Received: 9 April 2017 | Accepted: 15 June 2017 DOI: 10.1002/jcp.26059

ORIGINAL RESEARCH ARTICLE

Microtubule crosslinking factor 1 promotes osteoblast differentiation by promoting β-/TCF1/Runx2 signaling axis

Lifang Hu1,2,3 | Peihong Su1,2,3 | Chong Yin1,2,3 | Yan Zhang1,2,3 | Runzhi Li1,2,3 | Kun Yan1,2,3 | Zhihao Chen1,2,3 | Dijie Li1,2,3 | Ge Zhang2,3 | Liping Wang4 | Zhiping Miao1,2,3 | Airong Qian1,2,3 | Cory J. Xian4

1 Laboratory for Bone Metabolism, Key Laboratory for Space Bioscience and Osteoblast differentiation is a multistep process delicately regulated by many factors, Biotechnology, School of Life Sciences, including cytoskeletal dynamics and signaling pathways. actin crosslinking Northwestern Polytechnical University, Xi’an, Shaanxi, China factor 1 (MACF1), a key cytoskeletal linker, has been shown to play key roles in signal 2 Shenzhen Research Institute of transduction and in diverse cellular processes; however, its role in regulating osteoblast Northwestern Polytechnical University, Shenzhen, China differentiation is still needed to be elucidated. To further uncover the functions and 3 NPU-HKBU Joint Research Centre for mechanisms of action of MACF1 in osteoblast differentiation, we examined effects of Translational Medicine on Musculoskeletal MACF1 knockdown (MACF1-KD) in MC3T3-E1 osteoblastic cells on their osteoblast Health in Space, Northwestern Polytechnical University, Xi’an, China differentiation and associated molecular mechanisms. The results showed that 4 Sansom Institute for Health Research, School knockdown of MACF1 significantly suppressed mineralization of MC3T3-E1 cells, of Pharmacy and Medical Sciences, University down-regulated the expression of key osteogenic alkaline phosphatase (ALP), of South Australia, Adelaide, SA, Australia runt-related transcription factor 2 (Runx2) and type I collagen α1(ColIα1). Knockdown of Correspondence MACF1 dramatically reduced the nuclear translocation of β-catenin, decreased the Airong Qian, PhD and Zhiping Miao, PhD, School of Life Sciences, Northwestern transcriptional activation of T cell factor 1 (TCF1), and down-regulated the expression of Polytechnical University, Xi’an, Shaanxi TCF1, lymphoid enhancer-binding factor 1 (LEF1), and Runx2, a target of 710072, China. Email: [email protected] (AQ) and β-catenin/TCF1. In addition, MACF1-KD increased the active level of glycogen synthase [email protected] (ZM) kinase-3β (GSK-3β), which is a key regulator for β-catenin signal transduction. Moreover, Funding information the reduction of nuclear β-catenin amount and decreased expression of TCF1 and Runx2 Fundamental Research Funds for the Central were significantly reversed in MACF1-KD cells when treated with lithium chloride, an Universities, Grant number: 3102016ZY037; Shenzhen Science and Technology Project, agonist for β-catenin by inhibiting GSK-3β activity. Taken together, these findings suggest Grant number: JCYJ20160229174320053; that knockdown of MACF1 in osteoblastic cells inhibits osteoblast differentiation through Australian National Health and Medical Research Council, Grant number: 1094606; suppressing the β-catenin/TCF1-Runx2 axis. Thus, a novel role of MACF1 in and a new Australian NHMRC Senior Research mechanistic insight of osteoblast differentiation are uncovered. Fellowship, Grant number: 1042105; China Postdoctoral Science Foundation, Grant numbers: 2015T81051, KEYWORDS 2017M610653; National Natural Science β-catenin signaling, MACF1, osteoblast, osteoblast differentiation Foundation of China, Grant numbers: 31400725, 31570940, 81671928

1 | INTRODUCTION Liem, 2016; Hu et al., 2016; Suozzi, Wu, & Fuchs, 2012). It was first found as a novel member of the actin crosslinker superfamily and Microtubule actin crosslinking factor 1 (MACF1) is identified as a called actin crosslinking factor 7 (ACF7) (Byers, Beggs, McNally, & ∼600 kDa belonging to the spectraplakin family (Goryunov & Kunkel, 1995). Possessing both microtubule and actin binding

