http://orcid.org/0000-0002-5338-1295
Figures
Abstract
Irreversible cartilage collagen breakdown by the collagenolytic matrix metalloproteinases (MMPs)-1 and MMP-13 represents a key event in pathologies associated with tissue destruction such as arthritis. Inflammation is closely associated with such pathology and occurs in both rheumatoid and osteoarthritis making it highly relevant to the prevailing tissue damage that characterises these diseases. The inflammation-induced activating protein-1 (AP-1) transcription factor is an important regulator of both MMP1 and MMP13 genes with interplay between signalling pathways contributing to their expression. Here, we have examined the regulation of MMP1 expression, and using in vivo chromatin immunoprecipitation analyses we have demonstrated that cFos bound to the AP-1 cis element within the proximal MMP1 promoter only when the gene was transcriptionally silent as previously observed for MMP13. Subsequent small interfering RNA-mediated silencing confirmed however, that cFos significantly contributes to MMP1 expression. In contrast, silencing of ATF3 (a prime MMP13 modulator) did not affect MMP1 expression whilst silencing of the Wnt-associated regulator cysteine- serine-rich nuclear protein-1 (CSRNP1) resulted in substantial repression of MMP1 but not MMP13. Furthermore, following an early transient peak in expression of CSRNP1 at the mRNA and protein levels similar to that seen for cFOS, CSRNP1 expression subsequently persisted unlike cFOS. Finally, DNA binding assays indicated that the binding of CSRNP1 to the AP-1 consensus-like sequences within the proximal promoter regions of MMP1 and MMP13 was preferentially selective for MMP1 whilst activating transcription factor 3 (ATF3) binding was exclusive to MMP13. These data further extend our understanding of the previously reported differential regulation of these MMP genes, and strongly indicate that although cFos modulates the expression of MMP1/13, downstream factors such as CSRNP1 and ATF3 ultimately serve as transcriptional regulators in the context of an inflammatory stimulus for these potent collagenolytic MMPs.
Citation: Macdonald CD, Falconer AMD, Chan CM, Wilkinson DJ, Skelton A, Reynard L, et al. (2018) Cytokine-induced cysteine- serine-rich nuclear protein-1 (CSRNP1) selectively contributes to MMP1 expression in human chondrocytes. PLoS ONE 13(11): e0207240. https://doi.org/10.1371/journal.pone.0207240
Editor: Chunhong Yan, Augusta University, UNITED STATES
Received: August 2, 2018; Accepted: October 27, 2018; Published: November 15, 2018
Copyright: © 2018 Macdonald et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Microarray data are available with the GEO accession number GSE86578.
Funding: This work was supported by the Nuffield Foundation (Oliver Bird Rheumatism Programme), the JGW Patterson Foundation, Arthritis Research UK (grant 20199), the Medical Research Council and Arthritis Research UK Centre for Integrated Research into Musculoskeletal Ageing (CIMA), and a UK NIHR Biomedical Research Centre for Ageing and Age-related disease award to the Newcastle upon Tyne Hospitals NHS Foundation Trust. Clinical and translational research in the Skeletal Research Group is supported by the Northumberland, Tyne and Wear Comprehensive Local Research Network. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Degenerative joint diseases result in destruction of the articular cartilage, typically when a variety of pro-inflammatory mediators perpetuate the disease process (reviewed in [1]). There is now increasing evidence that such inflammatory mediators are involved the pathogenesis of both rheumatoid arthritis (RA) and osteoarthritis (OA) [1]. These mediators include interleukin (IL)-1, members of the IL-6 family (including IL-6 and oncostatin M (OSM)) as well as other inducers of signalling such as Wnt [2–4]. Cartilage collagen breakdown is well documented to be primarily due to the matrix metalloproteinases (MMPs), in particular the collagenases MMP-1 and MMP-13, which are responsible for the irreversible degradation of the collagenous matrix [5]. Although dogma suggests MMP-1 is the major collagenase in RA, whilst MMP-13 prevails in OA, the above mediators all drive the expression of both MMPs with MMP1 induction typically more marked [6, 7]. Furthermore, we have previously reported differential regulatory mechanisms for cytokine-induced MMP1 and MMP13 expression in human chondrocytes [8, 9], and recently confirmed that the most potent cytokine stimulus reported promotes many signalling pathways that are common to a wide variety of pro-inflammatory stimuli [10]. Indeed, we and others have demonstrated marked MMP expression in chondrocytes stimulated with numerous inflammatory mediators [11–15] indicating such potent expression occurs within most inflammatory situations associated with tissue destruction such as RA and OA. Consequently, the collagenases represent key therapeutic targets for disease-modifying agents [5, 16].
Investigations of the molecular mechanisms by which MMPs are regulated [17, 18] have highlighted a primary role for activator protein (AP)-1 transcription factor complexes in regulating MMP expression [9, 19–21]. AP-1 exists as a protein dimer found ubiquitously throughout different tissues and cell types and is comprised of FOS and JUN family heterodimers. Many studies [13, 22–24], including our own [9, 17, 25], have indicated that cFos/cJun AP-1 heterodimers act as critical regulators of MMP1 and MMP13 in chondrocytes following pro-catabolic stimuli such as IL-1+OSM. Interestingly, despite the importance of cFos (gene name FOS), differential regulation of MMP1 and MMP13 via specific cell signalling pathways [8, 9, 13–15, 24, 26] suggests more complex mechanism(s) underpin the transcriptional activation of these MMPs. In this context, we have recently shown that MMP13 expression is initially modulated by a transient expression of cFos, which subsequently results in the prolonged expression of activating transcription factor 3 (ATF3) to promote MMP13 expression via an AP-1 cis element within the MMP13 proximal promoter [10].
