Pei Y et al / Acta Pharmacol Sin 2003 Oct; 24 (10): 975-984

Expression of core binding factor 1 up-regulated by IGF-I, GM-CSF, and EGF through MAPK pathway in MC3T3-E1 and C2C12 cells¹

PEI Yu^2,3, MENG Xun-Wu², ZHOU Xue-Ying², XING Xiao-Ping², XIA Wei-Bo^2,4

²Department of Endocrinology, Peking Union Medical College Hospital, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100730

³Department of Endocrinology, the Second Artillery Forces General Hospital, PLA, Beijing 100088, China

¹ Project partly supported by Scientific Research Foundation of Beijing Medical Development (Capital ZD 199908).

⁴ Correspondence to XIA Wei-Bo MD. Phn/Fax 86-10-6529-6566. E-mail xiawb@csc.pumch.ac.cn

Received 2003-02-20 Accepted 2003-07-03

KEY WORDS transcription factors; osteoblast; insulin-like growth factor I; granulocyte-macrophage colony-stimulating factor; epidermal growth factor; mitogen-activated protein kinases

ABSTRACT

AIM: To study the regulating function and mechanism of insulin-like growth factor-I (IGF-I), granulocyte-macrophage colony-stimulating factor (GM-CSF), and epidermal growth factor (EGF) on murine core binding factor 1 (Cbfa1) gene expression. METHODS: Luciferase reporter gene method and RT-PCR technique were used to examine the effects of these growth factors on the promoter activity and mRNA expression of Cbfa1 gene in MC3T3-E1 and C2C12 cells. RESULTS: IGF-I (from 1 nmol/L to 1 µmol/L), GM-CSF (100 nmol/L), and EGF (1 µmol/L) increased the luciferase expression in MC3T3-E1 cells (P<0.05). And mitogen-activated protein kinase (MAPK) inhibitor, PD 98059 (10 µmol/L), completely blocked IGF-1, GM-CSF, and EGF-induced expression of Cbfa1 promoter activity (P<0.01). In C2C12 cells, IGF-I (from 1 nmol/L to 10 µmol/L), GM-CSF (100 nmol/L and 1 µmol/L), and EGF (100 nmol/L) enhanced the expression of luciferase reporter plasmid driven by mCbfa1 promoter (P<0.05). Addition of PD 98059 also blocked the stimulatory effects of these growth factors on Cbfa1 promoter activity (P<0.01). Moreover, Cbfa1 mRNA expression was significantly increased after treatment with IGF-I (1 nmol/L, 100 nmol/L), GM-CSF (100 nmol/L, 1 µmol/L), and EGF (1 µmol/L, 100 nmol/L) in MC3T3-E1 and C2C12 cells, respectively (P<0.05). These stimulatory effects of IGF-I, GM-CSF, and EGF on Cbfa1 mRNA expression were abolished by PD 98059. CONCLUSION: IGF-I, GM-CSF, and EGF could increase the promoter activity and the mRNA expression of murine Cbfa1 gene in MC3T3-E1 and C2C12 cells. These stimulatory effects might be mediated by activating the intracellular MAPK-dependent signaling pathway.

INTRODUCTION

Core binding factor 1 (Cbfa1), also known as Runx2, is an osteoblast-related transcription factor that is essential for bone formation^[1]. In 1995, Ducy and Karsenty^[2] characterized osteoblast-specific element 2 (OSE2), a cis-acting sequence in the promoter of the murine osteocalcin gene 2 (mOG2), which was required for osteocalcin gene expression in osteoblastic cells^[3]. The factor binding OSE2, initially termed osteoblast-specific factor 2 (Osf2), was subsequently identified as Cbfa1, a member of the Cbf/Runt family of transcription factors, which share a DNA binding motif that is homologous to the Drosophila protein, Runt^[1]. Cbfa1 is essential for the differentiation of osteoblasts from mesenchymal precursors, since homozygous Cbfa1-1- mice show a complete lack of functional osteoblasts^[4,5]. Moreover, this transcription factor is required for bone matrix synthesis by differentiated osteoblasts^[6], indicating that it regulates osteoblast gene expression at multiple levels.

