Rac1 promotes chondrogenesis by regulating STAT3 signaling pathway†

Keywords : chondrogenesis; micromass culture; aggregation; Rac1

Abbreviations: STAT3, transducer and activator of transcription 3; JAK2, janus kinase 2; BMP4, bone morphogenic protein 4; MAPK, mitogen-activated protein kinase; IL-6, interleukin 6; PNA, peanut agglutinin

†This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/cbin.10635].

This article is protected by copyright. All rights reserved Received 30 March 2016; Revised 9 June 2016; Accepted 12 June 2016.

Abstract

The small GTPase protein Rac1 is involved in a wide range of biological processes including cell differentiation. Previously, Rac1 was shown to promote chondrogenesis in micromass cultures of limb mesenchyme. However, the pathways mediating Rac1’s role in chondrogenesis are not fully understood. This study aimed to explore the molecular mechanisms by which Rac1 regulates chondrogenic differentiation. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) was increased as chondrogenesis proceeded in micromass cultures of chick wing bud mesenchyme. Inhibition of Rac1 with NSC23766, janus kinase 2 (JAK2) with AG490, or STAT3 with stattic, inhibited chondrogenesis and reduced phosphorylation of STAT3. Conversely, overexpression of constitutively active Rac1 (Rac L61) increased phosphorylation of STAT3. Rac L61 expression resulted in increased expression of interleukin 6 (IL-6), and treatment with IL-6 increased phosphorylation of STAT3. NSC23766, AG490, and stattic prohibited cell aggregation, whereas expression of Rac L61 increased cell aggregation, which was reduced by stattic treatment. Our studies indicate that Rac1 induces STAT3 activation through expression and action of IL-6. Overexpression of Rac L61 increased expression of bone morphogenic protein 4 (BMP4). BMP4 promoted chondrogenesis, which was inhibited by K02288, an activin receptor-like kinase-2 inhibitor, and increased phosphorylation of p38 MAP kinase. Overexpression of Rac L61 also increased phosphorylation of p38 MAPK, which was reduced by K02288. These results suggest that Rac1 activates STAT3 by expression of IL-6, which in turn increases expression and activity of BMP4, leading to the promotion of chondrogenesis.

1 Introduction

Chondrogenesis is a multi-step process that includes recruitment of cells, condensation of progenitors, and differentiation of the condensed cells into chondrocytes (DeLise et al., 2000). Chondrogenesis is marked by distinct changes in cellular morphology, correlated to specific changes in gene expression. This process is controlled by numerous growth and differentiation factors and environmental factors (Goldring et al., 2006) as well as adhesive events such as cell-cell adhesion and cell-matrix interactions (Woods et al., 2007). Several signaling molecules, such as Rho GTPases, are involved in adhesive signaling during chondrogenesis (Woods et al., 2007).

Rac1 belongs to the Rho family of GTPases, which includes Rho, Rac1, and Cdc42. These proteins control the assembly and disassembly of cytoskeletal elements (Hall, 1988). Rac1 has been implicated in a number of cellular processes such as phagocytosis, adhesion, cell motility, cell proliferation, and differentiation (Etienne-Manneville and Hall, 2002; Bustelo et al., 2007; Heasman and Ridley, 2008). Increasing evidence suggests that Rac1 plays an important role in the differentiation of mesenchymal cells and chondrocytes, and its activity is required for the maturation of chondrocytes (Wang and Beier, 2005; Kerr et al., 2008). Rac1 signaling stimulates mesenchymal condensation and chondrogenesis (Woods et al., 2007). Hydrostatic pressure enhances chondrogenic differentiation of bone marrow mesenchymal stem cells via upregulation of Rac1 (Zhao et al., 2015). However, the downstream molecular mechanisms by which Rac1 exerts its promoting effect on chondrogenesis have not been thoroughly elucidated.

Signal transducer and activator of transcription 3 (STAT3) is a member of a family of transcription factors comprising STAT1–4, 5A, 5B, and 6. STATs are phosphorylated by JAK tyrosine kinases in response to activation of cell surface receptor tyrosine kinases by cytokines, growth factors, and other stimuli (Levy et al., 2002; Sharma et al., 2014). The phosphorylated STATs form dimers and are localized to the nucleus where they bind to specific DNA sequences and activate their transcription (Heinrich et al., 1998; Germain and Frank, 2007). Activated STAT3 is involved in a variety of physiological processes including cell proliferation, inflammation, and differentiation (Qi and Yang, 2014).