In order to expand our understanding of MMP1 regulatory mechanisms, MMP1 regulation was assessed following the same potent pro-catabolic stimulus as detailed above for MMP13. Data herein indeed confirm that cFos silencing was sufficient to significantly inhibit MMP1 expression; however, ATF3 silencing did not affect MMP1 expression. Importantly, IL-1+OSM stimulation induced cysteine- serine-rich nuclear protein-1 (CSRNP1; also termed Axin upregulated-1 (AXUD1)), expression to selectively modulate MMP1 in human chondrocytes. CSRNP1 was first identified as being induced by the β-catenin regulator, AXIN1[27], and has subsequently been characterised as a transcription factor having a role in the regulation of Wnt signalling[28]. In this respect the Wnt/β-catenin signalling cascade pathway has recently been shown to play an important regulator in the onset and progression of OA[29].
This is the first report indicating a role for CSRNP1 in modulating the expression of a chondrogenic collagenase due to an inflammatory insult, and may be indicative of diverse regulatory moieties affecting specific MMP gene transcription as a consequence of inflammation associated with OA progression.
Materials and methods
Materials
All chemicals were of the highest purity available and obtained from Sigma Chemical Co (Poole, UK) unless otherwise stated. All cytokines were recombinant human. IL-1α was a generous gift from Dr Keith Ray (GlaxoSmithKline, Stevenage, UK). OSM was prepared in-house as described [30]. siRNA reagents were screened for toxicity using the Toxilight assay of adenylate kinase release (Lonza, Wokingham, UK).
Chondrocytes
Human chondrocytes were isolated by enzymatic digestion of macroscopically normal articular cartilage from OA patients undergoing joint replacement surgery as described [31]. All subjects gave informed consent and the study was approved by the Newcastle and North Tyneside Joint Ethics Committee. Chondrocytes were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal bovine serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, 40 U/mL nystatin.
Gene expression analyses
RNA was typically stabilised in cell lysates in a 96-well format and cDNA synthesised using the Cells-to-Signal kit (Life Technologies) as directed. SYBR Green real-time PCR or Taqman assays (using Universal Probe Library probes (Roche Applied Sciences)), referred to herein as qPCR, were conducted using primers and conditions described previously [8–10] except for MMP1: For, 5’-CGAATGGCTCATTAAATCAGTTATGG-3’ and Rev, 5’-TATTAGCTCTAGAATTACCACAGTTATCC-3’, Probe, FAM-CAGAGAGTACAACTTACA TCGTGTTGCGGCTC-TAMRA; MMP1 (proximal promoter/transcription start site): For, 5’-TCAGTACAGGTGCCGAACAG-3’ and Rev, 5’-CAAGATGTGTGCGAAGGAGA-3; MMP1 (3’ untranslated region): For, 5’-ACTGAATGGGCAAAAACTGG-3’ and Rev, 5’-TGCAAACAGGGACAATTTGA-3’; CSRNP1: For, 5’-CCTGCCTGACCGTGACTT-3’ and Rev, 5’-AGCCCGCTTCAGGATAGAC-3’, Probe: 57. Nascent hnRNA transcript expression of MMP1 was determined via reverse transcription and Taqman employing 5′-TTTCTTGATTGGCAGGGAA-3′ (For) and 5′-GGAAGCCAAAGGAGCTGTA-3′ (Rev) primers spanning the Exon8/Intron7 boundary with probe number 26.
Chromatin immunoprecipitation (ChIP)
ChIP experiments were performed according to the standard protocol detailed in the EZ-ChIP kit (Merck-Millipore). Briefly, human chondrocytes were cultured until 70–80% confluent at which point they were treated with IL-1 (0.05 ng/mL) in combination with OSM (10 ng/mL) for various durations. Cells were then subject to cross-linking with formaldehyde for 5 min with agitation, and the reaction quenched with 0.125 M glycine for 5 min (with agitation). Cells were then washed twice and scraped into PBS supplemented with protease inhibitors. Cells were pelleted, resuspended in lysis buffer and lysates sonicated in a Bioruptor Plus (Diagenode, Liege, Belgium) sonicating water bath (15 cycles of 30 sec on, 30 sec off at full power) to shear the chromatin. Samples were then pre-cleared with the appropriate agarose beads for 1 h and subject to an overnight immunoprecipitation with relevant antibodies (10 μg) or corresponding isotype controls; anti-phospho-RNA polymerase II CTD repeats YSPTSPS (Ser5; pRNA Pol II) was from Abcam (Cambridge, UK); anti-acetyl(Lys9/14)-histone H3 (AcH3; cat no. 06–599) was from Millipore (Dundee, UK). Antibody/protein/DNA complexes were extracted with agarose beads which were washed sequentially with kit-supplied buffers. DNA was then eluted and crosslinks reversed (4 h incubation at 65 °C with 5 M NaCl, and then 1 h incubation at 37 °C with RNase (1 IU) and proteinase K (1 IU) to degrade RNA and protein, respectively). DNA was purified using spin columns and then used as input for SYBR green qPCR using primers outlined above. Data were normalised to background using isotype control antibodies and variations in amount of genomic DNA were normalised using Input.
Gene promoter profiling
In silico analyses on ChIPSeq (cJun) MMP1/13 data [32] to identify the presence of putative CSRNP1 binding sites (AGAGTN [33]) were performed using ENCODE [34].
