Notch 1 Impairs Osteoblastic Cell Differentiation

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Endocrinology 144(12):5631–5639 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2003-0463

Notch 1 Impairs Osteoblastic Cell Differentiation MARIA SCIAUDONE, ELISABETTA GAZZERRO, LEAH PRIEST, ANNE M. DELANY, ERNESTO CANALIS

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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 Notch receptors are single pass transmembrane receptors activated by membrane-bound ligands with a role in cell proliferation and differentiation. As Notch 1 and 2 mRNAs are expressed by osteoblasts and induced by cortisol, we postulated that Notch could regulate osteoblastogenesis. We investigated the effects of retroviral vectors directing the constitutive expression of the Notch 1 intracellular domain (NotchIC) in murine ST-2 stromal and in MC3T3 cells. NotchIC overexpression was documented by increased Notch 1 transcripts and activity of the Notch-dependent Hairy Enhancer of Split promoter. In the presence of bone morphogenetic protein-2 (BMP-2), ST-2 cells differentiated toward osteoblasts forming mineralized nodules, and Notch 1 opposed this effect

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ONE MARROW STROMA contain pluripotential cells with the potential to differentiate into various cells of the mesenchymal lineage including osteoblasts and adipocytes (1, 2). The ultimate cellular phenotype depends on extracellular and intracellular signals. The Notch receptor is a single pass transmembrane receptor that is activated through direct contact with membrane-bound ligands (3). Four Notch receptors, termed Notch 1, 2, 3, and 4, have been identified, and they are activated by the Delta 1 and 2 and Serrate/Jagged 1 and 2 ligands (3). Notch ligand interaction results in proteolytic cleavage of the Notch receptor, with release of the intracellular domain and its translocation to the nucleus, where it interacts with the CSL (CBF1/RBP-J␬, 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 – 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 –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 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.

and decreased the expression of osteocalcin, type I collagen, and alkaline phosphatase transcripts and ⌬2⌬ FosB protein. Further, NotchIC decreased Wnt/␤-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. (Endocrinology 144: 5631–5639, 2003)

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. Materials and Methods Retroviral vectors and packaging cell lines A 2389-bp DNA fragment containing the intracellular domain of the murine Notch 1 (NotchIC) cDNA with a Myc epitope tag on the amino terminal end (provided by J. Nye, Chicago, IL) were cloned into the retroviral vector pLPCX (Clontech, Palo Alto, CA) (17). The vector contains a Moloney murine leukemia virus 5⬘ long terminal repeat to drive the packaging signal, a cytomegalovirus (CMV) promoter to drive the

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constitutive expression of NotchIC, and a puromycin resistance gene for positive selection. pLPCX and pLPCX NotchIC were transfected into Phoenix packaging cells [American Type Culture Collection (ATCC), Manassas, VA] by calcium phosphate/DNA coprecipitation and glycerol shock as previously described (18). Phoenix cells were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA) and stably transfected cell lines were generated by selection for puromycin resistance. Retrovirus-containing conditioned medium was harvested, filtered through a 0.45-␮m pore size membrane, and used to transduce ST-2 cells.

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 –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 ␮g/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 ␮g/ml ascorbic acid, and 5 mm ␤-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 ␮m (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 ␮g/ml ascorbic acid, and 5 mm ␤-glycerophosphate. To study the regulation of Notch 1, cells were cultured in the absence and presence of 1 nm BMP-2, 1 ␮m cortisol, or an adipogenic cocktail composed of 1 ␮m 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,

Sciaudone et al. • Notch and Cell Differentiation

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/EBP␤ 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 (PPAR␥2) 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 –33). DNAs were labeled with [␣-32P]deoxy-CTP (50 ␮Ci 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 1⫻ saline sodium citrate, and a wash at 65 C for 20–30 min in 0.1⫻ or 1⫻ 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 ␮g/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 ␤-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 ␮l FuGene/2 ␮g DNA) according to manufacturer’s instructions (Roche, Indianapolis, IN). Cotransfections with a construct containing the CMV promoter driving the ␤-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 ␤-galactosidase activities were measured using an Optocomp luminometer (MGM Instruments, Hamden, CT) as previously described (18). Luciferase activity was corrected for ␤-galactosidase activity to control for transfection efficiency.


