Downloaded from http://ard.bmj.com/ on November 18, 2014 - Published by group.bmj.com
ARD Online First, published on March 21, 2014 as 10.1136/annrheumdis-2013-204822 Basic and translational research
EXTENDED REPORT
Follistatin-like protein 1 regulates chondrocyte proliferation and chondrogenic differentiation of mesenchymal stem cells Yury Chaly,1 Harry C Blair,2,3 Sonja M Smith,1 Daniel S Bushnell,4 Anthony D Marinov,4 Brian T Campfield,4 Raphael Hirsch1 Handling editor Tore K Kvien ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ annrheumdis-2013-204822). 1
Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA 2 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA 3 VA Medical Center, Pittsburgh, Pennsylvania, USA 4 Department of Pediatrics, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA Correspondence to Dr Yury Chaly, Department of Pediatrics, University of Iowa Carver College of Medicine, 2191 ML, 500 Newton Rd, Iowa City, IA 52242, USA; [email protected] Received 1 November 2013 Revised 14 February 2014 Accepted 1 March 2014
ABSTRACT Objectives Chondrocytes, the only cells in the articular cartilage, play a pivotal role in osteoarthritis (OA) because they are responsible for maintenance of the extracellular matrix (ECM). Follistatin-like protein 1 (FSTL1) is a secreted protein found in mesenchymal stem cells (MSCs) and cartilage but whose function is unclear. FSTL1 has been shown to modify cell growth and survival. In this work, we sought to determine whether FSTL1 could regulate chondrogenesis and chondrogenic differentiation of MSCs. Methods To study the role of FSTL1 in chondrogenesis, we used FSTL1 knockout (KO) mice generated in our laboratory. Proliferative capacity of MSCs, obtained from skulls of E18.5 embryos, was analysed by flow cytometry. Chondrogenic differentiation of MSCs was carried out in a pellet culture system. Gene expression differences were assessed by microarray analysis and real-time PCR. Phosphorylation of Smad3, p38 MAPK and Akt was analysed by western blotting. Results The homozygous FSTL1 KO embryos showed extensive skeletal defects and decreased cellularity in the vertebral cartilage. Cell proliferation of FSTL1-deficient MSCs was reduced. Gene expression analysis in FSTL1 KO MSCs revealed dysregulation of multiple genes important for chondrogenesis. Production of ECM proteoglycans and collagen II expression were decreased in FSTL1-deficient MSCs differentiated into chondrocytes. Transforming growth factor β signalling in FSTL1 KO cells was significantly suppressed. Conclusions FSTL1 is a potent regulator of chondrocyte proliferation, differentiation and expression of ECM molecules. Our findings may lead to the development of novel strategies for cartilage repair and provide new disease-modifying treatments for OA.
INTRODUCTION
To cite: Chaly Y, Blair HC, Smith SM, et al. Ann Rheum Dis Published Online First: [please include Day Month Year] doi:10.1136/ annrheumdis-2013-204822
Osteoarthritis (OA) is the most common degenerative joint disease. It is characterised by loss of articular cartilage, changes in the subchondral bone, and osteophytosis. The exact cause of the disease remains unknown, and there is no effective therapy for OA, except joint replacement surgery. Typically, OA begins with aging-related disruption of the articular cartilage surface, although specific risk factors, such as joint trauma or metabolic disorders increase the risk of developing OA. Chondrocytes, the only cells in the articular cartilage, play a pivotal role in OA because they are responsible for maintenance of the extracellular matrix (ECM), the primary
