Mol Cell Biochem (2015) 399:201–208 DOI 10.1007/s11010-014-2246-5
Beta-arrestin-2 negatively modulates inflammation response in mouse chondrocytes induced by 4-mer hyaluronan oligosaccharide Giuseppe M. Campo • Angela Avenoso Angela D’Ascola • Michele Scuruchi • Alberto Calatroni • Salvatore Campo
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Received: 24 July 2014 / Accepted: 9 October 2014 / Published online: 16 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Beta-arrestin-2 is an adaptor protein that terminates G protein activation and seems to be involved in the modulation of the inflammatory response. Small hyaluronan (HA) fragments, such as 4-mer HA oligosaccharides, are known to interact with the toll-like receptor-4 (TLR-4) with consequent activation of the nuclear factor kappaB (NF-kB) that in turn stimulates the inflammation response. NF-kB activation is mediated by different pathways, in particular by the transforming growth factoractivated kinase-1 (TAK-1). Conversely, increased levels of protein kinase A (PKA), induced by cyclic adenosine monophosphate (cAMP), seem to inhibit NF-kB activation. We studied the involvement and role of beta-arrestin-2 in mouse chondrocytes stimulated with 4-mer HA fragments. The exposure of chondrocytes to 4-mer HA produced a significant up-regulation in TLR-4, cAMP, beta-arrestin-2, TAK-1, protein 38 mitogen-activated protein kinase (p38MAPK), and PKA, both in terms of mRNA expression and of the related protein levels. NF-kB was significantly activated, thereby producing the transcription of proinflammatory mediators, including tumor necrosis factor
G. M. Campo (&) A. Avenoso A. D’Ascola A. Calatroni S. Campo Department of Biomedical Sciences and Morphological and Functional Images, Section of Medical Biotechnologies and Preventive Medicine, School of Medicine, University of Messina, Policlinico Universitario, Torre Biologica, 5° piano, Via C. Valeria, 98125 Messina, Italy e-mail: [email protected] M. Scuruchi Department of Clinical and Experimental Medicine, School of Medicine, University of Messina, Policlinico Universitario, Torre Biologica, 5° piano, Via C. Valeria, 98125 Messina, Italy
alpha, interleukin-6, and interleukin-17. The treatment of 4-mer HA-stimulated chondrocytes with antibodies against beta-arrestin-2 and/or a specific PKA inhibitor, significantly increased the inflammatory response, while the treatment with a specific p38MAPK inhibitor significantly reduced the inflammatory response. Interestingly, the antiinflammatory action exerted by beta-arrestin-2 appeared to be mediated in part through the direct inhibition of p38MAPK, preventing NF-kB activation, and in part through cAMP and PKA activation primed by G protein signaling, which exerted an inhibitory effect on NF-kB. Taken together, these results could be useful for future anti-inflammatory strategies. Keywords Hyaluronan NF-kB TLR-4 Beta-arrestin2 Cytokines Inflammation Chondrocytes
Introduction b-arrestins are intracellular scaffolding proteins that modulate specific cell signaling pathways. The arrestin family consists of four isoforms, two expressed only in the visual system and two that are ubiquitously expressed, the latter named b-arrestins 1 and 2 [1]. They play a critical role in the regulation of the receptor signaling of seven-transmembrane receptors (7MSRs) phosphorylated by G protein-coupled receptor kinases [2]. More recently, b-arrestins have been shown to signal independently of heterotrimeric G protein coupling, suggesting that they represent a direct signal transducer of G protein-coupled receptors (GPCRs). However, the list of b-arrestin binding partners continues to expand. Among them, mitogen-activated-protein kinases (MAPKs), the serine/threonine kinase Akt, transforming growth factor-activated kinase-1 (TAK-1), and exchange
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proteins activated by cAMP (EPAC) appear to be deeply involved [3]. Recently, it has been reported that the expression of b-arrestins-1 and 2 is differentially regulated in immune cells and tissues in response to specific inflammatory stimuli; they, therefore, seem to play a critical regulatory role in the inflammatory response [4–6]. It has also been reported that b-arrestins-1 and 2 play a key role in experimental arthritis, and in particular that b-arrestin-1 positively regulates the inflammatory response while barrestin-2 inhibits inflammation [7]. Degeneration of joint cartilage is a key feature of arthritis. However, the disease is also associated with concomitant changes in synovium and subchondral bone metabolism, causing inflammation of the chondrocyte membrane in the affected joint [8]. There are no effective long-term treatments to counteract arthritis, and current research is focused on understanding how the imbalance between specific extracellular matrix (ECM) molecules may influence progression of the disease. Evidence is emerging to show that endogenous ECM molecules supply signals to damaged cartilage and synovial tissue in order to promote further cartilage degradation [9–11]. High molecular weight hyaluronan (HA) is found in normal, intact, healthy cartilage and contributes to local homeostasis by suppressing cell proliferation, migration, angiogenesis, inflammation, and immunogenicity [12]. Evidence from different studies has shown that the prime of inflammation resulting from cell damage produces an increased expression of growth factors and cytokines which promotes the degradation of the ECM [13–21]. HA degradation products of small size have been shown to stimulate inflammatory responses in different cell types. HA fragmentation also results in smaller oligosaccharides that are angiogenic, inflammatory, and immunogenic in a sizedependent manner [17, 18, 22]. Previous investigations have also shown that small fragments of HA are able to interact with the toll-like receptor-4 (TLR-4), thereby stimulating inflammation or increasing the inflammatory mechanism previously induced by other agents in different cell types [23, 24]. Hence, the generation of low molecular weight HA in pathologies may act as an endogenous danger signal leading to the activation of both innate and acquired immunity. HA fragments may prime inflammation via the TLR-4 which eventually stimulates nuclear factor kappaB (NF-kB) activation. NF-kB, in turn, translocates into the nucleus where it may prime the transcription of various inflammatory mediators, e.g., tumor necrosis-alpha (TNFa), interleukin-1beta (IL-1b), interleukin-6 (IL-6), interleukin-17 (IL-17), inducible nitric oxide synthase (iNOS), and matrix metalloproteases (MMPs) [17, 18, 23–25]. In this study, we investigated the role of b-arrestin-2 in a model of 4-mer HA oligosaccharide-induced inflammatory
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response on mouse chondrocytes obtained from normal DBA/J1 mice. We also studied the effects of the inhibition of b-arrestin-2 and/or protein kinase A (PKA) and/or p38MAPK in order to ascertain the actual role played by these proteins in the inflammatory pathway induced by 4-mer HA stimulation.
Materials and methods Animals Male mice DBA/1 J 6–7 weeks old with a mean weight of 25–30 g were used in our study. Mice, purchased from Charles River Laboratories Srl (Lecco, Italy), were maintained under climate-controlled conditions with a 12-h light/dark cycle. The animals were fed standard rodent chow and provided water ad libitum. The health status of the animal colony was monitored in accordance with Italian Veterinary Board guidelines.
