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Extracellular Calcium Stimulates Osteogenic Differentiation of Human Cell Calcium 83 (2019) 102058 Contents lists available at ScienceDirect Cell Calcium journal homepage: www.elsevier.com/locate/ceca Extracellular calcium stimulates osteogenic differentiation of human T adipose-derived stem cells by enhancing bone morphogenetic protein-2 expression Risa Yanaia, Fumi Tetsuob,h, Shinichi Itoc, Momoe Itsumid, Junko Yoshizumie, Tomoko Makif, ⁎ ⁎⁎ Yoshihide Moria, Yasutaka Kubotag, , Shunichi Kajiokah, a Department of Oral and Maxillofacial Surgery, Graduate School of Dental Science, Kyushu University, Japan b Department of Periodontology Division of Oral Rehabilitation, Graduate School of Dental Science, Kyushu University c Section of Dentistry, Fukuoka Tokushukai Medical Center, Japan d Department of Oral Microbiology and Immunology, Showa University School of Dentistry, Japan e Department of Oral and Maxillofacial Surgery, Fukuoka Dental College, Japan f Section of Urology, Spinal Injuries Center, Japan g Department of Oral and Maxillofacial Surgery, Sasebo Kyosai Hospital, Nabasaki, Japan h Department of Clinical Pharmacology, Graduate School of Medical Sciences, Kyushu University, Japan ARTICLE INFO ABSTRACT Keywords: Bone morphogenetic protein-2 (BMP-2) promotes the differentiation of non-osteogenic mesenchymal cells to Human adipose-derived stem cells osteogenic cells. In this study, we isolated human adipose-derived stem cells (hASCs) and investigated the effects BMP-2 2+ 2+ of recombinant human BMP-2 (rhBMP-2) and extracellular Ca concentration ([Ca ]out) on the osteogenic Osteogenic differentiation of hASCs. rhBMP-2 promoted calcium deposition in hASCs and stimulated the mRNA expressions Extracellular Ca2+ of six proteins known to be involved in the osteogenic differentiation of hASCs: Runx2, osterix, alkaline phos- 2+ phatase, osteonectin, bone sialoprotein and osteocalcin. Elevation of [Ca ]out enhanced the level of alkaline phosphatase enzyme, increased the mRNA expressions of Runx2 and osteocalcin and induced the expressions of 2+ 2+ BMP-2 mRNA and protein in hASCs. Elevation of [Ca ]out transiently increased the intracellular Ca con- 2+ 2+ centration ([Ca ]in) due to activation of the calcium-sensing receptor (CaSR). The Ca -induced expressions of BMP-2 mRNA and protein were inhibited by the calmodulin antagonist, W-7. Furthermore, elevation of 2+ [Ca ]out decreased the cytoplasmic level of phosphorylated nuclear factor of activated T-cell-2 (NFAT-2) and increased the nuclear level of dephosphorylated NFAT2. Taken together, these results suggest that rhBMP-2 2+ promotes the osteogenic differentiation of hASCs. Furthermore, an increase in[Ca ]out enhances the expression 2+ 2+ of BMP-2 via activation of the CaSR, elevation of [Ca ]in and stimulation of Ca /calmodulin-dependent NFAT-signaling pathways. 1. Introduction from adipose tissue and with a greater cellular yield than BMSCs [6]. Bone morphogenetic proteins (BMPs) are members of the trans- Adipose-derived stem cells (ASCs) have high multi-differentiative forming growth factor-β superfamily of polypeptides. BMPs bind to the potential [1,2]. ASCs retain their multipotency when expanded in cul- transmembrane heterotetrameric complexes formed by BMP receptor ture and can differentiate into a variety of cell types, including osteo- type IA (BMPR1A), type IB (BMPR1B) and type II (BMPR2). BMPs have blasts, chondrocytes, myocytes and adipocytes [3]. Both autologous unique functions in bone growth, including embryonic skeletal devel- ASCs [4] and autologous bone marrow-derived mesenchymal stem cells opment [7] and postnatal bone remodeling [8]. Among the BMP family (BMSCs) [5] have been used successfully for osteoregeneration in pa- members, BMP-2 has been shown to stimulate osteogenic differentia- tients with critical bone defects. However, ASCs can be obtained easily tion of mesenchymal progenitor cells [9]. Furthermore, exogenous ⁎ Corresponding author at: Department of Oral and Maxillofacial Surgery, Sasebo Kyosai Hospital, 10-17 Shimanji-Cho, Sasebo, Nagasaki, 857-8575, Japan. ⁎⁎ Corrsponding author at: Department of Clinical Pharmacology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi Higashiku, Fukuoka, 812-8582, Japan. E-mail addresses: [email protected] (Y. Kubota), [email protected] (S. Kajioka). https://doi.org/10.1016/j.ceca.2019.102058 Received 8 August 2018; Received in revised form 19 June 2019; Accepted 16 July 2019 Available online 08 August 2019 0143-4160/ © 2019 Elsevier Ltd. All rights reserved. R. Yanai, et al. Cell Calcium 83 (2019) 102058 application of BMP-2 also promotes the trans-differentiation of non- Chemical Industries Ltd), 100 U/mL penicillin, 100 μg/mL streptomycin osteogenic mesenchymal cells, such as myogenic cells [10], fibroblasts and various concentrations of Ca2+ [27]. [11] and ASCs [12], to osteogenic cells. Therefore, BMP-2 has attracted attention as a potential therapeutic agent, and recombinant human 2.2. Extraction of cytoplasmic and nuclear proteins BMP-2 (rhBMP2) has been used to treat degenerative lumbar disc dis- ease and tibial fractures [13]. However, the use of rhBMP-2 in the Cytoplasmic and nuclear proteins were extracted from cultured cells clinical setting could be problematic due to its high cost and potential using a ProteoExtract® Subcellular Proteome Extraction kit (Merck, risks of antigenicity and viral infection. Darmstadt, Germany) according to the manufacturer’s instructions. 