In the present study, we sought to characterize the signaling pathway by which Rac1 promotes chondrogenic differentiation. We demonstrate that Rac1 activates JAK/STAT3 signaling, which in turn stimulates the expression and signaling of bone morphogenic protein 4 (BMP4).

2 Materials and methods
2.1 Reagents

AG490 was purchased from TOCRIS Bioscience (Ellisville, MO). NSC23766 was obtained from Calbiochem (La Jolla,CA). Recombinant human BMP4 was from PROSPEC (Ness Ziona, Israel). Anti-type II collagen, -p38 MAPK, and -phospho-p38 MAPK antibodies and stattic were from Santa Cruz Biotechnology (Dallas, TX). Anti-Myc-tag, -phospho- STAT3 (T705), and -GAPDH antibodies were from Cell Signaling Technology (Danvers, MA). Anti-STAT3 antibody was obtained from BD Transduction Laboratories (Lexington, KY).

2.2 Micromass culture

Micromass cultures of chick wing bud mesenchymal cells were prepared as previously described (Ahrens et al., 1977). Briefly, wing buds were removed from stage 23/24 chick embryos and incubated for 10 min at 37°C in 0.1% trypsin- collagenase in Ca2+/Mg2+-free Hanks’ balanced salt solution. Following mechanical dissociation of cells, the cell suspension was centrifuged at 500 × g for 10 min and the cell pellets were resuspended in F12 medium containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 μg/mL streptomycin and filtered through eight layers of lens paper to remove cell clumps. Cells were counted with a hemocytometer and resuspended at a concentration of 2 × 107 cells/mL. The cells were plated in three drops (15 μL each) into 35-mm culture dishes. Cells were then allowed to adhere to the substratum for 1 h at 37°C, followed by the addition of 1.5 mL of F12 medium with 10% FBS. Cultures were fed every 24 h by a complete change of medium.

2.3 Expression of the constitutively active mutant Rac1 in mesenchymal cells

The pRK5-myc-tagged Rac1 L61 expression plasmid (plasmid 15903) was purchased from Addgene Inc. (Cambridge,MA). DNA transfection of the wing bud mesenchymal cells was performed by electroporation with a BTX-830 square wave generator (Gentronics, San Diego, CA). A 600-uL aliquot of the cell suspension (5 × 106 cells) was placed into an electroporation cuvette (Invitrogen, Grand Island, NY) with a 0.4-cm gap, followed by the addition of 10 µg of plasmid DNA. Cells were electroporated with a pulse of 200 V for 25 ms. Control cells were transfected under the same conditions with the pcDNA3.1+ plasmid (Invitrogen). Following electroporation, cells were micromass cultured as described above.

2.4 Alcian blue staining and quantification

The degree of chondrogenic differentiation was evaluated by staining sulfated cartilage glycosaminoglycans with Alcian blue. To obtain the photomicrographs, micromass cultures were rinsed twice with PBS, fixed in Kahle’s fixative for 20 min and incubated with 0.5% Alcian blue 8-GX (Sigma-Aldrich, St. Louis, MO) in 0.1 N HCl overnight. Images were acquired under a Nikon SMZ745T stereomicroscope. For quantitative analysis, cultures were fixed with acidified alcohol (2% glacial acetic acid in ethanol) for 30 min and rehydrated sequentially in 95% and 70% ethanol for 10 min each. Cells
were then stained in 0.5% Alcian blue (MP Biomedicals, Illkirch, France) overnight. Alcian blue-stained cultures were extracted with 4 M guanidine-HCl overnight at 4°C. The optical density of the extracted dye was measured using a microplate reader (Bio-Rad, Hercules, CA) at 595 nm.

2.5 Peanut agglutinin staining

Micromass cultures grown for up to 3 days were rinsed twice with PBS and fixed with 4% paraformaldehyde for 20 min. Fixed cultures were incubated with 3% H2O2 in tap water for 5 min and 1% bovine serum albumin for 30 min to block endogenous peroxidase activity and nonspecific binding reactions, respectively. Cultures were then incubated at room temperature for 30 min in biotinylated peanut agglutinin (PNA) (Sigma-Aldrich). PNA binding was detected using the Vectastain ABC and DAB substrate solution kit (Vector Laboratories Inc., Burlingame, CA) according to the manufacturer’s instructions.

