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Department of Research Saint Francis Hospital and Medical Center (M.S., E.G., L.P., A.M.D., E.C.), Hartford, Connecticut 06105-1299; and University of Connecticut School of Medicine (E.G., E.C.), Farmington, Connecticut 06030
Address all correspondence and requests for reprints to: Ernesto Canalis, M.D., Department of Research, Saint Francis Hospital and Medical Center, 114 Woodland Street, Hartford, Connecticut 06105-1299. E-mail: ecanalis{at}stfranciscare.org.
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Abstract |
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2 FosB protein. Further, NotchIC decreased Wnt/ß-catenin signaling. As cells differentiated in the presence of BMP-2, they underwent apoptosis, and Notch opposed this event. In the presence of cortisol, NotchIC induced the formation of mature adipocytes and enhanced the effect of cortisol on adipsin, peroxisome proliferator-activated receptor-
2 and CCAAT enhancer binding protein 
and 
mRNA levels. NotchIC also opposed MC3T3 cell differentiation and the expression of a mature osteoblastic phenotype. In conclusion, NotchIC impairs osteoblast differentiation and enhances adipogenesis in stromal cell cultures. |
Introduction |
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, SuH, and Lag-1) family of DNA binding proteins (4, 5). These nuclear proteins are C promoter binding factor (CBF)1 or recombination signal binding protein of the J Ig gene (RBP-J) in mammals, Suppressor of Hairless [Su(H)] in Drosophila, and Lag-1 in Caenorhabditis elegans (6, 7, 8). CSL transcription factors bind to the promoter region of the Enhancer of Split [E(spl)] or its mammalian homolog the Hairy Enhancer of Split-1 (HES-1) and HES-5 complex of genes and recruit a corepressor complex to regulate transcription (8). This complex contains several accessory proteins, including the silencing mediator for retinoid and thyroid hormone receptors and the Ski-interacting protein, both of which interact with the Notch intracellular domain (8, 9, 10).
There is a high degree of structural homology in the intracellular domain of the four Notch receptors, although their functional homology has not been explored in detail. The structural and functional properties of Notch 1 have been characterized to a greater extent than those of other Notch receptors. Notch 1 as well as CBF1/RBP-J null mutations result in embryonic lethality secondary to cellular death and significant developmental abnormalities (11, 12). Recently, our laboratory and others demonstrated the expression of the Notch receptors 1 and 2 and the Notch ligands Delta 1 and Jagged 1 in osteoblasts (13, 14). However, the mechanisms involved in Notch activation in osteoblasts and its role in osteoblastic differentiation and function are not well defined. Overexpression of Notch 1 in C2C12 cells blocks the osteogenesis induced by bone morphogenetic protein (BMP), indicating that Notch and BMPs have opposite effects on osteoblast differentiation (15). These observations would suggest that Notch plays a role in the differentiation of stromal cells directing them away from the osteoblastic pathway. However, recent reports have postulated that Notch 1 enhances differentiation of MC3T3 cells and the effects of BMP (16).
The intent of this study was to investigate the effects of Notch 1 on osteoblastic cell differentiation and function. For this purpose we used replication-incompetent retrovirus to create ST-2 stromal and MC3T3 cell lines that constitutively overexpress the intracellular domain of Notch 1, thus obviating the need for ligand interaction, and determined their phenotype during differentiation.
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Materials and Methods |
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Cell cultures
ST-2 cells, cloned stromal cells isolated from bone marrow of BC8 mice, and MC3T3-E1 osteoblastic cells derived from mouse calvariae were grown in a humidified 5% CO2 incubator at 37 C in MEM (Invitrogen), supplemented with 10% FBS (19, 20, 21). ST-2 and MC3T3 cells were transduced with pLPCX vector or pLPCX NotchIC by replacing the culture medium with retroviral conditioned medium from Phoenix packaging cells in the presence of 8 µg/ml polybrene (Sigma-Aldrich Corp., St. Louis, MO), followed by incubation for 16–18 h at 37 C. The culture medium was replaced with fresh MEM, and cells were grown, trypsinized, replated, and selected for puromycin resistance.
