Caffeine inhibits the viability and osteogenic differentiation of rat bone marrow-derived mesenchymal stromal cells

Abstract

Background and purpose: Caffeine is consumed extensively in Europe and North America. As a risk factor for osteoporosis, epidemiological studies have observed that caffeine can decrease bone mineral density, adversely affect calcium absorption and increase the risk of bone fracture. However, the exact mechanisms have not been fully investigated. Here, we examined the effects of caffeine on the viability and osteogenesis of rat bone marrow-derived mesenchymal stromal cells (rBMSCs).

Experimental approach: Cell viability, apoptosis and necrosis were quantified using thymidine incorporation and flow cytometry. Sequential gene expressions in osteogenic process were measured by real-time PCR. cAMP, alkaline phosphatase and osteocalcin were assessed by immunoassay, spectrophotometry and radioimmunoassay, respectively. Mineralization was determined by calcium deposition.

Key results: After treating BMSCs with high caffeine concentrations (0.1-1mM), their viability decreased in a concentration-dependent manner. This cell death was primarily due to necrosis and, to a small extent, apoptosis. Genes and protein sequentially expressed in osteogenesis, including Cbfa1/Runx2, collagen I, alkaline phosphatase and its protein, were significantly downregulated except for osteocalcin and its protein. Moreover, caffeine inhibited calcium deposition in a concentration- and time-dependent manner, but increased intracellular cAMP in a concentration-dependent manner.

Conclusions and implications: By suppressing the commitment of BMSCs to the osteogenic lineage and selectively inhibiting gene expression, caffeine downregulated some important events in osteogenesis and ultimately affected bone mass.

Figure 1

Viability of bone marrow-derived mesenchymal stromal cells (BMSCs) was decreased by caffeine. BMSCs were treated with different concentrations of caffeine (0, 0.1 and 1 mM) for 48 h and growth assessed by thymidine incorporation (shown as CPM per well). Data show that cell viability was decreased in a concentration-dependent manner. Bars represent the mean ± standard deviation (n = 9). Comparisons between groups were performed using anova. *P < 0.05, significantly different from the control group. CPM, counts per minute.

Figure 2

Caffeine inhibits bone marrow-derived mesenchymal stromal cell (BMSC) viability by inducing cell necrosis and apoptosis. BMSCs were treated with different concentrations of caffeine (0, 0.1, 1 mM) for 48 h. (A) Cell apoptosis and necrosis rates were analysed by flow cytometry (n = 3). PI−/AVF− represents viable cells. PI+/AVF+ represents necrotic cells. PI−/AVF+ represents apoptotic cells. (B,C) Data show the ratios of necrotic cells and apoptotic cells in each group. The percentage of apoptotic cells and necrotic cells increased significantly when exposed to 1 mM caffeine, but 0.1 mM caffeine was without effect on BMSC apoptosis and necrosis. Bars represent the mean ± standard deviation (n = 3). Comparisons between groups were performed using anova. *P < 0.01, significantly different from the control (0 mM caffeine) group. PI, propidium iodide. AVF, annexin-V-fluorescein.

Figure 3

Sequential gene expressions in osteogenesis were selectively inhibited by caffeine. The expressions of Cbfa1/Runx2, collagen I, ALP and osteocalcin were, respectively, detected at following time points: 3, 7, 11 and 16 days. (A–C) Cbfa1/Runx2, collagen I and ALP gene expressions were downregulated in a concentration-dependent manner. (D) Caffeine concentration dependently enhanced osteocalcin gene expression. Bars represent the mean ± standard deviation (n = 3). Comparisons between groups were performed using anova. *P < 0.01, **P < 0.05, significantly different from the control (0 mM caffeine) group. ALP, alkaline phosphatase.

Figure 4

ALP activity was negatively regulated by caffeine in a concentration- and time-dependent manner. ALP was assayed in cell lysates of BMSCs on 5, 10 and 15 days. Data show an attenuation of ALP activity after caffeine treatment at each time point. Each bar represents the mean ± standard deviation (n = 9). Comparisons between groups were performed using anova. *P < 0.01, significantly different from the control group. ALP, alkaline phosphatase; BMSCs, bone marrow-derived mesenchymal stromal cells.

Figure 5

Concentration and time response of osteocalcin upregulation by caffeine. Osteocalcin was assayed in cell lysates of BMSCs on 7, 14 and 21 days. Data show an accumulation of osteocalcin (OC) after caffeine treatment at each time point. Each bar represents the mean ± standard deviation (n = 9). Comparisons between groups were performed using anova. *P < 0.01, significantly different from the control group. BMSCs, bone marrow-derived mesenchymal stromal cells.

Figure 6

Time and concentration dependence of caffeine-induced inhibition of calcium deposition. Calcification of BMSCs was assayed on 7, 14 and 21 days of caffeine treatment. Data show that decreased calcium deposition was clearly observed at each time after caffeine treatment. Each bar represents the mean ± standard deviation (n = 9). Comparisons between groups were performed using anova. *P < 0.01, significantly different from the control group. BMSCs, bone marrow-derived mesenchymal stromal cells.

Figure 7

Caffeine concentration dependently increases intracellular cAMP. BMSCs were pre-treated for 5 min with 0.5 mM isobutyl methylxanthine and then treated with caffeine for 15 min. cAMP was measured by immunoassay. Data show an accumulation of cAMP after caffeine treatment. Each bar represents the mean ± standard deviation (n = 3). Comparisons between groups were performed using anova. *P < 0.01, significantly different from the control group. BMSCs, bone marrow-derived mesenchymal stromal cells.