The authors have no conflict of interest
Overexpression of Dlx5 in Chicken Calvarial Cells Accelerates Osteoblastic Differentiation†
Article first published online: 1 JUN 2002
Copyright © 2002 ASBMR
Journal of Bone and Mineral Research
Volume 17, Issue 6, pages 1008–1014, June 2002
How to Cite
Tadic, T., Dodig, M., Erceg, I., Marijanovic, I., Mina, M., Kalajzic, Z., Velonis, D., Kronenberg, M. S., Kosher, R. A., Ferrari, D. and Lichtler, A. C. (2002), Overexpression of Dlx5 in Chicken Calvarial Cells Accelerates Osteoblastic Differentiation. J Bone Miner Res, 17: 1008–1014. doi: 10.1359/jbmr.2002.17.6.1008
- Issue published online: 27 OCT 2009
- Article first published online: 1 JUN 2002
- Manuscript Accepted: 15 JAN 2002
- Manuscript Revised: 7 JAN 2002
- Manuscript Received: 19 MAR 2001
Our laboratory and others have shown that a homeodomain protein binding site plays an important role in transcription of the Col1a1 gene in osteoblasts. This suggests that homeodomain proteins have an important role in osteoblast differentiation. We have investigated the role of Dlx5 in osteoblastic differentiation. In situ hybridization studies indicated that Dlx5 is expressed in chick calvarial osteoblasts (cCOB) in vivo. Northern blot analysis indicated that Dlx5 expression in cultured cCOBs is induced concurrently with osteoblastic markers. To study the effect of overexpression of Dlx5 on osteoblast differentiation, we infected primary osteoblast cultures from 15-day-old embryonal chicken calvaria with replication competent retroviral vectors [RCASBP(A)] expressing Dlx5 or control replication competent avian splice acceptor brianhightiter polymerase subtype A [RCASBP(A)]. Expression of Col1a1, osteopontin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) occurred sooner and at higher levels in cultures infected with RCASBP(A)DLX5 than in RCASBP(A)-infected cultures. Mineralization of Dlx5-expressing cultures was evident by days 12-14, and RCAS-infected control osteoblasts did not begin to mineralize until day 17. Dlx5 also stimulated osteoblastic differentiation of calvarial cells that do not normally undergo osteoblastic differentiation in vitro. Our results suggest that Dlx5 plays an important role in inducing calvarial osteoblast differentiation.
THE DIFFERENTIATION of preosteoblasts to mature osteoblasts is a complex, multistage process in which control at the transcriptional level is not well understood. The recent observation that Cbfa1 is required for bone development is a significant discovery; however, it is probable that other transcription factors are involved in regulating this process. Our laboratory and others have shown that a homeodomain protein binding site in the Col1a1 promoter is necessary for high level expression of the gene in differentiated osteoblasts of transgenic mice; however, this site is less important in tendon or periosteal fibroblasts.(1,2) Because high-level expression of the Col1a1 gene is required for deposition of the bone matrix, proteins that regulate its expression are of fundamental importance to osteoblast differentiation. We have investigated the function of Dlx5, a homeodomain protein that is a potential regulator of the Col1a1 gene, in osteoblast differentiation.