Materials 4-mer HA oligosaccharide as sodium salt (cat CSR-11006) was obtained from Cosmo Bio Co Ltd (Tokyo, Japan). Mouse TLR-4 (cat. SEA753Mu) and mouse IL-17 (cat. SEA063Mu) commercial ELISA kits were purchased from USCN Life Science Inc (Wuhan, China). Mouse TNF-a (cat. IB49688) and IL-6 (cat. 27768) commercial ELISA kits were provided by Immuno-Biological Laboratories Inc (Minneapolis, MN, USA). The mouse b-arrestin-2 (cat. ABIN810988) commercial ELISA kit was supplied by Antibodies-online Inc (Atlanta, GA, USA). The mouse PKA kinase activity kit was obtained from Enzo Life Sciences International Inc (Plymouth Meeting, PA, USA). The P38-MAPK-a (cat. ab126425) commercial ELISA kit was supplied by Abcam plc (Cambridge, UK). Mouse b-arrestin-2 siRNA (cat. sc-29743) was supplied by Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Mouse PKA C-a siRNA (cat. 6406) was obtained from Cell Signaling Technology Inc (Danvers, MA, USA). Mouse p38MAPK-a siRNA SignalSilence was also supplied by Cell Signaling Technology Inc (Danvers, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Lglutamine, penicillin/streptomycin, trypsin–EDTA solution, and phosphate buffered saline (PBS) were obtained from Life Technologies Inc Gibco/Brl Division (Grand Island, NY, USA). All cell culture plastics were obtained from Falcon Plastics (Oxnard, CA, USA). RNase, proteinase K, protease inhibitor cocktail, sodium dodecylsulphate (SDS), and all other general laboratory chemicals were obtained from Sigma-Aldrich Srl (Milan, Italy).
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Chondrocyte cultures Mice were killed by cervical dislocation, and whole patellae were dissected from the hind knee joint. Chondrocytes were released from articular cartilage after being digested with 0.2 % pronase for 1 h. After washing with PBS, cartilage was further digested for 3 h with PBS containing 0.1 % collagenase in DMEM [26]. Chondrocytes were cultured in 75 cm2 plastic flasks containing 15 ml DMEM to which 10 % FBS, L-glutamine (2.0 mM), and penicillin/streptomycin (100 U/ml, 100 lg/ml) were added. siRNA treatment For siRNA experiments, 4 9 106 cells were transfected with b-arrestin-2, PKA C-a, and p38MAPK-a siRNAs following modified siRNA Transfection Protocols (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA, and Cell Signaling Technology Inc, Danvers, MA, USA). PKA C-a is a catalytic subunit of PKA that is indispensable for PKA activation; therefore, total inhibition of PKA activity occurs by inhibiting PKA C-a activity. P38MAPK-a is an active isoform of p38MAPK expressed by chondrocytes. Scrambled siRNA was used under the same conditions as a negative control. After transfection, cells were seeded in six-well plates in 10 % FCS/DMEM and allowed to adhere for 12 h. Culture medium was then replaced with fresh 10 % FCS/DMEM for a further 12-h period. Cells were serum starved for 24 h before 4-mer HA was added. Chondrocyte treatment Chondrocytes were cultured in six-well culture plates at a density of 1.3 9 105 cells/well. Twelve hours after plating (time 0), the culture medium was replaced with 2.0 mL of fresh medium containing 4-mer HA (40 lg/mL) and/or b-arrestin-2 siRNA (reported in Figures as b-ARR2siRNA), and/or PKA siRNA (reported in Figures as PKAsiRNA) and/or p38MAPK-a siRNA (reported in Figures as p38MAPKsiRNA). Finally, the cells and medium underwent biochemical evaluation 48 h after the last treatment. The study, therefore, included the following groups of chondrocytes: (1) CTRL; (2) b-ARR2siRNA; (3) PKAsiRNA; (4) p38MAPKsiRNA; (5) 4-mer HA; (6) 4-mer HA ? b-ARR2siRNA; (7) 4-mer HA ? PKAsiRNA; (8) 4-mer HA ? p38MAPKsiRNA; (9) 4-mer HA ? b-ARR2siRNA ? PKAsiRNA; (10) 4-mer HA ? b-ARR2siRNA ? p38-MAPKsiRNA; (11) 4-mer HA ? PKAsiRNA ? p38MAPKsiRNA; and (12) 4-mer HA ? b-ARR2siRNA ? PKAsiRNA ? p38MAPKsiRNA.
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RNA isolation, cDNA synthesis, and real-time quantitative PCR amplification Total RNA was isolated from chondrocytes for reversePCR real-time analysis of TLR-4, b-arrestin-2, PKA C-a, p38MAPK, TNF-a, IL-6, and IL-17 (RealTime PCR system, Mod. 7500, Applied Biosystems Inc, USA) using an Omnizol Reagent Kit (Euroclone Ltd, West York, UK). The amounts of specific mRNA in samples were calculated using the DDCT method. The mean value of normal cartilage target levels became the calibrator (one per sample), and the results are expressed as the n-fold difference relative to normal controls (relative expression levels). NF-kB p50/65 transcription factor assay NF-kB p50/65 DNA binding activity in nuclear extracts of chondrocytes was evaluated in order to measure the degree of NF-kB activation. Analysis was performed in line with the manufacturer’s protocol for a commercial kit (NF-kB p50/65 EZ-TFA Transcription Factor Assay Colorimetric, cat. n°70–510, Millipore Corporation, Billerica, MA, USA). Values are expressed as relative optical density (OD)/mg protein. TLR-4, b-arrestin-2, IL-17, p38MAPK-a, TNF-a, and IL-6 ELISA assay The analysis of TLR-4, b-arrestin-2, IL-17, p38MAPK, TNF-a, and IL-6 was carried out using a specific commercial kit in line with the manufacturer’s protocols. TLR4 values are expressed as ng/mg protein, while b-arrestin-2, IL-17, p38MAPK, TNF-a, and IL-6 values are expressed as pg/mg protein. PKA kinase assay PKA kinase activity was assayed using a commercial ELISA kit that utilizes a specific synthetic peptide as a substrate for PKA and a polyclonal antibody that recognizes the phosphorylated form of the substrate. PKA values are expressed as ng/mg protein. Protein analysis The amount of protein was determined using the Bio-Rad protein assay system (Bio-Rad Lab. Inc, Richmond, CA, USA) with bovine serum albumin as a standard.
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(n-fold
Data are expressed as means ± S.D. of no less than seven experiments for each test. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test. The statistical significance of differences was set at p \ 0.05. Statement of animal care The studies reported in this manuscript were carried out in accordance with the Declaration of Helsinki and NIH guidelines for the Care and Use of Laboratory Animals.
