Thrombospondin-1 inhibits osteogenic differentiation of human mesenchymal stem cells through latent TGF-β activation

Abstract

Transforming growth factor-β (TGF-β) is a critical regulator of bone development and remodeling. TGF-β must be activated from its latent form in order to signal. Thrombospondin-1 (TSP1) is a major regulator of latent TGF-β activation and TSP1 control of TGF-β activation is critical for regulation of TGF-β activity in multiple diseases. Bone marrow-derived mesenchymal stem cells (MSCs) have osteogenic potential and they participate in bone remodeling in injury and in response to tumor metastasis. Since both TSP1 and TGF-β inhibit osteoblast differentiation, we asked whether TSP1 blocks osteoblast differentiation of MSCs through its ability to stimulate TGF-β activation. TSP1 added to human bone marrow-derived MSCs under growth conditions increases active TGF-β. Cultured MSCs express TSP1 and both TSP1 expression and TGF-β activity decrease during osteoblast differentiation. TSP1 and active TGF-β block osteoblast differentiation of MSCs grown in osteogenic media as measured by decreased Runx2 and alkaline phosphatase expression. The inhibitory effect of TSP1 on osteoblast differentiation is due to its ability to activate latent TGF-β, since a peptide which blocks TSP1 TGF-β activation reduced TGF-β activity and restored osteoblast differentiation as measured by increased Runx2 and alkaline phosphatase expression. Anti-TGF-β neutralizing antibody also increased alkaline phosphatase expression in the presence of TSP1. These studies show that TSP1 regulated TGF-β activity is a critical determinant of osteoblast differentiation.

Figure 1. TSP1 increases TGF-β activity in MSCs

MSC were plated in 6-well plates in growth media until 50% confluent. Cells were serum starved for 48 hrs and then treated daily with increasing concentrations of TSP1 for 48 hrs. Levels of active and total TGF-β in the conditioned media were measured using the PAI-1 promoter reporter luciferase assay. The dashed line represents residual TGF-β activity in the purified TSP1 added to the reporter cells. Data are the means +/- SD of triplicate determinations.

Figure 2. TSP1 and active TGF-β are decreased in the MSCs under osteogenic conditions

Cells were grown to confluence and then treated every 2 days for 30 days with osteogenic media or maintained in growth media. After 30 days, the media and cell lysates were collected. (A) TSP1 in the conditioned media was determined by immunoblotting. In separate gels, cell lysates were resolved and immunoblotted for β-tubulin. Results were normalized to β-tubulin. Results of densitometric analysis of bands are expressed as the mean ± SD of triplicate samples. (B) Active and total TGF-β in the conditioned media were measured by PAI-1 promoter reporter luciferase assay To obtain total TGF-β levels, samples were heat activated for 3 minutes at 100°C. *p<0.05

Figure 3. TSP1 and TGF-β decrease osteoblast differentiation and TSP1 inhibitory peptide LSKL increases osteoblast differentiation by MSCs under osteogenic conditions

A) MSCs were grown to confluence in basal (control) media. Cells were treated with control growth media, osteogenic media, or osteogenic media with TGF-β (5 ng/ml) or stripped TSP1 (10 nM), TSP1 + LSKL (25 μM inhibitory peptide), TSP1 + SLLK (25 μM control peptide), or TSP1 + anti-TGF-β (5 μg/ml). Cultures were fed daily for 20 days. Alkaline phosphatase staining is representative of triplicate wells. B) Cells were grown in 6-well plates and treated every 2 days for 20 days with MSC growth media (growth), osteogenic media (control) or osteogenic media supplemented with TGF-β (5 ng/ml) or TSP1 (10 nM). Some TSP1 treated cultures were also treated daily with 25 μM LSKL or 25 μM SLLK control peptide. RNA was isolated from cells treated under these conditions and used for RT-PCR analysis of Runx2 expression. Samples were run in duplicate and each experimental treatment in triplicate.

Figure 4. LSKL peptide decreases active TGF-β in MSCs under osteogenic conditions

Cells were grown in 6-well plates and treated every 2 days for 20 days with osteogenic media (control) or with TGF- β (5 ng/ml) or TSP1 (10 nM). Some TSP1 treated cultures were also treated daily with 25 μM LSKL or 25 μM SLLK control peptide. Conditioned media were harvested and active TGF-β measured using the PAI-1 promoter reporter luciferase assay. Data are the means of triplicate determinations +/− SD. *p<0.05 vs. control