Cell fractionation and immunoblotting
Chondrocyte lysates were prepared as described previously [8, 31]. In some experiments chondrocytes were subjected to subcellular fractionation using NE-PER Nuclear and Cytoplasmic Protein Extraction Kit or Subcellular Protein Fractionation Kit (both from ThermoFisher Scientific, Loughborough, UK). Lysates or fractions were resolved by SDS-PAGE, transferred to PVDF membranes and subsequently probed with the following antibodies: ATF3 (sc188) and CSRNP1 (sc81191) (used on cellular fractionation and experiments utilising nuclear lysates) purchased from Santa Cruz Biotechnology (Santa Cruz, CA); and CSRNP1 (ab103708) (used on whole cell lysates) from Abcam (Cambridge, UK). The specificity of all antibodies was confirmed using chondrocyte lysates (see full-length blots in S1 Fig), whilst blots were cropped for clarity of comparison.
Cell transfection and RNA interference
Primary human chondrocytes were prepared and cultured as above, and transfected as described previously [8]. Dharmacon ON-TARGET plus SMARTpool (ThermoFisher Scientific, Loughborough, UK) of 4 specific siRNA duplexes (100 nM siRNA final concentration) were used throughout to assess IL-1+OSM-induced MMP1 expression. All siRNAs used were as previously described [10] except for CSRNP1 (Cat. L-013653; Dharmacon), BMP2 (Cat. L-011219; Dharmacon), IER2 (Cat. L-019884; Dharmacon) and IER5 (Cat. L-013467; Dharmacon). Following transfection, cells were stimulated for 1.25 h to measure transcriptional regulators, or 24 h for MMP1. Depletion of gene-specific mRNA levels was calculated by comparison of expression levels with cells transfected with 100 nM siCONTROL (siCon: non-targeting siRNA #2, Cat. 001210–02; Dharmacon).
DNA affinity precipitation assays (DAPA)
Chondrocyte nuclear lysates were generated as described above. Nuclear protein (100–300 μg) was incubated with double stranded biotinylated oligonucleotides (45 pmol) containing either the proximal AP-1 binding site for MMP1 (5’-[biotin]-TAAAGCATGAGTCAGACACCT-3’; 5’-AGGTGTCTGACTCATGCTTTA-3’) or MMP13 (5’-[biotin]-TAAGTGATGACTCACCATTGC-3’; 5’-GCAATGGTGAGTCATCCTTA-3’) in 500 μl of binding buffer (12 mM HEPES pH 7.9, 4 mM Tris-HCl, 60 mM KCl, 5% (w/v) glycerol, 0.5 mM EDTA, 1 mM DTT and 1 mini protease inhibitor cocktail tablet/10 mL of buffer) for 1 h at 4 °C with rotation. Streptavidin-coated magnetic dynabeads (ThermoFisher Scientific) were then added and incubated for 2 h with rotation at 4 °C. Dynabead-bound protein/DNA complexes were isolated using a Dynabead magnet (ThermoFisher Scientific). Beads were resuspended in 50 μl of SDS-PAGE sample buffer, incubated at 100 °C for 5 min, pelleted and supernatants removed for immunoblotting (as above) except that each individual blot was cut to provide two blots with proteins of molecular mass ≥35kDa or ≤35kDa for CSRNP1 and ATF3 probing, respectively. Specificity of binding was determined in the presence of 50x excess non-biotinylated AP-1 oligonucleotides.
Statistical analyses
Statistical differences between sample groups were assessed using one-way analysis of variance (ANOVA) with a post-hoc Bonferroni’s multiple comparison test or Student’s 2-tailed unpaired t-test, where ***p<0.001, **p<0.01, *p<0.05. For clarity, only selected comparisons are presented in some figures.
Results
MMP1 gene expression is temporally delayed in cytokine-stimulated chondrocytes
We have previously shown that the potent cytokine combination of IL-1+OSM demonstrates a time-dependency for collagenolytic MMP induction, typically reaching maximal induction at 24 h [6, 7, 35]. This dependency is confirmed here where primary human articular chondrocytes expressed MMP1 mRNA within 6 h post-stimulation (Fig 1A) in line with previous findings [7]. Importantly, nascent hnRNA mirrored the observed mRNA increase (Fig 1B) indicating MMP1 transcription occurred independently of mRNA synthesis/stability. Furthermore, previous data indicate that cFOS is rapidly, but transiently, expressed following IL-1+OSM stimulation [10, 25]; employing ChIP assays herein cFos was observed to be enriched at the proximal AP-1 element of the MMP1 promoter at 1 h post-stimulation (Fig 2A and 2B). However, ChIP assays also confirmed enhanced enrichment of phosphorylated RNA Pol II to the proximal MMP1 promoter 24 h post-stimulation, but not at 1 h (Fig 2C and 2D), whilst the time-dependent recruitment of acetylated histone H3 to the MMP1 promoter (Fig 2E) reflected the observed time course of MMP1 mRNA expression. Our previous data indicated a requirement for new protein synthesis for MMP13 induction [10] by employing the protein synthesis inhibitor emetine. Similarly, in this study chondrocytes were stimulated for 24 h with IL-1+OSM and emetine added subsequently for varying durations throughout the stimulation which significantly reduced cytokine-induced MMP1 expression and persisted for at least 6–8 h post-stimulation (Fig 3).
Chondrocytes were stimulated with IL-1 (0.05 ng/mL) in combination with OSM (10 ng/mL) for the indicated durations. Total RNA was isolated, reverse transcribed and subjected to qPCR for (A) MMP1 mRNA or (B) MMP1 nascent hnRNA as described in the Materials and Methods. Data are expressed relative to 18S rRNA and presented as fold increase compared to basal expression. qPCR data (n = 4) are representative of at least three separate chondrocyte populations. All data are presented as mean (± SD), where ***, p<0.001; **, p<0.01; *, p<0.05 IL-1+OSM-treated compared to control; ANOVA.
