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The p75 neurotrophin receptor regulates MC3T3-E1 osteoblastic differentiation
Differentiation 84 (2012) 392–399
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The p75 neurotrophin receptor regulates MC3T3-E1 osteoblastic differentiation Yoshikazu Mikami a,b, Shinnosuke Suzuki a, Yumiko Ishii c, Nobukazu Watanabe d, Tomihisa Takahashi a,b, Keitaro Isokawa a,b, Masaki J. Honda a,b,n a
Department of Anatomy, Nihon University School of Dentistry, Tokyo 101-8310, Japan Dental Research Center, Nihon University School of Dentistry, Tokyo 101-8310, Japan c FACS Core Laboratory, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan d Laboratory of Diagnostic Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 February 2012 Received in revised form 22 May 2012 Accepted 1 July 2012 Available online 17 August 2012
While the role of p75NTR signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75NTR on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75NTR-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75NTR in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75NTR, as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75NTR-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75NTR signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation. & 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.
Keywords: MC3T3-E1 Cells Osteoblast Osteogenic differentiation p75NTR Trk
1. Introduction Bone is a dynamic organ in which old and damaged bone is replaced by newly formed bone as a part of the process of remodeling that occurs throughout the life of an individual. Remodeling is carried out by both osteoblasts for bone formation and osteoclasts for bone resorption. Osteoblasts originate from mesenchymal stem cells (MSCs) and a subset of mature osteoblasts is embedded in the bone to become osteocytes. The process of osteocyte differentiation from MSC is complex and proceeds through multiple steps that involve both cell proliferation and differentiation. Many different signal transduction pathways govern the tightly-regulated expression of osteoblast-specific n Corresponding author at: Nihon University School of Dentistry Department of Anatomy 1-8-13 Kanda-Surugadai, Tokyo 101-8310, Japan. Tel.: þ81 3 3219 8121; fax: þ 81 3 3219 8319. E-mail addresses: [email protected] (Y. Mikami), [email protected] (S. Suzuki), [email protected] (Y. Ishii), [email protected] (N. Watanabe), [email protected] (T. Takahashi), [email protected] (K. Isokawa), [email protected] (M.J. Honda).
genes during the process of osteoblast differentiation. Runx2, Dlx5, Msx2, and Osterix are the major transcriptional factors that regulate osteoblast differentiation (Fu et al., 2007; Huang et al., 2007; Komori, 2002, 2006). In spite of the critical role of osteoblasts in bone formation, our understanding of the osteoblast differentiation program remains incomplete. MSCs are first identified in the bone marrow (BM) (Friedenstein et al., 1974), and are now recognized to be sustained throughout life and serve as a source of cell replacement within certain tissues in response to trauma and turnover (Jackson et al., 2007). A single MSC has the potential to differentiate into multiple lineages such as osteoblasts, chondrocytes, and adipocytes (Pittenger et al., 1999). More recently, MSCs have been evaluated for their potential therapeutic use in cell transplantation to repair musculoskeletal, neuronal, and cardiac tissues and to suppress immune responses in graft-versus-host diseases (Barry and Murphy, 2004; Le Blanc et al., 2004; Pittenger and Martin, 2004). The identification of suitable MSC molecular markers, such as surface antigens, is crucial for the manipulation of cells in vitro as well as to understand the mechanisms of stem cell regulation. One such marker is p75 neurotrophin receptor (p75NTR), which is a selective marker for the isolation and
0301-4681/$ - see front matter & 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved. Join the International Society for Differentiation (www.isdifferentiation.org) http://dx.doi.org/10.1016/j.diff.2012.07.001
Y. Mikami et al. / Differentiation 84 (2012) 392–399
phenotypic characterization of bone marrow-derived MSCs (Jones et al., 2002; Poloni et al., 2009; Quirici et al., 2002). p75NTR is a 75 kDa transmembrane protein, that is referred to as nerve growth factor receptor (NGFR), tumor necrosis factor receptor superfamily member 16 (TNFRSF16) or CD271 (Mischel et al., 2001). It modulates the expression of genes that are critically involved in the regulation of differentiation as well as cell adhesion, signal transduction, apoptosis, tumor cell invasion, and metastasis (Nalbandian et al., 2005). In spite of the low affinity binding of the precursors of neurotrophic factors to p75NTR, it is required for efficient trk receptor tyrosine kinase (trkA, trkB, trkC) activation and signaling (Kaplan and Miller, 2000; Mischel et al., 2001; Patapoutian and Reichardt, 2001). p75NTR functions as a ligand-stimulated apoptosis-inducing receptor that stimulates the activity of the JNK-p53-Bax pathway and suppresses Ras/PI-3k/Akt activation (Kaplan and Miller, 2000; Patapoutian and Reichardt, 2001). In contrast, trk receptors promote cell survival via several intracellular signaling cascades e.g. the Ras/PI-3k/Akt pathway (Chao, 2003; Kaplan and Miller, 2000; Patapoutian and Reichardt, 2001; Serafeim and Gordon, 2001). p75NTR has been used as a marker for the isolation of esophageal epithelial stem cells (Okumura et al., 2003), adipose tissue-derived MSCs (Yamamoto et al., 2007), and MSCs in the growth zones of regenerating fallow deer antlers and from the pedicle periosteum (Rolf et al., 2008). On the other hand, recently, there were reports that a role of CD271 may be influence to the cell differentiation. For instance, previous investigation demonstrated that when MSCs differentiated into a Schwann Cell lineage using glial growth factor, in the differentiation process, level of p75 protein was significantly elevated upon differentiation though p75 protein was not expressed in MSCs (Caddick et al., 2006). Furthermore, in the previous work, higher protein levels of CD271 were detected in mineralizing jaw periosteum-derived cells compared to nonmineralizing jaw periosteum-derived cells within the first five days of osteogenic differentiation process. (Alexander et al., 2009). Based on these previous results, we hypothesized that p75NTR might play an important role in MSCs, in particular, in the osteogenic lineage. In spite of its importance in neuronal development and regeneration, a specific functional role for p75NTR in osteogenic differentiation has not been demonstrated and no signaling cascade has been identified (Barde, 1989; Barker and Shooter, 1994; Chao, 2003). Therefore, we examined whether p75NTR modulates osteogenesis through the regulation of osteogenic gene expression in MC3T3-E1 pre-osteoblasts. The MC3T3-E1 cell line is derived from mouse calvaria and has been extensively studied with respect to bone matrix accumulation, mineralization, effects of growth factors, and changes in morphology and metabolism (Raouf and Seth, 2000). Our observations using MC3T3-E1 cells described here link p75NTR to osteoblast differentiation and provide one possible molecular mechanism by which p75NTR regulates osteogenesis.