Statistical analysis Data are expressed as the mean ⫾ sem. Statistical significance was determined by t test

Results

To investigate the impact of increased Notch 1 signaling on stromal cell differentiation, ST-2 stromal cells were transduced with pLPCX NotchIC, resulting in a cell line in which Notch signaling is constitutively active in the absence of ligands. ST-2 stromal cells overexpressing NotchIC were compared with cells transduced with pLPCX vector alone, and the behavior of these cells was similar to that of untransduced ST-2 cells (data not shown). Northern blot analysis demonstrated that the NotchIC transcripts, expressed under the control of the CMV promoter, were strongly expressed by ST-2 cells cultured from confluence to 4 wk post confluence (Fig. 1). 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). To monitor the impact of NotchIC on stromal cell differentiation, pLPCX and pLPCX NotchIC ST-2 cells were cultured to confluence; changed to medium containing ascorbic

FIG. 1. Overexpression of NotchIC in stably transduced ST-2 stromal cells. Upper panel, ST-2 cells transduced with pLPCX NotchIC (⫹) or with pLPCX vector (⫺) alone were grown to confluence (0) or for 1– 4 wk post confluence. Total RNA was extracted, subjected to Northern blot analysis, probed with ␣-32P-labeled Notch 1 and 18S cDNA, and visualized by autoradiography. Lower panel, Subconfluent cultures of ST-2 cells transduced with pLPCX vector or pLPCX NotchIC were transiently cotransfected with HES-5-tk-luc and a CMV ␤-galactosidase expression vector. Data shown are for luciferase activity/␤-galactosidase activity and are the mean ⫾ SEM for six observations. *, Significantly different from control, P ⬍ 0.05.

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acid, ␤-glycerophosphate, and 1 nm BMP-2; and cultured for 1- to 4-wk periods. Confirming previous observations, pLPCX ST-2 cells cultured under these conditions gave rise to mineralized nodules after 3 wk, as assessed by Alizarin Red staining (Fig. 2) (23). This mineralized nodule formation correlated with increased expression of the osteoblasticspecific 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 PPAR␥2 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 ⌬2⌬FosB isoform, in particular, was increased. However, this was not seen in pLPCX NotchIC cells, supporting the concept that Notch 1 signaling inhibited osteoblastogenesis. NotchIC overexpression caused modest changes in the number of viable ST-2 cells (Table 1). 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). To further assess the effect of NotchIC on adipogenesis, pLPCX ST-2 and pLPCX NotchIC ST-2 cells were cultured in the presence of the glucocorticoid cortisol, which is known to induce differentiation of ST-2 cells toward the adipocytic and not toward the osteoblastic pathway (23). Confirming prior observations, cortisol induced the expression of adipsin and PPAR␥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 PPAR␥2 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␣, -␤, and -␦ transcripts (23). Overexpression of NotchIC did not modify C/EBP␣, -␤, or -␦ mRNA levels, but enhanced the stimulatory effect of cortisol on C/EBP␣ and -␦ transcript expression (Fig. 6). To explore possible mechanisms involved in the effects of Notch 1 on stromal cell differentiation, we explored whether it modified the canonical Wnt/␤-catenin signaling pathway, as Notch and Wnt have opposite effects on adipogenesis and osteogenesis (42, 43). For this purpose, we tested the effect of

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FIG. 2. Effect of NotchIC in the presence of BMP-2 on the differentiation of ST-2 stromal cells. ST-2 cells transduced with pLPCX vector (⫺) or pLPCX NotchIC (⫹) were cultured to confluence (0) or for 1– 4 wk post confluence. Upper panel, Total RNA was extracted; subjected to Northern blot analysis; probed with ␣-32P-labeled osteocalcin (OC), ␣1(I)collagen [␣1(I) Coll.] alkaline phosphatase (Alk. Phos.), adipsin, PPAR␥2, and 18S cDNA; and visualized by autoradiography. The graph represents fold changes in NotchIC-overexpressing cultures/pLPCX vector for the expression of osteocalcin, ␣1(I) collagen, alkaline phosphatase, adipsin, and PPAR␥2 mRNAs. Values are the mean ⫾ SEM (n ⫽ 3). Lower panel, Cells were stained with Alizarin Red to detect mineralized nodule formation. There was no difference in the phenotype between untransfected ST-2 cells (not shown) and cells transduced with pLPCX vector.

TABLE 1. Effect of NotchIC on ST-2 stromal cell viability Wk

FIG. 3. Effect of NotchIC in the presence BMP-2 on the expression of FosB isoforms in ST-2 stromal cells. ST-2 cells transduced with pLPCX vector (⫺) or pLPCX NotchIC (⫹) were cultured for the indicated days post confluence. Nuclear extracts were subjected to Western blot analysis and probed with a primary antibody against FosB, which can detect all FosB isoforms. The ⌬FosB isoform results from alternative splicing, and the ⌬1 and ⌬2⌬FosB isoforms result from alternative translation initiation of the ⌬FosB transcript. n ⫽ 2 replicate cultures.

