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. 2013 Nov 15;8(11):e79463.
doi: 10.1371/journal.pone.0079463. eCollection 2013.

IL-17 inhibits chondrogenic differentiation of human mesenchymal stem cells

Masahiro Kondo  1 , Kunihiro Yamaoka, Koshiro Sonomoto, Shunsuke Fukuyo, Koichi Oshita, Yosuke Okada, Yoshiya Tanaka
Affiliations

IL-17 inhibits chondrogenic differentiation of human mesenchymal stem cells

Masahiro Kondo et al. PLoS One. .

Abstract

Objective: Mesenchymal stem cells (MSCs) can differentiate into cells of mesenchymal lineages, such as osteoblasts and chondrocytes. Here we investigated the effects of IL-17, a key cytokine in chronic inflammation, on chondrogenic differentiation of human MSCs.

Methods: Human bone marrow MSCs were pellet cultured in chondrogenic induction medium containing TGF-β3. Chondrogenic differentiation was detected by cartilage matrix accumulation and chondrogenic marker gene expression.

Results: Over-expression of cartilage matrix and chondrogenic marker genes was noted in chondrogenic cultures, but was inhibited by IL-17 in a dose-dependent manner. Expression and phosphorylation of SOX9, the master transcription factor for chondrogenesis, were induced within 2 days and phosphorylated SOX9 was stably maintained until day 21. IL-17 did not alter total SOX9 expression, but significantly suppressed SOX9 phosphorylation in a dose-dependent manner. At day 7, IL-17 also suppressed the activity of cAMP-dependent protein kinase A (PKA), which is known to phosphorylate SOX9. H89, a selective PKA inhibitor, also suppressed SOX9 phosphorylation, expression of chondrogenic markers and cartilage matrix, and also decreased chondrogenesis.

Conclusions: IL-17 inhibited chondrogenesis of human MSCs through the suppression of PKA activity and SOX9 phosphorylation. These results suggest that chondrogenic differentiation of MSCs can be inhibited by a mechanism triggered by IL-17 under chronic inflammation.

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Conflict of interest statement