Human chondrocytes were treated with IL-1 (0.05 ng/mL) in combination with OSM (10 ng/mL) for the indicated durations. Cells were then subject to DNA-protein cross-linking, lysis and DNA shearing. Immuno-precipitation for cFOS (A,B), pRNA Pol II (C,D) or AcH3 (E) was followed by isolation of complexed genomic DNA and qPCR for the proximal (closed bars) and 3’-UTR (used for normalization; open bars) regions of MMP1 as indicated. Data (mean ± SD, n = 4) are pooled from at least three separate chondrocyte populations from different donors. Statistical comparisons are: #, p<0.05 (1 h IL-1+OSM stimulation versus basal); *, p<0.05 (24 h IL-1+OSM stimulation versus basal).
Chondrocytes were treated with IL-1 (0.05 ng/mL) and OSM (10 ng/mL) for 24 h. Emetine (10 μM final concentration) was added at the indicated times after initiation of IL-1+OSM stimulation and qPCR performed on extracted RNA. Relative expression levels of MMP1 mRNA were normalized to 18S rRNA, where ###, p<0.001 (IL-1+OSM vs basal); ***, p<0.001 (IL-1+OSM+emetine vs basal). Data (mean ± SD, n = 6) are representative of three separate experiments each using chondrocyte cultures from different donors.
Given the discord between the transient increase of cFos by IL-1+OSM [9, 10, 25] and the requirement for new protein synthesis at times after cFos expression in terms of MMP1 expression, these findings strongly indicated the requirement of additional transcriptional regulator(s) as shown for the expression of MMP13 [10]. Employing a GEO-genome-wide analysis of IL-1+OSM-induced genes in chondrocytes (accession file GSE86578), we further confirmed that the temporal expression profile of MMP1 was maximal at 24 h (Fig 4A) whilst qPCR confirmed significant expression at 24h in these populations (Fig 4B). Moreover, this gene array analysis identified a number of transcriptional regulatory genes with an expression profile similar to ATF3 which has been shown to be an important AP-1-binding factor for MMP13 expression [10]. A siRNA screen of these regulators, including ATF3, to assess MMP1 repression confirmed a requirement for both FOS (cFos) and JUN (cJun) but not ATF3 for MMP1 expression (Fig 4C). Notably, only silencing of CSRNP1 selectively and significantly repressed MMP1 with no significant inhibition of MMP13 (S2 Fig).
Human chondrocytes were treated with IL-1 (0.05 ng/mL) in combination with OSM (10 ng/mL) for the indicated time points and total RNA isolated. (A) RNA from three separate populations (#1, #2 and #3) was profiled using the Human HT-12v4 Expression Beadchip. The expression profile for MMP1 from the microarray dataset for each chondrocyte population is shown. (B) RNA from six separate chondrocyte populations was subjected to qPCR for MMP1, and relative expression levels (normalized to 18S rRNA) at the indicated times determined. The plots show mean ± SD (n = 6). Data from the populations used in the microarray analyses are highlighted by the solid symbols (#1, ▲; #2 ■; #3, ●). (C) Prior to stimulation with IL-1 (0.05 ng/ml) in combination with OSM (10 ng/ml), human chondrocytes were transfected with siRNA specific for the indicated genes, or a non-targeting control (siCon; all 100 nM), and mRNA expression levels (mean ± S.D., n = 6) of MMP1 measured 24 h post-stimulation by qPCR, relative to siCon treated cells, normalized to 18S rRNA. Statistical comparisons are versus IL-1 + OSM + siCon (Student’s two-tailed unpaired t test), where ***, p<0.001; **, p <0.01; *, p<0.05; ns = not significant. The data are pooled from three separate experiments, each using chondrocyte cultures from different donors.
Sustained CSRNP1 expression contributes to MMP1 transcription
The siRNA data above implicated a selective role for CSRNP1 in contributing to the transcriptional activation of MMP1 (but not MMP13). Furthermore, expression profiling also revealed that CSRNP1 expression was evident by 1 h and peaked at 1.25 h (Fig 5A and 5B). In order to correlate CSRNP1 expression with FOS, a detailed time-course was performed which showed peak CSRNP1 expression to follow maximal FOS expression (Fig 5C). Importantly, CSRNP1 protein was present from 1 to 24 h (Fig 6A–6C) and appeared to be exclusively nuclear (both soluble and chromatin-bound fractions) following subcellular fractionation (Fig 6C), peaking at 3 h (Fig 6B and 6D).
Human chondrocytes were treated with IL-1 (0.05 ng/mL) in combination with OSM (10 ng/mL) for the indicated time points and total RNA isolated. (A) RNA from three separate populations (#1, #2 and #3) was profiled using the Human HT-12v4 Expression Beadchip. The expression profile for CSRNP1 from the microarray dataset for each chondrocyte population is shown. RNA from 6 (B) or 3 (C) separate chondrocyte populations was subjected to qPCR for FOS (C) or CSRNP1 (B and C), and relative expression levels (normalized to 18S rRNA or GAPDH as indicated) at the indicated times determined. The plots show mean ± SD. Data from the populations used in the microarray analyses in (A) are highlighted by the solid symbols (#1, ▲; #2 ■; #3, ●). (C) Statistical comparisons are: ***, p<0.001; **, p<0.01; *, p<0.05 versus basal.
