LncRNA H19 Regulates BMP2-Induced Hypertrophic Differentiation of Mesenchymal Stem Cells by Promoting Runx2 Phosphorylation

Abstract

Objectives: Bone morphogenetic protein 2 (BMP2) triggers hypertrophic differentiation after chondrogenic differentiation of mesenchymal stem cells (MSCs), which blocked the further application of BMP2-mediated cartilage tissue engineering. Here, we investigated the underlying mechanisms of BMP2-mediated hypertrophic differentiation of MSCs. Materials and Methods: In vitro and in vivo chondrogenic differentiation models of MSCs were constructed. The expression of H19 in mouse limb was detected by fluorescence in situ hybridization (FISH) analysis. Transgenes BMP2, H19 silencing, and overexpression were expressed by adenoviral vectors. Gene expression was determined by reverse transcription and quantitative real-time PCR (RT-qPCR), Western blot, and immunohistochemistry. Correlations between H19 expressions and other parameters were calculated with Spearman's correlation coefficients. The combination of H19 and Runx2 was identified by RNA immunoprecipitation (RIP) analysis. Results: We identified that H19 expression level was highest in proliferative zone and decreased gradually from prehypertrophic zone to hypertrophic zone in mouse limbs. With the stimulation of BMP2, the highest expression level of H19 was followed after the peak expression level of Sox9; meanwhile, H19 expression levels were positively correlated with chondrogenic differentiation markers, especially in the late stage of BMP2 stimulation, and negatively correlated with hypertrophic differentiation markers. Our further experiments found that silencing H19 promoted BMP2-triggered hypertrophic differentiation through in vitro and in vivo tests, which indicated the essential role of H19 for maintaining the phenotype of BMP2-induced chondrocytes. In mechanism, we characterized that H19 regulated BMP2-mediated hypertrophic differentiation of MSCs by promoting the phosphorylation of Runx2. Conclusion: These findings suggested that H19 regulates BMP2-induced hypertrophic differentiation of MSCs by promoting the phosphorylation of Runx2.

Silencing Smad7 potentiates BMP2-induced chondrogenic differentiation and inhibits endochondral ossification in human synovial-derived mesenchymal stromal cells

“…Total RNA was purified from cells in 60-mm dishes using TRIzol (Invitrogen) according to the manufacturer's instructions [35]. Then, cDNA was obtained from total RNA extracted from cells using a reverse transcription (RT) reaction kit (TAKARA, Japan) [13,36]. cDNA was completed with SYBR Premix Ex Taq™II (TaKaRa, Japan), and the program was carried out as follows: 95°C for 30 s for one cycle; then, 95°C for 5 s and 60°C for 30 s, followed by plate reading for 40 cycles.…”

“…For the chondrogenic differentiation and hypertrophic differentiation of hSMSCs, the micromass culture method was used as previously noted [9,10,13]. Then, the cells were cultured in chondrogenic medium for 1 week, and this medium was composed of DMEM with ITS (Sigma-Aldrich), 50 μg/mL ascorbate (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich), and 10 ng/mL transforming growth factor-β3 (Sigma-Aldrich).…”

“…We constructed recombinant adenovirus to stabilize overexpression of BMP2 and Smad7 using AdEasy technology, and we constructed another recombinant adenovirus that expressed small interfering RNA (siRNA) targeting the coding region of human Smad7 using the recently described established pSOS system [9,10,13]. To determine whether recombinant adenovirus can be effectively transduced into hSMSCs, we observed the expression of GFP and RFP by fluorescence microscopy 24 h after infection ( Fig.…”

“…We further examined the effect of combined PPCNg treatment on BMP2-induced chondrogenesis in vivo [9,10,13]. As a thermoresponsive macromolecule, PPCNg can provide a better in vivo microenvironment for hSMSCs [25].…”

“…However, the use of BMP2 for bioengineered cartilage construction is still under debate, because BMP2 promotes not only chondrogenic differentiation but also endochondral ossification. Previous studies showed that hypertrophic markers, such as COL10 and MMP13, were expressed in the BMP2-induced ectopic cartilagelike mass at 21 days [13]. Vascular invasion, matrix calcification, and endochondral ossification were also observed in BMP2-induced MSCs, leading to the destruction of chondrogenic phenotypes [11,14].…”