RGS12 Is a Novel Critical NF-κB Activator in Inflammatory Arthritis.

Rheumatoid arthritis (RA) is the most common inflammatory disease, which currently lacks effective treatment. Here, we discovered that the Regulator of G Protein Signaling 12 (RGS12) plays a key role in regulating inflammation. Transcriptional and protein analysis revealed that RGS12 was upregulated in human and mouse RA macrophages. Deletion of RGS12 in myeloid lineage or globally inhibits the development of collagen-induced arthritis including joint swelling and bone destruction. Mechanistically, RGS12 associates with NF-κB(p65) to activate its phosphorylation and nuclear translocation through PTB domain, and NF-κB(p65) regulates RGS12 expression in a transcriptional manner. The nuclear translocation ability of NF-κB(p65) and RGS12 can both be enhanced by cyclooxygenase-2 (COX2). Furthermore, ablation of RGS12 via RNA interference significantly blocks the inflammatory process in vivo and in vitro. These results demonstrate that RGS12 plays a critical role in the pathogenesis of inflammatory arthritis.

Introduction

Rheumatoid arthritis (RA) is the most common inflammatory disease with a marked expansion of the normally hypocellular synovium by infiltrating leukocytes, including synovial macrophages (SMs), which leads to the destruction of adjacent cartilage and bone (Smolen et al., 2016, Tang et al., 2011). Macrophages are one of the most abundant cell types in the RA synovium, and evidence indicates that macrophages are activated in RA and can directly drive the progression of RA (Choi et al., 2017). Macrophages are a major source of cytokines and chemokines, such as monocyte chemoattractant protein 1 (MCP1), granulocyte-macrophage-colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), and the interleukins (Sprague and Khalil, 2009). Enzyme cyclooxygenase-2 (COX2) produced from activated macrophages contributes to pain and swelling via promoting the production of inflammatory mediators such as prostaglandin E(2) (PGE2), which has been a major target of biological therapy (Bouta et al., 2018, Samad et al., 2001). Moreover, persistent activation of certain signaling pathways including NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) and PI3K-AKT (Phosphoinositide 3-kinases-Protein kinase B) enhances resistance to cell apoptosis or cytokine withdrawal (Pope, 2002). Therefore, targeting macrophages is a potentially effective way to inhibit inflammatory arthritis.

NF-κB plays a critical role in inflammation or RA owing to its principal function in transcriptional regulation of inflammatory cytokines (Yao et al., 2009). NF-κB is activated by receptor molecules located in the cell membrane such as TNF receptor (TNFR) superfamily members, ligands of various cytokine receptors, as well as pattern-recognition receptors (PRRs) (Liu et al., 2017). Activation of canonical NF-κB, a dimer comprising NF-κB1 (p50) and RelA (p65), is triggered by the phosphorylation-induced degradation of IκB and followed by the transcriptional activation of target genes in the nucleus (Vallabhapurapu and Karin, 2009). The phosphorylation of the NF-κB subunits has a profound effect on cell functions, which controls the interactions with other factors such as MYBBP1a (MYB Binding Protein 1a) and CBP (CREB-binding protein) (Christian et al., 2016, Milanovic et al., 2014). Activation of NF-κB steadily increases numerous known genes including cytokines, chemokines, cellular ligands, and adhesion molecules, which contributes to RA development and progression (Sundaram et al., 2009, Yuan et al., 2019). Thus, inhibition of NF-κB signaling or related pathways may have a huge potential for the treatment of RA (Yamamoto and Gaynor, 2001).

The Regulator of G-protein signaling (RGS) protein family contains a conserved RGS domain, which is often accompanied by other signaling regulatory domains (Neubig and Siderovski, 2002). RGS proteins accelerate the deactivation of G proteins to turn off GPCR signaling (Neubig and Siderovski, 2002). Recently, RGS proteins were found to play important roles in both innate and adaptive immune responses (Druey, 2017). RGS1 regulates myeloid cell accumulation in atherosclerosis through altered chemokine signaling (Patel et al., 2015). RGS10 expression is inhibited in activated microglia through altered histone deacetylase activity (Alqinyah et al., 2017). Therefore, RGS proteins may become important and new drug targets for the treatment of immune and inflammatory diseases.

Regulator of G Protein Signaling 12 (RGS12) was first found as the largest protein in the RGS family (Snow et al., 1997). Further studies found that RGS12 is located in both the cytosol and the nucleus (Willard et al., 2007) and contains an N-terminal PDZ (PSD-95, disc-large, zo-1) domain, PTB (phosphotyrosine binding) domain, RGS domain, tandem RAS-binding domain (RBD), and a GoLoco (Gαi/o-Loco) interaction motif, which play important roles through binding different proteins (Snow et al., 1998, Ponting, 1999, Siderovski et al., 1999, Kimple et al., 2001). RGS12 is involved in multiple signaling pathways, including G protein-coupled receptors (GPCRs) (Kimple et al., 2001), receptor tyrosine kinases (RTKs) (Willard et al., 2007), Ras GTPases (Snow et al., 1998, Willard et al., 2007), and MEK1/2-ERK1/2 coordinated signaling (Huang et al., 2016).

RGS12 has been reported to be closely associated with some diseases (Huang et al., 2016, Qi et al., 2017). The loss of RGS12 leads to the development of pathological cardiac hypertrophy and heart failure through MEK1/2-ERK1/2 signaling pathway (Huang et al., 2016). Our laboratory found that deletion of RGS12 in myeloid lineage caused mouse growth retardation with increased bone mass (Yang et al., 2013, Yuan et al., 2015). Notably, by analyzing FCGR2A functionally related genes in response to TNF-α antibody (adalimumab) treatment through microarray-based studies, Avila-Pedretti et al. found that there was a significant association between FCGR2A and the response to adalimumab, and DHX32 and RGS12 were the most consistently correlated genes with FCGR2A expression in RA synovial fluid macrophages (Avila-Pedretti et al., 2015). However, the function and mechanism of RGS12 involved in RA have not yet been uncovered.

