Regulation of chondrocyte gene expression by osteogenic protein-1
© Chubinskaya et al.; licensee BioMed Central Ltd 2011
Received: 5 January 2011
Accepted: 29 March 2011
Published: 29 March 2011
The objective of this study was to investigate which genes are regulated by osteogenic protein-1 (OP-1) in human articular chondrocytes using Affimetrix gene array, in order to understand the role of OP-1 in cartilage homeostasis.
Chondrocytes enzymatically isolated from 12 normal ankle cartilage samples were cultured in high-density monolayers and either transfected with OP-1 antisense oligonucleotide in the presence of lipofectin or treated with recombinant OP-1 (100 ng/ml) for 48 hours followed by RNA isolation. Gene expression profiles were analyzed by HG-U133A gene chips from Affimetrix. A cut-off was chosen at 1.5-fold difference from controls. Selected gene array results were verified by real-time PCR and by in vitro measures of proteoglycan synthesis and signal transduction.
OP-1 controls cartilage homeostasis on multiple levels including regulation of genes responsible for chondrocyte cytoskeleton (cyclin D, Talin1, and Cyclin M1), matrix production, and other anabolic pathways (transforming growth factor-beta (TGF-β)/ bone morphogenetic protein (BMP), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), genes responsible for bone formation, and so on) as well as regulation of cytokines, neuromediators, and various catabolic pathways responsible for matrix degradation and cell death. In many of these cases, OP-1 modulated the expression of not only the ligands, but also their receptors, mediators of downstream signaling, kinases responsible for an activation of the pathways, binding proteins responsible for the inhibition of the pathways, and transcription factors that induce transcriptional responses.
Gene array data strongly suggest a critical role of OP-1 in human cartilage homeostasis. OP-1 regulates numerous metabolic pathways that are not only limited to its well-documented anabolic function, but also to its anti-catabolic activity. An understanding of OP-1 function in cartilage will provide strong justification for the application of OP-1 protein as a therapeutic treatment for cartilage regeneration and repair.
Cartilage degeneration is one of the features of osteoarthritis (OA). In order to identify cellular mechanisms that drive OA progression, it is necessary to understand the interplay between anabolic and catabolic processes responsible for cartilage homeostasis under physiological and pathophysiological states. Osteogenic protein-1 (OP-1) or bone morphogenetic protein-7 (BMP-7) is one of the most potent growth factors for cartilage maintenance and repair identified thus far [1, 2]. A large number of in vivo and in vitro studies have shown a high synthetic potency of human recombinant OP-1 (rhOP-1; ). In earlier work, we found that the inhibition of OP-1 gene expression by antisense oligonucleotides (ODNs) caused a significant decrease in aggrecan expression, aggrecan core protein synthesis, and proteoglycan (PG) synthesis, which resulted in the depletion of PGs from the cartilage matrix . These findings suggest that OP-1 plays a key role in maintenance of cartilage integrity and homeostasis, but further work is needed to understand the mechanisms by which OP-1 acts at the molecular level.
In the current study, we used the Affymetrix GeneChip technology to monitor OP-1 regulation of 22,000 genes from the human genome with specific emphasis on genes that are relevant to adult articular cartilage. Those included matrix proteins, anabolic and catabolic gene products, as well as their intracellular regulators and receptors. Recently, applying the same methodology differential gene expression pattern in normal and OA cartilage tissue was identified . These analyses revealed numerous interesting gene expression profiles, but per se did not allow elucidating cellular reaction patterns in response to defined extracellular stimuli. The goal of the current project was to evaluate the role OP-1 plays in regulating human articular cartilage homeostasis by using a gene array approach under conditions where endogenous OP-1 gene expression was inhibited by antisense ODNs (; OP-1AS) or OP-1 signaling was activated and/or enhanced by rhOP-1. Key microarray findings were verified by real-time PCR and additional in vitro experiments of matrix synthesis and signal transduction. We found that OP-1/BMP-7 controls numerous metabolic pathways that are not limited to its direct anabolic or anti-catabolic function, but also related to cell growth, cell proliferation, differentiation, survival, apoptosis, and death.
