Potential Involvement of Twist2 and Erk in the Regulation of Osteoblastogenesis by HB-EGF-EGFR Signaling

Takashi Nakamura, Hidetoshi Toita, Akimasa Yoshimoto, Daigo Nishimura, Tomoyo Takagi, Takuya Ogawa, Tatsuo Takeya, and Norihiro Ishida-Kitagawa∗ Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan

ABSTRACT. Epidermal growth factor (EGF) family members play important roles in the skeletal system. In this study, we examined the role of EGF receptor (EGFR) signaling in osteoblastogenesis in vitro. The expression of HB-EGF and epiregulin (EPR) was transiently induced within 24 h after osteogenic stimulation, but when preosteoblastic MC3T3-E1 cells were incubated with HB-EGF or EPR, osteoblast differentiation was inhibited. These effects were Ras-dependent, and ERK modulated Runx2 activity through the localization of Smad1 and the induction of Twist2. PI3-kinase was also required for the induction of Twist2. However, the inhibition of individual signaling pathways was not sufficient to overcome HB-EGF-mediated inhibition of osteoblast differentiation. Additionally, HB-EGF treatment promoted the proliferation of preosteoblasts, and this was associated with the downregulation of p27 at the protein level. These results suggest that HB-EGF-EGFR signaling inhibits the differentiation of osteoblasts by suppression of Runx2 transcriptional activity and enhances proliferation of preosteoblasts by downregulation of expression of p27.

Key words: EGFR/osteoblasts/proliferation/differentiation/cell culture

Introduction Collagen type I and Osteocalcin. The activity of Runx2 is both positively and negatively regulated. In particular, C/ Bone homeostasis is tightly regulated by both systemic and EBPβ, Smad1, and Smad5 bind and activate Runx2, while local mechanisms (Harada and Rodan, 2003; Karsenty, other factors, e.g. Twist, inhibit the transcriptional activity 2006; Teitelbaum and Ross, 2003), and these signaling of Runx2 (Bialek et al., 2004; Franceschi et al., 2007). networks modulate the activity of two cell types within the Epidermal growth factor receptor (EGFR or ErbB1) sig- bone parenchyma: osteoclasts and osteoblasts. Osteoclasts naling modulates both the proliferation and differentiation are hematopoietic in origin and mediate bone resorption, but of osteoblasts (Canalis and Raisz, 1979; Hata et al., 1984; mesenchymally-derived osteoblasts play an essential role in Kumegawa et al., 1983). EGFR is a transmembrane glyco- skeletal development and bone formation (Karsenty et al., protein with intrinsic tyrosine kinase activity that becomes 2009). Many signaling pathways involved in osteoblast activated after binding EGF family members, including development have been identified. Bone morphogenic EGF, heparin-binding EGF-like growth factor (HB-EGF), protein (BMP) and several transcription factors including amphiregulin (AR), betacellulin (BTC), transforming growth Smad, Runx2, and Osterix, are essential for osteoblast factor-α (TGF-α), epiregulin (EPR) and epigen (Iwamoto formation (Bialek et al., 2004). Runx2 functions upstream and Mekada, 2006; Kochupurakkal et al., 2005). Osteoblast of Osterix and induces the expression of osteoblastic marker proliferation and differentiation are abnormal in mice lack- genes, including major bone matrix protein genes, such as ing EGFR, and this leads to impaired bone formation and skeletal structure (Sibilia et al., 2003; Wang et al., 2004). *To whom correspondence should be addressed: Norihiro Ishida-Kitagawa, Primary osteoblasts isolated from calvaria of mice lacking Graduate School of Biological Sciences, Nara Institute of Science and EGFR proliferate less and undergo maturation more readily Technology, Ikoma, Nara 630-0192, Japan. Tel: +81–743–72–5543, Fax: +81–743–72–5549 in vitro than those isolated from wild-type mice. Further- E-mail: [email protected] more, transgenic mice overexpressing EGFR ligands dem- Abbreviations: ALP, alkaline phosphatase; BMP, bone morphogenetic onstrate variable, ligand-specific effects on bone formation. protein; EGF, Epidermal growth factor; EGFR, EGF receptor; EPR, Epiregulin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HB- Overexpression of EGF led to osteoblast hyperproliferation EGF, Heparin-binding EGF-like growth factor. and hyperplasia and a reduction in cortical bone thickness

