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Article |
Critical role of the extracellular signal–regulated kinase–MAPK pathway in osteoblast differentiation and skeletal development
Correspondence to Renny T. Franceschi: rennyf{at}umich.edu
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The extracellular signal–regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway provides a major link between the cell surface and nucleus to control proliferation and differentiation. However, its in vivo role in skeletal development is unknown. A transgenic approach was used to establish a role for this pathway in bone. MAPK stimulation achieved by selective expression of constitutively active MAPK/ERK1 (MEK-SP) in osteoblasts accelerated in vitro differentiation of calvarial cells, as well as in vivo bone development, whereas dominant-negative MEK1 was inhibitory. The involvement of the RUNX2 transcription factor in this response was established in two ways: (a) RUNX2 phosphorylation and transcriptional activity were elevated in calvarial osteoblasts from TgMek-sp mice and reduced in cells from TgMek-dn mice, and (b) crossing TgMek-sp mice with Runx2+/– animals partially rescued the hypomorphic clavicles and undemineralized calvaria associated with Runx2 haploinsufficiency, whereas TgMek-dn; Runx2+/– mice had a more severe skeletal phenotype. This work establishes an important in vivo function for the ERK–MAPK pathway in bone that involves stimulation of RUNX2 phosphorylation and transcriptional activity.
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(adipose tissue; Adams et al., 1997). In bone, the ERK–MAPK pathway is a major conduit for conveying information about the extracellular environment to the nucleus, and has been implicated in the response of bone to a variety of signals, including hormone/growth factor stimulation (Hurley et al., 1996; Xiao et al., 2002b; Chen et al., 2004), extracellular matrix–integrin binding (Takeuchi et al., 1997; Xiao et al., 2002a), and mechanical loading (You et al., 2001).
Despite intensive investigation, the physiological role of the MAPK pathway in osteoblasts remains controversial, with some studies supporting a stimulatory role in osteoblast differentiation and others proposing that this pathway is inhibitory (for review see Schindeler and Little, 2006). For example, activation of 
2ß1 integrins by the type I collagen matrix of bone was reported to stimulate in vitro osteoblast differentiation via focal adhesion kinase activation of MAPK (Takeuchi et al., 1997; Xiao et al., 1998). Also, short-term pharmacological inhibition of MAPK signaling blocked osteoblast-specific gene expression in mature osteoblasts, whereas a constitutively active form of the MAPK intermediate, MEK1, was stimulatory (Xiao et al., 2002a). In related studies, we showed that RUNX2, which is an essential transcription factor for bone formation (Ducy et al., 1997), is required for cells to respond to MAPK in vitro. Both phosphorylation and transcriptional activity of RUNX2 were stimulated by the ERK–MAPK pathway, suggesting that this factor is a MAPK substrate and an important mediator of the MAPK response (Xiao et al., 2000; Franceschi et al., 2003). Also, treatment of MC3T3-E1 cells or primary cultures of marrow stromal cells with FGF2 induced RUNX2 phosphorylation and osteocalcin expression by a process requiring MAPK activation (Xiao et al., 2002b). In contrast, other in vitro studies reached an opposite conclusion, showing that the ERK–MAPK pathway can antagonize osteoblast functions. Thus, epidermal growth factor stimulation of ERK prevented SMAD1 activation by bone morphogenetic proteins in epithelial cells (Kretzschmar et al., 1997). This inhibition was explained by ERK-dependent phosphorylation of a distinct site in the linker region of SMAD1 that led to the exclusion of this molecule from the nucleus (Kretzschmar et al., 1999). Also, chronic treatment of osteoblast cultures with MAPK inhibitors was reported to actually stimulate osteoblast differentiation, whereas ERK activation was inhibitory (Higuchi et al., 2002; Nakayama et al., 2003).
The conflicting results of in vitro studies emphasize the need to address the role of the ERK–MAPK pathway in osteoblast function in vivo. To this end, a unique transgenic strategy was developed involving selective expression of constitutively active and dominant-negative forms of the MAPK intermediate MEK1 in osteoblasts using the osteocalcin promoter. With this approach, ERK–MAPK activation was found to stimulate osteoblast differentiation and skeletal development through a pathway involving RUNX2.
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In contrast to the results of proliferation studies, Mek-dn and -sp transgenes dramatically altered calvarial cell differentiation and gene expression (Fig. 2, d–j). Differentiation of TgMek-dn cells was reduced relative to wild type, as measured by von Kossa staining of mineralized nodules at day 14 (Fig. 2 d), alkaline phosphatase activity (Fig. 2 e), calcium accumulation as mineral (Fig. 2 f) and expression of OCN, bone sialoprotein (BSP), and alkaline phosphatase (ALP) mRNAs (Fig. 2, g–i). Time course studies revealed that the earliest effects of transgenes were seen at day 10, immediately after initial transgene expression was detected at day 7 (Fig. 2 a). Runx2 mRNA was not affected at day 7, but was reduced by 40% on day 10 and 14. Opposite results were obtained with TgMek-sp cells that accumulated twice the mineral of wild-type cells at d 14 and had higher levels of alkaline phosphatase and osteoblast marker mRNAs. Runx2 mRNA levels were only slightly stimulated on days 10 and 14.
