RORβ modulates a gene program that is protective against articular cartilage damage.

Osteoarthritis (OA) is the most prevalent chronic joint disease which increases in frequency with age eventually impacting most people over the age of 65. OA is the leading cause of disability and impaired mobility, yet the pathogenesis of OA remains unclear. Treatments have focused mainly on pain relief and reducing joint swelling. Currently there are no effective treatments to slow the progression of the disease and to prevent irreversible loss of cartilage. Here we demonstrate that stable expression of RORβ in cultured cells results in alteration of a gene program that is supportive of chondrogenesis and is protective against development of OA. Specifically, we determined that RORβ alters the ratio of expression of the FGF receptors FGFR1 (associated with cartilage destruction) and FGFR3 (associated with cartilage protection). Additionally, ERK1/2-MAPK signaling was suppressed and AKT signaling was enhanced. These results suggest a critical role for RORβ in chondrogenesis and suggest that identification of mechanisms that control the expression of RORβ in chondrocytes could lead to the development of disease modifying therapies for the treatment of OA.

Introduction

Osteoarthritis (OA) is the most prevalent chronic degenerative joint disease where the risk of disease increases in with age and obesity [1-3]. OA occurs more commonly later in life, after years of mechanical wear and tear on cartilage, a tissue that lines and cushions joints. Most therapies target pain relief and currently there are no effective treatments to slow the progression of the disease. Disease progression eventually results in irreversible loss of cartilage and when articular cartilage is significantly degraded or completely lost, joint replacement surgery is the only option [4]. Although the correlation between aging and the development of OA is not completely understood, it is becoming clear that age-related changes in the musculoskeletal system, combined with mechanical injury and genetic factors all contribute to the pathogenesis of OA [5, 6]. Thus, uncovering the molecular mechanism of joint degeneration requires analysis of cartilage metabolism, chondrocyte senescence, and inflammation [7]. Such studies should lead to the identification of therapeutic targets for treating and preventing OA. Chondrocytes are critical to maintenance of articular cartilage where loss of chondrocytes leads to cartilage damage and this damage is often irreversible. Chondrocyte progenitor cells would be an ideal experimental model for the proposed studies, but markers for such cells are still unclear, and the expression level of several candidate markers such as Notch1 and SOX9 are not consistent on superficial zone (SZ), middle zone (MZ), and deep zone (DZ) of normal tissue and OA tissue [8]. Therefore, in the studies presented here we utilized MG63 cells to overcome these issues. MG63 cells are derived from an established sarcoma cell line and is an osteoblastic model to study bone cell viability, adhesion, and proliferation. Importantly, these cells express the mesenchymal stem cell markers Notch1 and SOX9 and are a well characterized osteoblast-like cell line which can drive development of OA.

It has been shown that controlled fibroblast growth factor (FGF) signaling is essential for the balance of articular cartilage metabolism, as evidenced by the fact that aberrant FGF signaling contributes to progression of OA [9]. FGFR1 signaling triggers upregulation of pro-inflammatory mediators, matrix metalloproteinases (MMPs), and alters anabolic activities of articular cartilage by inhibition of extracellular matrix (ECM) production and autophagy. Whereas FGFR3 signaling is cartilage protective mainly through the inhibition of pro-inflammatory mediators and hypertrophic differentiation, as well as reduced MMP expression. For this reason, FGFR1 antagonists and FGFR3 agonists are being pursued as potential therapeutic strategies for OA [10-12].