Materials and methods
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), gentamicin, Ham's F-12, lipofectin, Opti-MEM, penicillin/streptomycin/fungizone (PSF), 1X Platinum Quantitative PCR SuperMix-UDG and SuperScript III reverse transcriptase with oligo (dT)12-18 were purchased from Invitrogen (Carlsbad, CA, USA). Phosphorothioate ODN was custom synthesized by Oligos Etc. (Wilsonville, OR, USA). RNeasy mini kit, QIA shredder, RNase-free DNase kit and QuantiTect Primer Assay were purchased from Qiagen (Valencia, CA, USA). Real time polymerase chain reaction (PCR) primers were custom synthesized by Integrated DNA Technologies (IDT), Coralville, IA, USA. 10,000 X SYBR Green 1 was purchased from Cambrex, Rockland, ME, USA. Recombinant human rhOP-1 was kindly provided by Stryker Biotech (Hopkinton, MA, USA).
Isolation and culture of chondrocytes
Full-thickness articular cartilage from the talus of the talocrural joint (ankle) from 12 human organ donors (age 55 to 70 years old, Collins grade 0 to 1 ) and from the femur of the tibiofemoral joint (knee) from two human organ donors (age 67 and 73 years old, Collins grade 2) was obtained from the Gift of Hope Organ and Tissue Donor Network (Elmhurst, IL, USA) with Institutional Review Board approval and appropriate consent within 24 hours of the donor's death. Knee cartilage was utilized for verification of the ankle cartilage results using real-time PCR. Chondrocytes were isolated by sequential digestion with pronase (2 mg/ml) for 60 minutes and collagenase P (0.25 mg/ml) overnight . Chondrocytes were plated in high density monolayer culture (4 × 106 cells/well in a six-well plate) and cultured for 24 hours in 50% DMEM/50% Ham's F-12 supplemented with 10% FBS, 1% PSF, and gentamicin (50 μg/ml) for attachment prior to treatment with either antisense (OP-1 AS) or recombinant OP-1 (rhOP-1). Both treatments were administered for 48 hours in the absence of serum.
Antisense ODNs were designed to be complementary to sequences in the 5'- and 3'-untranslated regions of the human OP-1 messenger RNA (mRNA) sequence (XM_030621, National Center for Biotechnology Information (NCBI)) as described . All verification experiments with appropriate negative controls (sense and scrambled probes) were performed in a previous study . For this study, the following antisense ODN was used: 5'-GGC-GAA-CGA-AAA-GGC-GAG-TGA-3' (position 237-257).
Chondrocyte cultures were divided into three experimental groups and treated for 48 hours as follows: 1) transfected with OP-1 AS in the presence of 10 μg/ml lipofectin ; 2) treated with 100 ng/ml of rhOP-1; and 3) culture control (no treatment, no serum).
Total cellular RNA was isolated using the RNeasy Mini Kit, following lysis of the cells with a Qia shredder  and included an on-column DNase digestion, according to the manufacturer's instructions (Qiagen). All samples were stored at -80°C until analyzed.
Microarray and pathway analysis
Gene expression profiles were analyzed by HG-U133A gene chips from Affimetrix (accession number: E-MTAB-571). At least 10 μg of RNA/per experimental group was required for analysis. Therefore, the RNA was pooled from donors in order to have sufficient RNA and to reduce donor-to-donor variations. Cells from all 12 donors were treated with each experimental condition. The microarray data collection was in compliance with the Minimum Information About Microarray Experiments standard . The quality of the RNA was checked by the Agilent Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA), and the quality of the hybridization image was checked by the affyPLM model . To deal with the technical variation, each gene was measured by 11 different probes on the Affymetrix U133A microarray. A statistical model at the probe-level was used to identify the differentially expressed genes. To estimate the variance more efficiently with a small sample size, we utilized an empirical Bayesian correction of the linear model . Statistical significance was considered with a P-value of P < 0.001 and fold change larger than 1.5-fold between the treatment group and corresponding control. All the data analysis was conducted using the Bioconductor/R package . To interpret the biological significance of differentially expressed genes, a gene ontology analysis was conducted using DAVID/EASE .
