pH-sensing G protein-coupled orphan receptor GPR68 is expressed in human cartilage and correlates with degradation of extracellular matrix during OA progression

RNA-sequencing data analysis

To define the role of proton sensing orphan GPCRs in the pathogenesis of OA, we analyzed multiple high throughput RNA-seq data available in public domain (NCBI-GEO) in the cartilage tissue during OA progression. The raw data were downloaded from healthy and OA cartilage tissues in human and mice (GSE106292, GSE110051, GSE114007) available from the NCBI-GEO database (Ferguson et al., 2018; Fisch et al., 2018). The human OA cartilage samples were resected from the weightbearing regions on medial and lateral femoral condyles, and scored as OA grade 4, whereas healthy cartilage samples were of grade 1 and had no history of joint disease or trauma (Fisch et al., 2018). BioJupies (http://biojupies.cloud), a web-based freely available application was used to analyze the differentially expressed genes (DEGs) to determine the expression of proton sensing receptors including GPR68 in healthy vs OA cartilage (Torre, Lachmann & Ma’ayan, 2018). Raw data were normalized to log10 counts per million (log CPM) and differentially expressed genes between the healthy and OA groups were determined using the R package limma (Ritchie et al., 2015). Multiple correction testing was performed using Benjamini-Hochberg method. The Jupyter Notebooks created for each RNA-seq raw data analysis provide differentially expressed gene lists in an interactive data visualizations format (Torre, Lachmann & Ma’ayan, 2018). Mapped read counts of selected genes from Jupyter Notebooks were used for the generation of heatmap using the ClustVis web tool as described previously (Metsalu & Vilo, 2015). The mapped read count was normalized using Z-score for generation of heatmap.

Microarray data analysis

To determine the level of GPR68 in experimental OA, we analyzed the microarray dataset of C57Bl/6 mouse cartilage with DMM and sham operated knee joints. Transcriptome profiling was performed in cartilage samples from mice at 1-, 2- and 6-weeks post-surgery using Agilent-014868 Whole Mouse Genome 4 × 44K G4122F Microarray available from the GEO database (GSE45793) (Bateman et al., 2013). The GEO datasets included RefSeq Gene ID, gene name, Gene symbol, microarray ID, adjusted P-value, and experimental group logFC relative to corresponding control groups. GEO2R was used to analyze the differential expression profiling between sham and DMM operated knee joints at 6-weeks. The FDR method was used to correct for multiple testing in GEO2R. The intensity value was used to represent the fold change of gene expression between sham and DMM operated knee joint cartilage.

450K DNA methylation analysis

To determine the relationship between the correlation of promoter DNA methylation level with gene expression of GPR68, we analyzed global DNA methylation analysis in human OA cartilage samples. To this end, we used publicly available Infinium 450K data sets from 12 knee OA patients (GSE63695) (Steinberg et al., 2017). Genome wide DNA methylation analysis was performed in chondrocytes isolated from two different region of each cartilage- high-grade degeneration (degraded/OA sample) vs low-grade degeneration (intact/healthy sample). All analyses were performed directly on the preprocessed data available online. We used the probe annotations from ChAMP (Morris et al., 2014) to assign promoter probes to gene. After computing the mean beta value in the promoter of GPR68 in each sample, we used a paired t-test to identify differential promoter-region methylation between degraded and intact samples. Significance was set at 5% FDR (false discovery rate).

Preparation of primary human OA chondrocytes

Prior to the initiation of the studies the study protocol was reviewed and approved by the Institutional Review Board (IRB) of Emory University, Atlanta, GA as a “not research involving human subjects” as defined by DHHS and FDA regulations and that no informed consent was needed. All the methods used in this study were carried out in accordance with the approved guidelines and all experimental protocols were approved by the IRB of Emory University, Atlanta, GA (#IRB ID: STUDY00003990). Discarded and de-identified knee joint cartilage samples were collected from the patients undergoing joint arthroplasty at the Emory University Orthopaedics and Spine Hospital (EUOSH), Atlanta, GA.

