Low-dose strontium stimulates osteogenesis but high-dose doses cause apoptosis in human adipose-derived stem cells via regulation of the ERK1/2 signaling pathway

Abstract

Background: Strontium is a widely used anti-osteoporotic agent due to its dual effects on inhibiting bone resorption and stimulating bone formation. Thus, we studied the dose response of strontium on osteo-inductive efficiency in human adipose-derived stem cells (hASCs).

Method: Qualitative alkaline phosphatase (ALP) staining, quantitative ALP activity, Alizarin Red staining, real-time polymerase chain reaction and Western blot were used to investigate the in vitro effects of a range of strontium concentrations on hASC osteogenesis and associated signaling pathways.

Results: In vitro work revealed that strontium (25-500 μM) promoted osteogenic differentiation of hASCs according to ALP activity, extracellular calcium deposition, and expression of osteogenic genes such as runt-related transcription factor 2, ALP, collagen-1, and osteocalcin. However, osteogenic differentiation of hASCs was significantly inhibited with higher doses of strontium (1000-3000 μM). These latter doses of strontium promoted apoptosis, and phosphorylation of ERK1/2 signaling was increased and accompanied by the downregulation of Bcl-2 and increased phosphorylation of BAX. The inhibition of ERK1/2 decreased apoptosis in hASCs.

Conclusion: Lower concentrations of strontium facilitate osteogenic differentiation of hASCs up to a point; higher doses cause apoptosis of hASCs, with activation of the ERK1/2 signaling pathway contributing to this process.

Keywords: ERK1/2 signaling pathway; Human adipose-derived mesenchymal stem cells; Osteogenesis; Strontium.

Fig. 1

hASCs had fibroblast-like morphology as observed under a phase-contrast microscope, at passage 0 (a) and 3 (b). Adipogenic differentiation was confirmed with positive Oil Red O staining (c). Osteogenic differentiation was confirmed with positive ALP (d) and Alizarin Red staining (e). Chondrogenic differentiation was verified with immunofluorescent staining for collagen type II (f). ALP activity of hASCs at indicated days after incubation with osteogenic differentiation medium (g)

Fig. 2

ALP staining and quantitative assay of osteo-induced hASCs in osteogenic differentiation medium with SrRan. SrRan was added at the indicated concentrations to hASCs in osteogenic differentiation medium induced for 10 days. a ALP expression was measured by staining. b Quantitative ALP activity determined with a colorimetric endpoint assay on the indicated days (d). ALP alkaline phosphatase, GM growth medium, OM osteogenic medium, Sr strontium

Fig. 3

Effect of SrRan on mineralization of hASCs. a Effect of SrRan added at the indicated concentrations to hASCs in osteogenic differentiation medium. Extracellular calcium deposition was measured with Alizarin Red S staining at day 21. b Extracellular calcium deposition was quantified with a colorimetric method on the indicated days (d). GM growth medium, OM osteogenic medium, Sr strontium

Fig. 4

Effect of SrRan on Cbfα1/RUNX2, ALP, COL I, and OCN gene expression in hASCs. Real-time PCR for osteogenic differentiation-related gene expression of hASCs treated with SrRan (25, 100, 250, and 500 μM) in OM for 10 days. Gene expression was measured using real-time PCR. a Core binding factor alpha 1 (Cbfα1); b alkaline phosphatase (ALP); c collagen type I (COLI); and d osteocalcin (OCN). Cells cultured in OM were used as controls. *p < 0.05, **p < 0.01 vs. OM. OM osteogenic medium, Sr strontium

Fig. 5

a Morphological features of hASCs exposed to SrRan at different time points. b hASCs were treated with SrRan (1000 μM) in GM and OM at the indicated days and viability was measured with a CCK-8 assay. GM growth medium, OM osteogenic medium, Sr strontium

Fig. 6

a Morphological features of hASCs exposed to SrRan. hASCs were treated with SrRan in GM and OM for 3 days. b hASCs exposed to SrRan in GM and OM, with cell viability measured with a CCK-8 assay. # p < 0.01 and * p < 0.05 compared with the control group

Fig. 7

SrRan-induced apoptotic and morphological changes and decreased hASC viability. a Apoptotic appearance (cell shrinkage) observed in hASCs after exposure to SrRan (1000, 2000, and 3000 μM) for 48 h. b High-dose SrRan-treated hASCs were fewer in number compared with the controls according to live/dead staining. c SrRan (1000, 2000, and 3000 μM) and apoptosis of hASCs were measured with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. d Quantification of TUNEL-positive hASCs. * p < 0.05 compared with the control group

Fig. 8

TEM analysis of hAMSC apoptosis induced by high-dose SrRan (1 mM) for 48 h. a–d Normal control hAMSCs. TEM of the hAMSC group showed intact cell membrane, nuclear membranes, and visible two unit-membranes (a and b); cytoplasmic organelle structures were integrated and there was a complete mitochondrial crest (c and d). e–h Apoptotic hAMSCs from the experimental groups. The cell membrane and nuclear membrane collapsed (e and f), and there was cellular content leakage, cytoplasmic organelle elimination, loss of chromatin and mitochondrial swelling, vacuolar degeneration, reduced matrix, and disappearing cristae seen with TEM (g and h). Scale bars = 2 meters

Fig. 9

SrRan activated MAPK pathways mediated by ERK, JNK, p38, and upregulated the expression of Bax and Bcl-2 in hASCs. a SrRan 1000 μM treatment downregulated Bcl-2 protein, and increased the expression of BAX and phosphorylation of P53in hASCs at 8 and 12 h. b The expression of total and phosphorylated ERK, p38, and JNK was measured by Western blotting in hASCs treated with SrRan 1000 μM at the indicated times. Phosphorylation of ERK was significantly increased at 8 and 12 h. No change occurred with phosphorylation of p38 and JNK after 0, 2, 4, 8, 12, and 24 h. c Results of densitometric scans of blots. Data are means ± SEM for three independent experiments

Fig. 10

ERK signaling contributed to SrRan-induced hASC apoptosis. The apoptosis of hASCs was measured by live/dead staining, AO/EB staining, and TUNEL assays in hASCs treated with strontium 1000 μM at 48 h. The hASC apoptosis caused by high-dose SrRan treatment was greatly attenuated by ERK-specific inhibitors (PD98059); however, JNK (SP600125) and p38 (SB203580) inhibitors showed little effects on hASC apoptosis induced by high-dose SrRan

Fig. 11

Diagram of high-dose SrRan-induced hASC apoptosis by ERK/p53-mediated pathways and subsequent Bcl-2 family-mediated mitochondrial apoptosis. SrRan activates ERK phosphorylation, which may increase phosphorylation of its target gene P53. Activation of P53, in turn, downregulates predicted target genes, anti-apoptotic protein, and Bcl-2, and upregulates the expression of the pro-apoptotic protein BAX in hASCs in OM, and causes mitochondrial apoptosis