target of osteoarthritic cartilage degradation. OA cartilage cells display dysregulation of anabolic and catabolic processes as well as profound reduction in cell number due to a decrease in cell proliferation and an increase in apoptotic cell death. Since articular cartilage has little or no regenerative capacity, the use of mesenchymal stem cells (MSCs) has become an attractive approach for cartilage repair.1 MSCs are capable of proliferating and differentiating into chondrocytes in culture; however, the process of in vitro chondrogenesis is not completely understood. For the future success of cartilage-regenerative medicine, it is critical to elucidate the regulatory mechanisms underlying chondrogenesis. A number of factors, such as insulin-like growth factor (IGF), transforming growth factor β (TGFβ), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), the sex determining region Y-type high-mobility group box (SOX) family of transcription factors, and other cell differentiationrelated signals, including Indian Hedgehog (IHH) and Wnt proteins have been implicated in the process of chondrocyte proliferation and differentiation.2–5 Proper cartilage development is linked to a delicate balance in the activity of the abovementioned factors. Their activities are controlled at multiple levels, both extracellularly and intracellularly. Despite the progress made in recent years in understanding signalling mechanisms, the role of different factors involved in the fine regulation of chondrogenic pathways remains to be elucidated. This study examined the contribution to articular cartilage homoeostasis of follistatin-like protein 1 (FSTL1), an extracellular protein whose functional role in physiological and pathological processes is still unclear. FSTL1 was cloned as a TGFβ-inducible protein from a mouse osteoblastic cell line.6 Our previous studies showed that FSTL1 is an important mediator in the pathogenesis of rheumatoid arthritis and other systemic autoimmune diseases.7–9 We also found that FSTL1 enhances the ability of T cells and monocytes/macrophages to respond to inflammatory signals.10–12 But, unlike most proinflammatory proteins, haematopoietic cells do not produce FSTL1.7 Rather, it is expressed in cells of the mesenchymal lineage, endothelial cells and neurones.13–16 It is noteworthy that FSTL1 is strongly expressed in the mesenchyme of multiple organs during embryogenesis.17–20 Loss-of-function studies in mice demonstrated that homozygous FSTL1 knockout
Chaly Y, etArticle al. Ann Rheum Dis 2014;0:1–7. doi:10.1136/annrheumdis-2013-204822 1 Copyright author (or their employer) 2014. Produced by BMJ Publishing Group Ltd (& EULAR) under licence.
Downloaded from http://ard.bmj.com/ on November 18, 2014 - Published by group.bmj.com
Basic and translational research (KO) animals die at birth due to abnormalities of the respiratory system, particularly, tracheomalacia.17 18 FSTL1 KO mice also display extensive skeletal defects, further supporting the role of FSTL1 in chondrogenesis.17 18 FSTL1 has been found to affect cell growth and survival. For example, FSTL1 inhibits apoptosis in cardiomyocytes and endothelial cells and promotes endothelial cell function and blood vessel growth.14 16 Recent studies17 18 21 also showed that FSTL1 may interact with key mediators of MSC chondrogenesis, proteins of the TGFβ superfamily or their receptors, and modulate their activities. Thus, we hypothesise that FSTL1 may play an important role in maintaining cartilage homeostasis by enhancing cell proliferation, survival and anabolic activity in articular chondrocytes. In the present work, we sought to determine whether FSTL1 could regulate chondrogenesis as well as to identify the molecular mechanisms for FSTL1-mediated regulation of differentiation of MSCs into chondrocytes.
METHODS Generation of FSTL1-deficient mice All animal experiments were approved by the University of Iowa Animal Care and Use Committee. The targeting vector and the FSTL1WT/flox mice were generated by Ozgene (Australia) (see online supplementary methods for details of the breeding strategies).
Skeletal preparations and histological examination Alcian blue/alizarin red staining of cartilage and bone in E18.5 embryos was performed following the standard protocol. For histological examination of lumbar vertebrae, FSTL1 KO and wild-type (WT) embryos at day E18.5 were fixed in 5% formaldehyde and then transferred to 70% ethanol the next day and subjected to paraffin embedding, sectioning and H&E staining using standard protocols.
Mouse MSC cultures and chondrogenic differentiation of MSCs MSCs were isolated from skulls of E18.5 embryos by collagenase digestion and differentiated into chondrocytes in a pellet culture system (see online supplementary methods for details).