To analyze the phenotypic impact of NotchIC, untransfected ST-2 or MC3T3 cells and cells transduced with pLPCX vector or pLPCX NotchIC were plated at a density of 104 cells/cm2 and cultured in MEM supplemented with 10% FBS until reaching confluence (2–4 d). At confluence (experimental d 0), ST-2 or MC3T3 cells were transferred to MEM containing 10% FBS, 100 µg/ml ascorbic acid, and 5 mM ß-glycerophosphate (Sigma-Aldrich Corp.) and cultured for an additional 1–4 wk. ST-2 cells were cultured in the presence or absence of recombinant human BMP-2 at 1 nM (a gift from Genetics Institute, Cambridge, MA) or cortisol at 1 µM (Sigma-Aldrich Corp.) as indicated in the text and legends. Cells were refed with fresh medium containing control or test solutions every 3–4 d.
To analyze for the endogenous expression of Notch 1 and 2 and their ligands, Delta and Jagged, ST-2 cells were grown to confluence and cultured for 1–4 wk in MEM containing 10% FBS, 100 µg/ml ascorbic acid, and 5 mM ß-glycerophosphate. To study the regulation of Notch 1, cells were cultured in the absence and presence of 1 nM BMP-2, 1 µM cortisol, or an adipogenic cocktail composed of 1 µM dexamethasone, 100 nM insulin, and 0.5 mM isobutylmethylxanthine (IBMX) (22).
Cytochemical analysis and cell number
For cytochemical analysis, ST-2 and MC3T3 cells were washed with PBS, fixed with 3.7% formaldehyde, and stained with 2% alizarin red (Sigma-Aldrich Corp.) (23, 24). To estimate the levels of cellular fat, ST-2 cells were air-dried for 1 h and stained with 0.5% Oil Red O in 60% isopropanol (Sigma-Aldrich Corp.) for 30–60 min (23, 25). To determine the number of viable cells, mitochondrial dehydrogenase activity was estimated using the CellTiter 96 AQueous One cell proliferation assay in accordance with manufacturer’s instructions (Promega, Madison, WI). Metabolically active cells are estimated by their ability to reduce the tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt to a formazan product measured at an absorbance of 490 nm. Data are expressed in arbitrary units of absorbance at 490 nm.
Northern blot analysis
Total cellular RNA was isolated using the RNeasy kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). RNA was quantified by spectrometry, and equal amounts of RNA were loaded on a formaldehyde agarose gel after denaturation. The gel was stained with ethidium bromide to visualize ribosomal RNA and to confirm equal RNA loading of the experimental samples. The RNA was blotted onto GeneScreen Plus charged nylon (PerkinElmer, Norwalk, CT), and uniformity of transfer was confirmed by revisualization of ethidium bromide-stained ribosomal RNA. A 1.2-kb murine Notch 1 cDNA (U. Lendhal, Stockholm, Sweden), an 800-bp human Notch 2 cDNA (ATCC), a 4.2-kb rat Jagged 1 cDNA (G. Weinmaster, Los Angeles, CA), a 400-bp mouse Jagged 2 cDNA (ATCC), a 2.1-kb mouse Delta 1 cDNA (R. Cordes, Hannover, Germany), a 2.1-kb mouse Delta 3 cDNA (A. Rana, London, UK), a 500-bp rat osteocalcin genomic DNA (J. Lian, Worcester, MA), a 2.5-kb rat alkaline phosphatase cDNA (Merck & Co., West Point, PA), a 1.6-kb rat type I collagen cDNA (B. Kream, Farmington, CT), a 1.8-kb rat CCAAT/enhancer-binding protein (C/EBP) cDNA, a 1.5-kb rat C/EBPß cDNA, a 1.0-kb rat C/EBP cDNA (all three from S. L. McKnight, Dallas, TX), a 900-bp human peroxisome proliferator-activated receptor 2 (PPAR2) cDNA, an 800-bp murine adipsin cDNA, and a 752-bp murine 18S ribosomal RNA (all three from ATCC) were purified by agarose gel electrophoresis (26, 27, 28, 29, 30, 31, 32, 33). DNAs were labeled with [-32P]deoxy-CTP (50 µCi at a specific activity of 3,000 Ci/mmol; PerkinElmer) using Ready-To-Go DNA labeling beads (-dCTP kit, Amersham Pharmacia Biotech, Piscataway, NJ) in accordance with the manufacturer’s instructions. Hybridizations were carried out at 42 C for 16–72 h, followed by two posthybridization washes at room temperature for 15 min in 1x saline sodium citrate, and a wash at 65 C for 20–30 min in 0.1x or 1x saline sodium citrate. The bound radioactive material was visualized by autoradiography on Kodak X-AR5 film (Eastman Kodak, Rochester, NY), employing Cronex Lightning Plus (PerkinElmer) or Biomax MS (Eastman Kodak Co., Rochester, NY) intensifying screens. Relative hybridization levels were determined by densitometry. Northern analyses shown are representative of two or three samples.