2. Materials and methods 2.1. Overexpression of p75NTR Plasmids were constructed by standard methods. Mouse p75NTR and deleted-p75NTR were amplified by PCR and cloned into pEGFP-N1 (Clontech/Takara bio, Tokyo, Japan). MC3T3-E1 cells were plated at a density of 5 105 cells/well on a 6-well cell culture dish (Iwaki, Chiba, Japan) and were allowed to grow in complete medium containing a-MEM medium (Gibco BRL, Grand Island, NY, USA) with 10% fetal bovine serum
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(FBS, Japan Bioserum Co., Ltd. Tokyo, Japan) and 1% penicillin– streptomycin (Gibco) for 18 h. Subsequently, the cells were incubated in 2 mL of a-MEM containing 6.25 mL of lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) with 2 mg of each plasmid. Empty pEGFP-N1 was also use for transfection as a control. After transfection, the cells with stable DNA integration were selected by treatment with 2 mg/mL G418 (Sigma, St. Louis, MO, USA). Furthermore, the cells expressing GFP were selected from each plasmid-transfected cultures by fluorescence-activated cell sorting (FACS) analysis using the BD FACS Aria (BD Biosciences, San Jose, CA, USA). 2.2. Cell culture and osteogenic differentiation protocol MC3T3-E1 cells were obtained from the Riken cell bank (Ibaragi, Japan). Cells were grown to confluence in 12-well cell culture dish in complete medium at 37 1C in the presence of 5% CO2. Subsequently, the cells were subcultured for various periods of time in an osteogenic induction medium. Osteogenic induction medium contained a-MEM supplemented with 10% FBS, 50 mg/ mL L-ascorbate phosphate (Sigma), 10 mM b-glycerophosphate (Sigma), and 100 ng/mL human recombinant BMP-2 (R&D Systems, Minneapolis, MN, USA). Cells were maintained with fresh osteogenic induction medium every two days for the indicated period. 2.3. Observation by fluorescent microscopy Cells were trypsinized and collected onto slides with a cytocentrifuge and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature. Nuclei were counterstained with DAPI at 0.2 mg/mL in Vectashield Antifade (Vector Laboratories). All fluorescence images were collected by fluorescence microscopy (Biozero BZ-8000, Keyence, Tokyo, Japan) and the images were analyzed with Keyence software, BZ analyzer. 2.4. Cell counting assay Cells were seeded onto 100 mm culture dishes (1 104 cells/ dish) and cultured for the indicated periods in the complete medium. After cells were cultured, the cells were trypsinized and resuspended in the media, and the number of cells was counted using a hemocytometer. 2.5. Cell cycle analysis Cell cycle analysis was performed by using Click-iTTM EdU Flow Cytometry Assay Kits (Invitrogen/Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions. In brief, cultured cells were treated with 10 mM EdU (5-ethynyl-20 - deoxyuridine) for 1 h. EdU is a nucleoside analog to thymidine and is incorporated into DNA during active DNA synthesis. EdU-incorporated cells were fixed with paraformaldehyde for 15 min, washed once with BSA/PBS buffer and permeabilized with a saponin-based buffer for 30 min. The cells were then washed once, treated with the click-reaction mixture containing Pacific BlueTM azide for 30 min, washed once, and resuspended in PBS buffer. Cells were analyzed using a FACS Aria (BD Biosciences). 2.6. ALP staining Cells were cultured in 12-well cell culture dish under the specified culture conditions before being rinsed twice and fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3) for 15 min. Subsequently, cultures were washed twice with PBS and stained with ALP staining solution, pH 9.5 (NBT/BCIP ready-to-use
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tablets; Roche Diagnostics, Penzberg, Germany). After incubation for 15 min, cells were washed with distilled water, and examined under a light microscope or an EPSON scanner, GT-X800 (Epson, Tokyo, Japan).
Table 2 Primers used for RT-PCR. Genes p75NTR
2.7. Alizarin red-S staining TrkA
Cells were cultured in a 12-well cell culture dish under the specified culture conditions. Thereafter, cultures were fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3) for 30 min, washed with 0.1 M cacodylate buffer (pH 7.3), and stained for 5 min with a saturated solution of Alizarin Red-S (pH 4.0). The wells were washed with sterilized distilled water, then dried and examined under a light microscope or an EPSON scanner, GT-X800.
TrkB TrkC NgR
b-actin
Primers 50 -TTG TGG CCT ATA TTG CTT TCA AGA-30 50 -TCC ACA GAG ATG CCG CTG TC-30 50 -ATG TGA CGT GCT GGG CAG-30 50 -AGA GAC GGT GCT GGC TGC-30 50 -GTT CAC GTG CTC CTG CGA C-30 50 -CTT TCC TTC CTC CAC GGT G-30 50 -GCT GAG CCT TCG GGA ATT G-30 50 -CAC TGA TCT CTG GGA GAT C-30 50 -GAC CCC GAA GAT GAA GAG-30 50 -CAG GAT AGT GAG ATT TCG-30 50 -CTT TCT ACA ATG AGC TGC GTG-30 50 -ATG GCT GGG GTG TTG AAG G-30
Amplicon length 141 bp 153 bp 189 bp 194 bp 268 bp 130 bp
2.8. Ca2 þ release assay Cells were cultured in 12-well cell culture dish under the specified culture conditions. After removal of the culture supernatant, cells were washed with 10 mM Tris–HCl (pH 7.2) before the addition of 1 N HCl solution to each well and incubated until the cells were dry. Then, 20 mL of distilled water was added to each well and the amount of Ca2 þ was determined using the Calcium E-test (Wako, Osaka, Japan) according to the manufacturer’s instructions. 2.9. Real-time reverse transcription-polymerase chain reaction First-stranded cDNA was synthesized from 1 mg of DNase I-treated total RNA in 20 mL of a solution containing 1x firststrand buffer, 50 ng random primer, 10 mM dNTP mixture, 1 mM DTT, and 0.5 units Super Script III RNase H reverse transcriptase (Invitrogen) at 42 1C for 1 h. Subsequently, cDNA was diluted 1/5 in sterile distilled water and a 2 mL aliquot of the diluted cDNA was subjected to real-time RT-PCR using SYBR Green I dye (Takara Bio). Real-time RT-PCR was performed in a 25 mL solution containing 1x PCR buffer, 1.5 mM dNTP mixture, 1x SYBR Green I, 15 mM Mg2 þ solution, 0.25 units Ex Taq R-PCR (Takara Bio), and 20 mM primers (sense and anti-sense). Assays were performed on a Smart Cycler (Cepheid, Sunnyvale, CA, USA) and analyzed with the accompanying Smart Cycler software (Ver. 1.2d). The cycling conditions were, 40 cycles of 95 1C for 3 s and 68 1C for 20 s, and measurements commenced at the end of the 68 1C annealing step. The primers were used for real-time RT-PCR as shown in Table 1. 2.10. Reverse transcription-polymerase chain reaction. First-stranded cDNA was synthesized as described above. Total RNA prepared from mouse brain (Takara Bio) was used as a Table 1 Primers used for real time RT-PCR. Genes
Primers
Amplicon length
p75NTR
50 -TTG TGG CCT ATA TTG CTT TCA AGA-30 50 -TCC ACA GAG ATG CCG CTG TC-30 50 -ACA ACC ACA GAA CCA CAA G-30 50 -TCT CGG TGG CTG GTA GTG A-30 50 -GGA GGT TTC ACT CCA TTC CA-30 50 -TAG AAG GAG CAG GGG ACA GA-30 50 -TGA AAC GGT TTC CAG TCC AG-30 50 -TGG TCT TCA TTC CCC TCA G-30 50 -GAC AAG TCC CAC ACA GCA GC-30 50 -GGA CAT GAA GGC TTT GTC AG-30 50 -CTT TCT ACA ATG AGC TGC GTG-30 50 -ATG GCT GGG GTG TTG AAG G-30
141 bp
Runx2 Osterix BSP OC
b-actin
positive control and 2 mL of synthesized cDNA solution was used for the PCR. PCR was performed on a Smart Cycler and the cycling conditions were, 40 cycles of 95 1C for 5 s and 68 1C for 25 s. Subsequently, the PCR products were separated by electrophoresis through a 2% agarose gel, which was stained with ethidium bromide, and photographed. The primers were used for RT-PCR as shown in Table 2. 2.11. Statistical analysis Results are presented as means7SD of triplicate cultures and statistical differences were assessed using the Student’s t-test. Significant differences (Po0.05) are indicated.