Notch 1 signaling on the transiently transfected ␤-cateninresponsive construct, pTOPFLASH, containing three Lef 1/Tcf-4 binding sequences upstream of the luciferase gene, or its mutant, pFOPFLASH (38). The activity of the pTOPFLASH reporter was decreased by about 50% in NotchICoverexpressing ST-2 cells, whereas the activity of the pFOPFLASH was not affected (Fig. 7). Similar to previous observations in MC3T3 cells, ST-2 cells expressed Notch 1 and 2, and Delta 1 and Jagged 1 mRNA, whereas Delta 3 and Jagged 2 transcripts were not detectable by Northern blot analysis (13) (Fig. 8, left panel). Cortisol

0 1 2 3 4

Absorbance at 490 nm pLPCX

pLPCX-NotchIC

379 ⫾ 8 1523 ⫾ 51 749 ⫾ 28 635 ⫾ 21 679 ⫾ 77

537 ⫾ 11a 1677 ⫾ 68 810 ⫾ 12 710 ⫾ 23a 709 ⫾ 37

ST-2 cells transfected with pLPCX or pLPCX-NotchIC were cultured to confluence (0) or for up to 4 wk postconfluence in the presence of BMP-2 at 1 nM, and cell viability was estimated by the conversion of MTS to formazan measured at 490 nm. Data are the mean ⫾ SEM for 6 –10 observations. a Significantly different from control, P ⬍ 0.05.

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). To confirm the effect of Notch 1 on osteoblastic cell differentiation, MC3T3-E1 osteoblastic cells were transduced with pLPCX or pLPCX NotchIC. The behavior of cells transduced with pLPCX vector alone was similar to that of untransduced MC3T3 cells (data not shown). Northern blot analysis demonstrated that NotchIC transcripts were ex-



FIG. 4. Effect of NotchIC in the presence of BMP-2 on apoptosis in ST-2 stromal cells. ST-2 cells transduced with pLPCX vector (⫺) or pLPCX NotchIC (⫹) were cultured to confluence (0) or for 1– 4 wk post confluence, fixed, stained with Acridine Orange, and examined by fluorescence microscopy. Upper panel, Percentage of apoptotic cells, determined by evaluation of nuclear condensation (f, pLPCX; 䡺, pLPCX NotchIC). Lower panel, Representative culture of cells grown 4 wk post confluence and stained with Acridine Orange. Note the increased abundance of nuclear condensation in cells transduced with pLPCX compared with pLPCX NotchIC.

FIG. 5. Effect of NotchIC in the presence or absence of cortisol (1 ␮M) on the differentiation of ST-2 stromal cells. ST-2 cells transduced with pLPCX vector (⫺) or pLPCX NotchIC (⫹) were cultured to confluence (0) or for 1– 4 wk post confluence. Upper panel, Total RNA was extracted; subjected to Northern blot analysis; probed with ␣-32P-labeled adipsin, PPAR␥2, osteocalcin (OC), and 18S cDNA; and visualized by autoradiography. The graph represents fold changes in NotchIC-overexpressing cultures/ pLPCX vector in the presence of cortisol for the expression of adipsin and PPAR␥2 mRNAs. Values are the mean ⫾ SEM (n ⫽ 3). Lower panel, Cells cultured for 1 wk post confluence in the presence of cortisol were stained with Oil Red O to detect intracellular lipid droplets and counterstained with hematoxylin.

pressed from confluence to 4 wk post confluence in cells transduced with pLPCX NotchIC (Fig. 9). 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).

Discussion

We used stromal and MC3T3 cells to study phenotypic changes associated with osteoblastic cell differentiation under conditions of constitutive Notch 1 signaling. Confirming previous observations, ST-2 stromal cells in the presence of BMP-2 differentiated toward the osteoblastic phenotype, and in the presence of cortisol they differentiated toward the adipocytic phenotype (23, 44). Overexpression of NotchIC opposed the effect of BMP-2 and prevented osteoblastic differentiation. Furthermore, NotchIC favored differentiation of ST-2 cells toward an adipocytic pathway, enhancing the

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FIG. 6. Effect of NotchIC in the presence or absence of cortisol (1 ␮M) on the expression of C/EBP␣, -␤, and -␦ mRNA in ST-2 stromal cells. ST-2 cells transduced with pLPCX vector (⫺) or pLPCX NotchIC (⫹) were cultured to confluence (0) or for 1– 4 wk post confluence. Total RNA was extracted, subjected to Northern blot analysis, probed with ␣-32P-labeled C/EBP␣, C/EBP␤, C/EBP␦, and 18S cDNA, and visualized by autoradiography. The graphs represent fold changes in NotchIC-overexpressing cultures/pLPCX vector in the absence (A) or presence (B) of cortisol for the expression of C/EBP␣, -␤, and -␦. Values are the mean ⫾ SEM (n ⫽ 3).