2.6 Western blot analysis

Cultures were washed in ice-cold PBS and lysed at 4°C in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 0.25% sodium deoxycholate, 1% Triton X-100, and 1 mM PMSF) supplemented with complete mini protease inhibitor cocktail (Roche Diagnostic Corp., Indianapolis, IN) and phosphatase inhibitor cocktail (Biotool, Houston, TX). Cell lysates were clarified by centrifugation at 10,000 × g for 10 min at 4°C, and the
protein concentration of the supernatant was measured using a Pierce BCA protein assay kit (Thermo Scientific,Rockford, IL). Thirty micrograms of protein were resolved in 10% SDS-PAGE and transferred to a nitrocellulose membrane (BA 85, Schleicher & Schuell, Keene, NH). Nonspecific binding sites on the membrane were blocked with 3% nonfat dry milk in TBS-T (Tris-buffered saline, 0.1% Tween 20) for 1 h and then probed with the indicated primary
antibodies overnight at 4°C. After washing three times for 10 min each with TBS-T, the membrane was exposed to horseradish peroxidase-conjugated antibody against mouse, rabbit, or goat (Cell Signaling Technology) for 1 h at room temperature. The immunoblotted proteins were visualized using the Super Signal West Femto kit (Pierce, Rockford, IL) and recorded on x-ray films. Some bands were quantified by densitometry using Image J software.

2.7 RNA isolation and reverse transcription

Total RNA was extracted from the cultures using TRIzol reagent (Invitrogen) and purified using a Direct-zol RNA miniprep kit (Zymo Research Corporation, Irvine, CA) according to the manufacturer’s instructions. First-strand cDNAs were prepared from 1 μg of purified total RNA using the EasyScript cDNA synthesis kit (ABM, Richmond, BC, Canada). cDNA products were amplified by PCR using PCR master mix (Takara, Otsu, Shiga, Japan) and the following gene- specific primers: BMP4, 5′-CCAAAGTGAACTCTTGC-3′ (forward) and 5′-GCTGAGGTTGAAGACGAAGC-3′ (reverse); interleukin 6 (IL-6), 5′-CCAAAGCCATGAACTCTTGC-3′ (forward) and 5′- GCTGAGGTTGAAGACGAAGC-3′ (reverse); GAPDH, 5′-AGTCATCCCTGAGCTGAATG-3′ (forward) and 5′-
ACCATCAAGTCCACAACACG-3′ (reverse). PCR conditions were as follows: 95°C for 10 min and then 29 cycles at 95°C for 1 min, 52°C (BMP4), 59°C (IL-6), or 55°C (GAPDH) for 1 min, and 72°C for 1 min, followed by 72°C for 10 min. The PCR products were resolved by 1% agarose electrophoresis and visualized using Midori Green Advanced DNA stain and a blue/green led box (Nippon Genetics, Tokyo, Japan).

2.8 Statistical analysis

The statistical analysis of Alcian blue staining and Western blot assay was performed using the paired Student’s t-test. Significance is defined as P < 0.05.

3 Results

3.1 Inhibition of Rac1 activity suppresses chondrogenesis via inhibiting phosphorylation of STAT3

To investigate whether Rac1 activity is associated with phosphorylation of STAT3, chick wing bud mesenchymal cells were micromass cultured in the absence or presence of NSC23766. NSC23766 inhibited Alcian blue staining (Figure 1A) and expression of type II collagen (Figure 1B), which is consistent with a previous study (Woods et al., 2007) that demonstrated Rac1 as a positive regulator of chondrogenesis. The phosphorylation of STAT3 was increased as chondrogenesis proceeded but was suppressed by treatment with NSC23766 (Figure 1B). We next determined whether STAT3 activity is required for chondrogenesis in micromass culture. Cultures were treated with stattic, a small-molecule inhibitor of STAT3 (Schust et al., 2006) and chondrogenesis was examined. Addition of stattic dose-dependently reduced Alcian blue staining (Figure 1C and D). AG490, a janus kinase 2 (JAK2) inhibitor, also inhibited chondrogenesis. Both stattic and AG490 decreased the phosphorylation of STAT3 (Figure 1E), suggesting the possibility that Rac1 may control STAT3 activity and that JAK/STAT3 signaling is involved in chondrogenesis.