Cbfa1 is an essential transcription factor for osteoblast differentiation and bone formation, however, the signaling pathways regulating Cbfa1 have not been clarified. Cbfa1 was phosphorylated and activated by the mitogen-activated protein kinases (MAPK) pathway^[7]. MAPK pathway is a major point of convergence for a variety of intracellular signals initiated by ECM-interin interaction, mechanical stimulation and many growth/differentiation factors binding to receptor tyrosine kinases^[8]. These studies led us to speculate that the growth factors such as IGF-I, GM-CSF, and EGF maybe regulate Cbfa1 gene expression through a pathway requiring MAPK activity. To test this hypothesis, the actions of IGF-I, GM-CSF, and EGF on Cbfa1 gene were examined in murine MC3T3-E1 preosteoblast cells and C2C12 myoblast cells.

MATERIALS AND METHODS

Reagents Recombinant murine IGF-I, GM-CSF, and EGF were purchased from R&D Systems (Inc Abingdon, UK). PD 98059, a specific inhibitor of MAPK pathway, was purchased from Sigma (St Louis, MO, USA). LipofectAMINE 2000 transfection reagent was obtained from Invitrogen (Life Technologies, Carlsbad, California).

Cell culture Murine preosteoblastic cell line MC3T3-E1 cells and myoblastic cell line C2C12 cells, purchased from Riken cell bank (Japan), were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco-BRL, Life Technologies, Grand island, NY), 10 % fetal bovine serum (Hyclone Laboratories, Logan, UT, USA), and supplemented with antibiotics (benzyl-penicillin 100 kU/L and streptomycin 100 kU/L) under a 5 % CO₂-95 % air atmosphere at 37 ºC.

Plasmid construction Mouse Cbfa1 promoter luciferase reporter plasmid (named mP696-Luc) contained 696 bp (-641_+55) murine Cbfa1 promoter region^[9] and this region was cloned in PGL3-basic vector (Promega Corp, Madison, WI). To generate a targeting vector, genomic clones containing Cbfa1 promoter (AB013129, nucleotides +6018/6713) region were isolated from the genome of C57BJ mouse white blood cells (Qiagen, Valencia, CA) by PCR. PCR reaction was amplified with the following primers: sense, 5'-AGCACTGTTGCTCAGAACGCCACACACCT-3'; anti-sense, 5'-TCCTGGAGAAAGTTTGCACCGCACTTG-3'. The gel-purified blunt-ending PCR fragment was ligated into T easy Vector (Promega Corp, Madison, WI), named mP696-Teasy. The PCR product and plasmid mentioned above were confirmed by sequencing. The fragment of Cbfa1 promoter region was then generated by PCR from mP696-Teasy using a 5' primer with MluI site (5'-ATAGacgcgtAGCACTGTTGCTCAGA 3')and a 3' primer with XhoI site (5'GTACctcgag-TCCTGGAGAAAGTTTGC-3'). After digesting with MluI and XhoI, the fragment was ligated into the MluI/XhoI sites of PGL3 Basic vector. The resulting product, mP696-Luc, contains nucleotides -641_+55 (696 bp) of the reported Cbfa1 promoter.