Pathway analysis and classification by gene ontology
Regulated genes (R > 1.5-fold, P < 0.001) were used as input for both analyses. The ingenuity pathway analysis system  was used to project genes onto known biological pathways (canonical pathways). The system determines a significance value for each pathway based on an F-statistics that the input-genes occur randomly within this pathway. Grouping of genes was done by computing over-representation of regulated genes in gene ontology (GO) classes . Statistical analysis consisted of 1) analysis of differentially expressed genes under a single experimental condition in comparison to the corresponding control (up- or down-regulated in the presence of OP-1 antisense or rhOP-1); 2) analysis of differentially expressed genes when comparison is made between two treatments (OP-1 antisense and rhOP-1); and 3) gene ontology, when changes were analyzed within a family of genes according to their function (comparison was made between single treatment and control or between both treatments). Selected gene array results were verified experimentally in vitro or by real-time PCR.
Validation experiments -quantitative real time PCR
Sequence of primers for quantitative real time PCR
Annealing temp and time
Qiagen QuantiTect Primer Assay
62°C, 40 sec
62°C, 40 sec
Qiagen QuantiTect Primer Assay
60°C, 40 sec
62°C, 40 sec
64°C, 40 sec
64°C, 40 sec
58°C, 30 sec
64°C, 30 sec
Statistical analysis for real-time
PCR Data are expressed as mean +/- standard deviation. Statistical significance was assessed by the Student t-test and P-values < 0.05 were considered significant.
Microarray analysis: overview of data
GeneChip (HG-U133A) expression data from un-stimulated, rhOP-1 and OP-1AS treated chondrocytes maintained in high-density monolayer culture were generated. For the analysis of the expression data we used a three step analytical strategy: (I) processing of raw intensity values and normalization of profiles, (II) examination of expression levels of gene categories that are relevant to articular cartilage, and (III) comparison of gene expression changes between the two treatments - OP-1AS to knockdown endogenous OP-1 expression vs. addition of exogenous rhOP-1.
Analysis of catabolic genes: cytokines and their regulators
Changes in chemokines, cytokines, and their receptors
rhOP-1 vs Cntr
OP-1AS vs Cntr
TNF-α induced protein 6
TNF-α induced protein-3
IL-1R accessory protein-like 1
Changes in the mediators of IL-6 signaling pathway
rhOP-1 vs Cntr
OP-1AS vs Cntr
Genes from IL-6 signaling pathway
IL-6 signal transducer (oncostatin M receptor)
Protein inhibitor of activated STAT3
MAP 3 kinase 7
In analyzing the relationship between treatments to modulate OP-1 and the expression of genes in the IL-6 signaling pathway, we found that OP-1 not only regulates expression of the IL-6 family of cytokines but also controls expression of their receptors and downstream intracellular mediators including signal transducers and activators of transcription (STATs), mitogen activated protein (MAP) kinases, and transcription factors. This suggests OP-1 inhibits IL-6 signaling at multiple levels (Table 3). Among other genes that either regulate cytokine activity or mediate their signaling, the most affected by OP-1 were the receptors for IL-1β and tumor necrosis factor alpha (TNF-α) (see Table 2) as well as TNF-α inducible protein. Although under the experimental conditions expression of TNF-α and IL-1β genes was not influenced by OP-1, previous studies showed that injection of OP-1 into nucleus pulposus inhibited production of autocrine TNF-α and IL-1β elevated in response to injurious compression of the intervertebral discs  proving an association between OP-1 and signaling pathways of the above mentioned cytokines. In addition, several other studies have provided evidence of an ability of OP-1 to regulate either IL-1β induced responses or IL-1β downstream signaling [16–18, 22, 23].