1574 | © 2017 Wiley Periodicals, Inc. wileyonlinelibrary.com/journal/jcp J Cell Physiol. 2018;233:1574–1584. HU ET AL. | 1575 domains, MACF1 regulates the dynamics of and actin sodium bicarbonate, 10% fetal bovine serum (FBS) (ExCell Biology to shape cell morphology and coordinate the cytostructure response Inc. Shanghai, China), 100 µg/ml streptomycin and 100 units/ml to environmental cues (Fassett et al., 2013; Kodama, Karakesisoglou, penicillin, in a humidified, 37°C, 5% CO2 incubator. The stable Wong, Vaezi, & Fuchs, 2003; Wu, Kodama, & Fuchs, 2008). MACF1 MACF1 knockdown (MACF1-KD) osteoblastic cell line was gener- has been shown to be ubiquitously expressed in numerous tissues, ated by lentivirus-mediated short-hairpin RNA (shRNA) technology including skin, neuron, heart, brain, lung, liver, stomach, kidney, and as previously reported (Hu, Su et al., 2015). In the pilot experiments, skeletal muscle (Bernier, Mathieu, De Repentigny, Vidal, & Kothary, four different shRNA constructs (MACF1 shRNA1, MACF1 shRNA2, 1996; Lin, Chen, Leung, Parry, & Liem, 2005; Sun et al., 1999). Our MACF1 shRNA3, and MACF1 shRNA4) targeting the murine MACF1 previous studies showed that MACF1 was expressed in MC3T3-E1 were designed (Supplementary Tables S1 and S2) and transfected osteoblastic cells (Qian et al., 2009) and recently, MACF1 was also into MC3T3-E1 cells. After selection, the stable, transfected MACF1 detected in bone tissues. Thus, as a widely expressed cytoskeletal shRNA1 MC3T3-E1 cell line was adopted. linker, MACF1 has shown critical functions in physiological and The MACF1-KD cells were also cultured in growth medium pathological processes. Lack of MACF1 resulted in delay of skin (α-MEM supplemented with 2.2 g/L sodium bicarbonate, 10% FBS, wound healing, defects of neural development, dysfunction of the 100 µg/ml streptomycin and 100 units/ml penicillin). For osteoblast heart, and decrease of colonic paracellular permeability (Fassett differentiation, confluent cells were cultured with osteogenic medium et al., 2013; Goryunov, He, Lin, Leung, & Liem, 2010; Liang et al., which contained α-MEM supplemented with 10% FBS, 100 µg/ml 2013; Wu et al., 2008). Recently, MACF1 has been shown to be streptomycin, 100 units/ml penicillin, 50 µg/ml ascorbic acid (AA) and involved in Parkinson’s disease and cancer development (Afghani, 10 mM β–glycerophosphate (β-GP) (Sigma-Aldrich, St Louis, MO). The Mehta, Wang, Tang, Skalli, & Quick, 2017; Chang, Huang, Yeh, & medium was changed every 2 days. For LiCl treatment experiments, Chang, 2017; Jorgensen, Mosbech, Faergeman, Graakjaer, Jacobsen, cells were cultured for 6 hr with the above medium also containing & Schroder, 2014; Wang et al., 2016). 30 mM lithium chloride (LiCl) (Sigma-Aldrich). MACF1 shows versatile functions in numerous cell types. MACF1 not only regulates directed skin stem (Wu et al., 2011) by 2.2 | Alizarin red staining guiding microtubules along stress fibers towards , but also controls neuronal migration and positioning by regulating Mineralized nodules were assessed using alizarin red (Sigma-Aldrich) microtubule dynamics (Ka, Jung, Mueller, & Kim, 2014; Ka, Moffat, staining as previously described (Hu, Li, Qian, Wang, & Shang, 2015). & Kim, 2016). In addition, MACF1 regulates dendritic arborization, In brief, cell cultures were fixed with 10% buffered formaldehyde for axon outgrowth and neurite differentiation (Ka & Kim, 2016). 15 min, rinsed with PBS and then stained with 0.5% alizarin red Furthermore, MACF1 has been shown to participate in responses of (pH 4.2) for 15 min at room temperature. After washing with tap osteoblasts to environmental stimuli and its deficiency was shown to water on a shaking platform for four times of 5 min each time, the inhibit osteoblast proliferation (Hu, Su, et al., 2015; Qian et al., 2009). mineralized nodules were imaged by a scanner (BenQ 5560B, BenQ However, the role of MACF1 in osteoblast differentiation is still Coporation, Suzhou, China) and were analyzed by Image J software unknown. (National Institutes of Health, Bethesda, MD) to determine the In the current work, we adopted a stable MACF1 knockdown mineralization area. osteoblastic cell line and conducted in vitro studies to investigate MACF1 functions and its action mechanisms in regulating osteoblast 2.3 | Real time PCR differentiation. This current study has studied effects of MACF1-KD on mineralization and expression of osteogenic genes in MC3T3-E1 Real time PCR assay was used to assess levels of mRNA expression of osteoblastic cells. In addition, this study has identified the osteogenic genes and some key components of the β-catenin signaling suppression of β-catenin signaling in mediating MACF1-KD effect pathway. Total RNA was extracted from cells using TRIzol reagent in regulating osteoblast differentiation. Uncovering the role of (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA). One µg of total MACF1 in osteoblast differentiation will be of significance for RNA sample was reverse transcribed into complementary DNA further elucidating the regulatory mechanisms for osteoblast (cDNA) using PrimeScript™ RT reagent kit (TaKaRa, Dalian, China). differentiation. The expression of genes was detected by real time PCR carried out in C1000™ Thermal Cycler (Bio-Rad Labs, Hercules, CA) with specific primers (Table 1) and SYBR Premix Ex TaqTM (TaKaRa). Samples were 2 | MATERIALS AND METHODS amplified using the following program: pre-denaturation at 95°C for 30 s, followed by 45 cycles of 95°C for 10 s, 58°C or 60°Cfor 30 s, and 2.1 | Cell cultures 72°C for 5 s. Fluorescence detection was performed at the annealing The murine MC3T3-E1 osteoblastic cells were provided by Dr. Hong phase at 80°C to exclude the interference of primer dimer. The relative Zhou (the University of Sydney). Cells were cultured in expression was calculated via 2−ΔΔCt method (Livak & Schmittgen, alpha-Minimum Essential Medium (α-MEM) (Life Technologies, 2001) by using β–actin or glyceraldehyde-3-phosphate dehydroge- Thermo Fisher Scientific, Carlsbad, CA) supplemented with 2.2 g/L nase (GAPDH) for normalization of the threshold cycle (Ct) value. 1576 | HU ET AL.