Transfections and luciferase analysis All cell lines were plated in 24-well plates at a density of 1×10⁵ cells/cm². After 24 h, cells were transiently transfected with 0.5 µg mP696-Luc plasmid using the DNA-lipid complex, LipofectAMINE 2000, according to the manufacture's protocol. To assess transfection efficacy, and to normalize the firefly luciferase signal expressed by mP696-Luc plasmid, 20 ng of PRL SV40 (Promega Corp, Madison, WI), which encodes a Renilla luciferase gene downstream of a minimal SV40 promoter, was systematically added to the transfection mix. In the positive and negative experiments, Cbfa1 constructs were replaced by pGL3-control plasmid (Promega Corp, Madison, WI) and empty pGL3-basic vector. Sixteen hours after transfection, cells were washed and culture medium was changed to serum free medium, HyQ-CCM5 (Hyclone, Logan, Utah), then cells were treated with growth factors and/or PD 98059 and cultured for an additional 24 h. Luciferase assays were performed with the Dual Luciferase Assay Kit (Promega Corp, Madison, WI), according to the manufacturer's instructions. A 50 µL quantity of cell lysate was assayed first for firefly luciferase and then for Renilla luciferase using a luminometer (Analytical luminoscencelab corp, USA). Firefly luciferase activity was normalized to Renilla luciferase activity. The mean corrected luciferase activity of the control group was defined as 1, and the relative luciferase activity (RLA) of the treat ment groups was obtained as compared to the control group.

Reverse transcriptase-polymerase chain reaction (RT-PCR) MC3T3-E1 and C2C12 cells were plated in 6-well plates at a density of 1×10⁵ cells/cm². After treatment with different growth factors and/or PD 98059 for 1-6 d, total RNA was isolated using Trizol reagent (Gibco-BRL, Life Technologies, Grand island, NY) following the manufacturer's instructions. For RT, a 25-µL reaction contained total RNA 2 µg, oligodT₍₁₅₎ primer 12.5 mg/L, each dNTP 500 µmol/L, 25 units of rRNasin ribonuclease inhibitor, and 200 units of M-MLV reverse transcriptase (Promega Corp, Madison, WI).

For PCR amplification, each 50-µL reaction contained 4 µL of RT aliquot, each dNTP 200 µmol/L, each primer 0.4 µmol/L, MgCl₂ 1.5 mmol/L, and 2.5 units of Taq DNA polymerase (TaKaRa Biotechnology Co, Ltd Dalian). The PCR conditions were as follows. After 5 min of preincubation at 95 ºC, amplification was performed for 35 cycles consisting of 45 s of denaturing at 95 ºC, 45 s of annealing at 60 ºC, and 2 min of extension at 72 ºC. The primers used for murine Cbfa1 were as follows. Sense: 5'-TTTAGGGCGCATTCCTCATC-3' (NM009820, nucleotides +969/988); antisense: 5'-TGTCCTTGTGGATTAAAAGGACTTG-3 (NM009820, nucleotides +1046/1070). As an internal control, the PCR analysis was also performed with the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) specific primers. Sense: 5'-TCCACTCACGGCAAATTCA-ACG-3' (M32599, nucleotides, +191/213); antisense: 5'-TAGACTCCACGACATACTCAGC-3' (M32599, nucleotides, +314/335). The RT-PCR products were separated by electrophoresis on 3 % agarose gels and the products of the expected size (102 bp of mCbfa1 and 145 bp of mGAPDH) were obtained and sequenced. The mCbfa1 signals were normalized to the GAPDH signals in the same reaction.

Statistical analysis All data were expressed as mean±SD of three wells per group. Each experiment was repeated at least three times. Statistical analyses were determined by Student's t-test.

RESULTS

IGF-I, GM-CSF, and EGF stimulate Cbfa1 promoter activity and the MAPK pathway is involved The effects of IGF-I, GM-CSF, and EGF on the promoter activity of murine Cbfa1 in MC3T3-E1 and C2C12 cell lines were examined using a Cbfa1 promoterluciferase reporter vector (mP696-Luc) by transient transfection assay (Fig 1, n=3).

Treatment of MC3T3-E1 cells with IGF-I (1 nmol/L-1 µmol/L) resulted in 83 % (P<0.01), 59 % (P< 0.05), 48 % (P<0.01), and 43 % (P<0.01) increases in Cbfa1 promoter activity over that found in control cells (Fig 1A). Compared with control group, the RLA of GM-CSF (100 nmol/L, Fig 1B) group increased by 31 % (P<0.05). Treatment of MC3T3-E1 cells with 1 µmol/L EGF resulted in a 51 % (P<0.05) increase in Cbfa1 promoter activity (Fig 1C).