Analysis of catabolic genes. Neuromediators
Changes in neuromediators and their receptors
rhOP-1 vs Cntr
OP-1AS vs Cntr
Nerve growth factor-β
(Substance K, Substance P)
Analysis of catabolic genes: Transcription factors
Changes in transcription factors
rhOP-1 vs Cntr
rhOP-1AS vs Cntr
Transcription factor 8 (represses IL-2 expression)
Transcription factor AP-2α
Suppression of tumorigenicity
Activating transcription factor 7
MADS box transcription enhancer factor 2-d
Upstream transcription factor 2, c-fos interacting
Transcription factor (p38 interacting protein)
MADS box transcription enhancer factor 2-C
Protein inhibitor of activated STAT3
Ubiquitin-like 1 (sentrin)
Analysis of catabolic genes: Matrix degrading proteases, cathepsins, and apoptosis-related genes
Changes in proteases and their inhibitors
rhOP-1 vs Cntr
rhOP-1AS vs Cntr
Caspase 4, apoptosis-related cysteine protease
Programmed cell death 8 (apoptosis-inducing factor)
MMPs and inhibitors
ADAM and ADAMTS
Tissue Plasminogen Activator
Among the proteases that were also regulated by OP-1 were cathepsins B, C, and S. So far, these lysosomal cysteine proteases have been less studied in cartilage, though cathepsin C appears to be a central coordinator for activation of many serine proteases in immune/inflammatory cells , while cathepsin B was thought to play an important role in the development of osteoarthritis . Expression of all three cathepsin genes was down-regulated under OP-1AS.
A previous study on acute impact injury in vivo  strongly suggested an anti-apoptotic effect of OP-1 in post-traumatic OA. Therefore, we expected that OP-1 may control genes involved in apoptosis-related processes. We found that rhOP-1 inhibited program cell death 8 gene (apoptosis-induced factor), Bcl-2 gene and the calpain-9 gene (Table 6). However, the key caspases that trigger and promote cell death by apoptosis were not affected. During the absence of OP-1 (antisense treatment), expression of caspases 8, 9, and 6 were inhibited and only caspase 2 was elevated (Table 6). The reason for a down-regulation of the apoptosis-related genes under conditions where OP-1 is lacking is not clear, but may be a response to help avoid cell death.
Analysis of anabolic genes: transforming growth factor-beta (TGF-β)/BMP family, their receptors and regulators of signaling
Changes in the expression of TGF-β/BMP family related genes, their receptors, and signaling regulators
rhOP-1 vs Cntr
rhOP-1AS vs Cntr
Inhibin-βa (activin A)
BMP-2 inducible kinase
Parathyroid hormone-like hormone
MAD interacting protein
Frizzled homolog 10 (Drosophila)
In addition, OP-1 modulated expression of the TGF-β/BMP receptors. With the exception of Activin-α RIB, which was inhibited by rhOP-1 and elevated under the lack of OP-1, expression of other receptors, Activin-α RIIB, BMPR1A, TGF-β RI, II, and III correlated positively with OP-1 expression (Table 7).
Analysis of anabolic genes: other growth factors
Association between OP-1 and other growth factors including igf-1, insulin, and tyrosine-kinase signaling
rhOP-1 vs Cntr
OP-1AS vs Cntr
Nerve growth factor-β
Endothelial cell growth factor 1 (platelet-derived)
Capillary morphogenesis protein 1
IRS2 (insulin receptor substrate 2)
DPYSL2 (dihydropyrimidinase-like 2)
MET (hepatocyte growth factor receptor)
SPRY2: sprouty homolog 2 (Drosophila)
SORBS1: sorbin and SH3 domain containing 1
PIK3R1 (Phosphoinositide-3-kinase, regulatory subunit 1)
MAP2K2 (mitogen-activated protein kinase kinase 2)
PDE3B (phosphodiesterase 3B, cGMP-inhibited)
SOCS3 (suppressor of cytokine signaling 3)
Modulation of OP-1 levels affected mRNA expression of growth factors and some of their receptors that belong to various families, such as Nerve Growth Factor-β, Vascular Endothelial Growth Factor, Endothelial Cell Growth Factor 1 (platelet-derived), Capillary Morphogenesis Protein-1, and Fibroblast Growth Factor (FGF)-7. Their expression was inhibited by rhOP-1 from 1.93- to 1.5-fold. Contrary, the expression of the FGF-R2 and 3 receptors, and α and β receptors of Platelet-Derived Growth Factor was stimulated by rhOP-1 Table 8).