Quantitative reverse transcriptase (RT)-PCR Total cDNA was produced from MSCs or chondrocyte pellets using the RNeasy Mini Kit (Qiagen) and the SuperScript II Reverse Transcriptase Kit (Invitrogen). PCR was performed in a LightCycler (ABI7000) using the Brilliant SYBR Green QPCR Master Mix (Agilent) as described.11 The copy number (number of transcripts) of amplified product was calculated from a standard curve obtained by plotting known input concentrations of plasmid DNA (see online supplementary table S1 for primer sequences).
was used as the starting material for further FSTL1 purification (see online supplementary methods).
RNA processing, microarray and data analysis Total RNA was extracted from MSCs isolated from WT and KO embryos using the RNeasy kit (Qiagen). Microarray analysis was performed with the MouseWG-6 V2.0 Expression BeadChip (Illumina) containing more than 45 200 transcripts by the Genomics and Proteomics Core Laboratories, University of Pittsburgh. Each sample was analysed using two separate arrays, and values for replicate arrays were averaged. For a comparison of FSTL1 KO MSC versus WT control, a threshold (cut-off point) >2 was selected.
Cell stimulation For the signalling study, MSCs were incubated with 5 ng/mL TGFβ for 1 h, followed by protein extraction for western blot analysis. In some experiments, the KO cells were incubated in the presence of FSTL1 (1 or 5 μg/mL) overnight before TGFβ stimulation.
Western blotting MSCs were lysed with Cell Lysis Buffer (Sigma) supplemented with proteinase inhibitor cocktail (Sigma) and phenylmethanesulfonyl fluoride following the manufacturer’s protocol. The amount of protein was measured by BCA assay (Sigma). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblotting were performed as described.11 Phospho-p38 MAPK, p38 MAPK, phospho-Smad3, Smad3, phospho-Akt, Akt and actin were detected with rabbit polyclonal antibodies (Cell Signalling). Blots were developed using horseradish peroxidaseconjugated secondary antibodies (Thermo) with a chemiluminescent substrate (Thermo). Membranes were scanned by the LAS-4000 imaging system (Fuji Film) and images were analysed by Multi Gauge software.
Statistical analysis The parametric Student’s t test was used to assess the significance of differences between groups. Data are presented as mean±SEM unless otherwise specified. p
ARD Online First, published on March 21, 2014 as 10.1136/annrheumdis-2013-204822 Basic and translational research
EXTENDED REPORT
Follistatin-like protein 1 regulates chondrocyte proliferation and chondrogenic differentiation of mesenchymal stem cells Yury Chaly,1 Harry C Blair,2,3 Sonja M Smith,1 Daniel S Bushnell,4 Anthony D Marinov,4 Brian T Campfield,4 Raphael Hirsch1 Handling editor Tore K Kvien ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ annrheumdis-2013-204822). 1
Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA 2 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA 3 VA Medical Center, Pittsburgh, Pennsylvania, USA 4 Department of Pediatrics, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA Correspondence to Dr Yury Chaly, Department of Pediatrics, University of Iowa Carver College of Medicine, 2191 ML, 500 Newton Rd, Iowa City, IA 52242, USA; [email protected] Received 1 November 2013 Revised 14 February 2014 Accepted 1 March 2014
ABSTRACT Objectives Chondrocytes, the only cells in the articular cartilage, play a pivotal role in osteoarthritis (OA) because they are responsible for maintenance of the extracellular matrix (ECM). Follistatin-like protein 1 (FSTL1) is a secreted protein found in mesenchymal stem cells (MSCs) and cartilage but whose function is unclear. FSTL1 has been shown to modify cell growth and survival. In this work, we sought to determine whether FSTL1 could regulate chondrogenesis and chondrogenic differentiation of MSCs. Methods To study the role of FSTL1 in chondrogenesis, we used FSTL1 knockout (KO) mice generated in our laboratory. Proliferative capacity of MSCs, obtained from skulls of E18.5 embryos, was analysed by flow cytometry. Chondrogenic differentiation of MSCs was carried out in a pellet culture system. Gene expression differences were assessed by microarray analysis and real-time PCR. Phosphorylation of Smad3, p38 MAPK and Akt was analysed by western blotting. Results The homozygous FSTL1 KO embryos showed extensive skeletal defects and decreased cellularity in the vertebral cartilage. Cell proliferation of FSTL1-deficient MSCs was reduced. Gene expression analysis in FSTL1 KO MSCs revealed dysregulation of multiple genes important for chondrogenesis. Production of ECM proteoglycans and collagen II expression were decreased in FSTL1-deficient MSCs differentiated into chondrocytes. Transforming growth factor β signalling in FSTL1 KO cells was significantly suppressed. Conclusions FSTL1 is a potent regulator of chondrocyte proliferation, differentiation and expression of ECM molecules. Our findings may lead to the development of novel strategies for cartilage repair and provide new disease-modifying treatments for OA.