RUNX2 phosphorylation and transcriptional activity
The Ocn, Bsp, Akp2 (ALP), and Runx2 genes shown to be regulated by Mek-sp and -dn in Fig. 2, g–h are all known to be directly or indirectly controlled by RUNX2. For Ocn, Bsp, and Runx2 itself, this regulation involves direct binding of RUNX2 to regulatory elements in the proximal promoter region (Ducy et al., 1997; Tou et al., 2003; Roca et al., 2005). In previous cell culture studies, we showed that MAPK activation by either transient transfection of the MC3T3-E1 osteoblast cell line with a MEK-SP expression vector or activation of endogenous ERK phosphorylation by treatment with FGF2 stimulated phosphorylation and transcriptional activity of RUNX2 (Xiao et al., 2000, 2002b).
Two types of experiments were conducted to determine if RUNX2 phosphorylation and transcriptional activity is modified in cells from TgMek-sp and -dn mice, and if changes in phosphorylation could explain differences in osteoblast differentiation. In the first experiment, cells were grown under differentiating conditions for 7 d and metabolically labeled with [32P]orthophosphate, and then total RUNX2 was immunoprecipitated from cell extracts using a specific polyclonal antibody. A replicate culture was labeled with [35S]Met/Cys and processed in the same way to normalize 32P incorporation to total RUNX2. As shown in Fig. 3 (a and b), steady-state RUNX2 phosphorylation was increased twofold in TgMek-sp cells versus wild-type controls, while phosphorylation in TgMek-dn cells was reduced by 50%. In the second study, cells were transfected with 1.3-kb mOG2-luc or 6OSE2-luc reporter genes, and luciferase activity was measured after 3 and 7 d. A previous work showed that both of these promoter constructs are highly responsive to RUNX2 (Ducy et al., 1997). The 1.3-mOG2 fragments, like 0.647-kb mOG2, contains all known elements necessary for osteoblast-specific expression of Ocn, including RUNX2 binding sites (osteoblast-specific element 2 [OSE2]) at –608 and 137, whereas 6OSE2-luc contains six copies of the OSE2 site in front of a minimal mOG2 promoter (from –47 to 13 bp; Ducy and Karsenty, 1995; Yang et al., 2004). After 7 d in culture, luciferase activity from both reporter genes was higher in TgMek-sp cells than wild-type cells, whereas activity in TgMek-dn cells was reduced (Fig. 3, c and d). In contrast, no differences in activity were seen at day 3, which is before Mek-sp and -dn transgenes become active.
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TgMek-sp and -dn alter skeletal development
Consistent with results obtained in osteoblast cultures, skeletal development was accelerated in TgMek-sp and delayed in TgMek-dn embryos (Fig. 4).
This was reflected by differences in skeletal size, body weight, and mineralization. Skeletons from TgMek-sp embryos were significantly larger (based on femur lengths at embryonic day (E) 15.5 [Fig. 4, c and f] and body weight [Fig. 4, a and d]) than wild-type littermates, and weights and embryo size were reduced in TgMek-dn mice. Moreover, differences in calvarial mineralization were apparent with mineralized area of calvarial preparations at E15.5 being reduced by 20% in TgMek-dn and stimulated by an equivalent amount in TgMek-sp embryos (Fig. 4, b and e).
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TgMek-sp or -dn mice were crossed with Runx2+/– mice, and skeletal phenotypes were determined using whole-mount skeletal preparations (Fig. 5). Fig. 5 (a–d) show the analysis of E18.5 animals from TgMek-sp x Runx2+/– breedings, and Fig. 5 (e–h) show results of the TgMek-dn x Runx2+/– cross. Runx2+/– mice exhibited the expected clavicular hypertrophy and calvarial hypomineralization of CCD, with a resultant reduction of clavicle cross-sectional area of 75 (Fig. 5, c and g), and 28, and 18%, respectively, in mineralized areas of calvaria (Fig. 5, d and h). In contrast, femur length was not significantly affected (Fig. 5, b and f).
The Mek-sp transgene was able to partially rescue both the clavicular hypertrophy and calvarial hypomineralization of Runx+/– mice. Transgene expression increased the cross-sectional area of clavicles by 53% in Runx2+/– animals, although it only increased this parameter by 12% in wild- type mice (Fig. 5, c). Restoration of calvarial mineralization was also observed with TgMek-sp, increasing this parameter in Runx2+/– animals to 90% of the wild-type control (a 23% increase relative to Runx2+/– mice; Fig. 5, d). In contrast, Mek-sp had no effect on femur length in Runx2+/– mice, but it did increase this parameter by 18% in wild-type littermates (Fig. 5, b).