2.4 | Immunocytochemistry and laser scanning membrane (PALL Corporation, Port Washington, NY). After incubation confocal microscopy with the blocking buffer (5% nonfat milk or 5% BSA), the membrane was incubated with appropriate primary antibodies against the For β-catenin immunostaining, cells were seeded on coverslips at a following in accordance with the manufacturer’s instructions: density of 1 × 104/cm2 and cultured for 24 hr. For LiCl treatment β-catenin, GSK-3β, phospho-GSK-3β (Ser 9), B1 (Cell Signaling experiments, cells were cultured for 6 more hours with 30 mM LiCl. Technology, Danvers, MA) and GAPDH (Merck, Kenilworth, NJ) at 4°C Then, immunocytochemical staining of β-catenin was conducted. overnight. The horseradish peroxidase (HRP) conjugated secondary After 15 min fixation with 4% paraformaldehyde and permeated with antibody was further used. Protein bands were visualized by 0.5% Triton X-100 TBS, cells were blocked with 3% bovine serum chemiluminescence using an ECL kit (Proteintech, Hubei, China) and albumin (BSA) in PBS for 30 min at room temperature. Then, cells exposed to X-ray film. Protein band intensities were quantified using were incubated with rabbit anti-β-catenin antibody (1:50, Cell Image-J software (National Institutes of Health, NIH). The GAPDH was Signaling Technology, Danvers, MA) in PBS containing 3% BSA used as the internal control for total protein and cytoplasmic protein, overnight at 4°C. After washing with PBST (0.05% Tween 20), cells and was used as the internal control for nuclear protein. were incubated with PE-conjugated goat anti-rabbit IgG secondary antibody (1:100, Hangzhou HuaAn Biotechnology, Hangzhou, China) for 90 min at room temperature. 4′, 6-Diamidino-2-phenylindole 2.6 | Transcriptional reporter assay (DAPI, 1 µg/ml) was used to counterstain cell nuclei for 5 min at A luciferase reportor assay was conducted to assess the transcription room temperature. Each sample was washed, enveloped with activity of TCF1. MACF1-KD cells and the corresponding control Fluoromount-G (Southern Biotech, Birmingham, AL) and examined (scrambled control) cells were plated into 24-well plates at 1.5 × 105 with a laser scanning confocal microscope (Leica TCS SP5, Wetzlar, cells/well and cultured with a-MEM medium supplemented with 10% Germany). PE (red fluorescence) was excited at a wavelength of FBS overnight. Then, cells were transfected with plasmid containing 543 nM and DAPI was excited at 405 nM. 1 µg Topflash luciferase construct (Promega) and 0.9 µg internal control pRL-TK vector (Promega) using Lipofectamine 2,000 (Life 2.5 | Western blot analysis Technologies, Shanghai, China). After transfection for 72 hr, cells were lysed using passive lysis buffer and the firefly and Renilla luciferase For Western blot protein detection and quantification, cellular total activities were detected using the dual luciferase reagent assay kit protein extracts were prepared on ice using Cell Lysis Buffer (Promega). Values for Topflash luciferase activity were normalized (Beyotime, Jiangsu, China) supplemented with 1% Protease Inhibitor with Renilla activity and the relative luciferase activity was shown as Cocktail Set III (Merck, Kenilworth, NJ). Nuclear and cytoplasmic percent of control. protein extracts were prepared with a Nuclear and Cytoplasmic Extraction Kit (Pioneer Biotechnology, Shaanxi, China). The protein 2.7 | Statistical analysis concentration was determined by using BCA protein assay kit (Pierce, Rockford, IL) and an equal amount of proteins for each sample was The statistical analysis of the data was performed with Prism software subjected to 10% SDS-PAGE and transblotted to nitrocellulose version 6.0 (Graphpad Software Inc. La Jolla, CA) and the Student’s

TABLE 1 Primer sequences used for real time PCR Gene name (Genebank no.) Primer sequences (5′–3′) Annealing temperature (°C) ALP (NM_007431.1) F: GTTGCCAAGCTGGGAAGAACAC 58 R: CCCACCCCGCTATTCCAAAC Runx2 (NM_001145920.1) F: TGCACCTACCAGCCTCACCATAC 58 R: GACAGCGACTTCATTCGACTTCC Col Iα1 (NM_007742.3) F: GAAGGCAACAGTCGATTCACC 58 R: GACTGTCTTGCCCCAAGTTCC TCF1 (NM_009331.3) F: CAGAATCCACAGATACAGCA 60 R: CAGCCTTTGAAATCTTCATC LEF1 (NM_010703.4) F: GATCCCCTTCAAGGACGAAG 60 R: GGCTTGTCTGACCACCTCAT GAPDH (NM_008084.2) F: TGCACCACCAACTGCTTAG 60 R: GGATGCAGGGATGATGTTC

ALP, alkaline phosphatase; Col Iα1, type I collagen alpha 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LEF1, lymphoid enhancer-binding factor 1; Runx2, runt-related transcription factor 2; TCF1, T cell factor 1. HU ET AL. | 1577 t-test was used for comparisons between two groups. The data are MACF1-KD2 cells (Supplement Figure S2b,c). Thus, in the following presented as mean ± standard deviation (SD). experiments, MACF1-KD1 (MACF1-KD) cell line was used. MACF1-KD cells were cultured with osteogenic medium for 7, 14, and 21 days, respectively, and levels of formation of mineralized 3 | RESULTS nodules in cells were detected. Compared to MC3T3-E1 cells or scrambled controls, the dramatic reduction in formation of mineralized 3.1 | Knockdown of MACF1 suppresses nodules was observed in MACF1-KD cells (Figure 1). In both parental mineralization of osteoblastic cells MC3T3-E1 cells and the scrambled control cells, mineralized nodules As a ubiquitously expressed cytoskeletal protein, MACF1 is expressed were detected on day 14 and increased further by 21 days’ culture, in MC3T3-E1 osteoblastic cells (Qian et al., 2009). Moreover, the while MACF1-KD cells showed almost no mineralized nodules MACF1 expression was detected in vivo in mouse bone tissues and (Figure 1). As the mineralization is one important step of osteoblast MACF1 levels were lower in bone tissues of older mice which were differentiation, this result indicates that knockdown of MACF1 inhibits associated with lower ALP levels (Supplement Figure S1). This suggests osteoblast differentiation. that MACF1 may play an important role in the differentiation of Recently, we generated a MACF1 conditional knockout (cKO) bone-forming osteoblasts. mouse model with specific deletion of MACF1 in bone mesenchymal The effect of MACF1 gene knockdown on osteoblast differentia- stem cells (MSCs) by using Cre-loxP conditional knockout technology. tion was determined using MACF1-KD MC3T3-E1 osteoblastic cells. In By adopting the primary MSCs, we found that MACF1-cKO MSCs the pilot experiments, four different shRNA constructs targeting murine showed an obvious decrease in mineralized nodule formation after 14 MACF1 were designed (Supplementary TableS S1 and S2) and trans- days’ osteogenic culture (Supplement Figure S3). All these results fected into MC3T3-E1 cells. After selection, two MACF1-KD osteo- together indicate that MACF1 deficiency inhibits osteoblastic blastic cell lines (MACF1-KD1 and MACF1-KD2) transfected with differentiation. MACF1 shRNA1 and shRNA2, respectively, were generated (Supple- ment Figure S2a). First, both MACF1-KD1 and MACF1-KD2 cell lines 3.2 | Knockdown of MACF1 down-regulates the were adopted to investigate the effects of MACF1 knockdown on expression of osteogenic genes osteoblast mineralization. After cultivation with osteogenic medium for 21 days, levels of formation of mineralized nodules in cells were Alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), examined by alizarin red staining. Compared to the untransfected and type I collagen α1(ColIα1) are critical osteogenic genes in regulating parental (MC3T3-E1) or scrambled shRNA-transfected controls (scram- osteoblast differentiation, and ALP is also one important marker for bled control), both MACF1-KD1 cells and MACF1-KD2 cells showed osteoblast differentiation. Thus, we investigated whether the gene significant decreases in formation of mineralized nodules, and formation expression patterns of ALP, Runx2, and Col Iα1 were altered by of mineralized nodules in MACF1-KD1 cells was lower than that in knockdown of MACF1. The was detected by real time

