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Address correspondence and reprint requests to: H. Meng, Division of Bioengineering, School of Engineering and Materials Science, Queen Mary University of London, London, Mile End Road, E1 4NS, UK. Tel: 44-7544983545 (mobile).
To clarify the role of YAP in modulating cartilage inflammation and degradation and the involvement of primary cilia and associated intraflagellar transport (IFT).
Isolated primary chondrocytes were cultured on substrates of different stiffness (6–1000 kPa) or treated with YAP agonist lysophosphatidic acid (LPA) or YAP antagonist verteporfin (VP), or genetically modified by YAP siRNA, all ± IL1β. Nitric oxide (NO) and prostaglandin E2 (PGE2) release were measured to monitor IL1β response. YAP activity was quantified by YAP nuclear/cytoplasmic ratio and percentage of YAP-positive cells. Mechanical properties of cartilage explants were tested to confirm cartilage degradation. The involvement of primary cilia and IFT was analysed using IFT88 siRNA and ORPK cells with hypomorphic mutation of IFT88.
Treatment with LPA, or increasing polydimethylsiloxane (PDMS) substrate stiffness, activated YAP nuclear expression and inhibited IL1β-induced release of NO and PGE2, in isolated chondrocytes. Treatment with LPA also inhibited IL1β-mediated inflammatory signalling in cartilage explants and prevented matrix degradation and the loss of cartilage biomechanics. YAP activation reduced expression of primary cilia, knockdown of YAP in the absence of functional cilia/IFT failed to induce an inflammatory response.
We demonstrate that both pharmaceutical and mechanical activation of YAP blocks pro-inflammatory signalling induced by IL1β and prevents cartilage breakdown and the loss of biomechanical functionality. This is associated with reduced expression of primary cilia revealing a potential anti-inflammatory mechanism with novel therapeutic targets for treatment of osteoarthritis (OA).
In articular chondrocytes, the stiffness of the extracellular environment modulates multiple aspects of cell behaviour, including cell spreading and cytoskeletal organisation, cellular mechanics, proliferation, extracellular matrix production, dedifferentiation, and chondrogenic phenotype
using a murine model of experimental OA showed that YAP activation attenuates cartilage degradation. The authors suggest that this occurs due to inhibition of the pro-inflammatory NF-κB pathway. However, conflicting in vivo studies in mice report that inhibition of YAP with Verteporfin (VP) reduced OA progression
. Consequently, whether YAP has a protective effect against OA remains controversial and the cellular mechanism is unclear.
Primary cilia are specialised, slender microtubule organelles that typically protrude from the cell membrane of most eukaryotic cells. Primary cilia are involved in cell signalling pathways, most notably Hedgehog, Wnt, and growth factor signalling
. However, no studies have investigated whether YAP induced inhibition of ciliogenesis modulates pro-inflammatory signalling.
This study tests the hypothesis that activation of YAP suppresses the inflammatory response to IL1β and that this involves a reduction in primary cilia expression. We examine this mechanism in articular chondrocytes and show that mechanical or pharmaceutical activation of YAP inhibits chondrocyte inflammatory signalling and prevents cartilage matrix breakdown and tissue softening. We show the anti-inflammatory response to YAP activation is accompanied by a reduced expression of chondrocyte primary cilia, the inflammatory response to IL1 and its regulation by YAP is dependent on cilia and IFT. These findings suggest a novel mechanistic pathway and providing potential therapeutic targets for treatment of inflammatory joint disease.
Method and materials
Cell and tissue culture
Our studies used isolated primary bovine articular chondrocytes, wild type and homozygous Tg737ORPK mutant mouse cell line and full depth bovine cartilage explants (for details see SI).
Preparation of polydimethylsiloxane (PDMS) substrates
According to the manufacturer's instructions and previous studies
, PDMS substrates were prepared using 184 Silicone Elastomer kit (1317318, SYLGARD) and 527 Silicone Dielectric kit (1696742, SYLGARD) and functionalised with 10 μg/ml Fibronectin (F0162, Sigma) before seeding with chondrocytes(for details see SI).
