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Arthritis Res Ther
2013 Sep 19;155:R126. doi: 10.1186/ar4306.
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GREM1, FRZB and DKK1 mRNA levels correlate with osteoarthritis and are regulated by osteoarthritis-associated factors.
Leijten JC, Bos SD, Landman EB, Georgi N, Jahr H, Meulenbelt I, Post JN, van Blitterswijk CA, Karperien M.
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INTRODUCTION: Osteoarthritis is, at least in a subset of patients, associated with hypertrophic differentiation of articular chondrocytes. Recently, we identified the bone morphogenetic protein (BMP) and wingless-type MMTV integration site (WNT) signaling antagonists Gremlin 1 (GREM1), frizzled-related protein (FRZB) and dickkopf 1 homolog (Xenopus laevis) (DKK1) as articular cartilage's natural brakes of hypertrophic differentiation. In this study, we investigated whether factors implicated in osteoarthritis or regulation of chondrocyte hypertrophy influence GREM1, FRZB and DKK1 expression levels.
METHODS: GREM1, FRZB and DKK1 mRNA levels were studied in articular cartilage from healthy preadolescents and healthy adults as well as in preserved and degrading osteoarthritic cartilage from the same osteoarthritic joint by quantitative PCR. Subsequently, we exposed human articular chondrocytes to WNT, BMP, IL-1β, Indian hedgehog, parathyroid hormone-related peptide, mechanical loading, different medium tonicities or distinct oxygen levels and investigated GREM1, FRZB and DKK1 expression levels using a time-course analysis.
RESULTS: GREM1, FRZB and DKK1 mRNA expression were strongly decreased in osteoarthritis. Moreover, this downregulation is stronger in degrading cartilage compared with macroscopically preserved cartilage from the same osteoarthritic joint. WNT, BMP, IL-1β signaling and mechanical loading regulated GREM1, FRZB and DKK1 mRNA levels. Indian hedgehog, parathyroid hormone-related peptide and tonicity influenced the mRNA levels of at least one antagonist, while oxygen levels did not demonstrate any statistically significant effect. Interestingly, BMP and WNT signaling upregulated the expression of each other's antagonists.
CONCLUSIONS: Together, the current study demonstrates an inverse correlation between osteoarthritis and GREM1, FRZB and DKK1 gene expression in cartilage and provides insight into the underlying transcriptional regulation. Furthermore, we show that BMP and WNT signaling are linked in a negative feedback loop, which might prove essential in articular cartilage homeostasis by balancing BMP and WNT activity.
Figure 1. GREM1, FRZB and DKK1 expression in healthy cartilage and osteoarthritic cartilage. Relative GREM1, FRZB and DKK1 mRNA expression levels of paired specimens of macroscopically preserved and degenerating osteoarthritic cartilage from a single osteoarthritic joint (n = 23) were assessed by quantitative PCR and were compared with healthy preadolescent (n = 4) and healthy adult (n = 3) articular cartilage specimen. Data expressed as fold-change relative to the specimen with the mRNA expression level on a log scale. *P <0.05. DKK1, dickkopf 1 homolog (Xenopus laevis); FRZB, frizzled-related protein; GADPH, glyceraldehyde 3-phosphate dehydrogenase; GREM1, Gremlin 1; OA, osteoarthritis.
Figure 2. Effects of bone morphogenetic protein signaling on the mRNA expression of GREM1, FRZB and DKK1. Chondrocytes were stimulated for 48 hours with different concentrations of BMP2 ranging from 0 to 200 ng/μl. Effects on gene expression of GREM1, FRZB, DKK1, AXIN2 and DKK1 were analyzed using quantitative PCR. Data expressed as fold-change relative to control and represents the mean of three donors ± standard deviation. *P <0.05 compared with 0 ng/ml BMP2. BMP2, bone morphogenetic protein 2; DKK1, dickkopf 1 homolog (Xenopus laevis); FRZB, frizzled-related protein; GADPH, glyceraldehyde 3-phosphate dehydrogenase; GREM1, Gremlin 1.