Western blot analysis
To determine the expression of FosB isoforms, nuclear extracts were prepared from transduced ST-2 stromal cells. At time of harvest, cells were washed with PBS; suspended in 10 mM HEPES/KOH (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, and 0.5 mM dithiothreitol buffer; allowed to swell on ice for 15 min; and lysed with 10% Nonidet P-40 (Sigma-Aldrich Corp.) (34). Nuclei were pelleted and extracted in HEPES/KOH buffer in the presence of protease inhibitors at 4 C for 30 min. After centrifugation, nuclear pellet was discarded, and the supernatant stored at -70 C, and protein concentrations were determined by DC Protein Assay in accordance with the manufacturer’s instructions (Bio-Rad Laboratories, Richmond, CA). Forty micrograms of protein, in reducing Laemmli sample buffer, were fractionated by electrophoresis in 15% polyacrylamide gels and were transferred to Immobilon P membranes (Millipore, Bedford, MA) (35). Membranes were blocked with 3% BSA and were exposed to 1 µg/ml rabbit anti-FosB in 1% BSA overnight (1:200 dilution; sc-48, Santa Cruz Biotechnology, Santa Cruz, CA) (34, 36). After incubation with primary antibody, blots were exposed to goat antirabbit IgG antiserum conjugated to horseradish peroxidase (Sigma-Aldrich Corp.), developed with a chemiluminescence detection reagent (PerkinElmer), and visualized by fluororadiography on Reflection x-ray film (DuPont, Wilmington, DE).
Transient transfections
To determine changes in HES-5 promoter activity, a construct containing a 3.7-kb murine HES-5 DNA fragment cloned upstream a thymidine kinase promoter and a luciferase-encoding gene (HES-5-tk-luc) provided by U. Lendhal was tested in transient transfection experiments (37). To determine changes in ß-catenin trans-activating activity, a pTOPFLASH reporter construct containing three copies of the lymphoid enhancer binding factor 1 (Lef 1)/T cell transcription factor 4 (Tef-4) binding sequences, CCTTTGATC, or its mutant, pFOPFLASH containing three copies of the mutant binding site, CCTTGGCC, both cloned upstream of a minimal c-fos promoter and a luciferase-encoding gene (Kitajewski, J., New York, NY) were tested in transient transfections (38). ST-2 stromal cells were cultured to 70% confluence and transiently transfected with the indicated constructs using FuGene 6 (3 µl FuGene/2 µg DNA) according to manufacturer’s instructions (Roche, Indianapolis, IN). Cotransfections with a construct containing the CMV promoter driving the ß-galactosidase gene (Clontech) were used to control for transfection efficiency. Cells were exposed to the Fugene-DNA mix for 16 h, transferred to serum containing medium for 48 h, washed twice with PBS, and harvested in a reporter lysis buffer (Promega). Luciferase and ß-galactosidase activities were measured using an Optocomp luminometer (MGM Instruments, Hamden, CT) as previously described (18). Luciferase activity was corrected for ß-galactosidase activity to control for transfection efficiency.