The vertebrate Dlx genes are related to the distal-less gene, which is involved in limb development in Drosophila and other invertebrates.(3) There are 6-7 vertebrate Dlx genes, which are expressed in overlapping but significantly divergent domains in the craniofacial region and the limb during development.(4,5) Dlx2 is required for bone morphogenetic protein (BMP) 2 induction of Col2a1 expression in a chondroblast cell line.(6)Dlx5 and - 6 are expressed in almost every developing skeletal element, including endochondral and membranous bone.(4,5,7) In the chick limb, Dlx5 is found in the developing cartilaginous skeletal elements, most strongly in the cells that will become the initial bony collar surrounding the cartilage(8) (R. Kosher, unpublished results, 1996). Thus, the expression pattern of Dlx5 is consistent with a role in skeletal development. However, the published in situ hybridization studies have not analyzed skeletal Dlx5 expression in sufficient detail to determine whether Dlx5 is expressed in differentiated osteoblasts or in osteoblast precursors. Dlx5 messenger RNA (mRNA) levels increase during in vitro rat calvarial osteoblast differentiation; however, overexpression of Dlx5 in ROS 17/2.8 osteosarcoma cells inhibited expression of osteocalcin, a marker of differentiated osteoblasts.(9)Dlx5 is induced by BMPs in the immortalized osteoblastic cell line MC3T3-E1, and overexpression of the protein stimulates expression of osteoblastic markers in these cells.(10) Mutation of mouse Dlx5 by homologous recombination causes craniofacial and sensory capsule skeletal defects.(11,12) In addition, calvarial ossification is delayed, and diaphyseal cortical bone is less well organized(12) in Dlx5 knockout mice. A homeodomain protein binding site within the mouse bone sialoprotein (BSP) promoter is important for osteoblast-specific expression in transfected cell lines. This site binds Dlx5 in gel shift analysis, and Dlx5 stimulates the BSP promoter in cotransfected COS7 monkey kidney cells (COS) cells.(13)
To assess the role of Dlx5 in osteoblastic differentiation, we wished to investigate the expression of Dlx5 in differentiated osteoblasts in vivo and in primary cells in vitro. We also wished to assess the function of Dlx5 in osteoblast differentiation using a primary culture system rather than an immortalized cell line. Thus, we studied the expression of Dlx5 in ossifying embryonic day 15 chick calvaria and in cultured primary osteoblast cultures. We also investigated the effect of Dlx5 on osteoblastic differentiation in primary osteoblastic cell cultures. Our results show that Dlx5 is expressed in chick calvarial osteoblasts (cCOBs) in vivo, and that its expression increases in primary cCOB cultures concurrently with markers of differentiation. Dlx5 overexpression stimulates osteoblastic differentiation of these cells in culture. In addition, Dlx5 induces osteoblastic differentiation of cultures enriched in chick calvarial periosteal cells, which do not undergo osteoblastic differentiation in vitro under the conditions used in our experiments.
MATERIALS AND METHODS
In situ hybridization
The 618-base pair (bp) antisense Dlx5 riboprobe was prepared by in vitro transcription of Nco I linearized chick Dlx5(8) complementary DNA (cDNA) clone using T7 RNA polymerase. Nonspecific hybridization signals and emulsion background were examined by hybridization of adjacent tissue sections with a nonspecific riboprobe probe (a 600-bp fragment of Bluescript vector prepared by in vitro transcription of Afl III linearized plasmid using T7 RNA polymerase.) or with no probe at all. Whole calvaria (with the overlying skin and underlying brain) from 15-day, mandibles from 10-day, and limbs from 7-day old chick embryos were isolated, fixed in freshly prepared 4% paraformaldehyde at 40C overnight, washed, and dehydrated using methanol. Tissues were embedded in paraffin, sectioned in 7-μm thickness and processed for in situ hybridization to tissue sections using [35 P]uridine triphosphate (UTP)-labeled riboprobes as described previously.(14) After in situ hybridization the sections were stained with hematoxylin, mounted, examined, and photographed using an E600 Nikon microscope and a Spot RT TM (Diagnostic Instruments, Inc., Sterling Heights, MI, USA) camera. Images were digitized and processed with Adobe PhotoShop 4.0 (Adobe Systems, San Jose, CA, USA) software. The silver grains in the dark-field image were selected, colored red, and then superimposed onto the bright-field image. Images in Figs. 1E,1F and 1G are PhotoShop pseudocolored superimpositions of the in situ hybridization signals and bright-field images.
