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Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-of-function mutation of TRPV4 associated with metatropic dysplasia
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-of-function mutation of TRPV4 associated with metatropic dysplasia Xu Han 1, Hiroki Kato 1, Hiroshi Sato, Yuta Hirofuji, Satoshi Fukumoto**, Keiji Masuda* Section of Oral Medicine for Children, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University, Maidashi 3-1-1, Higashi-Ku, Fukuoka, 812-8582, Japan
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Article history: Received 4 December 2019 Accepted 30 December 2019 Available online xxx
Metatropic dysplasia (MD) is a congenital skeletal dysplasia characterized by severe platyspondyly and dumbbell-like long-bone deformities. These skeletal phenotypes are predominantly caused by autosomal dominant gain-of-function (GOF) mutations in transient receptor potential vanilloid 4 (TRPV4), which encodes a nonselective Ca2þ-permeable cation channel. Previous studies have shown that constitutive TRPV4 channel activation leads to irregular chondrogenic proliferation and differentiation, and thus to the disorganized endochondral ossification seen in MD. Therefore, the present study investigated the role of TRPV4 in osteoblast differentiation and MD pathogenesis. Specifically, the behavior of osteoblasts differentiated from patient-derived dental pulp stem cells carrying a heterozygous single base TRPV4 mutation, c.1855C > T (p.L619F) was compared to that of osteoblasts differentiated from isogenic control cells (in which the mutation was corrected using the CRISPR/Cas9 system). The mutant osteoblasts exhibited enhanced calcification (indicated by intense Alizarin Red S staining), increased intracellular Ca2þ levels, strongly upregulated runt-related transcription factor 2 and osteocalcin expression, and increased expression and nuclear translocation of nuclear factor-activated T cell c1 (NFATc1) compared to control cells. These results suggest that the analyzed TRPV4 GOF mutation disrupts osteoblastic differentiation and induces MD-associated disorganized endochondral ossification by increasing Ca2þ/NFATc1 pathway activity. Thus, inhibiting the NFATc1 pathway may be a promising potential therapeutic strategy to attenuate skeletal deformities in MD. © 2020 Elsevier Inc. All rights reserved.
Keywords: Dental pulp stem cells Metatropic dysplasia Osteoblast differentiation Transient receptor potential vanilloid 4
1. Introduction Transient receptor potential vanilloid 4 (TRPV4) is a sixtransmembrane Ca2þ-permeable polymodal cation channel that localizes to the cell membrane, and that is activated by various endogenous and exogenous stimuli, including heat, osmotic pressure, mechanical stimulation, and arachidonic acid [1]. TRPV4 gene mutations cause congenital skeletal dysplasias and neurological
* Corresponding author. Section of Oral Medicine for Children, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University, Maidashi 3-1-1, Higashi-Ku, Fukuoka, 812-8582, Japan. ** Corresponding author. Section of Oral Medicine for Children, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University, Maidashi 3-1-1, Higashi-Ku, Fukuoka, 812-8582, Japan. E-mail addresses: [email protected] (S. Fukumoto), kemasuda@ dent.kyushu-u.ac.jp (K. Masuda). 1 These authors contributed equally to this study.
disorders [2,3], including metatropic dysplasia (MD) [2]. Patients with MD exhibit short limbs at birth due to a severe defect of long bone development, and progressive kyphoscoliosis after birth, which causes the trunk to become shorter than the limbs, resulting in a reversal of body proportions (i.e. from ‘short limbs’ to ‘short trunk’) [2]. They also frequently exhibit severe platyspondyly and/or dumbbell-like long-bone deformities. MD phenotypes vary widely in severity, and include both lethal and non-lethal subtypes [2]. Several autosomal dominant gain-of-function (GOF) TRPV4 mutations have been shown to cause MD [4e7]. Previous studies have suggested that constitutive TRPV4 Ca2þ channel activation disrupts the expression of critical factors including SOX9, and thereby leads to the aberrant chondrocyte differentiation and function from which MD-associated long-bone developmental defects arise [7e9]. During normal endochondral ossification, mesenchymal progenitors initially form aggregates, then differentiate to form chondrocytes, osteoblasts, and osteoclasts in a
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Please cite this article as: X. Han et al., Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-offunction mutation of TRPV4 associated with metatropic dysplasia, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.12.123
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temporally and spatially regulated manner, and finally undergo neovascular invasion into the developing bone [10e12]. A previously conducted histopathological analysis of bone tissues in aborted fetuses suggested that aberrant osteoblastic differentiation and ossification may underlie MD pathogenesis [5]. However, to date, only a small number of experimental models have been established to reproduce MD bone phenotypes; thus, the mechanisms that underlie MD osteoblastic defects with skeletal phenotypes remain poorly understood. The purpose of the present study was to investigate whether TRPV4 contributes to osteoblastic defects in MD. Dental pulpderived mesenchymal stem cells (DPSCs) were obtained from a patient that exhibited a severe MD phenotype, as well as the TRPV4 GOF mutation, c.1855C > T (p.L619F). The DPSCs were differentiated into osteoblasts, examined for MD-associated defects, and compared to isogenic control cells in which the c.1855C > T mutation was corrected using the CRISPR/Cas9 system.
2.4. RNA extraction and quantitative real-time polymerase chain reaction
2. Materials and methods
Total RNA was extracted from cells using an RNAeasy Mini Kit (Qiagen). First-strand cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). qRT-PCR was conducted using primers for runt-related transcription factor 2 (RUNX2) (forward (F), 50 -CAGTTCCCAAGCATTTCATCC-30 ; reverse (R), 50 -TCAATATGGTCGCCAAACAG-30 ); osteocalcin (OCN) (F, 50 -GCAAAGGTGCAGCCTTTGTG-30 ; R, 50 GGCTCCCAGCCATTGATACAG-30 ); early growth response 2 (EGR2) (F, 50 -GACCAGATGAACGGAGTGGC-30 ; R, 50 -GCAAAGCTGCTGGGATATGGG-30 ); 18S rRNA (F, 50 -CGGCTACCACATCCAAGGAA-30 ; R, 50 GCTGGAATTACCGCGGCT-30 ), as well as GoTaq qPCR Master Mix (Promega, WI, USA), and the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). The 18S rRNA (control) threshold cycle (Ct) value was subtracted from that of the target genes (DCt). Relative target gene expression levels are shown as fold changes determined using the 2DDCt method.