Characterization of cartilage mechanical properties and PDMS gels
Cartilage mechanical properties was quantified in uniaxial unconfined compressive using a universal mechanical testing machine with a 10 N load cell (Instron 3342, for details see SI).
Immunofluorescent staining and confocal microscopy
Samples were fixed for 10 min with 4% paraformaldehyde (PFA) and permeabilised with 0.5% Triton-X/PBS. Samples were blocked with 5% PBS for 1 h and incubated at 4°C overnight with primary antibodies to Arl13b and YAP (SI Table 3), followed by secondary fluorescent conjugated secondary antibodies. Cell nuclei were counterstained with DAPI (D9542, Sigma).
Primary cilia were visualised using a Zeiss LSM710 confocal microscope with x63/1,4 NA oil immersion objective. Additional imaging of YAP and actin was performed using a Leica DMi8 epifluorescence microscopy with a ×63/1.4 NA objective (for details see SI).
Quantification of YAP and primary cilia expression
Activities of YAP signalling was based on quantification of YAP immunofluorescence to determine the YAP Nuclear/Cytoplasmic ratio and percentage of YAP positive cells using Image J according to previous studies
. Primary cilia length and prevalence were also quantified using Image J as in previous studies (for details see SI).
Biochemical analysis of inflammatory signalling and matrix degradation
Our studies used the Griess assay for Nitric oxide (NO) quantification. Prostaglandin E2 (PGE2) was measured using an ELISA standard kit (KGE004B, R&D Systems) and sulfated glycosaminoglycan (sGAG) was quantified by Dimethylmethylene Blue Assay (DMMB, 341088 Sigma Aldrich) (for details see SI).
Atomic force microscopy
To measure cell stiffness as an indicator of actin tension, force–indentations curves were obtained on individual chondrocytes in monolayer culture using an atomic force microscopy (AFM) (JPK NanoWizard 4). These studies adopted well-established methodologies based on our previous studies
Cells were lysed in RIPA buffer (R0278, Sigma Aldrich) and total protein was quantified by bicinchoninic acid assay (BCA) assay. After SDS-PAGE, protein was transferred to polyvinylidene difluoride (PVDF) membranes followed by blocking and primary and secondary antibodies incubation. Blots were then imaged using iBright imaging system (FL1500, ThermoFisher). For details see SI.
Data analysis was performed using GraphPad Prism 8 (GraphPad Software Inc, CA). Parametric analyses were conducted based on normality test of data sets. Experiments replicates were based on cells isolated from at least two animals with more than three technical replicates (for detail see SI). Detailed statistical analysis methods and stats of each experiment were specified in figure legends.
Pharmaceutical activation of YAP blocks the IL1β response in isolated chondrocytes
Recent studies showed that Lysophosphatidic acid (LPA) can activate YAP through regulation of Rho GTPase activity and inhibition of LATS1/2
. Therefore, we examined the effect of actin tension and LPA in regulating chondrocyte YAP activity and the interaction between the two pathways. The mean YAP intensities in the nucleus and the cytoplasm were measured and YAP Nuclear/Cytoplasmic intensity ratio (YAP N/C ratio) was calculated for individual cells (n = 100). Chondrocytes with YAP N/C ratio larger than 1.5 were defined as YAP-positive (YAP+) similar to previous studies
For chondrocytes cultured on glass coverslips, both control group and 100 μM LPA (ab146430, Abcam) treatment group showed highly organised parallel F-actin filaments. By contrast, treatment with 100 nM Cyto D (Cyto D, C8273, Sigma Aldrich), either alone or in combination with LPA, caused loss of polymerized F-actin organisation [Fig. 1(A)].