In C2C12 cells, these growth factors had similar effects with MC3T3-E1 on the expression of luciferase reporter plasmid driven by the mp696-Luc. IGF-I (1 nmol/L-10 µmol/L) enhanced the RLA by 48 % (P<0.05), 68 % (P<0.01), 166 % (P<0.01), 55 % (P<0.05), and 46 % (P<0.01) compared with that of control group(Fig 1D). Treatment with GM-CSF (100 nmol/L and 1 µmol/L) resulted in 34 % (P<0.01) and 29 % (P<0.01) increases in Cbfa1 promoter activity over that found in control cells (Fig 1E). Compared with control group, the RLA of EGF (100 nmol/L) enhanced by 65 % (P<0.05, Fig 1F).

Treatment of PD 98059 (10 µmol/L), a specific inhibitor of MAPK, resulted in 20 %-30 % decreases in Cbfa1 promoter activity compared with that of control group (P<0.05). The RLA levels of growth factor+ PD 98059 groups were significantly lower than those of growth factor groups, respectively. These results indicated that PD 98059 suppressed basal and completely blocked IGF-I, GM-CSF, and EGF-induced expression of Cbfa1 promoter activity not only in MC3T3-E1 cells but also in C2C12 cells.

Fig 1. Relative luciferase activities (RLA) of Cbfal pomoter (mP696-Luc) in MC3T3-E1 and C2C12 cells treated with different concentrations of growth factors (GF) and/or PD 98059 (10 µmol/L). MC3T3-E1 (A, B, C) or C2C12 (D, E, F) cells were transfected with the mP696-Luc reported plasmid and PRL-SV40 expression plasmid. Sixteen hours after transfection, cells were cultured in various concentrations of IGF-I, GM-CSF, and EGF with or without PD 98059 for additional 24 h. Forty hours after transfection, cells were harvested and reported activities were measured. Values normalized for transfection efficiency are shown as fold induction relative to basal promoter activity as described in materials and methods. n=3. Mean±SD. ^bP<0.05, ^cP<0.01 vs control group (the cells without any treatment). ^fP<0.01 GF+PD 98059 groups vs GF groups.

Induction of Cbfa1 gene expression by IGF-I, GM-CSF, and EGF and the MAPK pathway is involved To assess the expression of murine Cbfa1 gene in response to growth factors, MC3T3-E1 and C2C12 cells were treated with IGF-I (1 nmol/L, 100 nmol/L), GM-CSF (100 nmol/L, 1 µmol/L), EGF (1 µmol/L, 100 nmol/L) respectively, and/or PD 98059 (10 µmol/L) from 1 to 6 d, then the mRNA levels of Cbfa1 gene were detected by RT-PCR at different time points (Fig 2, n=3). There were no significant differences in basal mRNA expression of Cbfa1 gene from 1 to 6 d both in MC3T3-E1 and C2C12 cells.

In MC3T3-E1 cells, Cbfa1 expression was induced significantly by IGF-I treatment from 1 to 6 d (d 1: 101±3; d 2: 176±14; d 3: 217±29; d 4: 191±19; d 5: 120±6; d 6: 112±12, P<0.01 vs d 0: 69±0.4, respectively). GM-CSF caused increases of Cbfa1 mRNA at d 4 and d 5 compared with d 0 (d 4: 87±7, P<0.01; d 5: 88±5, P<0.01 vs d 0: 68±2). Treatment with EGF resulted in increase of Cbfa1 gene expression at d 2 (d 2: 96±8 vs d 0: 70±2, P<0.01). These significant differences at each time points of those groups mentioned above were found not compared with d 0, but also with the control group (Fig 2 A, B, C, and Fig 3).

