MicroRNA-127-5p Regulates Matrix Metalloproteinase 13 Expression and Interleukin-1β–Induced Catabolic Effects in Human Chondrocytes

Authors

Errata

This article is corrected by:

  1. Errata: Incorrect Grant Number in the Article by Park et al (Arthritis Rheum, December 2013) Volume 66, Issue 5, 1394, Article first published online: 28 April 2014

Abstract

Objective

MicroRNAs (miRNAs), small noncoding RNA molecules, are involved in the pathogenesis of various diseases such as cancer and arthritis. The aim of this study was to determine whether miR-127-5p regulates interleukin-1β (IL-1β)–induced expression of matrix metalloproteinase 13 (MMP-13) and other catabolic factors in human chondrocytes.

Methods

Expression of miR-127-5p and MMP-13 by normal and osteoarthritic (OA) human cartilage was determined using real-time polymerase chain reaction. The effect of miR-127-5p on MMP-13 expression was evaluated using transient transfection of human chondrocytes or chondrogenic SW-1353 cells with miR-127-5p or its antisense inhibitor (anti–miR-127-5p). MMP-13 protein production was quantified by enzyme-linked immunosorbent assay, and the involvement of miR-127-5p in IL-1β–mediated catabolic effects was examined by immunoblotting. MicroRNA-127-5p binding with the putative site in the 3′-untranslated region (3′-UTR) of MMP-13 messenger RNA (mRNA) was validated by luciferase reporter assay.

Results

There was a significant reduction in miR-127-5p expression in OA cartilage compared with normal cartilage. Up-regulation of MMP-13 expression by IL-1β was correlated with down-regulation of miR-127-5p expression in human chondrocytes. MicroRNA-127-5p suppressed IL-1β–induced MMP-13 production as well as the activity of a reporter construct containing the 3′-UTR of human MMP-13 mRNA. In addition, mutation of the miR-127-5p binding site in the 3′-UTR of MMP-13 mRNA abolished miR-127-5p–mediated repression of reporter activity. Conversely, treatment with anti–miR-127-5p remarkably increased reporter activity and MMP-13 production. Interestingly, the IL-1β–induced activation of JNK, p38, and NF-κB and expression of MMP-1 and cyclooxygenase 2 were significantly inhibited by miR-127-5p.

Conclusion

MicroRNA-127-5p is an important regulator of MMP-13 in human chondrocytes and may contribute to the development of OA.

Osteoarthritis (OA) is a degenerative disease of articular cartilage characterized by loss of the cartilage matrix, mainly collagen and proteoglycans, leading to tissue destruction and loss of joint function. Although OA is regarded as a noninflammatory form of arthritis, considerable evidence suggests that proinflammatory cytokines derived from the synovium and chondrocytes play a role in cartilage destruction. The activity of the proinflammatory cytokine interleukin-1β (IL-1β) and its downstream mediators leads to up-regulation of matrix metalloproteinase (MMP) and a decrease in the synthesis of the cartilage extracellular matrix (ECM) ([1]). Among the target mediators of IL-1β, MMP-13 has gained the most interest, due to its capacity to degrade collagens along with a wide range of matrix molecules ([2]).

A variety of therapeutic strategies for OA have thus been developed to antagonize the activity of MMP-13; however, the toxicity of nonspecific MMP inhibitors has hampered their application in clinical settings. A detailed analysis of the mechanism of regulation of MMP-13 in chondrocytes is therefore indispensable to ensure safe and effective use of such strategies. MicroRNAs (miRNAs) are short endogenous oligonucleotides (∼22 bp) with a profound role in the regulation of posttranscriptional gene expression. The miRNAs regulate their targets by both translational suppression and acceleration of messenger RNA (mRNA) degradation ([3]). Target genes are determined by sequence complementarity between the 3′-untranslated region (3′-UTR) and the mature miRNA, particularly in a 6-bp “seed” region ([4]). Currently, 1,600 human miRNAs are registered in the miRBase database (release 19, August 2012), and each miRNA is expected to target ∼200 transcripts ([5]). The finding that miRNAs regulate multiple genes, with up to one-third of all human genes containing putative miRNA recognition elements, and the demonstration of differential expression of miRNAs in animal cells and tissues have led to speculation that control of gene expression by miRNAs is conceptually similar to the action of transcription factors ([6, 7]).

The physiologic and pathogenetic role of miRNAs in the maintenance of joint homeostasis and the development of arthritis is currently being elucidated. Dicer, a component for biogenesis of miRNAs, was found to have an essential function in skeletal development. Growth plates of dicer-null mice showed a progressive reduction in the proliferating pool of chondrocytes, leading to severe skeletal growth defects and premature death ([8]). In another study, miRNA-140 was found to contribute to the regulation of age-related OA-like changes, with miR-140−/− mice showing loss of proteoglycan and fibrillation of articular cartilage, and transgenic mice with cartilage overexpression of miR-140 showing resistance to antigen-induced arthritis ([9]). Recent reports have also described a correlation of MMP-13 with specific miRNAs, such as miR-9, miR-22, miR-27a, and miR-27b ([10-13]).