FIGURE 1 Knockdown of MACF1 inhibits mineralized nodule formation in MC3T3-E1 osteoblastic cells. (a) Mineralized nodule formation was detected by alizarin red staining at days 7, 14, and 21 during osteoblast differentiation. Representative pictures of mineralized nodules for parental MC3T3-E1 cells, scrambled control cells and MACF1-KD cells were shown. (b) Quantification of mineralized areas using Image J software (n =3) 1578 | HU ET AL.

PCR in a time course during osteoblast differentiation. As shown in As the nuclear translocation of β-catenin is the hallmark of Figure 2, knockdown of MACF1 significantly reduced ALP and Runx2 β-catenin signaling activation, the cellular localization and levels of expression levels throughout the cell differentiation period when β-catenin were examined by both immunocytochemistry staining and compared to the scrambled control. In the scrambled control cells, the Western blot analyses. As shown in Figure 3a, immunocytochemistry ALP expression firstly increased following osteogenic differentiation staining showed that in scrambled control cells, the β-catenin was with time (Day 1, Day 7), then, during the mineralized nodule formation mainly distributed around the cytoplasm and partially in the nucleus. period, ALP expression gradually decreased (Day 14, Day 21). The ALP However, in the MACF1-KD cells, β-catenin was mainly located at cell expression was significantly down-regulated in MACF1-KD cells on membrane and there was almost no β-catenin staining around the Day 1 (0.06-fold as scrambled control), Day 7 (0.44-fold as scrambled nuclear area. This result suggests that knockdown of MACF1 control), Day 14 (0.48-fold as scrambled control), and Day 21 (0.49-fold dramatically inhibited the nuclear translocation of β-catenin. Further as scrambled control) (Figure 2) (p < 0.01 or 0.001), although the Western blot analyses of the cytosolic and nuclear fractions of cellular expression trend was similar to that of the scrambled control. Runx2, a proteins also showed a significant decrease of the nuclear amount of β- key transcription factor regulating the initiation of osteogenic catenin in MACF1-KD cells ((Figure 3b,c) p < 0.001). Furthermore, differentiation, was dramatically down-regulated by knockdown of there was a 61% decrease in the transcription activity of TCF1 in MACF1 when compared to the scrambled controls at various time MACF1-KD cells compared to that in scrambled control cells points from the beginning of osteogenic differentiation (Figure 2) (Figure 3d). In addition, gene expression levels of both TCF1, LEF1 (p < 0.01 or 0.001). In addition, Col Iα1, a main extracellular matrix and Runx2 were significantly decreased in the MACF1-KD cells molecule that is important for osteoblastic mineralization regulation, compared to scrambled control (Figure 3-e,f,g). As Runx2 is a direct was down-regulated during the mineralization stage (Figure 2) (p < 0.05). target of β-catenin/TCF1, all these results indicate that knockdown of These results are consistent with the outcome of mineralized nodule MACF1 suppresses β-catenin signaling in osteoblasts and suggest that formation and further confirm the inhibitory effect of knockdown of MACF1 may positively regulate osteoblast differentiation through the MACF1 on osteoblast differentiation, suggesting a positive regulatory β-catenin/TCF1-Runx2 pathway. role of MACF1 for osteoblast differentiation. 3.4 | Knockdown of MACF1 activates GSK-3β by 3.3 | Knockdown of MACF1 inhibits β-catenin attenuating its Ser 9 inhibitory phosphorylation signaling in osteoblasts β-catenin signaling activation involves the accumulation of β-catenin in MACF1 has been found to participate in Wnt/β-catenin signaling cytoplasm without being degraded by active GSK-3β and thus allowing in embryo development and knockdown of MACF1 inhibited its translocation into nucleus to regulate the expression of target genes TCF/β-catenin-dependent transcriptional activation (Chen et al., (e.g. TCF1, Runx2). Thus, we wondered whether the activity of GSK-3β 2006). As β-catenin/TCF signaling plays a key role in regulating would be affected by MACF1 knockdown. The phosphorylation of osteoblast differentiation by targeting Runx2 (Gaur et al., 2005) and GSK-3β in Ser 9 ([pSer9]GSK-3β/P-GSK-3β), which results in GSK-3β the Runx2 expression was down-regulated by MACF1 knockdown as inhibition, was determined by Western blot. The level of phosphory- shown above, here we investigated whether knockdown of MACF1 lated GSK-3β ([pSer9]GSK-3β/P-GSK-3β) was significantly decreased would suppress β-catenin signaling, and thereby inhibiting osteoblast in MACF1-KD cells compared to that in scrambled controls (Figure 4). differentiation. This finding suggests that MACF1 knockdown attenuates the

