by goat anti-rabbit IgG (Alexa Fluor 488) and quantitation of fluorescence intensities (expressed as lumens/mm2) as described above.
Target gene expression was statistically evaluated by comparing DCt (Ct value of target gene- Ct value of GAPDH in the same sample) values of different experimental groups. Statistical significance comparing two groups or multiple groups with parametric data was assessed by Student’s t test or ANOVA followed by post-hoc multiple comparisons (Tukey’s post-hoc test). Statistical significance comparing two groups or multiple groups with nonparametric data was assessed by Rank Sum test or ANOVA on the ranks. A p value of < 0.05 was considered statistically significant. Data are presented as scatter plots with mean and standard deviations highlighted.