Primary cCOB culture
Calvarial cells were isolated by four sequential 15-minute digestions of 15-day-old embryonal chick calvariae in an enzyme mixture containing 0.05% trypsin (Gibco BRL, Grand Island, NY, USA) and 0.1% collagenase P (Roche Molecular Biochemicals, Indianapolis, IN, USA) at 37°C on a rocking platform. Fractions 2-4 were collected, resuspended in media, and grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS) for 8 days (until confluence). After the cells became confluent, differentiation media (BGJ containing 10% FCS, 50 μg/ml of ascorbic acid, and 5 mM of β-glycerophosphate; Gibco BRL) was used to maintain the cells for the duration of the experiment. Cells were harvested for analysis of bone markers at different stages of differentiation.
Retroviral vectors and infection of chick primary osteoblast cultures
The coding sequence of the chicken Dlx5 cDNA was cloned into the Cla I site of the RCASBP(A) helper-independent retroviral vector.(15) Control virus was RCASBP(A) vector without inserted cDNA. Vectors were transfected into producer cells (chicken embryonal fibroblasts [CEF]) using the calcium phosphate method. Reverse transcriptase activity was determined in the producer cell media as a measure of virus production. Conditioned media with high reverse transcriptase activity was collected and stored at −70°C until use. Primary cultures of cCOBs were infected three times with 500 μl of conditioned media containing RCAS-Dlx5, RCASBP(A), or no virus (MOCK) beginning on the day after plating. Virus was added to 2 ml of fresh media in a 35-mm dish.
Assessment of mineralization
Mineralization in cultures was determined by von Kossa staining. Briefly, after fixation cells were exposed for 45 minutes to bright light in a 5% solution of AgNO 3. The reaction was stopped with Na-thiosulphate; cells were washed and the cultures were photographed. The extent of mineralization was assessed by image analysis.
Northern blot analysis
RNA was isolated using TRI reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). Ten micrograms of total RNA was separated on a 1% agarose 1.1 M formaldehyde gel and transferred to nylon membrane (Extra strength Nytran; Schleicher and Schuell, Keene, NH, USA). Probes for chick Msx2, Dlx5, osteocalcin, osteopontin, and alkaline phosphatase represented full-length cDNAs. The probe for chick type I collagen was isolated from clone pcg54 containing a Col1a1 cDNA fragment.(16) The cDNA fragments were gel-purified using SpinBind DNA purification columns (FMC BioProducts, Rockland, ME, USA) and random primer-labeled. Hybridization was performed in 50% formamide (Gibco BRL) and 6× SSPE at 42°C. Evenness of loading was assessed by hybridization of the stripped blot to an 18S RNA probe or by evaluation of the ethidium bromide-stained gel or filter.
Dlx5 is expressed in developing skeletal elements during embryonic development; however, less is known about expression in bone at later stages. We analyzed expression of Dlx5 in 17-day chick calvaria by in situ hybridization. Expression of Dlx5 was detected in cells lining the bone surface (Fig. 1). Northern blot analysis of RNA from tissues derived from 17-day chick embryos showed expression of Dlx5 in calvaria, long bone, and brain but not in heart, muscle, liver, or skin (Fig. 2).
We examined endogenous Dlx5 expression during in vitro cCOB differentiation and found that levels were relatively low on day 4 and day 7 of culture, began to increase on day 12, were maximal on day 17, and continued high for the rest of the culture (Fig. 3A). Comparing this pattern to the normal sequence of osteoblastic development of these cultures (Fig. 3B), Dlx5 is induced at about the same time that Col1a1 and osteopontin mRNA, which are early markers of osteoblastic differentiation, are induced.
