Protein Inhibitor of Activated STAT3 Regulates Migration, Invasion, and Activation of Fibroblast-like Synoviocytes in Rheumatoid Arthritis

Minxi Lao, Maohua Shi, Yaoyao Zou, Mingcheng Huang, Yujin Ye, Qian Qiu, Youjun Xiao, Shan Zeng, Liuqin Liang, Xiuyan Yang and Hanshi Xu
J Immunol January 15, 2016, 196 (2) 596-606; DOI: https://doi.org/10.4049/jimmunol.1403254

Abstract

The aggressive phenotype displayed by fibroblast-like synoviocytes (FLSs) is a critical factor of cartilage destruction in rheumatoid arthritis (RA). Increased FLSs migration and subsequent degradation of the extracellular matrix are essential to the pathology of RA. Protein inhibitor of activated STAT (PIAS), whose family members include PIAS1, PIAS2 (PIASx), PIAS3, and PIAS4 (PIASy), play important roles in regulating various cellular events, such as cell survival, migration, and signal transduction in many cell types. However, whether PIAS proteins have a role in the pathogenesis of RA is unclear. In this study, we evaluated the role of PIAS proteins in FLSs migration, invasion, and matrix metalloproteinases (MMPs) expression in RA. We observed increased expression of PIAS3, but not PIAS1, PIAS2, or PIAS4, in FLSs and synovial tissues from patients with RA. We found that PIAS3 knockdown by short hairpin RNA reduced migration, invasion, and MMP-3, MMP-9, and MMP-13 expression in FLSs. In addition, we demonstrated that PIAS3 regulated lamellipodium formation during cell migration. To gain insight into molecular mechanisms, we evaluated the effect of PIAS3 knockdown on Rac1/PAK1 and JNK activation. Our results indicated that PIAS3-mediated SUMOylation of Rac1 controlled its activation and modulated the Rac1 downstream activity of PAK1 and JNK. Furthermore, inhibition of Rac1, PAK1, or JNK decreased migration and invasion of RA FLSs. Thus, our observations suggest that PIAS3 suppression may be protective against joint destruction in RA by regulating synoviocyte migration, invasion, and activation.

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by progressive destruction of cartilage and bone (1). Fibroblast-like synoviocytes (FLSs) in the synovial intimal lining play a key role in the progress of joint destruction (2). Stable activated FLSs in RA exhibit tumor-like characteristics, such as anchorage-independent growth and resistance to apoptosis (3, 4), and are essential in the development of pannus by migration and invasion toward cartilage and bone (5, 6). Increasing evidence suggests that modulation of activated FLS migration and invasion may be a novel therapeutic strategy targeting the destructive progress of RA (7). However, the precise molecular mechanisms regulating this pathogenic process are not clearly defined.

Cell migration is a complex cyclic process consisting of three major steps: the formation and protrusion of a leading lamellipodium, subsequent adhesion to the matrix, and finally tail retraction. These coordinated steps are controlled by multiple, integrated signal pathways from the extracellular environment and reorganization of the actin cytoskeleton (8–10). Mammalian protein inhibitor of activated STAT (PIAS), consisting of four members—PIAS1, PIAS2 (PIASx), PIAS3, and PIAS4 (PIASy)—play important roles in regulating many important cellular events (11). PIAS proteins contain four conserved structural domains and motifs: a SAP region for chromatin binding, RING (an interesting new gene) finger-like zinc-binding domain for E3-SUMO ligation, a SUMO-interacting motif for SUMO binding, and PINIT motif for localization (12). In addition to cell proliferation, apoptosis, and signal transduction (13–15), PIAS proteins also regulate cell migration and invasion of some cell lines. For example, PIASy enhances epithelial cell migration through alteration of C/EBPδ nuclear localization and reduction of C/EBPδ transcriptional activity (16). PIAS3 controls migration by SUMOylation of Rac1 (17). Although previous reports have shown the role of SUMO in synovial proliferation and inflammation of RA (18, 19), it is still unclear whether PIAS proteins are involved in the pathogenic FLSs behavior observed in RA.

In the current study, we evaluated whether PIAS was involved in FLS motility, and we demonstrated that PIAS3 knockdown by short hairpin RNA (shRNA) reduced migration and invasion of FLSs and matrix metalloproteinase (MMP) expression. We also showed that PIAS3-mediated SUMOylation of Rac1 regulated its GTP-bound levels and its downstream PAK1 and JNK activity. Thus, our results indicate that increased synovial PIAS3 protein expression contributes to the aggressive behavior of RA FLSs.

Materials and Methods

Reagents and Abs

Recombinant human TNF-α and IL-1β were obtained from R&D Systems (Minneapolis, MN). DMEM/F12, FBS, antibiotics, trypsin EDTA, PBS and other standard cell culture products were obtained from Invitrogen (Carlsbad, CA). Anti-PIAS1, anti-PIAS2, anti-PIAS3, anti-PIAS4, anti–SUMO-1, anti–SUMO-2/3, anti-Rac1, and anti–β-actin Abs were purchased from Abcam (Cambridge, UK). Anti-PAK1, anti–phospho-PAK1, anti-JNK, anti–phospho-JNK Abs were purchased from Cell Signaling Technology (Beverly, MA). Rac1 inhibitor, NSC23766, was purchased from Selleck (Houston, TX). PAK1 inhibitor, IPA-3, and JNK inhibitor, SP600125, were purchased from Sigma (St. Louis, MO).