2.1. Isolation and culture of DPSCs
2.5. Intracellular calcium level measurement
DPSCs were isolated from a 14-year-old patient with MD that harbored the TRPV4 GOF mutation, c.1855C > T (p.L619F) [7]. Written informed consent was obtained from the patient’s guardians for the patient’s participation in the study, which was approved by the Kyushu University Institutional Review Board for Human Genome/Gene Research (permission number: 678e01), and conducted in accordance with the Declaration of Helsinki. The TRPV4 1855C > T mutation was repaired using the CRISPR/ Cas9 system to produce isogenic control DPSCs, as previously described [7]. All DPSCs were cultured (37 C, 5% CO2) in Alpha Modification of Eagle’s Medium (aMEM; Nacalai Tesque, Kyoto, Japan) that was supplemented with 15% fetal bovine serum (SigmaAldrich, MO, USA), 100 mM L-ascorbic acid 2-phosphate (Wako Pure Chemical Industries, Osaka, Japan), 250 mg/mL fungizone (Thermo Fisher Scientific, MA, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Nacalai Tesque). Cells passaged seven times or less were used in experiments.
Cells were cultured in 96-well plates, and stained (45 min, 37 C) with 10 mM Fura-2 AM (Dojindo, Kumamoto, Japan), 0.05% (w/v) Pluronic F-127 (Sigma-Aldrich), and 500 mM probenecid (SigmaAldrich) in Hanks’ Balanced Salt Solution. Fluorescence signals were measured (at 37 C) by excitation at 340 and 380 nm, and emission at 510 nm, using an EnSight plate reader (PerkinElmer, MA, USA). Cells were administered 4a-phorbol 12,13-didecanoate (4aPDD) (1 mM; Wako Pure Chemical Industries) to stimulate TRPV4 activity. 2.6. Western blot analysis
DPSCs were seeded (4.5 104/cm2) in 6- or 96-well plates, and allowed to reach confluence. The culture medium was then replaced with differentiation medium comprising aMEM that was supplemented as above, but also with 10 nM dexamethasone (Sigma-Aldrich), 2 mM beta-glycerophosphate (Sigma-Aldrich), and 100 mM ascorbic acid (Nacalai Tesque). This medium was replaced twice wkly. All DPSCs were differentiated for 1 wk, except those that were stained with Alizarin Red S, which were instead differentiated for 4 wks.
Cells were lysed with sample buffer comprised of 62.5 mM TrisHCl buffer (pH 6.8) containing 2% sodium dodecyl sulfate (SDS), 5% b-mercaptoethanol, and 10% glycerol. Cell lysates were incubated (95 C), subjected to SDS-polyacrylamide gel electrophoresis, and immunoblotted using anti-TRPV4 (#ACC-034, Alomone labs, Jerusalem, Israel), anti-Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1; #649602, BioLegend, CA, USA), anti-Methyl-CpG-binding protein 2 (MeCP2; #3456S, Cell Signaling Technology, MA, USA), and anti-a-tubulin (#sc-32293, Santa Cruz Biotechnology, CA, USA) antibodies. Membranes were washed and incubated (1 h, room temperature) with HRP-conjugated secondary antibodies (TRPV4 and MeCP2, #7074S; NFATc1 and a-tubulin, #7076S; Cell Signaling Technology), and visualized with ECL prime detection reagent (GE Healthcare, Buckinghamshire, UK). Signals were detected and quantified using the LAS-1000 pro imager (Fuji Film, Tokyo, Japan) and Image Gauge software (Fuji Film). TRPV4 and NFATc1 expression was normalized to that of a-tubulin or MeCP2.
2.3. Alizarin Red S staining
2.7. Cell fractionation
Alizarin Red S staining was performed as previously described [13]. Briefly, cells were fixed (10 min, room temperature) with 4% paraformaldehyde (Wako Pure Chemical Industries) in 0.1 M sodium phosphate buffer (pH 7.4), and washed three times with phosphate-buffered saline. They were then rinsed with dH2O, and stained with 1% Alizarin Red S (pH 4.2; Sigma-Aldrich) for 30 s. The Alizarin Red S staining was extracted using 10% (v/w) cetylpyridinium chloride (Nakarai tesque) in 10 mM sodium phosphate buffer (pH 7.0), before its absorbance at 570 nm was measured using an Infinite 200 PRO plate reader (Tecan, Mannedorf, Switzerland).
After osteogenic differentiation for 1 wk, cells were harvested with trypsin-EDTA (Nakarai tesque), and separated into nuclear and cytosol fractions using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions.
2.2. Osteogenic differentiation of DPSC
2.8. Statistical analyses Data were analyzed using a student’s t-test that was conducted using JMP software version 13 (SAS Institute, NC, USA). P values < 0.05 were considered to indicate statistical significance.
Please cite this article as: X. Han et al., Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-offunction mutation of TRPV4 associated with metatropic dysplasia, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.12.123
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3. Results 3.1. Increased calcium-deposit accumulation and accelerated osteoblastic differentiation in patient derived mutant DPSCs
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groups after differentiation (Fig. 2C). Together, these results support that TRPV4 p.L619F is a GOF mutation that promotes intracellular Ca2þ influx during osteoblastic differentiation. 3.3. Enhanced activation of NFATc1 in MD-OBs
Patient-derived DPSCs carrying the TRPV4 p.L619F mutation were differentiated into osteoblasts (MD-OBs), and their behavior was compared with that of osteoblasts differentiated from isogenic control DPSCs (Ctrl-OBs). The MD-OBs showed a significant increase in calcium-deposit accumulation (indicated by Alizarin Red S staining) compared to Ctrl-OBs (Fig. 1A). Furthermore, the mRNA expression of both RUNX2 (a master regulator of early osteoblast differentiation), and OCN (a marker of mature-stage osteoblasts) were upregulated in MD-OBs compared to Ctrl-OBs (Fig. 1B and C). These results suggest that the p.L619F mutation accelerated both the differentiation of DPSCs to osteoblasts, and bone calcification. 3.2. Increased intracellular calcium levels in MD-OBs A previous study reported that the TRPV4 p.L619F mutation acts a GOF mutation in DPSCs and chondrocytes [7]. Herein, the Fura-2 Ca2þ indicator was used to measure intracellular Ca2þ levels, and thus examine TRPV4 channel function in mutant osteoblasts. Under baseline conditions, intracellular Ca2þ levels were significantly higher in the MD-OBs than the Ctrl-OBs (Fig. 2A), and this was also the case after cells were treated with the TRPV4 agonist, 4aPDD (Fig. 2B). Notably, the conducted western-blot analysis showed no significant difference in TRPV4 protein levels between the two
To investigate the potential association between the increased intracellular Ca2þ levels and the accelerated osteoblastic differentiation observed in MD-OBs, the expression of NFATc1 (which Ca2þdependently mediates osteoblastic differentiation) was next examined. Previous studies have reported that three different NFATc1 isoforms are generated via alternate splicing [14,15]. The conducted western-blot analysis showed that all three isoforms were expressed at significantly higher levels in both whole-cell and nuclear extracts of MD-OBs than Ctrl-OBs (Fig. 3A and B). This finding was supported by the fact that the expression of the direct NFATc1-transcriptional target EGR2 was upregulated in MD-OBs (Fig. 3C). Together, these results suggest that the accelerated osteoblastic differentiation observed in mutant DPSCs may be associated with enhanced NFATc1 transcriptional activity, likely caused either by increased NFATc1 expression and/or nuclear translocation. 4. Discussion The present study examined the effect of a heterozygous single base mutation in TRPV4, c.1855C > T (p.L619F), on osteoblastic differentiation. The behavior of mutant cells was compared to that of
Fig. 1. Accelerated osteoblastic differentiation in mutant osteoblasts (MD-OBs) (A) OBs were stained with Alizarin Red S, and this was then extracted, and its absorbance at 570 nm was measured. (B, C) Runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) expression levels in MD-OBs were measured by qRT-PCR, and normalized to those of Ctrl-OBs. All data are presented as the mean ± standard deviation from three experiments. *P < 0.05, **P < 0.01.