Because the regulation of MMP-13 conferred by IL-1β is considered one of the pivotal pathways leading to cartilage degeneration, we sought to identify MMP-13–regulating miRNAs among all of the miRNAs regulated by IL-1β in human chondrocytes. We conjectured that this would lead to elucidation of an important regulatory loop controlling IL-1β–induced MMP-13 up-regulation, which affects cartilage catabolism.

In a previous study, miR-127-5p was reported to be significantly down-regulated by IL-1β in human OA chondrocytes ([11]). MicroRNA-127-5p, which was initially identified in induced pluripotent mouse stem cells, belongs to imprinted miRNAs that are encoded by the Dlk1-Dio3 locus and that are related to multiple aspects of growth, differentiation, metabolism, and other developmental processes ([14]). Using bioinformatics, we found that miR-127-5p has a seed-matched sequence in the 3′-UTR of human MMP-13 mRNA.

In this study, we sought to determine whether miR-127-5p regulates MMP-13 expression and IL-1β–induced catabolic responses in human chondrocytes. Our results demonstrated that miR-127-5p could suppress MMP-13 and IL-1β responses in human chondrocytes, and alteration of its expression could affect the progression of cartilage destruction. Thus, miR-127-5p may be a novel regulator of cartilage homeostasis in OA.

MATERIALS AND METHODS

Materials

Recombinant human IL-1β and antibodies against MMP-1 and cyclooxygenase 2 (COX-2) were obtained from R&D Systems. Antibodies against p-JNK, p-p38, p-IκBα, and p-ERK were purchased from Cell Signaling Technology. Antibodies against MMP-13 and β-actin were purchased from Abcam and Sigma, respectively. Horseradish peroxidase (HRP)–conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology. PD98059 (a MEK-1/2 inhibitor), SB203580 (a p38 MAPK inhibitor), SP600125 (a JNK inhibitor), and SN50 (an NF-κB inhibitor) were purchased from Calbiochem.

Patients

OA cartilage samples were obtained at the time of total knee replacement surgery from patients with OA (n = 20, mean ± SD age 70.7 ± 6.9 years) who were diagnosed according to the American College of Rheumatology criteria ([15, 16]). Normal cartilage samples were obtained at the time of total hip replacement surgery from patients with femoral neck fracture who had no known history of OA or rheumatoid arthritis (RA) (n = 6, mean ± SD age 73.0 ± 10.6 years); the femoral head cartilage was carefully examined for any gross sign of degeneration, and only normal-appearing femoral heads were used for procuring cartilage. All of the normal cartilage samples obtained had a smooth surface, without fibrillation, and well-preserved proteoglycan staining in the matrix histologically.

Cell culture

Human chondrocytes were isolated from the OA knee joints from a relatively lesion-free area. Articular cartilage was dissected and subjected to sequential digestion with Pronase and collagenase in Dulbecco's modified Eagle's medium (DMEM; Life Technologies). Chondrocytes were maintained in DMEM containing 10% (volume/volume) fetal bovine serum and 1% penicillin/streptomycin. First-passage chondrocytes were obtained after the chondrocytes were released from the cartilage and cultured for 1 week at high density. All experiments were done within 3 days of passage 1 culture; by minimizing the duration of monolayer culture, we could minimize dedifferentiation. Human chondrogenic SW-1353 and C28I2 cells (kindly provided by Dr. Mary Goldring, Hospital for Special Surgery, New York, NY) were maintained in the same type of medium as that used for OA chondrocytes. During the culture period, cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air, and the medium was changed every 2–3 days.

Transfection of miRNAs

Human chondrocytes were transfected with mature-type hsa-miR-127-5p (5′-CUGAAGCUCAGAGGGCUCUGAU-3′) or its antisense inhibitor (anti–miR-127-5p) (both from Dharmacon) at 50 nM, using the calcium phosphate precipitation method ([17]). SW-1353 and C28I2 cells were transfected with 50 nM miR-127-5p or anti–miR-127-5p using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Nonspecific microRNA (miR-Control; Dharmacon) was used as a control. Forty-eight hours after transfection, the cells were used for the following experiments.

Quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR) analysis of MMP-13 and miR-127-5p expression

Total cellular RNA was extracted from cultured chondrocytes using TRIzol reagent (Invitrogen). MicroRNA was purified using the mirVana miRNA isolation kit (Ambion), according to the manufacturer's instructions. For some studies, cartilage tissue from the knees of normal and OA donors was crushed to a fine powder in liquid nitrogen, and total RNA and miRNA were prepared in the same manner as described above. The expression level of MMP-13 mRNA was quantified using a LightCycler-FastStart DNA Master SYBR Green I kit (Roche Diagnostics), using a LightCycler 2.0 instrument (Roche Diagnostics). GAPDH was used as an endogenous control. Primer sequences were as follows: for MMP-13, forward 5′-AAG-GAC-CCT-GGA-GCA-CTC-ATG-TTT-3′ and reverse 5′-TGG-CAT-CAA-GGG-ATA-AGG-AAG-GGT-3′; for GAPDH, forward 5′-TGA-TGA-CAT-CAA-GAA-GGT-GGT-GAA-G-3′ and reverse 5′-TCC-TTG-GAG-GCC-ATG-TGG-GCC-AT-3′.

Expression of mature miRNA was quantified using a TaqMan miRNA assay kit (Applied Biosystems). Purified miRNA was reverse transcribed using a TaqMan miRNA RT kit (Applied Biosystems) and miRNA-specific stem-loop RT primers (Applied Biosystems). Real-time PCR was performed using a StepOnePlus Real-time PCR System (Applied Biosystems) in a 10-μl PCR mixture containing 2 μl RT product, 5 μl TaqMan Universal PCR Master Mix, 0.2 μM TaqMan probe, and 10 μM forward and reverse primers. RNU6B was used as an internal control for miRNA detection.

Immunohistochemical analysis

Cartilage tissue specimens were fixed in 4% paraformaldehyde for paraffin embedding. For histologic evaluation, all sections were stained with Safranin O. The sections were deparaffinized, rehydrated, and treated with 0.05% trypsin/EDTA solution for 10 minutes at 37°C. Intrinsic peroxidase activity was blocked with 3% hydrogen peroxide, and sections were incubated with 1.5% normal goat serum for 30 minutes and then with 1:100 dilution of polyclonal antibody against MMP-13 for 16 hours at 4°C. The sections were rinsed and incubated sequentially with biotinylated secondary antibody for 30 minutes and Vectastain ABC reagent (Vector Laboratories) for 30 minutes at room temperature. All sections were developed with ImmPACT diaminobenzidine peroxidase substrate kit (Vector Laboratories) and counterstained with methyl green (Dako). Rabbit IgG was used as a negative control.

MMP-13 enzyme-linked immunosorbent assay (ELISA).

Cells were stimulated with IL-1β for 24 hours, and the MMP-13 protein level in the culture supernatants was quantified by ELISA using a proMMP-13 immunoassay kit (R&D Systems), according to the manufacturer's instructions. Plates were read at 450 nm using a Multiskan Go Microplate Spectrophotometer (Thermo Fisher Scientific), and the MMP-13 concentration in the samples was calculated using a standard curve.

Western blot analysis

Following stimulation with IL-1β, culture supernatants were collected and cells were washed with cold phosphate buffered saline and lysed in lysis buffer (50 mM Tris HCl [pH 7.4], 150 mM NaCl, 20 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], and protease inhibitors). Proteins were resolved on a 10% SDS–polyacrylamide gel electrophoresis gel and transferred to PVDF membranes (Millipore). After blocking with 5% nonfat milk in Tris buffered saline plus 0.1% Tween 20, the membranes were incubated with primary antibodies against MMP-1, COX-2, p-JNK, p-p38, p-IκBα, p-ERK, or β-actin. After washing, primary antibodies were detected using HRP-conjugated secondary anti-mouse or anti-rabbit antibodies and visualized using an enhanced chemiluminescence kit (Santa Cruz Biotechnology).

Luciferase constructs and reporter assay

For construction of the wild-type MMP-13 3′-UTR-Luc reporter plasmid, a fragment of the 3′-UTR of the MMP-13 gene, including the predicted miR-127-5p binding site, was PCR-amplified using the primer sets 5′-CTC-GAG-TAA-CCG-TAT-TGT-TCG-CGT-CAT-GCC-AGC-A-3′ (forward) and 5′-GCG-GCC-GCC-AGA-CCA-TGT-GTC-CCA-TTT-G-3′ (reverse), and then cloned into a psi-CHECK-2 vector downstream of firefly luciferase with Xho Ι and Not Ι sites. To produce constructs that bear mutations at a putative miR-127-5p binding site in the wild-type MMP-13 3′-UTR, site-directed mutagenesis was performed. The wild-type MMP-13 3′-UTR constructs were mutated with the following oligonucleotide primer sets: forward 5′-GGT-TCT-GTG-AAC-AAT-TGG-CAG-TAA-GTT-ATC-3′ and reverse 5′-GAT-AAC-TTA-CTG-CCA-ATT-GTT-CAC-AGA-ACC-3′ (underline denotes the substitutions). The PCR mixture had 0.7 μl of expand long-range enzyme mix (Roche), 10 μl of 5× expand long-range buffer, 100 ng of plasmid template, 100 nM of primers, 3 μl of DMSO, and 2.5 μl of dNTPs (10 mM). PCR cycling conditions were as follows: 92°C for 30 seconds, 55°C for 1 minute, 68°C for 10 minutes, and a final extension at 68°C for 10 minutes.