Figure 3. Effect of canonical WNT signaling on the mRNA expression of GREM1, FRZB and DKK1. (A) Primary human chondrocytes were exposed to 100 ng/ml WNT3A or three different concentrations of GIN. After 48 hours, AXIN2 mRNA expression was analyzed by quantitative PCR. (B) to (F) Chondrocytes were exposed to a single dose of 10 nM GIN or 100 ng/ml WNT3A. At indicated time points, mRNA expression was analyzed by quantitative PCR of AXIN2(B), FRZB(C), DKK1(D), GREM1(E) and ID1(F). Data expressed as fold-change relative to untreated time-point-matched control and represents the mean of three donors ± standard deviation. *P <0.05 compared with unstimulated cells (A) or 0 hours of stimulation (B) to (F). DKK1, dickkopf 1 homolog (Xenopus laevis); FRZB, frizzled-related protein; GADPH, glyceraldehyde 3-phosphate dehydrogenase; GREM1, Gremlin 1; WNT, wingless-type MMTV integration site.
Figure 4. Effects of IL-1β-mediated signaling on mRNA expression of GREM1, FRZB and DKK1. Chondrocytes received a single dose of 10 or 100 ng/ml IL-1β, or a daily medium refreshment containing 10 ng/ml IL-1β. Chondrocytes were stimulated up to 96 hours and mRNA expression was analyzed by quantitative PCR at the indicated time points for GREM1(A), FRZB(B), and DKK1(C). Data expressed as fold-change relative to untreated time point-matched control and represent the mean of three donors ± standard deviation. *P <0.05 compared with unstimulated cells. DKK1, dickkopf 1 homolog (Xenopus laevis); FRZB, frizzled-related protein; GADPH, glyceraldehyde 3-phosphate dehydrogenase; GREM1, Gremlin 1.
Figure 5. Effects of physiological factor-mediated signaling on mRNA expression of GREM1, FRZB and DKK1. Effects of mechanical loading, oxygen level and medium tonicity mediated signaling on mRNA expression of GREM1, FRZB and DKK1. Chondrocytes encapsulated in a hydrogel received no, intermittent or constant cyclical mechanical loading of 0.5 MPa with a frequency of 0.33 Hz and a loading phase of 50% (A). Chondrocytes were exposed to normoxic or hypoxic culture conditions (B), or were cultured in media with different tonicity with or without FK506 (C). All conditions were analyzed for the mRNA expression of GREM1, FRZB and DKK1 by quantitative PCR after 48 hours. Data expressed as fold-change relative to control and represent the mean of three donors ± standard deviation. *P <0.05 compared with unloaded samples or samples containing medium of 280 mOsm. DKK1, dickkopf 1 homolog (Xenopus laevis); FRZB, frizzled-related protein; GADPH, glyceraldehyde 3-phosphate dehydrogenase; GREM1, Gremlin 1.
Figure 6. Preliminary working model of WNT and BMP signaling feedback loop and perturbation by osteoarthritis factors. (A) WNT and BMP signaling reciprocally regulates the transcription of other antagonists. Exposure of WNT agonists leads to activation of WNT signaling [1]. This activation results in the downregulation of WNT antagonists (for example, FRZB and DKK1), leading to less inhibition of WNT signaling [2]. Additionally, BMP antagonists (for example, GREM1) are downregulated, leading to less inhibition of BMP signaling [3]. Stronger BMP signaling results in the upregulation of WNT antagonists [4], establishing a negative feedback mitigating WNT signaling [5]. This feedback loop allows for tight control of both BMP and WNT signaling in articular cartilage contributing to homeostasis. (B) Established factors that influence cartilage homeostasis also perturb this feedback loop. IL-1β, lack of mechanical stimulation and tonicity all decrease the mRNA levels of WNT and BMP antagonists, possibly resulting in a reset of the feedback loop, and contributing to the loss of cartilage homeostasis. BMP, bone morphogenetic protein; DKK1, dickkopf 1 homolog (Xenopus laevis); FRZB, frizzled-related protein; GADPH, glyceraldehyde 3-phosphate dehydrogenase; GREM1, Gremlin 1; WNT, wingless-type MMTV integration site.
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