Statistical analysis
Data are expressed as the mean ± SEM. Statistical significance was determined by t test
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Results |
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). Endogenous Notch 1 transcripts were detected in cells transduced with vector after longer exposure of the autoradiograms, but were masked by the NotchIC transcript driven by the viral long terminal repeat in pLPCX NotchIC cells (data not shown). To confirm that Notch signaling was increased by pLPCX NotchIC, ST-2 cells were transiently transfected with a HES-5 promoter-luciferase reporter construct. The activity of the HES-5 promoter was increased approximately 5-fold in NotchIC-overexpressing cells compared with control cells, documenting increased Notch signaling (Fig. 1).
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) (23). This mineralized nodule formation correlated with increased expression of the osteoblastic-specific gene osteocalcin. Overexpression of NotchIC prevented the formation of mineralized nodules and decreased the mRNA expression of the osteoblastic markers osteocalcin, alkaline phosphatase, and 1(I) collagen. Instead, pLPCX-NotchIC ST-2 cells cultured under these conditions displayed increased expression of adipsin transcripts, a marker of mature adipocytes, although mRNA levels for PPAR2 were not altered. As the FosB isoform of the transcription factor FosB stimulates osteoblastogenesis at the expense of adipogenesis, we examined the expression of FosB isoforms in pLPCX ST-2 and pLPCX NotchIC ST-2 cells cultured in the presence of BMP-2 (Fig. 3) (36). As pLPCX ST-2 cells underwent osteoblastic differentiation, expression of the 2FosB isoform, in particular, was increased. However, this was not seen in pLPCX NotchIC cells, supporting the concept that Notch 1 signaling inhibited osteoblastogenesis.
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). However, confirming previous observations, as ST-2 cells cultured in the presence of BMP-2 underwent osteoblastic differentiation and mineralized, they underwent apoptosis, an important event in cell differentiation (39). In accordance with the inhibitory effect of NotchIC on osteoblastic differentiation, there was a decrease in apoptosis, evaluated by nuclear condensation, in pLPCX NotchIC ST-2 cells compared with control pLPCX cells (Fig. 4).
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2 transcripts and decreased the expression of osteocalcin mRNA (Fig. 5). Overexpression of NotchIC accelerated the appearance of Oil Red O-stained intracellular lipid droplets and enhanced the stimulatory effects of cortisol on adipsin and PPAR2 mRNA expression. Individually, cortisol and NotchIC had dramatic inhibitory effects on osteocalcin mRNA, making it difficult to evaluate potential additive effects. As members of the C/EBP family of transcription factors play an essential role in adipocytic differentiation, we examined the effect of pLPCX NotchIC on the expression of these transcription factors in the absence and presence of cortisol (40, 41). Confirming previous observations, cortisol treatment increased C/EBP, -ß, and - transcripts (23). Overexpression of NotchIC did not modify C/EBP, -ß, or - mRNA levels, but enhanced the stimulatory effect of cortisol on C/EBP and - transcript expression (Fig. 6).
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).
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, left panel). Cortisol induced Notch 1 mRNA levels by 2- to 3-fold in ST-2 cells cultured for 2–4 wk after confluence, whereas BMP-2 did not have an appreciable effect on Notch 1 transcripts. The adipogenic cocktail of dexamethasone, insulin, and IBMX caused a 2-fold induction of Notch 1 after 1 wk and up to 8-fold after 3 and 4 wk of culture (Fig. 8, right panel).
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). pLPCX MC3T3 cell cultures gave rise to mineralized nodules after 3 wk, as assessed by Alizarin Red staining, and expressed the osteoblastic-specific gene osteocalcin. Overexpression of NotchIC prevented the formation of mineralized nodules and decreased the mRNA expression of the osteoblastic markers osteocalcin and 1(I) collagen. The expression of alkaline phosphatase mRNA was below the level of detection in control and Notch 1-overexpressing cultures (Fig. 9). NotchIC overexpression did not cause significant changes in the number of viable MC3T3 cells (Table 2).