Please cite this article as: X. Han et al., Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-offunction mutation of TRPV4 associated with metatropic dysplasia, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.12.123
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Fig. 2. Increased calcium levels in mutant osteoblasts (MD-OBs) (A) ‘Baseline’ intracellular calcium levels in OBs were measured using the Fura-2 Ca2þ indicator, and expressed as the 340/380 nm Fura-2 fluorescence ratio. (B) Relative intracellular calcium levels (indicated by the 340/380 nm Fura-2 fluorescence ratio) were measured 1 and 2 min after transient receptor potential vanilloid 4 (TRPV4) activation (i.e. via treatment with 1 mM 4aPDD), and expressed relative to those measured under baseline conditions (at 0 min). (C) TRPV4 expression in OBs was measured by western blotting, quantified, and graphed normalized to that a-tubulin. Data in (A) and (C) are presented as the mean ± standard deviation from three independent experiments. Data in (B) is represented as the mean ± standard deviation from four technical replicates. **P < 0.01, ***P < 0.001, n.s., not significant. Ctrl-OBs, control OBs.
Fig. 3. Increased nuclear factor-activated T cell c1 (NFATc1) signaling in mutant osteoblasts (MD-OBs) (A, B) NFATc1 expression in OBs was measured by western blotting, in whole-cell (A), nuclear (N) and cytosolic (C) fractions (B). Methyl CpG binding protein 2 (MeCP2) and a-tubulin were used as nuclear and cytosolic markers, respectively. The expression levels (i.e. band intensity) of all three known NFATc1 isoforms were combined and quantified. Total NFATc1 expression was normalized to that of a-tubulin (A) or MeCP2 (B). (C) Early growth response 2 (EGR2) expression levels in MD-OBs were measured by qRT-PCR, and expressed relative to those in Ctrl-OBs. All data are presented as the mean ± standard deviation from three experiments. **P < 0.01. Ctrl-OBs, control OBs.
isogenic control cells in which the mutation was repaired using a genome editing technology. Notably, the MD-OBs showed increased calcification and intracellular Ca2þ levels, as well as increased RUNX2, OCN, cellular and nuclear NFATc1, and EGR2 expression compared to Ctrl-OBs. Together, the data presented
herein suggest that the analyzed MD-associated TRPV4 GOF mutation induces a Ca2þ ‘overload’ that activates the Ca2þ-mediated NFATc1 pathway, and that this causes mesenchymal progenitors to undergo accelerated osteoblastic differentiation. Under normal conditions, phosphorylated NFATc1 localizes to
Please cite this article as: X. Han et al., Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-offunction mutation of TRPV4 associated with metatropic dysplasia, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.12.123
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the cytoplasm; however, once it is dephosphorylated by calcineurin, NFATc1 translocates to the nucleus to regulate target-gene transcription [16]. Importantly, calcineurin is activated in a Ca2þ/ calmodulin-dependent manner, and the TRPV4 carboxy-terminus contains a calmodulin-binding motif that has a pivotal role in Ca2þ/calmodulin signaling [17e19]. Moreover, TRPV4 has been shown to colocalize with calcineurin in rat-airway smooth muscle cells [20]; thus, TRPV4 has been suggested to mediate the Ca2þ/ calmodulin/calcineurin-mediated nuclear translocation and activation of NFATc1. Previous murine studies have demonstrated that nuclear NFATc1 is required for osteoblastic proliferation and bone formation [15]. A mechanical-stimulation model was used to show that increased TRPV4-mediated Ca2þ levels activate calcineurin and thus NFATc1 to drive the osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells [21]. These findings are consistent with the data generated by the present study, although notably, it is not yet clear whether or how each of the three NFATc1 isoforms individually modulate osteoblastic differentiation. The results presented herein also suggest a pathological association between accelerated osteoblastic differentiation and skeletal phenotypes in MD. As discussed, a previously conducted histological analysis of bone tissue collected from aborted fetuses with MD described proliferating mesenchymal cells and immature woven bone between the irregular cartilage nodules in the growth plate. That study suggested that these findings were indicative of ectopic ossification that occurred as a result of the direct osteoblastic differentiation of mesenchymal progenitors [5]. During endochondral ossification, osteoblast ossification begins in the perichondrium surrounding the cartilage primordia, and occurs prior to bone-collar formation [10e12]. Osteoprogenitors (and blood vessels) then infiltrate into the cartilage primordia, differentiate into osteoblasts, and replace mature hypertrophic cartilage to form trabecular bone in bone marrow, thereby producing the primary ossification center [22]. In contrast, chondrocytes continue to proliferate and differentiate in the growth plate at the epiphysis (without being ossified), thus allowing long bones to elongate [10e12]. Notably, the patient in the present study showed severe skeletal abnormalities, including dumbbell-shaped long-bone deformities, that were suggestive of a growth plate defect similar to that which has been previously reported in aborted fetuses with MD [5,7]. These findings suggest that the examined TRPV4 GOF mutation may contribute to MD pathogenesis by accelerating osteoblastic differentiation to produce disorganized endochondral ossifications. This study was somewhat limited in its capacity to examine the MD genotype-phenotype association in the examined patient. Firstly, it did not elucidate the molecular mechanism underlying the observed increased RUNX2 expression in the MD-OBs, despite that RUNX2 has not been confirmed as an NFATc1 target [15]. Therefore, additional (e.g. gene-expression profile) studies are required to identify signaling pathways by which TRPV4 activation alters RUNX2 expression. Secondly, the association between osteoclast differentiation and the p.L619F mutation remains unresolved. The osteoclast defects may involve disorganized endochondral ossification in MD, since NFATc1 has been shown to mediate osteoclast differentiation [23,24]; however, this hypothesis requires confirmation via additional studies using novel experimental systems to induce osteoclast differentiation in patientderived cells. Thirdly, the exact molecular mechanism by which TRPV4 p.L619F accelerates intracellular Ca2þ entry remains unclear. Further analysis (including biochemical and electrophysiological experiments) is required to resolve this issue. In conclusion, the pathological TRPV4, p.L619F GOF mutation was herein shown to accelerate the osteoblastic differentiation of mesenchymal stem cells, likely by activating the NFATc1 pathway.