53 T. Nakamura et al.

(Chan and Wong, 2000), whereas BTC overexpression 100 mM NaCl and 50 mM MgCl2) and staining with a NBT/BCIP resulted in increased cortical bone mass in transgenic mice solution. For Alizarin red staining, cells were fixed with cold 70% (Schneider et al., 2009a). Thus, EGFR ligands may promote ethanol and stained with a 40 mM Alizarin red solution. osteoblast proliferation and/or differentiation, leading to proper bone formation. Determining the mechanism by RNA isolation and RT-PCR which EGF family members differentially regulate intracellular signaling networks downstream of EGFR in osteo- Total RNA was extracted from cells using ISOGEN (Nippon blasts is essential to gain a better understanding of Gene), according to the manufacturer’s instructions. cDNA was osteoblastogenesis. Toward this end, in this study we exam- made by reverse transcription using SuperScript™II (Invitrogen) ined the role of EGFR signaling in osteoblast homeostasis. after DNase treatment of the RNA samples. PCR was performed using rTaq DNA Polymerase (Takara) with the gene-specific primer pairs as follows: Hb-egf, 5'-AAATACCGCAAGCTCG- Materials and Methods AGAA-3' (forward) and 5'-GGGATAGAGGACCAGGAAGC-3' (reverse); Egf, 5'-CCCAGGCAACGTATCAAAGT-3' (forward) α Cells and reagents and 5'-GGTCATACCCAGGAAAGCAA-3' (reverse); Tgf , 5'- CAGCATGTGTCTGCCACTCT-3' (forward) and 5'-CAAGCAG- MC3T3-E1 cells were maintained in minimum essential medium TCCTTCCCTTCAG-3' (reverse); Areg, 5'-TGGCAGTGAACT- alpha (α-MEM; Wako) supplemented with 10% FBS, penicillin, CTCCACAG-3' (forward) and 5'-CAATTGCATGTCACCACCTC-3' and streptomycin. The following antibodies were used: anti-β-actin (reverse); Epr, 5'-CGCTGCTTTGTCTAGGTTCC-3' (forward) monoclonal antibody (AC-74; Sigma Aldrich), rabbit anti-ERK, and 5'-CTCACATCGCAGACCAGTGT-3' (reverse); Btc, 5'- anti-pERK (Promega), anti-cyclin D1 (DCS-6; MBL), anti-Rb GCACAGGTACCACCCCTAGA-3' (forward) and 5'-TGAA- (BD Biosciences), anti-p21 (Santa Cruz), anti-p27 (MBL), anti- CACCACCATGACCACT-3' (reverse); Egfr, 5'-TACAGCTTT- phospho-Smad1/5/8 (Chemicon), HRP-conjugated anti-mouse GGTGCCACCTG-3' (forward) and 5'-GGCCAAGCCTGAAT- IgG, protein-A (GE Healthcare UK Ltd.), and anti-goat IgG CAGCAA-3' (reverse); ColI, 5'-CGAAAGGTGAACCTGGTGAT-3' (Chemicon). Ascorbic acid and glycerol 2-phosphate were obtained (forward) and 5'-TCCAGCAATACCCTGAGGTC-3' (reverse); from Sigma. Recombinant human HB-EGF protein (rHB-EGF), Alp, 5'-AACCCAGACACAAGCATTCC-3' (forward) and 5'- recombinant human epiregulin protein (rEpr) and anti-human GCCTTTGAGGTTTTTGGTCA-3' (reverse); Opn, 5'-TCTGAT- HB-EGF antibody were purchased from R&D