1 Bone Diseases Group, Department of Biotechnology, Hoechst-Marion-Roussel, 111 route de Noisy, 93230 Romainville, France
2 Dana-Farber Cancer Institute, 44 Binney St, Boston, MA 02115, USA
3 Departments of Cell Biology and Orthopaedics, Yale University School of Medicine, New Haven, CT 06510, USA
* Both authors equally contributed to this work
Author for correspondence (e-mail: sergio.romanroman{at}aventis.com)
Accepted March 7, 2001
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SUMMARY |
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Key words: Sonic hedgehog, Bone morphogenetic protein 2, Osteoblast, Adipocyte, Differentiation
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INTRODUCTION |
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Recently, members of the hedgehog gene family have been shown to regulate skeletal formation in vertebrates. Although Ihh was first identified as a regulator of chondrocyte differentiation (Vortkamp et al., 1996), Ihh signaling has been shown to be essential for normal osteoblast development in endochondral bones (St-Jacques et al., 1999). Mice in which Shh has been deleted fail to form vertebrae and display severe defects of distal limb skeletal elements (Chiang et al., 1996). Shh seems to affect both chondrocyte and osteoblast differentiation. Overexpression of Shh in in vitro chondrogenic cultures promoted characteristics of hypertrophic chondrocytes (Stott and Chuong, 1997) and Shh has been shown to mediate the survival of both myogenic and chondrogenic cell lineages in the somites (Teillet et al., 1998). In vitro, Shh has been demonstrated to induce alkaline phosphatase (ALP), a marker of osteoblast differentiation, in the mouse mesenchymal cell line C3H10T1/2 (Katsuura et al., 1999; Kinto et al., 1997; Murone et al., 1999; Nakamura et al., 1997) and the osteoblast cell line MC3T3-E1 (Nakamura et al., 1997). Interestingly, intramuscular transplantation of fibroblasts expressing Shh into athymic mice induced ectopic bone formation (Kinto et al., 1997).
In the present study, we analyzed the effects of Shh on osteoblastic and adipocytic differentiation by using either pluripotent cell lines or murine calvaria cultures. Interestingly, we found that Shh increases the commitment of mesenchymal cell lines and calvaria cells to the osteoblastic lineage in response to BMP-2. Shh also inhibits the ability of these cells to differentiate into adipocytes. Given that the bone loss occurring with aging is associated with reduced osteoblastic bone formation and an increased volume of marrow adipose tissue, the Shh pathway might constitute a good therapeutic target to treat osteopenic disorders.
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MATERIALS AND METHODS |
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Shh was then concentrated on a 5 ml Hitrap SP column (Pharmacia) and further purified using a gel filtration column (Superdex 200 pg, Pharmacia). Final fractions containing Shh were pooled then stored at -80°C until used.
Cell culture
C3H10T1/2 (obtained from ATCC) and MC3T3-E1 (kindly provided by R. Francesch) cell lines were cultured (5% CO2 at 37°C) in -MEM supplemented with 10% heat inactivated fetal calf serum. C2C12 cells (kindly provided by G. Karsenty) was maintained (5% CO2 at 37°C) in Dulbeccos modified Eagles medium supplemented with 15% fetal calf serum. For treatment or transient transfection, cells were plated at 2x104/cm2 and 24 hours later the culture medium was changed for that with 2% fetal calf serum. Treatment or transfections were carried out as indicated below.
Calvaria cell preparation
Murine calvaria cells were obtained from the calvariae of neonatal mice 1-2 days after birth by sequential collagenase digestion at 37°C. Calvariae were removed from the animals under aseptic conditions and incubated at 37°C in DMEM containing trypsin (0.5 mg/ml) and EDTA (1.5 mg/ml) under continuous agitation. Trypsin digests were discarded at 15 minutes and replaced with DMEM containing 1 mg/ml of collagenase. The collagenase digests were discarded at 20 minutes and replaced with fresh enzyme dilution. The cells released between 20-40 minutes were collected by a short passive sedimentation step, and two centrifugation steps (400 g, 10 minutes) and cultured in proliferation medium (DMEM supplemented with 20% FCS and 2 mM glutamine) at a density of 2.5x104 cells per cm2 in Petri dishes (100 mm diameter). Calvaria cells were cultured until 80% confluence and stocks were frozen. For the experiments described here, cells were thawed in proliferation medium; two days later, this medium was replaced by differentiation medium (MEM containing 10% FCS, 2 mM glutamine, 50 µg/ml ascorbic acid and 10 mM ß-glycerolphosphate) and stimulated with the different agents for the indicated times.