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Furthermore, the presented data suggest that osteoblastic defects may underlie the skeletal phenotype and disorganized endochondral ossification observed in MD. Thus, the NFATc1 pathway might be a promising potential therapeutic target to ameliorate the progression of MD skeletal phenotypes. Declaration of competing interest All authors declare no conflicts of interest. Acknowledgements We would like to thank all the members of the Department of Pediatric Dentistry and Special Needs Dentistry at Kyushu University Hospital for their valuable suggestions, technical support, and materials. We appreciate the technical assistance that was provided by the Research Support Center at the Research Center for Human Disease Modeling, Kyushu University Graduate School of Medical Sciences. This work was supported by the Japan Society for the Promotion of Science (KAKENHI; grant numbers, JP19K10387 and JP19K10406). References [1] W. Everaerts, B. Nilius, G. Owsianik, The vanilloid transient receptor potential channel TRPV4: from structure to disease, Prog. Biophys. Mol. Biol. 103 (2010) 2e17. [2] G. Nishimura, E. Lausch, R. Savarirayan, M. Shiba, J. Spranger, B. Zabel, S. Ikegawa, A. Superti-Furga, S. Unger, TRPV4-associated skeletal dysplasias, Am. J. Med. Genet. C Semin. Med. Genet. 160C (2012) 190e204. [3] B. Nilius, T. Voets, The puzzle of TRPV4 channelopathies, EMBO Rep. 14 (2013) 152e163. [4] D. Krakow, J. Vriens, N. Camacho, P. Luong, H. Deixler, T.L. Funari, C.A. Bacino, M.B. Irons, I.A. Holm, L. Sadler, E.B. Okenfuss, A. Janssens, T. Voets, D.L. Rimoin, R.S. Lachman, B. Nilius, D.H. Cohn, Mutations in the gene encoding the calcium-permeable ion channel TRPV4 produce spondylometaphyseal dysplasia, Kozlowski type and metatropic dysplasia, Am. J. Hum. Genet. 84 (2009) 307e315. [5] N. Camacho, D. Krakow, S. Johnykutty, P.J. Katzman, S. Pepkowitz, J. Vriens, B. Nilius, B.F. Boyce, D.H. Cohn, Dominant TRPV4 mutations in nonlethal and lethal metatropic dysplasia, Am. J. Med. Genet. 152A (2010) 1169e1177. [6] E. Andreucci, S. Aftimos, M. Alcausin, E. Haan, W. Hunter, P. Kannu, B. Kerr, G. McGillivray, R.J. McKinlay Gardner, M.G. Patricelli, D. Sillence, E. Thompson, , R. Savarirayan, TRPV4 related skeletal M. Zacharin, A. Zankl, S.R. Lamande dysplasias: a phenotypic spectrum highlighted byclinical, radiographic, and molecular studies in 21 new families, Orphanet J. Rare Dis. 6 (2011) 37. [7] K. Nonaka, X. Han, H. Kato, H. Sato, H. Yamaza, Y. Hirofuji, K. Masuda, Novel gain-of-function mutation of TRPV4 associated with accelerated chondrogenic differentiation of dental pulp stem cells derived from a patient with metatropic dysplasia, Biochem. Biophys. Rep. 19 (2019) 100648. [8] B. Saitta, J. Passarini, D. Sareen, L. Ornelas, A. Sahabian, S. Argade, D. Krakow, D.H. Cohn, C.N. Svendsen, D.L. Rimoin, Patient-derived skeletal dysplasia induced pluripotent stem cells display abnormal chondrogenic marker expression and regulation by BMP2 and TGFb1, Stem Cells Dev. 23 (2014) 1464e1478. [9] L. Hurd, S.M. Kirwin, M. Boggs, W.G. Mackenzie, M.B. Bober, V.L. Funanage, R.L. Duncan, A mutation in TRPV4 results in altered chondrocyte calcium signaling in severe metatropic dysplasia, Am. J. Med. Genet. 167A (2015) 2286e2293. [10] H.M. Kronenberg, Developmental regulation of the growth plate, Nature 423 (2003) 332e336. [11] V. Lefebvre, P. Bhattaram, Vertebrate skeletogenesis, Curr. Top. Dev. Biol. 90 (2010) 291e317. [12] E.J. Mackie, L. Tatarczuch, M. Mirams, The skeleton: a multi-functional complex organ: the growth plate chondrocyte and endochondral ossification, J. Endocrinol. 211 (2011) 109e121. [13] H. Kato, X. Han, H. Yamaza, K. Masuda, Y. Hirofuji, H. Sato, T.T.M. Pham, T. Taguchi, K. Nonaka, Direct effects of mitochondrial dysfunction on poor bone health in Leigh syndrome, Biochem. Biophys. Res. Commun. 493 (2017) 207e212. [14] S. Chuvpilo, E. Jankevics, D. Tyrsin, A. Akimzhanov, D. Moroz, M.K. Jha, €nig, A. Avots, J. Schulze-Luehrmann, B. Santner-Nanan, E. Feoktistova, T. Ko E. Schmitt, F. Berberich-Siebelt, A. Schimpl, E. Serfling, Autoregulation of NFATc1/A expression facilitates effector T cells to escape from rapid apoptosis, Immunity 16 (2002) 881e895. [15] M.M. Winslow, M. Pan, M. Starbuck, E.M. Gallo, L. Deng, G. Karsenty, G.R. Crabtree, Calcineurin/NFAT signaling in osteoblasts regulates bone mass,
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Please cite this article as: X. Han et al., Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-offunction mutation of TRPV4 associated with metatropic dysplasia, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.12.123
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Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-of-function mutation of TRPV4 associated with metatropic dysplasia Xu Han 1, Hiroki Kato 1, Hiroshi Sato, Yuta Hirofuji, Satoshi Fukumoto**, Keiji Masuda* Section of Oral Medicine for Children, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University, Maidashi 3-1-1, Higashi-Ku, Fukuoka, 812-8582, Japan
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a b s t r a c t
Article history: Received 4 December 2019 Accepted 30 December 2019 Available online xxx
