Overexpression of receptor for hyaluronan-mediated motility (RHAMM) in MC3T3-E1 cells induces proliferation and differentiation through phosphorylation of ERK1/2
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- Hatano, H., Shigeishi, H., Kudo, Y. et al. J Bone Miner Metab (2012) 30: 293. doi:10.1007/s00774-011-0318-0
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Receptor for hyaluronan (HA)-mediated motility (RHAMM) was first described as a soluble HA binding protein released by sub-confluent migrating cells. We previously found that RHAMM was highly expressed and plays an important role in proliferation in the human cementifying fibroma (HCF) cell line, which we previously established. HCF is a benign fibro-osseous neoplasm of the jaw and is composed of fibrous tissue containing varying amounts of mineralized material. However, the pathogenesis of HCF is not clear. In this paper, we examined the roles of RHAMM in osteoblastic cells. We generated RHAMM-overexpressing MC3T3-E1 cells and examined the cell proliferation and differentiation of osteoblastic cells. In MC3T3-E1 cells, overexpressing RHAMM was located intracellular and activated ERK1/2. Interestingly, the ERK1/2 activated by RHAMM overexpression promoted cell proliferation and suppressed the differentiation of osteoblastic cells. Our findings strongly suggest that RHAMM may play a key role in the osteoblastic differentiation process. The rupture of balance from differentiation to proliferation induced by RHAMM overexpression may link to the pathogenesis of bone neoplasms such as HCF.
Receptor for hyaluronan-mediated motility
Extracellular regulated kinase
Mitogen activated protein kinase
Human cementifying fibroma (HCF) is a benign fibro-osseous neoplasm of the jaw, and is composed of fibrous tissue containing varying amounts of mineralized material [1, 2]. However, the process of development consisting of proliferation and differentiation is not clear. We previously established immortalized cell lines from HCF of the jaw  and found by microarray analysis that receptor for hyaluronan (HA)-mediated motility (RHAMM) was highly expressed in comparison with normal osteoblasts obtained from normal human mandibular bone .
RHAMM was first described as a soluble hyaluronan binding protein released by sub-confluent migrating cells . The protein, which is called HMMR or CD168, is located intracellularly in the cytoplasm and the nuclei as well as on the cell surface . The RHAMM gene is located on chromosome 5q33.2 and contains 18 exons. The full-length RHAMM mRNA codes for a 84-kDa protein . Injured, subconfluent, or neoplastic cultured cells express some additional RHAMM proteins [8, 9]. These seem to be conformationally altered RHAMM and/or have a cellular or intracellular localization different from that of normal cells, which is known to be present on the cell surface as a glycosylphosphatidylinositol-linked receptor . Surface and intracellular forms of RHAMM are detected in multiple myeloma . Overexpression and modulation of the balance between soluble, surface, and intracellular RHAMM may control the regulation of key signaling molecules and the behavioral characteristics of premalignant cells, leading to constitutive stimulation and malignancy.
Cell-surface RHAMM, which is not an integral membrane protein, partners with CD44 and, in the presence of hyaluronan, activates ERK1/2, which results in the expression of genes that are required for motility and invasion [12, 13]. Extracellular expression of cytoplasmic proteins, such as RHAMM, results from a redistribution of intracellular pools to the extracellular compartment that may be associated in part with increased synthesis or stability of mRNA or protein. On the other hand, intracellular RHAMM binds to actin filaments, centrosomes, microtubules, and mitotic spindles [14, 15] and plays important roles in several cellular processes like signaling , mitosis, tumorigenesis, and cell proliferation [17, 18]. The cell growth mediated by RHAMM is considered to occur via signaling events leading to the phosphorylation of several intracellular proteins . In our previous reports, we found the EGFR–RHAMM/ERK signaling pathway to be implicated in the growth of HCF cells.
HCF usually consists of multiplied fibroblasts that produce extracellular collagen fibers and osteoblastic cells on the surface of spherules–bone/cementum granules. We found that RHAMM protein expression could be observed in most of the several cementifying fibroma cases examined. Moreover, expression of RHAMM protein was detected in cells from the fibrous region of the tissues. We assumed that ectopic-overexpressing RHAMM may be linked to the characterization of HCF: the promotion of proliferation and the suppression of differentiation. However, the biological behavior of ectopic-overexpressing RHAMM has not been fully investigated. To clarify the mechanism involved in RHAMM leading to HCF deviating from normal differentiation, we have further focused on the proliferation and differentiation of normal osteoblasts. Moreover, recent studies showed that ERK activity is associated with anti-osteogenesis in osteoblasts [20–22]. In this paper, we report the identification of a specific mechanism of growth and differentiation by RHAMM and highlight the novel signaling through RHAMM/ERK interaction in osteoblastic cells.