Macroscopic cartilage degeneration was determined by India ink staining and cartilage degeneration were graded according to Mankin scoring system (Mankin et al., 1971). In this system the scores are defined as follows: 0 = Normal, 1 = Superficial fibrillation, 2 = Pannus and superficial fibrillation, 3 = Fissures to the middle zone, 4 = Fissures to the deep zone and 5 = Fissures to the calcified zone. Cartilage area or sample with high-grade degeneration (Mankin score ≥ 3) signifying severe OA condition were classified as “High-grade OA cartilage”, whereas cartilage area with low-grade degeneration (Mankin score < 3) signifying healthy tissue were classified as “low-grade OA cartilage”. The cartilage surface from knee joint was resected using scalpel blade (Blade #10) and minced into small pieces and chondrocytes were prepared by enzymatic digestion as described previously (Khan et al., 2017a; Khan, Ansari & Haqqi, 2017; Khan, Ahmad & Haqqi, 2018; Khan et al., 2017b, 2017c). Chondrocytes phenotype during in vitro culture was maintained by plating the chondrocytes at high cell density (1 × 10⁶/well of 6-well plate) and phenotype was verified by analyzing the mRNA expression of chondrocytes phenotype markers such as COL2A1, ACAN, and COL10A1 using real time PCR analysis.

In vitro model of OA by stimulation of primary human OA chondrocytes with IL1β

Primary human OA chondrocytes were seeded at high confluency in six well plate (1 × 10⁶/ well) in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% fetal calf serum (FCS), penicillin (100 units/ml), and streptomycin (100 mg/ml) for 2–3 days. When cells reached 80% confluency, OA chondrocytes were serum starved overnight and then stimulated with IL1β (1 ng/ml) for 1, 3, 6, 12 and 24 h at 37 °C, in CO₂ incubator under 5% CO₂ and 95% air. Unstimulated chondrocytes were used as control.

Ogerin treatment of human chondrocytes and stimulation with IL1β

Human OA chondrocytes cultured in 6-well plate were treated with different concentration of Ogerin (1–10 µM) for 2 h followed by stimulation with IL1β (1 ng/ml) in incomplete DMEM/F-12 medium without presence of serum. Ogerin (OGR) is a positive allosteric modulator (PAM) of GPR68 and known to activate GPR68 at physiological pH. Chondrocytes which were treated with 0.1% DMSO served as control for this experiment.

Total RNA isolation and gene expression analysis using real time qPCR

Chondrocytes were harvested at end of incubation period and total RNA was isolated using TRIzol® method as previously described (Khan, Ahmad & Haqqi, 2018; Khan et al., 2017a; Khan, Ansari & Haqqi, 2017; Khan et al., 2017b, 2020a; Khan et al. 2023a). For gene expression analysis, cDNA was synthesized from 1 µg of total RNA using high-capacity cDNA reverse transcription kit (Life Technologies) and gene expression of GPR68, MMP13, and MMP3 was quantified using SYBR Green expression assay as previously described (Khan, Ahmad & Haqqi, 2018; Khan et al., 2017a; Khan, Ansari & Haqqi, 2017; Khan et al., 2017b; Khan et al. 2023a; Khan et al., 2020a, 2020b, 2023b). β-actin was used as housekeeping gene and relative expression levels were calculated using the 2^−ΔΔCT method (Livak & Schmittgen, 2001). Primer sequence for the genes used here were provided in Table 1.

Histological analysis of human OA cartilage

Cartilage matrix depletion due to IL1β-induced activation of matrix degrading proteases was determined by Safranin-O staining of cartilage explants. The cartilage explants stimulated with IL1β (25 ng/ml) for 72 h were fixed in 10% neutral buffer formalin for 24 h, decalcified in 10% EDTA (pH 7.4) for 72 h, dehydrated with series of alcohols and then embedded in paraffin wax, sectioned at 5 μm thickness were stained with Safranin-O dye as described previously (Khan et al., 2017b; Khan et al. 2023b). The cartilage sections were imaged using BioTek Lionheart LX Automated Microscope as described previously (Khan et al., 2020a, 2023a).

Animals and induction of experimental OA using surgical destabilization of the medial meniscus (DMM) in mice

Male C57BL/6 mice were obtained from Jackson Laboratory (ME, USA). Twenty mice were randomly divided into two groups (Sham and DMM) of ten animals per experimental group as described previously (Ansari et al., 2019) and each five mice were kept in one cage. All mice were housed under 12-h light/dark cycle at ambient temperature of 20–25 °C and humidity of 50 ± 10%, and they were given free access to food and water. The animal studies were approved by the Institutional Animal Use and Care Committee of the Atlanta VA medical center (#V002-22). Husbandry and environmental conditions in this facility comply with all governmental regulations. No excess experimental animals were used in this experiment. All mice were euthanized at end of experiment by CO₂ asphyxiation, followed by cervical dislocation as secondary physical method of euthanasia.