FIGURE 2 Knockdown of MACF1 down-regulates the expression of osteogenic genes. The expression of ALP, Runx2 and Col Iα1 was detected by real time PCR in a time course (1, 7, 14, and 21 days of osteogenic culture). Data represent mean values ± SD (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001, t-test HU ET AL. | 1579

FIGURE 3 Knockdown of MACF1 suppresses β-catenin nuclear translocation, TCF1 transcription activity, and down-regulates the expression of TCF1, LEF1 and Runx2. (a) Immunocytochemistry staining showed the location of β-catenin in the MACF1-KD cells and scrambled control cells. β-catenin was stained as red and nuclei were stained by DAPI showing blue. Yellow arrows indicate the around nuclear localization of β-catenin, while white arrows show the distribution of β-catenin. Bar: 50 µM. (b) Representative Western blots of the nuclear translocation of β-catenin. The nuclear (nucleus) and cytosolic (cytosol) fractions of proteins isolated from MACF1-KD cells and scrambled control cells were probed for β-catenin. Lamin B1 and GAPDH were used as internal controls for nuclear and cytosol fractions, respectively. (c) Quantification of nuclear and cytosol levels of β-catenin with Lamin B1 and GAPDH as internal control, respectively. Data represent mean values ± SD (n = 3), ***p < 0.001. (d) Transcription activity of TCF1 as assessed by the luciferase reportor assay. Data represent mean values ± SD (n = 3), **p < 0.01. (e–g) mRNA expression of TCF1 (e), LEF1 (f) and Runx2 (g) as detected by real time PCR (Data represent mean values ± SD (n = 3), ***p < 0.001 inhibitory phosphorylation of GSK-3β and increases GSK-3β activity, control cells as detected by Western blot (Figure 6a,ca,c). In addition, thus inhibiting β-catenin signal transduction. being consistent with the immunocytochemistry results (Figure 5), the amounts of β-catenin in the nuclei were remarkably increased in both MACF1-KD cells and the scrambled control cells being treated 3.5 | MACF1 acts upstream of GSK-3β/β-catenin with LiCl (Figure 6b,d). Furthermore, the decreased expression of signaling in osteoblasts TCF1 and Runx2 was almost fully restored in MACF1-KD cells LiCl is an agonist for β-catenin signaling by inhibiting GSK-3β kinase treated with LiCl (Figure 6e,f). These data suggest that MACF1 acts activity. To further determine if MACF1 acts upstream of GSK-3β to upstream of GSK-3β/β-catenin signaling to regulate osteoblast regulate β-catenin signaling in osteoblasts, MACF1-KD cells were differentiation through the TCF1-Runx2 axis. treated with LiCl and effects on β-catenin signaling were examined. As shown in Figure 5, the nuclear amount of β-catenin was obviously increased in MACF1-KD cells treated by LiCl comparing to the 4 | DISCUSSION untreated cells. Meanwhile, an obvious increase of the nuclear β-catenin amount was also observed in scrambled control cells There have been rapid advances in the understanding of MACF1’s treated by LiCl, although β-catenin was mainly located around in the functions in numerous tissues and the related cell types including the cytosol and in the nucleus in scrambled control cells (Figure 5, yellow skin (keratinocyte and skin stem cell), nerve tissues (neuron), heart arrows). The level of phosphorylated GSK-3β (P-GSK-3β)(Ser9), (cardiomyocyte), and colon (columnar epithelial cell), and consequently which represents the inactive status of GSK-3β, was obviously in providing new mechanistic understanding for specific physiological increased by LiCl treatment in both MACF1-KD cells and scrambled and pathological processes in these tissues and cells. However, 1580 | HU ET AL.

FIGURE 4 The effects of MACF1 knockdown on GSK-3β activity. (a) Representative Western blots visualized by enhanced chemiluminescence method. (b) Quantitative analyses of the P-GSK-3β/total GSK-3β ratios (mean values ± SD, n = 3), ***p < 0.001

functions of MACF1 in bone tissues and bone cells are relatively and the MACF1-cKO MSCs, we found that MACF1 deficiency unexplored. The current study observed that MACF1 expression was significantly inhibited the mineralized nodule formation and detected in mouse bone tissues and was decreased in older mice, down-regulated the expression of key osteogenic genes. These which was associated with decreased ALP expression. This finding indicate that MACF1 plays an important role in regulating osteoblastic suggests that MACF1 may play an important role in differentiation of differentiation. Further results have shown that knockdown of MACF1 bone-forming osteoblasts. Thus, the present study has focused our increased GSK-3β activity, suppressed β-catenin nuclear translocation, investigation on the role of MACF1 in osteoblastic cell differentiation. and thus inhibited expression of Runx2, a β-catenin target gene By adopting both the stable MACF1 knockdown osteoblastic cell lines downstream TCF1. Conversely, preserving GSK-3β activity and

FIGURE 5 LiCl treatment induces β-catenin nuclear translocation in MACF1-KD cells. MACF1-KD cells and scrambled control cells were treated with or without LiCl (30 mM) for 6 hr and processed for β-catenin immunofluorescence staining (red). Nuclei were counterstained using DAPI (blue). Yellow arrows indicate the nuclear localization of β-catenin, while white arrows show the cell membrane distribution of β-catenin. The β-catenin translocation into nuclei makes the nuclei appear as purple color (yellow arrows) when the two stains were merged. Bar: 50 µM HU ET AL. | 1581