INTRODUCTION
To cite: Chaly Y, Blair HC, Smith SM, et al. Ann Rheum Dis Published Online First: [please include Day Month Year] doi:10.1136/ annrheumdis-2013-204822
Osteoarthritis (OA) is the most common degenerative joint disease. It is characterised by loss of articular cartilage, changes in the subchondral bone, and osteophytosis. The exact cause of the disease remains unknown, and there is no effective therapy for OA, except joint replacement surgery. Typically, OA begins with aging-related disruption of the articular cartilage surface, although specific risk factors, such as joint trauma or metabolic disorders increase the risk of developing OA. Chondrocytes, the only cells in the articular cartilage, play a pivotal role in OA because they are responsible for maintenance of the extracellular matrix (ECM), the primary
target of osteoarthritic cartilage degradation. OA cartilage cells display dysregulation of anabolic and catabolic processes as well as profound reduction in cell number due to a decrease in cell proliferation and an increase in apoptotic cell death. Since articular cartilage has little or no regenerative capacity, the use of mesenchymal stem cells (MSCs) has become an attractive approach for cartilage repair.1 MSCs are capable of proliferating and differentiating into chondrocytes in culture; however, the process of in vitro chondrogenesis is not completely understood. For the future success of cartilage-regenerative medicine, it is critical to elucidate the regulatory mechanisms underlying chondrogenesis. A number of factors, such as insulin-like growth factor (IGF), transforming growth factor β (TGFβ), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), the sex determining region Y-type high-mobility group box (SOX) family of transcription factors, and other cell differentiationrelated signals, including Indian Hedgehog (IHH) and Wnt proteins have been implicated in the process of chondrocyte proliferation and differentiation.2–5 Proper cartilage development is linked to a delicate balance in the activity of the abovementioned factors. Their activities are controlled at multiple levels, both extracellularly and intracellularly. Despite the progress made in recent years in understanding signalling mechanisms, the role of different factors involved in the fine regulation of chondrogenic pathways remains to be elucidated. This study examined the contribution to articular cartilage homoeostasis of follistatin-like protein 1 (FSTL1), an extracellular protein whose functional role in physiological and pathological processes is still unclear. FSTL1 was cloned as a TGFβ-inducible protein from a mouse osteoblastic cell line.6 Our previous studies showed that FSTL1 is an important mediator in the pathogenesis of rheumatoid arthritis and other systemic autoimmune diseases.7–9 We also found that FSTL1 enhances the ability of T cells and monocytes/macrophages to respond to inflammatory signals.10–12 But, unlike most proinflammatory proteins, haematopoietic cells do not produce FSTL1.7 Rather, it is expressed in cells of the mesenchymal lineage, endothelial cells and neurones.13–16 It is noteworthy that FSTL1 is strongly expressed in the mesenchyme of multiple organs during embryogenesis.17–20 Loss-of-function studies in mice demonstrated that homozygous FSTL1 knockout
Chaly Y, etArticle al. Ann Rheum Dis 2014;0:1–7. doi:10.1136/annrheumdis-2013-204822 1 Copyright author (or their employer) 2014. Produced by BMJ Publishing Group Ltd (& EULAR) under licence.
