Hindawi BioMed Research International Volume 2019, Article ID 4714279, 10 pages https://doi.org/10.1155/2019/4714279 Research Article High Expression of Acid-Sensing Ion Channel 2 (ASIC2) in Bone Cells in Osteoporotic Vertebral Fractures Ching-Yu Lee,1,2,3 Tsung-Jen Huang ,1,2 Meng-Huang Wu ,1,2,3 Yen-Yao Li,4 and Kuan-Der Lee5,6 Department of Orthopedics, Taipei Medical University Hospital, Taipei, Taiwan Department of Orthopaedics, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan Department of Orthopedic Surgery, Chang Gung Memorial Hospital, Chiayi, Taiwan Division of Hematology and Oncology, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan Correspondence should be addressed to Tsung-Jen Huang; [email protected] Received 4 April 2019; Revised 11 July 2019; Accepted 28 July 2019; Published 19 August 2019 Academic Editor: Richard Tucker Copyright © 2019 Ching-Yu Lee et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Little is known about the function of acid-sensing ion channels (ASICs) in bone cells or osteoporotic vertebral fractures (OVF). Tis study delineated ASICs expression in adult human bone marrow-mesenchymal stem cells- (BM-MSC-) derived osteoblasts and in OVF bone cells. Adult BM-MSC-derived osteoblasts were isolated and cultured in diferent pH values. Osteogenic markers as alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OC) mRNA were assessed. Western blots method was applied + + to analyze ASICs protein expression in diferent pH values. Amiloride was added into the osteogenic media to analyze the Na /K ATPase change. We harvested the vertebral cancellous bone through a bone biopsy needle in 26 OVF patients when performing percutaneous vertebroplasty. Six vertebral bone specimens obtained from 4 patients with high-energy vertebral fractures were used as the control. Te reverse transcription polymerase chain reaction was performed to analyze the quantitative mRNA expression of ASICs. Osteogenic markers as ALP,OPN, and OC mRNA were higher expressed in increasing pH values throughout osteoblastogenesis. ASIC proteins were higher expressed in lower pH media, especially ASIC3, and ASIC4. Te highest protein + + expression at days 7, 14, and 21 was ASIC2, ASIC4, and ASIC3, respectively. Expression of Na /K ATPase was signifcantly decreased in cultured osteoblasts by addition of amiloride into the pH 6.9 osteogenic media. ASIC2 mRNA was most highly expressed with a 65.93-fold increase in the biopsied vertebral bone cells in OVF compared with the control. In conclusion, we found osteoblastogenesis was reduced in an acidic environment, and ASIC2, ASIC3, and ASIC4 were most highly expressed in turn during osteoblastogenesis within acidic media. ASIC2 was the most abundantly expressed gene in human bone cells in OVF compared with the control. ASIC2 could be crucial in the pathogenesis of osteoporosis and could serve as a therapeutic target for antiosteoporotic therapies. 1. Introduction and osteoblast-mediated formation of the bone. Te secretion of protons by osteoclasts during bone resorption leads to Osteoporosis is an age-related skeletal disease characterized acidifcation of the osteoclast-bone interface [4]. In addition, by decreased bone mass and deteriorated microarchitecture the activities of bone cells can be regulated by changes ofthebonetissuethatcontributetoanincreasedriskoffragile in pH. An increasing pH can induce mineralization and fractures [1, 2]. Te most common osteoporotic fractures are osteoblastic activity [5]. Acidosis, by contrast, stimulates vertebral, hip, and wrist fractures that result in morbidity and osteoclastic bone resorption [6]. An acidic microenviron- mortality in the elderly [3]. Biomechanism of osteoporosis ment can induce bone loss by increasing osteoclastogenesis is homeostatic imbalance in osteoclast-mediated resorption [7, 8], inducing autophagy in osteoblasts [9], and inhibiting 2 BioMed Research International osteoblast-mediated biomineralization [10]. With increas- Gibco BRL, Grand Island, NY) supplemented with 0.1 nM ing age, a signifcant increase in the steady-state blood dexamethasone(Sigma-Aldrich,St.Louis,MO),10mM�- hydrogen ion concentration and reduction in steady-state glycerophosphate (Sigma-Aldrich, St. Louis, MO), and 50 �M plasma bicarbonate concentration were observed, indicating L-ascorbic acid 2-phosphate (Sigma-Aldrich, St. Louis, MO). a progressively worsening low-level metabolic acidosis [11]. Osteoblastogenesis was evaluated by colorimetric semiquan- Hence, extracellular acidosis may play an important role in