Preparation of human synovial tissues and FLSs

The study was performed according to the recommendations of the Declaration of Helsinki and approved by the Medical Ethical Committee of the First Affiliated Hospital, Sun Yat-sen University, China. All patients gave informed consent to participate in the study. Synovial tissues (STs) were obtained from patients with RA (8 women and 2 men, 40–68 y old) who were undergoing synovectomy or joint replacement at the hospital. RA was diagnosed according to the 1987 revised criteria of the American College of Rheumatology (20). STs were cut into small pieces and digested with 1 mg/ml collagenase for 3 h at 37°C to isolate synoviocytes. All cells were cultured in DMEM/F12 with 10% FBS at 37°C and 5% CO2. In our experiments, cells were used from passages 4 to 6, during which time they were a homogeneous population of cells (<1% CD11b positive, <1% phagocytic, and <1% FcgRII and FcgRIII receptor positive).

Construction and infection of shRNA-expression lentivirus

Lentivirus-based shRNA vectors were constructed as described previously (21). The sequences of these shRNA oligonucleotides are listed as follows: PIAS3-1, 5′-GGAGCCAAATGTGATTATA-3′; PIAS3-2, 5′-CATCCAAGGTTTAGATTTA-3′; PIAS3-3, 5′-CAAGAAGGCTCCCTATGAA-3′; Scramble (control shRNA), 5′-GACGATGATTCGTATGTAA-3′. The shRNA-expression lentiviral vectors were generated by cloning gene-specific or scramble shRNA sequences into pLKO.1 vectors, and lentiviruses were produced by cotransfection of HEK 293T cells with expression vectors and helper plasmids pCMV-dR8.2-vprX and pCMV-VSVG using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Lentiviral particles were harvested from cell supernatants 48 and 72 h after transfection. For infection, FLSs were treated with the lentiviruses and polybrene (10 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) for 12 h at 37°C. Afterwards, the virus-containing medium was replaced with fresh medium. Infected cells were selected by addition of puromycin (5 mg/ml) for 48 h postinfection and then propagated for further use. The effect of these shRNA on PIAS3 protein expression was examined using Western blot analysis.

Western blot analysis

Protein concentrations were measured by the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Equal amounts of protein were solubilized in Laemmli buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, and 0.00625% bromophenol blue), boiled for 5 min, and then separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with primary Abs as indicated in TBS-Tween 20 containing 5% nonfat milk at 4°C overnight. The membranes were incubated with the appropriate secondary Abs for 1 h at room temperature. Immunoreactive bands were visualized using ECL (Amersham Pharmacia, Piscataway, NJ). Each blot was a representative of at least three similar independent experiments.

Cell migration and invasion assay

Chemotaxis assay of FLS was performed by the Boyden chamber method using a filter with a 6.5-mm diameter and 8.0-μm pore size (Transwell; Corning, Corning, NY, USA). Briefly, DMEM containing TNF-α (10 ng/ml) as a chemoattractant was placed in the lower chambers. FLSs (at a final concentration of 6 × 104 cells/ml) were suspended in serum-free DMEM in the upper chambers. The plates were incubated at 37°C under 5% CO2 for 8 h. After incubation, the nonmigrating cells were gently removed from the upper surface of the filter using a cotton swab. The filters were fixed in methanol for 15 min and stained with 0.1% crystal violet for 15 min. Chemotaxis was quantified with Image J software by counting the stained cells that migrated to the underside of the filter using an optical microscope (magnification ×100). The stained cells were counted as the mean number of cells per 10 random fields for each assay. For the in vitro invasion assay, similar experiments were performed using inserts coated with a Matrigel basement membrane matrix (BD Biosciences, Oxford, UK).

Wounding migration

RA FLSs, plated to confluence on 35-mm culture dishes, were wounded with 1-ml sterile micropipette tips and then washed three times with starving media to remove detached cells. The remaining cells were treated with or without TNF-α (10 ng/ml). After 48 h of incubation, migration was quantified with Image J software by counting the cells that moved beyond a reference line.

Immunohistochemistry analysis

For immunohistochemistry (IHC) analysis, formalin-fixed, paraffin-embedded ST sections were deparaffinized in xylene and rehydrated through alcohol to water. Heat-mediated Ag retrieval was performed in Tris-EDTA buffer for 15 min. After cooling, slides were incubated with 5% serum in PBS for 2 h to block nonspecific binding and incubated with 3% H2O2 for 10 min to block endogenous peroxidase activity. The expression of PIAS3 was determined by staining with polyclonal rabbit anti-human PIAS3 Ab overnight at 4°C. Irrelevant isotype-matched Abs were used as negative controls. Polyclonal goat anti-rabbit Abs labeled with HRP were used as secondary Abs and incubated for 1 h at room temperature. Results were revealed using diaminobenzidine chromogen staining.