Experimental OA was induced by performing surgical destabilization of the medial meniscus (DMM) in the left knee of 12 weeks old male mice as described previously (Ansari et al., 2019). Briefly, mice were anesthetized using isoflurane inhalational anesthesia (1.5–2%). The hind limb prepped under sterile conditions and a longitudinal skin incision were made anteriorly on the right knee. The knee joint exposed using a medial parapatellar capsulotomy. The patellar ligament (PL) was laterally displaced, and the knee brought into flexion and medial meniscotibial ligament were dissected with Jewelers forceps to induce destabilization of medial meniscus (DMM). Sham surgery was ed as control. The mice were euthanized at 12-week post-surgery and intact joints were used for the histological assessment of the severity of the induced OA. Knee joints were fixed in a 10% neutral buffered formalin, decalcified in 10% EDTA, embedded in paraffin, and sectioned at 5 µM thickness. Sections were deparaffinized and rehydrated with a series of graded ethanol and stained with Safranin-O/Fast Green to determine loss of proteoglycan content to analyze the loss of cartilage ECM during OA progression.

Immunohistochemical (IHC) analysis of GPR68 in human and mice cartilage

Full thickness human cartilage pieces resected from the human cartilage or intact knee joints harvested 12-week post-surgery from sham and DMM operated mice were fixed in 4% paraformaldehyde for 48 h. Samples were decalcified using 10% EDTA for 2 weeks and dehydrated cartilage pieces were embedded in paraffin and 5 μM thick sections were prepared. Deparaffinization of cartilage sections was performed by incubating histological section with xylene three time (5 min each) and then sections were rehydrated using various grade of alcohol in a decreasing concentration (100%, 90%, 70% and 50%). The sections were washed with 1X TBS for 10 min and antigen retrieval was performed by incubating histological sections in 10 mM citrate buffer (pH 6.0) at 65 °C for 1 h using water bath. To analyze the immunoreactivity of GPR68, the endogenous peroxidase activity was neutralized by 3% hydrogen peroxide in 1X TBS, sections were blocked in 10% normal goat serum for 30 min at room temperature, and incubated in rabbit polyclonal anti-GPR68 antibody (MyBiosource; MBS7044035) overnight at 4 °C. We also used recombinant rabbit IgG monoclonal antibody (Abcam; ab172730) as negative isotype control. Sections were washed with 1X TBS and incubated with HRP-conjugated secondary anti-rabbit antibody for 2 h followed by washing with 1X TBS, and developed using a SignalStain® DAB substrate kit (#8059S; Cell Signaling Technology) and images were captured BioTek Lionheart LX Automated Microscope as described previously (Diaz-Hernandez et al., 2020; Díaz-Hernández et al., 2022; Khan et al., 2023a, 2020a).

Statistical analyses

The data presented here represent the value of Mean ± SD. The statistical difference between control and experimental groups was performed by GraphPad PRISM (GraphPad, San Diego, CA, USA, V 9.0.) using one-way ANOVA with Tukey’s post hoc test. The significance was set at P < 0.05.