In this study, we investigated the role of RGS12 in the regulation of RA progression by creating and analyzing the RGS12 global and macrophage lineage conditional knockout (CMVCre;RGS12 and LysMCre;RGS12) mouse models. Our findings demonstrated that RGS12 is essential for NF-κB inflammatory signaling transduction and thereby plays a key role in the progression of RA.

Results

RGS12 Expression Is Upregulated in Inflammatory Arthritis

RA is characterized with an increased number of SMs. By using NIH GEO database (GSE49604) to analyze the human global expression of genes in synovial fluid macrophages from patients with RA and healthy macrophages differentiated from peripheral blood monocytes (You et al., 2014), we identified 1,576 genes that are mostly changed (with an FDR of less than 0.05 between RA SMs and healthy control macrophages) (Figure 1A and Table S5). Subsequently, we identified upregulated and downregulated RGS family genes (Figure S1). We found that RGS1 and RGS12 (isoform 2) were significantly upregulated, whereas RGS2 and RGS14 were significantly downregulated (Figure S1). Notably, RGS12 gene has three isoforms in human, and the expression levels of isoform 2 significantly increase during RA (Figure 1B). We then performed real-time PCR from human synovial tissue to confirm the GEO data, discovering that RGS12 isoform 1 and isoform 2 showed a significant change in human RA synovial tissue (Figure 1C). However, no significant changes were found in RGS12 isoform 3. Interestingly, among the three isoforms, RGS12 isoform 2 is the longest transcript with PDZ, PTB, and specialized C terminus, which showed the most severe increase in expression in human RA synovial tissue (Figure 1C). We then tested the expression level of the longest RGS12 gene in a mouse model of chicken type II collagen-induced arthritis (CIA), which shows the main features of RA (Figure 1D). We found that RGS12 mRNA expression level significantly increased in synovium and SMs of CIA mice when compared with the control mice (Figure 1D). Consistently, RGS12 protein level also increased in SMs by immunoblotting assay (Figure 1E). To further explore the inflammatory mechanisms of RGS12 in vitro, we tested the expression of RGS12 in response to inflammatory stimuli including TNF-α, LPS, and IL-1 and found RGS12 mRNA (Figure 1F) and protein expressions were upregulated with TNF-α (Figure 1G), LPS (Figure 1H), and IL1 (Figure 1I) stimulation in RAW264.7 cells.

Figure 1

RGS12 Expression Is Upregulated in Inflammatory Arthritis

(A) Identification of a human gene signature (Database: GSE49604) consisting of 1,576 most changed mRNAs (FDR<0.05) in macrophages from RA synovial fluid (red box, n = 6) compared with healthy control macrophages (gray box, n = 2) differentiated from peripheral blood monocytes. Red and green denote increase and decrease in mRNA expression, respectively. Each line on the heatmap represents a gene/gene family (see also Figure S1 and Table S5).

(B) Bar graphs illustrating the expression of the different RGS12 isoform genes (isoforms 1–3) associated with RA in human macrophages as described in Figure 1A (Control [gray] and RA [red]). Asterisks indicate significant differential expression (FDR<0.05).

(C) Real-time PCR showing altered expression of RGS12 isoforms in Control (gray) and RA synovium (red). Data are presented as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 versus the control group (n = 5).

(D) Study design for induction of CIA (collagen-induced arthritis) in an animal model. WT (wild-type, C57/BL6) mice were immunized with chicken type II collagen on day 0 and boosted on day 21. All mice were sacrificed on day 40.

Quantification by real-time PCR of RGS12 using total RNA isolated from spleen, cartilage, synovium, and SMs in Control (WT) and RA mice. Results are from five independent RNA preparations, analyzed by real-time PCR in triplicates with GAPDH as an internal control. Data are analyzed by unpaired t test, and asterisks on the graphs indicate p values of significance, ∗p < 0.05, ∗∗∗p < 0.001, (n = 5).

(E) Western blot analysis of RGS12 expression in SMs from Control (WT) and RA mice on day 40 (top panel). Quantitation of RGS12 protein levels (bottom panel). The data are normalized to β-Actin. ∗∗∗p < 0.001 versus the control group, (n = 3).

(F) RGS12 mRNA expression was increased in TNF-α (10 ng/mL), LPS (1 μg/mL), and IL-1 (10 ng/mL)-induced BMMs for 24 h ∗∗p < 0.01, ∗∗∗p < 0.001 versus the control group, (n = 5).

(G–I) Western blot analysis of RGS12 expression level in TNF-α (10 ng/mL) (G), LPS (1 μg/mL) (H), and IL-1 (10 ng/mL) (I)-treated BMMs for 24 h and statistical analysis of RGS12 in the indicated groups (n = 5). Data are presented as the mean ± SEM. ∗p < 0.05, ∗∗∗p < 0.001 versus the control group.

(J) Double immunofluorescence labeling showing that RGS12 is expressed in F4/80+ macrophages in synovial tissue. Merged image showing RGS12 and F4/80 labeling. Scale bar, 100 μm. Representative individual and overlaid images are shown. Quantitative results of Integrated Optical Density (IOD) of RGS12 per F4/80+ cell in the control and RA groups. ∗∗∗p < 0.001, (n = 5).

(K) Immunofluorescence showing that RGS12 and NF-κB(p65) show co-localization in human synovial tissue. Scale bar, 200 μm. Representative individual and overlaid images are shown.

(L) Quantitative results of IOD in sections of RGS12 and NF-κB(p65) in the control and RA groups as shown in (K). Note that RGS12 significantly increases in RA but not the total NF-κB(p65). ∗∗∗p < 0.001 (n = 5).

To explore the expression pattern and role of RGS12 in human RA, we performed immunofluorescence staining in human RA synovial tissue and found that RGS12 expression was significantly enhanced in RA SMs as compared with the healthy synovial tissue (Figure 1J). NF-κB, as a central inflammatory regulator, plays a key role in the orchestration of the multifaceted inflammatory response, not only in the pro-inflammatory phase but also in the regulation of the resolution of inflammation (Gasparini and Feldmann, 2012). Interestingly, our results showed that RGS12 and NF-κB(p65) co-localized in synovium of both non-RA and RA patients (Figure 1K), and the RGS12 expression level was significantly increased in the synovium of patients with RA compared with the control (Figure 1L).