TLR-4, b-arrestin-2, PKA, and p38MAPK-a mRNA expression (Fig. 1, panel a; Fig. 2, panels a, b, and c), and related protein concentrations (Fig. 1, panel b; Fig. 2, panels d, e, and f) were assayed in order to estimate the degree of TLR-4 activation in chondrocytes stimulated with 4-mer HA and/or treated with b-arrestin-2 siRNA, and/or PKA siRNA, and/or p38MAPK-a siRNA. The data showed a significant increase in both mRNA expression and protein synthesis of TLR-4 in all groups of cells exposed to 4-mer HA (Fig. 1, panels a and b). No changes in TLR-4 expression and synthesis were observed in chondrocytes treated with b-arrestin-2 siRNA, PKA siRNA, and with p38MAPK-a siRNA, both in cells treated with 4-mer HA or those not receiving this treatment. 4-mer HA treatment was also able to increase the mRNA expression and protein levels of b-arrestin-2, PKA, and p38MAPK-a to a significant extent (Fig. 2, panels a, b, c, d, e, and f). The increase in activity of b-arrestin-2, PKA, and p38MAPK-a induced by 4-mer HA was reduced when cells were treated with each specific siRNA (Fig. 2, panels a, b, c, d, e, and f). Notably, chondrocytes stimulated with 4-mer HA and pre-treated with b-Arrestin-2 siRNA showed reduced PKA activity and heightened activity of p38MAPK-a in terms of both mRNA expression and protein concentrations (Fig. 2, panels b, c and e, f), although the action of b-arrestin-2 siRNA on p38-MAPK-a seemed to be less marked than the one exerted on PKA. NF-kB activation Figure 3 shows the changes in the NF-kB p50/p65 heterodimer translocation over the course of the experiment in chondrocytes stimulated with 4-mer HA and/or treated
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Fig. 1 Effect of 4-mer HA and/or b-arrestin-2 siRNA (b-ARR2siRNA), and/or PKA C-a siRNA (PKAsiRNA), and/or p38MAPK-a (p38MAPKsiRNA) treatment on mouse articular chondrocyte TLR-4 mRNA expression (a) and related protein production (b). Biochemical evaluation was performed 24 h after the last treatment. Values are the mean ± SD of no less than seven experiments and are expressed as the n-fold increase with respect to Controls (a) and as ng/mg protein (b) for the TLR-4 levels. °p \ 0.001 versus control
with b-arrestin-2 siRNA, and/or PKA siRNA, and/or p38MAPK-a siRNA. The stimulation of chondrocytes with 4-mer HA produced a marked activation of the NF-kB (Fig. 3). The single treatment of chondrocytes with b-arrestin-2 siRNA or PKA siRNA or p38MAPK-a siRNA had no effect on NF-kB activation. In contrast, the silencing of these proteins in cells stimulated with 4-mer HA was able to modulate NF-kB activation in different ways. In particular, the silencing of b-arrestin-2 produced a significant increase in NF-kB activation. The silencing of PKA also increased NF-kB activation, but to a greater extent than barrestin-2 siRNA, while the silencing of p38MAPK-a significantly reduced NF-kB activation. The concomitant silencing of PKA and b-arrestin-2 increased NF-kB activation to a greater extent than the individual silencing, and produced the greatest increment in NF-kB activation (Fig. 3). The concomitant silencing of p38MAPK-a and b-
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Fig. 2 Effect of 4-mer HA and/or b-arrestin-2 siRNA (b-ARR2siRNA), and/or PKA C-a siRNA (PKAsiRNA), and/or p38MAPK-a (p38MAPKsiRNA) treatment on mouse articular chondrocyte barrestin-2, PKA C-a, and p38MAPK-a mRNA expression (a, b, and c) and related protein production (d, e, and f). Biochemical evaluation was performed 24 h after the last treatment. Values are the
mean ± SD of no less than seven experiments and are expressed as the n-fold increase with respect to controls (a, b, and c) and as pg/mg protein (d) for the b-arrestin-2 levels, as ng/mg protein (e) for the PKA C-a levels, and as pg/mg protein (f) for the p38MAPK-a levels. °p \ 0.001 versus controls; *p \ 0.001 versus 4-mer HA; #p \ 0.005 versus 4-mer HA
arrestin-2 or of p38MAPK-a and PKA still resulted in augmented NF-kB activation, although to a lesser extent (Fig. 3).
p38MAPK-a siRNA, prior to 4-mer HA exposure, produced a significant reduction in the transcription of the inflammatory cytokines. In contrast, the silencing of barrestin-2 resulted in a significant increase in NF-kB activation and inflammatory cytokines production. The silencing of PKA also increased NF-kB activation and inflammatory cytokines production, but to a greater extent with respect to that obtained by b-arrestin-2 silencing. The concomitant silencing of b-arrestin-2 and PKA produced the greatest effect in terms of increased cytokine transcription compared to 4-mer HA treatment alone. (Figure 4, panels a, b, c, d, e, and f).
TNF-a, IL-6, and IL-17 mRNA expression and protein levels TNF-a, IL-6, and IL-17 mRNA evaluation (Fig. 4, panels a, b, and c), and ELISA assay (Fig. 4, panels d, e, and f) confirmed the above findings obtained for NF-kB. The results showed a marked increase in the expression and protein synthesis of the three inflammatory cytokines in cells stimulated with 4-mer HA. In line with the results for NF-kB, the pre-treatment of chondrocytes with b-arrestin-2 siRNA, and/or PKA siRNA, and/or p38MAPK-a siRNA, prior to 4-mer HA exposure, markedly changed the effect of 4-mer HA on inflammatory cytokine transcription. In particular, the pre-treatment of chondrocytes with
Discussion There is accumulating evidence that b-arrestin-2 is functionally involved in different important immune responses
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Fig. 3 Effect of 4-mer HA and/or b-arrestin-2 siRNA (b-ARR2siRNA), and/or PKA C-a siRNA (PKAsiRNA), and/or p38MAPK-a (p38MAPKsiRNA) treatment on mouse articular chondrocyte NF-kB p50/65 transcription factor DNA binding activity. White bars represent the p/50 subunit, gray bars represent the p/65 subunit. Biochemical evaluations were performed 24 h after the last treatment. Values are the mean ± SD of no less than seven experiments and are expressed as optical density at k 450 nm/mg protein of nuclear extract. °p \ 0.001 versus controls; *p \ 0.001 versus 4-mer HA; § p \ 0.005 versus 4-mer HA; #p \ 0.001 versus 4-mer HA; £ p \ 0.001 versus 4-mer HA; & p \ 0.001 versus 4-mer HA; $ p \ 0.001 versus 4-mer HA; ^p \ 0.001 versus 4-mer HA
due to its adaptor and scaffold functions [5, 27]. b-arrestin2 may bind and prevent degradation of the factor IkBa that inhibits NF-kB activation [28, 29]. It has also been reported that b-arrestin-2 is involved in the regulation of C-X-C chemokine receptor type 4 (CXCR4)-induced chemotaxis by activating the ASK1/p38MAPK pathway [30]. Recent studies have reported that b-arrestin-2 negatively regulates polymicrobial sepsis-induced inflammation [4], as well as acting as an anti-inflammatory endogenous agent in experimental arthritis [7]. Activated fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) play a major role by activating chondrocytes and producing joint tissue destruction. FLS become activated through growth factors, chemokines, cytokines, and tissue degradation products such as endogenous TLR ligand HA [31, 32]. Consequently, activated chondrocytes secrete numerous pro-inflammatory mediators and degrading enzymes which greatly contribute to priming and maintaining the inflammation cascade and producing cartilage injury [33, 34]. In the present study, we obtained a marked increment in the mRNA expression and protein synthesis of TLR-4 in chondrocytes stimulated with 4-mer HA. The treatment of cells with 4-mer HA produced a significant increase in b-arrestin-2, PKA, and p38MAPK-a activities.