Treatment with LPA produced a statistically significant increase in YAP N/C ratio [P < 0.001, Fig. 1(A) and (B)] and percentage of YAP+ cells [P < 0.001, Fig. 1(C)]. By contrast, cytochalasin D treatment resulted in reduction of YAP N/C ratio [P < 0.001, Fig. 1(A) and (B)] and percentage of YAP+ cells [P < 0.001, Fig. 1(C)]. Additionally, Cyto D treatment caused a reduction in nucleus projected area [P < 0.001, Fig. 1(C)] consistent with a rounding of the cells in monolayer. Cyto D also induced a significant reduction in cell modulus measured by AFM [P < 0.001, Fig. 1(D)], associated with depolymerisation and loss of actin tension.
In chondrocytes pre-treated with Cyto D, LPA significantly increased the YAP N/C ratio [P < 0.001, Fig. 1(B)] and percentage of YAP+ cells [P < 0.001, Fig. 1(C)]. However, there was no significant recovery of the nuclear area [P = 0.9821, Fig. 1(D)] or the cell Young's modulus [P = 0.8901, Fig. 1(E)]. Thus, the inhibitory effect of Cyto D on YAP was successfully rescued by LPA treatment despite no rescue of the actin associated changes in cell morphology or biomechanics. These results confirm that loss of actin tension, in this case by Cyto D treatment, inhibits YAP and that LPA-induced upregulation of YAP occurs through an actin-independent pathway.
Having confirmed the effect on the expression of YAP for the pharmaceutical agonist, LPA, and the actin destabiliser, CytoD, we further examined whether YAP activity manipulated by pharmaceutical stimulation had the potential to regulate inflammatory signalling in isolated chondrocytes. We also introduced another YAP antagonist verteporfin (VP) because previous studies have shown that VP can bind with YAP and inhibit its reaction with TEADs via up-regulating 14-3–3σ sequestering YAP. Thus, VP provides a more specific long-term regulation of YAP than cyto D, and without interfering with the actin cytoskeleton and disrupting cell morphology
Treatment with IL1β (1 ng/ml, 24 h, 200-01B, PeproTech) reduced YAP activation as shown by significant differences in YAP N/C ratio [P < 0.001, Fig. 2(A) and (B)] and the percentage of YAP+ cells [P < 0.001, Fig. 2(C)]. This significant reduction in YAP activation occurred within 30–60 min of IL1β exposure [P < 0.001, Fig. S2]. In the presence of LPA, this inhibiting effect of IL1β on YAP was blocked such that there was no statistically significant difference in YAP N/C ratio or the percentage of YAP+ cells, ±IL1β.
We next examined the effect of the YAP agonist and antagonist on IL1β induced inflammatory signalling in isolated chondrocytes cultured in monolayer cells. Incubation with IL1β (1 ng/ml, 24 h) caused a 25% increase of NO production [P < 0.001, Fig. 2(D)]. This effect was completely inhibited by treatment with the YAP agonist, LPA, such that there was no significant difference ±IL1β. Conversely, treatment with YAP antagonist, VP, increased nitrite levels in both the presence and absence of IL1β. In this case, IL1β continued to induce a pro-inflammatory response with a statistically significant increase in nitrite levels [P < 0.001, Fig. 2(D)].
Together these results confirmed the role of LPA and CytoD in manipulating YAP activity and demonstrated that YAP expression activated by pharmaceutical manipulation of LPA blocks the pro-inflammatory response to IL1β in isolated chondrocytes.
Mechanical activation of YAP blocks the IL1β response in isolated chondrocytes
YAP plays an important role in mechanotransduction with previous studies showing that YAP can be regulated by a range of mechanical cues
.Thus, we next examined whether YAP activity and pro-inflammatory IL1β response can be regulated by substrate stiffness.
Isolated chondrocytes were cultured on PDMS substrates with modulus of 6, 20, 130 and 400 kPa confirmed by mechanical characterisation mentioned above (see SI, Fig. S3). Cells cultured on stiffer substrates had a greater level of YAP activation shown by higher YAP N/C ratios with statistically significant differences [P < 0.001, Fig. 3A–B] and higher percentage of YAP+ cells [P < 0.001, Fig. 3(C)]. Cells on stiffer substrates had significantly larger projected nuclear area compared to cells on softer substrates indicative of the cells becoming flatter [P < 0.001, Fig. 3(D)].