FIGURE 6 LiCl treatment partially restores the suppression of β-catenin signaling in MACF1-KD cells. (a) Representative Western blots showing the phosphorylation levels of GSK-3β (Ser 9) in MACF1-KD cells and scrambled control cells after 6 hr treatment of LiCl (30 mM). (b) Representative Western blots with isolated nuclear (nucleus) and cytosolic (cytosol) fractions of cellular proteins showing the nuclear translocation of β-catenin after LiCl treatment, with lamin B1 and GAPDH being used as internal controls for nuclear and cytosol fractions, respectively. (c) Quantitative analyses of the P-GSK-3β/Total GSK-3β ratios in MACF1-KD cells and scrambled control cells after treatment with LiCl (mean values ± SD, n = 3), *p < 0.05, **p < 0.01. (d) Quantitative analyses of nuclear and cytosol levels of β-catenin with Lamin B1 and GAPDH as internal control, respectively. Data represent mean ± SD (n = 3), *p < 0.05, **p < 0.01. (e) mRNA expression of TCF1 as regulated by β-catenin agonist (LiCl) and measured by real time PCR. Data represent mean values ± SD (n = 3), ***p < 0.001 compared to scrambled control cells, and ###p < 0.001 compared to MACF1-KD cells. (f) mRNA expression of Runx2 as regulated by β-catenin agonist LiCl. Data represent mean values ± SD (n = 3), ***p < 0.001 compared to scrambled control cells, and ##p < 0.01 compared to MACF1-KD cells

β-catenin signaling by LiCl treatment was found to partially reverse Runx2 and Col Iα1, which are the key regulators and markers for effects of MACF1 knockdown on β-catenin signaling suppression. osteoblast differentiation. Thus our study has now suggested a Thus, the current study has uncovered a novel role of MACF1 in positive regulatory role of MACF1 in both osteoblastic cell prolifera- osteoblasts and suggests that MACF1 plays a positive regulatory role tion and differentiation. As a versatile molecule, MACF1 not only in osteoblast differentiation via promoting β-catenin/TCF1-Runx2 regulates the dynamics of F-actin and microtubules, but also mediates signaling and acting upstream of GSK-3β/β-catenin signaling. cell signal transduction. F-actin and microtubules are known to play Previously, MACF1 has been found to be expressed in MC3T3-E1 key role in regulating cell cycle and cell proliferation by functioning osteoblastic cells and play a key role in regulating cytoskeletal during cytokinesis (Heng & Koh, 2010; Mishima, Pavicic, Gruneberg, distribution and cell proliferation (Hu, Su et al., 2015; Qian et al., 2009). Nigg, & Glotzer, 2004; Straight & Field, 2000). Besides, As a key cytoskeletal linker, MACF1 plays an important role in reorganization affects osteoblastic differentiation (Yourek, Hussain, & regulating the dynamics of F-actin and microtubules, which is critical Mao, 2007). The perturbation of the F-actin cytoskeleton organization for cytokinesis and cell proliferation. Our previous studies showed that abolished the osteoblast differentiation process (Zouani, Rami, Lei, & knockdown of MACF1 altered F-actin and microtubule distribution, Durrieu, 2013) and transient dynamic actin cytoskeletal reorganization causing alteration of cell cycle and thus inhibiting cell proliferation. The stimulated osteoblastic differentiation (Higuchi, Nakamura, current study has shown that knockdown of MACF1 inhibits Yoshikawa, & Itoh, 2009). Wang et al, have shown that cytoskeleton osteoblast differentiation as it dramatically suppressed mineralization reorganization is involved in lanthanum-promoted both osteoblast of osteoblasts and significantly down-regulated expression of ALP, proliferation and differentiation (Wang, Huang, Zhang, & Wang, 2009). 1582 | HU ET AL.

These findings together with our results indicate that MACF1 may regulate both osteoblast proliferation and differentiation through controlling cytoskeletal dynamics. Moreover, MACF1 is critical in activating Wnt/β-catenin signaling (Chen et al., 2006) and Wnt/β-catenin signaling is essential for both osteoblast precursor proliferation and differentiation (Baron & Kneissel, 2013). Thus, MACF1 may regulate osteoblastic proliferation and differentiation by both regulating cytoskeletal dynamics and cellular signaling including Wnt/β-catenin signaling. Previously, an essential role of MACF1 in activating Wnt/β-catenin signaling has been reported (Chen et al., 2006), which is one crucial pathway in regulating osteoblast differentiation (Baron & Kneissel, 2013; Krishnan, Bryant, & Macdougald, 2006). The translocation of β-catenin to the nucleus promotes osteoblast FIGURE 7 Proposed model depicting the mechanism of MACF1 differentiation by stimulating expression of its target gene Runx2 in in regulating osteoblast differentiation. MACF1 induces β-catenin a β-catenin/TCF1 dependent manner (Gaur et al., 2005). Results from accumulation and translocation into the nucleus. Subsequently, our current study showed that knockdown of MACF1 obviously β-catenin interacts with TCF1 and induces Runx2 expression, thus decreased the amount of β-catenin in the nuclei, which further promotes osteoblast differentiation. During this process, MACF1 inhibited β-catenin/TCF1 transcription activation, and down- may inactivate GSK-3β by phosphorylation and prevent β-catenin from being degraded, thereby allowing the accumulated β-catenin regulated the expression of TCF1 and LEF1. Moreover, Runx2, the to enter the nucleus essential transcription factor for osteoblast differentiation (Franceschi et al., 2003; Komori, 2010), was significantly down-regulated by knockdown of MACF1. It has been demonstrated that TCF1 but not activity. Then, β-catenin/TCF1 induces the expression of its target LEF1 binds directly to Runx2 promoter in MC3T3-E1 cells and that transcription factor Runx2 to regulate osteoblast differentiation. During Runx2 is a direct target of β-catenin/TCF1 for the stimulation of osteoblast differentiation process, we speculate that MACF1 acts osteoblast differentiation (Gaur et al., 2005). Thus, the current study upstream of GSK-3β and may inactivate GSK-3β by interaction, which suggests that MACF1 may regulate osteoblast differentiation through helps the release of β-catenin. However, the regulatory effect of MACF1 modulating the β-catenin/TCF1/Runx2 signal axis. on GSK-3β in osteoblasts needs further elucidation. Since β-catenin signaling activation involves the accumulation of β-catenin in cytoplasm without being degraded by active GSK-3β and thus allowing its translocation into nucleus, the expression and 5 | CONCLUSIONS activation status of GSK-3β were determined by Western blot analysis. The results showed that MACF1 knockdown activated In the current study, a novel role of MACF1 in osteoblast differentia- GSK-3β by attenuating the inhibitory phosphorylation of GSK-3β. tion was uncovered. MACF1 not only shows a positive regulatory role This indicates that MACF1 knockdown may increase GSK-3β in osteoblast differentiation, but also mediates or is important for the activity, thus inhibiting β-catenin signaling activation, and suggests nuclear translocation of β-catenin and the downstream signaling a possible upstream role of MACF1 in GSK-3β/β-catenin signaling. including Runx2 transcription. Thus, the current study has provided a Further important findings showed that the decreased nuclear new insight into MACF1 functions and a novel molecular mechanism translocation of β-catenin and reduced expression of downstream for osteoblast differentiation. TCF1 and Runx2 in MACF1-KD cells were remarkably restored by LiCl treatment which is known to inhibit activation of GSK-3β.These ’ suggest that MACF1 functions upstream of GSK-3β/β-catenin AUTHORS CONTRIBUTION signaling in osteoblasts, which is consistent with the findings of Wu et al. (Wu et al., 2011; Ka et al., 2014). They showed that MACF1 Study design: LH, ZM, and AQ. Study conduct: GZ, ZM, and AQ. Data possessed functional GSK-3β phosphorylation sites (Wu et al., 2011) collection: LH, PS, CY, KY, and DL. Data analysis: LH, PS, CY, YZ, RL, and mediated GSK-3 signaling in the developing neurons (Ka et al., KY, ZC, and DL. Data interpretation: LH, PS, CY, YZ, RL, KY, ZC, and DL. 2014). Besides, Chen et al. found that MACF1 acted upstream of Drafting manuscript: LH and LW, AQ. Revising manuscript: LH, LW, GSK-3β during embryo development Chen et al., 2006. CJX, and AQ. All authors have read and approved the final submitted Taken together, from the data in the current study, a model manuscript. illustrating the possible mechanism of MACF1 regulating osteoblast differentiation can be proposed (Figure 7). MACF1 is essential for the CONFLICTS OF INTEREST cytoplasmic accumulation and the nuclear translocation of β-catenin. Subsequently, β-catenin interacts with TCF1 and induces transcriptional The authors declare that they have no conflict of interest. HU ET AL. | 1583