Matrix proteins and their receptors
Changes in the expression of matrix proteins, their receptors, and integrins
rhOP-1 vs Cntr
OP-1AS vs Cntr
Chondroitin sulfate PG6 (bamacan)
Versican (chondroitin sulfate PG2)
Cartilage associated protein
Cadherin 11 (OB-cadherin (osteoblast))
Dermatan Sulfate PG3
Chondroitin sulfate PG4
Matrix protein receptors
To the best of our knowledge, this is the first report that uses microarray analysis to provide a comprehensive understanding of the role OP-1 plays in overall cartilage homeostasis. We found that OP-1 controls cartilage homeostasis on multiple levels including regulation of genes responsible for chondrocyte cytoskeleton (cyclin D, Talin1, and Cyclin M1, for example, and confirmed in ), matrix production and other anabolic pathways, as well as regulation of cytokines and various catabolic pathways responsible for matrix degradation and cell death. Importantly, in many of these cases, OP-1 modulated the expression of not only the ligands, but also their receptors, mediators of downstream signaling, kinases responsible for an activation of the pathways and transcription factors that induce transcriptional responses.
Due to high variability among human samples, only a few studies have utilized microarray analysis to test the entire human genome in primary adult articular chondrocytes [4, 37–39], and only one of Saas et al.  addressed in part the effect of BMP-7/OP-1. These analyses used the tissue from one or a maximum of two donor cartilage samples. In the present study, normal (grade 0) articular cartilage was collected from 12 donors within a similar age range. One of the limitations of the study is that we examined gene expression profiles only at one time point, after 48-hours of culture. Therefore, changes in early-response genes and late-response genes might have been missed. This could explain some results, as for example, the lack of changes in the expression of major cartilage matrix proteins. However, such an approach gave us a breath of the overall effects of OP-1 on cartilage homeostasis.
Due to the abundance of the results, we will discuss only the most relevant and those that could be explained by the current knowledge of the field. Perhaps most important was the finding that OP-1 is a unique growth factor in its capacity to display simultaneously pro-anabolic and anti-catabolic activities. It was previously shown that OP-1 stimulated expression and synthesis of collagen type II, aggrecan, hyaluronan, and CD44 [1, 2, 20, 40] as well as IGF-1, IGF-1 receptor, and responses to IGF-1 . In the current studies, we used high-density monolayers while in previous work explants or alginate beads were used with different media conditions (no serum vs serum or ITS-media). The finding that the microarray results shown here were comparable to the previous results suggest that the pro-anabolic effects of OP-1 in human articular chondrocytes are persistent. With regard to the anti-catabolic activity, the ability of OP-1 to counteract various pro-inflammatory/catabolic responses or directly inhibit expression of catabolic mediators was previously shown in primary chondrocyte cultures or in animal models of post-traumatic osteoarthritis or disc degeneration [17–19, 24, 31]. In this study, we found that OP-1 inhibits expression of IL-6 and members of the IL-6 family of chemokines as well as their receptors and signaling mediators. Furthermore, the tight association between these two classes of mediators (OP-1 and IL-6) was documented under both experimental conditions (plus or minus OP-1). Based on our new data on the role of IL-6 in acute post-traumatic responses , it is possible that OP-1 was able to protect cartilage from degenerative changes caused by acute trauma  not only due to its direct effect on matrix synthesis, but also because of its ability to inhibit IL-6, TNF-α, and the catabolic pathways induced by the fragments of the extracellular matrix: fibronectin , hyaluronan , and collagen telopeptides .