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Interestingly, chondrocytes stimulated with 4-mer HA and pre-treated with b-arrestin-2 siRNA showed reduced PKA activity and increased p38MAPK-a activity, although the action of b-arrestin-2 on p38-MAPK-a seems to be less marked than the one exerted on PKA. Based on these data, it is, therefore, conceivable that b-arrestin-2 exerted an inhibitory effect on p38MAPK-a and a stimulatory effect on PKA. Chondrocyte exposition to 4-mer HA resulted in a massive activation of the NF-kB. It is useful to measure NF-kB activity in order to evaluate the onset of the inflammatory process since the NF-kB factor can be activated by different pathways that in turn may converge in the transcription of several genes that contribute to prime/ amplify inflammation. The silencing of b-arrestin-2, PKA, and p38MAPK-a in chondrocytes exposed to 4-mer HA modulated NF-kB activation in different ways. Notably, the silencing of barrestin-2 produced a significant increase in NF-kB activation in addition to the increment exerted by the 4-mer HA. The silencing of the PKA also increased NF-kB activation, and to a greater extent than b-arrestin-2 siRNA. However, the silencing of p38MAPK-a significantly reduced NF-kB activation. This result could mean that, in this case, b-arrestin-2 exerts an inhibitory effect on the 4-mer HA-induced activation of NF-kB. In fact, the silencing of b-arrestin-2 increased NF-kB activation, as PKA was not activated, or partially activated, by other pathways, while p38MAPK was not inhibited. Hence, b-arrestin-2 could have mediated PKA stimulation and p38MAPK-a inhibition. The evaluation of the pro-inflammatory cytokines supported the above data obtained for NF-kB. In fact, at first a significant increment in the activity of all cytokines considered was found in chondrocytes exposed to 4-mer HA. Subsequently, the silencing of b-arrestin-2, PKA, and p38MAPK-a, prior to 4-mer HA treatment, was able to modulate the effect of 4-mer HA stimulation on inflammatory cytokine transcription. In particular, the silencing of p38MAPK-a siRNA, prior to 4-mer HA exposition, produced a significantly lower transcription of cytokines, compared to the effect of 4-mer HA alone. This is in line with the evidence that p38MAPK-a induced NF-kB activation. Conversely, the silencing of b-arrestin-2 produced an additional increase in cytokine transcription, indicating an inhibitory effect of b-arrestin-2 on NF-kB activation. The silencing of PKA was also able to increase cytokine production further, in addition to the effect of 4-mer HA alone. This effect was greater than that achieved by barrestin-2 silencing. This could be explained by the fact that PKA exerted an inhibitory effect on NF-kB, and its activation may be due to different pathways besides barrestin-2.
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Fig. 4 Effect of 4-mer HA and/or b-arrestin-2 siRNA (b-ARR2siRNA), and/or PKA C-a siRNA (PKAsiRNA), and/or p38MAPK-a (p38MAPKsiRNA) treatment on mouse articular chondrocyte TNF-a, IL-6, and IL-17 mRNA expression (a, b and c) and related protein production (d, e and f). Biochemical evaluation was performed 24 h after the last treatment. Values are the mean ± SD of no less than seven experiments and are expressed as the n-fold increase with
respect to controls (a, b and c) and as pg/mg protein (d, e and f) for the TNF-a, IL-6, and the IL-17 levels. °p \ 0.001 versus controls; *p \ 0.001 vs 4-mer HA; §p \ 0.005 versus 4-mer HA; #p \ 0.001 versus 4-mer HA; £p \ 0.001 versus 4-mer HA; & p \ 0.001 versus 4-mer HA; $p \ 0.001 versus 4-mer HA; ^p \ 0.001 versus 4-mer HA
Taken together, the body of evidence accumulated so far suggests the functional involvement of b-arrestin-2 in many inflammatory and autoimmune diseases. This evidence could be considered a strength of this study, while the different multitude of pathways that may activate the NF-kB could represent a limit of this paper, as other not investigated mechanism in the present study could take part in the experiment. However, although it is not yet known precisely how extensively b-arrestin-2 is involved, this topic could be further explored by viewing b-arrestin-2 as a potential diagnostic biomarker or even a prognostic indicator for different diseases. This would contribute to efforts to provide new anti-inflammatory strategies and tools and allow their development so that they are able to modulate these intricate inflammation pathways simultaneously. Such an approach could be useful in devising future strategies in order to tackle inflammation more effectively than the currently available therapeutic drugs.
Acknowledgments This study was supported by a Grant COFIN 2009 from the MIUR, Italy [Grant n° 20094C2H2M_003].
References 1. Tian X, Kang DS, Benovic JL (2014) b-arrestins and G proteincoupled receptor trafficking. Handb Exp Pharmacol 219:173–186 2. Violin JD, Lefkowitz RJ (2007) b-arrestin-biased ligands at seven-transmembrane receptors. Trends Pharmacol Sci 28:416– 422 3. DeFea KA (2011) Beta-arrestins as regulators of signal termination and transduction: how do they determine what to scaffold? Cell Signal 23:621–629 4. Fan H, Bitto A, Zingarelli B, Luttrell LM, Borg K, Halushka PV, Cook JA (2010) Beta-arrestin-2 negatively regulates sepsisinduced inflammation. Immunology 130:344–351 5. Fan H (2013) b-arrestins 1 and 2 are critical regulators of inflammation. Innate Immun 20:451–460 6. Jiang D, Xie T, Liang J, Noble PW (2013) Beta-Arrestins in the immune system. Prog Mol Biol Transl Sci 118:359–393
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208 7. Li P, Cook JA, Gilkeson GS, Luttrell LM, Wang L, Borg KT, Halushka PV, Fan H (2011) Increased expression of beta-arrestin 1 and 2 in murine models of rheumatoid arthritis: isoform specific regulation of inflammation. Mol Immunol 49:64–74 8. Ospelt C, Gay S (2008) The role of resident synovial cells in destructive arthritis. Best Pract Res Clin Rheumatol 22:239–252 9. Maquart FX, Bellon G, Pasco S, Monboisse JC (2005) Matrikines in the regulation of extracellular matrix degradation. Biochimie 87:353–360 10. Adair-Kirk TL, Senior SM (2008) Fragments of extracellular matrix as mediators of inflammation. Int J Biochem Cell Biol 40:1101–1110 11. Jiang D, Liang J, Noble PW (2011) Hyaluronan as an immune regulator in human diseases. Physiol Rev 91:221–264 12. Jiang D, Liang J, Noble PW (2007) Hyaluronan in tissue injury and repair. Annu Rev Cell Dev Biol 23:435–461 13. Termeer CC, Hennies J, Voith U, Ahrens T, Weiss JM, Prehm P, Simon JC (2000) Oligosaccharides of hyaluronan are potent activators of dendritic cells. J Immunol 165:1863–1870 14. Campo GM, Avenoso A, Campo S, D’Ascola A, Traina P, Calatroni A (2009) Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA. Biochim Biophys Acta 1790:1353–1367 15. Li Z, Potts EN, Piantadosi CA, Foster WM, Hollingsworth JW (2010) Hyaluronan fragments contribute to the ozone-primed immune response to lipopolysaccharide. J Immunol 185:6891– 6898 16. Campo GM, Avenoso A, Campo S, D’Ascola A, Nastasi G, Calatroni A (2010) Small hyaluronan oligosaccharides induce inflammation by both toll-like-4 and CD44 receptors in human chondrocytes. Biochem Pharmacol 80:480–490 17. Campo GM, Avenoso A, D’Ascola A, Scuruchi M, Prestipino V, Calatroni A, Campo S (2012) 6-Mer hyaluronan oligosaccharides increase IL-18 and IL-33 production in mouse synovial fibroblasts subjected to collagen-induced arthritis. Innate Immun 18:675–684 18. Campo GM, Avenoso A, D’Ascola A, Prestipino V, Scuruchi M, Nastasi G, Calatroni A, Campo S (2012) Hyaluronan differently modulates TLR-4 and the inflammatory response in mouse chondrocytes. BioFactors 38:69–76 19. Campo GM, Avenoso A, D’Ascola A, Prestipino V, Scuruchi M, Nastasi G, Calatroni A, Campo S (2012) The stimulation of adenosine A2 receptor reduces inflammatory response in mouse articular chondrocytes treated with hyaluronan oligosaccharides. Matrix Biol 31:338–351 20. Campo GM, Avenoso A, D’Ascola A, Prestipino V, Scuruchi M, Nastasi G, Calatroni A, Campo S (2013) 4-Mer hyaluronan oligosaccharides stimulate inflammation response in synovial fibroblasts in part via TAK-1 and in part via P38-MAPK. Curr Med Chem 20:1162–1172 21. Campo GM, Avenoso A, D’Ascola A, Scuruchi M, Nastasi G, Micali A, Puzzolo D, Pisani A, Calatroni A, Campo S (2013) The SOD mimic MnTM-2-PyP(5 ?) reduces hyaluronan degradationinduced inflammation in mouse articular chondrocytes stimulated with Fe(II) plus ascorbate. Int J Biochem Cell Biol 45:1610–1619