Nuclear receptors (NRs) represent a druggable superfamily of ligand-dependent transcription factors. The NR1F subfamily of NRs contains the retinoic acid receptor-related orphan receptors (RORs), which have homology to both the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) [13]. The ROR subfamily includes three major isoforms, RORα, RORβ, and RORγ. The physiological functions of RORα and RORγ have been well characterized and they have been shown to play roles in regulation of metabolism and inflammation while RORβ (NR1F2) has been significantly less well studied. RORβ is expressed in regions of the CNS that are involved in processing of sensory information and components of the mammalian clock, the suprachiasmatic nuclei, the retina, and the pineal gland. RORβ has been shown to play a critical role for the proliferation and differentiation of retinal cells in addition to the maintenance of circadian rhythms [14-16]. The clock gene BMAL1 (brain and muscle Arnt-like protein 1), a target gene of the RORs, was reported that contributed to the maintenance of cartilage homeostasis [17, 18].

Recently, it was shown that RORβ plays a role in osteogenesis by impacting Runx2 target gene expression. Levels of RORβ inversely correlated with osteogenic potential suggesting that suppression of RORβ may drive osteoblastic mineralization. It was shown that RORβ and a subset of RORβ-regulated genes were increased in bone biopsies from post-menopausal women compared to pre-menopausal women suggesting a role for RORβ in human age-related bone loss [19]. Additionally, it was shown that the miR-219a-5p regulates RORβ during osteoblast differentiation and in age-related bone loss [20]. While RORβ-/- mice display bone abnormalities, it was shown that deletion of RORβ was osteoprotective [21].

The main goal of this study was to determine if overexpressing RORβ in an osteoblast-like cell would inhibit the osteoblast phenotype while inducing a chondrogenic phenotype. Here we demonstrate that stable expression of the nuclear receptor RORβ in cultured cells results in alteration of a gene program that is supportive of chondrogenesis and protective against development of OA. Specifically, RORβ balances the expression of genes implicated in cartilage homeostasis such as altering FGFR signaling towards that favorable for cartilage stability. While it remains unclear how RORβ regulates the balance of FGFR1/3 expression and downstream signaling, the results presented here suggest RORβ is an important transcription factor involved in the control of a gene program that could prevent articular cartilage damage. Understanding the mechanism of RORβ control of this gene program could lead to the identification of novel therapeutics or drug targets for the prevention and/or treatment of OA.

Materials and methods

hRORβ/pLPCX construction and stable expressing transfection

Full length wild type human RORβ (NM_006914.3) was subcloned into the pLPCX retroviral vector (Clontech) using XhoI and NotI restriction enzymes (NEB) with forward primer 5’- CGCGCTCGAGATGCGAGCACAAATTGAAGTGATAC-‘3 and reverse primer 5’-GCGGCGGCCGCTCATTTGCAGCCGGTGGCAC-‘3. PCR product was obtained with Maxime PCR Premix (i-pfu) (Intron Biotechnologies). pLPCX vector was digested and then dephosphorylated before ligation using the Rapid DNA Dephos & Ligation Kit (Roche). Ampicillin resistance clones were verified using a 5’ sequencing primer 5’-AGCTGGTTTAGTGAACCGTCAGATC-3’ and 3’ sequencing primer 5’-ACCTACAGGTGGGGTCTTTCATTCCC-3’. Selected clone was linearized with AseI (NEB) restriction digestion and dephosphorylated with Antarctic Phosphatase (NEB) prior usage in transfection.

Human osteoblast-like MG-63 cells (ATCC CRL-1427TM) were maintained in EMEM (Eagle’s Minimum Essential Medium, ATCC) with 10% heat-inactivated fetal bovine serum (Invitrogen). Linearized hRORβ/pLPCX plasmid was transfected using lipid mediated transfection method (MG-63 transfection kit, Altogen Biosystems). 0.5 ug/ml of Puromycin was added into complete media for selection of stable expressing hRORβ/pLPCX in MG-63 cells. A clone expression high levels of RORβ and a mock-vector clone were used for the following experiments.