ACKNOWLEDGMENTS Higuchi, C., Nakamura, N., Yoshikawa, H., & Itoh, K. (2009). Transient dynamic actin cytoskeletal change stimulates the osteoblastic. Journal The authors would like to thank Dr. Hong Zhou (The University of of Bone and Mineral Metabolism, 27, 158–167. Sydney) for providing MC3T3-E1 cell line and Dr. Pengsheng Zheng Hu, L., Su, P., Li, R., Yan, K., Chen, Z., Shang, P., & Qian, A. (2015). (Xi’an Jiaotong University) for providing the Topflash luciferase Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells. BMB Reports, 48, plasmid and Renilla luciferase vector. This work was supported by 583–588. the National Natural Science Foundation of China (31400725, Hu, L., Su, P., Li, R., Yin, C., Zhang, Y., Shang, P., ...Qian, A. (2016). Isoforms, 31570940, and 81671928), the China Postdoctoral Science Founda- structures, and functions of versatile spectraplakin MACF1. BMB – tion (2015T81051, 2017M610653), the Fundamental Research Funds Reports, 49,37 44. Hu,L.F.,Li,J.B.,Qian,A.R.,Wang,F.,&Shang,P.(2015). for the Central Universities (3102016ZY037), and the Shenzhen Mineralization initiation of MC3T3-E1 preosteoblast is sup- Science and Technology Project (JCYJ20160229174320053). LW is pressed under simulated microgravity condition. Cell Biology supported by Australian NHMRC grant (1094606), and CJX is International, 39,364–372. supported by Australian NHMRC Senior Research Fellowship Jorgensen, L. H., Mosbech, M. B., Faergeman, N. J., Graakjaer, J., Jacobsen, S. V., & Schroder, H. D. (2014). Duplication in the microtubule-actin (1042105). cross-linking factor 1 gene causes a novel neuromuscular condition. Scientific Reports, 4, 5180. Ka, M., Jung, E. M., Mueller, U., & Kim, W. Y. (2014). MACF1 regulates the migration of pyramidal neurons via microtubule dynamics and GSK-3 REFERENCES signaling. Developmental Biology, 395,4–18. Afghani, N., Mehta, T., Wang, J., Tang, N., Skalli, O., & Quick, Q. A. (2017). Ka, M., & Kim, W. Y. (2016). Microtubule-actin crosslinking factor 1 is Microtubule actin cross-linking factor 1, a novel target in glioblastoma. required for dendritic arborization and axon outgrowth in the International Journal of Oncology, 50, 310–316. developing brain. Molecular Neurobiology, 53, 6018–6032. Baron, R., & Kneissel, M. (2013). WNT signaling in bone homeostasis and Ka, M., Moffat, J. J., & Kim, W. Y. (2016). MACF1 controls migration and disease: From human mutations to treatments. Nature Medicine, 19, positioning of cortical GABAergic interneurons in mice. Cerebral Cortex. 179–192. https://doi.org/10.1093/cercor/bhw309 Bernier, G., Mathieu, M., De Repentigny, Y., Vidal, S. M., & Kothary, R. Kodama, A., Karakesisoglou, I., Wong, E., Vaezi, A., & Fuchs, E. (2003). (1996). Cloning and characterization of mouse ACF7, a novel member ACF7: An essential integrator of microtubule dynamics. Cell, 115, – of the dystonin subfamily of actin binding proteins. Genomics, 38, 343 354. 19–29. Komori, T. (2010). Regulation of osteoblast differentiation by Runx2. – Byers, T. J., Beggs, A. H., McNally, E. M., & Kunkel, L. M. (1995). Novel actin Advances in Experimental Medicine and Biology, 658,43 49. crosslinker superfamily member identified by a two step degenerate Krishnan, V., Bryant, H. U., & Macdougald, O. A. (2006). Regulation of bone PCR procedure. FEBS Letters, 368, 500–504. mass by Wnt signaling. The Journal of Clinical Investigation, 116, – Chang, Y. S., Huang, H. D., Yeh, K. T., & Chang, J. G. (2017). 1202 1209. ... Identification of novel mutations in endometrial cancer patients by Liang, Y., Shi, C., Yang, J., Chen, H., Xia, Y., Zhang, P., Qin, H. (2013). whole-exome sequencing. International Journal of Oncology, 50, ACF7 regulates colonic permeability. International Journal of Molecular – 1778–1784. Medicine, 31, 861 866. Chen,H.J.,Lin,C.M.,Lin,C.S.,Perez-Olle, R., Leung, C. L., & Liem, R. K. Lin, C. M., Chen, H. J., Leung, C. L., Parry, D. A., & Liem, R. K. (2005). (2006). The role of microtubule actin cross-linking factor 1 (MACF1) Microtubule actin crosslinking factor 1b: A novel that localizes to – in the . Genes & Development, 20, the Golgi complex. Journal of Cell Science, 118, 3727 3738. 1933–1945. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Fassett, J. T., Xu, X., Kwak, D., Wang, H., Liu, X., Hu, X., ...Chen, Y. (2013). Methods, 25, 402–408. Microtubule actin cross-linking factor 1 regulates cardiomyocyte Mishima, M., Pavicic, V., Gruneberg, U., Nigg, E. A., & Glotzer, M. (2004). microtubule distribution and adaptation to hemodynamic overload. Cell cycle regulation of central spindle assembly. Nature, 430, 908–913. PLoS ONE, 8, e73887. Qian, A. R., Hu, L. F., Gao, X., Zhang, W., Di, S. M., Tian, Z. C., ...Shang, P. Franceschi, R. T., Xiao, G., Jiang, D., Gopalakrishnan, R., Yang, S., & Reith, E. (2009). Large gradient high magnetic field affects the association of (2003). Multiple signaling pathways converge on the Cbfa1/Runx2 MACF1 with actin and microtubule cytoskeleton. Bioelectromagnetics, transcription factor to regulate osteoblast differentiation. Connective 30, 545–555. Tissue Research, 44, 109–116. Straight, A. F., & Field, C. M. (2000). Microtubules, membranes and Gaur, T., Lengner, C. J., Hovhannisyan, H., Bhat, R. A., Bodine, P. V., Komm, cytokinesis. Current Biology, 10, R760–R770. B. S., ... Lian, J. B. (2005). Canonical WNT signaling promotes Sun,Y.,Zhang,J.,Kraeft,S.K.,Auclair,D.,Chang,M.S.,...Chen, L. B. osteogenesis by directly stimulating Runx2 gene expression. The (1999). Molecular cloning and characterization of human trabe- Journal of Biological Chemistry, 280, 33132–33140. culin-alpha, a giant protein defining a new family of actin-binding Goryunov, D., He, C. Z., Lin, C. S., Leung, C. L., & Liem, R. K. (2010). Nervous- proteins. The Journal of Biological Chemistry, 274, 33522–33530. tissue-specific elimination of microtubule-actin crosslinking factor 1a Suozzi, K. C., Wu, X., & Fuchs, E. (2012). Spectraplakins: Master results in multiple developmental defects in the mouse brain. Molecular orchestrators of cytoskeletal dynamics. The Journal of Cell Biology, and Cellular Neurosciences, 44,1–14. 197, 465–475. Goryunov, D., & Liem, R. K. (2016). Microtubule-actin cross-linking factor 1: Wang, X., Huang, J., Zhang, T., & Wang, K. (2009). Cytoskeleton Domains, interaction partners, and tissue-specific functions. Methods in reorganization and FAK phosphorylation are involved in lanthanum Enzymology, 569, 331–353. (III)-promoted proliferation and differentiation in rat osteoblasts. Heng, Y. W., & Koh, C. G. (2010). Actin cytoskeleton dynamics and the cell Progress in Natural Science, 19, 331–335. division cycle. The International Journal of Biochemistry & Cell Biology, 42, Wang,X.,Li,N.,Xiong,N.,You,Q.,Li,J.,Yu,J.,... Lin, Z. (2016). – 1622 1633. Genetic variants of microtubule actin cross-linking factor 1 1584 | HU ET AL.