Systems. AG1478, GAGACCTGCACTGC-3' (forward) and 5'-TCTCCTGGCTCTC- PD98059, and LY294002 were purchased from Calbiochem. Cell TTTGGAA-3' (reverse); Ocn, 5'-CTTCATGTCCAAGCAGGAG-3' Counting Kit-8 (Dojindo) was used for the WST assay. (forward) and 5'-AGCTGCTGTGACATCCATAC-3' (reverse); cyclin D1, 5'-GGCGGATGAGAACAAGCAGA-3' (forward) and kip1 Vector construction and retroviral infection 5'-ACCAGCCTCTTCCTCCACTT-3' (reverse); p27 , 5'-CACT- GCCGGGATATGGAAGA-3' (forward) and 5'-AGTAGAACT- cDNA encoding mouse full-length p27kip1, ERK1, ERK2, MEK1, CGGGCAAGCTG-3' (reverse); p21cip1, 5'-CCTGGTGATGTCC- Twist2 was obtained by RT-PCR and cloned into the retroviral GACCTG-3' (forward) and 5'-CCATGAGCGCATCGCAATC-3' expression vector pCX4-puro (Dr. T. Akagi; KAN Research Insti- (reverse); and Gapdh, 5'-ATGGTGAAGGTCGGTCAACGGA-3' tute, Kyoto, Japan). Constitutively active MEK1 (MEK1 S218E: (forward) and 5'-ACTCCTTGGAGGCCATGTAG-3' (reverse). S222E, MEK CA) was generated by site-directed mutagenesis with Glu substitutions at Ser218 and Ser222. The dominant- Western blotting and subcellular fractionation negative form of Ha-Ras (Ras S17N, DN-Ras) was kindly provided by Dr. K. Kaibuchi (Nagoya University, Japan) and was Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM subcloned into pCX4-puro. For the production of retroviruses, NaCl, 2 mM EDTA, 2 mM PMSF, 2 mM Na3VO4, 20 mM NaF, Plat-E cells were co-transfected with pGL, pE-Eco (Takara), and 100 KIU/ml of aprotinin, and 1% Triton X-100). Proteins were retroviral vectors using FuGene6 (Roche Diagnostics), according separated by SDS-PAGE, transferred to a PVDF membrane, and to the manufacturer’s directions. MC3T3-E1 cells were infected detected by Western blotting using an ECL detection kit (GE and were selected two days later in media containing puromycin. Healthcare UK Ltd.). Nuclear and cytoplasmic extracts were prepared using NE-PERTM nuclear and cytoplasmic extraction In vitro osteoblastogenesis and cell staining reagents (PIERCE), according to the manufacturer’s instructions. α μ MC3T3-E1 cells were cultured in -MEM containing 50 g/ml of Immunofluorescence microscopy ascorbic acid and 10 mM glycerol 2-phosphate or BMP2. rHB- EGF and rEpr were added to a final concentration of 30 ng/ml as Cells were grown on coverslips and stimulated with BMP2 for 6 h indicated. For alkaline phosphatase (ALP) staining, cells were with or without rHB-EGF and PD98059. Cells were then fixed washed with PBS and fixed with 3.7% formaldehyde, followed with 3.7% paraformaldehyde for 10 min at room temperature (RT), by pre-treatment with ALP buffer (100 mM Tris-HCl pH 9.5, permeabilized with 0.2% Triton X-100/PBS, and blocked with 5%