Immunofluorescence staining

RA FLSs, growing on glass coverslips at 90% confluence, were wounded with micropipette tips and then treated with 10% FBS. After 3 h of incubation, cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. The cells were incubated with anti-PIAS3 Ab (diluted 1:200) for 1 h at room temperature and then incubated with FITC-conjugated secondary Ab (Santa Cruz Biotechnology). For detection of pseudopodia organization, cells were incubated with Alexa Fluor-546 rhodamine-phalloidin (Molecular Probes; Invitrogen, Eugene, OR) and nuclei were visualized using 0.25 mg/ml DAPI. The coverslips were mounted on glass slides with antifade mounting media and examined using fluorescence microscopy.

Quantitative real-time PCR

Total RNA from RA FLSs were prepared by Takara PrimeScript RT reagent kit according to the manufacturer’s protocol. Quantitative real-time PCR was performed using the Bio-Rad CFX96 system. The following primers were used: MMP-1 Sense 5′-CTCTGGAGTAATGTCACACCTCT-3′, Antisense 5′-TGTTGGTCCACCTTTCATCTTC-3′; MMP-3 Sense 5′-TGTAAAGAAACCTTCCTGCAA-3′, Antisense 5′-TTTAAAACACAGTATGCCCAA-3′; MMP-9 Sense 5′-AGACCTGGGCAGATTCCAAAC-3′, Antisense 5′-CGGCAAGTCTTCCGAGTAGT-3′; MMP-13 Sense 5′-ACTGAGAGGCTCCGAGAAATG-3′, Antisense 5′-GAACCCCGCATCTTGGCTT-3′; and GAPDH Sense 5′-GCACCGTCAAGGCTGAGAAC-3′, Antisense 5′-TGGTGAAGACGCCAGTGGA-3′.

Gene expression was normalized to GAPDH as endogenous control, and data were analyzed using the ΔΔCT method. All experiments were performed in triplicate.

Measurement of MMPs production

The levels of MMP-3, MMP-9, and pro–MMP-13 secreted in culture media by TNF-α–stimulated RA FLSs were measured with ELISA (R&D Systems).

FLSs proliferation assays

RA FLSs were cultured for 24 h at a density of 1 × 104 cells/well in 96-well plates in serum-free medium. After starvation, the cells were incubated with TNF-α (10 ng/ml) for 72 h and then incubated with 10 mM BrdU for 1 h. BrdU incorporation was assessed in triplicate, using a cell proliferation ELISA kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions.

FLSs apoptosis assays

RA FLSs were cultured on glass coverslips to 90% confluency. Cell apoptosis assays were then performed using The DeadEnd Fluorometric TUNEL System (Promega, Madison, WI), according to the manufacturer’s instructions.

Measurement of Rac1 activity

RA FLSs were cultured for 24 h at a density of 1 × 105 cells/well in 35-mm culture dishes in serum-free medium. After starvation, the cells were incubated with TNF-α (10 ng/ml) for 10 min and then harvested for Rac1 activity detection, using a G-LISA Rac1 activation assay kit (Cytoskeleton, Denver, CO) according to the manufacturer’s instructions.

Immunoprecipitation assay

Immunoprecipitation (IP) was performed using Pierce Classical IP Kit (Thermo Fisher, Waltham, MA) according to the manufacturer’s instructions. Cell lysates were prepared in IP lysis buffer containing protease and phosphatase inhibitor cocktails. Equivalent amounts of protein were either denatured with SDS-PAGE sample buffer for Western blot or incubated with 2–5 μg specified Ab and 20 μl protein A/G-agarose beads for IP analysis. Immunoprecipitated proteins were eluted with SDS-PAGE loading buffer, resolved by SDS-PAGE, and transferred to nitrocellulose membranes for subsequent immunoblotting procedures.

Statistical analysis

Data were expressed as mean ± SEM. Student t test or ANOVA was used to evaluate differences between experimental groups. A p value ≤0.05 was considered significant.

Results

Elevated expression of PIAS3 in FLSs and STs from RA patients

The expression of PIAS proteins were determined by Western blot analysis. As shown in Fig.1A, PIAS3 expression was increased in FLSs from patients with RA compared with those with osteoarthritis (OA) patients and healthy controls; however, PIAS2 was expressed but did not exhibit significant difference among the three groups. Both PIAS1 and PIAS4 were expressed at low levels. We also observed subcellular distribution of PIAS3 in FLSs by immunofluorescence and found that RA FLSs exhibited a markedly enhanced staining for PIAS3, which was localized in both the nucleus and cytoplasm (Fig.1B). Furthermore, we detected PIAS3 protein expression in STs by using IHC. As shown in Fig.1C, PIAS3 protein expression was prominent in STs from RA patients and mostly localized in the synovial lining and sublining cells, whereas its expression was less prominent in STs from OA patients.

FIGURE 1.
FIGURE 1.