3. Results 3.1. Overexpression of p75NTR-GFP in MC3T3-E1 cells In this study, we established a stably transfected MC3T3-E1 cell line expressing p75NTR fused to GFP (p75GFP-E1), to investigate the effect of p75NTR on osteoblastic differentiation. As a negative control, a non-fused GFP-expressing MC3T3-E1 cell line (GFP-E1) was also generated. The p75NTR mRNA expression level in each cell line was measured and mouse brain RNA was the positive control for p75NTR mRNA. Expression of p75NTR was measured by real-time RT-PCR and was seen in GFP-E1 cells as well as p75GFP-E1 cells. The mRNA expression levels of p75NTR in GFP-E1 were much lower (approximately 1/200 of brain) than either p75GFP-E1 or brain (Fig. 1A), whereas p75GFP-E1 expression of p75NTR was approximately 1.5 fold greater than brain (Fig.1 A). The localization of p75NTR protein in the cell membrane was confirmed by immunofluorescence microscopy in p75GFP-E1 whereas GFP was localized to the cytoplasm in GFP-E1 cells. In contrast, no proteins were detected in the cell membrane of GFPE1 cells by immunofluorescence (Fig. 1B). Essentially no morphologic difference was observed between p75GFP-E1 and GFP-E1 cells in the growth phase by phase contrast microscopy (Fig. 1C).
106 bp 103 bp 139 bp 150 bp 130 bp
3.2. Cell proliferation and cell cycle progression To examine the effect of p75NTR on osteoblastic cell proliferation, p75GFP-E1 and GFP-E1 cells were cultured in complete medium and cell numbers were measured. Both p75GFP-E1 and GFP-E1 rapidly proliferated from day 4 to day 6 but the rate of proliferation of p75GFP-E1 was higher than GFP-E1 on both days 4 and 6. The cell cultures were almost confluent on day 6 (Fig. 2A).
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Fig. 1. Overexpression of p75NTR -GFP in MC3T3-E1 cells. (A) p75NTR/ p75NTR-GFP mRNA expression. Cells were cultured with standard medium before confluence, and mRNA expression levels was measured by real-time RT-PCR (upper panel) and RT-PCR (lower panel). b-actin was used as an internal control. *Significantly different from GFP-E1. Mean7 S.D. (n¼ 3, Po 0.05). (B) Cell morphology. p75GFPE1 and GFP-E1 cells were observed by phase-contrast microscopy. (C) Localization of p75NTR-GFP. Typical images for fluorescence are shown. GFP signals appear green. DNA was counterstained with DAPI (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 3. Effect of p75NTR overexpression on ALP activity and mineralization in MC3T3-E1 cells. (A) ALP staining. Cells were cultured with standard medium before confluence (pre-culture). After confluence, cells were cultured with osteogenic medium for the indicated number of days before ALP staining was performed. The stained cultures on day 3 were observed by phase-contrast microscopy (right panel). (B) Alizarin Red-S staining. Cells were cultured under the same conditions as indicated in (A). After culture, Alizarin Red-S staining was performed. The stained cultures on day 14 were observed by phase-contrast microscopy (right panel). (C) Quantification of calcium deposition. Cells were cultured under the same conditions as described for (B), and the calcium content of the cell layers was then measured using the o-cresolphthalein complexone method. Data are the mean 7 S.D. (n¼ 3). *P o 0.05 compared with GFP-E1 at each time point.
3.3. Formation of the mineralized nodules and expression of osteogenic marker genes
Fig. 2. Effect of p75NTR overexpression on proliferation and cell cycle progression in MC3T3-E1 cells. (A) Cell proliferation. Cells were cultured in the growth media for indicated number of days, and then cells were counted. Mean 7S.D. (n¼3, P o0.05); * Significantly different from GFP-E1 at each time point. (B) Cell cycle progression. Cells in logarithmic growth phase were treated with EdU for 1 h. EdUincorporated cells were labeled by Pacific BlueTM azide and then they were detected by FACS analysis using 407 nm excitation with a 450/50 nm bandpass.
An analysis of cell cycle progression revealed that overexpression of p75NTR led to an increase in the number of the cells entering S-phase.
To analyze the osteogenic differentiation of p75GFP-E1 cells, the cells were cultured in osteogenic medium and ALP staining and Alizarin Red-S staining were conducted. After three days of induction, p75GFP-E1 cells showed more intense ALP staining than GFP-E1 cells (Fig. 3A). Mineralized matrix aggregates were observed in p75GFP-E1 cells after 14 day of culture and the time course of mineralization revealed that p75NTR stimulated mineralization as identified by Alizarin Red-S staining (Fig. 3B). A Ca2 þ release assay was used to determine the mineralization volume in p75GFP-E1and GFP-E1 cultures. Ca2 þ release from the mineralized nodules was increased in p75GFP-E1 cultures compared to GFP-E1 cultures after 14 days of osteogenic induction (Fig. 3C). To establish if osteoblast marker gene expression was affected by p75NTR overexpression during osteoblastic differentiation, p75GFP-E1 and GFP-E1 cells were cultured with osteogenic medium for the indicated number of days and then mRNA were analyzed by real-time RT-PCR. No difference was observed in mRNA expression levels of Rux2, OSX, BSP, and OC in between p75GFP-E1 and GFP-E1 in pre-induced cells or at the early phase of induction (day 3). At the late phase (day 14), the mRNA of OSX,
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Fig. 5. mRNA expression of trks and NgR in MC3T3-E1 cells. Cells were cultured with standard medium before confluence (pre-culture). After confluence, cells were cultured in osteogenic medium for 24 day before mRNA was extracted. RTPCR was used to analyze mRNA expression of trkA, trkB, trkC, and NgR. b-actin mRNA expression was measured as an internal control. Brain was analyzed as positive control.
Fig. 4. Effect of p75NTR overexpression on osteogenic markers in MC3T3-E1 cells. Cells were cultured with standard medium before confluence (pre-culture). After confluence, cells were cultured in osteogenic medium the indicated number of days before mRNA was extracted. Real-time RT-PCR was used to analyze mRNA expression of indicated osteogenic markers. Data are the mean 7 S.D. (n¼3). *Po 0.05 compared with GFP-E1 at each time point.
BSP, and OC were increased by p75NTR. The expression of Runx2 was same between p75GFP-E1 and GFP-E1 (Fig. 4). 3.4. Signaling pathway in the p75NTR-induced osteoblast differentiation It is not clear which signaling cascade is responsible for the p75NTR -mediated induction of osteogenic marker expression. p75NTR acts via two different signaling pathways by forming heterodimers with members of the trk family of receptors or with NgR (Fig. 7). When p75NTR formed the heterodimerizes with trks, then tyrosine kinase activity was stimulated but when p75NTR formed the heterodimerizes with NgR, GTP kinase activity was stimulated via RhoA. To investigate the signaling cascade used by p75NTR in osteogenesis, we initially assayed the expression of trks and NgR in p75GFP-E1 and GFP-E1 cells by RT-PCR. Expression of trkA, trkB, and trkC mRNAs was detectable in both p75GFP-E1 and GFP-E1 cells although their expression levels were lower than those of the brain positive control (Fig. 5). No difference was observed in the mRNA expression of trks between p75GFP-E1 and GFP-E1 cells (Fig. 5). NgR mRNA expression was detected in brain only but not in either p75GFP-E1 or GFP-E1 cells (Fig. 5). To further elucidate whether RhoA-GTP kinase signaling was involved in the p75NTR -induced osteoblast differentiation, we generated a cell line stably expressing a p75NTR derivative (p75Del.GFP-E1) in which the GDI binding domain was deleted in p75NTR (Fig.6A). p75GFP-E1, p75Del.GFP-E1, and GFP-E1 were cultured with osteogenic medium for 14 days, and Alizarin Red-S staining for mineralization was performed. Alizarin Red-Spositive mineralization was increased in p75GFP-E1 and p75Del.GFP-E1 compared to that in GFP-E1 culture (Fig. 6B). To identify the signaling pathway involved in the enhancement of osteogenic differentiation, we treated p75GFP-E1 and GFP-E1 cells with K252a, an inhibitor for trk tyrosine kinase. p75GFP-E1 and GFP-E1 cells were cultured with osteogenic medium in the presence or absence of K252a for 14 days. Alizarin
Fig. 6. Effect of GDI binding domain deletion and tyrosine kinase inhibitor on p75NTR-induced mineralization. (A) Map of the deletion of the GDI binding domain in p75NTR-GFP. (B) Effect of GDI binding domain deletion on the mineralization. Cells were cultured with standard medium before confluence. After confluence, cells were cultured with osteogenic medium for 14 days before Alizarin Red-S staining was performed. The stained cultures were observed by phase-contrast microscopy (lower panel). (C) Effect of a tyrosine kinase inhibitor, K252a, on the mineralization. Cells were cultured with standard medium before confluence. After confluence, cells were cultured with osteogenic medium with or without K252a for 14 day before Alizarin Red-S staining was performed. The stained cultures were observed by phase-contrast microscopy (lower panel).
