BackgroundStrontium 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).MethodQualitative 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.ResultsIn 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.ConclusionLower 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.

The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to participate in maintenance and switches of smooth muscle cell (SMC) phenotypes. However, which isoform of CaMKII is involved in differentiation of adult mesenchymal stem cells into contractile SMCs remains unclear. In the present study, we detected γ isoform of CaMKII in differentiation of human adipose derived stem cells (hASCs) into SMCs that resulted from treatment with TGF-β1 and BMP4 in combination for 7 days. The results showed that CaMKIIγ increased gradually during differentiation of hASCs as determined by real-time PCR and western blot analysis. The siRNA-mediated knockdown of CaMKIIγ decreased the protein levels and transcriptional levels of smooth muscle contractile markers (a-SMA, SM22a, calponin, and SM-MHC), while CaMKIIγ overexpression increases the transcriptional and protein levels of smooth muscle contractile markers. These results suggested that γ isoform of CaMKII plays a significant role in smooth muscle differentiation of hASCs.

Understanding the molecular mechanisms underlying human adipose-derived stem cell (hASC) differentiation to smooth muscle may contribute to the development of effective therapies for relevant muscle defects, such as bladder wall and urethral defects. A previous study described the differentiation of hASCs to smooth muscle cells (SMCs) by transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein‑4 (BMP4) treatment. The present study investigated whether microRNA-145 (miR‑145) may be involved in the process of hASC differentiation. The expression of miR‑145 was significantly increased during differentiation of ASCs to SMCs. SMC‑specific genes and proteins, including a‑smooth muscle actin (α‑SMA), smooth muscle protein‑22α(SM22α), calponin and myosin heavy chain (SM‑MHC) were upregulated by transfection of a miR‑145 mimic. By contrast, these factors were downregulated following introduction of antisense oligonucleotides. In addition, Krüppel‑like factor 4 (KLF4) levels, which decreased during the differentiation of hASCs, were downregulated when the cells were transfected miR‑145 mimics. Futhermore, inhibition of KLF4 by treatment with short‑interfering‑RNA against KLF4, resulted in increased expression of SMC‑specific genes and proteins. In conclusion, the results of the present study demonstrated that by regulating KLF4, miR‑145 may be involved in regulating smooth muscle differentiation of ASCs induced by TGF‑β1 and BMP4.