Downloaded from http://ard.bmj.com/ on November 18, 2014 - Published by group.bmj.com
Basic and translational research (KO) animals die at birth due to abnormalities of the respiratory system, particularly, tracheomalacia.17 18 FSTL1 KO mice also display extensive skeletal defects, further supporting the role of FSTL1 in chondrogenesis.17 18 FSTL1 has been found to affect cell growth and survival. For example, FSTL1 inhibits apoptosis in cardiomyocytes and endothelial cells and promotes endothelial cell function and blood vessel growth.14 16 Recent studies17 18 21 also showed that FSTL1 may interact with key mediators of MSC chondrogenesis, proteins of the TGFβ superfamily or their receptors, and modulate their activities. Thus, we hypothesise that FSTL1 may play an important role in maintaining cartilage homeostasis by enhancing cell proliferation, survival and anabolic activity in articular chondrocytes. In the present work, we sought to determine whether FSTL1 could regulate chondrogenesis as well as to identify the molecular mechanisms for FSTL1-mediated regulation of differentiation of MSCs into chondrocytes.
METHODS Generation of FSTL1-deficient mice All animal experiments were approved by the University of Iowa Animal Care and Use Committee. The targeting vector and the FSTL1WT/flox mice were generated by Ozgene (Australia) (see online supplementary methods for details of the breeding strategies).
Skeletal preparations and histological examination Alcian blue/alizarin red staining of cartilage and bone in E18.5 embryos was performed following the standard protocol. For histological examination of lumbar vertebrae, FSTL1 KO and wild-type (WT) embryos at day E18.5 were fixed in 5% formaldehyde and then transferred to 70% ethanol the next day and subjected to paraffin embedding, sectioning and H&E staining using standard protocols.
Mouse MSC cultures and chondrogenic differentiation of MSCs MSCs were isolated from skulls of E18.5 embryos by collagenase digestion and differentiated into chondrocytes in a pellet culture system (see online supplementary methods for details).
Quantitative reverse transcriptase (RT)-PCR Total cDNA was produced from MSCs or chondrocyte pellets using the RNeasy Mini Kit (Qiagen) and the SuperScript II Reverse Transcriptase Kit (Invitrogen). PCR was performed in a LightCycler (ABI7000) using the Brilliant SYBR Green QPCR Master Mix (Agilent) as described.11 The copy number (number of transcripts) of amplified product was calculated from a standard curve obtained by plotting known input concentrations of plasmid DNA (see online supplementary table S1 for primer sequences).
was used as the starting material for further FSTL1 purification (see online supplementary methods).
RNA processing, microarray and data analysis Total RNA was extracted from MSCs isolated from WT and KO embryos using the RNeasy kit (Qiagen). Microarray analysis was performed with the MouseWG-6 V2.0 Expression BeadChip (Illumina) containing more than 45 200 transcripts by the Genomics and Proteomics Core Laboratories, University of Pittsburgh. Each sample was analysed using two separate arrays, and values for replicate arrays were averaged. For a comparison of FSTL1 KO MSC versus WT control, a threshold (cut-off point) >2 was selected.
Cell stimulation For the signalling study, MSCs were incubated with 5 ng/mL TGFβ for 1 h, followed by protein extraction for western blot analysis. In some experiments, the KO cells were incubated in the presence of FSTL1 (1 or 5 μg/mL) overnight before TGFβ stimulation.
Western blotting MSCs were lysed with Cell Lysis Buffer (Sigma) supplemented with proteinase inhibitor cocktail (Sigma) and phenylmethanesulfonyl fluoride following the manufacturer’s protocol. The amount of protein was measured by BCA assay (Sigma). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblotting were performed as described.11 Phospho-p38 MAPK, p38 MAPK, phospho-Smad3, Smad3, phospho-Akt, Akt and actin were detected with rabbit polyclonal antibodies (Cell Signalling). Blots were developed using horseradish peroxidaseconjugated secondary antibodies (Thermo) with a chemiluminescent substrate (Thermo). Membranes were scanned by the LAS-4000 imaging system (Fuji Film) and images were analysed by Multi Gauge software.
Statistical analysis The parametric Student’s t test was used to assess the significance of differences between groups. Data are presented as mean±SEM unless otherwise specified. p