Deletion of RGS12 Inhibits the Spontaneous Development of Inflammatory Arthritis in CIA Mice

To examine the role of RGS12 during inflammatory arthritis development in vivo, we created RGS12 global knockout (CMVCre;RGS12) and the conditional knockout of RGS12 in myeloid cell-line (LysMCre;RGS12) mouse models and investigated the clinical and histopathological features of these RGS12-deficient mice in CIA development (Figures 2A and S2). Both CMVCre;RGS12 and LysMCre;RGS12 developed much less severe joint swelling and bone destruction compared with the control mice (Figures 2B and 2C). Additionally, the arthritis severity score (Figure 2D), the paw thickness (Figure 2E), and the incidence of arthritis were also significantly decreased in RGS12-deficient mice compared with the control (Figure 2F). Histological and quantitative analysis of H&E-stained joint sections of whole ankles demonstrated a significant decrease in synovitis (CMVCre;RGS12, 62%; LysMCre;RGS12 70%, p < 0.001), pannus formation (CMVCre;RGS12, 65%; LysMCre;RGS12, 79%, p < 0.001), and destruction of bone and cartilage (CMVCre;RGS12, 73%; LysMCre;RGS12, 77%, p < 0.001) in RGS12-deficient mice compared with those in control mice (Figures 2G and 2H). Hallmark genes of arthritis, such as TNFα, IL6, COX2 (Figure 2I) in SMs, and the COX2 enzymatic product PGE2 in serum were decreased in both CMVCre;RGS12 and LysMCre;RGS12 mice with RA on day 40 after immunization (Figure 2J).

Figure 2

Deletion of RGS12 in Macrophages Inhibits the Spontaneous Development of Inflammatory Arthritis in CIA Mice

(A) Graphical representation of the experimental strategy. CIA was induced in RGS12 (gray), CMVCre; RGS12 (blue), and LysMCre; RGS12 (red) mice derived from C57BL/6 strain by subcutaneous injection of chicken type II collagen in enriched CFA (First immunization, day 0) and subsequent challenge with type II collagen in incomplete Freund's adjuvant (IFA) (Second immunization) on day 21. After the immunization for 40 days, RA mice were sacrificed (see also Figure S2).

(B) Paws of RGS12, CMVCre; RGS12 and LysMCre; RGS12 mice derived from C57BL/6 strain (n = 5 mice/group) were immunized with chicken type II collagen for 40 days.

(C) Radiography of ankle joints of RGS12, CMVCre; RGS12 and LysMCre; RGS12 CIA mice. White arrow shows severe joint destruction in RGS12 CIA mice compared with the RGS12-deficient mice.

(D) Clinical arthritis scores in RGS12, CMVCre; RGS12 and LysMCre; RGS12 mice with CIA. Data are presented as the mean ± SEM. ∗∗p < 0.01 versus the control RGS12 group (n = 5).

(E) Changes of paw thickness in the indicated groups.

(F) Incidence of arthritis in the indicated groups.

(G) Hematoxylin and eosin (H&E) staining for RA synovial tissue samples.

(H) Quantitative evaluation of synovitis, pannus formation, and erosion of tarsal joints in (G). Scale bar, 200 mm. Values are mean ± SEM. ∗∗∗p < 0.001 versus the control RGS12 group, (n = 5).

(I) Real-time PCR analysis for the expression of TNFα, IL6, and COX2 in synovium tissue relative to RGS12 group (n = 5).

(J) ELISA test for serum PGE2 levels in RGS12, CMVCre+; RGS12 and LysMCre; RGS12 mice with CIA. Data are presented as the mean ± SEM. ∗∗∗p < 0.001 versus the control RGS12 group (n = 5).

RGS12 Is Essential for the Phosphorylation of NF-κB(p65) and IκBα

To understand the mechanism of RGS12 in RA, we first identify the subcellular localization of RGS12 by immunofluorescence staining in RAW264.7 cells (Figure 3A). Our data showed that RGS12 was located mainly in the cytoplasm in non-inflammatory condition (Figure 3A). TNF-α or LPS stimulation increased the expression level and nuclear translocation of RGS12 (Figures 3A–3C) in a dose-dependent manner. TNF-α is one of the most potent physiological inducers of the nuclear transcription factor NF-κB(p65). It has been proved that RGS12 is associated with the response to anti-TNF therapy (Avila-Pedretti et al., 2015) and co-localized with NF-κB(p65) (Figure 1K). To get further insights into the potential association, we treated the RAW264.7 cells with TNF-α at different doses and time points (Figures 3D–3G and S6). We found that TNF-α upregulated the expression of RGS12, pNF-κB (phospho-NF-κB p65 (Ser536)), and pIκBα (phospho-IκBα (Ser32)) in both dose- and time-dependent manners (Figures 3D–3G). To further confirm whether RGS12 directly regulates the phosphorylation of NF-κB(p65) and IκBα, BMMs were isolated from RGS12 and the LysmCre;RGS12 and treated with TNF-α for 0, 15, 30, and 60 min. We found the deletion of RGS12 inhibited TNF-α stimulated the phosphorylation of NF-κB(p65) and pIκB (Figures 3H–3J). Moreover, forced expression of RGS12 upregulated pNF-κB(p65) expression but had no effect on NF-κB(p65) mRNA and total protein levels (Figures S3A and S3B). These findings suggest that RGS12 plays an essential role in inflammation-stimulated activation of NF-κB(p65) and IκB.

Figure 3

RGS12 Is Essential for the Phosphorylation of NF-κB(p65) and IκBα

(A) Left: Immunofluorescence staining for RGS12 expression in BMMs treated with different doses of TNF-α (0, 5, and 10 ng/mL) and LPS (0, 0.5, and 1 μg/mL). Scale bar, 50 μm. Representative individual and overlaid images are shown.