These data confirm biomechanical regulation of chondrocyte YAP expression with increased YAP activation on stiffer PDMS substrates over the range of 6–400 kPa.
We next examined whether substrate stiffness alteration regulates the response to IL1β and the potential role of YAP in this pro-inflammatory pathway. We cultured chondrocytes on the softest (6 kPa) and stiffest (400 kPa) of the PDMS substrates analysed for YAP activation in Fig. 3. In addition, we also introduced a stiffer PDMS substrate with a modulus of 1000 kPa.
For isolated cells on all three substrates, incubation with IL1β (10 ng/ml, 24 h) caused release of the inflammatory mediator NO, resulting in significantly greater nitrite levels for IL1β treated cells compared to untreated controls [P < 0.001 in all cases, Fig. 4(A)]. Similarly, there was negligible release of PGE2 in untreated samples with IL1β inducing a significant increase on all three substrates [P < 0.001 in all cases, Fig. 4(B)]. These pro-inflammatory responses were significantly greater on softer substrates, such that cells cultured on the stiffer substrates produced 22% (400 kPa) and 34% (1000 kPa) less nitrite compared to those on the softest 6 kPa substrates [P < 0.001 in both cases, Fig. 4(A)]. Similarly, PGE2 release was 73% less on 400 kPa substrates and 92% less on the stiffest 1000 kPa substrates, compared to that on the softest 6 kPa substrates [P < 0.001 in both cases, Fig. 4(B)]. These findings demonstrate a significant reduction in the pro-inflammatory response to IL1β with increasing substrate stiffness (Fig. 4) associated with greater YAP activation (Fig. 1).
We then investigated whether the increased inflammatory response on the softest 6 kPa substrate could be reversed by the YAP agonist, LPA. Conversely, we tested whether the reduction of the inflammatory response on the stiffest 1000 kPa substrate could be rescued by inhibition of YAP with cytochalasin D.
Treatment with LPA (100 μM) produced a 24% reduction in IL1β-mediated nitrite levels [P < 0.001, Fig. 4(C)] and a 90% reduction in PGE2 levels [P < 0.001, Fig. 4(D)] for chondrocytes cultured on 6 kPa substrates. Thus, YAP activation with LPA on soft substrates was able to reduce pro-inflammatory signalling mimicking the effects of increased substrate stiffness.
Treatment with cytochalasin D (100 nM) of cells on stiff, 1000 kPa, substrates increased IL1β-mediated levels of nitrite [97%, P < 0.001, Fig. 4(E)] and PGE2 [75%, P < 0.001, Fig. 4(F)]. Thus, cytochalasin D and associated inhibition of YAP, reversed the anti-inflammatory effects of the stiff 1000 kPa substrate, producing a response to IL1β which was even greater than that on the softest 6 kPa substrates.
To further investigate the role of YAP in these substrate stiffness-regulated inflammatory response alterations, we then introduced small interfering ribonucleic acid (siRNA) experiments to knockdown YAP in chondrocytes cultured on soft (6 kPa) and stiff (1000 kPa) substrates. Knockdown of YAP was confirmed by Western blot [Fig. 4(G)]. YAP knockdown increased IL1β-mediated nitride release of cells on soft (6 kPa) [301%, P < 0.001, Fig. 4(H)] and stiff (1000 kPa) [439%, P < 0.001, Fig. 4(H)] substrates. In addition, knockdown of YAP lead to no statistical significance in IL1β-mediated nitride level between cells cultured on soft substrate (6 kPa) and stiff substrate (1000 kPa) [P = 0.966, Fig. 4(H)], indicating that substrate stiffness lost the ability of regulating inflammatory response when YAP was knocked down. Thus, YAP is required in the changes of inflammatory response regulated by substrate stiffness.