Proton-sensing GPCRs are expressed in human and mice cartilage

To examine the role of proton sensing orphan GPCRs in the pathogenesis of OA, we first examined the expression levels of all family members in human and mice cartilage. We analyzed high throughput RNA-seq dataset originally generated for molecular mapping of human skeletal ontogeny and pluripotent stem cell-derived articular chondrocyte (GSE106292) (Ferguson et al., 2018). After stringent quality control, reads per kilobase pair per million mapped reads (RPKM) value (expression) for all four gene members (GRP68, GPR88, GPR132 and GPR4) were determined in human cartilage across four different stages of human ontogeny including (1) embryonic cartilage isolated from limb bud condensations at embryonic 5-6 Week Post Conception (WPC), (2) presumptive cartilage derived from 17 WPC, (3) healthy juvenile cartilage, and (4) adult articular chondrocytes. In addition to human in vivo chondrogenesis, we also analyzed two stages of in vitro pluripotent stem cell (PSC) differentiation into chondrocytes at 14 and 60 days. Our analysis demonstrates that all there four members of proton-sensing orphan GPCR genes expressed in human cartilage, and GPR68 expression was highest in all the human cartilages analyzed (Fig. 1A). We next determined the expression levels of these orphan GPCRs in murine cartilage by further analyzing a different RNA-Seq dataset (GSE110051) generated in immature and mature chondrocytes isolated from the mouse embryo and adult histological section using laser capture microdissection. Our analysis demonstrates variable degree of expression levels of all four GPCRs, with Gpr68 showing highest expression level in both immature and mature cartilage (Fig. 1B). These results suggest that GPR68 was robustly expressed in both murine and human cartilage during course of chondrogenic differentiation in vivo and in vitro suggesting a potential role for GPR68 in articular cartilage. Owing to the robust and intensified expression of GPR68 among other members of proton sensing receptors in human and mice cartilage, we focus our study on GPR68 for in depth analysis of its functional role in cartilage during OA progression.

GPR68 expression is profoundly increased in human OA cartilage

To determine the role of GPR68 in OA pathogenesis, we assessed the expression level in human cartilage isolated from healthy donors and OA patients. OA cartilage showed macroscopic cartilage degeneration as compared to healthy cartilage and exhibited reduced proteoglycan content as analyzed by Safranin-O staining indicating the loss of cartilage ECM in OA patients (Fig. 2A). To investigate the comparative expression profile of GPR68, we analyzed RNA-seq data performed on performed on 38 individuals including 18 healthy and 20 OA human knee cartilage tissues (GSE114007) (Fisch et al., 2018). Our results as shown in Fig. 2B demonstrate that GPR68 is expressed in both healthy and OA cartilage and its mRNA levels are significantly increased in OA cartilage (N = 20) compared to healthy cartilage (N = 18) (# P < 0.01) (Fig. 2B). These results suggest that GPR68 might be playing a role OA pathogenesis.

GPR68 expression positively correlates with OA severity in human

To further confirm the role of GPR68 in OA pathogenesis, we analyzed its expression in human cartilage isolated from different stages of OA. High-grade OA cartilage (Mankin score ≥ 3;) showed severe cartilage degeneration as compared to low-grade OA cartilage (Mankin score ≤ 3) and exhibited loss of proteoglycan content as analyzed by Safranin-O and Fast green staining indicating the loss of cartilage ECM during OA progression (Fig. 3A). Our IHC and qPCR analyses showed that expression of GPR68 was significantly higher in cartilage with high-grade OA (N = 10) compared to the cartilage with low-grade OA (N = 10) (Fig. 3A, B; P < 0.01). The increased expression of the GPR68 positively correlated with the severity of OA further reinforcing our hypothesis that GPR68 plays a unique role in the pathogenesis of OA.

Gpr68 expression is increased in murine cartilage of experimentally induced OA

To further confirm of role of Gpr68 in OA pathogenesis, we examined the expression level of GPR68 in murine cartilage of surgically induced OA. DMM surgery, which is transection of medial meniscotibial ligament, is a gold standard approach to model human post-traumatic OA (PTOA) (de Hooge et al., 2005; Ma et al., 2007) by inducing experimental OA in murine knee joint. This model reproduces most of the pathogenic events leading to PTOA following injuries that impact load distribution in the knee. Safranin-O/Fast Green staining of knee joints after 12-week post-DMM surgery demonstrated significant cartilage erosion and cartilage matrix depletion as shown by fibrillation of cartilage surface and reduced proteoglycan level in the medial femoral condyle and medial tibial plateau of DMM mice as compared to sham surgery (Fig. 4A). It has been used in dozens of previous studies including ours (Ansari et al., 2019), which aimed at determining the importance and roles of specific genes on OA initiation or progression. Moreover, IHC analysis using anti-GPR68 antibody in murine cartilage showed that protein levels of GPR68 were higher in DMM cartilage and meniscus as compared to sham cartilage (Fig. 4B). Interestingly, the meniscus cells in the histological section of DMM mice had stronger immunoreaction with anti-GPR68 antibody. However, as shown in Zoomed image (indicated by red arrows) increased immunoreactivity was observed in both cartilage and meniscus region of DMM mice as compared to sham control suggesting that GPR68 expression markedly increased during OA progression in mice. Further, negative antibody control which include the nonspecific rabbit IgG antibody used at same concentration did not show any immunoreactivity in the histological sections of murine joint (Fig. 4B). These results were further corroborated by transcriptome analysis of microarray dataset demonstrating increased Gpr68 expression in mice cartilage of DMM joints as compared to sham operated knee joints (GSE45793) (Bateman et al., 2013). (N = 4/group) (Fig. 4C; P < 0.01). Collectively, data support our hypothesis that Gpr68 play a significant role in cartilage erosion during OA progression in mice.