54 EGFR Signaling in Osteoblasts skim milk/PBS for 1 h at RT. Cells were then incubated with anti- in the culture medium (Fig. 1E). Thus, rHB-EGF stimula- pSmad1/5/8 for 1 h and visualized with goat-anti-rabbit Alexa tion downregulates Alp and Ocn expression downstream of fluor 546 (Molecular Probes). Nuclei were counterstained with EGFR-initiated signaling. Hoechst dye. All images were obtained using a LSM510 laser We next examined the role of Ras signaling on the inhibi- scanning microscope (Carl Zeiss, Inc.). tion of osteoblast differentiation following EGFR ligation. We prepared MC3T3-E1 cells expressing a dominant- Transient transfection and luciferase assay negative form of Ras (DN-Ras) and stimulated the cells with BMP2 (Fig. 1F). At baseline, ALP activity was enhanced The mouse ALP promoter (–1838/+81) in the pGL3 vector in these cells compared to mock-transfected cells, and the (Promega) was kindly provided by Dr. T. Komori (Nagasaki addition of HB-EGF to the mock-transfected cells led to University, Japan). MC3T3-E1 cells were transfected with the decreased ALP activity. In contrast, no change in ALP plasmids using LipofectAMINE2000 reagent (Invitrogen), accord- activity was seen following HB-EGF treatment of DN-Ras ing to the manufacturer’s instructions. Cells were harvested 48 h expressing cells. Thus, Ras inhibits osteoblastogenesis and after transfection, and luciferase activity was determined using a is required for the function of EGFR signaling. Dual-Luciferase Reporter Assay System (Promega). EGFR signaling inhibits Runx2 activity through Twist2 and Smad Results Runx2 is a key regulator of osteoblast differentiation, and EGFR signaling inhibits osteoblastogenesis in Alp and Ocn mRNA expression are controlled by Runx2. As a Ras-dependent manner shown in Fig. 1C, treatment of cells with rHB-EGF down- regulated the expression of Alp and Ocn mRNA, we exam- To better understand the role of EGFR signaling in osteo- ined the expression profiles of Runx2. However, Runx2 blast homeostasis, we first examined the expression of EGF mRNA levels were unchanged (Fig. 2A) even in rHB-EGF- family members in MC3T3-E1 cells, a well characterized treated cells, suggesting that HB-EGF stimulation regulates osteoblast-like cell line. When cultured in media containing the activity of Runx2 rather than its expression. Runx2 ascorbic acid and glycerol 2-phosphate that promotes osteo- activity is directly modulated by transcriptional regulators blastic differentiation, Hb-egf and Epiregulin (Epr) mRNA such as ATF4, SATB2, and Twist 1/2. We therefore exam- expression were transiently increased within 24 h (Fig. 1A). ined the expression of these genes in rHB-EGF-treated EGFR mRNA was constitutively expressed under the same cells. In the presence of rHB-EGF, Twist2 mRNA was tran- conditions (data not shown). ALP activity is a marker of siently induced at day 1, but there was no change in Twist2 osteoblast differentiation, and, under the culture conditions expression under control conditions (Fig. 2A). Twist2 is a used, it became detectable after 3–5 days and continued to negative regulator of Runx2 activity, and the timing of the increase until 10 days after the addition of differentiation expression of Twist2 after rHB-EGF treatment coincides stimuli. Additionally, mineralizing activity, a marker of with the changes in Alp mRNA expression in differentiating mature osteoblasts, became apparent after 20 days (Fig. 1B). MC3T3-E1 cells in the presence or absence of HB-EGF We next wished to examine whether the observed upreg- (Fig. 1C). In contrast, most of the other genes examined ulation of HB-EGF and Epr affected osteoblast maturation were constitutively expressed in differentiating cells, sug- and/or differentiation using recombinant human HB-EGF gesting that Twist2 could be involved in the downregulation (rHB-EGF) and Epr (rEpr). When MC3T3-E1 cells were of Alp and Ocn downstream of EGFR signaling. Accord- grown in osteogenic media supplemented with rHB-EGF, ingly, Twist2 mRNA expression was suppressed by the both ALP and mineralizing activities were substantially treatment of cells with either a MEK (PD98059) or PI3- decreased, and there was an associated downregulation of kinase inhibitor (LY294002) (Fig. 2B). These data suggest Alp and Ocn mRNA expression (Fig. 1B and 1C). ALP that the induction of Twist2 expression is mediated by the activity was also decreased when MC3T3-E1 cells were MEK and/or PI3K pathway(s). Further analysis was carried differentiated in the presence of rEpr (data no shown), indi- out to examine the effect of EGFR signaling on Runx2 cating that EGFR signaling downregulates differentiation activity using a reporter plasmid in which luciferase expres- signals in MC3T3-E1 cells. All ErbB-family receptors can sion is driven by the Alp promoter, which contains a Cbfa1/ activate Ras and the downstream MAPK signaling cascade Runx2-binding site (pALP(–1838/+81)-luc) (Harada et al., consisting of Raf, MEK, and ERK (Montagut and Settleman, 1999). Treatment of cells with rHB-EGF significantly sup- 2009). Accordingly, rHB-EGF stimulation induced ERK pressed luciferase activity (Fig. 2C). When cells were trans- phosphorylation in MC3T3 cells (Fig. 1D). The ability of fected with ERK1/2, Constitutively active MEK1 (MEK HB-EGF to inhibit osteoblast differentiation was abrogated CA) and Twist2, luciferase activity was also suppressed, when either a neutralizing antibody against human HB-EGF suggesting that HB-EGF induced Twist2 and MAPK signal- (αHB-EGF) or an EGFR inhibitor (AG1478) were included ing inhibited osteoblast differentiation by suppressing