(B) Quantification of RGS12 expression in (A), measured by the ImageJ densitometry method. Data are presented as the mean ± SEM. ∗∗∗p < 0.001 versus the control group, n = 5.

(C) TNF-α and LPS induce nuclear translocation of RGS12 in BMMs as depicted in (A). Quantitative data showed the subcellular location of RGS12. Gray, nucleus; blue, cytoplasm and nucleus; red, cytoplasm.

(D) Protein expression levels of RGS12, pNF-κB (Phospho-NF-κB p65 (Ser536)), NF-κB(p65), pIκBα (Phospho-IκBα (Ser32)), IκBα, and β-Actin following treatment of TNF-α with different doses (0, 5, 10, and 20 ng/mL) in RAW264.7 cells for 60 min.

(E) Blots underwent semi-quantitative analysis of RGS12, pNF-κB(p65), and pIκBα in (D) (n = 3). Data are the mean ± SEM. ∗∗p < 0.01 versus TNF-α untreated group.

(F) Protein expression levels of RGS12, pNF-κB(p65), NF-κB(p65), pIκBα, IκBα, and β-Actin following treatment of TNF-α with 10 ng/mL in RAW264.7 cells for 0, 15, 30, and 60 min.

(G) Quantitative analysis of RGS12, pNF-κB(p65), and pIκBα in (F) (n = 3). Data are mean ± SEM. ∗∗p < 0.01 versus TNF-α untreated group.

(H) RGS12 deficiency in macrophages inhibits pNF-κB(p65) and pIκBα expression in TNF-α-mediated inflammation in vitro. The BMMs were isolated from LysMCre; RGS12 and RGS12 mice and then incubated with TNF-α (10 ng/mL) for 0, 15, 30, and 60 min (see also Figure S6).

(I) The expression of pNF-κB(p65) in (H) was analyzed by western blots. NF-κB(p65) as an internal control. Note that the loss of RGS12 decreases the pNF-κB(p65) after TNF-α treatment. Data are mean ± SEM. ∗∗p < 0.01 versus RGS12 group. The representative of three experiments.

(J) The expression of pIκBα in (H) was analyzed by western blots. IκBα as an internal control. Note that the loss of RGS12 decreases the pIκBα expression after TNF-α treatment. Data are mean ± SEM. ∗∗p < 0.01 versus RGS12 group, (n = 3).

RGS12 Associates with NF-κB(p65) to Promote NF-κB(p65) Phosphorylation and Nuclear Translocation

To further identify the relationship between RGS12 and NF-κB(p65), we performed co-immunoprecipitation (Co-IP) and found that RGS12 associated with NF-κB(p65) in RAW 264.7 cells (Figure 4A). RGS12 contains six functional domains (PDZ, PTB, RGS, RBD1, RBD2, and GoLoco) (Figure 4B). To further determine which RGS12 domain affects the association with NF-κB (p65), we created six RGS12 mutations by deleting the specific functional domain as shown in Figure 4B. We found that the deletion of the RGS12 PTB domain impaired the association between RGS12 and NF-κB(p65) (Figure 4C), demonstrating that PTB domain promotes the association of RGS12 and NF-κB(p65). Moreover, to explore whether RGS12 regulates NF-κB(p65) nuclear translocation during inflammation, RAW264.7 cells were treated with LPS at different time points (0, 30 and 60 min). Interestingly, we found that RGS12 co-localized with NF-κB(p65), and both proteins were translocated to the nucleus within 60 min following LPS stimulation (Figures 4D and 4E). To further characterize the function of the PTB domain, stable cells that express RGS12 or RGS12 were treated with LPS. The loss of the PTB domain reduced phosphorylation (Figure 4F) and nuclear translocation of NF-κB(p65) (Figure 4G). Consistently, overexpression of the RGS12 PTB domain in RAW264.7 cell increased the level of pNF-κB(p65) (Figure S4) but not the total NF-κB(p65), suggesting that RGS12 regulates NF-κB activation through its PTB domain.

Figure 4

RGS12 Associates with NF-κB(p65) to Promote NF-κB(p65) Phosphorylation and Nuclear Translocation

(A) RGS12 interacts with NF-κB(p65) in RAW264.7 cells (Co-IP assay). The cell lysates were incubated with anti-RGS12 or control immunoglobulin G (IgG) antibodies, and the bound protein was examined by western blotting (WB) with the corresponding antibodies.

(B) Schematic diagram of RGS12 domain mutations. Functional domains are shown in the top. FLAG-tagged RGS12 and FLAG-tagged RGS12 domain mutations (lack of one functional domain) are constructed into the p3xFLAG-Myc-CMV-26 vector.

(C) RGS12 PTB domain is required for the interaction with NF-κB(p65). RAW264.7 cells were co-transfected with a series of expression vectors as described in the method section. NF-κB(p65) protein was immunoprecipitated by FLAG antibody, and abundance of coimmunoprecipitated proteins was determined by western blotting using anti-NF-κB(p65) or anti-FLAG antibodies.

(D) Co-localization of RGS12 (green) and NF-κB(p65) (red) in RAW264.7 cells treated with LPS (1 μg/mL) across different time points (0, 30, and 60 min). The merged images showed co-localization (yellow to orange). Scale bar, 20 μm. Representative individual and overlaid images are shown.

(E) Quantitation of the subcellular distribution of RGS12 (left) and NF-κB(p65) (right). The subcellular localization was categorized as nuclear (gray), cytoplasmic and nuclear (blue), and cytoplasmic (red).

(F) Left panel: the expressions of pNF-κB(p65) and pIκBα in RAW264.7 cells with and without RGS12 or RGS12 overexpression (OE) or LPS treatment. Western blotting was performed to analyze the expression of pNF-κB(p65) and pIκBα in RAW264.7 cells. RAW264.7 cells were transfected with vector plasmid DNA (p3xFLAG-Myc-CMV-26) (3 μg) and RGS12 (3 μg) or RGS12 plasmid (3 μg) for 48 h, respectively, and cells of LPS groups were treated with LPS (1 μg/mL) for 1 h before harvest. Right panel: Densitometric evaluation of pNF-κB(p65) and pIκBα in RAW264.7 cells transfected with RGS12 or RGS12 and exposed to LPS (n = 3). ∗p < 0.05, ∗∗p < 0.01, Values are means ± SEM. Data are representative of three separate experiments. Note that RGS12 without the PTB domain cannot increase the pNF-κB(p65) and pIκBα expression (see also Figure S4).