Increased expression of PIAS3 in FLSs and the STs from patients with RA. (A) Expression of PIAS1, 2, 3, and 4 was detected by Western blot in FLSs from RA patients, OA patients, and NCs. A representative blot of three independent experiments is shown. Data are expressed as mean ± SEM of densitometry quantification (lower panel) and presented as fold changes over controls after normalization by β-actin. *p < 0.05 versus NCs. (B) For cellular IF staining, PIAS3 (green) and nuclei (blue) were evaluated using confocal microscopy and representative pictures from three independent experiments were shown. Original magnification ×400. (C) Expression of PIAS3 assessed by IHC in STs isolated from RA and OA patients. Original magnification ×400. (D) Confluent FLSs were treated with TNF-α (10 ng/ml) or IL-1β (10 ng/ml) for 24 h, and expression of PIAS3 was measured with Western blot analysis. Data were expressed as mean ± SEM of densitometry quantification (lower panel) from three independent experiments. *p < 0.05 versus Basal.

Because proinflammatory cytokines, particularly TNF-α and IL-1β, play an important role in the transformation of RA FLSs to a more invasive phenotype, we examined the effect of these cytokines on the PIAS3 expression in confluent RA FLSs. As shown in Fig.1D, PIAS3 protein expression was upregulated in the cells treated with TNF-α (10 ng/ml) or IL-1β (10 ng/ml).

PIAS3 knockdown decreases migration of RA FLSs

To determine the role of PIAS3 in regulating directed migration, we examined chemotaxis migration of FLSs using a trans-well chamber assay in shRNA-mediated PIAS3-knockdown FLSs. To rule out nonspecific interference, we constructed three different sequences of shRNA oligonucleotides for PIAS3. As shown in Supplemental Fig. 1, transfection with all three shRNA oligonucleotides downregulated PIAS3 protein expression; however, the inhibitory effect of PIAS3 shRNA-3 was the most prominent. Accordingly, PIAS3 shRNA-3 (PIAS3 shRNA) was used for subsequent experiments. We found that transfection with PIAS3 shRNA-3 resulted in a decrease in the migration of RA FLSs as compared with scramble control (SC) toward chemoattractant TNF-α (Fig.2A). Furthermore, we used a monolayer wound scratch assay to detect the role of PIAS3 in cell migration. As expected, RA FLSs transfected with PIAS3 shRNA displayed significantly lower cell migration compared with scramble control (Fig. 2B). Interestingly, PIAS3 shRNA had no effect on the migration of OA FLSs or normal control (NC) FLSs in the chemotaxis and wound healing assays (Fig. 2A, 2B, respectively).

FIGURE 2.
FIGURE 2.

Targeted depletion of PIAS3 decreases migration of RA FLSs. (A) RA FLSs, NC FLSs, or OA FLSs transfected with PIAS3 shRNA (SPIAS3) (oligonucleotide no. 3) or control shRNA (SC) were serum starved overnight at 24 h. Chemotaxis migration was performed in a Boyden chamber. The RA FLSs, NC FLSs, or OA FLSs were seeded in a Boyden upper chamber, allowed to migrate for 8 h, fixed, and stained with crystal violet. TNF-α (10 ng/ml) was used as chemoattractant. The migration index represents the number of migrated cells normalized to TNF-α–containing media. Data are presented as mean ± SEM of five independent experiments. The images are representative of migration through the membrane after staining. Original magnification ×100. (B) Effect of PIAS3 shRNA on the wounding migration of RA FLSs, NC FLSs, or OA FLSs. Representative light microscopy images of RA FLSs, NC FLSs, or OA FLSs monolayers 48 h after wounding are shown. Original magnification ×50. Cells migrating beyond the reference line were imaged and counted. Data shown are representative of experiments from three different RA patients. *p < 0.05, **p < 0.01 versus control.

PIAS3 knockdown impairs invasion of RA FLSs

The ability to invade cartilage is a critical pathogenic behavior of RA FLSs. In vitro invasion potential of RA FLSs has previously been well related to the rate of joint destruction in patients with RA (22). Therefore, we evaluated the effect of PIAS3 knockdown on regulating invasive behavior of RA FLSs through Matrigel-coated trans-well membranes. As shown in Fig. 3A, transfection with PIAS3 shRNA decreased Matrigel invasion compared with transfection with control shRNA in RA FLSs. Consistent with its effect on migration, PIAS3 shRNA transfection did not affect the ability of invasion of either OA FLSs or NC FLSs (Fig. 3A).

FIGURE 3.
FIGURE 3.

Effect of PIAS3 knockdown on invasion and proliferation of RA FLSs. (A) In vitro invasion assay were performed using inserts coated with a Matrigel basement membrane matrix in Boyden chambers. Cells were allowed to invade through Matrigel toward media containing TNF-α (10 ng/ml) for 8 h. The number of invading cells was averaged from three ×10 field-of-view images, and invasion index was calculated by normalizing the mean of invaded cells to media containing TNF-α. Shown are representative images of stained cells that invaded through Matrigel invasion chambers. Data are presented as mean ± SEM of five independent experiments. Original magnification ×100. (B and C) Effect of PIAS3 knockdown on proliferation (B) and apoptosis (C) of RA FLSs. RA FLSs were stimulated with or without TNF-α for 72 h. Cell proliferation and apoptosis were measured by BrdU incorporation and TUNEL assay, respectively. Data are presented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 versus control.