55 T. Nakamura et al.

Fig. 1. EGFR signaling suppresses osteoblast differentiation through Ras. A) Expression of EGF family members mRNA in osteoblasts. MC3T3-E1 cells were induced to differentiate into osteoblasts, and the mRNA expression of EGF family members was monitored by RT-PCR at the indicated time points. GAPDH was used as an internal control. Hb-egf, heparin-biding EGF-like growth factor; Tgfα, transforming growth factor-α; Areg, amphiregulin; Epr, epiregulin; Btc, betacellulin. P, positive control. mRNA from mouse heart, liver, and lung was used as a positive control. B) Effect of rHB-EGF on osteoblastogenesis. MC3T3-E1 cells were seeded on 24-well plates, and osteoblast differentiation was induced. Alkaline phosphatase (ALP) activity and mineralizing activity were measured by ALP staining (upper panel) and Alizarin red staining (lower panel), respectively, at the indicated time points. C) Expression of osteoblastic marker genes in MC3T3-E1 cells. Cells were cultured in osteogenic media in the presence of rHB-EGF, and the mRNA expression of the indicated genes was monitored by RT-PCR. ColI, Collagen type I; Alp, alkaline phosphatase; Opn, osteopontin; Ocn, osteocalcin. GAPDH was used as an internal control. D) Activation of ERK in osteoblast differentiation. The phosphorylated form of ERK was visualized by Western blotting. MC3T3-E1 cells were stimulated with rHB-EGF for 5 min in the presence of the indicated inhibitors. A.A., ascorbic acid; HB, rHB-EGF; αHB, anti-human HB-EGF neutralizing antibody; PD, PD98059, MEK inhibitor; AG, AG1478, EGFR inhibitor. E) MC3T3-E1 cells were cultured in osteogenic media stimulated with rHB-EGF and indicated inhibitors as shown in D) for 3 days and ALP staining was carried out. F) Involvement of Ras in osteoblast differentiation. A dominant-negative form of Ras was expressed in MC3T3-E1 cells, and ALP staining was performed three days after osteogenic stimulation in the presence or absence of rHB-EGF. Mock, empty vector.