After PCR, 20 μl of the reaction was digested with Dpn I at 37°C for 1 hour, and 10 μl was transformed into Escherichia coli DH5α to prepare the mutant construct plasmids. All constructs were confirmed by sequencing (Cosmogenetech). Cells were transfected with combinations of wild-type or mutant-type MMP-13 3′-UTR-Luc reporter plasmid and miR-Control or miR-127-5p, using Lipofectamine Plus reagent. Forty-eight hours after transfection, luciferase activity was determined with a Dual-Glo Luciferase Assay system (Promega), according to the manufacturer's instructions.

Statistical analysis

Results are expressed as the mean ± SD. Statistical analysis was carried out using the Mann-Whitney U test or Wilcoxon's signed rank test. P values less than 0.05 were considered statistically significant. All experiments were performed using samples from at least 3 different donors, with duplicate or triplicate replication.

RESULTS

Lower expression of miR-127-5p in OA cartilage versus normal cartilage, and down-regulation by IL-1β in human chondrocytes

To identify the miRNAs targeting the 3′-UTR of MMP-13 mRNA, a potent degrader of type II collagen in OA cartilage, miRNAs reported to be regulated by IL-1β in chondrocytes were analyzed using bioinformatic searches ([11, 18, 19]). We evaluated numerous miRNAs reported to be regulated by IL-1β in chondrocytes, using miRNA target prediction algorithms, including miRanda (available at http://www.microrna.org), TargetScan (available at http://www.targetscan.org), and PicTar (available at http://pictar.mdc-berlin.de/). Interestingly, among the IL-1β–responsive miRNAs, miR-127-5p was found to have the potential to regulate MMP-13.

To determine whether miR-127-5p expression changes during the progression of OA, we compared its expression level between normal and OA cartilage. As shown in Figure 1A, miR-127-5p expression was significantly reduced in OA cartilage compared with normal cartilage. Conversely, OA cartilage exhibited higher levels of MMP-13 mRNA (Figure 1B) and protein (Figure 1C) compared with normal cartilage. In addition, an inverse correlation between miR-127-5p expression and MMP-13 production was observed when human chondrocytes were stimulated with IL-1β, a potent proinflammatory cytokine in OA cartilage degradation. IL-1β stimulation of chondrocytes resulted in decreased miR-127-5p expression (Figures 1D and G) and increased levels of MMP-13 mRNA (Figures 1E and H) and protein (Figures 1F and I), in a time- and dose-dependent manner. These results suggest that miR-127-5p is closely related to the regulation of MMP-13 production induced by IL-1β.

Figure 1.

Expression of microRNA-127-5p (miR-127-5p) and matrix metalloproteinase 13 (MMP-13) in normal and osteoarthritic (OA) human cartilage and in interleukin-1β (IL-1β)–stimulated human chondrocytes. A and B, Relative expression levels of miR-127-5p mRNA (A) and MMP-13 mRNA (B) were assessed in normal and OA cartilage (n = 6 donors per group). Symbols represent individual cartilage donors; bars show the mean and 95% confidence interval for each group. C, Immunohistochemistry was performed on paraffin-embedded sections of normal and OA cartilage using a polyclonal antibody against MMP-13. Representative results are shown for 1 of 3 cartilage samples from each group. Original magnification × 200. D, E, G, and H, Human chondrocytes were deprived of serum for 24 hours, and then left untreated or treated with various concentrations of IL-1β for the indicated times. Relative expression levels of miR-127-5p (D and G) and MMP-13 mRNA (E and H) were measured by TaqMan miRNA assay and SYBR Green–based real-time polymerase chain reaction, respectively. RNU6B and GAPDH were used as endogenous controls. Results are the mean ± SD relative ratio, with values for the untreated cells designated as 1 in each experiment. F and I, IL-1β–induced MMP-13 production in human chondrocytes was determined by enzyme-linked immunosorbent assay, after 3–12 hours of stimulation (F) or 24 hours of stimulation (I) with IL-1β. Results are the mean ± SD of duplicate experiments in samples from 3 different donors. ∗ = P < 0.05 versus untreated control.

Regulatory IL-1β signaling pathways involved in miR-127-5p and MMP-13 expression in human chondrocytes

To investigate which signaling pathways of IL-1β regulate the expression of miR-127-5p and MMP-13, human chondrocytes were pretreated for 2 hours with the NF-κB inhibitor SN50 or various MAPK inhibitors, the MEK-1/2 inhibitor PD98059, p38 MAPK inhibitor SB203580, and JNK inhibitor SP600125. The chondrocytes were then stimulated with IL-1β for 6 hours (Figures 2A and B) or 24 hours (Figure 2C).