A previous report indicated that PIAS3 participates in regulating proliferation of tumor cells (23); therefore, to rule out whether the inhibitory effect of PIAS3 knockdown on migration and invasion was associated with either proliferation or apoptosis, we examined the role of PIAS3 in proliferation and apoptosis of RA FLSs. Our results showed that PIAS3 was not involved in proliferation or apoptosis of RA FLSs (Fig. 3B, 3C), suggesting that the effect of PIAS3 knockdown on migration and invasion was not a function of aberrant proliferation or apoptosis.

PIAS3 knockdown impairs lamellipodia formation in RA FLSs

Dynamic reorganization of the actin cytoskeleton is critical for optimal cell migration. To evaluate the role of PIAS3 proteins in regulating actin reorganization in RA FLSs, we used fluorescent phalloidin staining to visualize polymerized actin in migrating cells shortly after wounding in PIAS3 shRNA or control shRNA-transfected RA FLSs. As shown in Fig. 4A, FLSs transfected with control shRNA displayed flat or ruffling lamellipodia at their leading edge; however, cells transfected with PIAS3 shRNA had significantly suppressed lamellipodia formation. Furthermore, we also observed that PIAS3 colocalized with Rac1 in ruffling lamellipodia at the leading edge of RA FLSs (Fig. 4B), suggesting that PIAS3 regulated the formation of membrane protrusions in migrating cells.

FIGURE 4.
FIGURE 4.

PIAS3 knockdown impairs lamellipodia formation in RA FLSs. (A) Effect of PIAS3 knockdown on lamellipodia formation in RA FLSs. Cells were plated overnight on coverslips, and then fixed and stained with fluorescent phalloidin 3 h after wounding to visualize polymerized actin in migrating cells. Arrows indicate lamellipodia formation. (B) Localization of PIAS3 (green) and Rac1 (red) in ruffling lamellipodia in migrating cells. PIAS3 was detected with a rabbit anti-PIAS3 Ab and visualized with Alexa Fluor 488 goat anti-rabbit Ab (green), and Rac1 was detected with a mouse anti-Rac1 Ab and visualized with Alexa Fluor 568 goat anti-mouse Ab (red). The results presented are representative of three independent experiments. Original magnification ×400.

PIAS3 knockdown inhibits MMPs expression

The aggressive phenotype of RA FLSs is characterized by the upregulated expression of MMPs. To determine the role of PIAS3 in regulating MMPs mRNA expression, RA FLSs transfected with PIAS3 shRNA or control shRNA were stimulated with TNF-α. As shown in Fig. 5A, transfection with PIAS3 shRNA markedly suppressed TNF-α–induced mRNA expression of MMP-3, MMP-9, and MMP-13, but not MMP-1, compared with control shRNA transfection. Next, we determined the inhibitory effect of PIAS3 knockdown on the secretion of MMPs, as measured by ELISA. As expected, PIAS3 shRNA transfection also reduced TNF-α–induced levels of MMP-3, MMP-9, and MMP-13 in culture supernatants (Fig. 5B–D).

FIGURE 5.
FIGURE 5.

PIAS3 knockdown reduces TNF-α–binduced expression of MMPs in RA FLSs. RA FLSs, transfected with control shRNA (SC) or PIAS3 shRNA (SPIAS3), were treated with TNF-α (10 ng/ml) for 24 h. (A) mRNA expression of MMP-1, MMP-3, MMP-9, and MMP-13 was determined with quantitative real-time PCR. Data were normalized to GAPDH. The levels of (B) MMP-3, (C) MMP-9, and (D) pro-MMP-13 in culture supernatants were measured with ELISA. The data expressed as mean ± SEM were repeated with RA FLSs from five patients. *p < 0.05 versus SC, #p < 0.05 versus SC plus TNF-α.

PIAS3 knockdown reduces TNF-α–induced Rac1 activation

The small guanosine triphosphatase (GTPase) Rac1 is a central regulator of cell motility. Activation of Rac1 promotes actin cytoskeletal reorganization at the leading edge, particularly, formation of lamellipodia (24, 25). Because Rac1 has been previously suggested as a possible target of PIAS3 (17), we evaluated the inhibitory effect of PIAS3 inhibition on activation of Rac1. As shown in Fig. 6A, compared with control shRNA, PIAS3 shRNA transfection inhibited TNF-α–induced activation of Rac1, but had no effect on the expression of total Rac1 protein. Furthermore, immunofluorescence double staining also showed that PIAS3 colocalized with Rac1 in ruffling lamellipodia at the leading edge of RA FLSs (Fig. 4B). These data imply that PIAS3 interacts with Rac1 at sites of membrane protrusion to promote lamellipodia formation to affect cell motility.

FIGURE 6.
FIGURE 6.