Results with the TgMek-dn x Runx2+/– cross were even more striking. First, after four consecutive breedings, we failed to obtain viable newborn pups with the TgMek-dn;Runx2+/– genotype. This is in contrast to the TgMek-sp;Runx2+/– genotype that was present in the expected Mendelian ratio. When embryos were harvested by Caesarean section at E18.5, TgMek-dn;Runx2+/– embryos were obtained in the expected ratios, indicating that this genotype cannot survive the birth process. Embryos were smaller and exhibited selective worsening of CCD-associated features. Specifically, Mek-dn reduced clavicular cross-sectional area by 67% in Runx+/– embryos, but only reduced this value by 17% in Runx+/+ mice (Fig. 5, g). Also, Mek-dn reduced calvarial mineralization by 31% in Runx2+/– embryos, but only reduced this value by 15% in wild type (Fig. 5, h). Collectively, these results show that in Runx2+/– mice, the clavicles, and calvaria are selectively sensitive to manipulation of ERK–MAPK activity in osteoblasts, which is consistent with the concept that RUNX2 is an in vivo target of this pathway.
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These results are consistent with previous cell culture studies from this and other laboratories. For example, we reported that MAPK activation, either by transfection of osteoblasts with constitutively active MEK1 or treatment with FGF2, rapidly increased RUNX2 phosphorylation and transcriptional activity through a process that was blocked by MAPK inhibition, whereas acute inhibition of MAPK in differentiated cells blocked osteoblast-specific gene expression (Xiao et al., 2000, 2002a,b). In related studies, Lai et al. (2001) showed that expression of a dominant-negative ERK1 blocked differentiation of human osteoblasts. Similarly, differentiation of human marrow stromal cells to osteoblasts is accompanied by sustained ERK1/2 phosphorylation; pharmacological MAPK inhibition or dominant-negative ERK blocked osteoblast formation in this system and stimulated adipogenesis (Jaiswal et al., 2000). Also, several reports support our hypothesis that MAPK actions in osteoblasts are, at least in part, mediated by RUNX2. During osteoblast differentiation of human marrow stromal cells, RUNX2 type II levels remain relatively unchanged, but binding to OSE2 DNA increased, as did RUNX2 phosphorylation (Shui et al., 2003). Also, mechanical loading of osteoblasts, which is known to be mediated, in part, through integrin activation, induces MAPK (Pavalko et al., 1998; Schmidt et al., 1998); loading of periodontal ligament cells (which are osteoprogenitor-like cells associated with tooth cementum) increased RUNX2 phosphorylation and binding to OSE2 DNA via an ERK–MAPK-dependent process (Ziros et al., 2002). In osteoblast-like prostate cancer cells, differentiation is accompanied by ERK1/2 activation, increased RUNX2-OSE2 binding, and Ocn expression, responses that are all blocked by MAPK inhibition (Zayzafoon et al., 2004). Lastly, IGF-1, which activates P13K and, subsequently, the ERK–MAPK pathway, stimulates RUNX2-OSE2 binding and phosphorylation in vascular endothelial cells (Qiao et al., 2004), as well as differentiation of marrow stromal cells (Celil and Campbell, 2005; Celil et al., 2005).
This work is to be contrasted with other in vitro studies that reported antagonism between the ERK–MAPK pathway and BMPs in epithelial cells (Kretzschmar et al., 1997, 1999) and inhibitory effects of this pathway on osteoblast differentiation (Higuchi et al., 2002; Nakayama et al., 2003; Schindeler and Little, 2006). Possible explanations for these disparate results may be related to differences in the magnitude and duration of MAPK activation and/or stage of cell maturation that are both known to affect MAPK responsiveness. For example, treatment of PC12 neuronal progenitor cells with EGF transiently stimulated ERK1/2 phosphorylation and cell proliferation, whereas NGF led to sustained ERK activation and neuronal differentiation, a result explained by the formation of transient versus long-lived complexes of MAPK-activating factors (Kao et al., 2001). Similarly, in studies with MC3T3-E1 preosteoblasts, EGF suppressed BMP-dependent Smad 1 activation, whereas sustained ERK–MAPK activation by extracellular matrix synthesis or constitutively active Ras stimulated BMP activity (Suzawa et al., 2002). In contrast, the aforementioned studies, indicating an inhibitory role for MAPK in osteoblast differentiation, generally either treated cells with MAPK inhibitors for extended times or stably transfected cells with expression vectors that produced high levels of constitutively active or dominant-negative MAPK intermediates during all stages of growth and differentiation.