(G) The lack of a functional PTB domain in RGS12 inhibits nuclear translocation of RGS12. The cells were transiently transfected with construct encoding either RGS12 (top panels) or FLAG-tagged RGS12 (bottom panels) for 48 h and treated with LPS (1μg/mL) for 60 min. Scale bar, 10 μm.

COX2 Promotes the Nuclear Translocation of NF-κB(p65) and RGS12 in Macrophages

Previous studies have reported that COX2 expression increases in human RA and COX2 regulates NF-κB activity (Rao et al., 2005, Tipton et al., 2007). We found that LPS increases both RGS12 and COX2 expression in a dose-dependent manner (Figure S5). To determine whether COX2 affects RGS12/NF-κB(p65) signaling in RAW264.7 cells, we performed IP and found both RGS12 and NF-κB(p65) associated with COX2 (Figures 5A and 5B). Upon LPS treatment, COX2 co-localized with RGS12 and NF-κB(p65) in the exterior of the nucleus (Figure 5C). To further explore the relationship between COX2 and RGS12/NF-κB(p65), we overexpressed COX2 in macrophages and found that overexpression of COX2 enhanced the nuclear translocation of RGS12 and NF-κB(p65) in a dose-dependent manner (Figures 5D and 5E). This result was further confirmed by immunofluorescence staining results (Figures 5G and 5H). Moreover, we found that COX2 promoted the association of RGS12 and NF-κB(p65) (Figure 5F). In contrast, treatment of RAW264.7 cells with the different concentrations (0, 5, and 10μM) of COX2 inhibitor celecoxib and LPS (1μg/mL) decreased the association of RGS12 and NF-κB(p65) (Figure 5I) and downregulated the expression of RGS12, TNFα, and IL6 (Figure 5J). Furthermore, we found that RGS12 overexpression increased PGE2 level, which was inhibited by the COX2 inhibitor celecoxib (Figure 5K). To further explore whether PGE2 also regulates RGS12 expression, we treated RAW264.7 cells with PGE2 in different doses (0, 5, 10, and 20 nM) and at different times (0, 6, 12, and 24 h) (Figure 5L). We found that PGE2 significantly promotes the expression of RGS12 in a dose- and time-dependent manner (Figure 5L). Moreover, blocking one of the PGE2 receptors, EP4 significantly impaired the PGE2-mediated increase of the RGS12 expression as well as nuclear translocation (Figures 5M and 5N). Blocking EP2 and EP3 antagonists minorly inhibited the RGS12 level and nuclear translocation, whereas EP1 antagonist did not affect the RGS12 expression (Figures 5M and 5N). Thus, our results demonstrated that COX2 may regulate RGS12 mainly through the PGE2-EP4 pathway.

Figure 5

COX2 Promotes NF-κB(p65) and RGS12 Nuclear Translocation in Macrophages

(A and B) COX2 interacts with RGS12 and NF-κB(p65) in macrophages. The RAW264.7 cells were co-transfected with COX2/RGS12 (A) or COX2/NF-κB(p65) (B), respectively. Cell lysates of macrophages were incubated with anti-RGS12, anti-NF-κB(p65), or control immunoglobulin G (IgG) antibodies, and bound protein was examined by western blotting (WB) with the corresponding antibodies.

(C) Co-localization of RGS12 (green), NF-κB(p65) (red), and COX2 (blue) in RAW264.7 cells treated with LPS (1 μg/mL) for 30 min. The merged images showed the co-localization (yellow to orange). Scale bar, 10 μm.

(D) Western blot analysis of nuclear translocation of RGS12 and NF-κB(p65) after RAW264.7 cells transfected with COX2at the indicated concentrations (0, 5, 15, and 20 μg) for 24 h.

(E) Densitometric evaluation of RGS12 and NF-κB(p65) for nuclear translocation in RAW264.7 cells as shown in (D). ∗p < 0.05, ∗∗p < 0.01 versus COX2 untreated group. Data are representative of three separate experiments. Note that nuclear translocation of RGS12 and NF-κB(p65) are enhanced by COX2 overexpression.

(F) RAW264.7 cells were transfected with/without pCMV-COX2 (3 μg) and pcDNA3.1-RGS12-FLAG (3 μg). The cell extracts and the immunoprecipitates were analyzed by western blot. ∗p < 0.05, values are means ± SEM (n = 3).

(G) COX2-dependent nucleocytoplasmic shuttling of NF-κB(p65) and RGS12. RAW264.7 cells transiently transfected with control vector (pCMV) or pCMV-COX2 (3 μg) for 24 h. The cells were then fixed and incubated with the anti-NF-κB(p65) or anti-RGS12 antibody followed by incubation with fluorescein isothiocyanate-conjugated secondary antibody. Note that the transfection of COX2 constructs enhanced RGS12 and NF-κB(p65) nuclear translocation. Scale bar, 20 μm.

(H) Quantitative data on the subcellular localization of NF-κB(p65) (left) and RGS12 (right). Gray, nucleus; blue, cytoplasm/nucleus; red, cytoplasm.

(I) Ectopic RGS12 pulls down endogenous NF-κB(p65). RAW264.7 cells were transfected with RGS12-FLAG plasmid (10 μg) for 24 h and treated with LPS (1 μg/mL) and celecoxib (COX2 inhibitor) at the indicated concentrations (0, 5, and 10 μM) for 24 h. Cell lysates were immunoprecipitated with anti-FLAG antibody and analyzed for NF-κB(p65) by western blot. ∗∗∗p < 0.001 and ∗∗p < 0.01, values are means ± SEM (n = 3).