2+ Changes in intracellular calcium level ([Ca ]in) act as a universal signal that intersects with many pathways regulating gene expression 2.3. Alizarin red S staining 2+ [14]. Elevated [Ca ]in activates the phosphatase, calcineurin, by in- teracting with Ca2+/calmodulin [15], and in turn, activated calcineurin Cells were cultured on 35-mm plates for 3, 7 or 14 days and then dephosphorylates a set of substrates that include nuclear factor of ac- stained with Alizarin red S (Sigma-Aldrich) for 15 min [28]. Densito- tivated T-cells (NFAT) [16]. Dephosphorylated NFAT translocates from metric analysis of the staining was performed using ImageJ 1.48 [29]. the cytoplasm to the nucleus [17] and activates gene expression in cooperation with other transcriptional regulators [18]. NFATs are a 2.4. von Kossa staining family of four transcription factors, NFAT1, NFAT2, NFAT3 and NFAT4. Reportedly, NFAT2 regulates bone mass by affecting both osteoblasts Cells were cultured on chamber slides to reach 80% confluence. 2+ and osteoclasts [19]. Furthermore, elevated [Ca ]in induces chon- After stimulation with rhBMP-2 (R&D Systems Inc., Minneapolis, MN, drogenesis in mesenchymal cells by stimulating BMP-2 expression via a USA) for 3, 7 or 14 days, the cells were stained with a calcium-staining calcineurin/NFAT pathway [20]. Although there is substantial evidence kit (ScyTek Laboratories, Inc., West Logan, UT, USA) according to the 2+ for the importance of [Ca ]in in the regulation of gene expression, the manufacturer’s instructions. Briefly, after fixation in 10% formalin, the 2+ role of [Ca ]in in ASCs remains to be investigated. cells were incubated first with 5% silver nitrate solution under ultra- The aim of this study was to examine how BMP-2 and Ca2+ regulate violet light for 1 h and then with Nuclear Fast Red solution (Sigma- osteogenic differentiation in isolated human ASCs (hASCs) and de- Aldrich) for 5 min to remove unreacted silver. The cells were washed termine whether Ca2+/NFAT2 signaling pathways might contribute to with 100% ethanol. Densitometric analysis of the staining was per- the underlying mechanisms. To achieve this aim, we manipulated formed using ImageJ 1.48. 2+ 2+ [Ca ]in in hASCs by varying extracellular Ca concentration 2+ 2+ ([Ca ]out), which has been shown in fibroblasts to influence [Ca ]in 2.5. ALP staining via a calcium-sensing receptor (CaSR) [21]. The osteogenic differ- entiation of hASCs was assessed by measuring the expressions of two ALP staining was performed after 3, 14 and 21 days of cell culture. transcription factors involved in osteoblastic differentiation, namely Briefly, the cells were fixed in 10% formalin for 2 min and thenin- runt-related transcription factor-2 (Runx2) and osterix [22–24], as well cubated with ALP staining solution (Takara Bio, Shiga, Japan) for as several bone proteins secreted by osteoblasts, namely osteonectin, 15 min. osteocalcin, alkaline phosphatase (ALP) and bone sialoprotein (BSP) [23,25,26]. 2.6. ALP activity 2. Materials and methods ALP activity was measured using an ALP assay kit (Wako Pure Chemical Industries) in accordance with the manufacturer’s instruc- 2.1. hASC isolation and culture tions. Briefly, cells were cultured in 48-well culture plates4 (3×10 cells/well) and lysed with 0.1% (v/v) Triton-X100. The cell lysates hASCs were isolated according to the modified protocol of Zuk et al. (20 μL) were incubated with 100 μL of a solution of the ALP substrate, [1]. Briefly, abdominal subcutaneous adipose tissue was collected from p-nitrophenol phosphate, at 37 °C for 15 min. The reaction was stopped 11 patients (6 males and 5 females; mean age, 69.1 ± 11.5 years) by the addition of 50 μL of 0.2 M NaOH, and the absorbance at 405 nm admitted to Kyushu University Hospital, Japan. The Ethics Committee was measured [28]. of Kyushu University approved the study protocol (No. 2019-130), and informed consent was obtained from all patients. The adipose tissues 2.7. Real-time polymerase chain reaction (qPCR) were minced into small pieces and washed extensively with sterile phosphate-buffered saline (PBS) to remove
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