MiR-365: a mechanosensitive microRNA stimulates chondrocyte differentiation through targeting histone deacetylase 4

Abstract

Mechanical stress plays an essential role in tissue development and remodeling. In this study, we determined the role of microRNA in chondrocyte mechanotransduction. Using microarray, we identified miR-365 as a mechanoresponsive microRNA in parallel to mechanical induction of Indian hedgehog (Ihh) in primary chicken chondrocytes cultured in 3-dimensional collagen scaffoldings under cyclic loading (1 Hz, 5% elongation). Interestingly, expression of miR-365 is elevated in the prehypertrophic zone of the growth plate, coinciding with the Ihh expression region in vivo. MiR-365 significantly stimulates chondrocyte proliferation and differentiation. MiR-365 increases expression of Ihh and the hypertrophic marker type X collagen, whereas anti-miR-365 inhibits the expression of these genes. We identified histone deacetylase 4 (HDAC4), an inhibitor of chondrocyte hypertrophy, as a target of miR-365. MiR-365 inhibits both endogenous HDAC4 protein levels as well as the activity of a reporter gene bearing the 3'-untranslated region of HDAC4 mRNA. Conversely, inhibition of endogenous miR-365 relieves the repression of HDAC4. Mutation of the miR-365 binding site in HDAC4 mRNA abolishes miR-365-mediated repression of the reporter gene activity. Overexpression of HDAC4 reverses miR-365 stimulation of chondrocyte differentiation markers including Ihh, Col X, and Runx2. Moreover, inhibition of miR-365 abolishes mechanical stimulation of chondrocyte differentiation. Taken together, miR-365 is the first identified mechanically responsive microRNA that regulates chondrocyte differentiation via directly targeting HDAC4.

Figure 1.

Regulation of miR-365 expression levels by mechanical loading. Proliferating chondrocytes isolated from the caudal part of sterna of 17 d chicken embryos were cultured in 3-D collagen sponges. Cyclic loading was applied to induce 5% deformation of the sponge at 1 Hz, 15 min/h for 24 and 48 h. Expression levels of miR-365 (A) and Ihh and ColX mRNA (B) were quantified by real-time RT-PCR. Values are means ± sd (n=3 cell culture experiments). *P < 0.01 vs. nonload.

Figure 2.

Expression of miR-365 in embryonic chicken sterna and tibia growth plate. A1–3) Total RNA was isolated from the caudal part (P, proliferating) and the cephalic part (PH, prehypertrophy) of sterna of 17-d chicken embryos. Expression levels of miR-365 (A1); Runx2 and Ihh (A2); and HDAC4, Sox9, Col II, and Col X (A3) were quantified by real time RT-PCR. B1–3) Tibia growth plate was isolated by dissection of 17-d chicken embryos. Three zones (P, proliferating; PH, prehypertrophy; and H, hypertrophy) of the growth plate were separated under dissection microscope as described previously (37). Total RNA was isolated from the cartilage pieces of the 3 zones, respectively. Expression levels of miR-365 (B1); HDAC4, Runx2, Sox9, Ihh, and Col II (B2); and Col X (B3) were quantified by real-time RT-PCR. Values are means ± sd (n=6). *P < 0.05.

Figure 3.

MiR-365 stimulates chondrocyte proliferation. A) Expression of miR-365 promotes chondrocyte proliferation. Primary chicken proliferating chondrocytes were transfected with a miR-365 mimic (miR-365) or a control miRNA as negative control (conmiR). After transfection of miRNAs at 100 nmol, cells were incubated in F12 medium containing 10% FBS for 15, 24, 36, 48, and 72 h. Cell growth was measured by an MTT-based cell proliferation assay. Values are means ± sd (n=3). *P < 0.05. B) A miR-365 inhibitor inhibits chondrocyte growth in a dose-dependent manner. Primary chicken proliferating chondrocytes were transfected with a miR-365 inhibitor (anti-miR-365) or a control miRNA inhibitor (control) at indicated concentrations. After incubation for 48 h, cell growth was measured by an MTT-based cell proliferation assay. Values are means ± sd (n=3). *P < 0.05, **P < 0.01 vs. control; ^#P < 0.05 vs. 50 nmol.

Figure 4.

MiR-365 enhances chondrocyte differentiation. A) Expression of miR-365 increases chondrocyte differentiation. Primary chicken proliferating chondrocytes were transfected with a miR-365 mimic (miR-365) or control miRNA (conmiR) at 100 nmol, respectively. Total RNA was extracted 48 h post-transfection. The mRNA levels of Ihh and ColX were quantified by real-time RT-PCR. Values are means ± sd (n=3). *P < 0.05. B) Inhibition of miR-365 down-regulates mRNA levels of Ihh and Col X. Primary chicken prehypertrophic chondrocytes were transfected with a miR-365 inhibitor (anti-miR-365) or a control miRNA inhibitor (control) at 100 nmol, respectively. At 48 h post-transfection, mRNA levels of Ihh and Col X were quantified by real-time RT-PCR. Values are means ± sd (n=3). *P < 0.05. C) Expression of miR-365 increases Ihh protein levels. Whole-cell lysates were collected and subjected to Western blot with an antibody against Ihh.