mRNA sequencing analysis

Total RNA was extracted using a Qiagen Kit-74106. Total RNA was quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and run on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) for quality assessment. RNA samples of good quality with RNA integrity number (RIN) > 8.0 were further processed. A RNase-free working environment was maintained, and RNase-free tips, Eppendorf tubes, and plates were utilized for the subsequent steps. Messenger RNA was selectively isolated from total RNA (300 ng input) using poly-T oligos attached to magnetic beads and converted to sequence-ready libraries using the TruSeq stranded mRNA sample prep protocol (cat. #: RS-122-2101, Illumina, San Diego, CA). The final libraries were validated on the bioanalyzer DNA chips, and qPCR was quantified using primers that recognize the Illumina adaptors. The libraries were then pooled at equimolar ratios, quantified using qPCR, and loaded onto the NextSeq 500 flow cell at 1.8 pM final concentration. They were sequenced using the high-output, paired-end, 75-bp chemistry. Demultiplexed and quality filtered raw reads (fastq) generated from the NextSeq 500 were trimmed (adaptor sequences) using Flexbar 2.4 and aligned to the mouse reference database (mm9) using TopHat version 2.0.9 (Trapnell, et al.). HTseq-count version 0.6.1 was used to generate gene counts and differential gene expression analysis was performed using Deseq2 (Anders and Huber). We then identified genes that were significantly downregulated or upregulated (adjusted P < 0.05) in each comparison. The RNA-seq data is deposited in https://www.ncbi.nlm.nih.gov/geo/ GSE208277.

Protein expression analysis

Protein was purified using RIPA lysis and extraction buffer (ThermoFisher Scientific) from MG-63 clones. HaltTM Protease and phosphatase inhibitor cocktail (100X, ThermoFisher Scientific) were added into lysis buffer. To normalize the amount of cell lysate run on SDS-PAGE, BCA (Thermo Fisher Scientific) assay was used. Anti-ROR beta antibody (rabbit monoclonal EPR15552, abcam), anti-phospho ERK1/2 (D13.14.4E, CST), anti-ERK1/2 (L34F12, CST), anti-phospho AKT (D9E, CST), anti-AKT (40D4, CST), and anti-beta Actin (8H10D10, CST) antibodies were used for primary antibodies. IRDye 680RD and IRDye800CW were used for secondary antibodies. A LI-COR Odyssey system was used for fluorescence imaging. Anti- aggrecan Ab (6-B-4, abcam) was used for primary antibody and FITC-anti mouse Ab was used for secondary antibody. Cell sorting was performed using LSRII (BD Bioscience).

Quantitative RT-PCR

Total RNA was extracted from RORβ OE/MG63 cells using RNeasy Plus Micro Kit (Qiagen), and the RNA was reverse transcribed using the ABI reverse transcription kit (Applied Biosystems/Thermo Fisher Scientific, Waltham MA). Quantitative PCR was performed with a 7900HT Fast Real Time PCR System (Applied Biosystems) using SYBR green (Roche). A list of primers used for these studies is shown in . To measure the effect of IL1β stimulation, 2 nM ILβ (recombinant human IL-1β/IL-1F2, R&D system) was added to WT and RORB OE cells. After 24hr, total RNA was isolated and analyzed by qRT-PCR.

Results and discussion

Stable expression of human RORβ in MG63 cells

To determine if overexpressing RORβ in an osteoblast-like cell line would inhibit the osteoblast phenotype while inducing a chondrogenic phenotype MG63 cells were transfected with a pLPCX vector harboring the puromycin-resistance gene and human RORβ cDNA. The transfected cells were selected with puromycin and the expression of RORβ was analyzed by western blotting using an anti-human RORβ specific rabbit EPR1552 mAb with fluorescence imaging using a LI-COR system (). Anti-beta actin (8H10D10) was used as a protein loading control. Relative mRNA expression of hRORβ was determined for clones that survived selection (). One clone demonstrating high expression of RORβ was selected for all subsequent studies. The expression level of RORβ in this clone was confirmed monthly. A control cell line was generated by transfection of MG63 cells with the pLPCX vector devoid of RORβ and selection with puromycin to generate the wild-type (WT) clone. The WT clone line was used as a control for all studies presented.