To examine the in vivo actions of the ERK–MAPK pathway in osteoblasts, this study used the mOG2 promoter to drive expression of constitutively active/dominant-negative MEK1 in developing mice. Because this promoter is only active in mature osteoblasts that are largely postmitotic (Owen et al., 1990), we were able to discriminate between effects of MAPK on cell proliferation versus differentiation. Furthermore, transgenic modification led to sustained, but quite modest, changes in ERK1/2 phosphorylation (
50% increased/decreased levels). Using this approach, ERK–MAPK activation was shown to stimulate osteoblast differentiation of calvarial osteoblasts from transgenic animals and to accelerate bone accumulation in developing embryos without affecting cell proliferation. In contrast, dominant-negative MAPK inhibition slowed osteoblast differentiation in cell culture and delayed bone development. In vivo effects of transgenes were most striking in calvaria and long bones. Of particular interest, long bones of TgMek-dn mice displayed delayed bone collar formation, whereas TgMek-sp was associated with an increase in metaphyseal bone without any obvious effects on cartilage. This is consistent with an in situ analysis of transgene expression that was unable to detect transgene expression in growth plates (Fig. 1).
In agreement with our in vitro studies, we also obtained evidence that MAPK regulates RUNX2 transcriptional activity. Calvarial osteoblasts from transgenic mice exhibited the predicted changes in RUNX2 phosphorylation and transcriptional activity, with these parameters being stimulated in TgMek-sp cells and inhibited with Mek-dn. Of note, changes in RUNX2 were coincident with initial activation of Mek-sp and -dn transgenes and preceded any changes in osteoblast differentiation markers or RUNX2 mRNA that were all equivalent in wild-type, TgMek-sp, and -dn groups at the 7-d time point examined (Figs. 2 and 3). Thus, it is unlikely that changes in RUNX2 phosphorylation/activity are secondary to changes in osteoblast differentiation induced by Mek-sp/dn transgenes. Studies that examined genetic interactions between Runx2 and Mek-sp and -dn transgenes provided further evidence for in vivo regulation of RUNX2 by MAPK (Fig. 5). Because clavicles and calvaria are preferentially sensitive to Runx2 haploinsufficiency, they are good markers for changes in RUNX2 transcriptional activity. The observation that TgMek-sp is able to rescue the CCD phenotype of Runx2+/– embryos, whereas TgMek-dn exacerbated clavicular and calvarial defects provides compelling in vivo evidence for our hypothesis that the ERK–MAPK pathway regulates RUNX2.
Studies in other differentiating systems, such as muscle, fat, and cartilage, suggest that the ERK–MAPK pathway, acting through tissue-specific transcription factors, may generally control progenitor/stem cell lineage decisions in mesenchymal tissues. For example, in X. laevis muscle differentiation, MAPK increases levels of XMyoD, possibly by decreasing protein turnover (Zetser et al., 2001). In fat, phosphorylation of PPAR by ERK1 inhibits transcriptional activity of this factor to block adipogenesis (Adams et al., 1997). Lastly, MAPK activation by FGFs, constitutively active FGFR3, or MEK1 mutants in cartilage increases levels of Sox9 to keep chondrocytes in a prehypertrophic state (Murakami et al., 2000, 2004).
Studies in cartilage are of particular interest in that they used the Col2a1 promoter to express the same constitutively active MEK1 mutant used in this study. In contrast to our results with osteoblasts, Col2a1-mediated expression led to a chondrodysplasia phenotype that is characterized by shortened limbs and inhibition of hypertrophy without affecting chondrocyte proliferation. The observed limb shortening was explained by a reduction in the size of chondrocytes in the hypertrophic zone. Because the ERK–MAPK pathway is known to activate Sox9 (Murakami et al., 2000), the authors postulated that this transcription factor prevents hypertrophy, which normally requires Sox9 down-regulation. In contrast, our use of the mOG2 promoter to express constitutively active MEK1 resulted in expression in osteoblasts rather than cartilage, acceleration of bone development and skeletal mineralization, and no obvious changes in cartilage. As was the case for stimulation of Sox9 in cartilage, we find that ERK–MAPK can activate RUNX2-dependent transcriptional activity in osteoblasts.