Metatropic dysplasia (MD) is a congenital skeletal dysplasia characterized by severe platyspondyly and dumbbell-like long-bone deformities. These skeletal phenotypes are predominantly caused by autosomal dominant gain-of-function (GOF) mutations in transient receptor potential vanilloid 4 (TRPV4), which encodes a nonselective Ca2þ-permeable cation channel. Previous studies have shown that constitutive TRPV4 channel activation leads to irregular chondrogenic proliferation and differentiation, and thus to the disorganized endochondral ossification seen in MD. Therefore, the present study investigated the role of TRPV4 in osteoblast differentiation and MD pathogenesis. Specifically, the behavior of osteoblasts differentiated from patient-derived dental pulp stem cells carrying a heterozygous single base TRPV4 mutation, c.1855C > T (p.L619F) was compared to that of osteoblasts differentiated from isogenic control cells (in which the mutation was corrected using the CRISPR/Cas9 system). The mutant osteoblasts exhibited enhanced calcification (indicated by intense Alizarin Red S staining), increased intracellular Ca2þ levels, strongly upregulated runt-related transcription factor 2 and osteocalcin expression, and increased expression and nuclear translocation of nuclear factor-activated T cell c1 (NFATc1) compared to control cells. These results suggest that the analyzed TRPV4 GOF mutation disrupts osteoblastic differentiation and induces MD-associated disorganized endochondral ossification by increasing Ca2þ/NFATc1 pathway activity. Thus, inhibiting the NFATc1 pathway may be a promising potential therapeutic strategy to attenuate skeletal deformities in MD. © 2020 Elsevier Inc. All rights reserved.
Keywords: Dental pulp stem cells Metatropic dysplasia Osteoblast differentiation Transient receptor potential vanilloid 4
1. Introduction Transient receptor potential vanilloid 4 (TRPV4) is a sixtransmembrane Ca2þ-permeable polymodal cation channel that localizes to the cell membrane, and that is activated by various endogenous and exogenous stimuli, including heat, osmotic pressure, mechanical stimulation, and arachidonic acid [1]. TRPV4 gene mutations cause congenital skeletal dysplasias and neurological
* Corresponding author. Section of Oral Medicine for Children, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University, Maidashi 3-1-1, Higashi-Ku, Fukuoka, 812-8582, Japan. ** Corresponding author. Section of Oral Medicine for Children, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University, Maidashi 3-1-1, Higashi-Ku, Fukuoka, 812-8582, Japan. E-mail addresses: [email protected] (S. Fukumoto), kemasuda@ dent.kyushu-u.ac.jp (K. Masuda). 1 These authors contributed equally to this study.
disorders [2,3], including metatropic dysplasia (MD) [2]. Patients with MD exhibit short limbs at birth due to a severe defect of long bone development, and progressive kyphoscoliosis after birth, which causes the trunk to become shorter than the limbs, resulting in a reversal of body proportions (i.e. from ‘short limbs’ to ‘short trunk’) [2]. They also frequently exhibit severe platyspondyly and/or dumbbell-like long-bone deformities. MD phenotypes vary widely in severity, and include both lethal and non-lethal subtypes [2]. Several autosomal dominant gain-of-function (GOF) TRPV4 mutations have been shown to cause MD [4e7]. Previous studies have suggested that constitutive TRPV4 Ca2þ channel activation disrupts the expression of critical factors including SOX9, and thereby leads to the aberrant chondrocyte differentiation and function from which MD-associated long-bone developmental defects arise [7e9]. During normal endochondral ossification, mesenchymal progenitors initially form aggregates, then differentiate to form chondrocytes, osteoblasts, and osteoclasts in a
https://doi.org/10.1016/j.bbrc.2019.12.123 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: X. Han et al., Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-offunction mutation of TRPV4 associated with metatropic dysplasia, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.12.123
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temporally and spatially regulated manner, and finally undergo neovascular invasion into the developing bone [10e12]. A previously conducted histopathological analysis of bone tissues in aborted fetuses suggested that aberrant osteoblastic differentiation and ossification may underlie MD pathogenesis [5]. However, to date, only a small number of experimental models have been established to reproduce MD bone phenotypes; thus, the mechanisms that underlie MD osteoblastic defects with skeletal phenotypes remain poorly understood. The purpose of the present study was to investigate whether TRPV4 contributes to osteoblastic defects in MD. Dental pulpderived mesenchymal stem cells (DPSCs) were obtained from a patient that exhibited a severe MD phenotype, as well as the TRPV4 GOF mutation, c.1855C > T (p.L619F). The DPSCs were differentiated into osteoblasts, examined for MD-associated defects, and compared to isogenic control cells in which the c.1855C > T mutation was corrected using the CRISPR/Cas9 system.
2.4. RNA extraction and quantitative real-time polymerase chain reaction
2. Materials and methods
Total RNA was extracted from cells using an RNAeasy Mini Kit (Qiagen). First-strand cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). qRT-PCR was conducted using primers for runt-related transcription factor 2 (RUNX2) (forward (F), 50 -CAGTTCCCAAGCATTTCATCC-30 ; reverse (R), 50 -TCAATATGGTCGCCAAACAG-30 ); osteocalcin (OCN) (F, 50 -GCAAAGGTGCAGCCTTTGTG-30 ; R, 50 GGCTCCCAGCCATTGATACAG-30 ); early growth response 2 (EGR2) (F, 50 -GACCAGATGAACGGAGTGGC-30 ; R, 50 -GCAAAGCTGCTGGGATATGGG-30 ); 18S rRNA (F, 50 -CGGCTACCACATCCAAGGAA-30 ; R, 50 GCTGGAATTACCGCGGCT-30 ), as well as GoTaq qPCR Master Mix (Promega, WI, USA), and the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). The 18S rRNA (control) threshold cycle (Ct) value was subtracted from that of the target genes (DCt). Relative target gene expression levels are shown as fold changes determined using the 2DDCt method.