Human chondrocytes were stimulated with IL-1 (0.05 ng/mL) in combination with OSM (10 ng/mL) for the indicated durations. Protein from either whole cell lysates (A) or subcellular fractions (C: a, cytosolic; b, membrane-bound; c, soluble nuclear; d, chromatin-bound; e, cytoskeletal) were resolved using SDS-PAGE and immunoblotted with an antibody to CSRNP1. Combined densitometric scans of separate blots for CSRNP1 in cell lysates (n = 3; B) or pooled soluble and chromatin-bound nuclear fractions (n = 5; D), relative to t = 0 h, are shown and were obtained from separate chondrocyte populations. All data are presented as mean (± SD), where **, p<0.01, IL-1+OSM-treated compared to control; ANOVA.
In silico analyses using the ENCODE database [34] indicated that a consensus-like CSRNP1 motif AGAGTN [33] was present within the proximal ChIPSeq AP-1 (cJun) binding site within the MMP1 promoter, with a 1 bp mismatch. We therefore performed DAPA analyses which, importantly, confirmed specific CSRNP1 binding to this sequence, compared to the equivalent motif within MMP13 (Fig 7), thus further confirming the selective specificity of CSRNP1 for MMP1. Finally, we also assessed the ability of the MMP1/13 AP-1 sequences to bind ATF3 and only observed binding to the MMP13 AP-1 cis element (Fig 7) in line with previous findings [10].
Human chondrocytes were treated with IL-1 (0.2 ng/mL) in combination with OSM (10 ng/mL) for 3 h. Nuclear lysates were prepared and subjected to either Western blotting alone (input) or DAPA with CSRNP1 or ATF3 antibodies, as described in the Materials and Methods. Non-biotinylated AP-1 oligonucleotides (50x excess; Comp) were used to confirm specificity of binding in the DAPAs. The data are representative of 3–5 separate experiments each using chondrocyte cultures from different donors.
Discussion
Recent evidence indicates that IL-1+OSM-induces MMP13 expression via transcriptional modulation by initial AP-1 (cFos) expression and then by subsequent ATF3 transcriptional activity [10]. In this context, the transcriptional regulation of MMP1, which is also potently induced by this stimulus [6–9, 35–37], has remained undefined. Although MMP-13 is the most potent collagenolytic proteinase against type II collagen [38], the abundance of MMP-1 compared to MMP-13 following pro-inflammatory stimuli [35, 37, 39] also results in significant cleavage of type II collagen resulting in irreversible cartilage damage [40]. Since MMP1 and MMP13 appear to be differentially regulated following IL-1+OSM stimulation [8, 9, 26], in the study presented herein we sought to identify transcriptional modulators that are selective for MMP1 in comparison to our previous observations for MMP13 [10].
Employing ChIP assays with cFos, phosphorylated RNA Pol II and acetylated histone H3 antibodies, we demonstrated that cFos transcriptional control was similarly important for regulating MMP1 expression as observed for MMP13 [10]. cFOS was recruited to the MMP1 promoter 1 hour post-stimulation, however pRNA pol II was not detected at this timepoint, rather being recruited to the promoter 24 hours post-stimulation. These data indicate that despite cFOS recruitment to the MMP1 promoter, MMP1 transcription does not occur at this early time point. Furthermore, the inhibition of protein synthesis after IL-1+OSM stimulation demonstrates that when protein synthesis is inhibited within the first 4 hours of stimulation, no MMP1 expression is observed 24 hours post-stimulation. However if protein synthesis is not inhibited until after 6 hours of IL-1+OSM, MMP1 expression is observed following 24 hour stimulation. The requirement for the de novo synthesis of transcriptional regulators following cell activation has been previously reported in the literature [10, 41, 42]. Taken together, these data strongly suggest that MMP1 expression is dependent on de novo protein synthesis of additional factors post-FOS but prior to MMP1 expression.
In order to identify such factors, GEO-genome-wide microarray analyses with subsequent siRNA assays were performed. These studies identified several transcriptional regulators which had an elevated expression level 1–1.25 hours post IL-1+OSM stimulation. In comparing the effect of siRNA knock down of these transcriptional regulators on MMP expression [10], only CSRNP1 was found to be selective and specific for the regulation of MMP1 expression over MMP13. This study indicated that MMP1 expression was indeed dependent on cFos but that other FOS family members also affected MMP1 expression via promoting AP-1-dependent gene expression. In this respect, FOSL1 and FOSB were induced (see S3 Fig) and gene silencing of both resulted in significant MMP1 repression. These analyses are also in agreement with other studies indicating a role for cytokine induction of the NFκB component RelA (p65) for MMP1 expression in chondrocytes [15, 43–45]. We show here that siRNA silencing of RELA leads to a significant decrease in MMP1 expression although to a much lesser extent than seen for FOS or FOSL1.
Little is known about the role of CSRNP1 as a transcriptional regulator. Here we provide the first evidence that CSRNP1 has an important modulatory role in affecting cytokine-induced MMP1 expression in human articular chondrocytes. The consensus sequence for CSRNP1 binding is an AP-1-like binding motif 5’-AGAGTN-3’ [33] and both MMP1 and MMP13 have similar AP-1-like binding motifs within their proximal promoter regions. The MMP1 sequence is 5’-TGAGTCA-3’ and the MMP13 sequence has a C instead of a G (underlined) resulting in 5’-TGACTCA-3’. This G to C switch indicates preferential binding of CSRNP1 to the MMP1 compared to the MMP13 AP-1-like cis element as observed via DAPA analyses. This may be indicative that the proximal MMP1 AP-1-like sequence is a specific cis element capable of binding CSRNP1 in chondrocytes. Furthermore, in silico analysis indicates the presence of other putative CSRNP1 binding sites located within the MMP1 proximal promoter, but not within MMP13, that may also contribute to the expression of MMP1. Since no evidence for reproducible ATF3 binding to the MMP1 AP-1 cis element was observed employing DAPAs in contrast to MMP13, and gene silencing of ATF3 resulted in no significant decrease in MMP1 expression, MMP1 appears unlikely to be an ATF3-responsive gene. Thus, CSRNP1 and ATF3 may respectively have mutually exclusive roles in the cytokine-mediated expression of MMP1 and MMP13 in chondrocytes.