Although our studies focused on RUNX2 as the primary mediator of MAPK signals, other transcription factors may also participate in this response, functioning together with RUNX2 to control gene expression. Of primary interest, ERK is known to phosphorylate and activate a second kinase, RSK2, in the cytoplasm of many cells (Fisher and Blenis, 1996). RSK2 phosphorylates ATF4, a transcription factor that, together with RUNX2, controls the osteoblast-specific expression of the osteocalcin gene (Yang et al., 2004). Mice deficient in either RSK2 or ATF4 have similar bone phenotypes characterized by delayed cranial and long-bone development (Yang et al., 2004). Furthermore, RUNX2 and ATF4 physically and functionally interact in osteoblasts, although it is not known whether phosphorylation of RUNX2 and/or ATF4 affects this interaction (Xiao et al., 2005). While this work was under review, Elefteriou et al. (2006) reported that the high bone-mass phenotype of mice harboring an osteoblast-specific deletion of the Ras GTPase-activating protein (GAP), Nf1, involves activation of the ERK–MAPK pathway. Although these authors attributed the phenotype of Nf1–/– mice exclusively to activation of ATF4 via RSK2, the involvement of Runx2 was not directly examined. To address the possible involvement of ATF4 in TgMek-sp and -dn mice, we transfected calvarial osteoblasts with an ATF4 reporter-luciferase construct containing oligomerized copies of the ATF4-responsive enhancer OSE1 (Ducy and Karsenty, 1995; Yang et al., 2004). Unlike results with the RUNX2 reporters (Fig. 3), this construct was not affected by transgene activity (not depicted). Based on this result, the involvement of ATF4 in the osteogenic response seen with transgenic ERK–MAPK activation appears to be secondary to that of RUNX2. However, this issue still needs to be examined in greater detail.
This study establishes an important role for the ERK–MAPK pathway in RUNX2 regulation, osteoblast differentiation, and fetal bone development. Furthermore, the animal models developed will be extremely useful for assessing roles of ERK–MAPK signaling during bone remodeling in adult and ageing mice, as well as for evaluating the possible therapeutic value of MAPK manipulation as a means of regulating bone mass in diseased states.
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Histological examination and in situ hybridization
Tissues were fixed in 10% formalin and embedded in paraffin, and 7-µm-thick sections were cut and stained with hematoxylin and eosin. RNA in situ hybridization was done using a digoxigenin-labeled riboprobe specific for SV40 sequences in the transgene (Roche). Sections were viewed with a microscope (Eclipse 50i; Nikon) using a 10x objective. Images were captured with a digital camera (Stereo TLRC; Leica) using Image-Pro Plus 5.1 (Media Cybernetics) acquisition software without further manipulation.
Whole-mount skeletal preparations
Skeletal morphology was analyzed by alizarin red and alcian blue staining, followed by tissue clarification with KOH, as previously described (Yang et al., 2004). To measure bone length, isolated bones were laid next to a ruler and photographed using a digital camera and stereo dissection scope. Bone area was calculated using ImagePro Plus software.
Cell culture and Western blot analysis
Primary calvarial osteoblasts were isolated from 4-wk-old transgenic mice and littermates, as previously described (Ducy and Karsenty, 1995). Cells were grown in -MEM/10% FCS until confluent. Cells were then replated at a density of 5 x 103 cells/cm2 in 35-mm dishes and grown for up to 2 wk in differentiation medium (-MEM, 10% FCS, 50 µg/ml ascorbic acid, and 5 mM ß-glycerol phosphate). Quantitative RT-PCR was performed using an ABI PRISM 7700 Sequence Detection System. Optimized primers and probes for Ocn, Bsp, Akp2, Runx2, and Gapdh were purchased from Applied Biosystems. Total and phospho-ERK1/2 were measured on Western blots using specific antibodies at a dilution of 1:2,000 (Cell Signaling Technology).
Cell transfections and metabolic labeling
Primary calvaria osteoblasts were isolated from newborn mice and cultured in 75-cm flasks for 3 d until confluent. Cells were then trypsinized and replated on 35-mm dishes at a density of 2.5 x 105 cells/cm2. After 24 h, cells were transfected with Fugene6 (Roche) according to the manufacturer's instructions. Each transfection contained 0.5 µg of p1.3mOG2-luc or p6OSE2-luc (Ducy and Karsenty, 1995) and 0.05 µg of pRL-SV40 (encodes Renilla reformis luciferase to control for transfection efficiency). Cells were then cultured in differentation medium for an additional 6 d before harvesting and assaying for luciferase activity using a Dual Luciferase Assay kit (Promega) on a luminometer (Monolight 2010; BD Biosciences).
For cell labeling, calvarial cells were cultured for 6 d, as described in the previous paragraph, and transferred to phosphate or cysteine and methionine-free medium containing 0.1% dialyzed FCS (Sigma-Aldrich). After an overnight preincubation, cells were labeled for 5 h in the same media containing either 150 µCi/ml of [32P]orthophosphate or [35S]cysteine/methionine (MP Biomedical). Preparation of nuclear extracts and immunoprecipitation of Runx2 were described previously (Xiao et al., 2000). 32P/35S incorporation was measured using an A2024 InstantImager (Packard).