Red-S staining revealed that p75NTR -mediated induction of osteogenic differentiation was strongly inhibited by K252a (Fig. 6C).
4. Discussion In this study, we demonstrated that p75NTR plays a critical role in pre-osteoblast differentiation. p75NTR is a key marker for the isolation and characterization of bone marrow-derived MSCs (Jones et al., 2002) and of adipose tissue-derived MSCs
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(Yamamoto et al., 2007). A recent study has shown that the mineralization capacity of p75NTR-expressing cells derived from the periosteum was higher than similar periosteum-derived cells without p75NTR expression (Alexander et al., 2009). Therefore, it is possible that p75NTR may have a functional role in osteogenic cells, which has not yet been elucidated. We confirmed that p75NTR mRNA is expressed in the osteogenic MC3T3E-1 cell line and that p75NTR protein was localized to the cell surface membrane, but at generally low or undetectable levels. Stable transfection of cells to generated gene overexpression has been widely used to investigate the role of gene products in gain-of-function experiments. To examine whether p75NTR might directly regulate pre-osteoblast proliferation and differentiation, we transfected an expression construct of p75NTR into MC3T3-E1 cells to obtain cells stably expressing p75NTR. Flow cytometric analysis revealed that the purified p75NTR -positive cell population could be isolated from the transfected MC3T3-E1 cells (p75GFP-E1). Overexpression of p75NTR significantly increased expression of p75NTR protein on the cell membrane of p75GFP-E1. We determined that p75GFP-E1 has strongly enhanced cell proliferation at the indicated time during in vitro cultivation and the rate of S-phase cell population in p75GFP-E1 cells increased in comparison with that of GFP-E1. p75NTR stimulates increased proliferation of hepatic stellate cells during the proliferative stage (Asai et al., 2006). Furthermore, there is evidence that p75NTR is involved in controlling the fate of keratinocyte stem cells (Botchkarev et al., 2000) and that it is a marker for esophageal keratinocyte stem cells in vitro (Okumura et al., 2003). Together these data suggest the p75NTR is involved in the cell proliferation associated with cell cycle progression in pre-osteoblasts. Osteoblast differentiation occurs through a multistep molecular pathway regulated by different signal transduction pathways, but the full spectrum of events has not been elucidated. Therefore, to determine whether p75NTR overexpression in p75GFP-E1 cells affected osteoblast differentiation, we assayed tissue nonspecific ALPase, which serves as an early marker of calcification. ALPase activity in p75GFP-E1 strongly increased in comparison with that of GFP-E1 consistent with a functional role for p75NTR in osteogenesis. Moreover, a quantitative investigation of the osteoblast marker gene expression in vitro showed that osteoblastic maturation is strongly promoted as assessed by expression of Osterix, BSP and the mature osteoblast marker, OCN (Power and Fottrell, 1991). In addition, p75GFP-E1 cells showed enhanced osseous nodule formation. The expression of p75 mRNA was maintained during the osteogenic differentiation process as shown in a supplemental figure. These results indicate that osteoblast differentiation was induced as a result of p75NTR overexpression. Osteoblasts differentiation is controlled predominantly through the transcription factors, Runx2 and Osterix (Ducy et al., 1997; Komori, 2006; Komori et al., 1997; Nakashima et al., 2002). In this study, expression of Osterix, BSP and OCN was significantly increased by p75NTR overexpression but the expression of Runx2 was not altered. These results are consistent with a previous report demonstrating that Runx2 is an upstream regulator of Osterix (Nakashima et al., 2002), which in turn regulates BSP and OCN (Igarashi et al., 2004; Nakashima et al., 2002). Our findings are the first to show that p75NTR is an important regulator of both cell proliferation and osteogenic differentiation in pre-osteoblasts. This is consistent withprior studies indicating the importance of the p75NTR in osteogenesis (Alexander et al., 2009). How does the p75NTR participate in osteoblast differentiation? Numerous adaptor proteins that bind to p75NTR have been reported. The findings from the current study are consistent with the model illustrated in Fig. 7, which shows the sites and
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Fig. 7. Two different signaling pathways containing p75NTR.
mechanisms of action of p75NTR used in the present study. p75NTR contributes to several signaling pathways and has different biological functions (Barker, 2004; Chao, 2003; Teng and Hempstead, 2004; Zampieri and Chao, 2006). In the absence of neurotrophins, p75NTR or the intracellular domain (ICD) of p75NTR alone can induce apoptosis (Majdan et al., 1997; Rabizadeh et al., 1993) and activation of phosphatidylinositol 3-kinase (Roux et al., 2001) and Rho (Yamashita et al., 1999). Even though we have identified a role for p75NTR in osteoblast differentiation, questions remains concerning the identity of its binding partner during osteogenesis. The association of p75NTR and trk receptors and signal transduction is well documented in the nervous system. In non-neuronal tissue, trkA has been shown to preferentially localize to limbal basal epithelial cells and was proposed as a potential marker for corneal limbal stem cells. (Lambiase et al., 1998; Stepp and Zieske, 2005; Touhami et al., 2002). Human keratinocytes express trkA and p75NTR and the specific binding by these receptors modulate either proliferation or differentiation (Di Marco et al., 1993). We confirmed the expression of three trk receptors (trkA, trkB, trkC) in MC3T3E-1 cells, which is consistent with previous observations (Mogi et al., 2000; Nakanishi et al., 1994). The neurotrophin/trk receptor autocrine signaling pathway is required for the survival of proliferating for human osteoblasts (Roux and Barker, 2002). To identify the binding partner of p75NTR in p75GFP-E1 during osteoblast differentiation, we conducted a pharmacological study using K252a, an inhibitor of trk tyrosine kinase. The inhibitory effect of K252a has been confirmed by the suppression of NGF induced neurite formation in PC12 cells (Pinski et al., 2002). The osteoblastic differentiation of p75GFPE1 cells was inhibited by treatment with K252a as determined by inhibition of ALPase activity. The trkA inhibitor K252a functions as a JNK inhibitor and stimulates cell survival by activating ERK1/ 2 and Akt (Koizumi et al., 1988). JNK is usually associated with p75NTR activation and the induction of cell death (Kaplan and Miller, 2000; Roux and Barker, 2002; Roux et al., 2002). A link between JNKs and trk receptors has not been shown before for osteoblast differentiation. On the other hand, p75NTR acts in combination with other receptors, such as the Nogo receptor, NgR (Dechant, 2001; Lee et al., 2001; Mischel et al., 2001). The activation of the NgR- p75NTR complex of the neurotrophin receptor leads to activation of RhoA and the RhoA kinase pathway, which results in growth cone collapse (Al Halabiah et al., 2005; Shao et al., 2005; Wong et al., 2002). Conversely, the expression of NgR was not observed in MC3T3-E1 cells. In addition, we have demonstrated that the GDI-domain in p75NTR, which is the binding motif for RhoA, is not involved in the osteoblast differentiation. Taken together, our findings suggest that the binding partner of p75NTR are the trk receptors and that p75NTR signaling is associated with trk tyrosine kinase receptor promotion of pre-osteoblast proliferation and differentiation.
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Acknowledgments This work was supported in part by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology [Kakenhi (822791778) to YM; (21390528 and 20659305) to MH], and Dental Research Center and Sato Fund, Nihon University School of Dentistry.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.diff. 2012.07.001.