Materials and methods
Immortalized human cementifying fibroma (HCF) cell lines were established by co-transfection with simian virus-40 (SV40) T-antigen and hTERT . HCF cells were maintained in α-modified Eagle medium (α-MEM; Sigma) supplemented with penicillin/streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Invitrogen) under 5% CO2 in air at 37°C. MC3T3-E1 cells were provided by the Japanese Collection of Research Bioresources Cell Bank. They were maintained in α-MEM supplemented with penicillin/streptomycin and 10% heat-inactivated FBS under 5% CO2 in air at 37°C. The culture medium was changed every 4 days. They were then cultured with the same medium and used for the following analyses. For the proliferation assay, 5.0 × 104 cells were plated on 6-well plates (Falcon), and trypsinized cells were counted by Cell Counter. To examine the differentiation, 2.0 × 106 cells were plated on 6-well plates, and after reaching confluence the medium was changed to the osteogenic medium containing l-ascorbic acid (vitamin C) (50 μg/ml), β-glycerophosphate (10 mM), and dexamethasone (100 nm). In addition to the prior medium, the treated group was incubated with synthesized 160-kDa HA (Hyalose) at 10 μg/ml, anti-CD44 (Thermo Fisher Scientific), and the commercial phosphorylation of ERK1/2 inhibitor PD98059 at 50 μM final concentration.
Generation of RHAMM-overexpressing MC3T3-E1 cells
The recombinant vector was produced by Origene Technologies. The pCMV6-RHAMM-GFP or pCMV6-GFP plasmid was introduced into MC3T3-E1 cells, and the stable clones were obtained by G418 selection (500 μg/ml, Life Technologies) in the culture medium. We obtained pool and stable clones. Cell transfection was done using FuGENE 6 HD (Roche) according to the manufacturer’s instruction.
Small interfering RNA (siRNA)
ERK1 siRNA, ERK2 siRNA, and negative control siRNA were purchased from Santa Cruz Biotechnology. Cells were transiently transfected with the indicated combinations of the siRNAs using Lipofectamine™ 2000 transfection reagent (Invitrogen), according to the manufacturer’s recommendations.
Quantitative reverse transcription-PCR
Total RNA was isolated from cultures of confluent cells using the RNeasy mini kit (Qiagen). Preparations were quantified, and their purity was determined by standard spectrophotometric methods. cDNA was synthesized from 1 μg total RNA using the ReverTra Dash kit (Toyobo Biochemicals). The quantification of mRNA levels was carried out using a real-time fluorescence detection method. The fluorescence was detected using a ABI7000 (Applied Biosystems) by measuring the binding of a fluorescence dye, SYBR Green I, to double-stranded DNA. The reaction mixture contained 1.0 μg of cDNA, 10 μl of SYBR Green PCR Master Mix (Toyobo Biochemicals), and 10 pmol of each pair of oligonucleotide primers. The primer sequences were: human RHAMM: 5′-TCTAAACAAAATCTTAATGTTGACAAA-3′ (sense), 5′-TCTTTCTCTAATATCTTCAAATCTTTA-3′ (antisense) ; mouse G3PDH: 5′-CACCATGGAGAAGGCCGGGG-3′ (sense), 5′-GACGGACACATTGGGGGTAG-3′ (antisense); mouse ALP: 5′-GTTGCCAAGCTGGGAAGAACA-3′ (sense), 5′-CCCACCCCGCTATTCCAAAC-3′ (antisense); mouse bone sialoprotein (BSP): 5′-ACCCCAAGCACAGACTTTTGA-3′ (sense), 5′-CTTTCTGCATCTCCAGCCTTCT-3′ (antisense). The PCR program was as follows: initial melting at 95°C for 60 s followed by 40 cycles at 95°C for 15 s, 58°C for 40 s. The quantification of mRNA expression relative to an internal control, G3PDH, was performed by the ΔCt method .
For extracting total lysate, cultured cells were extracted using TBSN(+) buffer consisting of 20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA (pH 8.0), 5 mM EGTA (pH 8.0), 0.5 mM Na3VO4, 20 mM p-nitrophenyl phosphate, 1 mM PMSF, 0.5% NP-40, various protease inhibitor cocktails (Sigma-Aldrich), and various phosphatase inhibitor cocktails (Sigma-Aldrich). Each sample was frozen in liquid nitrogen and thawed at 4°C, and then vortexed and centrifuged. The supernatant was then used for the immunoblot experiments.