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Mol Cell Biochem (2015) 399:201–208 22. Taylor KR, Yamasaki K, Radek KA, Di Nardo A, Goodarzi H, Golenbock D, Beutler B, Gallo RL (2007) Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on toll-like receptor 4, CD44, and MD-2. J Biol Chem 282:18265–18275 23. Jiang D, Liang J, Fan J, Yu S, Chen S, Luo Y, Prestwich GD, Mascarenhas MM, Garg HG, Quinn DA, Homer RJ, Goldstein DR, Bucala R, Lee PJ, Medzhitov R, Noble PW (2005) Regulation of lung injury and repair by Toll-like receptors and hyaluronan. Nat Med 11:1173–1179 24. Campo GM, Avenoso A, Campo S, D’Ascola A, Nastasi G, Calatroni A (2010) Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes. Biochimie 92:204–215 25. Campo GM, Avenoso A, D’Ascola A, Nastasi G, Micali A, Puzzolo D, Pisani A, Prestipino V, Scuruchi M, Calatroni A, Campo S (2013) Combined treatment with hyaluronan inhibitor Pep-1 and a selective adenosine A2 receptor agonist reduces inflammation in experimental arthritis. Innate Immun 19:462–478 26. Sun JS, Wu CX, Tsuang YH, Chen LT, Sheu SY (2006) The in vitro effects of dehydroepiandrosterone on chondrocyte metabolism. Osteoarthr Cartil 14:238–249 27. Walker JK, Fong AM, Lawson BL, Savov JD, Patel DD, Schwatz DA, Lefkowitz RJ (2003) Beta-arrestin-2 regulates the development of allergic asthma. J Clin Invest 112:566–574 28. Gao H, Sun Y, Wu Y, Luan B, Wang Y, Qu B, Pei G (2004) Identification of b-arrestin2 as a G-protein-coupled receptorstimulated regulator of NF-kB pathways. Mol Cell 14:303–317 29. Witherow DS, Garrison TR, Miller WE, Lefkowitz RJ (2004) bArrestin inhibits NF-kB activation by means of its interaction with the NF-kB inhibitor IkBa. Proc Natl Acad Sci USA 101: 8603–8607 30. Sun Y, Cheng Z, Ma L, Pei G (2002) b-arrestin-2 is critically involved in CXCR4-mediated chemotaxis and this is mediated by its enhancement of p38MAPK activation. J Biol Chem 277: 49212–49219 31. Noss EH, Brenner MB (2008) The role and therapeutic implications of fibroblast-like synoviocytes in inflammation and cartilage erosion in rheumatoid arthritis. Immunol Rev 223:252–270 32. Campo GM, Avenoso A, D’Ascola A, Scuruchi M, Prestipino V, Nastasi G, Calatroni A, Campo S (2012) Adenosine A2A receptor activation and hyaluronan fragment inhibition reduce inflammation in mouse articular chondrocytes stimulated with interleukin1b. FEBS J 279:2120–2133 33. Campo GM, Avenoso A, D’Ascola A, Scuruchi M, Prestipino V, Nastasi G, Calatroni A, Campo S (2012) The inhibition of hyaluronan degradation reduced pro-inflammatory cytokines in mouse synovial fibroblasts subjected to collagen-induced arthritis. J Cell Biochem 113:1852–1867 34. Campo GM, Avenoso A, D’Ascola A, Prestipino V, Scuruchi M, Nastasi G, Calatroni A, Campo S (2012) Inhibition of hyaluronan synthesis reduced inflammatory response in mouse synovial fibroblasts subjected to collagen-induced arthritis. Arch Biochem Biophys 518:42–52
Beta-arrestin-2 negatively modulates inflammation response in mouse chondrocytes induced by 4-mer hyaluronan oligosaccharide Giuseppe M. Campo • Angela Avenoso Angela D’Ascola • Michele Scuruchi • Alberto Calatroni • Salvatore Campo
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Received: 24 July 2014 / Accepted: 9 October 2014 / Published online: 16 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Beta-arrestin-2 is an adaptor protein that terminates G protein activation and seems to be involved in the modulation of the inflammatory response. Small hyaluronan (HA) fragments, such as 4-mer HA oligosaccharides, are known to interact with the toll-like receptor-4 (TLR-4) with consequent activation of the nuclear factor kappaB (NF-kB) that in turn stimulates the inflammation response. NF-kB activation is mediated by different pathways, in particular by the transforming growth factoractivated kinase-1 (TAK-1). Conversely, increased levels of protein kinase A (PKA), induced by cyclic adenosine monophosphate (cAMP), seem to inhibit NF-kB activation. We studied the involvement and role of beta-arrestin-2 in mouse chondrocytes stimulated with 4-mer HA fragments. The exposure of chondrocytes to 4-mer HA produced a significant up-regulation in TLR-4, cAMP, beta-arrestin-2, TAK-1, protein 38 mitogen-activated protein kinase (p38MAPK), and PKA, both in terms of mRNA expression and of the related protein levels. NF-kB was significantly activated, thereby producing the transcription of proinflammatory mediators, including tumor necrosis factor
G. M. Campo (&) A. Avenoso A. D’Ascola A. Calatroni S. Campo Department of Biomedical Sciences and Morphological and Functional Images, Section of Medical Biotechnologies and Preventive Medicine, School of Medicine, University of Messina, Policlinico Universitario, Torre Biologica, 5° piano, Via C. Valeria, 98125 Messina, Italy e-mail: [email protected] M. Scuruchi Department of Clinical and Experimental Medicine, School of Medicine, University of Messina, Policlinico Universitario, Torre Biologica, 5° piano, Via C. Valeria, 98125 Messina, Italy
alpha, interleukin-6, and interleukin-17. The treatment of 4-mer HA-stimulated chondrocytes with antibodies against beta-arrestin-2 and/or a specific PKA inhibitor, significantly increased the inflammatory response, while the treatment with a specific p38MAPK inhibitor significantly reduced the inflammatory response. Interestingly, the antiinflammatory action exerted by beta-arrestin-2 appeared to be mediated in part through the direct inhibition of p38MAPK, preventing NF-kB activation, and in part through cAMP and PKA activation primed by G protein signaling, which exerted an inhibitory effect on NF-kB. Taken together, these results could be useful for future anti-inflammatory strategies. Keywords Hyaluronan NF-kB TLR-4 Beta-arrestin2 Cytokines Inflammation Chondrocytes
Introduction b-arrestins are intracellular scaffolding proteins that modulate specific cell signaling pathways. The arrestin family consists of four isoforms, two expressed only in the visual system and two that are ubiquitously expressed, the latter named b-arrestins 1 and 2 [1]. They play a critical role in the regulation of the receptor signaling of seven-transmembrane receptors (7MSRs) phosphorylated by G protein-coupled receptor kinases [2]. More recently, b-arrestins have been shown to signal independently of heterotrimeric G protein coupling, suggesting that they represent a direct signal transducer of G protein-coupled receptors (GPCRs). However, the list of b-arrestin binding partners continues to expand. Among them, mitogen-activated-protein kinases (MAPKs), the serine/threonine kinase Akt, transforming growth factor-activated kinase-1 (TAK-1), and exchange
