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MicroRNA-381 Regulates Chondrocyte Hypertrophy by Inhibiting Histone Deacetylase 4 Expression

View Article: PubMed Central - PubMed

ABSTRACT

Chondrocyte hypertrophy, regulated by Runt-related transcription factor 2 (RUNX2) and matrix metalloproteinase 13 (MMP13), is a crucial step in cartilage degeneration and osteoarthritis (OA) pathogenesis. We previously demonstrated that microRNA-381 (miR-381) promotes MMP13 expression during chondrogenesis and contributes to cartilage degeneration; however, the mechanism underlying this process remained unclear. In this study, we observed divergent expression of miR-381 and histone deacetylase 4 (HDAC4), an enzyme that directly inhibits RUNX2 and MMP13 expression, during late-stage chondrogenesis of ATDC5 cells, as well as in prehypertrophic and hypertrophic chondrocytes during long bone development in E16.5 mouse embryos. We therefore investigated whether this miRNA regulates HDAC4 expression during chondrogenesis. Notably, overexpression of miR-381 inhibited HDAC4 expression but promoted RUNX2 expression. Moreover, transfection of SW1353 cells with an miR-381 mimic suppressed the activity of a reporter construct containing the 3′-untranslated region (3′-UTR) of HDAC4. Conversely, treatment with a miR-381 inhibitor yielded increased HDAC4 expression and decreased RUNX2 expression. Lastly, knockdown of HDAC4 expression resulted in increased RUNX2 and MMP13 expression in SW1353 cells. Collectively, our results indicate that miR-381 epigenetically regulates MMP13 and RUNX2 expression via targeting of HDAC4, thereby suggesting the possibilities of inhibiting miR-381 to control chondrocyte hypertrophy and cartilage degeneration.

No MeSH data available.


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Evaluation of the expression patterns of miR-381 and HDAC4, and of the hypertrophic markers RUNX2 and MMP13 during the chondrogenesis of ATDC5 cells. ATDC5 cells were cultured with/without ITS (insulin (10 μg/mL), transferrin (10 μg/mL), and sodium selenite (3 × 10−8 M)) and harvested on the indicated days. (A–E) Quantitative real-time reverse transcription (qRT)-PCR and Western blot analyses were utilized to evaluate the mRNA expression levels of (A) miR-381, (D) RUNX2, and (E) MMP13, and the protein expression levels of (B) HDAC4 during chondrogenesis, respectively; (C) graphic depiction of the protein expression levels quantified from the data presented in panel B. Data in panels A, C, D, and E are presented as means ± standard deviations of the results of three independent experiments. Negative control (NC) indicates cells cultured without ITS. * p < 0.05; ** p < 0.001. miR-381, microRNA-381; HDAC4, histone deacetylase 4; RUNX2, Runt-related transcription factor 2; MMP13, matrix metalloproteinase 13.
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ijms-17-01377-f001: Evaluation of the expression patterns of miR-381 and HDAC4, and of the hypertrophic markers RUNX2 and MMP13 during the chondrogenesis of ATDC5 cells. ATDC5 cells were cultured with/without ITS (insulin (10 μg/mL), transferrin (10 μg/mL), and sodium selenite (3 × 10−8 M)) and harvested on the indicated days. (A–E) Quantitative real-time reverse transcription (qRT)-PCR and Western blot analyses were utilized to evaluate the mRNA expression levels of (A) miR-381, (D) RUNX2, and (E) MMP13, and the protein expression levels of (B) HDAC4 during chondrogenesis, respectively; (C) graphic depiction of the protein expression levels quantified from the data presented in panel B. Data in panels A, C, D, and E are presented as means ± standard deviations of the results of three independent experiments. Negative control (NC) indicates cells cultured without ITS. * p < 0.05; ** p < 0.001. miR-381, microRNA-381; HDAC4, histone deacetylase 4; RUNX2, Runt-related transcription factor 2; MMP13, matrix metalloproteinase 13.

Mentions: To characterize the expression patterns of miR-381 and HDAC4 during chondrogenesis, ATDC5 cells were cultured in the presence of ITS ((insulin (10 μg/mL), transferrin (10 μg/mL), and sodium selenite (3 × 10−8 M)) to induce differentiation into chondrocytes. There was significantly greater expression of miR-381 in cells treated with ITS at 21 and 28 days post-induction than in those cultured in the absence of ITS (Figure 1A). In contrast, HDAC4 expression peaked at day 21 and decreased significantly at day 28 post-induction (Figure 1B,C). Meanwhile, the expression patterns of the two chondrocyte-hypertrophy markers RUNX2 and MMP13 were similar to that of miR-381 during chondrogenesis of ATDC5 cells (Figure 1D,E).


MicroRNA-381 Regulates Chondrocyte Hypertrophy by Inhibiting Histone Deacetylase 4 Expression
Evaluation of the expression patterns of miR-381 and HDAC4, and of the hypertrophic markers RUNX2 and MMP13 during the chondrogenesis of ATDC5 cells. ATDC5 cells were cultured with/without ITS (insulin (10 μg/mL), transferrin (10 μg/mL), and sodium selenite (3 × 10−8 M)) and harvested on the indicated days. (A–E) Quantitative real-time reverse transcription (qRT)-PCR and Western blot analyses were utilized to evaluate the mRNA expression levels of (A) miR-381, (D) RUNX2, and (E) MMP13, and the protein expression levels of (B) HDAC4 during chondrogenesis, respectively; (C) graphic depiction of the protein expression levels quantified from the data presented in panel B. Data in panels A, C, D, and E are presented as means ± standard deviations of the results of three independent experiments. Negative control (NC) indicates cells cultured without ITS. * p < 0.05; ** p < 0.001. miR-381, microRNA-381; HDAC4, histone deacetylase 4; RUNX2, Runt-related transcription factor 2; MMP13, matrix metalloproteinase 13.
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ijms-17-01377-f001: Evaluation of the expression patterns of miR-381 and HDAC4, and of the hypertrophic markers RUNX2 and MMP13 during the chondrogenesis of ATDC5 cells. ATDC5 cells were cultured with/without ITS (insulin (10 μg/mL), transferrin (10 μg/mL), and sodium selenite (3 × 10−8 M)) and harvested on the indicated days. (A–E) Quantitative real-time reverse transcription (qRT)-PCR and Western blot analyses were utilized to evaluate the mRNA expression levels of (A) miR-381, (D) RUNX2, and (E) MMP13, and the protein expression levels of (B) HDAC4 during chondrogenesis, respectively; (C) graphic depiction of the protein expression levels quantified from the data presented in panel B. Data in panels A, C, D, and E are presented as means ± standard deviations of the results of three independent experiments. Negative control (NC) indicates cells cultured without ITS. * p < 0.05; ** p < 0.001. miR-381, microRNA-381; HDAC4, histone deacetylase 4; RUNX2, Runt-related transcription factor 2; MMP13, matrix metalloproteinase 13.
Mentions: To characterize the expression patterns of miR-381 and HDAC4 during chondrogenesis, ATDC5 cells were cultured in the presence of ITS ((insulin (10 μg/mL), transferrin (10 μg/mL), and sodium selenite (3 × 10−8 M)) to induce differentiation into chondrocytes. There was significantly greater expression of miR-381 in cells treated with ITS at 21 and 28 days post-induction than in those cultured in the absence of ITS (Figure 1A). In contrast, HDAC4 expression peaked at day 21 and decreased significantly at day 28 post-induction (Figure 1B,C). Meanwhile, the expression patterns of the two chondrocyte-hypertrophy markers RUNX2 and MMP13 were similar to that of miR-381 during chondrogenesis of ATDC5 cells (Figure 1D,E).

View Article: PubMed Central - PubMed

ABSTRACT

Chondrocyte hypertrophy, regulated by Runt-related transcription factor 2 (RUNX2) and matrix metalloproteinase 13 (MMP13), is a crucial step in cartilage degeneration and osteoarthritis (OA) pathogenesis. We previously demonstrated that microRNA-381 (miR-381) promotes MMP13 expression during chondrogenesis and contributes to cartilage degeneration; however, the mechanism underlying this process remained unclear. In this study, we observed divergent expression of miR-381 and histone deacetylase 4 (HDAC4), an enzyme that directly inhibits RUNX2 and MMP13 expression, during late-stage chondrogenesis of ATDC5 cells, as well as in prehypertrophic and hypertrophic chondrocytes during long bone development in E16.5 mouse embryos. We therefore investigated whether this miRNA regulates HDAC4 expression during chondrogenesis. Notably, overexpression of miR-381 inhibited HDAC4 expression but promoted RUNX2 expression. Moreover, transfection of SW1353 cells with an miR-381 mimic suppressed the activity of a reporter construct containing the 3&prime;-untranslated region (3&prime;-UTR) of HDAC4. Conversely, treatment with a miR-381 inhibitor yielded increased HDAC4 expression and decreased RUNX2 expression. Lastly, knockdown of HDAC4 expression resulted in increased RUNX2 and MMP13 expression in SW1353 cells. Collectively, our results indicate that miR-381 epigenetically regulates MMP13 and RUNX2 expression via targeting of HDAC4, thereby suggesting the possibilities of inhibiting miR-381 to control chondrocyte hypertrophy and cartilage degeneration.

No MeSH data available.


Related in: MedlinePlus