Selection of MG63 clone which is consistently over expressing hRORβ in MG63 cells.

hRORβ was subcloned into pLPCX vector which was transfected into MG63 cells and selected by 1ug/mL of puromycin. hRORβ was consistently overexpressing in MG63 that was confirmed by a) protein expression level and b) relative mRNA expression level. WT: mock vector (pLPCX) transfected MG63 cells, OE: Consistently over expressing of hRORβ in MG63 cells.

Differential gene expression (DEG) analysis

To investigate the biological function of RORβ in MG63 cells, RORβ OE-MG63 cells (cells overexpressing RORβ) and mock vector transfected MG63 cells (WT clone) were analyzed by mRNAseq. Three independent biological replicates were processed and analyzed. Principle component analysis results and a heatmap of the sample to sample distance are shown in . To investigate molecular pathways altered by RORβ expression, we analyzed log2 fold change of mRNA sequencing data using QIAGEN ingenuity pathway analysis (IPA) software with a P-value cutoff of 0.05. As shown in , cells overexpressing RORβ showed alteration in genes involved in signaling pathways associated with OA when compared with WT cells. As further detailed below, the expression profiling results suggest that RORβ may play a protective role in chondrocytes to prevent development of OA.

Differential gene expression sequence.

A) principle component analysis (PCA) of wild type (213–1, 213–2, 213–3) and over expressing RORβ clone (213–4, 213–5, 213–6) in NextSeq 500. Each condition is labeled with a separate color allowing for a visual method for identifying sample outliers. RORβOE cells and WT cells are colored in orange and green, respectively. B) Heatmap of the sample to sample distance. The scale is the Euclidean distance between two samples in multi-dimensional space which is also represented in different shades of blue. Each sample is most closely related to itself (with a Euclidian distance of 0) which explains the perfect relatedness (dark blue) along the diagonal. c) Suppression of osteoarthritis signaling pathway. mRNAseq data was analyzed using IPA program. The green color of genes represents significantly repressed fold changing genes on OE compared with WT and the red color of genes represent significantly increased fold changing genes on OE compared with WT in OA pathway signaling.

RORβ expression suppresses osteoarthritis inducing genes

Aging and chronic inflammation are associated with increase in several critical genes associated with the induction of OA. Matrix metalloproteinases (MMPs) including ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS4) and MMP3 and the proinflammatory cytokine IL6 are well characterized pathogenic genes for OA. Interestingly, increased expression of RORβ in MG63 cells suppressed the expression of these genes () and we demonstrate that the cartilage damaging factor MMP3 was also reduced at the protein level (). These results suggest that RORβ may play an inhibitory role to prevent cartilage damage by suppression of chondrocyte degradation by blocking chondrocyte catabolic pathways.

mRNA expression level for cartilage structure genes and cartilage destruction related genes.

a) Relative mRNA expression for ADAMTS4, MMP3, and IL6 which are direct/indirect cartilage destructive genes and b) the protein expression of MMP3 in OE/MG63 clone. c) Relative mRNA expression for aggrecan (ACAN) and COL2A1 which is main structure of cartilage maintain and d) the protein expression of ACAN on OE/MG63 clone. TC28α2 cells were used for positive control and MG63 cells were used for negative control. Graph with gray color is isotype control.

RORβ induces expression of articular cartilage structural genes

Articular cartilage is composed of collagens, proteoglycans, and non-collagenous proteins. Extracellular matrix proteoglycans and collagens play a critical role in the maintenance of articular cartilage structure by regulating chondrocyte proliferation and promoting cartilage repair [22]. Induction of OA is initiated by mechanical forces and inflammatory events that destabilize the normal coupling of synthesis and degradation of extracellular matrix proteins in both articular cartilage and subchondral bone. In the study presented here, we observed that the expression of the core structural genes in articular cartilage, aggrecan (ACAN) and collagen type II alpha (COL2A1), were increased by overexpressing RORβ in MG63 cells (). Additionally, we demonstrate that ACAN protein level was also increased when compared to the WT clone (). The levels of ACAN protein in RORβ OE MG63 cells was similar to that observed in TC28a2 cells, which are normal chondrocyte cells. Thus, RORβ inhibits the production of MMPs and increases the expression of extracellular matrix proteins, both of which would be protective to articular cartilage degradation.