Figure 2.

Negative correlation between miR-127-5p expression and MMP-13 expression via IL-1β–induced signaling pathways. Human chondrocytes were pretreated for 2 hours with MAPK inhibitors (SB203580 at 1 μM, PD98059 at 10 μM, or SP600125 at 10 μM) or an NF-κB inhibitor (SN50 at 5 μM), and then left unstimulated or stimulated with IL-1β for 6 hours (A and B) or 24 hours (C). Human chondrocytes were analyzed by TaqMan miRNA assay for miR-127-5p expression (A), by SYBR Green real-time polymerase chain reaction for MMP-13 mRNA expression (B), and by enzyme-linked immunosorbent assay for production of MMP-13 protein in culture supernatants (C). In A and B, RNU6B and GAPDH were used as endogenous controls. Results are the mean ± SD of triplicate determinations in samples from 3 different donors. ∗ = P < 0.05 versus unstimulated control; # = P < 0.05 versus IL-1β–stimulated control. See Figure 1 for definitions.

Compared with the expression in unstimulated controls, miR-127-5p expression in IL-1β–stimulated human chondrocytes was decreased by 73%, and pretreatment with the MAPK inhibitors and the NF-κB inhibitor attenuated the inhibitory effects of IL-1β on miR-127-5p expression (Figure 2A). In contrast, stimulation of the cells with IL-1β resulted in significant up-regulation of MMP-13 mRNA and protein expression, and treatment with the NF-κB inhibitor and all MAPK inhibitors, except MEK-1/2 inhibitor PD98059, resulted in significant suppression of the IL-1β–induced MMP-13 expression in human chondrocytes (Figures 2B and C). This indicates that the down-regulation of miR-127-5p expression by activation of IL-1β signaling pathways has an inverse correlation with the production of MMP-13 in human chondrocytes.

Role of miR-127-5p as an endogenous attenuator of MMP-13 production in human chondrocytes, SW-1353 cells, and C28I2 cells

To assess whether IL-1β–responsive miR-127-5p itself regulates MMP-13 expression, human chondrocytes and chondrogenic SW-1353 cells were transfected with mature miR-127-5p, anti–miR-127-5p, or miR-Control. Transfection with miR-127-5p led to a 1,700-fold increase and 35,000-fold increase in miR-127-5p expression in human chondrocytes and SW-1353 cells, respectively, relative to control treatment, while transfection with anti–miR127-5p led to significant down-regulation of miR-127-5p expression (Figures 3A and C), as expected.

Figure 3.

Effects of miR-127-5p on IL-1β–induced MMP-13 mRNA expression. Human chondrocytes and SW-1353 cells were transfected with miR-127-5p or nonspecific control microRNA (miR-Control), or with anti–miR-127-5p or anti–miR-Control, and then left unstimulated or stimulated with IL-1β for 6 hours. Forty-eight hours after transfection with miR-127-5p or miR-Control (A and B) or with anti–miR-127-5p or anti–miR-Control (C and D), expression levels of miR-127-5p were measured by TaqMan miRNA assay (A and C), and expression levels of MMP-13 mRNA were analyzed by SYBR Green–based real-time polymerase chain reaction (B and D). RNU6B and GAPDH were used as endogenous controls. Results are the mean ± SD of triplicate determinations in samples from 3 different donors (for human OA chondrocytes) or the mean ± SD of duplicate determinations in samples from 3 different experiments (for SW-1363 cells). ∗ = P < 0.05 versus unstimulated control; # = P < 0.05 versus IL-1β–stimulated control. See Figure 1 for other definitions.

Forty-eight hours after transfection, cells were stimulated with IL-1β for 6 hours, and MMP-13 mRNA expression was determined by real-time RT-PCR. Overexpression of miR-127-5p, both in human OA chondrocytes and in SW-1353 cells, significantly decreased the IL-1β–induced expression of MMP-13 mRNA, while suppression of endogenous miR-127-5p with anti–miR-127-5p led to a significant up-regulation of MMP-13 (Figures 3B and D). In addition, miR-127-5p overexpression significantly decreased the IL-1β–induced production of MMP-13 protein, as measured with ELISA, while transfection with anti–miR-127-5p significantly up-regulated MMP-13 protein production in human chondrocytes, SW-1353 cells, and C28I2 chondrocytes (Figure 4). Importantly, transfection with anti–miR-127-5p led to an elevation in MMP-13 production in unstimulated chondrocytes (Figures 3D and 4B). These results suggest that miR-127-5p possesses the ability to suppress MMP-13 production in human chondrocytes.

Figure 4.