Statistical analysis
Statistical analyses were performed using Instat 3.0 (GraphPAD Software, Inc.). For cell culture studies (Figs. 2 and 3), values are the means ± the SD of triplicate independent samples. In studies with mice, n = 8 mice/group. Analysis of variance followed by Tukey-Kramer multiple comparisons test was used to assess significance between groups as indicated.
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Acknowledgments |
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This work was supported by National Institutes of Health/National Institute of Dental and Craniofacial Research grants DE11723 and DE12211 (to R.T. Franceshi).
The authors declare they have no competing financial interests.
Submitted: 10 October 2006
Accepted: 23 January 2007
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References |
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Adams, M., M.J. Reginato, D. Shao, M.A. Lazar, and V.K. Chatterjee. 1997. Transcriptional activation by peroxisome proliferator-activated receptor gamma is inhibited by phosphorylation at a consensus mitogen-activated protein kinase site. J. Biol. Chem. 272:5128–5132.
Celil, A.B., and P.G. Campbell. 2005. BMP-2 and insulin-like growth factor-I mediate Osterix (Osx) expression in human mesenchymal stem cells via the MAPK and protein kinase D signaling pathways. J. Biol. Chem. 280:31353–31359.
Celil, A.B., J.O. Hollinger, and P.G. Campbell. 2005. Osx transcriptional regulation is mediated by additional pathways to BMP2/Smad signaling. J. Cell. Biochem. 95:518–528.[CrossRef][Medline]
Chen, C., A.J. Koh, N.S. Datta, J. Zhang, E.T. Keller, G. Xiao, R.T. Franceschi, N.J. D'Silva, and L.K. McCauley. 2004. Impact of the mitogen-activated protein kinase pathway on parathyroid hormone-related protein actions in osteoblasts. J. Biol. Chem. 279:29121–29129.
Ducy, P., and G. Karsenty. 1995. Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene. Mol. Cell. Biol. 15:1858–1869.[Abstract]
Ducy, P., R. Zhang, V. Geoffroy, A.L. Ridall, and G. Karsenty. 1997. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. 89:747–754.[CrossRef][Medline]
Elefteriou, F., M.D. Benson, H. Sowa, M. Starbuck, X. Liu, D. Ron, L. Parada, and G. Karsenty. 2006. ATF4 mediation of NF1 functions in osteoblast reveals a nutritional basis for congenital skeletal dysplasiae. Cell Metab. 4:1–11.[CrossRef][Medline]
Fisher, T.L., and J. Blenis. 1996. Evidence for two catalytically active kinase domains in pp90rsk. Mol. Cell. Biol. 16:1212–1219.[Abstract]
Franceschi, R.T., G. Xiao, D. Jiang, R. Gopalakrishnan, S. Yang, and E. Reith. 2003. Multiple signaling pathways converge on the Cbfa1/Runx2 transcription factor to regulate osteoblast differentiation. Connect. Tiss. Res. 44:109–116.[Medline]
Frendo, J.L., G. Xiao, S. Fuchs, R.T. Franceschi, G. Karsenty, and P. Ducy. 1998. Functional hierarchy between two OSE2 elements in the control of osteocalcin gene expression in vivo. J. Biol. Chem. 273:30509–30516.
Hall-Jackson, C.A., P.A. Eyers, P. Cohen, M. Goedert, F.T. Boyle, N. Hewitt, H. Plant, and P. Hedge. 1999. Paradoxical activation of Raf by a novel Raf inhibitor. Chem. Biol. 6:559–568.[CrossRef][Medline]
Higuchi, C., A. Myoui, N. Hashimoto, K. Kuriyama, K. Yoshioka, H. Yoshikawa, and K. Itoh. 2002. Continuous inhibition of MAPK signaling promotes the early osteoblastic differentiation and mineralization of the extracellular matrix. J. Bone Miner. Res. 17:1785–1794.[CrossRef][Medline]
Hurley, M.M., K. Marcello, C. Abreu, and M. Kessler. 1996. Signal transduction by basic fibroblast growth factor in rat osteoblastic Py1a cells. J. Bone Miner. Res. 11:1256–1263.[Medline]
Jaiswal, R.K., N. Jaiswal, S.P. Bruder, G. Mbalaviele, D.R. Marshak, and M.F. Pittenger. 2000. Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J. Biol. Chem. 275:9645–9652.
Kao, S., R.K. Jaiswal, W. Kolch, and G.E. Landreth. 2001. Identification of the mechanisms regulating the differential activation of the MAPK cascade by epidermal growth factor and nerve growth factor in PC12 cells. J. Biol. Chem. 276:18169–18177.