titative assessment of alkaline phosphatase activity as well osteoporosis development. as Alizarin Red S staining. In addition, amiloride (Sigma- Acid-sensing ion channels (ASICs) are a subfamily of Aldrich, St. Louis, MO), an ASICs antagonist, the ASIC epithelial sodium channel/degenerin and are recognized channel can be blocked by amiloride. An 0.2mM amiloride as tissue pH sensors [12, 13]. ASICs are transcripted and will be added into osteogenic culture median with diferent translated from 5 genes that encode 7 subunits of ASIC pH(pH6.9,7.4),inwhichtheMSCsareincubatedfor3,6,and + + noted (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4, and 12 hours to analyze the diferences of Na /K ATPase change. ASIC5). Most of ASICs were activated by rapidly increasing extracellular concentration of hydrogen ion (acidic pH) while .. Osteoporotic and Nonosteoporotic Bone Tissue Collection ASIC5 appears to be sensitive to bile acids rather than protons and Processing. Osteoporosis was defned as bone marrow [14]. In 1997,Waldmann et al. identifed the ASIC as a proton- density (BMD) at the hip or lumbar spine less than or equal gatedcationchannelinvolvedinacidsensingandfound to 2.5 standard deviations (SDs) below the mean BMD of a that the ASIC is expressed in the dorsal root ganglia and young adult reference population or a fragility fracture of a is distributed widely throughout the brain [15]. Multiple vertebra or hip and BMD between 1.0 and 2.0 SDs below the studies have focused on the relevance between ASICs and mean BMD of a young adult reference population [24]. Bone the pathophysiology of nonneuronal tissue diseases, such as density was measured through dual energy X-ray absorp- pulmonary cystic fbrosis [16], infammatory bowel disease tiometry. A bone biopsy needle (Stryker 11G Match Ground �� [17], liver fbrogenesis [18], immunobiology of dendritic cells Bevel Tip Introduction Needle 5 ,StrykerCorporation, [19], arthritis [20, 21], and intervertebral disc degeneration Michigan, USA) was inserted into the vertebral body through [22].However,littleisknownaboutwhethertheexpressionof the transpedicular route to obtain the vertebral cancellous ASICs is relevant to the pathophysiology of osteoporosis. Tis bone tissue. Te biopsied cancellous bone specimens from study aimed at delineating the expression pattern of ASICs in OVF were collected during spine surgery of 26 patients (22 adult human bone marrow-mesenchymal stem cells- (BM- females and 4 males, with a mean age of 78 years) with MSC-) derived osteoblasts and the expression pattern of low-energy trauma. Of the 26 patients with fragile vertebral ASICs in human bone cells in osteoporotic vertebral fractures fractures, the T-score of −2.5 SDs or lower was noted in 23 (OVF). patients and the T-score between −1and−2.5 SDs was noted in 3 patients. Te six biopsied cancellous bone specimens at 2. Materials and Methods nonfracture vertebral levels from 4 patients (2 females and 2 males, with a mean age of 45 years), who underwent surgery .. Human Bone Marrow MSC Isolation and Culture. To for high-energy spine fractures, were used as the control. Te isolate human MSCs, bone marrow aspirates were taken from 4 patients in the control group had a T-score of −1SDor the iliac crest of normal adult donors afer informed consent above, which was considered as normal bone density. Te and under a protocol approved by an Institutional Review biopsied cancellous bone specimen was fushed with saline to Board (IRB/CGMH reference no. 97-0230B). remove blood clots and then was immediately frozen in liquid ∘ Te techniques for isolation and culture of the adult bone nitrogen following surgery and stored at −80 Cuntilfurther marrow MSCs were previously described by our colleague, use. Approval for this study was obtained from the ethics Lee KD [23]. MSCs are cultured and maintained in expansion committee and institutional review board of the institution medium consists of Iscove’s modifed Dulbecco’s medium (CMRP IRB no. 101-0135C). (IMDM, Gibco BRL, Grand Island, NY) with 10% fetal bovine serum (Hyclone, Logan, UT) supplemented with 10 ng/mL .. RNA Extraction and Reverse Transcription Polymerase epidermal growth factor (R&D Systems), 10 ng/mL beta Chain Reaction (RT-PCR). Total cellular RNA was extracted fbroblast growth factor (R&D Systems), and 1% penicillin- from the mesenchymal stem cells afer diferentiation at days streptomycin- glutamine (Gibco BRL). Cells are allowed to 7, 14, and 21 or the biopsied cancellous bone cells using the adhere overnight, and nonadherent