Figure 5.

HDAC4 is a direct target of miR-365. A) Diagram of predicted miR-365 sequential pairing with a putative target region (miR-365 binding site) in the 3′-UTR of HDAC4 mRNA. The 5′ end of miR-365 contains a base pair sequence complementary to the putative seed site in HDAC4 mRNA. B, C) MiR-365 inhibits endogenous HDAC4 protein levels in mouse chondrocytes (B) and embryonic chicken chondrocytes (C). Primary mouse or embryonic chicken chondrocytes were transfected with a miR-365 mimic (miR-365) or with a control miRNA (conmiR) at 100 nmol, respectively. At 48 h post-transfection, total protein was extracted from cell lysates. Western blot analysis was performed with an antibody against HDAC4. D) Inhibition of miR-365 enhances the protein levels of HDAC4 in mouse chondrocytes. Primary mouse chondrocytes were transfected with a miR-365 inhibitor (anti-miR-365) or a control miRNA inhibitor (control) at 100 nmol, respectively for 48 h. After total protein was extracted from cell lysates, Western blot analysis was performed with an antibody against HDAC4. Equal amounts of protein were loaded in each lane, and actin was used as a loading control. Values of quantitation of HDAC4 protein levels normalized to actin are presented. E) Inhibition of pGL-HDAC4 reporter gene activity requires the miR-365 seed site. Mouse chondrocytes were transfected with 0.5 μg pGL-HDAC4 reporter gene or pGL-HDAC4 mutant gene (pGL-HDAC4mut) with firefly luciferase and 0.1 μg PRL-TK gene with Renilla luciferase as internal control. At 48 h after transfection, cells were harvested for quantification of dual luciferase activities. Ratio of firefly vs. Renilla luciferase is the relative value of pGL-HDAC4 or pGL-HDAC4mut reporter gene activities. Values are means± sd (n=3). *P < 0.05.

Figure 6.

Overexpression of HDAC4 inhibits miR-365 stimulation of Ihh and Col X mRNA levels. A) Primary mouse chondrocytes were transfected with following 3 groups of constructs. Control: a control miRNA (conmiR) and a cDNA plasmid containing empty vector (EV); miR-365: a miR-365 mimic (miR-365) and a cDNA plasmid containing EV; and miR-365+HDAC4: a miR-365 mimic (miR-365) and a cDNA plasmid containing HDAC4 expression vector (HDAC4). At 48 h post-transfection, total RNA was isolated, and the mRNA levels of Ihh and Col X were quantified by real-time RT-PCR. Values are means± sd (n=3). *P < 0.05 vs. control; ^#P < 0.05 vs. miR-365. B) HDAC4 overexpression increased HDAC4 protein level. EV, transfection with empty vector. Western blot analysis was performed with cell lysates collected at 48 h post-transfection, with an antibody against HDAC4.

Figure 7.

MiR-365 increases Runx2 gene expression and its luciferase reporter activity. A) Primary chicken chondrocytes were transfected with a miR-365 mimic (miR-365) or a control miRNA (conmiR). At 48 h post-transfection, total RNA was extracted, and the mRNA levels of Runx2 was quantified by real-time PCR. B) Primary chicken chondrocytes were transfected with the Runx2 promoter reporter gene with firefly luciferase and an internal control gene with Renilla luciferase. They were cotransfected with the following 3 groups of constructs. Control: a control miRNA (conmiR) and a cDNA plasmid containing empty vector (EV); miR-365: a miR-365 mimic (miR-365) and a cDNA plasmid containing EV; and miR-365+HDAC4: a miR-365 mimic (miR-365) and a cDNA plasmid containing HDAC4 expression vector (HDAC4). The Runx2 luciferase reporter construct was transfected into primary chicken chondrocytes together with miR-365 or negative control miRNA (conmiR), as well as an internal control plasmid for Renilla luciferase. At 48 h after transfection, cells were harvested for quantification of dual luciferase activities. Ratio of firefly vs. Renilla luciferase is the relative value of Runx2 promoter reporter gene activities. Values are means± sd (n=3). *P < 0.05 vs. control; ^#P < 0.05 vs. miR-365.

Figure 8.

Inhibition of miR-365 abolishes mechanical stimulation of Ihh (A) and Col X (B) mRNA levels. Primary chicken chondrocytes were transfected with a miR-365 inhibitor (anti-miR-365) or a control miRNA inhibitor (control) for 48 h followed by cyclic loading for 24 h. Total RNA was extracted, and expression of ColX and Ihh mRNA was quantified by real-time RT-PCR. Ihh and Col X mRNA levels were normalized against the endogenous control, 18S RNA. Values are means ± sd (n=3). *P < 0.05 vs. nonload.

Figure 9.

Model for mechanical activation of chondrocyte proliferation and differentiation in a growth plate. A proposed mechanotransduction pathway activating chondrocyte proliferation and differentiation is shown. Cyclic loading induces proliferation and differentiation through regulating several key genes expressed in the prehypertrophic zone of the growth plate, including miR-365, HDAC4, Ihh, and Runx2.