For the preparation of the nuclear extracts, cells were suspended in 10–20 volume of ice-cold hypotonic buffer N, 10 mM HEPES pH 7.5, 2 mM MgCl2, 25 mM KCl, 1 mM DTT, various protease inhibitor cocktails (Sigma-Aldrich), and various phosphatase inhibitor cocktails (Sigma-Aldrich), centrifuged at 1500 rpm for 10 min and the supernatant discarded. The pellet was resuspended in 10–20 volume of hypotonic buffer N and incubated on ice for 30 min. Cells were transferred to a Dounce homogenizer and lysed with 15 strokes. Cell lysis was confirmed by the addition of trypan blue and examination under a microscope. When cells were lysed, 125 μl of 2 M sucrose solution per milliliter of lysate was added, mixed well by inversion, and centrifuged at 1000 rpm for 10 min. After decanting supernatant, the pellet containing nuclei was resuspended in 10–20 volume of ice-cold buffer N; 10 mM HEPES pH 7.5, 250 mM sucrose, 2 mM MgCl2, 25 mM KCl, 1 mM DTT, various protease inhibitor cocktails, and various phosphatase inhibitor cocktails, centrifuged at 1000 rpm for 10 min, and the supernatant discarded. The pellet was resuspended in one volume of buffer N.
For extraction of membranous and cytoplasmic proteins, cells were suspended in TM-PEK Extraction Buffer 1 containing Protease Inhibitor Cocktail SET III from TM-PEK (Merck KGaA) for 10 min. Membranous and cytoplasmic proteins were separated into pellet and supernatant fractions, respectively, by centrifuging at 1000g. Membranous protein was incubated for 45 min at 22°C in TM-PEK Reagent. After centrifuging at 15000g, the supernatant was transferred to fresh tubes which were enriched with integral membrane protein.
Western blot analysis
Protein concentrations were measured using Protein Assay Reagent (BIO-RAD). Protein samples (10 μg) were solubilized in sample buffer by boiling, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) followed by electrotransfer onto Protran® Nitrocellulose Membranes (Whatman). We detected the band of Western blotting using enhanced chemiluminescence (ECL) Western blotting reagent (GE Healthcare). Images were captured with a cooled CCD camera system (LAS-4000) from Fujifilm (Japan). Anti-GFP mouse monoclonal antibody (Wako), anti-RHAMM mouse monoclonal antibody (Monosan), anti-G3PDH mouse monoclonal antibody (Millipore), anti-CENP rabbit polyclonal antibody (Santa Cruz Biotechnology), anti-tubulin rabbit monoclonal antibody, anti-cadherin rabbit monoclonal antibody, anti-Raf rabbit polyclonal antibody, anti-phosphorylated-Raf rabbit polyclonal antibody, anti-MEK1/2 rabbit polyclonal antibody, anti-phosphorylated-MEK1/2 rabbit polyclonal antibody, and anti-ERK1/2 rabbit polyclonal antibody, and anti-phosphorylated-ERK1/2 rabbit polyclonal antibody (all Cell Signaling Technology) were used. For detecting the phosphorylation of ERK1/2 correlated with RHAMM, we performed an immunoprecipitation assay. Antibodies were allowed to bind to protein A-Sepharose (Sigma-Aldrich) and then incubated with equal amounts of protein (0.5 mg of total protein in 400 μl) for 12 h at 4°C. Beads were washed three times with PBS. Each pellet was boiled in 20 μl of SDS–PAGE sample buffer at 95°C for 3 min, and the entire volume was loaded onto a gel for Western blotting.
Cell cycle analysis
The distribution of cells at different stages in the cell cycle was estimated by flow cytometric analysis. Briefly, 5.0 × 105 cells were incubated at 37°C. To synchronize them in G0, the cells were starved by serum deprivation for 48 h. After restimulation with medium containing 10% FBS, cells passed through the cell cycle synchronously and were incubated for 0, 6, 12, 18, 24, or 30 h. Cells from the different conditions were trypsinized, washed in PBS, fixed in 70% ethanol, and stored at 4°C for 2 h. An aliquot (1 ml) of the fixed cell suspension was washed twice in PBS. The fixed cells were treated for 30 min at 4°C in the dark with 40 μg of propidium iodide and 0.1 mg of RNase A, and then analyzed by flow cytometry. The percentage of cells in each cell cycle phase (G0/G1, S, or G2/M) was calculated by using ModFit LT software (Becton–Dickinson).