Another important effect of OP-1 may be an ability to inhibit expression of neuromediators and their receptors. Previously, an anti-pain effect of OP-1 was documented in the rat models of herniated disc or disc degeneration induced by injurious compression. In these studies, OP-1 injections reduced hyperalgesia and inhibited elevation of IL-1, TNF-α, substance P, bradykinin and their receptors in various disc tissues including spinal cord and dorsal ganglion [21, 24, 26]. In chondrocytes, it is the first report that indicates a connection between OP-1 and various neuromediators, though substance P and its NK-1 receptor were already identified in cartilage in the model of mechanical stress . Very recently, our findings received another proof in phase I placebo-controlled clinical studies on OP-1 treatment for osteoarthritis patients , in which a single injection of OP-1 reduced pain even after six months of treatment.
Interesting results were found with regard to the ability of OP-1 to regulate the TGF-β/BMP signaling pathway. The levels of OP-1 expression positively correlated with the expression of activin-like kinase (ALK)-3 or BMP-RIA, transcription factors Id proteins 2 to 4, and a binding protein Gremlin, indicating that this could be a primary route for OP-1 signaling. We also found that another binding protein, Follistatin, exhibited a negative correlation with the levels of OP-1. Thus, our results suggest a differential regulation of these two binding proteins by OP-1, which could mean that Gremlin and Follistatin perform distinct functions in the mediating BMP responses or they are involved in different stages of signaling. This point of view is supported by studies of Tardif et al.  who reported their differential expression and spatial distribution in the dog OA model. One of the most surprising results was the finding that OP-1 inhibits expression of another member of the BMP family, BMP-2, which shares the same signaling machinery and in many cases exhibits similar anabolic activities [23, 46]. This result was confirmed by real-time PCR and definitely warrants further studies in understanding the responses induced by homologous, yet very different members of the same family .
Finally, another unexpected result was the inhibition of TIMP-3 expression by rhOP-1; while previously, TGF-β has been shown to induce this inhibitor . The differences in the results could be attributed to distinct culture conditions (primary chondrocytes in our case and passaged chondrocytes as in Qureshi et al. , assessment at various time points, or distinct signaling mechanisms between TGF-β and OP-1 that are responsible for induction of TIMP-3 expression. However, on the protein level it has already been reported that OP-1 inhibits TIMP-3 protein . Furthermore, changes in TIMP-3 were parallel to changes in certain genes responsible for apoptosis, which supports the notion that in cancer cells TIMP-3 may promote cell death by apoptosis . On the other hand, TIMP-3 has been shown to inhibit aggrecanase-mediated release of glycosaminoglycans in bovine nasal cartilage . At this point, the role of TIMP-3 in human chondrocytes and its regulation by various mediators remains to be investigated.
This analysis of gene array data strongly suggests a critical role of OP-1 in human cartilage homeostasis. OP-1 regulates numerous metabolic pathways that are not only limited to its anabolic function, but also to its anti-catabolic activity. Understanding of OP-1 function in cartilage will provide strong justification for the application of OP-1 protein as therapeutic treatment for cartilage regeneration and repair.
a disintegrin and metalloproteinase
B-cell lymphoma 2
bone morphogenetic proteins
Dulbecco's modified Eagle's medium
fetal bovine serum
fibroblast growth factor
growth differentiation factor
insulin-like growth factor
leukemia inhibitory factor
- MAP kinase:
mitogen activated protein kinase
National Center for Biotechnology Information
nuclear factor kappa-light-chain-enhancer of activated B cells
polymerase chain reaction
runt-related transcription factor 1
signal transducers and activators of transcription
transforming growth factor-beta
tissue inhibitor of metalloproteinases
tumor necrosis factor-alpha
vascular endothelial growth factor.
The authors would like to acknowledge the Gift of Hope Organ & Tissue Donor Network and donors' families as well as Dr. Margulis for tissue procurement. The work was supported by the NIH grant AR 47654, Stryker Biotech grant SC-001, and Ciba-Geigy Endowed Chair (SC). RFL was supported by NIH grant AG016697.
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