GPR68 DNA promoter shows hypomethylation in OA cartilage

DNA methylation plays a major regulatory role in gene transcription mainly via a negative regulation way (Ehrlich, 2019; Rauluseviciute, Drablos & Rye, 2020; Smith et al., 2020). The hypomethylated DNA facilitates the binding by transcription factors and promotes gene transcriptional activation (Ehrlich, 2019; Rauluseviciute, Drablos & Rye, 2020; Smith et al., 2020). To examine whether increased expression of GPR68 observed in OA cartilage was associated with decreased level of DNA methylation, we analyzed DNA methylome of cartilage in knee and hip OA. Genome-wide methylation was performed in chondrocyte DNA extracted from 12 high vs low grade OA cartilage using llumina Infinium Human Methylation450K BeadChip array (GSE63695) (Steinberg et al., 2017). We analyzed all 20 CpG sites for assessing the average DNA methylation levels of GPR68 in OA cartilage. Our analysis demonstrates the presence of total 5 CpG sites in the promoter (TSS1500 and TSS200) region of GPR68 (Fig. 5A). Interestingly, average promoter CpG methylation was significantly lower in high grade OA cartilage compared with the level in low grade OA cartilage (Fig. 5B, # P < 0.01). These results demonstrating hypomethylation of GPR68 promoter in severe OA cartilage are in concordance with increased GPR68 expression in high grade OA cartilage as shown in Fig. 3B. Taken together our data further highlights the role of GPR68 in OA progression.

GPR68 expression positively correlates with matrix degeneration in human and mice cartilage during OA progression

Since GPR68 expression robustly increased in high grade human OA cartilage and DMM operated knee joints in mice as shown in Figs. 3 and 4, we next determined whether GPR68 expression correlates with the degeneration of extracellular matrix. For this, we analyzed cartilage ECM content by Safranin-O staining which stain the sulfated GAG and proteoglycan levels of cartilage matrix in human cartilage explants and murine knee joint sections. Our analysis of human cartilage as shown in Fig. 3A revealed that as compared to low grade OA, high grade OA cartilage showed reduced proteoglycan content indicating the higher rate of matrix degeneration. Furthermore, IHC analysis of same cartilage explants showed that high grade OA cartilages exhibited increased immunoreactivity (Fig. 3A) demonstrating that during OA progression, human cartilage that displayed increased matrix degeneration showed positive correlation with protein level of GPR68. Similarly, during OA progression in mice, reduced Safranin-O staining indicating loss of proteoglycans in DMM operated mice knee joints at 12-week post-surgery also showed increased immunoreactivity with GPR68 antibody (Fig. 4B). These data further demonstrates that GPR68 level positively correlates with ECM degeneration in human and mice model indicating the plausible role of GPR68 in degeneration cartilage matrix during OA progression.

IL1β stimulation of primary human OA chondrocytes represents an in vitro model of OA

Enhanced production of matrix degrading proteases by activated chondrocytes is hallmark feature of OA and IL1β is key player of catabolism in OA joints which leads to progressive and irreversible cartilage degradation. To demonstrate IL1β as a key mediator of OA, we analyzed proteoglycan content as an indication of cartilage ECM degradation using Safranin-O and fast green staining of cartilage explant stimulated with or without IL1β. Our results demonstrate that IL1β stimulation of OA cartilage explants resulted in reduced Safranin-O staining, indicating loss of proteoglycans, in comparison to untreated explants demonstrating increased production of cartilage ECM degrading proteinases (Fig. 6A). To further demonstrate whether IL1β treatment induced matrix degrading protease gene expression in primary human OA chondrocytes, we prepared chondrocytes using full thickness cartilage using enzymatic digestion, cultured in monolayer, stimulated with IL1β (1 ng/ml) for 24 h, then assessed the expression profile of catabolic mediators mRNA levels using qPCR array. Our qPCR array data showed that IL1β treatment increased the mRNA expression of a plethora of catabolic genes, proinflammatory molecules, several inflammatory signaling genes (NF-κβ) and also reduced the expression of cartilage anabolic genes such COL2A1, ACAN etc., (Fig. 6B) in primary human OA chondrocytes. These data indicate that human chondrocyte cultures derived from pathologic cartilage samples produced matrix degrading protease repertoire mirroring that of OA joints, therefore monolayer culture of primary human OA chondrocytes stimulated with IL1β represent an ideal cellular model to study the pathogenesis of OA.