PIAS3 knockdown inhibits TNF-α-induced Rac1/PAK1 and JNK activation. RA FLSs were transfected with shRNAs specific for PIAS3 (SPIAS3) or control shRNA (SC). (A) Effect of PIAS3 knockdown on Rac1 activity. FLSs were serum starved for 24 h and stimulated with TNF-α for 10 min. Cells were lysed, and Rac1 activity was detected by G-LISA Rac1 activation assay. Rac1 protein was measured by Western blotting. Data are presented as mean ± SEM of five independent experiments. (B and C) Effect of PIAS3 knockdown on phosphorylation of (B) PAK1 and (C) JNK. Western blot analysis for p-PAK1, total-PAK, p-JNK, and total JNK following TNF-α stimulation is shown in cells transfected with PIAS3 shRNA or control shRNA. A representative blot of three independent experiments is shown. Data are shown as the ratio of arbitrary absorption units of p-PAK1 or p-JNK to total PAK1 or JNK (mean ± SEM; lower panel). (D and E) Effect of Rac1 inhibition on (D) PAK1 and (E) JNK phosphorylation. RA FLSs were preincubated with Rac1 inhibitor NSC23766 for 1 h prior to TNF-α stimulation. PAK1 and JNK phosphorylation was assessed with Western blot analysis. (F) Effect of PAK1 inhibition on JNK phosphorylation. RA FLSs were preincubated with PAK1 inhibitor IPA-3 for 1 h prior to TNF-α stimulation. A representative blot of three independent experiments is shown. Data are shown as the ratio of arbitrary absorption units of p-JNK to total JNK (mean ± SEM; lower panel). *p < 0.05 versus Basal, #p < 0.05 versus TNF-α stimulation.

PIAS3 knockdown decreases TNF-α–induced activation of PAK1 and JNK

Numerous studies showed that phosphorylated p21-activated kinase 1 (PAK1) is involved in cell migration (26–29). Because PAK1 is considered a key downstream effector of Rac1 signal pathway, we sought to determine the role of PIAS3 in PAK1 activation in RA FLSs. As shown in Fig. 6B, transfection with PIAS3 shRNA decreased TNF-α–induced phosphorylation of PAK1, but had no effect on the expression of total PAK1.

JNK also plays an important role in regulating cell migration (30), and Rac1 mediates IL-1β–induced activation of JNK (31). Therefore, we evaluated the role of PIAS3 in the activation of JNK. PIAS3 knockdown with shRNA markedly decreased TNF-α–induced JNK phosphorylation (Fig. 6C). We also found that specific Rac1 inhibitor (NSC23766) or PAK1 inhibitor (IPA-3) inhibited TNF-α–induced JNK phosphorylation (Fig. 6D), indicating that PAK1 mediated JNK activation by Rac1 in RA FLSs.

Inhibition of Rac1, PAK1, and JNK activity decreases migration and invasion of RA FLSs

To confirm whether activation of Rac1, PAK1, and JNK was required for PIAS3-mediated migration and invasion in RA FLS, we treated RA FLSs or OA FLSs with the pharmacologic Rac1 inhibitor NSC23766, PAK1 inhibitor IPA-3, or JNK inhibitor SP600125. We found that these specific inhibitors significantly decreased migration and invasion of RA FLS; however, migration and invasion of OA FLSs was not affected by treatment with these inhibitors (Fig. 7A, 7B).

FIGURE 7.
FIGURE 7.

Rac1, PAK1, or JNK inhibition reduces migration and invasion of RA FLSs. (A) Chemotaxis was evaluated using a Boyden chamber migration assay. RA FLSs or OA FLSs in serum-free condition in presence of Rac1 inhibitor (NSC23766) or PAK1 inhibitor (IPA-3), or JNK inhibitor (SP600125) were placed in the upper chambers. Cells were allowed to migrate for 8 h, fixed, and stained with crystal violet staining kit. The migration index represents the number of migrated cells normalized to FBS containing media. (B) In vitro invasion assay was performed using inserts coated with a Matrigel basement membrane matrix in Boyden chambers. Cells were allowed to invade through Matrigel toward FBS-containing media for 8 h. Invasion index was calculated by normalizing the mean of invaded cells to FBS-containing media. Data are presented as mean ± SEM from five independent experiments. *p < 0.05 versus control.

PIAS3 knockdown inhibits TNF-α–induced SUMOylation of Rac1 in RA FLSs

PIAS3 is an important regulator of SUMOylation in several cell lines. To determine whether PIAS3 was involved in protein SUMOylation in RA FLSs, we initially evaluated the effect of PIAS3 knockdown on the expression of SUMO-1 and SUMO-2/3, as assessed with Western blot analysis. The expression of SUMO-1 and SUMO-2/3 in RA FLSs was higher than in OA FLSs (Fig. 8A, 8B). PIAS3 shRNA knockdown inhibited the free and protein-conjugated expression of SUMO-1 compared with control shRNA in RA FLSs, but it did not affect SUMO-2/3 expression (Fig. 8C). Because PIAS3-dependent SUMOylation of Rac1 has been demonstrated previously in other cells (17), we examined whether PIAS3 modulated SUMOylation of Rac1 in RA FLSs. Endogenous Rac1–SUMO-1 was expressed in control cells following stimulation with TNF-α, but not in PIAS3 shRNA-transfected cells (Fig. 8D). These data suggested that PIAS3 was involved in regulating SUMOylation of Rac1 in RA FLSs.