(J) The mRNA expression of RGS12, TNFα, and IL6 after treating with indicated concentration of celecoxib (0, 5, and 10 μM). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, mean values are shown unless otherwise specified, and error bars represent ±SEM (n = 5).

(K) The RAW264.7 cells were forced overexpression of COX2, RGS12 and treated with/without celecoxib (10 μM) for 24 h for detecting PGE2 level. ∗∗p < 0.01, ∗∗∗p < 0.001 versus the control group (n = 5).

(L) mRNA expression levels of RGS12 following treatment of PGE2 with different doses (0, 5, 10, and 20 ng/mL) (left) and time (0, 6, 12, and 24 h) (right) were determined. Data are mean ± SEM. ∗∗p < 0.01 versus PGE2 untreated group (n = 3).

(M) mRNA expression levels of RGS12 following treatment of PGE2 (20 ng/mL) for 24 h without/with different EP antagonists (EP1, 17-phenyl trinor Prostaglandin E2 [10 μM]; EP2 and EP3, AH 6809 [10 μM]; EP4, AH 23848 [10 μM]). Data are mean ± SEM. ∗∗p < 0.01 versus PGE2 untreated group (n = 3).

(N) Nuclear translocation of RGS12 following treatment of PGE2 (20 ng/mL) for 24 h without/with different EP antagonists as described in (J). Note that EP4 antagonist (10 μM) inhibits nuclear translocation of RGS12 induced by PGE2 (20 ng/mL).

NF-κB(p65) Transcriptionally Regulates RGS12 Expression in Macrophages

We first tested whether the inhibition of NF-κB affects RGS12 expression level in the LPS-treated RAW264.7 cells. As expected, inhibition of NF-κB(p65) using inhibitor JSH23 reduced the expression levels of RGS12 and COX2 (Figures 6A–6C). To investigate the transcriptional regulation role of NF-κB(p65), we overexpressed or silenced NF-κB(p65) in RAW264.7 cells. The results showed that overexpression of NF-κB(p65) increased RGS12 expression; in contrast, the silence of NF-κB(p65) decreased the expression of RGS12 (Figures 6D and 6E). To further test whether RGS12 acts as a transcriptional target of NF-κB(p65), 293T cells were co-transfected with the RGS12-luciferase reporter vector and different concentrations of NF-κB(p65) vectors. The results showed that NF-κB(p65) promoted RGS12 expression in 293T cells in a dose-dependent manner (Figure 6F). We then analyzed the sequences of RGS12 and found the κB-binding motifs (GGGGCTTCC) locate in the RGS12 promoter region. To determine whether NF-κB(p65) binds to the κB motif of the RGS12 promoter region, chromatin immunoprecipitation (ChIP) assay was performed in SMs isolated from wild-type (WT) mice without or with RA. We found that NF-κB(p65) directly bound to this κB motif (GGGGCTTCC) and that the binding level was increased in RA mice compared with the control (Figure 6G). This enhanced binding was also found in the TNF-α (Figure 6H), IL1 (Figure 6I), and LPS (Figure 6J)-induced RAW264.7 cells.

Figure 6

NF-κB Transcriptionally Regulates RGS12 Expression in Macrophages

(A) RGS12 and COX2 mRNA levels in RAW264.7 cells treated with LPS (1 μg/mL) and JSH23 (NF-κB inhibitor) at indicated concentrations (0, 10, and 20 μM) for 24 h ∗∗p < 0.01, ∗∗∗p < 0.001 versus the control group (n = 5).

(B) Western blot analysis of RGS12 and COX2 expression profile in RAW264.7 cells treated by LPS (1 μg/mL) and JSH23 at indicated concentration (0, 10, and 20 μM) for 24h.

(C) Quantitation of RGS12 and COX2 protein levels as shown in (B). The data are normalized to β-Actin. ∗p < 0.05, ∗∗p < 0.01 versus the control group (n = 3).

(D) The relative gene expression levels showing that overexpression of NF-κB(p65) (NF-κB(p65) OE) increased NF-κB(p65) and RGS12 levels, NF-κB(p65) shRNA decreased the expression of NF-κB(p65) and RGS12. The t test showed significant differences between the two groups, ∗∗∗p < 0.001 versus control (n = 5).

(E) Immunoblot showed the NF-κB(p65) and RGS12 protein level in NF-κB(p65) overexpression (NF-κB(p65) OE) or NF-κB(p65) knockdown (NF-κB(p65) shRNA) condition. Quantitative data showed the relative levels of RGS12 (quantified with NIH ImageJ software). A t test showed significant differences between the two groups, ∗p < 0.05, ∗∗p < 0.01 versus Control. Data are representative of three separate experiments.

(F) 293T cells were co-transfected with the RGS12-LUC reporter (1 μg) and combinations of plasmids expressing NF-κB(p65) (1, 3, and 5 μg) for 24 h. The overexpression of NF-κB(p65) results in higher RGS12 activation (p < 0.001).

(G) ChIP assays from Mock and RA SMs showing RGS12 and IgG control at κB site. ChIP was analyzed by quantitative PCR. A t test showed significant differences between the two groups, ∗∗p < 0.01, ∗∗∗p < 0.001 versus Control (n = 5).

(H–J) ChIP assays from RAW264.7 cells showing NF-κB(p65) at RGS12 κB site after introduction of control PBS or TNF-α (10 ng/mL) (H), IL-1(10 ng/mL) (I), and LPS (1 μg/mL) (J). ∗∗p < 0.01, ∗∗∗p < 0.001 versus the control group (n = 5).