RORβ expression balances FGFR signaling

Fibroblast growth factor (FGF) signaling has a role in growth and homeostasis of joint related cells, including articular chondrocytes, synovial cells, and osteogenic cells and aberrant FGF signaling contributes to the progression of OA [23]. Genetic inhibition of FGFR1 in knee cartilage attenuates cartilage degradation by the RAF-MEK-ERK and PKCd-p38 pathways [24] and conditional deletion of FGFR3 in mice aggravated DMM-induced cartilage degeneration [25]. In addition, FGF18 attenuates cartilage degradation through FGFR3/PI3K-AKT signaling [26]. As shown in , stable expression of RORβ reduced the ratio of FGFR1/FGFR3 towards a protective profile. This alteration of FGFR signaling correlates with changes in phosphorylation of ERK1 (decreased) and AKT (increased) as expected (.

Reduction of FGFR1 expression and Erk signaling and enhanced FGFR3 expression and Akt signaling in RORβ OE cells.

a) Relative mRNA expression of FGFR1 and FGFR3 along with RORβ. b) The expression of phosphorylated Erk1/2 and phosphorylated Akt in RORβ over expressing MG63 cells.

RORβ protects against IL-1β mediated inflammation and inhibits basal protease production

IL-1β is an essential mediator of acute joint inflammation induced by physical injuries and it plays a critical role in cartilage degradation. Induction of IL-1β increases the expression of catabolic matrix enzymes such as MMPs and ADAMTS, as well as the pro-inflammatory cytokine IL-6 which ultimately leads to cartilage matrix degradation [27-29]. Here we demonstrate that stable expression of RORβ suppressed IL-1β induced expression of IL6, MMP3 and ADAMTS4. Additionally, expression of RORβ downregulates expression of MMP3 and ADAMTS4 as compared to WT control cells ().

Suppression of IL1β stimulated genes in hRORβ/MG63 clone.

Inflammatory cytokine IL1β was treated into WT or OE RORβ/MG63 cells for 24 hr with 2nM. The mRNA expression level of IL6, MMP3 and ADAMTS4 was significantly increased by IL1 stimulation but it was protected in RORβ OE cells. The RORβ expression did not affect by IL1β stimulation.

Conclusions

Development of OA impairs the biomechanical properties of articular cartilage eventually leading to irreversible loss of cartilage resulting in debilitating joint pain and swelling. Owing to the limited regenerative capacity of articular cartilage, advanced surgical techniques have been usually required for repair of damaged cartilage [30, 31]. Risk factors for the development of OA include age, obesity, physical injury, and low-grade systemic inflammation where pro-inflammatory mediators such as IL-6 and TNFα can exacerbate cartilage erosion [32, 33]. While the pathogenesis of OA remains unclear, hypertrophic chondrocyte differentiation, reduced proliferation, dysregulated apoptosis, combined with the loss of collagen, proteoglycans and cartilage integrity are associated with the development of OA [3].

An important observation in patients with OA is the upregulation of FGFR1 expression which appears to accelerate matrix degradation by inducing the expression of RUNX2 /ELK1 and consequent downregulation of FGFR3 in articular cartilage. Furthermore, pharmacological inhibition of FGFR1 attenuates progression of disease in a mouse model of OA [10] and FGFR3 deficiency in myeloid cells enhances CXCRL12 dependent chemotaxis via CXCR7 (CXC-chemokine receptor 7), thereby leading to the exacerbation of joint destruction [34]. In this study we demonstrate that the nuclear receptor RORβ balances FGFR1/R3 signaling towards that favorable for cartilage stability by suppressing FGFR1 expression and amplifying that of FGFR3. While it remains unclear how RORβ regulates the balance of FGFR1/3 expression, the results presented here suggest RORβ is an important transcription factor controlling a gene program that is protective against articular cartilage damage. Future studies focused on understanding the mechanism of RORβ’s control of FGFR1/3 modulation in chondrocytes and murine models of OA could lead to the identification of novel therapeutics and drug targets for the prevention and or treatment of OA.