Komori, T., H. Yagi, S. Nomura, A. Yamaguchi, K. Sasaki, K. Deguchi, Y. Shimizu, R.T. Bronson, Y.H. Gao, M. Inada, et al. 1997. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 89:755–764.[CrossRef][Medline]
Kretzschmar, M., J. Doody, and J. Massague. 1997. Opposing BMP and EGF signalling pathways converge on the TGF-beta family mediator Smad1. Nature. 389:618–622.[CrossRef][Medline]
Kretzschmar, M., J. Doody, I. Timokhina, and J. Massague. 1999. A mechanism of repression of TGFbeta/Smad signaling by oncogenic Ras. Genes Dev. 13:804–816.
Lai, C.F., L. Chaudhary, A. Fausto, L.R. Halstead, D.S. Ory, L.V. Avioli, and S.L. Cheng. 2001. Erk is essential for growth, differentiation, integrin expression, and cell function in human osteoblastic cells. J. Biol. Chem. 276:14443–14450.
Murakami, S., M. Kan, W.L. McKeehan, and B. de Crombrugghe. 2000. Up-regulation of the chondrogenic Sox9 gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway. Proc. Natl. Acad. Sci. USA. 97:1113–1118.
Murakami, S., G. Balmes, S. McKinney, Z. Zhang, D. Givol, and B. de Crombrugghe. 2004. Constitutive activation of MEK1 in chondrocytes causes Stat1-independent achondroplasia-like dwarfism and rescues the Fgfr3-deficient mouse phenotype. Genes Dev. 18:290–305.
Nakayama, K., Y. Tamura, M. Suzawa, S. Harada, S. Fukumoto, M. Kato, K. Miyazono, G.A. Rodan, Y. Takeuchi, and T. Fujita. 2003. Receptor tyrosine kinases inhibit bone morphogenetic protein-Smad responsive promoter activity and differentiation of murine MC3T3-E1 osteoblast-like cells. J. Bone Miner. Res. 18:827–835.[CrossRef][Medline]
Otto, F., A.P. Thornell, T. Crompton, A. Denzel, K.C. Gilmour, I.R. Rosewell, G.W. Stamp, R.S. Beddington, S. Mundlos, B.R. Olsen, et al. 1997. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell. 89:765–771.[CrossRef][Medline]
Owen, T.A., M. Aronow, V. Shalhoub, L.M. Barone, L. Wilming, M.S. Tassinari, M.B. Kennedy, S. Pockwinse, J.B. Lian, and G.S. Stein. 1990. Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J. Cell. Physiol. 143:420–430.[CrossRef][Medline]
Pavalko, F.M., N.X. Chen, C.H. Turner, D.B. Burr, S. Atkinson, Y.F. Hsieh, J. Qiu, and R.L. Duncan. 1998. Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions. Am. J. Physiol. 275:C1591–C1601.
Qiao, M., P. Shapiro, R. Kumar, and A. Passaniti. 2004. Insulin-like growth factor-1 regulates endogenous RUNX2 activity in endothelial cells through a phosphatidylinositol 3-kinase/ERK-dependent and Akt- independent signaling pathway. J. Biol. Chem. 279:42709–42718.
Roca, H., M. Phimphilai, R. Gopalakrishnan, G. Xiao, and R.T. Franceschi. 2005. Cooperative interactions between RUNX2 and homeodomain protein-binding sites are critical for the osteoblast-specific expression of the bone sialoprotein gene. J. Biol. Chem. 280:30845–30855.
Schindeler, A., and D. Little. 2006. Ras-MAPK signaling in osteogenic differentiation: friend or foe? J. Bone Miner. Res. 21:1331–1338.[CrossRef][Medline]
Schmidt, C., H. Pommerenke, F. Durr, B. Nebe, and J. Rychly. 1998. Mechanical stressing of integrin receptors induces enhanced tyrosine phosphorylation of cytoskeletally anchored proteins. J. Biol. Chem. 273:5081–5085.
Shui, C., T.C. Spelsberg, B.L. Riggs, and S. Khosla. 2003. Changes in Runx2/Cbfa1 expression and activity during osteoblastic differentiation of human bone marrow stromal cells. J. Bone Miner. Res. 18:213–221.[CrossRef][Medline]
Stokoe, D., S.G. Macdonald, K. Cadwallader, M. Symons, and J.F. Hancock. 1994. Activation of Raf as a result of recruitment to the plasma membrane. Science. 264:1463–1467.
Sugimoto, T., S. Stewart, M. Han, and K.L. Guan. 1998. The kinase suppressor of Ras (KSR) modulates growth factor and Ras signaling by uncoupling Elk-1 phosphorylation from MAP kinase activation. EMBO J. 17:1717–1727.[CrossRef][Medline]
Suzawa, M., Y. Tamura, S. Fukumoto, K. Miyazono, T. Fujita, S. Kato, and Y. Takeuchi. 2002. Stimulation of Smad1 transcriptional activity by Ras-extracellular signal-regulated kinase pathway: a possible mechanism for collagen-dependent osteoblastic differentiation. J. Bone Miner. Res. 17:240–248.[CrossRef][Medline]
Takeuchi, Y., M. Suzawa, T. Kikuchi, E. Nishida, T. Fujita, and T. Matsumoto. 1997. Differentiation and transforming growth factor-beta receptor down-regulation by collagen-alpha2beta1 integrin interaction is mediated by focal adhesion kinase and its downstream signals in murine osteoblastic cells. J. Biol. Chem. 272:29309–29316.