FIGURE 8.
FIGURE 8.

PIAS3 knockdown inhibits TNF-α–induced SUMOylation of Rac1 in RA FLSs. (A and B) Expression of (A) SUMO-1 and (B) SUMO-2/3 in FLSs from patients with RA and OA. SUMO expression was detected with Western blotting. (C) Effect of PIAS3 knockdown on the free and protein-conjugated SUMO-1 and SUMO-2/3. SUMO expression was measured with Western blotting. A representative blot of three independent experiments is shown. (D) Effect of PIAS3 knockdown on SUMOylation of Rac1. Cell lysates were harvested and immunoprecipitated (IP) with an anti-Rac1 or control IgG Ab. Immunoprecipitated samples (upper panel) or whole cell lysates (Input, lower panels) were subjected to SDS-PAGE and Western blotting (WB) using an anti-SUMO-1 or anti-Rac1 or anti-PIAS3 Ab. A representative blot of three independent experiments is shown.

Discussion

In this study, we showed that the expression of PIAS3, but not PIAS1, PIAS2, or PIAS4, was elevated in FLSs and STs derived from patients with RA. To our knowledge, we also demonstrated for the first time that specific shRNA for PIAS3 decreased migration and invasion of RA FLSs, as well as expression of MMP-3, MMP-9, and MMP-13. Furthermore, our studies indicated that PIAS3-mediated SUMOylation of Rac1 controlled its GTP-bound levels. PIAS3 shRNA also suppressed activation of PAK1 and JNK, which are downstream proteins of Rac1. Inhibition of Rac1, PAK1, or JNK activity by their respective pharmacologic inhibitors significantly reduced in vitro migration and invasion, indicating that PIAS3 regulates migration and invasion through the Rac1/PAK1/JNK pathway in RA FLSs. Combined, these data suggest an important role of the increased PIAS3 expression in the maintenance of the activated phenotype of FLSs from patients with RA.

The migration of FLSs to cartilage and bone has been considered a key process in cartilage destruction in RA. Arriving at the cartilage or bone, FLSs can destroy cartilage and activate osteoclasts (5, 32, 33). Increasing evidence indicates the potential importance of FLSs-mediated joint damage in RA; however, to date, no effective therapies have been found to target FLSs-mediated joint destruction directly. Clarifying the precise mechanisms of the signal pathways that control migration and invasion of RA FLSs could ultimately result in novel therapies.

As a family of multifunctional proteins, PIAS proteins not only exhibit their DNA and protein binding ability; they also function as SUMO-E3 ligases. Thus, PIAS can interact with diverse proteins and regulate multiple signaling cascades, which modulate varied cellular functions, such as survival, proliferation, and migration (13–15). Therefore, PIAS proteins act as regulators of transcription, either positively or negatively (12).

Recent evidence indicates that PIAS3 has been associated with tumor cell migration and survival in glioblastoma multiforme and prostate cancer (34, 35), and abnormal expression of PIAS3 in tumors is associated with malignant properties of tumor cells (36–38). The role of PIAS3 in tumor cells indicates the relation between PIAS3 and the aggressive nature of RA FLSs. In the current study, we demonstrated that inhibition of PIAS3 by shRNA significantly reduced migration as examined by wound healing and chemotaxis assays. Similar results were obtained in modulating invasive behavior of RA FLSs through Matrigel-coated trans-well membranes. These results suggest that overexpression of PIAS3 may contribute to aberrant migration and invasion of RA FLSs. Consistent with our findings, PIAS3 knockdown decreased directional migration of HEK293T cells (17). PIASy also enhanced migration of epithelial cells (16). However, in contrast to these studies, a previous report shows that PIAS3 negatively regulates the migration of glioma cells by stimulating vimentin 354 SUMOylation (34). This report indicates that the role of PIAS3 in regulating migration is context-dependent, as it seems to be associated with difference in cell types. In addition, a previous study showed that SUMO-1 expression was maintained at high levels in invading RA FLSs (39), and in this study we found that SUMO-1 expression was inhibited by PIAS3 knockdown in RA FLSs, further supporting the notion that PIAS3 positively regulates the migration and invasion of RA FLSs. Interestingly, we found that not only was PIAS3 expression higher in cultured RA FLSs and RA synovial lining than in OA; PIAS3 knockdown did not affect the migration and invasion of OA FLSs, indicating that PIAS3 seems to be especially relevant in regulating the invasive behavior of FLSs in RA.

Adhesion, polarization, and cytoskeleton reorganization are critical steps that promote directional cell migration. The formation of a lamellipodium at the leading edge results from extended membrane protrusions and directs cell movement. Localized actin polymerization at the lamellipodium is therefore necessary for driving cell movement forward (26, 40). Therefore, we further evaluated the relationship between PIAS3 and cell membrane protrusions. Localization of PIAS3 in lamellipodium protrusions and reduced lamellipodium formation with PIAS3 knockdown suggest that PIAS3 is involved in protrusion formation and migration of RA FLSs.