In conclusion, our data presented here indicate that a potent catabolic stimulus such as IL-1+OSM induces the expression of specific transcriptional regulators that contribute to MMP1 gene activation which may well reflect the markedly enhanced levels of expression that are observed compared to either cytokine alone [35, 39]. Since multiple pro-inflammatory mediators are likely to be prevalent in most inflammatory situations, this study further highlights the complexity of MMP gene regulation that occurs in human chondrocytes when subjected to inflammatory cues.
Supporting information
S1 Fig. Confirmation of antibody specificity using human chondrocyte lysates.
The specificity of each antibody used in the study was confirmed using nuclear lysates prepared as described in the Methods from primary human articular chondrocytes either unstimulated or stimulated with IL-1 (0.2 ng/mL) in combination with OSM (10 ng/mL). Following SDS-PAGE, proteins were transferred to PVDF membranes and probed with the indicated antibodies. Full-length blots are presented to highlight the specific immuno-reactivity of each antibody with an arrow indicating the expected molecular mass.
https://doi.org/10.1371/journal.pone.0207240.s001
(TIF)
S2 Fig. Multiple transcriptional regulators impact on MMP13 gene expression.
Following transfection with siRNA specific for the indicated genes or non-targeting control (siCon; all 100 nM), Human chondrocytes were treated with IL-1 (0.05 ng/ml) in combination with OSM (10 ng/ml) for 24 hours. RNA isolated from three separate populations were subjected to qPCR, relative to siCon control normalised to 18S rRNA. Statistical comparisons are: ***, p<0.001; **, p<0.01; *, p<0.05; ns = not significant, versus siCon. The effect of silencing ATF3, FOS, FOSB, FOSL1, IER3, IRF1, JUN and RELA on MMP13 expression was originally published in the Journal of Biological Chemistry. Chan et al. (2017)[10], Copyright the American Society for Biochemistry and Molecular Biology.
https://doi.org/10.1371/journal.pone.0207240.s002
(TIF)
S3 Fig. Multiple transcriptional regulators are induced following IL-1+OSM stimulation of human chondrocytes.
Human chondrocytes were treated with IL-1 (0.05 ng/mL) in combination with OSM (10 ng/mL) for 1, 1.25 or 24 h and total RNA isolated. RNA from three separate populations (#1, #2 and #3 as presented in Figs 4 and 5) was profiled using the Human HT-12v4 Expression Beadchip. The MMP1 expression profile reported in Fig 4 is presented here for comparison with the profiles of the indicated genes from the microarray dataset for each chondrocyte population.
https://doi.org/10.1371/journal.pone.0207240.s003
(TIF)
Acknowledgments
We thank all the collaborators mentioned for their kind gifts, and the surgeons and research nurses of the Newcastle Hospitals Trust for the generous supply of human tissue.
References
- 1. Livshits G, Kalinkovich A. Hierarchical, imbalanced pro-inflammatory cytokine networks govern the pathogenesis of chronic arthropathies. Osteoarthritis Cartilage. 2018;26(1):7–17. pmid:29074297.
- 2. van den Bosch MH, Blom AB, van de Loo FA, Koenders MI, Lafeber FP, van den Berg WB, et al. Brief Report: Induction of Matrix Metalloproteinase Expression by Synovial Wnt Signaling and Association With Disease Progression in Early Symptomatic Osteoarthritis. Arthritis Rheumatol. 2017;69(10):1978–83. pmid:28678406.
- 3. Landman EB, Miclea RL, van Blitterswijk CA, Karperien M. Small molecule inhibitors of WNT/beta-catenin signaling block IL-1beta- and TNFalpha-induced cartilage degradation. Arthritis Res Ther. 2013;15(4):R93. pmid:23965253.
- 4. Zhu M, Tang D, Wu Q, Hao S, Chen M, Xie C, et al. Activation of beta-catenin signaling in articular chondrocytes leads to osteoarthritis-like phenotype in adult beta-catenin conditional activation mice. J Bone Miner Res. 2009;24(1):12–21. pmid:18767925.
- 5. Page-McCaw A, Ewald AJ, Werb Z. Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol. 2007;8(3):221–33. Epub 2007/02/24. pmid:17318226.