Treatment with Nanoparticles Containing RGS12 shRNA Blocks the Progression of Chronic Arthritis in Mice

To determine whether inhibition of RGS12 can block the inflammatory response in macrophages, we constructed RGS12 shRNA silence vectors (psi-nU6.1-shRGS12), transfected shRGS12 into RAW264.7 cells, and then treated them with LPS. The silence of RGS12 inhibited transcript and protein expression of enzymes or inflammatory factors such as COX2, TNF-α, and IL-6 in vitro (Figures 7A and 7B). To determine whether shRGS12 can inhibit inflammatory responses through the suppression of NF-κB(p65) nuclear translocation, we transfected the shRGS12 plasmid into BMMs with or without LPS treatment. The result showed that the silence of RGS12 impaired NF-κB(p65) nuclear translocation during the inflammatory state (Figure 7C). We next sought to examine the therapeutic efficacy of shRGS12 on inhibiting RA progression. By delivering shRGS12-nanoparticles to mice with established mild arthritis (clinical score 8–10) every 3 days for 14 days (Figure 7D), we found that shRGS12-nanoparticles reduced RGS12 protein levels in control and RA groups (Figure 7E). Treatment of RA mice with shRGS12-nanoparticles for 14 days resulted in the elimination of visual signs of arthritis (Figure 7F), a significant reduction of arthritis severity score and paw swelling, (Figure 7G) and reduced inflammatory cell infiltration in the synovium (Figure 7H). Taken together, these data demonstrated that inhibition or silence of RGS12 is a promising strategy for the treatment of inflammatory arthritis.

Figure 7

Treatment with Nanoparticles Containing RGS12 shRNA Blocks the Progression of Chronic Arthritis in Mice

(A) The mRNA expression of RGS12, COX2, IL6, and TNFα in RAW264.7 cells after treating with LPS (1 μg/mL) and shRGS12 at indicated concentrations (0, 1.5, and 3 μg) were evaluated by qPCR. Data were analyzed by unpaired t test, and asterisks on the graphs indicate p values of significance, ∗p < 0.05, ∗∗∗p < 0.001.

(B) Left: western blot analysis for RGS12, TNF-α, COX2, and IL-6 expression in BMMs transfected with shRGS12 (3 μg) for 24 h and treated with or without LPS (1 μg/mL) for 24 h. Right: densitometric evaluation of RGS12, TNF-α, COX2, and IL-6. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus shControl group. Data are representative of three separate experiments.

(C) Left panel: BMMs were transfected without or with shRGS12 (3 μg) for 48 h and treated with LPS (1 μg/mL) for 24 h. Note that the transfection of shRGS12 inhibits NF-κB(p65) nuclear translocation. Right panel: quantitative data on the subcellular localization of NF-κB(p65). Scale bar, 50 μm. Representative individual and overlaid images are shown.

(D) Graphical representation of the experimental strategy. CIA was induced in wild-type mice (C57BL/6) by subcutaneous injection of chicken type II collagen in enriched CFA and subsequent challenge with chicken type II collagen in incomplete Freund's adjuvant (IFA) on day 21. After the first immunization for 30 days, the mild RA mice (clinical score 8–10) received 10 μg of shRGS12 nanoparticles or shControl nanoparticles into both the ankle joint cavities every 3 days during a 14-day course of therapy.

(E) Left: western blot analysis of RGS12 expression in synovial tissue from RA + shControl and RA + shRGS12 treatment mice. Right: quantitation of RGS12 protein levels. The data are normalized to β-Actin. ∗∗p < 0.01 versus the control group (n = 3).

(F) Photographs showing the paw swelling in RA + shControl and RA + shRGS12 nanoparticles-treated mice (n = 5/group).

(G) Clinical arthritis scores and paw thickness in the indicated groups. Data are presented as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 versus the control.

(H) H&E staining shows a decrease in the local recruitment of inflammatory cells within synovial tissue (black arrows) in the shRGS12 treatment group (B, bone area). ∗∗p < 0.01, n = 5. Scale bar, 200 μm.

Discussion

For the first time, we found a critical role of RGS12 in regulating inflammation through activating a RGS12-NF-κB feedback loop (Figure 8). The major findings of this study are: first, LPS, TNF-α, and PGE2 associate with their receptors such as TNFR/TLR and EP receptors to trigger the RGS12 expression; second, the increased RGS12 promotes the phosphorylation of NF-κB and IκB, IκB proteolysis, and nuclear translocation of NF-κB; third, deletion or silence of RGS12 in macrophages inhibits the release of pro-inflammatory cytokines in RA, through the reduced activation of NF-κB(p65); fourth, PTB domain promotes the association of RGS12 and NF-κB(p65), and NF-κB(p65) inversely regulates the transcription of the RGS12 gene, which forms an auto-regulatory feedback loop; last but not least, COX2 enhances nuclear translocation of RGS12/NF-κB(p65) in inflammatory conditions. Thus, RGS12 is a critical NF-κB activator in the pathogenesis of inflammatory arthritis.

Figure 8

Proposed Model of RGS12 Regulation of Inflammation via Activating a RGS12-NF-κB Positive Feedback Loop

First, TNF-α and LPS or PGE2 associate with their receptors to promote the RGS12 expression. RGS12 associates with NF-κB(p65) to activate its phosphorylation and nuclear translocation of both proteins, which can be enhanced by association with COX2 on the nuclear membrane. Inhibition of COX2 by celecoxib reduces the RGS12/NF-κB(p65) complex association and inhibits their activation. Then, nuclear NF-κB(p65) increases the expression of inflammatory cytokines such as IL1, IL6, and TNFα, as well as enhances the expression of RGS12 and COX2 through transcriptional activation. Meanwhile, enhanced RGS12 further activates the phosphorylation and nuclear translocation of NF-κB(p65) in the cytoplasm, which forms a positive feedback loop. In addition, enhanced COX2 further increases the RGS12/NF-κB(p65) nuclear translocation and increases PGE2 production. PGE2 then promotes RGS12 activation through binding the membrane receptor EP4.