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proteins activated by cAMP (EPAC) appear to be deeply involved [3]. Recently, it has been reported that the expression of b-arrestins-1 and 2 is differentially regulated in immune cells and tissues in response to specific inflammatory stimuli; they, therefore, seem to play a critical regulatory role in the inflammatory response [4–6]. It has also been reported that b-arrestins-1 and 2 play a key role in experimental arthritis, and in particular that b-arrestin-1 positively regulates the inflammatory response while barrestin-2 inhibits inflammation [7]. Degeneration of joint cartilage is a key feature of arthritis. However, the disease is also associated with concomitant changes in synovium and subchondral bone metabolism, causing inflammation of the chondrocyte membrane in the affected joint [8]. There are no effective long-term treatments to counteract arthritis, and current research is focused on understanding how the imbalance between specific extracellular matrix (ECM) molecules may influence progression of the disease. Evidence is emerging to show that endogenous ECM molecules supply signals to damaged cartilage and synovial tissue in order to promote further cartilage degradation [9–11]. High molecular weight hyaluronan (HA) is found in normal, intact, healthy cartilage and contributes to local homeostasis by suppressing cell proliferation, migration, angiogenesis, inflammation, and immunogenicity [12]. Evidence from different studies has shown that the prime of inflammation resulting from cell damage produces an increased expression of growth factors and cytokines which promotes the degradation of the ECM [13–21]. HA degradation products of small size have been shown to stimulate inflammatory responses in different cell types. HA fragmentation also results in smaller oligosaccharides that are angiogenic, inflammatory, and immunogenic in a sizedependent manner [17, 18, 22]. Previous investigations have also shown that small fragments of HA are able to interact with the toll-like receptor-4 (TLR-4), thereby stimulating inflammation or increasing the inflammatory mechanism previously induced by other agents in different cell types [23, 24]. Hence, the generation of low molecular weight HA in pathologies may act as an endogenous danger signal leading to the activation of both innate and acquired immunity. HA fragments may prime inflammation via the TLR-4 which eventually stimulates nuclear factor kappaB (NF-kB) activation. NF-kB, in turn, translocates into the nucleus where it may prime the transcription of various inflammatory mediators, e.g., tumor necrosis-alpha (TNFa), interleukin-1beta (IL-1b), interleukin-6 (IL-6), interleukin-17 (IL-17), inducible nitric oxide synthase (iNOS), and matrix metalloproteases (MMPs) [17, 18, 23–25]. In this study, we investigated the role of b-arrestin-2 in a model of 4-mer HA oligosaccharide-induced inflammatory
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response on mouse chondrocytes obtained from normal DBA/J1 mice. We also studied the effects of the inhibition of b-arrestin-2 and/or protein kinase A (PKA) and/or p38MAPK in order to ascertain the actual role played by these proteins in the inflammatory pathway induced by 4-mer HA stimulation.
Materials and methods Animals Male mice DBA/1 J 6–7 weeks old with a mean weight of 25–30 g were used in our study. Mice, purchased from Charles River Laboratories Srl (Lecco, Italy), were maintained under climate-controlled conditions with a 12-h light/dark cycle. The animals were fed standard rodent chow and provided water ad libitum. The health status of the animal colony was monitored in accordance with Italian Veterinary Board guidelines.
Materials 4-mer HA oligosaccharide as sodium salt (cat CSR-11006) was obtained from Cosmo Bio Co Ltd (Tokyo, Japan). Mouse TLR-4 (cat. SEA753Mu) and mouse IL-17 (cat. SEA063Mu) commercial ELISA kits were purchased from USCN Life Science Inc (Wuhan, China). Mouse TNF-a (cat. IB49688) and IL-6 (cat. 27768) commercial ELISA kits were provided by Immuno-Biological Laboratories Inc (Minneapolis, MN, USA). The mouse b-arrestin-2 (cat. ABIN810988) commercial ELISA kit was supplied by Antibodies-online Inc (Atlanta, GA, USA). The mouse PKA kinase activity kit was obtained from Enzo Life Sciences International Inc (Plymouth Meeting, PA, USA). The P38-MAPK-a (cat. ab126425) commercial ELISA kit was supplied by Abcam plc (Cambridge, UK). Mouse b-arrestin-2 siRNA (cat. sc-29743) was supplied by Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Mouse PKA C-a siRNA (cat. 6406) was obtained from Cell Signaling Technology Inc (Danvers, MA, USA). Mouse p38MAPK-a siRNA SignalSilence was also supplied by Cell Signaling Technology Inc (Danvers, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Lglutamine, penicillin/streptomycin, trypsin–EDTA solution, and phosphate buffered saline (PBS) were obtained from Life Technologies Inc Gibco/Brl Division (Grand Island, NY, USA). All cell culture plastics were obtained from Falcon Plastics (Oxnard, CA, USA). RNase, proteinase K, protease inhibitor cocktail, sodium dodecylsulphate (SDS), and all other general laboratory chemicals were obtained from Sigma-Aldrich Srl (Milan, Italy).
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Chondrocyte cultures Mice were killed by cervical dislocation, and whole patellae were dissected from the hind knee joint. Chondrocytes were released from articular cartilage after being digested with 0.2 % pronase for 1 h. After washing with PBS, cartilage was further digested for 3 h with PBS containing 0.1 % collagenase in DMEM [26]. Chondrocytes were cultured in 75 cm2 plastic flasks containing 15 ml DMEM to which 10 % FBS, L-glutamine (2.0 mM), and penicillin/streptomycin (100 U/ml, 100 lg/ml) were added. siRNA treatment For siRNA experiments, 4 9 106 cells were transfected with b-arrestin-2, PKA C-a, and p38MAPK-a siRNAs following modified siRNA Transfection Protocols (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA, and Cell Signaling Technology Inc, Danvers, MA, USA). PKA C-a is a catalytic subunit of PKA that is indispensable for PKA activation; therefore, total inhibition of PKA activity occurs by inhibiting PKA C-a activity. P38MAPK-a is an active isoform of p38MAPK expressed by chondrocytes. Scrambled siRNA was used under the same conditions as a negative control. After transfection, cells were seeded in six-well plates in 10 % FCS/DMEM and allowed to adhere for 12 h. Culture medium was then replaced with fresh 10 % FCS/DMEM for a further 12-h period. Cells were serum starved for 24 h before 4-mer HA was added. Chondrocyte treatment Chondrocytes were cultured in six-well culture plates at a density of 1.3 9 105 cells/well. Twelve hours after plating (time 0), the culture medium was replaced with 2.0 mL of fresh medium containing 4-mer HA (40 lg/mL) and/or b-arrestin-2 siRNA (reported in Figures as b-ARR2siRNA), and/or PKA siRNA (reported in Figures as PKAsiRNA) and/or p38MAPK-a siRNA (reported in Figures as p38MAPKsiRNA). Finally, the cells and medium underwent biochemical evaluation 48 h after the last treatment. The study, therefore, included the following groups of chondrocytes: (1) CTRL; (2) b-ARR2siRNA; (3) PKAsiRNA; (4) p38MAPKsiRNA; (5) 4-mer HA; (6) 4-mer HA ? b-ARR2siRNA; (7) 4-mer HA ? PKAsiRNA; (8) 4-mer HA ? p38MAPKsiRNA; (9) 4-mer HA ? b-ARR2siRNA ? PKAsiRNA; (10) 4-mer HA ? b-ARR2siRNA ? p38-MAPKsiRNA; (11) 4-mer HA ? PKAsiRNA ? p38MAPKsiRNA; and (12) 4-mer HA ? b-ARR2siRNA ? PKAsiRNA ? p38MAPKsiRNA.
