TGF β 1-Induced Differentiation of Human Bone Marrow-Derived MSCs Is Mediated by Changes to the Actin Cytoskeleton

Abstract

TGFβ is a potent regulator of several biological functions in many cell types, but its role in the differentiation of human bone marrow-derived skeletal stem cells (hMSCs) is currently poorly understood. In the present study, we demonstrate that a single dose of TGFβ1 prior to induction of osteogenic or adipogenic differentiation results in increased mineralized matrix or increased numbers of lipid-filled mature adipocytes, respectively. To identify the mechanisms underlying this TGFβ-mediated enhancement of lineage commitment, we compared the gene expression profiles of TGFβ1-treated hMSC cultures using DNA microarrays. In total, 1932 genes were upregulated, and 1298 genes were downregulated. Bioinformatics analysis revealed that TGFβl treatment was associated with an enrichment of genes in the skeletal and extracellular matrix categories and the regulation of the actin cytoskeleton. To investigate further, we examined the actin cytoskeleton following treatment with TGFβ1 and/or cytochalasin D. Interestingly, cytochalasin D treatment of hMSCs enhanced adipogenic differentiation but inhibited osteogenic differentiation. Global gene expression profiling revealed a significant enrichment of pathways related to osteogenesis and adipogenesis and of genes regulated by both TGFβ1 and cytochalasin D. Our study demonstrates that TGFβ1 enhances hMSC commitment to either the osteogenic or adipogenic lineages by reorganizing the actin cytoskeleton.

Figure 1

TGFβ1 induces osteogenic and adipogenic differentiation. MSCs underwent osteogenic or adipogenic differentiation by culturing cells in the appropriate medium for 7 days. (a) Micrographs showing the degree of mineralized calcium deposition in noninduced cells (NI), osteoinduced cells (OS), osteoinduced cells + SB-431542 (OS + SB), and osteoinduced cells + TGFβ1 (OS + TGFB), as assessed by alizarin red S staining (20x magnification). (b) Quantification of mineralization in the alizarin red S stained groups shown in (a). Data are shown as the mean ± SD of three independent experiments (^∗∗∗ p < 0.005). (c) mRNA expression of the osteogenic markers ALPL, RUNX2, and OCN, normalized to GAPDH, as determined by RT-PCR. Data are shown as the mean ± SD of three independent experiments (^∗ p < 0.05; ^∗∗∗ p < 0.0005). (d) Micrographs showing the accumulation of lipid droplets in noninduced cells (NI), adipoinduced cells (AD), adipoinduced cells + SB-431542 (AD + SB), and adipoinduced cells + TGFβ1 (AD + TGFB), as determined by Oil red O staining (20x magnification). (e) Quantification of mature adipocytes in the NI, AD, AD + SB, and AD + TGFB groups, as determined by Nile red fluorescence intensity. Data are shown as the mean ± SD of three independent experiments (^∗∗∗ p < 0.005). (f) mRNA expression of the adipogenic markers LPL, aP2, PPARG-2, and ADIPOQ, normalized to GAPDH, as determined by RT-PCR. Data are shown as the mean ± SD of three independent experiments (^∗ p < 0.05; ^∗∗∗ p < 0.0005).

Figure 2

TGFβ1 does not affect hMSC proliferation or viability. (a) Chart showing the relative hMSC viability in the absence (CNT) or presence (TGFB1) of TGFβ1, as determined by alamarBlue assay reagent. Shown are cell viabilities on days 2 (D2), 3 (D3), and 4 (D4) of culture. (b) Real-time proliferation assay data using the xCELLigence RTCA DP system for hMSC cells with and without TGFβ1 treatment. Lower panel: cell proliferation was measured at 15-minute intervals for a total duration of 24 hours. Upper panel: summary data showing cellular proliferation after 24 hours. Data are shown as the mean ± SD of two independent experiments (n = 6). NS: not significant.