Runx2 transcriptional activity. When cells were treated with study EGFR signaling induced Smad1 phosphorylation at PD98059, the suppression of luciferase activity by rHB- its linker region through the ERK/MAPK pathway, and EGF was not restored (Fig. 2C). LY294002 inhibited Runx2 BMP signaling inhibited the nuclear translocation of Smad1 transcriptional activity even in the absence of rHB-EGF, in R-1B/L17 cells transiently co-transfected with Smad1 suggesting that PI3K was essential for the osteoblast differ- and BMP receptors (Kretzschmar et al., 1997). Consistent entiation. These data suggested that ERK and Twist2 are with these results, BMP2 stimulation of MC3T3-E1 cells sufficient to inhibit Runx2 activity, MAPK signaling is not enhanced the nuclear localization of Smad1, and this was essential for the inhibition of differentiation by HB-EGF. suppressed by rHB-EGF treatment (Fig. 3A). Inhibition of Runx2 integrates BMP/Smad1 signaling through the for- MEK signaling with PD98059 restored Smad1 nuclear mation of a Runx2-Smad1 complex in a specific region of localization to a level comparable to that observed in differ- the nucleus (Lian et al., 2006). Additionally, in a previous entiating cells (Fig. 3A), and this was confirmed by sub-

56 EGFR Signaling in Osteoblasts

Fig. 2. EGFR signaling inhibits Runx2 activity through the induction of Twist2. A) Induction of Runx2 regulatory genes by rHB-EGF. The expression of genes known to be under the control of Runx2 was monitored in rHB-EGF-treated MC3T3-E1 cells by RT-PCR. GAPDH was used as an internal control. B) The effect of inhibitors of Twist2 induction. MC3T3-E1 cells were cultured for one day with the indicated inhibitors, and the expression of Twist2 was measured by RT-PCR. PD, PD98059, MEK inhibitor; LY, LY294002, PI3K inhibitor. C) A reporter assay using a plasmid containing the mouse Alp promoter (–1838/+81). Cells were transfected with the pALP(–1830/+81)-luc plasmid and cultured for 48 h in the absence (AA–) or presence (AA+) of ascorbic acid (AA), and the effect of rHB-EGF (HB+) on luciferase activity was quantified. ERK1 and ERK2 (ERK1/2), constitutively active MEK1 (MEK CA) and Twist2 (Twi2) were co-transfected with pALP-luc plasmid and cells were treated with PD98059 (PD) and LY294002 (LY) when needed. All experiments were performed in triplicate. Bars represent the means±SEM (n=3). *p<0.05 versus AA+. Vec: vector cellular fractionation experiments (Fig. 3B). osteoblast differentiation is inhibited by ERK signaling These findings suggest that the normal induction of downstream of HB-EGF. However, treatment of cells with twist2 expression and Smad translocation that occurs during both PD98059 and rHB-EGF did not lead to greater osteo-

57 T. Nakamura et al.

Fig. 3. EGFR signaling inhibits Runx2 activity by inhibiting Smad nuclear localization. A) Intracellular localization of Smad1. Localization of Smad1 in control and rHB-EGF-treated cells was examined by fluorescent microscopy using anti-Phospho-Smad1. Nuclei were counterstained with Hoechst dye. Bar, 100 μm. B) Nuclear localization of Smad1 determined by subcellular fractionation. Nuclear extracts and cytoplasmic fractions were prepared from the cells 6 h after stimulation, and were immunoblotted using anti-Phospho-Smad1 antibody. AA, ascorbic acid; HB, rHB-EGF; PD, PD98059. C) ALP activity in PD98059-treated cells. MC3T3-E1 cells were cultured with the indicated concentrations of PD98059 in the presence of rHB-EGF (HB+). ALP staining was performed after three days of culture.