Rho GTPases function as molecular switches and play critical roles in regulating cytoskeleton reorganization during cell migration. Rac1 can stimulate lamellipodium formation and cell migration in part through modulating actin polymerization (9, 41). In the current study, we demonstrated that PIAS3 inhibition by shRNA reduced Rac1 activity and its downstream effector PAK1 in RA FLSs, indicating that PIAS3-mediated Rac1 activation contributes to the role of PIAS3 proteins in the invasive behavior of RA-FLSs. Consistent with our results, PIAS3 activates Rac1 to promote lamellipodium dynamics and migration in other cell lines (17). Our findings that PIAS3 inhibition exhibited inhibitory effects on JNK phosphorylation and the specific inhibitor for Rac1 or PAK1 suppressed JNK phosphorylation suggest that Rac1/PAK1/JNK pathway is involved in PIAS3-mediated migration and invasion of RA FLSs.

The manner in which PIAS3 activates Rac1 in FLSs is still unclear. In other cell types, however, PIAS3 can bind to Rac1 and lead to SUMOylation within the polybasic regions of Rac1 in response to hepatocyte growth factor stimulation (17). In our work, we demonstrated that PIAS3 knockdown decreased SUMO-1 expression and interaction of Rac1 and SUMO-1. We also found colocalization of PIAS3 and Rac1 in ruffling lamellipodia at the leading edge of RA FLSs. These data strongly imply that PIAS3 might regulate Rac1 activity through its SUMOylation in RA FLSs at sites of membrane protrusion. However, we cannot rule out the possibility that PIAS3 may modulate Rac1 activity independently of its SUMO ligase activity, because PIAS3 can also regulate transcriptional activity through SUMOylation-independent pathways, such as recruiting histone deacetylase 1 to the promoter regions of their target genes (42, 43). Further study is thus needed to evaluate whether there are alternative mechanisms of Rac1 activity regulated by PIAS3 in RA FLSs.

MMPs, mainly produced by FLSs in RA, are proteases that regulate the remodeling of the extracellular matrix and play an important role in the progressive destruction of joints in RA. MMPs can be induced by proinflammatory cytokines, such as TNF-α and IL-1β in RA FLSs. The signaling mechanisms that modulate MMPs expression in RA FLSs remain to be identified. In the current study, for the first time to our knowledge, we demonstrate that in RA FLSs, PIAS3 knockdown decreased TNF-α–induced mRNA expression and secretion of MMP-3, MMP-9, and MMP-13, suggesting an important role of PIAS3 in the maintenance of the activated phenotype of RA FLSs via regulating MMPs expression. Consistent with our results, a previous study showed that PAK1, a downstream effector of PIAS3 that has been demonstrated in the present work, also regulated MMP-13 expression in RA FLSs (44). SENP1, which served as a deSUMOylation enzyme, participated in the regulation of MMP-1 expression in RA FLSs (45). These results therefore suggest that one of the mechanisms of PIAS3 involved in pathogenesis of RA is regulating MMPs production.

In addition, we found that the Rac1 inhibitor NSC23766 reduced the migration of FLSs from patients with RA but not those with OA. We propose that two non–mutually exclusive interpretations might explain this puzzle. One possibility is due to low baseline migration of OA FLSs as compared with RA FLSs. Indeed, in our experiments, we also found that NSC23766 treatment resulted in slight but not significant reduction of migration and invasion of OA FLSs. On the other hand, as we know, Rac1 can be activated by a number of guanine nucleotide exchange factors (GEFs) such as Trio, Tiaml, Lbc, Dock, and Vav depending on the different situations or cell types. However, NSC23766 only blocks the activation of Rac1 through binding to Trio and Tiam1 (46); therefore, it is likely that NSC23766-sensitive Rac-GEFs such as Trio and Tiaml are not involved in the regulation of Rac1 activation in OA FLSs. In the future, it would be interesting to explore the role of different Rac-GEFs in regulating migration of RA FLSs and OA FLSs.

Our findings show that PIAS3 plays an important role in regulating invasive behavior of RA FLSs, suggesting that elevated synovial PIAS3 protein might contribute to cartilage invasion and destruction in RA.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

The authors thank Jinjin Fan for technical assistance.

Footnotes

  • This work was supported by grants from the National Natural Science Foundation of China (81373182 and U1401222), the Guangdong Natural Science Foundation (S2011020002358 and S2013010015363), and the Guangdong Project of Science and Technology (2012B031800375 and 2011B050300009).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    FLS
    fibroblast-like synoviocyte
    GEF
    guanine nucleotide exchange factor
    IHC
    immunohistochemistry
    MMP
    matrix metalloproteinase
    NC
    normal control
    OA
    osteoarthritis
    PIAS
    protein inhibitor of activated STAT
    RA
    rheumatoid arthritis
    SC
    scramble control
    shRNA
    short hairpin RNA
    ST
    synovial tissue.

  • Received January 12, 2015.
  • Accepted November 17, 2015.

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