Effects of miR-127-5p on IL-1β–induced MMP-13 protein expression. Human chondrocytes, SW-1353 cells, and C29I2 cells were transfected with miR-127-5p or nonspecific control microRNA (miR-Control) (A), or with anti–miR-127-5p or anti–miR-Control (B). Forty-eight hours after transfection, cells were left unstimulated or stimulated with IL-1β for 24 hours, and MMP-13 production was analyzed by enzyme-linked immunosorbent assay. Results are the mean ± SD of triplicate determinations in samples from 3 different donors (for human OA chondrocytes) or the mean ± SD of triplicate determinations in samples from 4 different experiments (for cell lines). ∗ = P < 0.05 versus unstimulated control; # = P < 0.05 versus IL-1β–stimulated control. See Figure 1 for other definitions.

To analyze the effects of simultaneous transfection with both sense and antisense miRNA, we used SW-1353 and C28I2 cell lines, because the transfection efficiency of primary chondrocytes was not good. The results showed that simultaneous transfection led to a null effect in both cell lines (results available from the corresponding author upon request).

Binding of miR-127-5p to the 3′-UTR of MMP-13 mRNA leading to down-regulation of MMP-13 expression

To further clarify the molecular mechanism underlying the suppressive effect of miR-127-5p on MMP-13 expression, we analyzed the sequences in the 3′-UTR of human MMP-13 mRNA in detail. Notably, the 3′-UTR of human MMP-13 mRNA contains a putative miR-127-5p binding site with 7-mer seeds, and the putative miR-127-5p binding site and its adjacent sequences are highly conserved among diverse species (Figure 5A).

Figure 5.

MicroRNA-127-5p targets the 3′-untranslated region (3′-UTR) of MMP-13 mRNA. A, Sequence alignment of a putative miR-127-5p binding site within the 3′-UTR of MMP-13 mRNA shows a high level of sequence conservation and complementarity with miR-127-5p. Asterisks indicate the sequences that were changed to make mutant MMP-13 3′-UTR. CDS = coding sequence. B–D, The wild-type MMP-13 3′-UTR reporter plasmid (B and C) or mutant-type MMP-13 3′-UTR reporter plasmid (D) was cotransfected with miR-127-5p or nonspecific control microRNA (miR-Control) (B and D), or with anti–miR-127-5p or anti–miR-Control (C), into human OA chondrocytes, SW-1353 cells, and C28I2 cells. Untreated cells were used for comparison. Forty-eight hours after transfection, luciferase activity was determined, calculated as the ratio of reporter (firefly) to control (Renilla) activity. Results are the mean ± SD of triplicate determinations in samples from 3 different donors (for human OA chondrocytes) or the mean ± SD of duplicate determinations in samples from 3 different experiments (for cell lines). ∗ = P < 0.05. See Figure 1 for other definitions.

In order to determine the direct interaction between miR-127-5p and the 3′-UTR of MMP-13 mRNA, we generated a wild-type MMP-13 3′-UTR construct containing a putative miR-127-5p binding sequence. In accordance with the results of the MMP-13 ELISA, the luciferase reporter assay revealed that treatment with miR-127-5p significantly inhibited reporter activity, while anti–miR-127-5p enhanced reporter activity, in human OA chondrocytes, SW-1353 cells, and C28I2 cells (Figures 5B and C). Furthermore, we found that the construct bearing mutations at a putative miR-127-5p binding site completely abolished the inhibitory effect of miR-127-5p (Figure 5D). Taken together, these findings suggest that miR-127-5p acts as a direct suppressor of MMP-13 in human chondrocytes.

Regulatory effects of miR-127-5p on diverse catabolic pathways induced by IL-1β in human chondrocytes and SW-1353 cells

Since miR-127-5p is responsive to IL-1β, a potent catabolic stimulus of cartilage degradation, we further investigated the involvement of miR-127-5p in the IL-1β–induced catabolic effects in chondrocytes. For these studies, human chondrocytes and SW-1353 cells were transfected with mature miR-127-5p or miR-Control, and then stimulated with IL-1β for various amounts of time (ranging from 15 minutes up to 60 minutes). The regulatory effects of miR-127-5p on IL-1β–induced catabolic pathways were then determined by Western blot analysis.

Overexpression of miR-127-5p significantly inhibited the phosphorylation of JNK, p38, and IκBα in human chondrocytes (Figure 6A). However, miR-127-5p had no effect on IL-1β–induced activation of ERK (Figure 6A). In SW-1353 cells transfected with miR-127-5p, the phosphorylation of p38 and IκBα was significantly suppressed, whereas the activation of JNK and ERK was unchanged (Figure 6A).

Figure 6.