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Differentiation journal homepage: www.elsevier.com/locate/diff
The p75 neurotrophin receptor regulates MC3T3-E1 osteoblastic differentiation Yoshikazu Mikami a,b, Shinnosuke Suzuki a, Yumiko Ishii c, Nobukazu Watanabe d, Tomihisa Takahashi a,b, Keitaro Isokawa a,b, Masaki J. Honda a,b,n a
Department of Anatomy, Nihon University School of Dentistry, Tokyo 101-8310, Japan Dental Research Center, Nihon University School of Dentistry, Tokyo 101-8310, Japan c FACS Core Laboratory, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan d Laboratory of Diagnostic Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 February 2012 Received in revised form 22 May 2012 Accepted 1 July 2012 Available online 17 August 2012
While the role of p75NTR signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75NTR on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75NTR-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75NTR in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75NTR, as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75NTR-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75NTR signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation. & 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.
Keywords: MC3T3-E1 Cells Osteoblast Osteogenic differentiation p75NTR Trk
1. Introduction Bone is a dynamic organ in which old and damaged bone is replaced by newly formed bone as a part of the process of remodeling that occurs throughout the life of an individual. Remodeling is carried out by both osteoblasts for bone formation and osteoclasts for bone resorption. Osteoblasts originate from mesenchymal stem cells (MSCs) and a subset of mature osteoblasts is embedded in the bone to become osteocytes. The process of osteocyte differentiation from MSC is complex and proceeds through multiple steps that involve both cell proliferation and differentiation. Many different signal transduction pathways govern the tightly-regulated expression of osteoblast-specific n Corresponding author at: Nihon University School of Dentistry Department of Anatomy 1-8-13 Kanda-Surugadai, Tokyo 101-8310, Japan. Tel.: þ81 3 3219 8121; fax: þ 81 3 3219 8319. E-mail addresses: [email protected] (Y. Mikami), [email protected] (S. Suzuki), [email protected] (Y. Ishii), [email protected] (N. Watanabe), [email protected] (T. Takahashi), [email protected] (K. Isokawa), [email protected] (M.J. Honda).
genes during the process of osteoblast differentiation. Runx2, Dlx5, Msx2, and Osterix are the major transcriptional factors that regulate osteoblast differentiation (Fu et al., 2007; Huang et al., 2007; Komori, 2002, 2006). In spite of the critical role of osteoblasts in bone formation, our understanding of the osteoblast differentiation program remains incomplete. MSCs are first identified in the bone marrow (BM) (Friedenstein et al., 1974), and are now recognized to be sustained throughout life and serve as a source of cell replacement within certain tissues in response to trauma and turnover (Jackson et al., 2007). A single MSC has the potential to differentiate into multiple lineages such as osteoblasts, chondrocytes, and adipocytes (Pittenger et al., 1999). More recently, MSCs have been evaluated for their potential therapeutic use in cell transplantation to repair musculoskeletal, neuronal, and cardiac tissues and to suppress immune responses in graft-versus-host diseases (Barry and Murphy, 2004; Le Blanc et al., 2004; Pittenger and Martin, 2004). The identification of suitable MSC molecular markers, such as surface antigens, is crucial for the manipulation of cells in vitro as well as to understand the mechanisms of stem cell regulation. One such marker is p75 neurotrophin receptor (p75NTR), which is a selective marker for the isolation and
0301-4681/$ - see front matter & 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved. Join the International Society for Differentiation (www.isdifferentiation.org) http://dx.doi.org/10.1016/j.diff.2012.07.001
Y. Mikami et al. / Differentiation 84 (2012) 392–399
phenotypic characterization of bone marrow-derived MSCs (Jones et al., 2002; Poloni et al., 2009; Quirici et al., 2002). p75NTR is a 75 kDa transmembrane protein, that is referred to as nerve growth factor receptor (NGFR), tumor necrosis factor receptor superfamily member 16 (TNFRSF16) or CD271 (Mischel et al., 2001). It modulates the expression of genes that are critically involved in the regulation of differentiation as well as cell adhesion, signal transduction, apoptosis, tumor cell invasion, and metastasis (Nalbandian et al., 2005). In spite of the low affinity binding of the precursors of neurotrophic factors to p75NTR, it is required for efficient trk receptor tyrosine kinase (trkA, trkB, trkC) activation and signaling (Kaplan and Miller, 2000; Mischel et al., 2001; Patapoutian and Reichardt, 2001). p75NTR functions as a ligand-stimulated apoptosis-inducing receptor that stimulates the activity of the JNK-p53-Bax pathway and suppresses Ras/PI-3k/Akt activation (Kaplan and Miller, 2000; Patapoutian and Reichardt, 2001). In contrast, trk receptors promote cell survival via several intracellular signaling cascades e.g. the Ras/PI-3k/Akt pathway (Chao, 2003; Kaplan and Miller, 2000; Patapoutian and Reichardt, 2001; Serafeim and Gordon, 2001). p75NTR has been used as a marker for the isolation of esophageal epithelial stem cells (Okumura et al., 2003), adipose tissue-derived MSCs (Yamamoto et al., 2007), and MSCs in the growth zones of regenerating fallow deer antlers and from the pedicle periosteum (Rolf et al., 2008). On the other hand, recently, there were reports that a role of CD271 may be influence to the cell differentiation. For instance, previous investigation demonstrated that when MSCs differentiated into a Schwann Cell lineage using glial growth factor, in the differentiation process, level of p75 protein was significantly elevated upon differentiation though p75 protein was not expressed in MSCs (Caddick et al., 2006). Furthermore, in the previous work, higher protein levels of CD271 were detected in mineralizing jaw periosteum-derived cells compared to nonmineralizing jaw periosteum-derived cells within the first five days of osteogenic differentiation process. (Alexander et al., 2009). Based on these previous results, we hypothesized that p75NTR might play an important role in MSCs, in particular, in the osteogenic lineage. In spite of its importance in neuronal development and regeneration, a specific functional role for p75NTR in osteogenic differentiation has not been demonstrated and no signaling cascade has been identified (Barde, 1989; Barker and Shooter, 1994; Chao, 2003). Therefore, we examined whether p75NTR modulates osteogenesis through the regulation of osteogenic gene expression in MC3T3-E1 pre-osteoblasts. The MC3T3-E1 cell line is derived from mouse calvaria and has been extensively studied with respect to bone matrix accumulation, mineralization, effects of growth factors, and changes in morphology and metabolism (Raouf and Seth, 2000). Our observations using MC3T3-E1 cells described here link p75NTR to osteoblast differentiation and provide one possible molecular mechanism by which p75NTR regulates osteogenesis.