In C2C12 cells, Cbfa1 expression was increased significantly by IGF-I treatment from 1 to 6 d (d 1: 121±6; d 2: 194±7; d 3: 110±5; d 4: 108±5; d 5: 105±2; d 6: 106±5, P<0.01 vs d 0: 30±3, repectively). GM-CSF enhanced the Cbfa1 mRNA level from 1 to 6 d (d 1: 85±5; d 2: 73±5; d 3: 71±7; d 4: 67±4; d 5: 69±6; d 6: 69±9, P<0.01 vs d 0: 30±4, respectively). Treatment of EGF resulted in increases in Cbfa1 gene expression at d 2 and d 3 (d 2: 40±2; d 3: 39±1, P<0.05 vs d 0: 30±5). The Cbfa1 mRNA levels of these groups mentioned above were significantly higher than those of control group (Fig 2 D, E, F, and Fig 4).

Treatment with PD 98059 alone resulted in decreases of murine Cbfa1 gene expression both in MC3T3-E1 cells (d 1: 44±5; d 2: 50±8; d 3: 49±10; d 4: 47±9; d 5: 46±7; d 6: 46±10, P<0.05 or P<0.01 vs d 0: 69±0.4) and in C2C12 cells (d 1: 19±2; d 2: 22±3; d 3: 22±5; d 4: 21±4; d 5: 20±3; d 6: 20±4, P<0.05 or P<0.01vs d 0: 30±3) compared with d 0. Moreover, The Cbfa1 mRNA levels of growth factors plus PD 98059 groups were significantly lower than those of growth factors groups. These results indicated that PD 98059 blocked IGF-I, GM-CSF, and EGF-induced Cbfa1 gene expression not only in MC3T3-E1 cells but also in C2C12 cells.

Fig 2. The mRNA expression of Cbfa1 gene in MC3T3-E1 and C2C12 cells treated with growth factors (GF) and/or PD 98059 (10 µmol/L). MC3T3-E1 (A, B, C) or C2C12 (D, E, F) cells were cultured in DMEM (2 % FBS) with or without IGF-I, GM-CSF, EGF, and PD 98059 for 0-6 d. Then total RNA was isolated using Trizol reagent and the mRNA expression levels of Cbfa1 gene were detected by RT-PCR. The RT-PCR products were separated by electrophoresis on 3 % agarose gels and mCbfa1 signals (102 bp) were normalized to the GAPDH signals (146 bp) in the same reaction. ^bP<0.05 vs d0 of GF or PD 98059 groups. ^eP<0.05 vs control groups. ⁱP<0.05 vs GF groups. ^lP<0.05 vs PD 98059 groups.

Fig 3. mRNA Expression of Cbfa1 gene in MC3T3-E1 cells treated with growth factors (GF) and/or PD 98059 (10 µmol/L). MC3T3-E1 cells were cultured in DMEM (2 % FBS) with or without IGF-I, GM-CSF, EGF, and PD 98059 for 0-6 d. Then total RNA was isolated using TriZol reagent and the mRNA expression of Cbfa1 gene was detected by RT-PCR. The RT-PCR products were separated by electrophoresis on 3 % agarose gels and mCbfa1 signals (102 bp) were normalized to the GAPDH signals (146 bp) in the same reaction. M: marker.

Fig 4. mRNA expression of Cbfa1 gene in MC3T3-E1 cells treated with growth factors (GFs) and/or PD 98059 (10 µmol/L). C2C12 cells were cultured in DMEM (2 % FBS) with or without IGF-I, GM-CSF, EGF, and PD 98059 for 0-6 d. Then total RNA was isolated using Trizol reagent and the mRNA expression of Cbfa1 gene was detected by RT-PCR. The RT-PCR products were separated by electrophoresis on 3 % agarose gels and mCbfa1 signals (102 bp) were normalized to the GAPDH signals (146 bp) in the same reaction. M: marker.