We then used an RCASBP(A)-Dlx5 expression vector to induce increased expression of Dlx5 during the first week of cCOB culture, when endogenous levels are relatively low. Control experiments indicated that expression of RCASBP(A) alone had no effect on differentiation of these cultures (Fig. 3B). Northern blot analysis showed that retrovirally produced Dlx5 mRNA was highly expressed on day 12 of culture compared with the endogenous Dlx5 mRNA and was even further increased on day 22 (Fig. 4). Note that only the lowest band can be translated to produce Dlx5 protein. The largest band is the unspliced RNA containing the retroviral gag-pol and env sequences in addition to Dlx5 sequences. The intermediate band contains env and Dlx5. Both can be translated only to produce retroviral proteins.(15) The diffuse nature of the hybridization pattern is characteristic of RCAS vectors in our hands. In this culture the endogenous Dlx5 band on day 22 of culture was less intense compared with day 12, presumably, because this culture differentiated relatively rapidly, and, thus, maximal levels of Dlx5 appeared relatively early. In the culture shown in Fig. 3, the induction of differentiation was later, between days 12 and 17.
By day 7, Col1a1 was highly induced in the Dlx5 expressing cultures, and alkaline phosphatase, osteopontin, and osteocalcin were beginning to be expressed. In the control cultures, Col1a1 and alkaline phosphatase were low and osteopontin and osteocalcin were not present at all. By day 12 all osteoblast markers examined were highly expressed in the Dlx5 cultures. In 12-day control cultures, osteocalcin and osteopontin were very weakly expressed, alkaline phosphatase was moderately expressed, and Col1a1 was maximal (Fig. 5). Examination of mineralization by Von Kossa staining showed that Dlx5 expressing cultures began to mineralize by day 12 and were strongly mineralized by day 14 and day 27. In contrast, control cultures were not mineralized until day 27, at which time they resembled the Dlx5 cultures (Fig. 6). These results suggested that forced expression of Dlx5 at high levels beginning around days 2-3 of culture, at a time when endogenous Dlx5 is low, causes premature differentiation; however, the endogenous levels produced at later times are enough to produce maximal expression of Col1a1 and mineralization.
During the course of our experiments, which were repeated several times, we found that some lots of FCS did not support differentiation of control cCOB cultures. However, cultures infected with the Dlx5 expressing vector consistently differentiated. Thus, Dlx5 was able to induce osteoblastic differentiation under suboptimal conditions. This led us to test whether fraction 1 cells from collagenase-digested calvaria, which are presumed to be primarily periosteal fibroblasts, could be induced to undergo osteoblastic differentiation by Dlx5. We found that control fraction 1 cells (referred to as cCPeri) coalesced into large cell aggregates that detached from the plate after they had been confluent for several days. This behavior is similar to our experience with primary body cavity chick embryo fibroblasts. Thus, for these experiments we do not have control cultures older than day 12. However, the Dlx5 expressing cultures remained attached to the plate and exhibited a similar morphology to differentiating cCOB cultures. By day 12, almost all cells in these cultures expressed high levels of alkaline phosphatase, compared with controls that expressed very little (Fig. 7). By day 26, the Dlx5 cultures had many Von Kossa-positive nodules. Northern blot analysis showed that control day 7 and day 12 cultures expressed low Col1a1 mRNA and no osteopontin or osteocalcin mRNA. Day 7 Dlx5 expressing cultures were similar to controls; however, by day 12 the Dlx5 cultures expressed very high Col1a1, osteopontin, and osteocalcin (Fig. 8). Thus, we concluded that Dlx5 promotes osteoblastic differentiation of cells, which normally do not become osteoblastic in culture.
We have shown that Dlx5 is expressed in fetal chick calvaria in vivo and is induced in concert with markers of osteoblastic differentiation in cultured calvarial osteoblasts. Forced overexpression of Dlx5 in calvarial osteoblasts during the proliferative phase of in vitro culture causes premature induction of osteoblastic markers. Our previous studies using the same system showed that overexpression of the related homeodomain protein Msx2 inhibited osteoblastic differentiation,(17) supporting the specificity of Dlx5. Thus, we believe that Dlx5 plays a significant role in induction of calvarial osteoblast differentiation. Our studies are consistent with those of Miyama et al.,(10) which suggest that Dlx5 is downstream of BMPs in the osteoblast phenotype induction pathway. However, their studies used an immortalized osteoblastic cell line MC3T3-E1, and our studies used primary calvarial cells.