Our previous study has shown that cyclic mechanical loading can suppress inflammatory response induced by IL1β in chondrocytes
. Thus, to strengthen the evidence that mechanical activation of YAP is anti-inflammatory, we introduced another mechanical stimulation in the form of cyclic tensile strain (CTS). Results showed that YAP was activated when 10% CTS was applied for 24 h [P < 0.001, Fig. S4], indicating that YAP might be involved in CTS induced inflammatory alteration.
Together, these findings suggest that mechanical activation of YAP blocks chondrocyte pro-inflammatory response to IL1β.
Activation of YAP by LPA reduces inflammatory signalling and matrix degradation in response to IL1β in cartilage explants
Having shown that activation of YAP by LPA inhibited inflammatory signalling in response to IL1β in isolated chondrocytes, we next examined whether this also occurred in cartilage explants over a 12 day culture period. Cell viability within the explants was confirmed for all treatment groups based on Calcein-AM and Ethidium Homodimer staining (Fig. S5). Cartilage explants treated with IL1β (10 ng/ml) for 12 days produced an inflammatory response, similar to that observed in isolated cells, with a sustained release of the inflammatory marker, nitric oxide [Fig. 5(A) and (B)]. As in isolated cells, this response was significantly inhibited in the presence of LPA at the second day of culture (Fig. S6) and the cumulative NO release caused by IL1β was reduced by 48% [P < 0.001, Fig. 5(B)].
We next measured matrix degradation by the release of sGAG and associated changed in tissue mechanical properties, as used in previous studies
. Treatment with IL1β increased the release of sGAG from the extracellular matrix into the culture media with differences detectable after 2 days in culture [Fig. S7 and Fig. 5(C)]. This degradation was significantly inhibited by LPA treatment, with 7% reduction in sGAG release [P < 0.001, Fig. 5(D)].
Inhibition of cartilage matrix degradation was further confirmed by analysis of tissue mechanics after the 12-day culture period. The IL1β treatment resulted in significant reductions in tangent modulus (53%), relaxation modulus (64%), relaxation half-life (54%) and an increase in percentage relaxation (9%) [P < 0.001 in all cases, Fig. 6(C)–(F)]. Treatment with LPA completely blocked these changes in cartilage mechanical properties such that IL1β had no significant effect on cartilage mechanics in the presence of LPA.
In summary, the YAP agonist, LPA, significantly inhibited IL1β-induced inflammatory signalling in cartilage tissue and prevented the associated mechanical breakdown of the extracellular matrix.
YAP activation reduces primary cilia expression as a potential mechanism mediating the anti-inflammatory response
We have previously shown that primary cilia and/or associated IFT proteins play an important role in regulating chondrocyte inflammatory signalling. Hypermorphic mutation of IFT88 and associated reduction in cilia expression, inhibited the pro-inflammatory response to IL1β
. Thus, we examined whether YAP activation could supress chondrocyte primary cilia expression as a potential mechanism inhibiting inflammatory signalling.
Based on confocal immunofluorescence [Fig. 7(A)], activation of YAP, by both increasing substrate stiffness and treatment with 100 μM LPA, decreased cilia prevalence [Fig. 7(B)–(D)] and cilia length [Fig. 7(E)–(G)]. Treatment with LPA induced a 13% reduction in cilia prevalence [P = 0.0313, Fig. 7(B)] and an 11% reduction in median cilia length [P < 0.001, Fig. 7(E)]. Similarly, an increase of substrate stiffness from 6 to 400 kPa reduced cilia prevalence by 16% [P = 0.0336, Fig. 7(C)] and length by 21% [P < 0.001, Fig. 7(F)]. In addition to this behaviour on PDMS gels, we found the same response on polyacrylamide gels with significant reductions in cilia length and prevalence with increasing substrate stiffness (see SI, Table S2 and Fig. S8 for further details). On all four PDMS substrates YAP negative cells had 12% more ciliated cells [P = 0.0230, Fig. 7(D)] with cilia that were 11% longer [P < 0.001, Fig. 7(G)] compared to the YAP+ group. We then performed YAP siRNA experiments to further test the relationship between YAP and cilia (see Fig. S9 or details). Here we show that knockdown of YAP by siRNA increased inflammatory response to IL1β with nitride release increased by 300% [P < 0.05, Fig. 8(C)]. Knockdown of YAP increased cilia length [+17%, P < 0.01, Fig. 8(A)] and prevalence [+19%, P < 0.05, Fig. 8(B)]. Treatment with IL1β further elongated cilia [+18%, P < 0.01, Fig. 8(B) and Fig. S10] in the presence of YAP siRNA.