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RNA isolation, cDNA synthesis, and real-time quantitative PCR amplification Total RNA was isolated from chondrocytes for reversePCR real-time analysis of TLR-4, b-arrestin-2, PKA C-a, p38MAPK, TNF-a, IL-6, and IL-17 (RealTime PCR system, Mod. 7500, Applied Biosystems Inc, USA) using an Omnizol Reagent Kit (Euroclone Ltd, West York, UK). The amounts of specific mRNA in samples were calculated using the DDCT method. The mean value of normal cartilage target levels became the calibrator (one per sample), and the results are expressed as the n-fold difference relative to normal controls (relative expression levels). NF-kB p50/65 transcription factor assay NF-kB p50/65 DNA binding activity in nuclear extracts of chondrocytes was evaluated in order to measure the degree of NF-kB activation. Analysis was performed in line with the manufacturer’s protocol for a commercial kit (NF-kB p50/65 EZ-TFA Transcription Factor Assay Colorimetric, cat. n°70–510, Millipore Corporation, Billerica, MA, USA). Values are expressed as relative optical density (OD)/mg protein. TLR-4, b-arrestin-2, IL-17, p38MAPK-a, TNF-a, and IL-6 ELISA assay The analysis of TLR-4, b-arrestin-2, IL-17, p38MAPK, TNF-a, and IL-6 was carried out using a specific commercial kit in line with the manufacturer’s protocols. TLR4 values are expressed as ng/mg protein, while b-arrestin-2, IL-17, p38MAPK, TNF-a, and IL-6 values are expressed as pg/mg protein. PKA kinase assay PKA kinase activity was assayed using a commercial ELISA kit that utilizes a specific synthetic peptide as a substrate for PKA and a polyclonal antibody that recognizes the phosphorylated form of the substrate. PKA values are expressed as ng/mg protein. Protein analysis The amount of protein was determined using the Bio-Rad protein assay system (Bio-Rad Lab. Inc, Richmond, CA, USA) with bovine serum albumin as a standard.
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Data are expressed as means ± S.D. of no less than seven experiments for each test. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test. The statistical significance of differences was set at p \ 0.05. Statement of animal care The studies reported in this manuscript were carried out in accordance with the Declaration of Helsinki and NIH guidelines for the Care and Use of Laboratory Animals.
TLR-4, b-arrestin-2, PKA, and p38MAPK-a mRNA expression (Fig. 1, panel a; Fig. 2, panels a, b, and c), and related protein concentrations (Fig. 1, panel b; Fig. 2, panels d, e, and f) were assayed in order to estimate the degree of TLR-4 activation in chondrocytes stimulated with 4-mer HA and/or treated with b-arrestin-2 siRNA, and/or PKA siRNA, and/or p38MAPK-a siRNA. The data showed a significant increase in both mRNA expression and protein synthesis of TLR-4 in all groups of cells exposed to 4-mer HA (Fig. 1, panels a and b). No changes in TLR-4 expression and synthesis were observed in chondrocytes treated with b-arrestin-2 siRNA, PKA siRNA, and with p38MAPK-a siRNA, both in cells treated with 4-mer HA or those not receiving this treatment. 4-mer HA treatment was also able to increase the mRNA expression and protein levels of b-arrestin-2, PKA, and p38MAPK-a to a significant extent (Fig. 2, panels a, b, c, d, e, and f). The increase in activity of b-arrestin-2, PKA, and p38MAPK-a induced by 4-mer HA was reduced when cells were treated with each specific siRNA (Fig. 2, panels a, b, c, d, e, and f). Notably, chondrocytes stimulated with 4-mer HA and pre-treated with b-Arrestin-2 siRNA showed reduced PKA activity and heightened activity of p38MAPK-a in terms of both mRNA expression and protein concentrations (Fig. 2, panels b, c and e, f), although the action of b-arrestin-2 siRNA on p38-MAPK-a seemed to be less marked than the one exerted on PKA. NF-kB activation Figure 3 shows the changes in the NF-kB p50/p65 heterodimer translocation over the course of the experiment in chondrocytes stimulated with 4-mer HA and/or treated
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Fig. 1 Effect of 4-mer HA and/or b-arrestin-2 siRNA (b-ARR2siRNA), and/or PKA C-a siRNA (PKAsiRNA), and/or p38MAPK-a (p38MAPKsiRNA) treatment on mouse articular chondrocyte TLR-4 mRNA expression (a) and related protein production (b). Biochemical evaluation was performed 24 h after the last treatment. Values are the mean ± SD of no less than seven experiments and are expressed as the n-fold increase with respect to Controls (a) and as ng/mg protein (b) for the TLR-4 levels. °p \ 0.001 versus control
with b-arrestin-2 siRNA, and/or PKA siRNA, and/or p38MAPK-a siRNA. The stimulation of chondrocytes with 4-mer HA produced a marked activation of the NF-kB (Fig. 3). The single treatment of chondrocytes with b-arrestin-2 siRNA or PKA siRNA or p38MAPK-a siRNA had no effect on NF-kB activation. In contrast, the silencing of these proteins in cells stimulated with 4-mer HA was able to modulate NF-kB activation in different ways. In particular, the silencing of b-arrestin-2 produced a significant increase in NF-kB activation. The silencing of PKA also increased NF-kB activation, but to a greater extent than barrestin-2 siRNA, while the silencing of p38MAPK-a significantly reduced NF-kB activation. The concomitant silencing of PKA and b-arrestin-2 increased NF-kB activation to a greater extent than the individual silencing, and produced the greatest increment in NF-kB activation (Fig. 3). The concomitant silencing of p38MAPK-a and b-
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Fig. 2 Effect of 4-mer HA and/or b-arrestin-2 siRNA (b-ARR2siRNA), and/or PKA C-a siRNA (PKAsiRNA), and/or p38MAPK-a (p38MAPKsiRNA) treatment on mouse articular chondrocyte barrestin-2, PKA C-a, and p38MAPK-a mRNA expression (a, b, and c) and related protein production (d, e, and f). Biochemical evaluation was performed 24 h after the last treatment. Values are the
mean ± SD of no less than seven experiments and are expressed as the n-fold increase with respect to controls (a, b, and c) and as pg/mg protein (d) for the b-arrestin-2 levels, as ng/mg protein (e) for the PKA C-a levels, and as pg/mg protein (f) for the p38MAPK-a levels. °p \ 0.001 versus controls; *p \ 0.001 versus 4-mer HA; #p \ 0.005 versus 4-mer HA
arrestin-2 or of p38MAPK-a and PKA still resulted in augmented NF-kB activation, although to a lesser extent (Fig. 3).
p38MAPK-a siRNA, prior to 4-mer HA exposure, produced a significant reduction in the transcription of the inflammatory cytokines. In contrast, the silencing of barrestin-2 resulted in a significant increase in NF-kB activation and inflammatory cytokines production. The silencing of PKA also increased NF-kB activation and inflammatory cytokines production, but to a greater extent with respect to that obtained by b-arrestin-2 silencing. The concomitant silencing of b-arrestin-2 and PKA produced the greatest effect in terms of increased cytokine transcription compared to 4-mer HA treatment alone. (Figure 4, panels a, b, c, d, e, and f).