58 EGFR Signaling in Osteoblasts

Fig. 4. EGFR signaling enhances osteoblast proliferation. A) Cell proliferation assay. The number of viable cells after rHB-EGF treatment in the absence or presence of ascorbic acid (AA– or AA+, respectively) was counted by a WST assay. Bars represent the means±SEM (n=3). *p<0.01. B) Expression profiles of cell cycle regulatory genes in HB-EGF-treated cells. The expression of Cyclin D1, p21, p27, and Alp mRNA was monitored by RT-PCR at the indicated times (left panel). Alp was used as a marker of differentiation. Western blot analysis using antibodies against the indicated gene products (right panel). GAPDH and β-actin were used as internal controls. C) The effect of p27 on cell proliferation. MC3T3-E1 cells were infected with either an empty retroviral vector (mock) or a vector expressing p27, and cell proliferation was examined by a WST assay. Bars represent the mean±SEM (n=3). *p<0.01. D) The effect of exogenous p27 expression on differentiation. Cells prepared as in C) were stimulated by BMP2, rHB-EGF, or both as indicated, and ALP staining was performed after three days. blast differentiation (Fig. 3C). Thus, ERK signaling contrib- stream of receptors other than EGFR is involved in the utes to the regulation of Twist2 expression and Smad inhibition of osteoblast differentiation. localization, but this is not sufficient to explain the inhibitory effects of EGFR signaling on osteoblast differentiation. EGFR signaling modulates cell cycle regulation and Interestingly, cells treated with PD98059 and grown in enhances cell proliferation osteogenic media demonstrate enhanced Runx2 and ALP activity (Fig. 3C), suggesting that ERK signaling down- EGFR signaling exhibits mitogenic activity, and we next