Having shown that primary cilia expression is correlated with YAP activity, we then examined the involvement of cilia and associated IFT in YAP-induced suppression of inflammatory responses. Here we introduced IFT88 siRNA to genetically knockdown cilia/IFT88 in primary chondrocytes and homozygous Tg737ORPK mutant mouse cell line to disrupt cilia/IFT (see SI, Fig. S11 for details). Cilia disruption by both IFT88 siRNA [Fig. 8(D)] and IFT88 hypermorphic mutation [Fig. 8(E)–(G) and Fig. S12], inhibited inflammatory signalling in response to IL1β. Treatment with IFT88 siRNA also inhibited restoration of inflammatory signalling by YAP siRNA, although there was still a significant difference in IL1β induced NO release [P = 0.036, Fig. 8(D)]. This possibly reflects that IFT88 knockdown was only partial using siRNA [Fig. S11(A)]. In ORPK cells with complete disruption of IFT88 and cilia expression, YAP modification by either YAP siRNA, agonist or antagonist resulted in no significant differences in NO release from IL1β compared to scrambled siRNA [Fig. 8(E)–(G) and Fig. S12(B)–(C)]. Taken together, these data show that YAP regulation of inflammatory signalling is dependent on the presence of primary cilia and/or IFT88.
Recent in vivo studies on the effect of YAP on osteoarthritis are contradictory with one study reporting that YAP activation inhibits cartilage degradation
. Both these previous studies injected alginate beads loaded with either YAP agonist (LPA) or YAP antagonist (VP), into OA mice, and measured YAP activity and Osteoarthritis Research Society International (OARSI) score against vehicle controls. Deng et al.
found VP prevented OA. However, these in vivo studies have potential limitations in that it is difficult to estimate the effective intra-articular concentration and how this changes with time. Furthermore, cartilage degradation and OA progression are likely to be influenced by the complex in vivo environment and the presence of other cell types within the joint including macrophages and synoviocytes
We therefore used isolated cartilage cells and tissue explants to determine the direct effect of YAP regulation on the response to the inflammatory cytokine, IL1β. The use of these carefully controlled in vitro models enabled us to decouple some of the complex interactions within the in vivo joint environment. For these studies, we regulated YAP through pharmaceutical modulation and through changes in the mechanical environment achieved through alternations in substrate stiffness and CTS.
Within articular cartilage tissue, chondrocytes interact with their surrounding pericellular matrix which is known to regulate various aspects of cell behaviour (for review see Ref.
. Consequently, we developed PDMS gels with modulus values in the range 6–1000 kPa to reflect the range of matrix stiffness experienced by cells in situ.
We first confirmed the effect of pharmaceutical agonist (LPA) and antagonists (CytoD and VP) in regulating YAP and inflammatory response for chondrocytes on relatively stiff glass or tissue culture plastic. We confirmed that LPA activates YAP with increasing YAP nuclear expression whilst Cyto D inhibits YAP by disrupting F-actin polymerisation. Activation of YAP by LPA was found to be independent of disruption of actin tension induced by Cyto D (Fig. 2). We then found that LPA can inhibit nitride release induced by IL1β associated with YAP activation. Further results showed that NF-κB P65 nuclear translocation is inversely correlated with YAP expression when treated with IL1β (Fig. S12). Together, these results suggest that YAP activation suppresses inflammatory signalling induced by IL1β.