Measurement of alkaline phosphatase activity
Cells were treated for the indicated time with BMP-2, Shh or BMP-2/Shh, and alkaline phosphatase (ALP) activity was determined in cell lysates using Alkaline Phosphatase Opt kit (Roche Molecular Biochemicals). Cell lysates were analyzed for protein content using a micro-BCA Assay kit (Pierce), and ALP activity was normalized for total protein concentration. For histochemical analysis of plasma-membrane-associated ALP, after stimulation with BMP-2, Shh or BMP-2/Shh for the time indicated, cells were washed three times with PBS and stained using Alkaline Phosphatase Leukocyte Staining Kit (Sigma, St Louis, MO), according to the manufacturers protocol.
Plasmids, cell transfection and assay for luciferase activity
The gal4-Smad1 construct was provided by A. Atfi. The aP2 promoter luciferase construct (paP2/luc) was made by subcloning the 5.4 kb KpnI-SmaI insert isolated from aP2-pBSKII+ into the pGL3-basic vector (Promega). Gli1 and Noggin were isolated by RT-PCR and clones were confirmed by DNA sequence analysis. Gli1 and Noggin were subcloned into pcDNA3.1 vector (Invitrogen). pRSV-PKI and pRSV-PKImut were kindly provided by R. A. Maurer.
C3H10T1/2 cells plated in 24-well plates, as indicated above, were transiently transfected with the indicated construct (1 µg) using DNA-lipid complex Fugene 6 (Boehringer Mannheim) according to the manufactures protocol. To assess transfection efficacy 20 ng of pRL-TK (Promega, Madison, WI), which encodes a Renilla luciferase gene downstream of a minimal HSV-TK promoter, was systematically added to the transfection mix. In experiments using pGli1 and pNog constructs, controls were carried out by replacing constructs with empty pcDNA3. 16 hours after transfection, cells were washed, cultured in medium at 2% fetal calf serum and either left unstimulated or stimulated with Shh for an additional 48 hours. ALP activity was determined in cell lysates as indicated above. When luciferase reporter constructs were used, luciferase assays were performed with the Dual Luciferase Assay Kit (Promega) according to the manufacturers instructions. 10 µl of cell lysate was assayed first for firefly luciferase and then for Renilla luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity.
RNA purification and gene expression analysis by real-time TaqMan PCR
Cells were treated as indicated above and total RNA was isolated from cultured cells using total isolation kit from Quantum Appligene (Illkirch, France). ALP, PPAR, C/EBP1
, aP2 and leptin mRNA expression was determined by RT followed by real-time TaqMan PCR analysis. Optimal oligonucleotide primers and TaqMan probes (Table 1) were designed using Primer Express V1.0 (Perkin-Elmer Applied Biosystems Inc.) using murine ALP (Accession J02980), PPAR
, C/EBP1
, aP2 and leptin sequences from the GenBank database. Glyceraldehyde phosphate dehydrogenase (GAPDH) primers and TaqMan probe (labeled with Vic fluorochrome) were from Perkin-Elmer Applied Biosystems.
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Determination of triglyceride
C3H10T1/2 cells were cultured as indicated above, then either left unstimulated or stimulated with Shh. The accumulation of intracellular triglyceride droplets was visualized by staining with Oil Red O. Triglyceride release into culture supernatant was measured using Sigma diagnosis glycerol-triglyceride (GPO-Trinder) kit according to the manufactures specification (Sigma).
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RESULTS |
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Together, our results indicate that N-Shh differentially regulates the osteoblastic and adipocytic commitment in a pluripotent cell line. It was therefore very interesting to investigate whether comparable effects are found using primary cells in culture. Calvaria cells constitute a good cellular model because these cells differentiate into osteoblasts in the presence of serum, ascorbate and ß-glycerolphosphate. Moreover, in these conditions, adipogenesis can also be observed. In fact, most of the adipocyte markers are induced during the culture (data not shown). Mouse calvaria cells prepared as described in Materials and Methods were cultured in the presence of N-Shh, BMP-2 or both. After 5 days of culture, ALP activity was measured and RNA expression levels of aP2, PPAR2 and C/EBP
were determined and compared with control unstimulated cells. As shown in Fig. 10A, N-Shh alone did not affect the ALP activity of calvaria cells but it significantly increased the ALP activity displayed by calvaria cells at day 5 of culture in the presence of BMP-2. N-Shh alone dramatically reduced the adipocyte markers in calvaria cells (Fig. 10B).