TNF-a, IL-6, and IL-17 mRNA expression and protein levels TNF-a, IL-6, and IL-17 mRNA evaluation (Fig. 4, panels a, b, and c), and ELISA assay (Fig. 4, panels d, e, and f) confirmed the above findings obtained for NF-kB. The results showed a marked increase in the expression and protein synthesis of the three inflammatory cytokines in cells stimulated with 4-mer HA. In line with the results for NF-kB, the pre-treatment of chondrocytes with b-arrestin-2 siRNA, and/or PKA siRNA, and/or p38MAPK-a siRNA, prior to 4-mer HA exposure, markedly changed the effect of 4-mer HA on inflammatory cytokine transcription. In particular, the pre-treatment of chondrocytes with
Discussion There is accumulating evidence that b-arrestin-2 is functionally involved in different important immune responses
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Mol Cell Biochem (2015) 399:201–208 NF-kB activation (OD450nm /mg protein)
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Fig. 3 Effect of 4-mer HA and/or b-arrestin-2 siRNA (b-ARR2siRNA), and/or PKA C-a siRNA (PKAsiRNA), and/or p38MAPK-a (p38MAPKsiRNA) treatment on mouse articular chondrocyte NF-kB p50/65 transcription factor DNA binding activity. White bars represent the p/50 subunit, gray bars represent the p/65 subunit. Biochemical evaluations were performed 24 h after the last treatment. Values are the mean ± SD of no less than seven experiments and are expressed as optical density at k 450 nm/mg protein of nuclear extract. °p \ 0.001 versus controls; *p \ 0.001 versus 4-mer HA; § p \ 0.005 versus 4-mer HA; #p \ 0.001 versus 4-mer HA; £ p \ 0.001 versus 4-mer HA; & p \ 0.001 versus 4-mer HA; $ p \ 0.001 versus 4-mer HA; ^p \ 0.001 versus 4-mer HA
due to its adaptor and scaffold functions [5, 27]. b-arrestin2 may bind and prevent degradation of the factor IkBa that inhibits NF-kB activation [28, 29]. It has also been reported that b-arrestin-2 is involved in the regulation of C-X-C chemokine receptor type 4 (CXCR4)-induced chemotaxis by activating the ASK1/p38MAPK pathway [30]. Recent studies have reported that b-arrestin-2 negatively regulates polymicrobial sepsis-induced inflammation [4], as well as acting as an anti-inflammatory endogenous agent in experimental arthritis [7]. Activated fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) play a major role by activating chondrocytes and producing joint tissue destruction. FLS become activated through growth factors, chemokines, cytokines, and tissue degradation products such as endogenous TLR ligand HA [31, 32]. Consequently, activated chondrocytes secrete numerous pro-inflammatory mediators and degrading enzymes which greatly contribute to priming and maintaining the inflammation cascade and producing cartilage injury [33, 34]. In the present study, we obtained a marked increment in the mRNA expression and protein synthesis of TLR-4 in chondrocytes stimulated with 4-mer HA. The treatment of cells with 4-mer HA produced a significant increase in b-arrestin-2, PKA, and p38MAPK-a activities.
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Interestingly, chondrocytes stimulated with 4-mer HA and pre-treated with b-arrestin-2 siRNA showed reduced PKA activity and increased p38MAPK-a activity, although the action of b-arrestin-2 on p38-MAPK-a seems to be less marked than the one exerted on PKA. Based on these data, it is, therefore, conceivable that b-arrestin-2 exerted an inhibitory effect on p38MAPK-a and a stimulatory effect on PKA. Chondrocyte exposition to 4-mer HA resulted in a massive activation of the NF-kB. It is useful to measure NF-kB activity in order to evaluate the onset of the inflammatory process since the NF-kB factor can be activated by different pathways that in turn may converge in the transcription of several genes that contribute to prime/ amplify inflammation. The silencing of b-arrestin-2, PKA, and p38MAPK-a in chondrocytes exposed to 4-mer HA modulated NF-kB activation in different ways. Notably, the silencing of barrestin-2 produced a significant increase in NF-kB activation in addition to the increment exerted by the 4-mer HA. The silencing of the PKA also increased NF-kB activation, and to a greater extent than b-arrestin-2 siRNA. However, the silencing of p38MAPK-a significantly reduced NF-kB activation. This result could mean that, in this case, b-arrestin-2 exerts an inhibitory effect on the 4-mer HA-induced activation of NF-kB. In fact, the silencing of b-arrestin-2 increased NF-kB activation, as PKA was not activated, or partially activated, by other pathways, while p38MAPK was not inhibited. Hence, b-arrestin-2 could have mediated PKA stimulation and p38MAPK-a inhibition. The evaluation of the pro-inflammatory cytokines supported the above data obtained for NF-kB. In fact, at first a significant increment in the activity of all cytokines considered was found in chondrocytes exposed to 4-mer HA. Subsequently, the silencing of b-arrestin-2, PKA, and p38MAPK-a, prior to 4-mer HA treatment, was able to modulate the effect of 4-mer HA stimulation on inflammatory cytokine transcription. In particular, the silencing of p38MAPK-a siRNA, prior to 4-mer HA exposition, produced a significantly lower transcription of cytokines, compared to the effect of 4-mer HA alone. This is in line with the evidence that p38MAPK-a induced NF-kB activation. Conversely, the silencing of b-arrestin-2 produced an additional increase in cytokine transcription, indicating an inhibitory effect of b-arrestin-2 on NF-kB activation. The silencing of PKA was also able to increase cytokine production further, in addition to the effect of 4-mer HA alone. This effect was greater than that achieved by barrestin-2 silencing. This could be explained by the fact that PKA exerted an inhibitory effect on NF-kB, and its activation may be due to different pathways besides barrestin-2.
Mol Cell Biochem (2015) 399:201–208
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Fig. 4 Effect of 4-mer HA and/or b-arrestin-2 siRNA (b-ARR2siRNA), and/or PKA C-a siRNA (PKAsiRNA), and/or p38MAPK-a (p38MAPKsiRNA) treatment on mouse articular chondrocyte TNF-a, IL-6, and IL-17 mRNA expression (a, b and c) and related protein production (d, e and f). Biochemical evaluation was performed 24 h after the last treatment. Values are the mean ± SD of no less than seven experiments and are expressed as the n-fold increase with
respect to controls (a, b and c) and as pg/mg protein (d, e and f) for the TNF-a, IL-6, and the IL-17 levels. °p \ 0.001 versus controls; *p \ 0.001 vs 4-mer HA; §p \ 0.005 versus 4-mer HA; #p \ 0.001 versus 4-mer HA; £p \ 0.001 versus 4-mer HA; & p \ 0.001 versus 4-mer HA; $p \ 0.001 versus 4-mer HA; ^p \ 0.001 versus 4-mer HA
Taken together, the body of evidence accumulated so far suggests the functional involvement of b-arrestin-2 in many inflammatory and autoimmune diseases. This evidence could be considered a strength of this study, while the different multitude of pathways that may activate the NF-kB could represent a limit of this paper, as other not investigated mechanism in the present study could take part in the experiment. However, although it is not yet known precisely how extensively b-arrestin-2 is involved, this topic could be further explored by viewing b-arrestin-2 as a potential diagnostic biomarker or even a prognostic indicator for different diseases. This would contribute to efforts to provide new anti-inflammatory strategies and tools and allow their development so that they are able to modulate these intricate inflammation pathways simultaneously. Such an approach could be useful in devising future strategies in order to tackle inflammation more effectively than the currently available therapeutic drugs.
Acknowledgments This study was supported by a Grant COFIN 2009 from the MIUR, Italy [Grant n° 20094C2H2M_003].
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