Primer sequences.

(DOCX)

Click here for additional data file.

5 Jul 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Ewa Tomaszewska, DVM Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

"Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

4. Please include a copy of Table 1 which you refer to in your text on page 6.

Additional Editor Comments:

Authors have to justificate the use of MG63 osteoblast-like cells derived from osteosarcoma.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study reports the results of overexpressing retinoic acid recepter-related orphan receptor beta (RORβ) in human osteoblast-like cells (MG63, derived from osteosarcoma), transfecting via pLPCX retroviral vector. A differential effect for RORβ-overexpressing cells versus control was shown using mRNAseq. Several pro-inflammatory genes were reduced in osteoblast cultures overexpressing RORβ. Extracellular matrix genes such as aggrecan and type II collagen were increased. FGFR1 was decreased and FGFR3 was increased. With the assistance of pathway analysis, additional inferences were collected. The ratio of FGFR1/FGFR3 was affected in what is deemed by the authors to be a beneficial response which could balance and mitigate matrix degradation associated with their individual expression. This inference is not clear from the data (Figure 4a,b). Upon induction via IL-1β, catabolic matrix enzymes MMP3, ADAMTS4, and IL-6 were suppressed in RORβ OE culture.

The report is somewhat short. Several important details of methods are omitted, such as no mention of the time course in culture and when assays were conducted. The title and full introduction of the manuscript speaks to chondrocytes and osteoarthritis, and then without mention of justification jumps to utilizing MG63 osteoblast-like cells derived from osteosarcoma. The inferences regarding FGFR ratio do not appear to be supported by the data, warranting full explanation.

The results section includes introductory and methods which would seemingly apply to those sections. The paper does not reference Figure 2a or 2b.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

12 Aug 2022

Dear Editor

Thank you for allowing us to submit a revised manuscript. Below you will see that we have thoughtfully and carefully responded to all concerns raised during review. I trust they will find our responses satisfactory.

Sincerely,

Patrick R. Griffin

Editor’s Comments:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Author’s Response: We have made edits to the manuscript to adhere to the PLOS ONE style requirements.

2. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found.

Author’s Response: We have added the following statement “All relevant data are within the paper and its Supporting Information files and the RNA-seq data is deposited in https://www.ncbi.nlm.nih.gov/geo/ GSE208277.

4. Please include a copy of Table 1 which you refer to in your text on page 6.

Author’s Response: We apologize for this oversight. Table 1 is now “S1 Table” in the supplemental material.

Additional Editor Comments:

Authors have to justify the use of MG63 osteoblast-like cells derived from osteosarcoma.

Author’s Response: We have added the following text to justify the use of MG63 osteoblast-like cells.

“Chondrocyte progenitor cells would be an ideal experimental model for the proposed studies, but markers for such cells are still unclear, and the expression level of several candidate markers such as Notch1 and SOX9 are not consistent on superficial zone (SZ), middle zone (MZ), and deep zone (DZ) of normal tissue and OA tissue [8]. Therefore, in the studies presented here we utilized MG63 cells to overcome these issues. MG63 cells are derived from an established sarcoma cell line and is an osteoblastic model to study bone cell viability, adhesion, and proliferation. Importantly, these cells express the mesenchymal stem cell markers Notch1 and SOX9 and are a well characterized osteoblast-like cell line which can drive development of OA.”

Reviewer(s)' Comments to Author:

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Author’s Response: We address this concern below in response to specific comments from the review.