Suppression by miR-127-5p of IL-1β–mediated catabolic effects in human chondrocytes and SW-1353 cells. A, To investigate the effect of miR-127-5p on IL-1β–induced signaling pathways, human chondrocytes and SW-1353 cells were transfected with miR-127-5p or nonspecific control microRNA (miR-Control). After transfection, cells were left unstimulated or stimulated with IL-1β for the indicated times, and total cell lysates were analyzed by Western blotting for phosphorylation of JNK, p38, IκBα, and ERK. B and C, Forty-eight hours after transfection with miR-127-5p or miR-Control (B) or with anti–miR-127-5p or anti–miR-Control (C), cells were left unstimulated or stimulated with IL-1β for 24 hours, and the expression levels of MMP-1 and cyclooxygenase 2 (COX-2) protein were determined by Western blotting. The protein content was normalized to that of β-actin. Results are representative of 3 independent experiments. See Figure 1 for other definitions.

We then examined the relative expression levels of MMP-1 and COX-2, both of which are important mediators of the IL-1β–induced catabolic effects in cartilage. Interestingly, transfection with miR-127-5p dramatically reduced the expression of MMP-1 and COX-2, while anti–miR-127-5p caused a significant increase in the expression of MMP-1 and COX-2, in IL-1β–stimulated human chondrocytes and SW-1353 cells (Figures 6B and C). Taken together, these results suggest that miR-127-5p plays an important role in the maintenance of cartilage homeostasis, after the proinflammatory cytokine IL-1β has perturbed this process.

DISCUSSION

MMP overexpression can play an important role in the destruction of cartilage in chronic joint diseases such as OA or RA. The objective of this study was to investigate the regulation of MMP-13 production by miR-127-5p, a recently identified miRNA regulated by IL-1β. Our results showed that the expression of miR-127-5p is significantly decreased in OA cartilage compared with normal cartilage. The overexpression of miR-127-5p suppressed IL-1β–induced MMP-13 production, while transfection of the cells with anti–miR-127-5p remarkably increased MMP-13 production. Mutation of the miR-127-5p binding site in the 3′-UTR of MMP-13 mRNA abolished miR-127-5p–mediated repression of reporter activity, indicating that miR-127-5p constitutes a bona fide regulatory mechanism of MMP-13.

Recent studies reported the role of specific miRNAs in the pathogenesis of OA. MicroRNA-140 was found to be involved in chondrocyte hypertrophy, osteoblast differentiation, and maintenance of articular cartilage, by potently suppressing histone deacetylase 4 (HDAC-4), a known corepressor of RUNX-2 ([20]). MicroRNA-146a, which was found to be expressed in OA cartilage, is induced by a variety of microbial components and proinflammatory cytokines, such as interferon-γ, IL-1β, and tumor necrosis factor α (TNFα) ([21, 22]). MicroRNA-146a has gained interest because it was found to regulate key signaling intermediates of the proinflammatory Toll-like receptor/myeloid differentiation factor 88 pathway, including IL-1 receptor–associated kinase 1 and TNF receptor–associated factor 6 ([21]). In OA cartilage, an association between decreased expression of miR-146a and increased expression of MMP-13 was observed ([22]); however, whether MMP-13 is a direct target of miR-146a was not validated.

Consistent with the recognized importance of the role of cartilage as a load-bearing tissue, mechanoresponsive miRNAs in chondrocytes have also been identified. With the use of an miRNA microarray approach, one study explored the miRNAs differentially expressed in the different functional zones and in the anterior weight-bearing and posterior non–weight-bearing regions of bovine articular cartilage, and miRNA-221 and miR-222 were found to be up-regulated in the greater weight-bearing location ([23]). Furthermore, in a study of primary chicken chondrocytes, expression of miRNA-365 was found to be elevated in parallel with the mechanical induction of Indian hedgehog under cyclic loading, leading to significant stimulation of chondrocyte proliferation and differentiation through targeting of HDAC-4 ([24]).

Recent reports have described the regulation of MMPs by distinct miRNAs. MicroRNA-22 was shown to act indirectly on MMP-13 through 2 other factors, peroxisome proliferator–activated receptor γ and bone morphogenetic protein 7 ([13]). A study by Stanczyk et al demonstrated that the overexpression of miR-155 in RA synovial fibroblasts induced the repression of MMP-3, but not MMP-13 ([25]); however, MMP-3 was not validated as a direct target of miR-155. Jones and colleagues reported that miR-9 could modulate MMP-13 expression, but MMP-13 was not validated as a direct target of this miRNA ([10]).

MicroRNA-127 is expressed as a part of a cluster with miR-136, miR-431, miR-432, and miR-433, both in normal tissue and in cultured mouse and human fibroblasts. It has been suggested to function as a tumor suppressor because it is down-regulated in primary tumors and various cancer cell lines, and because it targets the mRNA of the protooncogene Bcl-6 to inhibit its expression ([26, 27]). In addition, miR-127 has been suggested to play a role in the control of cellular differentiation ([28]). However, its role as a regulator of inflammation and tissue degeneration has not been reported. In the present study, when human chondrocytes were stimulated with IL-1β, the expression of miR-127-5p was significantly down-regulated, and enhanced production of MMP-13 protein was noted.