Figure 3

Molecular phenotype of TGFβ1-treated hMSCs. (a) Hierarchical clustering of genes that were differentially expressed in TGFβl-treated and untreated control (CNT) hMSCs. Rows represent individual gene expression for duplicate treated and untreated samples, as indicated. Columns represent individual transcripts. Relative expression levels are presented colorimetrically, according to the scale shown in the color bar. (b) Pie chart showing the pathways with the highest enrichment of genes significantly upregulated in TGFβ1-treated cells. (c) qRT-PCR validation of selected genes that were upregulated in the microarray data (n = 3, ^∗ p < 005; ^∗∗∗ p < 0001). Cells treated with vehicle (DMSO) were used as controls.

Figure 4

Transmission electron microscopy of MSCs with and without treatment with SB-431542, TGFβ1, or CYD. TEM ultrastructural analysis of MSCs following no treatment (CNT) or treatment with SB-431542 (SB), TGFβ1, or CYD. Increasing levels of magnification are indicated by scale bars. N: nucleus; Nu: nucleolus; AC: actin filaments; V: microvilli; M: mitochondria; PL: primary lysosome; SL: secondary lysosome; rER: rough endoplasmic reticulum; G: Golgi bodies; B: cell blebs; P: cell processes; IF: nuclear membrane infolding; EV: endocytotic vacuole.

Figure 5

Inhibition of actin polymerization promotes adipogenic differentiation but inhibits osteogenic differentiation in MSCs. MSCs underwent osteogenic or adipogenic differentiation by culturing cells in the appropriate medium for 7 days. Cells also underwent the indicated treatments. (a) Mineralized calcium deposition, as determined by alizarin red S staining in MSCs that were osteoinduced (OS), osteoinduced with TGFβ1 treatment two days prior to induction (OS + TGFβ1), osteoinduced with CYD treatment at the onset of induction (OS + CYD), or osteoinduced with both TGFβ1 and CYD treatment at the time points described above (OS + TGFβ1 + CYD). Lower panel: micrograph of stained wells. Upper panel: quantification of mineralized matrix formation under the indicated treatment conditions. Data are shown as the mean ± SD of three independent experiments (^∗ p < 0.05; ^∗∗∗ p < 0.005). (b) Gene expression of the osteogenic markers ALPL, RUNX2, and OCN, normalized to GAPDH, as determined by qRT-PCR. Cells were either not induced (NI) or induced under the conditions described in (a). Data are shown as the mean ± SD of three independent experiments (^∗ p < 0.05; ^∗∗ p < 0.005, ^∗∗∗ p < 0.0005). (c) Adipogenic differentiation of MSCs that were adipoinduced (AD), adipoinduced with TGFβ1 treatment 2 days prior to induction (AD + TGFβ1), adipoinduced with CYD treatment, initiated at the onset of induction (AD + CYD), or adipoinduced with both TGFβ1 and CYD treatment at the time points described above (AD + TGFβ1 + CYD). Lower panel: Oil red O staining of the indicated cells. Upper panel: Nile red quantification of oil content under the indicated conditions. (d) Gene expression of the adipogenic marker genes PPARG and LPL, determined by qRT-PCR and normalized to GAPDH, under the indicated treatment regimens. Data are shown as the mean ± SD of three independent experiments (^∗∗ p < 0.005, ^∗∗∗ p < 0.0005). All controls were treated with vehicle only.

Figure 6

Molecular phenotype of CYD-treated hMSCs. (a) Hierarchical clustering of genes that were differentially expressed in CYD-treated and untreated control (CNT) hMSCs. Rows represent individual gene expression for duplicate treated and untreated samples, as indicated. Columns represent individual transcripts. Relative expression levels are presented colorimetrically, according to the scale shown in the color bar. (b) Pie chart showing the pathways with the highest enrichment of genes significantly upregulated in CYD-treated cells. (c) Venn diagram depicting the overlap between the upregulated genes in TGFβ1-treated cells (UP TGFβ1 versus CNT) and the downregulated genes in CYD-treated cells (DOWN CYD versus CNT).

Figure 7

TGFβ1 signaling in hMSC differentiation. Schematic showing how TGFβ and CYD affect hMSC osteogenic and adipogenic differentiation through the modulation of genes associated with the actin cytoskeletal pathway. Suggested downstream targets are also shown.