2.1. Isolation and culture of DPSCs
2.5. Intracellular calcium level measurement
DPSCs were isolated from a 14-year-old patient with MD that harbored the TRPV4 GOF mutation, c.1855C > T (p.L619F) [7]. Written informed consent was obtained from the patient’s guardians for the patient’s participation in the study, which was approved by the Kyushu University Institutional Review Board for Human Genome/Gene Research (permission number: 678e01), and conducted in accordance with the Declaration of Helsinki. The TRPV4 1855C > T mutation was repaired using the CRISPR/ Cas9 system to produce isogenic control DPSCs, as previously described [7]. All DPSCs were cultured (37 C, 5% CO2) in Alpha Modification of Eagle’s Medium (aMEM; Nacalai Tesque, Kyoto, Japan) that was supplemented with 15% fetal bovine serum (SigmaAldrich, MO, USA), 100 mM L-ascorbic acid 2-phosphate (Wako Pure Chemical Industries, Osaka, Japan), 250 mg/mL fungizone (Thermo Fisher Scientific, MA, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Nacalai Tesque). Cells passaged seven times or less were used in experiments.
Cells were cultured in 96-well plates, and stained (45 min, 37 C) with 10 mM Fura-2 AM (Dojindo, Kumamoto, Japan), 0.05% (w/v) Pluronic F-127 (Sigma-Aldrich), and 500 mM probenecid (SigmaAldrich) in Hanks’ Balanced Salt Solution. Fluorescence signals were measured (at 37 C) by excitation at 340 and 380 nm, and emission at 510 nm, using an EnSight plate reader (PerkinElmer, MA, USA). Cells were administered 4a-phorbol 12,13-didecanoate (4aPDD) (1 mM; Wako Pure Chemical Industries) to stimulate TRPV4 activity. 2.6. Western blot analysis
DPSCs were seeded (4.5 104/cm2) in 6- or 96-well plates, and allowed to reach confluence. The culture medium was then replaced with differentiation medium comprising aMEM that was supplemented as above, but also with 10 nM dexamethasone (Sigma-Aldrich), 2 mM beta-glycerophosphate (Sigma-Aldrich), and 100 mM ascorbic acid (Nacalai Tesque). This medium was replaced twice wkly. All DPSCs were differentiated for 1 wk, except those that were stained with Alizarin Red S, which were instead differentiated for 4 wks.
Cells were lysed with sample buffer comprised of 62.5 mM TrisHCl buffer (pH 6.8) containing 2% sodium dodecyl sulfate (SDS), 5% b-mercaptoethanol, and 10% glycerol. Cell lysates were incubated (95 C), subjected to SDS-polyacrylamide gel electrophoresis, and immunoblotted using anti-TRPV4 (#ACC-034, Alomone labs, Jerusalem, Israel), anti-Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1; #649602, BioLegend, CA, USA), anti-Methyl-CpG-binding protein 2 (MeCP2; #3456S, Cell Signaling Technology, MA, USA), and anti-a-tubulin (#sc-32293, Santa Cruz Biotechnology, CA, USA) antibodies. Membranes were washed and incubated (1 h, room temperature) with HRP-conjugated secondary antibodies (TRPV4 and MeCP2, #7074S; NFATc1 and a-tubulin, #7076S; Cell Signaling Technology), and visualized with ECL prime detection reagent (GE Healthcare, Buckinghamshire, UK). Signals were detected and quantified using the LAS-1000 pro imager (Fuji Film, Tokyo, Japan) and Image Gauge software (Fuji Film). TRPV4 and NFATc1 expression was normalized to that of a-tubulin or MeCP2.
2.3. Alizarin Red S staining
2.7. Cell fractionation
Alizarin Red S staining was performed as previously described [13]. Briefly, cells were fixed (10 min, room temperature) with 4% paraformaldehyde (Wako Pure Chemical Industries) in 0.1 M sodium phosphate buffer (pH 7.4), and washed three times with phosphate-buffered saline. They were then rinsed with dH2O, and stained with 1% Alizarin Red S (pH 4.2; Sigma-Aldrich) for 30 s. The Alizarin Red S staining was extracted using 10% (v/w) cetylpyridinium chloride (Nakarai tesque) in 10 mM sodium phosphate buffer (pH 7.0), before its absorbance at 570 nm was measured using an Infinite 200 PRO plate reader (Tecan, Mannedorf, Switzerland).
After osteogenic differentiation for 1 wk, cells were harvested with trypsin-EDTA (Nakarai tesque), and separated into nuclear and cytosol fractions using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions.
2.2. Osteogenic differentiation of DPSC
2.8. Statistical analyses Data were analyzed using a student’s t-test that was conducted using JMP software version 13 (SAS Institute, NC, USA). P values < 0.05 were considered to indicate statistical significance.
Please cite this article as: X. Han et al., Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-offunction mutation of TRPV4 associated with metatropic dysplasia, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.12.123
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3. Results 3.1. Increased calcium-deposit accumulation and accelerated osteoblastic differentiation in patient derived mutant DPSCs
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groups after differentiation (Fig. 2C). Together, these results support that TRPV4 p.L619F is a GOF mutation that promotes intracellular Ca2þ influx during osteoblastic differentiation. 3.3. Enhanced activation of NFATc1 in MD-OBs
Patient-derived DPSCs carrying the TRPV4 p.L619F mutation were differentiated into osteoblasts (MD-OBs), and their behavior was compared with that of osteoblasts differentiated from isogenic control DPSCs (Ctrl-OBs). The MD-OBs showed a significant increase in calcium-deposit accumulation (indicated by Alizarin Red S staining) compared to Ctrl-OBs (Fig. 1A). Furthermore, the mRNA expression of both RUNX2 (a master regulator of early osteoblast differentiation), and OCN (a marker of mature-stage osteoblasts) were upregulated in MD-OBs compared to Ctrl-OBs (Fig. 1B and C). These results suggest that the p.L619F mutation accelerated both the differentiation of DPSCs to osteoblasts, and bone calcification. 3.2. Increased intracellular calcium levels in MD-OBs A previous study reported that the TRPV4 p.L619F mutation acts a GOF mutation in DPSCs and chondrocytes [7]. Herein, the Fura-2 Ca2þ indicator was used to measure intracellular Ca2þ levels, and thus examine TRPV4 channel function in mutant osteoblasts. Under baseline conditions, intracellular Ca2þ levels were significantly higher in the MD-OBs than the Ctrl-OBs (Fig. 2A), and this was also the case after cells were treated with the TRPV4 agonist, 4aPDD (Fig. 2B). Notably, the conducted western-blot analysis showed no significant difference in TRPV4 protein levels between the two
To investigate the potential association between the increased intracellular Ca2þ levels and the accelerated osteoblastic differentiation observed in MD-OBs, the expression of NFATc1 (which Ca2þdependently mediates osteoblastic differentiation) was next examined. Previous studies have reported that three different NFATc1 isoforms are generated via alternate splicing [14,15]. The conducted western-blot analysis showed that all three isoforms were expressed at significantly higher levels in both whole-cell and nuclear extracts of MD-OBs than Ctrl-OBs (Fig. 3A and B). This finding was supported by the fact that the expression of the direct NFATc1-transcriptional target EGR2 was upregulated in MD-OBs (Fig. 3C). Together, these results suggest that the accelerated osteoblastic differentiation observed in mutant DPSCs may be associated with enhanced NFATc1 transcriptional activity, likely caused either by increased NFATc1 expression and/or nuclear translocation. 4. Discussion The present study examined the effect of a heterozygous single base mutation in TRPV4, c.1855C > T (p.L619F), on osteoblastic differentiation. The behavior of mutant cells was compared to that of
Fig. 1. Accelerated osteoblastic differentiation in mutant osteoblasts (MD-OBs) (A) OBs were stained with Alizarin Red S, and this was then extracted, and its absorbance at 570 nm was measured. (B, C) Runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) expression levels in MD-OBs were measured by qRT-PCR, and normalized to those of Ctrl-OBs. All data are presented as the mean ± standard deviation from three experiments. *P < 0.05, **P < 0.01.