Tou, L., N. Quibria, and J.M. Alexander. 2003. Transcriptional regulation of the human Runx2/Cbfa1 gene promoter by bone morphogenetic protein-7. Mol. Cell. Endocrinol. 205:121–129.[CrossRef][Medline]
Umbhauer, M., C.J. Marshall, C.S. Mason, R.W. Old, and J.C. Smith. 1995. Mesoderm induction in Xenopus caused by activation of MAP kinase. Nature. 376:58–62.[CrossRef][Medline]
Wu, X., S.J. Noh, G. Zhou, J.E. Dixon, and K.L. Guan. 1996. Selective activation of MEK1 but not MEK2 by A-Raf from epidermal growth factor-stimulated Hela cells. J. Biol. Chem. 271:3265–3271.
Xiao, G., Y. Cui, P. Ducy, G. Karsenty, and R.T. Franceschi. 1997. Ascorbic acid-dependent activation of the osteocalcin promoter in MC3T3-E1 preosteoblasts: requirement for collagen matrix synthesis and the presence of an intact OSE2 sequence. Mol. Endocrinol. 11:1103–1113.
Xiao, G., D. Wang, M.D. Benson, G. Karsenty, and R.T. Franceschi. 1998. Role of the alpha2-integrin in osteoblast-specific gene expression and activation of the Osf2 transcription factor. J. Biol. Chem. 273:32988–32994.
Xiao, G., D. Jiang, P. Thomas, M.D. Benson, K. Guan, G. Karsenty, and R.T. Franceschi. 2000. MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1. J. Biol. Chem. 275:4453–4459.
Xiao, G., R. Gopalakrishnan, D. Jiang, E. Reith, M.D. Benson, and R.T. Franceschi. 2002a. Bone morphogenetic proteins, extracellular matrix, and mitogen-activated protein kinase signaling pathways are required for osteoblast-specific gene expression and differentiation in MC3T3-E1 cells. J. Bone Miner. Res. 17:101–110.[CrossRef][Medline]
Xiao, G., D. Jiang, R. Gopalakrishnan, and R.T. Franceschi. 2002b. Fibroblast growth factor 2 induction of the osteocalcin gene requires MAPK activity and phosphorylation of the osteoblast transcription factor, Cbfa1/Runx2. J. Biol. Chem. 277:36181–36187.
Xiao, G., D. Jiang, C. Ge, Z. Zhao, Y. Lai, H. Boules, M. Phimphilai, X. Yang, G. Karsenty, and R.T. Franceschi. 2005. Cooperative interactions between activating transcription factor 4 and Runx2/Cbfa1 stimulate osteoblast-specific osteocalcin gene expression. J. Biol. Chem. 280:30689–30696.
Yang, X., K. Matsuda, P. Bialek, S. Jacquot, H.C. Masuoka, T. Schinke, L. Li, S. Brancorsini, P. Sassone-Corsi, T.M. Townes, et al. 2004. ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology; implication for Coffin-Lowry Syndrome. Cell. 117:387–398.[CrossRef][Medline]
Yao, Y., W. Li, J. Wu, U.A. Germann, M.S. Su, K. Kuida, and D.M. Boucher. 2003. Extracellular signal-regulated kinase 2 is necessary for mesoderm differentiation. Proc. Natl. Acad. Sci. USA. 100:12759–12764.
You, J., G.C. Reilly, X. Zhen, C.E. Yellowley, Q. Chen, H.J. Donahue, and C.R. Jacobs. 2001. Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3-E1 osteoblasts. J. Biol. Chem. 276:13365–13371.
Zayzafoon, M., S.A. Abdulkadir, and J.M. McDonald. 2004. Notch signaling and ERK activation are important for the osteomimetic properties of prostate cancer bone metastatic cell lines. J. Biol. Chem. 279:3662–3670.
Zetser, A., D. Frank, and E. Bengal. 2001. MAP kinase converts MyoD into an instructive muscle differentiation factor in Xenopus. Dev. Biol. 240:168–181.[CrossRef][Medline]
Ziros, P.G., A.P. Gil, T. Georgakopoulos, I. Habeos, D. Kletsas, E.K. Basdra, and A.G. Papavassiliou. 2002. The bone-specific transcriptional regulator Cbfa1 is a target of mechanical signals in osteoblastic cells. J. Biol. Chem. 277:23934–23941.
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