DISCUSSION

Osteoblasts originate from common progenitors, which are capable of differentiating into other mesenchymal cell lineages such as chondrocytes, myoblasts, bone marrow stromal cells, etc^[10-13]. And various hormones and cytokines participate this process^[14-18]. Among those, IGF-I, GM-CSF, and EGF are the most potent inducers and stimulators, since they are synthesized by osteoblast lineage and present in substantial concentration in bone tissue ^[19-21]. Because none of these growth factors is specific for cells of the osteoblast lineage. Therefore, mechanisms to induce skeletal tissue specificity might be mediated by some specific intracellular molecules.

During the past several years, our molecular understanding of osteoblast biology has made rapid progress due to the characterization of the function of one molecule, Cbfa1. This member of the runt/Cbfa family of transcription factors was first identified as the nuclear protein binding to an osteoblast-specific cis-acting element activating the expression of osteocalcin, the most osteoblast-specific gene^[2]. Cbfa1 was then shown to regulate the expression of all the major genes expressed by osteoblasts, such as osteocalcin, alkaline phosphatase (ALP)^[22], 1 and 2(I) collagen^[23], osteopontin^[1], and osteoprotegerin ligand^[24,25]. Indeed, Cbfa1 is a critical molecule not only for osteoblast differentiation but also for osteoblast function. However, The regulation of Cbfa1 gene expression and Cbfa1 protein activity by systemic hormones and local factors are just beginning to be understood. It has been shown that Cbfa1 expression and activity are regulated by PTH^[26], 1,25-(OH)₂-vitamin D₃^[27], dexamethasone^[28], bone morphogenetic protein (BMP)^[29], transforming growth factor-beta (TGF-)^[30], and inflammatory cytokine tumor necrosis factor alpha (TNF)^[31], etc.

Considering of the stimulatory effects of IGF-I, GM-CSF, and EGF on osteoblast differentiation, we speculated that Cbfa1 might play an important role in osteoblast differentiation regulated by these growth factors. So Cbfa1 promoter activity and mRNA expression induced by IGF-I, GM-CSF, and EGF were determined in the present study.

To investigate whether IGF-I, GM-CSF, and EGF regulate the promoter activity of Cbfa1 gene, we firstly cloned a mouse Cbfa1 promoter-luciferase reporter vector, mP696-Luc. Using the mP696-Luc vector, we provided evidence that Cbfa1 promoter activity was stimulated by IGF-I, GM-CSF, and EGF in both pre-osteoblastic (MC3T3-E1) and myoblastic (C2C12) cells by transient transfection assay.

To confirm the stimulatory effects of IGF-I, GM-CSF, and EGF on Cbfa1 gene expression, we next examined the mRNA expression of murine Cbfa1 gene in response to these growth factors. RT-PCR analysis indicated that these three growth factors could increase the levels of Cbfa1 mRNA respectively.

The skeleton is both a source and a repository for many growth factors, including IGF-I, which plays a key role in skeletal growth and remodeling. IGF-I plays an essential role in longitudinal bone growth in response to growth hormone exposure^[32,33]. Moreover, IGF-I is mitogenic for less differentiated bone cells, whereas it increases the synthesis of collagen and perhaps other matrix proteins by osteoblasts^[34,35]. Our results showed that treatment with IGF-I resulted in increases of Cbfa1 promoter activity and mRNA levels in both MC3T3-E1 and C2C12 cells. These findings suggest that the Cbfa1 may be involved in the process of osteoblasts differentiation induced by IGF-I. In contrast to our results with MC3T3-E1 and C2C12 cells, Ducy et al^[1] showed that IGF-I had no effect on Cbfa1 gene expression in the rat osteoblast-like osteosarcoma cell line, ROS17/2.8. These inconsistent results can be attributed to the use of different osteogenic cell lines at different stages of osteoblast differentiation. ROS17/2.8 cells are phenotypically more mature than MC3T3-E1 and C2C12 cells, which both require inductive signals provided by ECM before they will express osteoblast marker genes.