- 6. Koshy PJT, Lundy CJ, Rowan AD, Porter S, Edwards DR, Hogan A, et al. The modulation of matrix metalloproteinase and ADAM gene expression in human chondrocytes by interleukin-1 and oncostatin M: A time-course study using real-time quantitative reverse transcription–polymerase chain reaction. Arthritis & Rheumatism. 2002;46(4):961–7. pmid:11953973
- 7. Barksby HE, Hui W, Wappler I, Peters HH, Milner JM, Richards CD, et al. Interleukin-1 in combination with oncostatin M up-regulates multiple genes in chondrocytes: Implications for cartilage destruction and repair. Arthritis & Rheumatism. 2006;54(2):540–50. pmid:16447230
- 8. Litherland GJ, Dixon C, Lakey RL, Robson T, Jones D, Young DA, et al. Synergistic Collagenase Expression and Cartilage Collagenolysis Are Phosphatidylinositol 3-Kinase/Akt Signaling-dependent. Journal of Biological Chemistry. 2008;283(21):14221–9. pmid:18332138
- 9. Litherland GJ, Elias MS, Hui W, MacDonald CD, Catterall JB, Barter MJ, et al. Protein kinase C isoforms zeta and iota mediate collagenase expression and cartilage destruction via STAT- and ERK-dependent c-fos induction. J Biol Chem. 2010:M110.120121. pmid:20463008
- 10. Chan CM, Macdonald CD, Litherland GJ, Wilkinson DJ, Skelton A, Europe-Finner GN, et al. Cytokine-induced MMP13 Expression in Human Chondrocytes Is Dependent on Activating Transcription Factor 3 (ATF3) Regulation. J Biol Chem. 2017;292(5):1625–36. pmid:27956552.
- 11. Koshy PJ, Henderson N, Logan C, Life PF, Cawston TE, Rowan AD. Interleukin 17 induces cartilage collagen breakdown: novel synergistic effects in combination with proinflammatory cytokines. Ann Rheum Dis. 2002;61(8):704–13. Epub 2002/07/16. pmid:12117676.
- 12. Zhang Q, Hui W, Litherland GJ, Barter MJ, Davidson R, Darrah C, et al. Differential Toll-like receptor-dependent collagenase expression in chondrocytes. Ann Rheum Dis. 2008;67(11):1633–41. Epub 2008/02/09. pmid:18258708.
- 13. Selvamurugan N, Chou WY, Pearman AT, Pulumati MR, Partridge NC. Parathyroid hormone regulates the rat collagenase-3 promoter in osteoblastic cells through the cooperative interaction of the activator protein-1 site and the runt domain binding sequence. J Biol Chem. 1998;273(17):10647–57. Epub 1998/05/30. pmid:9553127.
- 14. Mengshol JA, Vincenti MP, Brinckerhoff CE. IL-1 induces collagenase-3 (MMP-13) promoter activity in stably transfected chondrocytic cells: requirement for Runx-2 and activation by p38 MAPK and JNK pathways. Nucleic Acids Res. 2001;29(21):4361–72. Epub 2001/11/03. pmid:11691923.
- 15. Vincenti MP, Coon CI, Brinckerhoff CE. Nuclear factor κB/p50 activates an element in the distal matrix metalloproteinase 1 promoter in interleukin-1β-stimulated synovial fibroblasts. Arthritis & Rheumatism. 1998;41(11):1987–94. pmid:9811054
- 16. Rowan AD, Litherland GJ, Hui W, Milner JM. Metalloproteases as potential therapeutic targets in arthritis treatment. Expert Opin Ther Targets. 2008;12(1):1–18. Epub 2007/12/14. pmid:18076366.
- 17. Catterall JB, Carrere S, Koshy PJ, Degnan BA, Shingleton WD, Brinckerhoff CE, et al. Synergistic induction of matrix metalloproteinase 1 by interleukin-1alpha and oncostatin M in human chondrocytes involves signal transducer and activator of transcription and activator protein 1 transcription factors via a novel mechanism. Arthritis Rheum. 2001;44(10):2296–310. Epub 2001/10/23. pmid:11665970.
- 18. Chinenov Y, Kerppola TK. Close encounters of many kinds: Fos-Jun interactions that mediate transcription regulatory specificity. Oncogene. 2001;20(19):2438–52. Epub 2001/06/13. pmid:11402339.
- 19. Auble DT, Brinckerhoff CE. The AP-1 sequence is necessary but not sufficient for phorbol induction of collagenase in fibroblasts. Biochemistry. 1991;30(18):4629–35. Epub 1991/05/07. pmid:1850629.
- 20. White LA, Brinckerhoff CE. Two activator protein-1 elements in the matrix metalloproteinase-1 promoter have different effects on transcription and bind Jun D, c-Fos, and Fra-2. Matrix Biol. 1995;14(9):715–25. Epub 1995/12/01. pmid:8785586.
- 21. Benbow U, Brinckerhoff CE. The AP-1 site and MMP gene regulation: what is all the fuss about? Matrix Biol. 1997;15(8–9):519–26. pmid:9138284.
- 22. Hess J, Porte D, Munz C, Angel P. AP-1 and Cbfa/runt physically interact and regulate parathyroid hormone-dependent MMP13 expression in osteoblasts through a new osteoblast-specific element 2/AP-1 composite element. J Biol Chem. 2001;276(23):20029–38. Epub 2001/03/29. pmid:11274169.
- 23. Sun Y, Wenger L, Brinckerhoff CE, Misra RR, Cheung HS. Basic calcium phosphate crystals induce matrix metalloproteinase-1 through the Ras/mitogen-activated protein kinase/c-Fos/AP-1/metalloproteinase 1 pathway. Involvement of transcription factor binding sites AP-1 and PEA-3. J Biol Chem. 2002;277(2):1544–52. Epub 2001/10/30. pmid:11682465.
- 24. Schmucker AC, Wright JB, Cole MD, Brinckerhoff CE. Distal interleukin-1beta (IL-1beta) response element of human matrix metalloproteinase-13 (MMP-13) binds activator protein 1 (AP-1) transcription factors and regulates gene expression. J Biol Chem. 2012;287(2):1189–97. Epub 2011/11/22. pmid:22102411.