(MACF1) confer risk for Parkinson’sdisease.Molecular Neurobiology, SUPPORTING INFORMATION 54, 2878–2888. Wu, X., Kodama, A., & Fuchs, E. (2008). ACF7 regulates cytoskeletal-focal Additional Supporting Information may be found online in the adhesion dynamics and migration and has ATPase activity. Cell, 135, supporting information tab for this article. 137–148. Wu, X., Shen, Q. T., Oristian, D. S., Lu, C. P., Zheng, Q., Wang, H. W., & Fuchs, E. (2011). Skin stem cells orchestrate directional migration by regulating microtubule-ACF7 connections through GSK3beta. Cell, 144, 341–352. How to cite this article: Hu L, Su P, Yin C, et al. Microtubule Yourek, G., Hussain, M. A., & Mao, J. J. (2007). Cytoskeletal changes of actin crosslinking factor 1 promotes osteoblast mesenchymal stem cells during differentiation. American Society for differentiation by promoting β-catenin/TCF1/Runx2 Artificial Internal Organ, 53, 219–228. signaling axis. J Cell Physiol. 2018;233:1574–1584. Zouani, O. F., Rami, L., Lei, Y., & Durrieu, M. C. (2013). Insights into the osteoblast precursor differentiation towards mature osteoblasts. https://doi.org/10.1002/jcp.26059 Biology Open, 2, 872–881.