Competing Interests: Yoshiya Tanaka has read the journal’s policy and has the following conflicts: YT received consulting fees, lecture fees, and/or honoraria from Mitsubishi-Tanabe Pharma, Eisai, Chugai Pharma, Abbott Japan, Astellas Pharma, Daiichi-Sankyo, Abbvie, Janssen Pharma, Pfizer, Takeda Pharma, Astra-Zeneca, Eli Lilly Japan, GlaxoSmithKline, Quintiles, MSD, Asahi-Kasei Pharma, and received research grants from Bristol-Myers, Mitsubishi-Tanabe Pharma, Abbvie, MSD, Chugai Pharma, Astellas Pharma, Daiichi-Sankyo. MK and KO are employees of the Mitsubishi Tanabe Pharma Corporation. KY received consulting fees from Pfizer. The authors declare that there has been no significant financial support for this work which could have influenced its outcome, and this does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. IL-17 inhibits TGF-β3-induced chondrogenic differentiation.
A, Human mesenchymal stem cells (MSCs) were cultured in pellets, with or without TGF-β3 (10 ng/mL). Macro-images of the aggregates cultured with IL-17, TNF-α or IL-1β at the indicated concentrations for 14 days. Scale bar represents 1 mm. B, Aggregates cultured in the presence of the indicated cytokines for 21 days were fixed and paraffin embedded, then sections were stained with Safranin O and anti-type II collagen antibody. Original magnification×10. Scale bars represent 500 µm. (A and B) Data are representative of two independent experiments with similar findings. C, Wet weight of aggregates treated with IL-17 for 14 days. Values are mean±SD of 6–7 aggregates per group from three independent experiments with similar tendencies. ## P<0.01, compared to no cytokine (−TGF-β3), by Student’s t-test. *P<0.05; **P<0.01, compared to no cytokine (+TGF-β3), by Dunnett’s multiple comparison test. D, Sulfated glycolaminoglycan (sGAG) content in aggregates treated with IL-17 for 21 days. Values are mean±SD of 3–4 aggregates per group from two independent experiments with similar findings. ## P<0.01, compared to no cytokine (−TGF-β3), by Student’s t-test. **P<0.01, compared to no cytokine (+TGF-β3), by Dunnett’s multiple comparison test. E, IL-17 receptor A (IL-17RA) mRNA levels in aggregates were determined by real-time PCR for the indicated time points. Values are normalized to β-actin expression and expressed as mean±SD of 6 aggregates per group from three independent experiments with similar tendencies. **P<0.01, compared to day 0 (undifferentiated MSC), by Dunnett’s multiple comparison test.
Figure 2
Figure 2. IL-17 suppressed the expression of chondrogenic marker genes.
Human MSCs were cultured as aggregates in TGF-β3-containing medium with the indicated concentrations of IL-17. After 14 days, type II collagen (COL2A1), aggrecan (ACAN), type X collagen (COL10A1), and alkaline phosphatase (ALP) mRNA levels were determined by real-time PCR and expressed relative to β-actin expression level. Values are mean±SD of 3 aggregates from 1 of 3 independent experiments with similar findings.
Figure 3
Figure 3. IL-17 does not affect SAMD2 activation, but attenuates SOX9 phosphorylation induced by chondrogenic culture.
Human MSCs were cultured in monolayer (DMEM containing 5% FBS) with indicated concentrations of IL-17 throughout the culture period. After 16 hr culture with serum-starved medium (0% FBS), cells were stimulated with TGF-β3 (10 ng/mL) for 15 min and A, whole cell lysates or B, cytoplasmic (upper panel) and nuclear (lower panel) fraction were analyzed for SMAD2 and phospho-SMAD2 expression by western blotting. β-actin and TBP were used as loading controls. C, Human MSCs were cultured on cover-glass slides and SMAD2 localization was determined by immunofluorescence microscopy. Original magnification×20. Scale bars represent 100 µm. D, Human MSCs were cultured as aggregates in chondrogenic induction medium supplemented with TGF-β3 and aggregate lysates were evaluated at the indicated time points by western blot analysis for total and phosphorylated SOX9. β-actin was used as a loading control. (A, B, C and D) Data are representative of two independent experiments with similar findings. E, IL-17 or 10 µM H89 was added at the indicated concentrations and analysis carried out at day 2 and 7 by western blotting (top: day 2, bottom: day 7). F, Densitometric analysis on day 7 was performed with CS Analyzer, version 3.0 (bottom). Values represent the mean±SD of three independent experiments. *P<0.05, compared to no cytokine, by Dunnett’s multiple comparison test. NS: not significant.
Figure 4
Figure 4. IL-17 treatment reduces PKA activity during chondrogenesis.
A, Human MSCs were cultured as aggregates with TGF-β3 in the presence of IL-17 (100 ng/mL) or H89 (10 µM). After 7 days, 3 aggregates were pooled and lysed in each group, and PKA activity within the soluble protein fraction was determined using PepTag non-radioactive PKA assay (top). Recombinant PKA catalytic subunit (2 µg/mL) was used as a positive control and water was used as a negative control. Densitometric analyses of the band intensities were performed and results were expressed as the test band intensity relative to that of the “no TGF-β3” sample (bottom). B, The DNA content of three aggregates in each group was measured after 7-day culture. Values shown in A and B are mean±SD of three independent experiments. **P<0.01 compared to no cytokine (+TGF-β3), by Dunnett’s multiple comparison test.
Figure 5
Figure 5. PKA activation is required for chondrogenic differentiation of human MSCs.
A, Paraffin sections from aggregates cultured in the presence of 10 µM H89 for 21 days were stained with Safranin O and anti-type II collagen antibody. Original magnification×10. Scale bars represent 500 µm. B, COL2A1, ACAN, COL10A1, and ALP mRNA levels in aggregates treated with the indicated concentrations of H89 for 21 days were determined by real-time PCR. Values are mean±SD of three aggregates from 1 of 2 independent experiments with similar findings. See Figures 1 and 2 for the definition of other symbols.
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Supported in part by a Research Grant-In-Aid for Scientific Research by the Ministry of Health, Labor and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the University of Occupational and Environmental Health, Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.