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DISCUSSION |
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One of the most interesting observations of this study is the synergistic effect of BMP-2 and N-Shh on the osteoblast differentiation. Although BMP-2 was shown to be dispensable for a slight induction of ALP by N-Shh in C3H10T1/2 cells, cooperation between BMP-2 and N-Shh was necessary to promote a strong induction of the osteoblast marker. This synergy might be explained by a modulation exerted by N-Shh on the signaling by BMP-2 or vice-versa. Currently, the intracellular signals induced by Hh proteins in mammal cells are poorly understood. Smo has been demonstrated to be the signaling component of the Hh receptor complex, whereas patched is considered as a ligand-regulated inhibitor of Smo (Murone et al., 1999). Smo activity requires the third intracellular loop of Smo, a domain typically involved in the coupling of seven transmembrane receptors to G protein effectors. However, so far there is no evidence that second messengers implicated in G-protein-coupled receptor signalling take part in the Hh response (Murone et al., 1999). Members of the Ci/Gli family of DNA-binding proteins are the major downstream transcriptional effectors mediating Hh signaling (Ruiz i Altaba, 1997). Studies conducted in Drosophila have shown that, in the absence of Hh, full-length Ci, which is localized in the cytoplasm, is processed into an N-terminal nuclear repressor form. Phosphorylation of Ci by PKA may target this protein for ubiquitination and processing by the 26S proteasome. In the presence of Hh, phosphorylation of Ci is suppressed, possibly a consequence of inhibiting PKA (reviewed by Ingham, 1998). Concerning BMP signaling, it is well established that the main intracellular signaling mediators are the different members of the Smad protein family. Receptor-regulated Smad1, Smad5 and Smad8 are the targets of BMP receptors. After phosphorylation, receptor-regulated Smads associate with the common Smad, Smad4, and the heteromeric complex is translocated into the nucleus where it activates specific genes through cooperative interactions with DNA and other DNA-binding proteins such as FAST1, FAST2 and Fos/Jun (reviewed by Derynck et al., 1998). Recently, two examples of crosstalking between BMP and Hh signaling have been reported. First, truncated Gli3 proteins have been demonstrated to associate with Smads (Liu et al., 1998). Second, Shh has been shown to promote somitic chondrogenesis by altering the cellular response to BMP signaling (Murtaugh et al., 1999).
Concerning the synergy between BMP-2 and N-Shh described here, we have demonstrated that a short pre-treatment (30-60 minutes) with N-Shh followed by BMP-2 stimulation is sufficient to obtain the synergy between these proteins. Interestingly, no synergy was demonstrated with a longer pre-treatment with Shh (24 hours). This suggests that Shh provides a transient window of competence during which time BMP signals induce osteoblastic differentiation in cells otherwise refractory to osteoblast commitment in response to BMP-2. A similar mechanism has been recently proposed to explain the effects of Shh in the promotion of somitic chondrogenesis (Murtaugh et al., 1999). Shh can alter BMP responsiveness by directly affecting one or several proteins in the cascade of BMP signaling events. We have demonstrated that the synergistic N-Shh/BMP-2 effect is in part mediated by an N-Shh modulation of the BMP signaling pathway component Smad1. Indeed, N-Shh increases BMP-2-mediated Smad1 transcriptional activity in both C3H10T1/2 and ST2 cells. In addition, this modulation seems to be independent of the well known Hh pathway transcription factor Gli1, because transfection with Gli1 did not alter the transcriptional activity of Smad1 (Fig. 6), suggesting that Hh signaling components upstream of Gli are integrated with the BMP signaling complex to affect Smad activity. The enhancement of Smad1 transcriptional activity may be the result of a positive effect in the life span/availability, phosphorylation or nuclear accumulation of Smad1 or an indirect effect on a co-activator or co-repressor involved in the transcriptional machinery of Smad1. We are currently investigating whether N-Shh modifies the expression of a series of proteins described as capable of interacting with BMP signaling.