3.2 Rac1 regulates the phosphorylation of STAT3 by increasing the expression of IL-6

In order to confirm that Rac1 regulates phosphorylation of STAT3, mesenchymal cells were transfected with Myc-tagged Rac1 L61-expressing vectors and micromass cultured. The cell lysates were immunoblotted with anti-Myc tag antibody to detect transfected Myc-tagged Rac1 expression. As shown in Figure 2A, cells transfected with Myc-tagged Rac1 L61 expressed Myc. Overexpression of constitutively active Rac1 L61 increased the phosphorylation of STAT3 without changes in the expression of STAT3 and promoted chondrogenesis, based on Alcian blue staining, which was significantly inhibited by stattic (Figure 2B and C).

It has been proposed that Rac1 mediates STAT3 activation by IL-6 (Faruqi et al., 2001). To examine whether Rac1 regulates STAT3 activity through IL-6, level of IL-6 mRNA was measured in the Rac1 L61-transfected cells.

Overexpression of Rac1 L61 increased the expression of IL-6 (Figure 2D). Treatment of the cultures with IL-6 increased the phosphorylation of STAT3 and Alcian blue staining (Figure 2E and F). These results suggest that Rac1 activates STAT3 through upregulation of IL-6.

3.3 STAT3 activity is required for cell aggregation

It has been reported that Rac1 stimulates cell condensation, leading to the promotion of chondrogenesis (Woods et al., 2007). Thus, we determined whether STAT3 activity is associated with cell aggregation. Mesenchymal cells were incubated with NSC23766, AG490, or stattic for 3 days and stained with PNA to detect condensing chondroprogeniton cells. NSC23766, stattic, and AG490 decreased PNA staining of micromass cultures as compared to control cultures (Figure 3A).

To confirm whether STAT3 activity is required for stimulation of cell aggregation by Rac1, cells were transfected with constitutively active Rac1 L61, micromass cultured in the absence or presence of AG490 or stattic, and stained with PNA.As shown in Figure 3B, overexpression of Rac1 L61 enhanced PNA staining intensity as compared to empty vector-infected cultures. AG490 and stattic reduced PNA staining that was increased by Rac1 L61 transfection. These results suggest that Rac1 promotes cell aggregation through JAK/STAT3 activity.

3.4 Rac1-regulated STAT3 activates BMP4 signaling

We examined downstream signaling of STAT3 during chondrogenesis. Mesenchymal cells were incubated with NSC23766, AG490, or stattic for 2 days and expression of BMP4 mRNA was examined by RT-PCR. As shown in Figure 4A, all the reagents used in this study suppressed the expression of BMP4. Overexpression of Rac1 L61 significantly induced the expression of BMP4 (Figure 4B).

To evaluate the effect of BMP4 on chondrogenesis, cells were treated with BMP-4 for 3 days and chondrogenesis was examined by Alcian blue staining and Western blotting with anti-type II collagen antibody. As shown in Figure 4C and D,BMP4 increased Alcian blue staining and expression of type II collagen, which were suppressed by K02288, an inhibitor of activin receptor-like kinase-2. We further examined downstream signaling of BMP4. Cells were treated with BMP4 for 3 days and the phosphorylation of p38 MAPK was examined. BMP4 increased the phosphorylation of p38 MAPK (Figure 4E). Overexpression of active L61 increased the phosphorylation of p38, which was suppressed by stattic (Figure 4F). These results suggest that Rac1-activated STAT3 stimulates expression of BMP4 and its signaling pathway and thereby enhances chondrogenesis.

4 Discussion

Rac1, a member of the Ras homolog family, not only plays a role in the control of cytoskeletal dynamics, but also is currently known for its involvement in diverse cellular functions, including the cell cycle, polarity, morphogenesis, and migration. Rac1 interacts with many downstream effector molecules to engage signaling cascades (Bishop and Hall, 2000; Jaffe and Hall, 2005). STAT3 is one of the Rac1 effectors (Bustelo et al., 2007), and tyrosine phosphorylation of STAT3 is promoted by Rac1 (Raptis et al., 2011). These observations prompted us to determine whether Rac1 activity is associated with activation of STAT3 during chondrogenesis of mesenchymal cells in micromass culture. Inhibition of Rac1 with NSC23766 suppressed chondrogenesis of wing bud mesenchymal cells and phosphorylation of STAT3.