59 T. Nakamura et al. examined the effect of EGFR signaling on MC3T3-E1 cell MC3T3-E1 cells was not changed in osteogenic media for proliferation. rHB-EGF enhanced MC3T3-E1 cell prolifera- 10 days (Varanasi et al., 2009), suggesting that ColI is not tion (Fig. 4A), and RT-PCR and immunoblotting analyses suitable as a marker gene in MC3T3-E1 cells at least for 10 of cell cycle regulatory genes in rHB-EGF-treated MC3T3 days after differentiation stimulation. cells showed that CycD1, Rb, and p21 expression were HB-EGF induces Ras-mediated intracellular signaling induced, while p27 was suppressed at the protein level (Fig. to block osteoblast differentiation and maturation. ERK, 4B). While exogenous expression of p27 in MC3T3-E1 which is downstream of Ras, functions in multiple signaling cells suppressed proliferation (Fig. 4C), it did not affect the pathways, and Runx2 is a transcription factor with essential ability of BMP2 or rHB-EGF to promote or inhibit osteo- roles in osteoblast differentiation during intramembranous blast differentiation, respectively (Fig. 4D). Thus, our data and endochondrial bone formation (Karsenty and Wagner, indicate that EGFR signaling enhances cell proliferation, 2002). In this study, we showed that ERK downregulates but inhibition of proliferation per se is not sufficient to Runx2 transcriptional activity, and that the inhibition of induce differentiation. ERK reduced the induction of Twist2 and restored impaired pSmad nuclear localization by HB-EGF. Twist2 was also shown to suppress Runx2 activity. Expression of DN-Ras Discussion completely abrogated the HB-EGF-induced inhibition of differentiation, but the inhibition of ERK signaling with the EGFR family signaling networks have been implicated in MEK inhibitor PD98059 did not restore ALP activity, sug- regulating bone homeostasis, but the detailed mechanism by gesting that ERK and Twist2 are sufficient for the inhibition which they act in this compartment has remained elusive. of osteoblast differentiation; however, ERK is not essential Contributing to the difficulty is the presence of multiple for the inhibition of osteoblast differentiation by HB-EGF. genes belonging to the EGF and EGFR families, and differ- This raises the possibility that an additional signaling ent receptor-ligand combinations may have different activi- molecule/pathway contributes to HB-EGF function. Indeed, ties. This is more than a theoretical possibility because treatment of cells with the PI3K/AKT inhibitor LY294002 EGF, TGF-α, HB-EGF, BTC, and EPR expression are completely blocked the induction of Twist2 mRNA by induced in the rat osteoblastic cell line UMR106 and pri- rHB-EGF. These data suggested that osteoblast differentia- mary rat osteoblasts (Qin et al., 2003; Qin et al., 2005). tion is controlled by multiple signaling pathways down- However, when UMR106 cells are stimulated with the stream of different stimuli. bone-forming agent parathormone (PTH), only AR, HB- EGF family members are prototypical growth factors EGF, and TGF-α were rapidly induced (Qin et al., 2005; and induce cell proliferation and migration. In the present Schneider et al., 2009a). In this study, we found that only study, HB-EGF treatment of MC3T3-E1 preosteoblast cells Hb-egf and Epr were induced in the mouse preosteoblast enhanced cell proliferation, and this was accompanied by cell line MC3T3-E1, and we focused our analysis on EGFR modulated expression of the cell cycle regulatory genes signaling in preosteoblasts and analyzed its role in osteo- p21, cyclin D1, and p27. Expression of p21 and cyclin D1 blastogenesis. Using an in vitro differentiation system, we was upregulated, whereas p27 expression was downregu- showed that EGFR signaling induces two distinct signals in lated. These data are consistent with a previous study show- preosteoblasts: differentiation-suppressing and proliferation- ing that p21 is expressed in proliferating cells during promoting signals. osteoblast development in vitro, but p27 is induced during The transient induction of HB-EGF and Epr suppressed the immediate post-proliferative stage of osteoblast matura- the expression of Alp and Ocn through osteoblast differen- tion and acts as a key regulator of the transition from osteo- tiation, suggesting that they may participate in negative blast cell proliferation to differentiation (Drissi et al., 1999). feedback during osteoblast differentiation. Although the We found that p27 was reduced at the protein level follow- expression of Alp and Ocn was suppressed by HB-EGF, the ing BMP2 or HB-EGF treatment, but p27 mRNA levels expression of ColI and Opn, which are also known as osteo- were unchanged, suggesting that p27 is regulated post- blastic marker genes, was not affected. In this study, we translationally in preosteoblasts. However, the exogenous focused on the early stage of differentiation and Opn is expression of p27 suppressed cell proliferation, and its over- induced in the late stage of differentiation. Furthermore, expression was not sufficient to induce differentiation. different from ALP and Ocn which are known to be tran- Thus, the functional relationship between proliferation and scriptional target of Runx2, Opn expression was regulated differentiation in osteoblastogenesis remains unclear. by Ets transcription factors (Vary et al., 2000). Taken In this study, our data show that EGFR signaling nega- together, constitutive expression of Opn would be due to the tively regulates osteoblast differentiation via multiple path- differentiation stage which we observed, and the difference ways downstream of Ras, and that proliferation is affected of transcriptional factor would be a reason that the expres- by the downregulation of p27. In vivo, EGFR knockout mice sion of Opn was not affected by HB-EGF. ColI is also well and amphiregulin knockout mice showed the phenotype of known for the osteoblastic marker, but the expression in decreased bone mass. These results suggest three possibili-

60 EGFR Signaling in Osteoblasts ties to the role of EGFR signaling in bone formation: Endocrinology, 115: 867–876. 1) EGFR signaling promotes preosteoblast proliferation. Iwamoto, R. and Mekada, E. 2006. ErbB and HB-EGF signaling in heart 2) EGFR signaling promotes osteoblast differentiation. development and function. Cell Struct. Funct., 31: 1–14. Karsenty, G. and Wagner, E.F. 2002. Reaching a genetic and molecular 3) EGFR signaling promotes both proliferation and dif- understanding of skeletal development. Dev. Cell, 2: 389–406. ferentiation of osteoblast. In vitro study showed that EGF Karsenty, G. 2006. Convergence between bone and energy homeostases: suppressed osteoblast differentiation and enhanced pre- leptin regulation of bone mass. Cell Metab., 4: 341–348. osteoblast proliferation (Schneider et al., 2009b). It is possi- Karsenty, G., Kronenberg, H.M., and Settembre, C. 2009. Genetic control ble that this discrepancy of the results between in vivo and of bone formation. 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