We observed that miR-127-5p targets the seed site (1,556–1,562 bp) in human MMP-13 mRNA, and the seed site and adjacent sequences are highly conserved in the 3′-UTR of human, chimpanzee, mouse, rat, rabbit, and dog MMP-13 mRNA. These observations, in addition to the results of our luciferase reporter assay, indicate that miR-127-5p is most likely to interact with the 3′-UTR of MMP-13 mRNA and down-regulate its expression at the posttranscriptional level. Most animal miRNAs bind with mismatches and bulges, but a key feature of target recognition involves Watson–Crick base-pairing of miRNA nucleotides 2–8. This type of complementarity excludes AGO-mediated cleavage of mRNA while promoting the repression of mRNA translation ([29]); this constitutes the predominant mechanism of regulation by animal miRNA.

In our experiments, the IL-1β–induced suppression of miR-127-5p was reversed with an NF-κB inhibitor and several MAPK inhibitors. In addition, the phosphorylation of IκB, p38, and JNK induced by IL-1β was significantly down-regulated by miR-127-5p, suggesting that there is a reciprocal regulatory loop between NF-κB, MAPKs, and miR-127-5p in the modulation of IL-1β–induced MMP-13 expression. A similar negative regulation of miRNA in chondrocytes by activated MAPKs and NF-κB has also been reported previously ([11, 30]). These results suggest that miRNAs may amplify their impact by targeting a set of genes that are in a common pathway or protein complex. Many studies have shown that the proinflammatory signaling pathways regulated by miRNAs involve NF-κB and MAPK ([31-35]). For example, the role played by MAPK in the regulation of miRNA expression was previously demonstrated in a study in which regulation of miR-155–mediated inflammatory responses was observed in macrophages ([35]). In addition, a recent experiment in chondrocytes found that miR-34a, an miRNA regulated by JNK, is involved in chondrogenic differentiation by regulating the RhoA/Rac1 cross-talk signaling complex ([36]).

In the present study, although pretreatment of the cells with PD98059 reversed the suppressive effects of IL-1β on miR-127-5p, it did not exert any significant effect on the expression of either MMP-13 mRNA or MMP-13 protein. Thus, we speculate that there is an additional regulatory loop in IL-1–ERK–miR-127–MMP-13 signaling, such as additional miRNAs that may negate the influence of miR-127-5p on MMP-13 expression. Considering the results from recent research showing that the regulation conferred by miRNAs is usually redundant and complex, it would not be totally unexpected that the regulatory loop of miR-127-5p suppression of IL-1β–induced MMP-13 production does not work in the same direction in all MAPK pathways.

The potential role of miR-127-5p as an important regulator of the catabolic response in cartilage degradation was further suggested by its down-regulation of MMP-1, a major protease responsible for the degradation of the ECM, mainly collagen, and COX-2, the crucial enzyme in prostaglandin E2 production ([37, 38]). Although we did not find seed sequences for either MMP-1 or COX-2 in miR-127-5p, it is plausible that miRNAs constitute a regulatory motif by targeting a major transcription factor that regulates inflammatory and catabolic signaling. A recent report identified Smad4 as a direct target of miR-146a in chondrocytes ([39]). The findings demonstrated that miR-146a up-regulated vascular endothelial growth factor and reduced cellular responsiveness to transforming growth factor β via the suppression of Smad4 ([39]). These results further suggest that miRNAs can augment their impact by targeting diverse components in a signaling pathway, thus highlighting the importance of elucidating the pathways regulated by miRNAs in a broader perspective.

One of the important caveats of the current study is that there was no clarification of the significance of miR-127-5p in vivo. Considering the wide range of biologic influences that could be attributed to a single miRNA, great caution should be made in the application of miRNA technology in the treatment of a disease. In addition to biologic redundancy and possible off-target effects, the efficiency of miRNA delivery is also an important issue in the treatment of OA. With the hypothesis-generating in vitro results of this study in hand, we are currently planning an animal experiment that could prove its efficacy in vivo, incorporating the optimal miRNA delivery method.

In summary, we found that one additional crucial microRNA, miR-127-5p, which is down-regulated by IL-1β, acts as an important regulator of the MMP-13 and catabolic signaling pathways in human chondrocytes. Further research into the network of mediators involved in miR-127-5p regulation could facilitate the development of a novel therapeutic and/or preventive approach in OA.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Kim had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Park, Cheon, Lee, Kim.

Acquisition of data. Park, Cheon, Lee, Kim.

Analysis and interpretation of data. Park, Cheon, Lee, Kim.

Acknowledgments

We thank In Young Park for assistance with the histologic processing.

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