Please cite this article as: X. Han et al., Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-offunction mutation of TRPV4 associated with metatropic dysplasia, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.12.123
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Fig. 2. Increased calcium levels in mutant osteoblasts (MD-OBs) (A) ‘Baseline’ intracellular calcium levels in OBs were measured using the Fura-2 Ca2þ indicator, and expressed as the 340/380 nm Fura-2 fluorescence ratio. (B) Relative intracellular calcium levels (indicated by the 340/380 nm Fura-2 fluorescence ratio) were measured 1 and 2 min after transient receptor potential vanilloid 4 (TRPV4) activation (i.e. via treatment with 1 mM 4aPDD), and expressed relative to those measured under baseline conditions (at 0 min). (C) TRPV4 expression in OBs was measured by western blotting, quantified, and graphed normalized to that a-tubulin. Data in (A) and (C) are presented as the mean ± standard deviation from three independent experiments. Data in (B) is represented as the mean ± standard deviation from four technical replicates. **P < 0.01, ***P < 0.001, n.s., not significant. Ctrl-OBs, control OBs.
Fig. 3. Increased nuclear factor-activated T cell c1 (NFATc1) signaling in mutant osteoblasts (MD-OBs) (A, B) NFATc1 expression in OBs was measured by western blotting, in whole-cell (A), nuclear (N) and cytosolic (C) fractions (B). Methyl CpG binding protein 2 (MeCP2) and a-tubulin were used as nuclear and cytosolic markers, respectively. The expression levels (i.e. band intensity) of all three known NFATc1 isoforms were combined and quantified. Total NFATc1 expression was normalized to that of a-tubulin (A) or MeCP2 (B). (C) Early growth response 2 (EGR2) expression levels in MD-OBs were measured by qRT-PCR, and expressed relative to those in Ctrl-OBs. All data are presented as the mean ± standard deviation from three experiments. **P < 0.01. Ctrl-OBs, control OBs.
isogenic control cells in which the mutation was repaired using a genome editing technology. Notably, the MD-OBs showed increased calcification and intracellular Ca2þ levels, as well as increased RUNX2, OCN, cellular and nuclear NFATc1, and EGR2 expression compared to Ctrl-OBs. Together, the data presented
herein suggest that the analyzed MD-associated TRPV4 GOF mutation induces a Ca2þ ‘overload’ that activates the Ca2þ-mediated NFATc1 pathway, and that this causes mesenchymal progenitors to undergo accelerated osteoblastic differentiation. Under normal conditions, phosphorylated NFATc1 localizes to
Please cite this article as: X. Han et al., Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-offunction mutation of TRPV4 associated with metatropic dysplasia, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.12.123
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the cytoplasm; however, once it is dephosphorylated by calcineurin, NFATc1 translocates to the nucleus to regulate target-gene transcription [16]. Importantly, calcineurin is activated in a Ca2þ/ calmodulin-dependent manner, and the TRPV4 carboxy-terminus contains a calmodulin-binding motif that has a pivotal role in Ca2þ/calmodulin signaling [17e19]. Moreover, TRPV4 has been shown to colocalize with calcineurin in rat-airway smooth muscle cells [20]; thus, TRPV4 has been suggested to mediate the Ca2þ/ calmodulin/calcineurin-mediated nuclear translocation and activation of NFATc1. Previous murine studies have demonstrated that nuclear NFATc1 is required for osteoblastic proliferation and bone formation [15]. A mechanical-stimulation model was used to show that increased TRPV4-mediated Ca2þ levels activate calcineurin and thus NFATc1 to drive the osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells [21]. These findings are consistent with the data generated by the present study, although notably, it is not yet clear whether or how each of the three NFATc1 isoforms individually modulate osteoblastic differentiation. The results presented herein also suggest a pathological association between accelerated osteoblastic differentiation and skeletal phenotypes in MD. As discussed, a previously conducted histological analysis of bone tissue collected from aborted fetuses with MD described proliferating mesenchymal cells and immature woven bone between the irregular cartilage nodules in the growth plate. That study suggested that these findings were indicative of ectopic ossification that occurred as a result of the direct osteoblastic differentiation of mesenchymal progenitors [5]. During endochondral ossification, osteoblast ossification begins in the perichondrium surrounding the cartilage primordia, and occurs prior to bone-collar formation [10e12]. Osteoprogenitors (and blood vessels) then infiltrate into the cartilage primordia, differentiate into osteoblasts, and replace mature hypertrophic cartilage to form trabecular bone in bone marrow, thereby producing the primary ossification center [22]. In contrast, chondrocytes continue to proliferate and differentiate in the growth plate at the epiphysis (without being ossified), thus allowing long bones to elongate [10e12]. Notably, the patient in the present study showed severe skeletal abnormalities, including dumbbell-shaped long-bone deformities, that were suggestive of a growth plate defect similar to that which has been previously reported in aborted fetuses with MD [5,7]. These findings suggest that the examined TRPV4 GOF mutation may contribute to MD pathogenesis by accelerating osteoblastic differentiation to produce disorganized endochondral ossifications. This study was somewhat limited in its capacity to examine the MD genotype-phenotype association in the examined patient. Firstly, it did not elucidate the molecular mechanism underlying the observed increased RUNX2 expression in the MD-OBs, despite that RUNX2 has not been confirmed as an NFATc1 target [15]. Therefore, additional (e.g. gene-expression profile) studies are required to identify signaling pathways by which TRPV4 activation alters RUNX2 expression. Secondly, the association between osteoclast differentiation and the p.L619F mutation remains unresolved. The osteoclast defects may involve disorganized endochondral ossification in MD, since NFATc1 has been shown to mediate osteoclast differentiation [23,24]; however, this hypothesis requires confirmation via additional studies using novel experimental systems to induce osteoclast differentiation in patientderived cells. Thirdly, the exact molecular mechanism by which TRPV4 p.L619F accelerates intracellular Ca2þ entry remains unclear. Further analysis (including biochemical and electrophysiological experiments) is required to resolve this issue. In conclusion, the pathological TRPV4, p.L619F GOF mutation was herein shown to accelerate the osteoblastic differentiation of mesenchymal stem cells, likely by activating the NFATc1 pathway.