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Discussion |
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The effects of NotchIC overexpression on stromal cell differentiation are similar to those of glucocorticoids, as glucocorticoids impair osteoblastic differentiation and induce the appearance of an adipocytic phenotype. Further, Notch 1 transcripts are induced by glucocorticoids in ST-2 stromal and MC3T3 osteoblastic cells (13). Notch 1 may play a role in the mechanisms of adipogenesis induced by glucocorticoids, enhancing the effects of these steroids, as shown in this study. However, glucocorticoids have additional effects on adipocyte cell differentiation and induce transcription factors that play an integral role in adipogenesis. Glucocorticoids induce the expression of C/EBP, -ß, and - and PPAR2, and these nuclear factors play a central role in adipogenesis (23, 25, 40, 41, 47, 48). In the present studies we found that NotchIC overexpression enhanced the effects of cortisol on the expression of PPAR2 and C/EBP and -, confirming that Notch 1 potentiates the actions of glucocorticoids on adipogenesis. It is not clear whether the induction of adipogenic differentiation by Notch 1 is a default mechanism, related to the suppression of osteogenesis, or is due to a direct stimulation of stromal cell differentiation toward the adipocytic pathway. However, the described interactions with cortisol, enhancing the expression of genes directly related to adipogenesis and the marked induction of Notch 1 mRNA levels by an adipogenic cocktail, would suggest a direct role in this process.
Members of the activating protein-1 family of transcription factors, consisting of Fos- and Jun-related proteins, are differentially regulated during osteoblast differentiation (49). Overexpression of FosB, a Fos-related protein generated from alternative splicing of FosB transcripts, results in increased bone formation and osteoblastogenesis and suppressed adipogenesis (36). Furthermore, FosB, but not FosB, levels increase as osteoblastic cells differentiate in culture. In accordance with these observations, we demonstrated that the induction of adipogenesis and suppression of osteogenesis by NotchIC overexpression is associated with decreased expression of FosB, particularly 2FosB.
The present studies indicate that Notch 1 signaling in marrow stromal cells inhibits their osteoblastic differentiation, although the mechanisms involved were not fully characterized. We provide evidence, however, that the CBF1/RBP-J pathway was activated and the canonical Wnt pathway was suppressed in NotchIC-transduced ST-2 stromal cells, suggesting possible mechanisms of action for the Notch effect. Notch signaling is important in the determination of cell fate pathways, including neurogenesis, myogenesis, pancreatic cell differentiation, and nephrogenesis (15, 50, 51, 52). Our studies confirm that Notch signaling is important in the specification of cell fate favoring adipogenesis in stromal cells. It is of interest that Notch 1 and Wnt have opposite effects on cell differentiation, as Wnt signaling inhibits adipogenesis and has the potential to induce osteoblastic differentiation by regulating BMP signaling pathways (42, 43). Consequently, it is not surprising that NotchIC suppressed ß-catenin signaling. These results confirm that NotchIC in stromal cells, as in keratinocytes, represses Wnt signaling (53). Additional interactions between Notch and Wnt signaling are possible and could involve Dishevelled, a molecule implicated in the implementation of Wnt signaling that interacts and blocks Notch signaling in Drosophila (54).
In conclusion, our studies demonstrate that NotchIC overexpression favors stromal cell differentiation toward adipocytes and prevents osteoblastic differentiation.
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Acknowledgments |
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Footnotes |
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M.S. and E.G. contributed equally to this work.
Abbreviations: BMP-2, Bone morphogenetic protein-2; CBF, C promoter binding factor; C/EBP, CCAAT/enhancer-binding protein; CMV, cytomegalovirus; FBS, fetal bovine serum; HES, Hairy Enhancer of Split; IBMX, isobutylmethylxanthine; NotchIC, Notch 1 intracellular domain; RBP-J, recombination signal binding protein of the J Ig gene.
Received April 14, 2003.
Accepted for publication August 25, 2003.
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gene results in early embryonic death. Development 121:3291–3301[Abstract]
induces the insulin-dependent glucose transporter GLUT4 in the absence of C/EBP during the conversion of 3T3 fibroblasts into adipocytes. J Clin Invest 101:22–32[Medline]
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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, pLPCX; 
, pLPCX NotchIC). Lower panel, Representative culture of cells grown 4 wk postconfluence and stained with Acridine Orange. Note the increased abundance of nuclear condensation in cells transduced with pLPCX compared with pLPCX NotchIC.