FIG. 7. Effect of NotchIC overexpression on ␤-catenin signaling. Subconfluent cultures of ST-2 cells transduced with pLPCX vector or pLPCX NotchIC were transiently cotransfected with the ␤-cateninresponsive Lef 1 reporter plasmid, pTOPFLASH, containing three binding sites for Lef 1/Tcf-4, or its mutant, pFOPFLASH, containing three mutated binding sites for Lef 1, cloned into a luciferase reporter plasmid and a CMV ␤-galactosidase expression vector. Data shown are for luciferase activity/␤-galactosidase activity and are the mean ⫾ SEM for six observations. *, Significantly different from vector, P ⬍ 0.05.

effects of cortisol. These results indicate that Notch 1 signaling causes a shift in the differentiation of the cell population from osteoblasts to adipocytes. The inhibitory effects of Notch 1 on osteoblastic cell differentiation were confirmed in cultures of MC3T3 cells, a cell line considered preosteoblastic. Furthermore, our results are in agreement with those of previous investigations demonstrating an inhibitory effect of NotchIC on osteogenesis in C2C12 cells (15). However, they contrast with recent reports demonstrating that NotchIC, delivered by an adenoviral vector, induces the differentiation

of MC3T3 and C3H10T1/2 cells and potentiates the effect of BMP (16). The differences in the results may be due to the use of different cell lines and culture conditions or to the shortlived overexpression of NotchIC induced by the adenoviral vector delivery system. This could have resulted in an early increase in cell replication, leading to a larger pool of cells that eventually differentiated in the presence of BMP. Notch 1 could affect cell replication, as it increases the transcription of cyclin D1, and in selected cell systems the expression of Notch mRNA is induced by cell mitogens (45, 46). In the present studies NotchIC had only minor effects on cell number in the initial phases of the culture. Although NotchIC overexpression decreased apoptosis, this event appeared to be related to its inhibitory effect on terminal osteoblastic differentiation of stromal cells, which is associated with apoptosis (39). 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␣, -␤, and -␦ and PPAR␥2, 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 PPAR␥2 and C/EBP␣ and -␦,



FIG. 8. Expression of Notch 1 and 2, Delta 1, and Jagged 1 mRNA in ST-2 stromal cells. ST-2 cells were cultured to confluence (0) and for 1– 4 wk post confluence (upper panel) and in the presence or absence of BMP-2 (1 nM), cortisol (1 ␮M), or an adipogenic cocktail [A.C.; dexamethasone (1 ␮M), insulin (100 nM), and IBMX (0.5 mM); lower panel]. Total RNA was extracted, subjected to Northern blot analysis; probed with ␣-32Plabeled Notch 1, Notch 2, Delta 1, Jagged 1, and 18S cDNAs (left panel) or Notch 1 and 18S cDNAs (right panel); and visualized by autoradiography.

FIG. 9. Effect of NotchIC on the differentiation of MC3T3 cells. MC3T3 cells transduced with pLPCX vector (⫺) or pLPCX NotchIC (⫹) were cultured to confluence (0) or for 1– 4 wk post confluence. Upper panel, Total RNA was extracted; subjected to Northern blot analysis; probed with ␣-32P-labeled osteocalcin (OC), ␣1(I) collagen [␣1(I)Coll.], Notch 1, and 18S cDNAs; and visualized by autoradiography. The graph represents fold changes in NotchIC-overexpressing cultures/pLPCX vector for osteocalcin and ␣1(I) collagen mRNAs. Values are the mean ⫾ SEM (n ⫽ 3). Lower panel, Cells were stained with Alizarin Red to detect mineralized nodule formation. There was no difference in the phenotype between untransfected MC3T3 cells (not shown) and cells transduced with pLPCX vector.

TABLE 2. Effect of NotchIC on MC3T3 cell viability Wk

0 1 2 3 4

Absorbance at 490 nm pLPCX

pLPCX-NotchIC

1088 ⫾ 42 1180 ⫾ 75 1835 ⫾ 49 1885 ⫾ 64 2191 ⫾ 160

967 ⫾ 31a 1372 ⫾ 94 1933 ⫾ 67 1811 ⫾ 120 2509 ⫾ 56

MC3T3 cells transfected with pLPCX or pLPCX-NotchIC were cultured to confluence (0) or for up to 4 wk post confluence, and cell viability was estimated by the conversion of MTS to formazan measured at 490 nm. Data are the mean ⫾ SEM for 10 observations. a Significantly different from control, P ⬍ 0.05.

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 ⌬2⌬FosB.

5638


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 –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 ␤-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. Acknowledgments


11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

21. 22.

23.

We thank Ms. Nancy Wallach for helpful secretarial assistance. 24.

Received April 14, 2003. Accepted August 25, 2003. 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: [email protected]. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-42424 and DK-45227 and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-44877. M.S. and E.G. contributed equally to this work.

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