miR-483 targets SMAD4 to suppress chondrogenic differentiation of human mesenchymal stem cells

Abstract

MicroRNAs (miRNAs) can regulate cellular differentiation processes by modulating multiple pathways simultaneously. Previous studies to analyze in vivo miRNA expression patterns in developing human limb cartilage tissue identified significant downregulation of miR-483 in hypertrophic chondrocytes relative to proliferating and differentiated chondrocytes. To test the function of miR-483 during chondrogenesis, lentiviral strategies were used to overexpress miR-483 during in vitro chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). While the in vivo expression patterns led us to hypothesize that miR-483 may enhance chondrogenesis or suppress hypertrophic marker expression, surprisingly, miR-483 overexpression reduced chondrocyte gene expression and cartilage matrix production. In addition, cell death was induced at later stages of the chondrogenesis assay. Mechanistic studies revealed that miR-483 overexpression resulted in downregulation of the TGF-β pathway member SMAD4, a known direct target of miR-483-3p. From these studies, we conclude that constitutive overexpression of miR-483 in hBM-MSCs inhibits chondrogenesis of these cells and does not represent an effective strategy to attempt to enhance chondrocyte differentiation and anabolism in this system in vitro. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2369-2377, 2017.

Keywords: SMAD4; TGF-β signaling; chondrogenesis; mesenchymal stem cell; miR-483; microRNA.

Figure 1

miR-483 is differentially expressed during in vivo cartilage development but not during in vitro chondrogenic differentiation. (A) Relative abundance of miR-483-5p and miR-483-3p relative to the reference gene RNU44 in proliferating (PC), differentiated (DC), and hypertrophic (HYP) chondrocytes isolated from developing human limb tissue by laser capture microdissection. Fold changes described by McAlinden et al., 2013 are PC > HYP, 8.19x; DC > HYP, 4.53x. *** indicates q = 0 based on Significance of Microarray (SAM) analysis; no asterisks indicates q ≥ 0.05. Data are replotted with permission from McAlinden et al., 2013 (20). (B, C) Differential expression of miR-483-5p (B) and miR-483-3p (C) during in vitro chondrogenesis of hBM-MSCs, calculated relative to the reference gene RNU44 and normalized to day 0 (hBM-MSCs in monolayer culture; n = 3 for all time points). Data were analyzed using a two-way ANOVA and significant changes compared to Day 0 were analyzed using a post-hoc Dunnett’s multiple comparisons test; no changes were statistically significant (p ≥ 0.05) after correction for multiple comparisons. (D) Differential expression of the miR-483 host gene IGF2, calculated relative to the reference gene B2M and normalized to day 0 as previously indicated. Statistical testing is as described in (B).

Figure 2

Physiologically-relevant miR-483 overexpression reduces cartilage matrix production and induces cell death. (A) Relative abundance of miR-140, miR-34a, miR-483-5p, and miR-483-3p compared to the reference gene RNU44 at day 14 of chondrogenic differentiation (n = 3 for all time points). Triangles indicate cells transduced with pTight-NS/rtTA, circles indicate cells transduced with pTight-miR-483/rtTA, and lines connect triangles and circles corresponding to the same donor. Data were analyzed using a Student’s t-test with a Bonferroni correction for multiple comparisons; no asterisks indicate p ≥ 0.05. (B) Representative image of Safranin O stained sections of day 14 chondrogenic pellets. Bar beneath image represents 100 μm. (C) Representative image of day 14 chondrogenic pellets stained for type II collagen (green); blue indicates nuclei stained with DAPI. Scale bar is as described in (B). (D) Representative image of NucGreen488-stained sections of day 14 chondrogenic pellets. Green indicates dead cells marked by NucGreen488, and blue indicates all nuclei, marked by DAPI staining. Scale bar is as described in (B). (D) Assessment of mRNA expression in miR-483-overexpressing pellets and NS-transduced controls at day 14 of chondrogenic differentiation. Data are calculated relative to the control gene B2M and are normalized to the NS-transduced controls (n = 3). Statistical testing was performed using a Student’s t-test using a Bonferroni correction for multiple comparisons. No asterisks indicates p ≥ 0.05, while *** indicates p < 0.001.

Figure 3

miR-483 overexpression decreases SMAD4 protein but does not affect mRNA levels of SMAD4 or the anti-apoptotic genes API5 or RAN. (A) Diagram depicting miR-483-3p binding sites within the SMAD4 3′ UTR as described in (37). Lines indicate complimentary base pairing. (B, C) Assessment of mRNA expression in miR-483-overexpressing pellets relative to NS-transduced controls at day 4 (B; n = 2) and day 14 (C; n = 3) of chondrogenic differentiation in the presence of TGF-β3. Data representation and statistical tests were performed and are represented as described in Figure 2D. (D–F) Quantification of SMAD4 and β-actin protein levels in pellets cultured in the presence (+ TGF-β) or absence (− TGF-β) of TGF-β3 and in pellets transduced with miR-483 or the NS control at day 4 of chondrogenic differentiation in the presence of TGF-β3. (D) Western blots of SMAD4 or β-actin from two independent donors, represented in grayscale. (E) Quantification of SMAD4 relative to β-actin, represented as percent of control (+ TGF-β). (F) Quantification of SMAD4 relative to β-actin, represented as percent of control (NS). For Figures D–F, experiments are shown separately to show variable responses to omission of TGF-β3; analysis of statistical significance was performed by Student’s t test and showed no significant result (p ≥ 0.05).