We next found that an increase in substrate stiffness upregulates chondrocyte YAP activation quantified by increases in relative nuclear expression (Fig. 3). This is in agreement with studies in a variety of other cell types
. Activation of YAP by substrate stiffness was associated with flattening of the cells and changes in actin organisation consistent with increased intracellular tension which has previously been reported to regulate YAP
. We showed that increased YAP activation on stiffer substrates, reduced the inflammatory response to IL1β, quantified by the release of the pro-inflammatory markers, NO and PGE2, similar to that seen with YAP agonist, LPA (Fig. 4). This anti-inflammatory effect of stiffer substrates was reversed by disruption of actin with Cyto D. Conversely the increased inflammatory response of softer substrates was reversed by treatment with LPA. The anti-inflammatory effect of YAP activation was further confirmed by knock down of YAP expression with siRNA which prevented modulation of inflammatory signalling by changes in substrate stiffness. These results confirm that YAP activation directly disrupts pro-inflammatory NF-κB signalling in isolated chondrocytes.
We next examined the response in intact cartilage tissue. Here we also found that LPA treatment completely blocked the inflammatory response to IL1β (Fig. 5) and effectively protected cartilage against extracellular matrix degradation and associated loss of mechanical integrity (Fig. 6). These findings are consistent with the in vivo studies of Deng et al. and indicate that the anti-inflammatory effects of LPA are mediated via direct modulation of YAP
. Consistent with this, activation of YAP through either increased substrate stiffness or LPA, inhibits inflammatory signalling associated with reduced chondrocyte primary cilia expression (Fig. 7). Conversely, the exacerbated inflammatory response induced by YAP knockdown with siRNA was accompanied by an increased ciliogenesis. These results are supported by previous findings that demonstrate that YAP activation suppresses ciliogenesis in epithelial cells
. In addition, inhibition of YAP with siRNA or VP treatment was unable to restore the inflammatory response in the absence of functional cilia/IFT. Thus our studies suggest that YAP regulates pro-inflammation NF-κB signalling through inhibition of primary cilia expression.
In summary, this study reveals an anti-inflammatory pathway activated by YAP signalling which blocks IL1β-induced cartilage matrix degradation and is associated with suppression of primary cilia. Consequently, this study provides new mechanistic understanding of how the stiffness of the extracellular environment regulates cartilage inflammation and degradation, which are key factors in OA progression. The findings also suggest the possibility of novel pharmacological therapeutic strategies using YAP activation and/or cilia modulation, to reduce cartilage degradation in inflammatory joint disease such as OA. Indeed, the anti-inflammatory effects of YAP activation and reduced ciliogenesis may also have application in treating inflammation in other tissues and diseases.
All authors aided in revising this manuscript for intellectual content and approved the final version to be published.
Study design: Huan Meng, Martin M. Knight and Oliver M. Pearce and Nuria Gavara.
Data acquisition: Huan Meng, Su Fu, Marta B. Ferreira and Yu Hou.
Data analysis and interpretation: Huan Meng.
Drafting article: Huan Meng and Martin M. Knight.
Declaration of competing interest
The authors declare no competing interests.
Role of funding source
Huan Meng was funded on a PhD studentship from the China Scholarship Council (CSC, China) . Some consumable funding was provided by a grant from the UK Medical Research Council (PI: Martin Knight, MRC Project Ref: MR/L002876/1, United Kingdom ).
The authors gratefully acknowledge the support of Dr Thomas Iskratsch (Bioengineering, Queen Mary University of London) for his help in preparation of PDMS substrates, Dr Clare Thompson for support with cilia studies, Haidi Gao for assistance with confocal microscopy and Alexandra Ionescu for assistance with mechanical testing of cartilage explants. The authors wish to thank Prof Courtney Haycraft and Prof Sue McGlashan for the establishment of the ORPK cell line.
Appendix A. Supplementary data
The following is the Supplementary data to this article: