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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 miR-381, HDAC4, and RUNX2 expression in E16.5 mouse radius bones. In situ hybridization analysis of (A) the negative control and (B) miR-381 expression are shown; (C) enlarged view of the boxed area in panel B; white arrows show areas of positive staining for miR-381; (D) sections were stained with safranin O/Fast Green for observation of chondrocyte morphology; and (E–G) sections were subjected to immunohistochemistry (brown) analysis using normal (E) IgG (negative control), (F) HDAC4-specific, and (G) RUNX2-specific antibodies. Scale bar = 20 μm in panel C and 50 μm in the other panels. miR-381, microRNA-381; HDAC4, histone deacetylase 4; RUNX2, Runt-related transcription factor 2. P, proliferating chondrocytes; PH, prehypertrophic chondrocytes; H, hypertrophic chondrocytes.
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ijms-17-01377-f002: Evaluation of miR-381, HDAC4, and RUNX2 expression in E16.5 mouse radius bones. In situ hybridization analysis of (A) the negative control and (B) miR-381 expression are shown; (C) enlarged view of the boxed area in panel B; white arrows show areas of positive staining for miR-381; (D) sections were stained with safranin O/Fast Green for observation of chondrocyte morphology; and (E–G) sections were subjected to immunohistochemistry (brown) analysis using normal (E) IgG (negative control), (F) HDAC4-specific, and (G) RUNX2-specific antibodies. Scale bar = 20 μm in panel C and 50 μm in the other panels. miR-381, microRNA-381; HDAC4, histone deacetylase 4; RUNX2, Runt-related transcription factor 2. P, proliferating chondrocytes; PH, prehypertrophic chondrocytes; H, hypertrophic chondrocytes.

Mentions: To assess the levels of miR-381 expression during different stages of cartilage development, the forelimbs of mouse embryos at embryonic day 16.5 (E16.5) were subjected to in situ hybridization analysis. While low levels of miR-381 expression were observed in proliferating chondrocytes, expression of this miRNA increased significantly in pre-hypertrophic and hypertrophic chondrocytes (Figure 2A–C). Notably, this expression pattern was similar to that of RUNX2 (Figure 2G). In contrast, HDAC4 expression peaked in pre-hypertrophic chondrocytes and decreased in hypertrophic chondrocytes, as determined by immunohistochemistry analysis (Figure 2D–F). These results are therefore consistent with those obtained using chondrogenic ATDC5 cells.


MicroRNA-381 Regulates Chondrocyte Hypertrophy by Inhibiting Histone Deacetylase 4 Expression
Evaluation of miR-381, HDAC4, and RUNX2 expression in E16.5 mouse radius bones. In situ hybridization analysis of (A) the negative control and (B) miR-381 expression are shown; (C) enlarged view of the boxed area in panel B; white arrows show areas of positive staining for miR-381; (D) sections were stained with safranin O/Fast Green for observation of chondrocyte morphology; and (E–G) sections were subjected to immunohistochemistry (brown) analysis using normal (E) IgG (negative control), (F) HDAC4-specific, and (G) RUNX2-specific antibodies. Scale bar = 20 μm in panel C and 50 μm in the other panels. miR-381, microRNA-381; HDAC4, histone deacetylase 4; RUNX2, Runt-related transcription factor 2. P, proliferating chondrocytes; PH, prehypertrophic chondrocytes; H, hypertrophic chondrocytes.
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Related In: Results  -  Collection

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ijms-17-01377-f002: Evaluation of miR-381, HDAC4, and RUNX2 expression in E16.5 mouse radius bones. In situ hybridization analysis of (A) the negative control and (B) miR-381 expression are shown; (C) enlarged view of the boxed area in panel B; white arrows show areas of positive staining for miR-381; (D) sections were stained with safranin O/Fast Green for observation of chondrocyte morphology; and (E–G) sections were subjected to immunohistochemistry (brown) analysis using normal (E) IgG (negative control), (F) HDAC4-specific, and (G) RUNX2-specific antibodies. Scale bar = 20 μm in panel C and 50 μm in the other panels. miR-381, microRNA-381; HDAC4, histone deacetylase 4; RUNX2, Runt-related transcription factor 2. P, proliferating chondrocytes; PH, prehypertrophic chondrocytes; H, hypertrophic chondrocytes.
Mentions: To assess the levels of miR-381 expression during different stages of cartilage development, the forelimbs of mouse embryos at embryonic day 16.5 (E16.5) were subjected to in situ hybridization analysis. While low levels of miR-381 expression were observed in proliferating chondrocytes, expression of this miRNA increased significantly in pre-hypertrophic and hypertrophic chondrocytes (Figure 2A–C). Notably, this expression pattern was similar to that of RUNX2 (Figure 2G). In contrast, HDAC4 expression peaked in pre-hypertrophic chondrocytes and decreased in hypertrophic chondrocytes, as determined by immunohistochemistry analysis (Figure 2D–F). These results are therefore consistent with those obtained using chondrogenic ATDC5 cells.

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.


Related in: MedlinePlus