The other two growth factors, GM-CSF and EGF had similar effects with IGF-I on the expression of luciferase reporter plasmid driven by the mP696-Luc and the levels of Cbfa1 mRNA. Many studies have shown that GM-CSF is a stimulating factor of osteoclast^[36,37]. However, the stimulatory effect of GM-CSF on osteoblast differentiation has not been widely accepted. Postiglione et al^[38] found that SaOS-2 cells expressed GM-CSF receptor, in vitro treatment of SaOS-2 cells with recombinant human GM-CSF caused a decreased cell proliferation and an increased production of osteopontin, ALP and most but not all ECM components. But Modrowski et al^[39] reported that GM-CSF promoted the proliferation of human osteoblastic cells. Our results showed that GM-CSF stimulated Cbfa1 gene promoter activity and increased Cbfa1 mRNA levels, indicating that GM-CSF maybe regulate osteoblast differentiation through induction of Cbfa1 gene expression. The expression of EGF receptor on osteogenic cells indicates that EGF maybe regulate osteoblast differentiation directly. In vitro studies have demonstrated that treatment with EGF stimulates the proliferation of osteoprogenitor cells, inhibits collagen synthesis, down- regulates ALP activity of matured osteoblasts^[21]. In our study, two well-characterized cell lines of early differing stage of differentiation were chosen as experimental cell models. We found that EGF enhanced Cbfa1 gene expression in MC3T3-E1 and C2C12 cells. However, additional experiments should be done to gain further insight into the role of EGF on fully differentiated osteoblastic cells.

The present study showed that IGF-I, GM-CSF, and EGF stimulated Cbfa1 promoter activity and increased Cbfa1 mRNA levels. But little is known about the mechanisms regulating Cbfa1 gene expression by these growth factors. Cbfa1 protein was phosphorylated and activated by MAPK pathway^[7,40]. Since Cbfa1 gene expression is up-regulated by Cbfa1 itself^[1]. And a major route for IGF-I, GM-CSF, and EGF receptor signaling involves activation of the MAPK ERK kinases (MEK)/ERK branch of the MAPK pathway. We speculated that MAPK pathway might be required for the stimulatory effects of IGF-I, GM-CSF, and EGF on Cbfa1 gene. To test this hypothesis, PD 98059 was used in our experiment. PD 98059 is a potent, cell permeable and selective inhibitor of MAPK. It blocks the activation of MEK1, therefore inhibiting the subsequent phosphorylation and activation of MAPK. The present study demonstrated that the cbfa1 promoter activity and mRNA expression were both completely abolished by supplement with PD 98059 both in basic or after stimulation with IGF-I, GM-CSF, and EGF. These results raise the possibility that MAPK pathway is necessary for stimulation of Cbfa1 gene expression by IGFI, GM-CSF, and EGF. But two questions still remain regarding the regulation mechanisms of MAPK on Cbfa1 gene expression. Firstly, we do not yet know whether MAPK-dependent phosphorylation cascade regulates Cbfa1 gene expression directly. Further experiments should be done for resolving this question. For example, MAPK stimulator may be used, or we can examine directly whether Cbfa1 mRNA and promoter activity will be increased after transfection of cells with constitutively active MEK1, the kinase immediately before ERK1/2 in the MAPK cascade, thus more evidence can be obtained. Secondly, it is not currently clear whether the other subfamilies of MAPK are also involved in regulating Cbfa1 gene. Multicellular organisms have three well-characterized subfamilies of MAPK that control a vast array of physiological processes^[41]. These MAPK include the ERK, the c-Jun NH₂-terminal kinases (JNK), and the four p38 enzymes, p38, p38, p38, and p38. In the present study, only a specific MEK1/ERK inhibitor, PD98059, was used. So further studies should be conducted to clarify the role of JNK and P38 MAPK in regulating Cbfa1 gene expression.

All the data together, our results indicated that IGF-I, GM-CSF, and EGF could increase the promoter activity and the mRNA expression of murine Cbfa1 gene both in preosteoblastic cells (MC3T3-E1) and myoblastic cells (C2C12). And the MAPK pathway might be responsible for these growth factors' stimulatory effects.

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