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Furthermore, the presented data suggest that osteoblastic defects may underlie the skeletal phenotype and disorganized endochondral ossification observed in MD. Thus, the NFATc1 pathway might be a promising potential therapeutic target to ameliorate the progression of MD skeletal phenotypes. Declaration of competing interest All authors declare no conflicts of interest. Acknowledgements We would like to thank all the members of the Department of Pediatric Dentistry and Special Needs Dentistry at Kyushu University Hospital for their valuable suggestions, technical support, and materials. We appreciate the technical assistance that was provided by the Research Support Center at the Research Center for Human Disease Modeling, Kyushu University Graduate School of Medical Sciences. This work was supported by the Japan Society for the Promotion of Science (KAKENHI; grant numbers, JP19K10387 and JP19K10406). References [1] W. Everaerts, B. Nilius, G. Owsianik, The vanilloid transient receptor potential channel TRPV4: from structure to disease, Prog. Biophys. Mol. Biol. 103 (2010) 2e17. [2] G. Nishimura, E. Lausch, R. Savarirayan, M. Shiba, J. Spranger, B. Zabel, S. Ikegawa, A. Superti-Furga, S. Unger, TRPV4-associated skeletal dysplasias, Am. J. Med. Genet. C Semin. Med. Genet. 160C (2012) 190e204. [3] B. Nilius, T. Voets, The puzzle of TRPV4 channelopathies, EMBO Rep. 14 (2013) 152e163. [4] D. Krakow, J. Vriens, N. Camacho, P. Luong, H. Deixler, T.L. Funari, C.A. Bacino, M.B. Irons, I.A. Holm, L. Sadler, E.B. Okenfuss, A. Janssens, T. Voets, D.L. Rimoin, R.S. Lachman, B. Nilius, D.H. Cohn, Mutations in the gene encoding the calcium-permeable ion channel TRPV4 produce spondylometaphyseal dysplasia, Kozlowski type and metatropic dysplasia, Am. J. Hum. Genet. 84 (2009) 307e315. [5] N. Camacho, D. Krakow, S. Johnykutty, P.J. Katzman, S. Pepkowitz, J. Vriens, B. Nilius, B.F. Boyce, D.H. Cohn, Dominant TRPV4 mutations in nonlethal and lethal metatropic dysplasia, Am. J. Med. Genet. 152A (2010) 1169e1177. [6] E. Andreucci, S. Aftimos, M. Alcausin, E. Haan, W. Hunter, P. Kannu, B. Kerr, G. McGillivray, R.J. McKinlay Gardner, M.G. Patricelli, D. Sillence, E. Thompson, , R. Savarirayan, TRPV4 related skeletal M. Zacharin, A. Zankl, S.R. Lamande dysplasias: a phenotypic spectrum highlighted byclinical, radiographic, and molecular studies in 21 new families, Orphanet J. Rare Dis. 6 (2011) 37. [7] K. Nonaka, X. Han, H. Kato, H. Sato, H. Yamaza, Y. Hirofuji, K. Masuda, Novel gain-of-function mutation of TRPV4 associated with accelerated chondrogenic differentiation of dental pulp stem cells derived from a patient with metatropic dysplasia, Biochem. Biophys. Rep. 19 (2019) 100648. [8] B. Saitta, J. Passarini, D. Sareen, L. Ornelas, A. Sahabian, S. Argade, D. Krakow, D.H. Cohn, C.N. Svendsen, D.L. Rimoin, Patient-derived skeletal dysplasia induced pluripotent stem cells display abnormal chondrogenic marker expression and regulation by BMP2 and TGFb1, Stem Cells Dev. 23 (2014) 1464e1478. [9] L. Hurd, S.M. Kirwin, M. Boggs, W.G. Mackenzie, M.B. Bober, V.L. Funanage, R.L. Duncan, A mutation in TRPV4 results in altered chondrocyte calcium signaling in severe metatropic dysplasia, Am. J. Med. Genet. 167A (2015) 2286e2293. [10] H.M. Kronenberg, Developmental regulation of the growth plate, Nature 423 (2003) 332e336. [11] V. Lefebvre, P. Bhattaram, Vertebrate skeletogenesis, Curr. Top. Dev. Biol. 90 (2010) 291e317. [12] E.J. Mackie, L. Tatarczuch, M. Mirams, The skeleton: a multi-functional complex organ: the growth plate chondrocyte and endochondral ossification, J. Endocrinol. 211 (2011) 109e121. [13] H. Kato, X. Han, H. Yamaza, K. Masuda, Y. Hirofuji, H. Sato, T.T.M. Pham, T. Taguchi, K. Nonaka, Direct effects of mitochondrial dysfunction on poor bone health in Leigh syndrome, Biochem. Biophys. Res. Commun. 493 (2017) 207e212. [14] S. Chuvpilo, E. Jankevics, D. Tyrsin, A. Akimzhanov, D. Moroz, M.K. Jha, €nig, A. Avots, J. Schulze-Luehrmann, B. Santner-Nanan, E. Feoktistova, T. Ko E. Schmitt, F. Berberich-Siebelt, A. Schimpl, E. Serfling, Autoregulation of NFATc1/A expression facilitates effector T cells to escape from rapid apoptosis, Immunity 16 (2002) 881e895. [15] M.M. Winslow, M. Pan, M. Starbuck, E.M. Gallo, L. Deng, G. Karsenty, G.R. Crabtree, Calcineurin/NFAT signaling in osteoblasts regulates bone mass,
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Please cite this article as: X. Han et al., Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-offunction mutation of TRPV4 associated with metatropic dysplasia, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.12.123