IL1β is a potent inducer of GPR68 expression in human OA cartilage and chondrocytes

Since IL1β is a key inducer of catabolic events in OA, we determined whether IL1β induced expression of GPR68 in primary chondrocytes. We prepared chondrocytes from knee cartilage of OA patients undergoing joint arthroplasty and monolayer cultures were stimulated with IL1β (1 ng/ml) for different time points. Our qPCR analysis showed that IL1β treatment induced the expression of GPR68 in a time dependent manner in human OA chondrocytes (Fig. 7A; P < 0.01). Consistent with the increased mRNA expression levels, immunostaining of histological sections of human cartilage revealed that the GPR68 protein level was also markedly high upon stimulation with IL1β (25 ng/ml, 72 h) (Fig. 7B). These results demonstrate that stimulation of both human chondrocytes and cartilage with IL1β, which is known to be associated with OA pathogenesis and cartilage matrix destruction, caused significant upregulation of the GPR68 mRNA and protein expression.

IL1β induced GPR68 expression positively correlates with the expression of matrix degrading proteases in human OA chondrocytes

Our analysis demonstrates that IL1β is potent inducer of GPR68 expression in human OA chondrocytes and cartilage (Fig. 7A). We next determined whether IL1β induced expression of GPR68 correlates with the expression of matrix degrading proteases in human OA chondrocytes. We stimulated the monolayer culture of human OA chondrocytes with IL1β (1 ng/ml) for different time points and analyzed the gene expression of matrix metalloproteases such as MMPs and ADAMTSs. Our qPCR analysis revealed that as compared to untreated control, stimulation of OA chondrocytes with IL1β resulted in significantly high-level mRNA expression of several matrix degrading proteases such as MMP3, MMP9, MMP13, ADAMTS4 and ADAMT5 (Fig. 7C). These data indicate that IL1β is a potent inducer of GPR68 and proteases mRNA expression in human OA chondrocytes. Furthermore, OA chondrocytes that express high level of GPR68 under stimulation with IL1β showed positive correlation with expression level of MMP13 (Fig. 7D) and other matrix degrading proteases (Fig. 7E). Altogether, these data suggest that GPR68 may mediate catabolic and matrix degrading role of IL1β in human OA chondrocytes.

Ogerin mediated GPR68 activation repressed the expression of MMPs in human OA chondrocytes

Our correlation studies as shown in Figs. 7D and 7E demonstrate a clear correlation between mRNA level of GPR68 and MMPs in human chondrocytes. We next determined whether increased GPR68 activation modulate the expression of MMPs. To this end, we used small molecule activator of GPR68 in human chondrocytes and analyzed whether increased GPR68 had any effect on the expression of MMPs in presence of IL1β stimulation. Ogerin (OGR) is a synthetic GPR68 selective agonist, which activate GPR68 at physiological pH and enhanced the receptor activity (Huang et al., 2015). Human OA chondrocytes were treated with Ogerin (1–10 μM) for 2 h and then stimulated with IL1 β and mRNA expression of MMP3 and MMP13 was determined by SYBR Green gene expression assay. As reported previously (Khan et al., 2017a; Khan, Ansari & Haqqi, 2017), our data showed that treatment of OA chondrocytes with IL1β significantly increased the mRNAs expression of MMP3 and MMP13 as compared to untreated control OA chondrocytes (Figs. 7F and 7G). Interestingly, pretreatment of OA chondrocytes with Ogerin which in turn activates GPR68 significantly inhibited the mRNA expression of these matrix degrading proteases in a dose dependent manner (Figs. 7F and 7G). These results indicate that GPR68 activation by Ogerin in human chondrocytes exhibits anti-catabolic effects through repression of MMP3 and MMP13 mRNA expression under OA pathological conditions.