Inhibition of JAK2 or STAT3 also decreased chondrogenesis and phosphorylation of STAT3. These results suggest that Rac1 activity is linked to activation of STAT3 and JAK/STAT3 signaling pathways in chondrogenesis.Additional direct evidence that Rac1 regulates STAT3 activity comes from the experiment that uses a constitutively active Rac1 L61-expressing plasmid. Transfection of active Rac1 L61 increased the phosphorylation of STAT3. Moreover, inhibition of STAT3 with stattic reduced the phosphorylation of STAT3 in Rac1 L61-transfected cells, confirming that the activity of STAT3 is regulated by Rac1.

Several mechanisms conferring activation of STAT3 by Rac1 have been described. First, activated Rac1 directly binds to STAT3 and stimulates its phosphorylation (Simon et al., 2000). Second, Rac1 indirectly activates STAT3 through mRNA expression and autocrine production of IL-6 (Faruqi et al., 2001; Arulanandam et al., 2010). Third, the Rho GTPases are able to induce STAT3 activation independently of the IL-6 autocrine pathway (Debidda et al., 2005). It has been reported that the activation of IL-6/STAT3 signaling positively regulates the chondrogenic differentiation of human mesenchymal cells (Kondo et al., 2015). Thus, the possibility of IL-6 as a functional link between Rac1 and STAT3 was examined. Overexpression of active Rac1 L61 significantly induced the gene expression of IL-6. In addition, IL-6 increased the phosphorylation of STAT3 and promoted chondrogenesis. These findings suggest that Rac1 regulates the phosphorylation of STAT3 by stimulating the expression of IL-6.

Cell condensation is a prerequisite for chondrogenesis (Oberlender and Tuan, 1994), and Rac1 promotes chondrogenesis by stimulating cell condensation (Woods et al., 2007). Therefore, we examined whether STAT3 is similarly involved in cell condensation. Inhibition of Rac1, JAK, and STAT3 suppressed cell aggregation. Overexpression of active Rac1 L61 increased cell aggregation, which was decreased by inhibition of JAK and STAT3. These results suggest that Rac1 promotes chondrogenesis by increasing cell aggregation through JAK/STAT3 signaling.

STAT3 plays essential roles in a diverse array of cellular processes including the cell cycle, development, and differentiation by interaction with its target genes including BMP4 (Snyder et al., 2007). It has been well documented that BMPs play a fundamental role in the regulation of bone organogenesis. BMP signaling is necessary for mesenchymal condensation and the initiation of chondrogenesis. During limb development, the BMP receptor, type IB is necessary and sufficient for cartilage condensation (Zou et al., 1997). Noggin, a potent BMP inhibitor, suppressed mesenchymal condensation of early-stage chick limb buds (Pizette and Niswander, 2000). Treatment with BMP4 promotes chondrogenesis of mouse limb bud mesenchymal cells (Hoffman et al., 2006), and overactivity of BMP4 in the skeleton caused an increase in cartilage production and enhanced chondrocyte differentiation (Tsumaki et al., 2002). In the current study, overexpression of Rac1 increased BMP4 gene expression, and inhibition of JAK/STAT3 signaling reduced BMP4 expression. We also showed that BMP4 enhanced chondrogenesis, which was almost completely blocked by K02288, an inhibitor of the BMP receptor (Sanvitale et al., 2013). Binding by BMPs induces phosphorylation of their receptors and activates diverse intracellular downstream pathways including p38 MAPK (Rahman et al., 2015). In this study, BMP4 increased phosphorylation of p38 MAPK, which was suppressed by K02288. Overexpression of active Rac1 L61 increased phosphorylation of p38 MAPK, which was suppressed by stattic.

In conclusion, our results suggest that Rac1 exerts its promoting effect on chondrogenesis by activating JAK/STAT3 signaling, leading to stimulation of BMP4 expression and its signaling. Because Rac1 interacts with many downstream effector molecules, further studies are needed to determine the exact role of Rac1 in chondrogenesis.