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

Author’s Response: All relevant data are within the paper and its Supporting Information files and the RNA-seq data is deposited in https://www.ncbi.nlm.nih.gov/geo/ GSE208277.

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Authors’ Response: We have carefully reviewed the manuscript to correct typos and grammatical errors.

Response to Reviewer #1

This study reports the results of overexpressing retinoic acid recepter-related orphan receptor beta (RORβ) in human osteoblast-like cells (MG63, derived from osteosarcoma), transfecting via pLPCX retroviral vector. A differential effect for RORβ-overexpressing cells versus control was shown using mRNAseq. Several pro-inflammatory genes were reduced in osteoblast cultures overexpressing RORβ. Extracellular matrix genes such as aggrecan and type II collagen were increased. FGFR1 was decreased and FGFR3 was increased. With the assistance of pathway analysis, additional inferences were collected. The ratio of FGFR1/FGFR3 was affected in what is deemed by the authors to be a beneficial response which could balance and mitigate matrix degradation associated with their individual expression. This inference is not clear from the data (Figure 4a,b). Upon induction via IL-1β, catabolic matrix enzymes MMP3, ADAMTS4, and IL-6 were suppressed in RORβ OE culture.

Author’s response: Our responses are below.

The report is somewhat short. Several important details of methods are omitted, such as no mention of the time course in culture and when assays were conducted.

Author’s response: We have added additional experimental detail to the methods and figure legends.

The title and full introduction of the manuscript speaks to chondrocytes and osteoarthritis, and then without mention of justification jumps to utilizing MG63 osteoblast-like cells derived from osteosarcoma.

Author’s response: We have added the following text to justify the use of MG63 osteoblast-like cells.

“Chondrocyte progenitor cells would be an ideal experimental model for the proposed studies, but markers for such cells are still unclear, and the expression level of several candidate markers such as Notch1 and SOX9 are not consistent on superficial zone (SZ), middle zone (MZ), and deep zone (DZ) of normal tissue and OA tissue [8]. Therefore, in the studies presented here we utilized MG63 cells to overcome these issues. MG63 cells are derived from an established sarcoma cell line and is an osteoblastic model to study bone cell viability, adhesion, and proliferation. Importantly, these cells express the mesenchymal stem cell markers Notch1 and SOX9 and are a well characterized osteoblast-like cell line which can drive development of OA.”

The inferences regarding FGFR ratio do not appear to be supported by the data, warranting full explanation.

Author’s response: We have edited the text as follows to clarify the observations related to FGF receptors.

“We determined that ROR�  alters the ratio of expression of the FGF receptors FGFR1 (associated with cartilage destruction) and FGFR3 (associated with cartilage protection). Additionally, ERK1/2-MAPK signaling was suppressed and AKT signaling was enhanced.”

The results section includes introductory and methods which would seemingly apply to those sections.

Author’s response: Part of this issue is addressed now as the Results section is now “Results and Discussion” to comply with the PLOS ONE style requirements.

The paper does not reference Figure 2a or 2b.

Author’s response: We apologize for this oversight. We have added text to the document referencing Figures 2a and 2b.

“Principle component analysis results and a heatmap of the sample to sample distance are shown in Fig. 2a and 2b.”

Submitted filename: Response to review final.docx

Click here for additional data file.

23 Sep 2022

RORβ modulates a gene program that is protective against articular cartilage damage

PONE-D-22-12514R1

Dear Dr. Patrick Robert Griffin ,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Ewa Tomaszewska, DVM Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors took into account all comments of the reviewer. Individual ambiguities are explained in the text in the appropriate places. The manuscript can be accepted for publication.

Reviewers' comments:

29 Sep 2022

PONE-D-22-12514R1

RORβ modulates a gene program that is protective against articular cartilage damage

Dear Dr. Griffin:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Ewa Tomaszewska

Academic Editor

PLOS ONE