- 25. Proctor CJ, Macdonald C, Milner JM, Rowan AD, Cawston TE. A computer simulation approach to assessing therapeutic intervention points for the prevention of cytokine-induced cartilage breakdown. Arthritis Rheumatol. 2014;66(4):979–89. Epub 2014/04/24. pmid:24757149.
- 26. Vincenti MP, Brinckerhoff CE. Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex signaling pathways for the recruitment of gene-specific transcription factors. Arthritis Res. 2002;4(3):157–64. Epub 2002/05/16. pmid:12010565.
- 27. Ishiguro H, Tsunoda T, Tanaka T, Fujii Y, Nakamura Y, Furukawa Y. Identification of AXUD1, a novel human gene induced by AXIN1 and its reduced expression in human carcinomas of the lung, liver, colon and kidney. Oncogene. 2001;20(36):5062–6. pmid:11526492
- 28. Simoes-Costa M, Stone M, Bronner ME. Axud1 Integrates Wnt Signaling and Transcriptional Inputs to Drive Neural Crest Formation. Dev Cell. 2015;34(5):544–54. pmid:26256212.
- 29. Lietman C, Wu B, Lechner S, Shinar A, Sehgal M, Rossomacha E, et al. Inhibition of Wnt/beta-catenin signaling ameliorates osteoarthritis in a murine model of experimental osteoarthritis. JCI Insight. 2018;3(3). pmid:29415892.
- 30. Staunton D, Hudson KR, Heath JK. The interactions of the cytokine-binding homology region and immunoglobulin-like domains of gp130 with oncostatin M: implications for receptor complex formation. Protein Engineering. 1998;11(11):1093–102. pmid:9876932
- 31. Cleaver CS, Rowan AD, Cawston TE. Interleukin 13 blocks the release of collagen from bovine nasal cartilage treated with proinflammatory cytokines. Ann Rheum Dis. 2001;60(2):150–7. Epub 2001/01/13. pmid:11156549.
- 32. List K, Bugge TH, Szabo R. Matriptase: Potent proteolysis on the cell surface. Molecular Medicine. 2006;12(1–3):1–7. pmid:16838070
- 33. Gingras S, Pelletier S, Boyd K, Ihle JN. Characterization of a family of novel cysteine- serine-rich nuclear proteins (CSRNP). PLoS One. 2007;2(8):e808. pmid:17726538.
- 34. Consortium EP. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012;489(7414):57–74. pmid:22955616.
- 35. Cawston TE, Curry VA, Summers CA, Clark IM, Riley GP, Life PF, et al. The role of oncostatin M in animal and human connective tissue collagen turnover and its localization within the rheumatoid joint. Arthritis & Rheumatism. 1998;41(10):1760–71. pmid:9778217
- 36. Rowan AD, Hui W, Cawston TE, Richards CD. Adenoviral gene transfer of interleukin-1 in combination with oncostatin M induces significant joint damage in a murine model. Am J Pathol. 2003;162(6):1975–84. Epub 2003/05/22. pmid:12759253.
- 37. Rowan AD, Koshy PJ, Shingleton WD, Degnan BA, Heath JK, Vernallis AB, et al. Synergistic effects of glycoprotein 130 binding cytokines in combination with interleukin-1 on cartilage collagen breakdown. Arthritis Rheum. 2001;44(7):1620–32. Epub 2001/07/24. pmid:11465713.
- 38. Knauper V, Lopez-Otin C, Smith B, Knight G, Murphy G. Biochemical characterization of human collagenase-3. J Biol Chem. 1996;271(3):1544–50. pmid:8576151.
- 39. Morgan TG, Rowan AD, Dickinson SC, Jones D, Hollander AP, Deehan D, et al. Human nasal cartilage responds to oncostatin M in combination with interleukin 1 or tumour necrosis factor alpha by the release of collagen fragments via collagenases. Ann Rheum Dis. 2006;65(2):184–90. pmid:15975972.
- 40. Karsdal MA, Madsen SH, Christiansen C, Henriksen K, Fosang AJ, Sondergaard BC. Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity. Arthritis Res Ther. 2008;10(3):R63. Epub 2008/06/03. pmid:18513402.
- 41. Schwenger GTF, Kok CC, Arthaningtyas E, Thomas MA, Sanderson CJ, Mordvinov VA. Specific activation of human interleukin-5 depends on de novo synthesis of an AP-1 complex. Journal of Biological Chemistry. 2002;277(49):47022–7. pmid:12354764
- 42. Li B, Power MR, Lin TJ. De novo synthesis of early growth response factor-1 is required for the full responsiveness of mast cells to produce TNF and IL-13 by IgE and antigen stimulation. Blood. 2006;107(7):2814–20. pmid:16317093
- 43. Raymond L, Eck S, Hays E, Tomek I, Kantor S, Vincenti M. RelA is required for IL-1beta stimulation of Matrix Metalloproteinase-1 expression in chondrocytes. Osteoarthritis Cartilage. 2007;15(4):431–41. pmid:17097317.
- 44. Wang CM, Brennan VC, Gutierrez NM, Wang X, Wang L, Yang WH. SUMOylation of ATF3 alters its transcriptional activity on regulation of TP53 gene. J Cell Biochem. 2013;114(3):589–98. Epub 2012/09/20. pmid:22991139.
- 45. Li N, Xu Q, Liu Q, Pan D, Jiang Y, Liu M, et al. Leonurine attenuates fibroblast-like synoviocyte-mediated synovial inflammation and joint destruction in rheumatoid arthritis. Rheumatology (Oxford). 2017;56(8):1417–27. pmid:28431044.