2. Materials and methods 2.1. Overexpression of p75NTR Plasmids were constructed by standard methods. Mouse p75NTR and deleted-p75NTR were amplified by PCR and cloned into pEGFP-N1 (Clontech/Takara bio, Tokyo, Japan). MC3T3-E1 cells were plated at a density of 5 105 cells/well on a 6-well cell culture dish (Iwaki, Chiba, Japan) and were allowed to grow in complete medium containing a-MEM medium (Gibco BRL, Grand Island, NY, USA) with 10% fetal bovine serum
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(FBS, Japan Bioserum Co., Ltd. Tokyo, Japan) and 1% penicillin– streptomycin (Gibco) for 18 h. Subsequently, the cells were incubated in 2 mL of a-MEM containing 6.25 mL of lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) with 2 mg of each plasmid. Empty pEGFP-N1 was also use for transfection as a control. After transfection, the cells with stable DNA integration were selected by treatment with 2 mg/mL G418 (Sigma, St. Louis, MO, USA). Furthermore, the cells expressing GFP were selected from each plasmid-transfected cultures by fluorescence-activated cell sorting (FACS) analysis using the BD FACS Aria (BD Biosciences, San Jose, CA, USA). 2.2. Cell culture and osteogenic differentiation protocol MC3T3-E1 cells were obtained from the Riken cell bank (Ibaragi, Japan). Cells were grown to confluence in 12-well cell culture dish in complete medium at 37 1C in the presence of 5% CO2. Subsequently, the cells were subcultured for various periods of time in an osteogenic induction medium. Osteogenic induction medium contained a-MEM supplemented with 10% FBS, 50 mg/ mL L-ascorbate phosphate (Sigma), 10 mM b-glycerophosphate (Sigma), and 100 ng/mL human recombinant BMP-2 (R&D Systems, Minneapolis, MN, USA). Cells were maintained with fresh osteogenic induction medium every two days for the indicated period. 2.3. Observation by fluorescent microscopy Cells were trypsinized and collected onto slides with a cytocentrifuge and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature. Nuclei were counterstained with DAPI at 0.2 mg/mL in Vectashield Antifade (Vector Laboratories). All fluorescence images were collected by fluorescence microscopy (Biozero BZ-8000, Keyence, Tokyo, Japan) and the images were analyzed with Keyence software, BZ analyzer. 2.4. Cell counting assay Cells were seeded onto 100 mm culture dishes (1 104 cells/ dish) and cultured for the indicated periods in the complete medium. After cells were cultured, the cells were trypsinized and resuspended in the media, and the number of cells was counted using a hemocytometer. 2.5. Cell cycle analysis Cell cycle analysis was performed by using Click-iTTM EdU Flow Cytometry Assay Kits (Invitrogen/Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions. In brief, cultured cells were treated with 10 mM EdU (5-ethynyl-20 - deoxyuridine) for 1 h. EdU is a nucleoside analog to thymidine and is incorporated into DNA during active DNA synthesis. EdU-incorporated cells were fixed with paraformaldehyde for 15 min, washed once with BSA/PBS buffer and permeabilized with a saponin-based buffer for 30 min. The cells were then washed once, treated with the click-reaction mixture containing Pacific BlueTM azide for 30 min, washed once, and resuspended in PBS buffer. Cells were analyzed using a FACS Aria (BD Biosciences). 2.6. ALP staining Cells were cultured in 12-well cell culture dish under the specified culture conditions before being rinsed twice and fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3) for 15 min. Subsequently, cultures were washed twice with PBS and stained with ALP staining solution, pH 9.5 (NBT/BCIP ready-to-use
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tablets; Roche Diagnostics, Penzberg, Germany). After incubation for 15 min, cells were washed with distilled water, and examined under a light microscope or an EPSON scanner, GT-X800 (Epson, Tokyo, Japan).
Table 2 Primers used for RT-PCR. Genes p75NTR
2.7. Alizarin red-S staining TrkA
Cells were cultured in a 12-well cell culture dish under the specified culture conditions. Thereafter, cultures were fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3) for 30 min, washed with 0.1 M cacodylate buffer (pH 7.3), and stained for 5 min with a saturated solution of Alizarin Red-S (pH 4.0). The wells were washed with sterilized distilled water, then dried and examined under a light microscope or an EPSON scanner, GT-X800.
TrkB TrkC NgR
b-actin
Primers 50 -TTG TGG CCT ATA TTG CTT TCA AGA-30 50 -TCC ACA GAG ATG CCG CTG TC-30 50 -ATG TGA CGT GCT GGG CAG-30 50 -AGA GAC GGT GCT GGC TGC-30 50 -GTT CAC GTG CTC CTG CGA C-30 50 -CTT TCC TTC CTC CAC GGT G-30 50 -GCT GAG CCT TCG GGA ATT G-30 50 -CAC TGA TCT CTG GGA GAT C-30 50 -GAC CCC GAA GAT GAA GAG-30 50 -CAG GAT AGT GAG ATT TCG-30 50 -CTT TCT ACA ATG AGC TGC GTG-30 50 -ATG GCT GGG GTG TTG AAG G-30
Amplicon length 141 bp 153 bp 189 bp 194 bp 268 bp 130 bp
2.8. Ca2 þ release assay Cells were cultured in 12-well cell culture dish under the specified culture conditions. After removal of the culture supernatant, cells were washed with 10 mM Tris–HCl (pH 7.2) before the addition of 1 N HCl solution to each well and incubated until the cells were dry. Then, 20 mL of distilled water was added to each well and the amount of Ca2 þ was determined using the Calcium E-test (Wako, Osaka, Japan) according to the manufacturer’s instructions. 2.9. Real-time reverse transcription-polymerase chain reaction First-stranded cDNA was synthesized from 1 mg of DNase I-treated total RNA in 20 mL of a solution containing 1x firststrand buffer, 50 ng random primer, 10 mM dNTP mixture, 1 mM DTT, and 0.5 units Super Script III RNase H reverse transcriptase (Invitrogen) at 42 1C for 1 h. Subsequently, cDNA was diluted 1/5 in sterile distilled water and a 2 mL aliquot of the diluted cDNA was subjected to real-time RT-PCR using SYBR Green I dye (Takara Bio). Real-time RT-PCR was performed in a 25 mL solution containing 1x PCR buffer, 1.5 mM dNTP mixture, 1x SYBR Green I, 15 mM Mg2 þ solution, 0.25 units Ex Taq R-PCR (Takara Bio), and 20 mM primers (sense and anti-sense). Assays were performed on a Smart Cycler (Cepheid, Sunnyvale, CA, USA) and analyzed with the accompanying Smart Cycler software (Ver. 1.2d). The cycling conditions were, 40 cycles of 95 1C for 3 s and 68 1C for 20 s, and measurements commenced at the end of the 68 1C annealing step. The primers were used for real-time RT-PCR as shown in Table 1. 2.10. Reverse transcription-polymerase chain reaction. First-stranded cDNA was synthesized as described above. Total RNA prepared from mouse brain (Takara Bio) was used as a Table 1 Primers used for real time RT-PCR. Genes
Primers
Amplicon length
p75NTR
50 -TTG TGG CCT ATA TTG CTT TCA AGA-30 50 -TCC ACA GAG ATG CCG CTG TC-30 50 -ACA ACC ACA GAA CCA CAA G-30 50 -TCT CGG TGG CTG GTA GTG A-30 50 -GGA GGT TTC ACT CCA TTC CA-30 50 -TAG AAG GAG CAG GGG ACA GA-30 50 -TGA AAC GGT TTC CAG TCC AG-30 50 -TGG TCT TCA TTC CCC TCA G-30 50 -GAC AAG TCC CAC ACA GCA GC-30 50 -GGA CAT GAA GGC TTT GTC AG-30 50 -CTT TCT ACA ATG AGC TGC GTG-30 50 -ATG GCT GGG GTG TTG AAG G-30
141 bp
Runx2 Osterix BSP OC
b-actin
positive control and 2 mL of synthesized cDNA solution was used for the PCR. PCR was performed on a Smart Cycler and the cycling conditions were, 40 cycles of 95 1C for 5 s and 68 1C for 25 s. Subsequently, the PCR products were separated by electrophoresis through a 2% agarose gel, which was stained with ethidium bromide, and photographed. The primers were used for RT-PCR as shown in Table 2. 2.11. Statistical analysis Results are presented as means7SD of triplicate cultures and statistical differences were assessed using the Student’s t-test. Significant differences (Po0.05) are indicated.