Ectopic expression of Dlx5 in the first fraction of cells released by collagenase/trypsin digestion from chick calvaria induces expression of osteoblastic markers. In our experiments, this cell population did not normally undergo osteoblastic differentiation, instead the cells coalesced into a large aggregate, which detached from the dish. This behavior is very similar to that of body cavity CEF (data not shown). Dlx5 prevents both CEF (data not shown) and fraction 1 calvarial cells from detaching from culture dishes. Fraction 1 cells are not well defined but presumably contain some cells that are in the osteoblast developmental lineage but are earlier than cells in fractions 2-4. Therefore, it is possible that the major effect of Dlx5 on fraction 1 cells is to prevent aggregation and detachment and stimulate osteoblastic differentiation similarly to its effect on fraction 2-4 cells.
Dlx5 knockout mice have significant skeletal defects suggestive of a role for Dlx5 in skeletal development.(11,12) In particular, the undermineralization or absence of the parietal and interparietal calvarial bones of these animals is consistent with a deficit of differentiated osteoblasts. However, many skeletal elements appeared normal, and those that were affected clearly contained differentiated osteoblasts. This indicates that Dlx5 is not required for osteoblastic differentiation. There are two possible hypotheses to reconcile our data and the Dlx5 knockout phenotype. One is that, although Dlx5 is a modulator of osteoblastic differentiation in which importance is variable in different types of osteoblasts, the signal delivered by Dlx5 is not necessary for differentiation. A second possibility is that the signaling mediated by Dlx5 is necessary for differentiation in many or all types of osteoblasts, but other Dlx family members can deliver the same signal and thus compensate for the absence of Dlx5. In support of the latter possibility, Dlx6 is expressed in the same pattern as Dlx5,(4,5,7) and our preliminary studies indicate that Dlx3 is expressed in cultured mouse calvarial osteoblasts (I. Ercig, unpublished results, 2000). Acampora et al.(12) reported that Dlx5 knockout mice express osteocalcin in limb periosteum at an earlier stage than wild-type mice. Although this could be interpreted to suggest that Dlx5 may inhibit osteoblastic differentiation, osteocalcin is not consistently detected during early mouse bone development(18) and it is not required for bone synthesis.(19) Given the consistency of the overall bone phenotype of the Dlx5 knockout with a stimulatory role for Dlx5 in osteoblast differentiation, it is possible that there may be a dissociation between osteocalcin expression and osteoblastic differentiation in the knockout mice.
Redundant function of Dlx1 and −2 has been evoked to explain the phenotypes resulting from inactivation of these genes. However, little could be concluded from these studies about the specific differentiation processes affected by lack of Dlx 1 or −2. The hypothesis that Dlx proteins play a necessary and redundant role in osteoblast differentiation predicts that those skeletal elements in which a given Dlx gene constitutes the great majority of total Dlx gene expression in differentiating osteoblasts would be affected by a knockout of that gene. Skeletal elements in which several Dlx genes are expressed at similar levels may not be affected by inactivation of any one Dlx gene. Little precise information about the relative expression of the members of the Dlx family in different types of differentiating osteoblasts is available to test this hypothesis. However, it is interesting that the Dlx5 knockout has a severe effect on the calvaria, which our studies indicate express significant levels of Dlx5 mRNA.
We thank Dr. S. Hughes for the RCASBP(A) vector. We also thank Dr. L. Gerstenfeld for sending us the chick osteocalcin, osteopontin, and Col1a1 probes, and Dr. J.L. Millan for the chick alkaline phosphatase probe. This work was supported by the following grants from the National Institutes of Health (NIH): AR29983 (to A.C.L.) and HD22610 (to R.A.K. and A.C.L.).
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