Cells were seeded onto glass Lab-Tek II Chamber Slides (Thermo Fisher Scientific) at a density of 5.0 × 104 cells/well and incubated for one day. The growth medium was then removed, and cell monolayers were washed three times with a 10% PBS solution and fixed with 3.5% paraformaldehyde for 10 min at room temperature. Cells were washed three times with PBS and permeabilized by 0.2% Triton X-100 for 10 min at room temperature. Nonspecific binding sites were blocked by treatment at room temperature for 30 min with PBS containing 1% BSA. The cells were washed three times with PBS and incubated with anti-RHAMM mouse monoclonal antibody, anti-GFP mouse monoclonal antibody, and anti-phosphorylated-ERK1/2 rabbit polyclonal antibody in PBS with 1% BSA, for 60 min at room temperature. RHAMM and GFP staining was revealed by incubation with an Alexa-Fluor dye-labeled goat anti-mouse antibody (Invitrogen) for 60 min at room temperature. Phospho-ERK staining was revealed by incubation with an Alexa-Fluor dye-labeled goat anti-rabbit antibody (Invitrogen) for 60 min at room temperature. After three rinses in PBS, the slides were mounted in Vectashield (Vecto Laboratories) and examined using a Leica TCS STED (Leica Microsystems).
Alkaline phosphatase activity
Cells were plated in a 6-well plate, then the alkaline phosphatase (ALP) activity was evaluated, as described below. The confluent cells were grown in osteogenic medium for 7 days. The ALP activity of the lysate was determined using p-nitrophenyl phosphate (pNPP; Wako) using the Lowry method. After 30 min incubation at 37°C, absorbance of pNPP at 405 nm was measured using a Multiskan JX microplate reader (Thermo Fisher Scientific).
Staining for mineralization
The mineralization of MC3T3-E1 cells was determined in 6-well plates using von Kossa staining and Alizarin red staining, respectively. The confluent cells were grown in osteogenic medium for 3 weeks, and the cells were fixed with 95% ethanol and stained with AgNO3 by the Von Kossa method to detect phosphate deposits in bone nodules. At the same time, the other plates were fixed with ice-cold 70% ethanol and stained with Alizarin red S (Sigma) to detect calcification.
The statistical analysis was performed using one-way ANOVA and Student’s t test. P values less than 0.05 were regarded as statistically significant.
Cell-cycle-dependent expression of RHAMM in HCF cells
Overexpressing RHAMM promotes the phosphorylation of ERK1/2 in nuclei
RHAMM promotes proliferation and suppresses differentiation
The function of phosphorylated-ERK1/2 induced by overexpressing RHAMM is not inhibited by anti-CD44
The function of phosphorylated-ERK1/2 induced by overexpressing RHAMM is inhibited by ERK inhibitor
We previously identified RHAMM as a proliferation factor of HCF . Previous studies also show the overexpression of RHAMM in tumor development and the prognostic significance of its expression [27–29]. On the other hand, the signaling mediated by mitogen-activated protein kinases (MAPKs) has been shown to have critical roles in the regulation of cell growth and differentiation . In addition, recent studies have raised the possibility that RHAMM could regulate MAPKs such as ERK . In HCF cells, we found the EGFR–RHAMM/ERK signaling pathway to be implicated in the regulation of growth .
For the normal development of the jaw, the balance of proliferation and differentiation of osteoblasts is important, but the mechanism of its regulation is not yet elucidated. We assumed that ectopic RHAMM overexpression make the balance incline to proliferation in HCF, and it may be linked to its pathogenesis. To prove this hypothesis, we generated ectopic RHAMM overexpression in osteoblastic cells, MC3T3-E1, that showed low expression of RHAMM, and examined the proliferation and its mechanism.
Interestingly, ectopic overexpressing RHAMM is expressed intracellularly. As in previous reports, the RHAMM localized to nuclei interacted with the phosphorylation of ERK1/2 more directly. It may be that the increased proliferation associated with cancer cells facilitates the secretion of intracellular protein and provides for outside-in and inside-out control of genetic stability. As we expected, RHAMM overexpression promoted cell proliferation and suppressed cell differentiation to osteoblasts of MC3T3-E1 cells. We also found that RHAMM overexpression activated ERK1/2.
Our present study suggests that ectopic overexpression of RHAMM breaks down the balance of cell proliferation and differentiation. Above all, we showed that the localization of overexpressing RHAMM to nuclei may be important in the pathogenesis of disease. Further studies are required to clarify the mechanism of up-regulation of RHAMM. However, our results provide the possibility of controlling HCF by regulating the RHAMM protein localized to nuclei. In conclusion, our studies have revealed a critical role for RHAMM in cell proliferation and differentiation in osteoblastic cells. These findings provide new and important information on the metabolism of bone and pathogenesis of HCF.
This work was supported by a Grant-in-Aid for JSPS fellows (No. 22-6035) from the Japan Society for the Promotion of Science (JSPS).
Conflict of interest
The authors declare no conflict of interest.