3. Results 3.1. Overexpression of p75NTR-GFP in MC3T3-E1 cells In this study, we established a stably transfected MC3T3-E1 cell line expressing p75NTR fused to GFP (p75GFP-E1), to investigate the effect of p75NTR on osteoblastic differentiation. As a negative control, a non-fused GFP-expressing MC3T3-E1 cell line (GFP-E1) was also generated. The p75NTR mRNA expression level in each cell line was measured and mouse brain RNA was the positive control for p75NTR mRNA. Expression of p75NTR was measured by real-time RT-PCR and was seen in GFP-E1 cells as well as p75GFP-E1 cells. The mRNA expression levels of p75NTR in GFP-E1 were much lower (approximately 1/200 of brain) than either p75GFP-E1 or brain (Fig. 1A), whereas p75GFP-E1 expression of p75NTR was approximately 1.5 fold greater than brain (Fig.1 A). The localization of p75NTR protein in the cell membrane was confirmed by immunofluorescence microscopy in p75GFP-E1 whereas GFP was localized to the cytoplasm in GFP-E1 cells. In contrast, no proteins were detected in the cell membrane of GFPE1 cells by immunofluorescence (Fig. 1B). Essentially no morphologic difference was observed between p75GFP-E1 and GFP-E1 cells in the growth phase by phase contrast microscopy (Fig. 1C).
106 bp 103 bp 139 bp 150 bp 130 bp
3.2. Cell proliferation and cell cycle progression To examine the effect of p75NTR on osteoblastic cell proliferation, p75GFP-E1 and GFP-E1 cells were cultured in complete medium and cell numbers were measured. Both p75GFP-E1 and GFP-E1 rapidly proliferated from day 4 to day 6 but the rate of proliferation of p75GFP-E1 was higher than GFP-E1 on both days 4 and 6. The cell cultures were almost confluent on day 6 (Fig. 2A).
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Fig. 1. Overexpression of p75NTR -GFP in MC3T3-E1 cells. (A) p75NTR/ p75NTR-GFP mRNA expression. Cells were cultured with standard medium before confluence, and mRNA expression levels was measured by real-time RT-PCR (upper panel) and RT-PCR (lower panel). b-actin was used as an internal control. *Significantly different from GFP-E1. Mean7 S.D. (n¼ 3, Po 0.05). (B) Cell morphology. p75GFPE1 and GFP-E1 cells were observed by phase-contrast microscopy. (C) Localization of p75NTR-GFP. Typical images for fluorescence are shown. GFP signals appear green. DNA was counterstained with DAPI (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 3. Effect of p75NTR overexpression on ALP activity and mineralization in MC3T3-E1 cells. (A) ALP staining. Cells were cultured with standard medium before confluence (pre-culture). After confluence, cells were cultured with osteogenic medium for the indicated number of days before ALP staining was performed. The stained cultures on day 3 were observed by phase-contrast microscopy (right panel). (B) Alizarin Red-S staining. Cells were cultured under the same conditions as indicated in (A). After culture, Alizarin Red-S staining was performed. The stained cultures on day 14 were observed by phase-contrast microscopy (right panel). (C) Quantification of calcium deposition. Cells were cultured under the same conditions as described for (B), and the calcium content of the cell layers was then measured using the o-cresolphthalein complexone method. Data are the mean 7 S.D. (n¼ 3). *P o 0.05 compared with GFP-E1 at each time point.
3.3. Formation of the mineralized nodules and expression of osteogenic marker genes
Fig. 2. Effect of p75NTR overexpression on proliferation and cell cycle progression in MC3T3-E1 cells. (A) Cell proliferation. Cells were cultured in the growth media for indicated number of days, and then cells were counted. Mean 7S.D. (n¼3, P o0.05); * Significantly different from GFP-E1 at each time point. (B) Cell cycle progression. Cells in logarithmic growth phase were treated with EdU for 1 h. EdUincorporated cells were labeled by Pacific BlueTM azide and then they were detected by FACS analysis using 407 nm excitation with a 450/50 nm bandpass.
An analysis of cell cycle progression revealed that overexpression of p75NTR led to an increase in the number of the cells entering S-phase.
To analyze the osteogenic differentiation of p75GFP-E1 cells, the cells were cultured in osteogenic medium and ALP staining and Alizarin Red-S staining were conducted. After three days of induction, p75GFP-E1 cells showed more intense ALP staining than GFP-E1 cells (Fig. 3A). Mineralized matrix aggregates were observed in p75GFP-E1 cells after 14 day of culture and the time course of mineralization revealed that p75NTR stimulated mineralization as identified by Alizarin Red-S staining (Fig. 3B). A Ca2 þ release assay was used to determine the mineralization volume in p75GFP-E1and GFP-E1 cultures. Ca2 þ release from the mineralized nodules was increased in p75GFP-E1 cultures compared to GFP-E1 cultures after 14 days of osteogenic induction (Fig. 3C). To establish if osteoblast marker gene expression was affected by p75NTR overexpression during osteoblastic differentiation, p75GFP-E1 and GFP-E1 cells were cultured with osteogenic medium for the indicated number of days and then mRNA were analyzed by real-time RT-PCR. No difference was observed in mRNA expression levels of Rux2, OSX, BSP, and OC in between p75GFP-E1 and GFP-E1 in pre-induced cells or at the early phase of induction (day 3). At the late phase (day 14), the mRNA of OSX,
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Fig. 5. mRNA expression of trks and NgR in MC3T3-E1 cells. Cells were cultured with standard medium before confluence (pre-culture). After confluence, cells were cultured in osteogenic medium for 24 day before mRNA was extracted. RTPCR was used to analyze mRNA expression of trkA, trkB, trkC, and NgR. b-actin mRNA expression was measured as an internal control. Brain was analyzed as positive control.
Fig. 4. Effect of p75NTR overexpression on osteogenic markers in MC3T3-E1 cells. Cells were cultured with standard medium before confluence (pre-culture). After confluence, cells were cultured in osteogenic medium the indicated number of days before mRNA was extracted. Real-time RT-PCR was used to analyze mRNA expression of indicated osteogenic markers. Data are the mean 7 S.D. (n¼3). *Po 0.05 compared with GFP-E1 at each time point.
BSP, and OC were increased by p75NTR. The expression of Runx2 was same between p75GFP-E1 and GFP-E1 (Fig. 4). 3.4. Signaling pathway in the p75NTR-induced osteoblast differentiation It is not clear which signaling cascade is responsible for the p75NTR -mediated induction of osteogenic marker expression. p75NTR acts via two different signaling pathways by forming heterodimers with members of the trk family of receptors or with NgR (Fig. 7). When p75NTR formed the heterodimerizes with trks, then tyrosine kinase activity was stimulated but when p75NTR formed the heterodimerizes with NgR, GTP kinase activity was stimulated via RhoA. To investigate the signaling cascade used by p75NTR in osteogenesis, we initially assayed the expression of trks and NgR in p75GFP-E1 and GFP-E1 cells by RT-PCR. Expression of trkA, trkB, and trkC mRNAs was detectable in both p75GFP-E1 and GFP-E1 cells although their expression levels were lower than those of the brain positive control (Fig. 5). No difference was observed in the mRNA expression of trks between p75GFP-E1 and GFP-E1 cells (Fig. 5). NgR mRNA expression was detected in brain only but not in either p75GFP-E1 or GFP-E1 cells (Fig. 5). To further elucidate whether RhoA-GTP kinase signaling was involved in the p75NTR -induced osteoblast differentiation, we generated a cell line stably expressing a p75NTR derivative (p75Del.GFP-E1) in which the GDI binding domain was deleted in p75NTR (Fig.6A). p75GFP-E1, p75Del.GFP-E1, and GFP-E1 were cultured with osteogenic medium for 14 days, and Alizarin Red-S staining for mineralization was performed. Alizarin Red-Spositive mineralization was increased in p75GFP-E1 and p75Del.GFP-E1 compared to that in GFP-E1 culture (Fig. 6B). To identify the signaling pathway involved in the enhancement of osteogenic differentiation, we treated p75GFP-E1 and GFP-E1 cells with K252a, an inhibitor for trk tyrosine kinase. p75GFP-E1 and GFP-E1 cells were cultured with osteogenic medium in the presence or absence of K252a for 14 days. Alizarin
Fig. 6. Effect of GDI binding domain deletion and tyrosine kinase inhibitor on p75NTR-induced mineralization. (A) Map of the deletion of the GDI binding domain in p75NTR-GFP. (B) Effect of GDI binding domain deletion on the mineralization. Cells were cultured with standard medium before confluence. After confluence, cells were cultured with osteogenic medium for 14 days before Alizarin Red-S staining was performed. The stained cultures were observed by phase-contrast microscopy (lower panel). (C) Effect of a tyrosine kinase inhibitor, K252a, on the mineralization. Cells were cultured with standard medium before confluence. After confluence, cells were cultured with osteogenic medium with or without K252a for 14 day before Alizarin Red-S staining was performed. The stained cultures were observed by phase-contrast microscopy (lower panel).