Another interesting finding described in this study is the fact that the ability to respond to N-Shh in terms of ALP expression seems to be dependent on the stage of differentiation of cells. N-Shh displayed synergistic effect on stromal cell line ST2 (described as a pre-adipocyte cell line but able to differentiate into an osteoblast in the presence of BMP-2) and mesenchymal pluripotent C3H10T1/2 cells. On the contrary, the pre-osteoblastic MC3T3-E1 cells and the mature osteoblastic cell lines ROS 17/2.8 and ROB-C26 (data not shown) are insensitive to N-Shh in terms of ALP induction in the presence of BMP-2. This suggests that only immature pluripotent cells respond to N-Shh. A synergistic effect of N-Shh and BMP-2 was clearly demonstrated in calvaria, in which there is a heterogeneous population including cells at different stage of differentiation.
It is important to point out that, in the absence of BMP-2, only C3H10T1/2 was shown to significantly respond to N-Shh in terms of ALP expression. This response could be mimicked by transfecting cells with a Gli1 expression vector, thus suggesting that the activity is dependent on the activity of this transcription factor. Our results suggest that there are at least two mechanisms by which Shh regulates osteogenic differentiation of cells: directly via a Gli-dependent manner and indirectly via a Gli-independent modulation of BMP signaling via Smad, as discussed above. In addition, our results suggest that these two mechanisms are triggered at the same time only in cells displaying a particular stage of differentiation, because only the more immature cells studied here, C3H10T1/2, respond to N-Shh alone. Although N-Shh alone did not affect the osteoblastic commitment in calvaria cells, one could not exclude the existence of a subpopulation of calvaria cells that respond to N-Shh in the absence of BMP-2 but, if they exist, the fraction of responding cells is very small.
Hh molecules have been shown to control the differentiation of different cell types. Here, we present evidence that N-Shh dramatically compromises the adipocytic commitment of both the pluripotent mesenchymal cell line C3H10T1/2 and calvaria cells. The decrease in the number of mature adipocytes was evaluated by looking at the presence of lipidic vacuoles in cells, by measuring the lipase activity and also by looking at the gene expression of a number of adipocytic markers. Thus, Shh dramatically reduced the levels of PPAR2 and C/EBP
, two transcription factors playing a central role in adipogenesis. Furthermore, we have shown that overexpression of the transcription factor Gli1 in the cells mimics this activity. How Gli1 can affect adipocytic differentiation needs to be explored in detail. Gli1 has not been described as directly repressing any gene transcription, therefore one possibility is that Shh via Gli1 induces the expression of molecules that inhibit adipocytic commitment. One candidate could be PTHrP, which can be induced in mesenchymal cells and osteoblasts and has been described as negatively affecting adipogenesis. However, our data demonstrate that PTHrP is not involved in the effects described here. We are currently using genome-wide expression analysis to study the regulation of genes by N-Shh in C3H10T1/2 to select candidate genes potentially involved in the activity of N-Shh on adipocytic differentiation.
The potential in vivo relevance of our results remains to be elucidated. Concerning the expression of Hh proteins in vivo, Shh signal is observed in the posterior mesoderm at the initial stage of limb development, and remains distant from the area where skeletal elements appear. By contrast, the distribution of Ihh signals is closely related to cartilage-forming regions. Little information exists about the expression of hedgehog molecules in osteoblastic or adipocytic lineages in adult tissues, but the expression of Shh in cells surrounding the periostium after fracture has recently been reported in mice (Kuriyama et al., 2000). None of the different cell lines used in our study expresses Shh mRNA (data not shown). These data suggest that the source of Hh proteins in vivo must be other than the osteoblasts or their precursors. Concerning the effect of Shh on adipocytic commitment, no relevant finding has been described in mice lacking Shh gene function (Chiang et al., 1996).
Total marrow fat increases with age, and there is an inverse relationship between marrow adipocytes and osteoblasts with aging (Beresford et al., 1992; Burkhardt et al., 1987). The number of mesenchymal stem cells with osteogenic potential decreases early during aging in humans and may be responsible for the age-related reduction in osteoblast number (DIppolito et al., 1999). In addition, it has been demonstrated that cells cultured from human trabecular bone are not only osteogenic, but undergo adipocytic differentiation under defined culture conditions (Nuttall et al., 1998). A better understanding of the pathways triggered by Hh proteins is necessary to elucidate the mechanisms by which these proteins modulate osteogenesis/adipogenesis in vitro and in vivo. The elucidation of these mechanisms could be crucial to consider new approaches to treat osteopenic disorders.
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