Macrophages are one of the most abundant cell types in the RA synovium and are critical drivers in the progression of RA (Sack et al., 1994). Interestingly, after analyzing the GEO data (Database: GSE49604) from control subjects and patients with RA (You et al., 2014), we found that the expression of the longest isoform of RGS12 (isoform 2, GenBank: NM_002926.3), which contains PDZ-PTB domains and specialized C terminus, showed a more significant change in RA macrophages (Figure 2). Similarly, our real-time PCR data showed the long RGS12 isoforms (isoform 1 and 2) with the PTB domain significantly increased in RA synovium. The longest isoform 2 was increased the most in RA, which may be due to the specialized C terminus enhancing its function. However, RGS12 isoform 3 (short isoform) without the PTB domain did not show any significant change (Figure 1C), suggesting that isoform 3 may not be able to regulate inflammation in RA. Additionally, these findings were consistent with our results showing that the RGS12 longest isoform was highly expressed in synovial RA macrophages in human RA samples and CIA mouse models (Figures 1C, 1D, and 1J–1L).

Our results showed that global deletion of RGS12 alleviated the RA phenotype in mice (Figure 2), which was replicated in the RGS12 conditional knockout model targeted to monocytes/macrophages, indicating that RGS12 plays an important role in macrophages of RA. However, besides macrophages, T cells, B cells, and the orchestrated interaction of pro-inflammatory cytokines also play important roles in the pathophysiology of RA (Matsumoto et al., 1999). Whether RGS12 plays a role in T/B cells or other cells needs to be further studied. Although the DBA/1 strain was found to be more susceptible to collagen type II arthritis and developed more severe arthritis than the C57BL/6 strain, the C57BL/6 strain was also suitable for RA research (Inglis et al., 2007). Our results showed that chicken type II collagen method was able to induce arthritis in C57BL/6 mice with an average incidence of 53.8% on day 40 after primary immunization, which was able to replicate the typical pathological process of RA. Those results were supported by other reports showing that chicken type II collagen could induce arthritis in C57BL/6 mice (Asquith et al., 2011, Chrobak et al., 2015, Park et al., 2015), with a maximum incidence of 61.7% and mean day of onset of 29.4 ± 1.3 days after primary immunization (Inglis et al., 2007).

RA is a complex immune system disease that is regulated by multiple factors. We observed that RGS12 expression in SMs increased during joint inflammation (Figure 2), which contributes to the enhanced nuclear translocation and activity of NF-κB (Figure 3). It has been proven that RGS12 directly interacts with the tyrosine-phosphorylated calcium channel through its PTB domain in neuronal cells (Schiff et al., 2000). Interestingly, the PTB domain of RGS12 is unique, which does not contain any of the conserved Arg or Lys residues required by Shc (Src homology 2 domain containing) or IRS (Insulin receptor substrate) for phosphotyrosine coordination (Uhlik et al., 2005). Most recently, Schroer et al. reported that the RGS12 PTB domain binds to the specific phosphoinositides and then is localized to early endosomes in cultured myoblasts (Schroer et al., 2019, Willard et al., 2007). Here, we found that the PTB domain is essential for association of RGS12 and NF-κB(p65) to further promote the NF-κB(p65) activation. NF-κB contains different phosphorylation sites, which can be affected by various kinases. For example, c-Src and Lck are involved in the tyrosine phosphorylations of IκB-α and p65 NF-κB under silica induction (Kang et al., 2006). Phosphorylation of NF-κB is regulated by BDNF via its receptor tyrosine kinase TrkB (Gavalda et al., 2009). Thus, it is possible that the PTB domain of RGS12 can promote phosphorylation of NF-κB via enhancing the activity of a certain kinase. Meanwhile, the RGS12 PTB domain may promote NF-κB to expose more phosphorylation sites by binding and changing the spatial structure. Although the precise mechanism needs to be further defined, our results suggest that the PTB domain of RGS12 may be a potential therapeutic target for inflammation. Importantly, NF-κB activation and its downstream inflammatory response can be inhibited by the introduction of shRGS12 both in vivo and in vitro, suggesting inhibition of RGS12 is a promising new therapeutic strategy for RA or other inflammatory diseases.

COX2 is a key intracellular enzyme in RA (Rao et al., 2005), which is monotopically inserted in the nuclear membrane to take up released arachidonic acid (Funk, 2001) and produce PGE2. Our results showed that COX2 acts as a chaperone to translocate the RGS12/NF-κB complex from the cytoplasm to the nucleus, suggesting COX2 plays an important role in RGS12/NF-κB signaling transduction. PGE2 acts in an autocrine and paracrine manner via a family of GPCR receptors termed EP1–EP4. However, whether RGS12 regulates Eps-mediated signaling regulation is unknown. Some studies have reported that EP1 regulates Ca2+ mobilization via Gαq and EP3 inhibits adenylyl cyclase via Gαi (Goulet et al., 2004). EP2 and EP4 regulate intracellular adenosine-3′,5′-cyclic monophosphate (cAMP) through Gαs. Moreover, EP4 associates with a pertussis toxin-sensitive inhibitory G-protein, Gαi (Fujino and Regan, 2006). In our study, we found that PGE2 can increase in RGS12 expression and NF-κB(p65) nuclear translocation and activation through EP4. Moreover, activated NF-κB(p65) further promotes the production of RGS12 and COX2/PGE2. Thus, these findings suggest that COX2-mediated PGE2/EP4 signaling and RGS12/NF-κB(p65) signaling may form a positive feedback regulation loop for inflammatory regulation (Figure 8).

In conclusion, our work provides strong evidence and critical mechanistic insights into a key role of RGS12 in inflammation. These findings will enable future studies to facilitate the design of clinical treatments for difficult-to-treat inflammatory diseases such as RA.

Limitations of the Study

Our studies were mainly performed on mouse models. Although mouse models are heavily used in preclinical studies, the onset time, intensity, and pathophysiological characteristics of rheumatoid arthritis are different from human clinical conditions. We refined our methods and mouse models to closely reflect human conditions; however, the differences in mouse anatomy and physiology may potentially limit the translational value of our conclusions. In addition, our study was limited only to the long isoforms of RGS12 on mouse models; therefore, further clinical trials are needed to validate our findings acquired from mouse models.

Resource Availability

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Shuying Yang (shuyingy@upenn.edu).

Materials Availability

This study did not generate new unique reagents.

Data and Code Availability

The whole-genome microarray data were obtained using accession numbers GSE49604 at Gene Expression Omnibus (GEO).

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.