Red-S staining revealed that p75NTR -mediated induction of osteogenic differentiation was strongly inhibited by K252a (Fig. 6C).
4. Discussion In this study, we demonstrated that p75NTR plays a critical role in pre-osteoblast differentiation. p75NTR is a key marker for the isolation and characterization of bone marrow-derived MSCs (Jones et al., 2002) and of adipose tissue-derived MSCs
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(Yamamoto et al., 2007). A recent study has shown that the mineralization capacity of p75NTR-expressing cells derived from the periosteum was higher than similar periosteum-derived cells without p75NTR expression (Alexander et al., 2009). Therefore, it is possible that p75NTR may have a functional role in osteogenic cells, which has not yet been elucidated. We confirmed that p75NTR mRNA is expressed in the osteogenic MC3T3E-1 cell line and that p75NTR protein was localized to the cell surface membrane, but at generally low or undetectable levels. Stable transfection of cells to generated gene overexpression has been widely used to investigate the role of gene products in gain-of-function experiments. To examine whether p75NTR might directly regulate pre-osteoblast proliferation and differentiation, we transfected an expression construct of p75NTR into MC3T3-E1 cells to obtain cells stably expressing p75NTR. Flow cytometric analysis revealed that the purified p75NTR -positive cell population could be isolated from the transfected MC3T3-E1 cells (p75GFP-E1). Overexpression of p75NTR significantly increased expression of p75NTR protein on the cell membrane of p75GFP-E1. We determined that p75GFP-E1 has strongly enhanced cell proliferation at the indicated time during in vitro cultivation and the rate of S-phase cell population in p75GFP-E1 cells increased in comparison with that of GFP-E1. p75NTR stimulates increased proliferation of hepatic stellate cells during the proliferative stage (Asai et al., 2006). Furthermore, there is evidence that p75NTR is involved in controlling the fate of keratinocyte stem cells (Botchkarev et al., 2000) and that it is a marker for esophageal keratinocyte stem cells in vitro (Okumura et al., 2003). Together these data suggest the p75NTR is involved in the cell proliferation associated with cell cycle progression in pre-osteoblasts. Osteoblast differentiation occurs through a multistep molecular pathway regulated by different signal transduction pathways, but the full spectrum of events has not been elucidated. Therefore, to determine whether p75NTR overexpression in p75GFP-E1 cells affected osteoblast differentiation, we assayed tissue nonspecific ALPase, which serves as an early marker of calcification. ALPase activity in p75GFP-E1 strongly increased in comparison with that of GFP-E1 consistent with a functional role for p75NTR in osteogenesis. Moreover, a quantitative investigation of the osteoblast marker gene expression in vitro showed that osteoblastic maturation is strongly promoted as assessed by expression of Osterix, BSP and the mature osteoblast marker, OCN (Power and Fottrell, 1991). In addition, p75GFP-E1 cells showed enhanced osseous nodule formation. The expression of p75 mRNA was maintained during the osteogenic differentiation process as shown in a supplemental figure. These results indicate that osteoblast differentiation was induced as a result of p75NTR overexpression. Osteoblasts differentiation is controlled predominantly through the transcription factors, Runx2 and Osterix (Ducy et al., 1997; Komori, 2006; Komori et al., 1997; Nakashima et al., 2002). In this study, expression of Osterix, BSP and OCN was significantly increased by p75NTR overexpression but the expression of Runx2 was not altered. These results are consistent with a previous report demonstrating that Runx2 is an upstream regulator of Osterix (Nakashima et al., 2002), which in turn regulates BSP and OCN (Igarashi et al., 2004; Nakashima et al., 2002). Our findings are the first to show that p75NTR is an important regulator of both cell proliferation and osteogenic differentiation in pre-osteoblasts. This is consistent withprior studies indicating the importance of the p75NTR in osteogenesis (Alexander et al., 2009). How does the p75NTR participate in osteoblast differentiation? Numerous adaptor proteins that bind to p75NTR have been reported. The findings from the current study are consistent with the model illustrated in Fig. 7, which shows the sites and
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Fig. 7. Two different signaling pathways containing p75NTR.
mechanisms of action of p75NTR used in the present study. p75NTR contributes to several signaling pathways and has different biological functions (Barker, 2004; Chao, 2003; Teng and Hempstead, 2004; Zampieri and Chao, 2006). In the absence of neurotrophins, p75NTR or the intracellular domain (ICD) of p75NTR alone can induce apoptosis (Majdan et al., 1997; Rabizadeh et al., 1993) and activation of phosphatidylinositol 3-kinase (Roux et al., 2001) and Rho (Yamashita et al., 1999). Even though we have identified a role for p75NTR in osteoblast differentiation, questions remains concerning the identity of its binding partner during osteogenesis. The association of p75NTR and trk receptors and signal transduction is well documented in the nervous system. In non-neuronal tissue, trkA has been shown to preferentially localize to limbal basal epithelial cells and was proposed as a potential marker for corneal limbal stem cells. (Lambiase et al., 1998; Stepp and Zieske, 2005; Touhami et al., 2002). Human keratinocytes express trkA and p75NTR and the specific binding by these receptors modulate either proliferation or differentiation (Di Marco et al., 1993). We confirmed the expression of three trk receptors (trkA, trkB, trkC) in MC3T3E-1 cells, which is consistent with previous observations (Mogi et al., 2000; Nakanishi et al., 1994). The neurotrophin/trk receptor autocrine signaling pathway is required for the survival of proliferating for human osteoblasts (Roux and Barker, 2002). To identify the binding partner of p75NTR in p75GFP-E1 during osteoblast differentiation, we conducted a pharmacological study using K252a, an inhibitor of trk tyrosine kinase. The inhibitory effect of K252a has been confirmed by the suppression of NGF induced neurite formation in PC12 cells (Pinski et al., 2002). The osteoblastic differentiation of p75GFPE1 cells was inhibited by treatment with K252a as determined by inhibition of ALPase activity. The trkA inhibitor K252a functions as a JNK inhibitor and stimulates cell survival by activating ERK1/ 2 and Akt (Koizumi et al., 1988). JNK is usually associated with p75NTR activation and the induction of cell death (Kaplan and Miller, 2000; Roux and Barker, 2002; Roux et al., 2002). A link between JNKs and trk receptors has not been shown before for osteoblast differentiation. On the other hand, p75NTR acts in combination with other receptors, such as the Nogo receptor, NgR (Dechant, 2001; Lee et al., 2001; Mischel et al., 2001). The activation of the NgR- p75NTR complex of the neurotrophin receptor leads to activation of RhoA and the RhoA kinase pathway, which results in growth cone collapse (Al Halabiah et al., 2005; Shao et al., 2005; Wong et al., 2002). Conversely, the expression of NgR was not observed in MC3T3-E1 cells. In addition, we have demonstrated that the GDI-domain in p75NTR, which is the binding motif for RhoA, is not involved in the osteoblast differentiation. Taken together, our findings suggest that the binding partner of p75NTR are the trk receptors and that p75NTR signaling is associated with trk tyrosine kinase receptor promotion of pre-osteoblast proliferation and differentiation.
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Acknowledgments This work was supported in part by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology [Kakenhi (822791778) to YM; (21390528 and 20659305) to MH], and Dental Research Center and Sato Fund, Nihon University School of Dentistry.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.diff. 2012.07.001.
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