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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Bone morphogenetic proteins (BMPs) were originally identified by their ability to induce ectopic bone formation and have been shown to promote both chondrogenesis and chondrocyte hypertrophy. BMPs have recently been found to activate a membrane serine/threonine kinase signaling mechanism in a variety of cell types, but the downstream effectors of BMP signaling in chondrocyte differentiation remain unidentified. We have previously reported that BMP-2 markedly stimulates type X collagen expression in prehypertrophic chick sternal chondrocytes, and that type X collagen mRNA levels in chondrocytes cultured under serum-free (SF) conditions are elevated 3- to 5-fold within 24 h. To better define the molecular mechanisms of induction of chondrocyte hypertrophy by BMPs, we examined the effect of BMPs on type X collagen production by 15-day chick embryo sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. Two populations of chondrocytes were used: one representing resting cartilage isolated from the caudal third of the sterna and the second representing prehypertrophic cartilage from the cephalic third of the sterna. BMP-2, BMP-4, and BMP-7 all effectively promoted chondrocyte maturation of cephalic sternal chondrocytes as measured by high levels of alkaline phosphatase, diminished levels of type II collagen, and induction of the hypertrophic chondrocyte-specific marker, type X collagen. To test whether BMP control of type X collagen expression occurs at the transcriptional level, we utilized plasmid constructs containing the chicken collagen X promoter and 5′ flanking regions fused to a reporter gene. Constructs were transiently transfected into sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. A 533 bp region located 2.4–2.9 kb upstream from the type X collagen transcriptional start site was both necessary and sufficient for strong BMP responsiveness in cells destined for hypertrophy, but not in chondrocytes derived from the lower sterna.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

FORMATION OF BONE occurs by one of two developmental programs: intramembranous or endochondral ossification. Intramembranous ossification is characterized by differentiation of mesenchymal cells directly into osteoblasts. During endochondral ossification, mesenchymal cells differentiate into chondrocytes before being replaced by osteoblasts. Endochondral bone formation is therefore a multistep process involving chondrocyte proliferation, hypertrophy, calcification, degradation, and replacement of cartilage by bone. This process is responsible for all long bone formation and is involved in pathological processes such as osteoarthritis1,2 and fracture repair.3 As chondrocytes progress toward this terminal differentiation, morphological and biosynthetic changes occur. Proliferating chondrocytes become flattened before fully maturing into round, hypertrophic chondrocytes. These cells also secrete and organize a different extracellular matrix characterized by high levels of alkaline phosphatase (ALP), diminished levels of collagens type II and IX and appearance of type X collagen, which is specific for hypertrophic chondrocytes.4,5 Cultured chondrocytes from regions of cartilage destined for hypertrophy can be induced to express type X collagen by bone morphogenetic protein (BMP).6,7

For over 30 years, it has been known that BMPs are capable of inducing the formation of new cartilage and bone when implanted extraskeletally.8 More than 20 members of the BMP family have now been identified and have been subdivided according to their homology within their mature carboxyl terminus as well as similarity to Drosophila counterparts.9,10 BMP-2 and BMP-4 are the most closely related of the categorized BMPs, sharing 92% identity of amino acids in their mature region and forming a subgroup with their Drosophila counterpart, decapentaplegic (dpp).11 BMP-5, BMP-6, BMP-7, and BMP-8 share ∼90% identity and are categorized as another subgroup along with Drosophila wingless and 60A proteins. Although BMP-2, BMP-4, BMP-5, and BMP-7 have all been shown to possess the ability to promote bone formation in vivo,9,11–13 their individual roles in skeletal formation and maintenance have yet to be defined. The temporal and spatial patterns of expression of BMPs in embryonic mouse forelimbs support roles for BMP-2, BMP-4, BMP-6, and BMP-7 in skeletal formation.14–16

BMPs, like other members of the transforming growth factor β (TGF-β) superfamily, have been shown to transduce signals through heteromeric transmembrane serine-threonine kinase receptors,16–19 but relatively little is known about the downstream signal transduction activated by BMP which leads to transcriptional activation of target genes. Two Drosophila genes, schnurri and mothers-against-dpp (Mad) which encode a zinc finger protein and a novel intracellular protein, respectively, have been proposed to play a role in the signaling cascade of dpp.20–24 Eight vertebrate signaling molecules have been identified and named Smads for their similarities to Drosophila Mad and related Caenorhabditis Sma proteins. In response to the TGF-β family member signaling, Smads form functional heteromeric complexes. Smad1 and Smad5 mediate signaling by BMP-2 and BMP-4 but not activin or TGF-β. Smad1 is phosphorylated directly by the BMP type I receptor on serine residues found in its carboxyl terminus, leading Smad1 to associate with Smad4 and translocate to the nucleus as a heterodimer.25 Although Smad2, a mediator of TGF-β signaling has been found to associate with the winged-helix DNA binding protein member FAST-1, no DNA binding partner has been found for Smad1 or Smad5 in BMP signaling.26 Despite recent advances in the identification of prospective candidate proteins which act downstream of activated TGF-β family receptors, a gap remains in our understanding of how these proteins convert the signal induced by interaction of BMP with its heteromeric receptor to transcriptional changes in vertebrate genes involved in differentiation.

In this paper, we describe a BMP responsive region in the chicken type X collagen gene. A 533 bp fragment localized 2.4–2.9 kb upstream from the transcriptional start site is sufficient to produce a 15- to 20-fold increase in luciferase reporter activity in response to BMP-4. A comparison of BMP-2, BMP-4, and BMP-7 shows that all are effective in inducing elevated ALP activity, diminished levels of collagen type II, and elevated levels of type X collagen mRNA in prehypertrophic chicken sternal chondrocytes. Furthermore, the 533 bp BMP responsive region is active with all three BMPs in cells destined for hypertrophy but is inactive in chondrocytes of the permanent cartilagenous region of the sterna.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Cell culture

Cells were isolated from the cephalic and caudal portions of sternae from 15-day chick embryos (B&E Eggs, Stevens, PA, U.S.A.) by digestion for 3.5 h at 37°C, 5% CO2 in calcium- and magnesium-free Hank's balanced salt solution (CMF-HBSS) containing 0.6 mg/ml collagenase and 0.04% trypsin. The cells were resuspended in a complete medium containing high glucose Dulbecco's modified Eagle's medium (DMEM) with 10% NuSerum IV (Collaborative Biomedical Products/Becton-Dickinson, Bedford, MA, U.S.A.) and 100 U/ml penicillin/streptomycin. The chondrocytes were plated at three sternae per 100-mm plate and maintained on Falcon tissue culture dishes at 37°C, 5% CO2 (Becton Dickinson, Lincoln Park, NJ, U.S.A.). After 5 days, the floating chondrocytes were harvested, counted, and replated at 4.8 × 104 cells/cm2 on tissue culture dishes in complete medium supplemented with 4 U/ml hyaluronidase to promote attachment. After 24 h, cultures were transferred to serum-free (SF) conditions to assess more accurately the direct effects of BMPs. SF medium contained DMEM (with Pen/Strep and hyaluronidase) supplemented with 10 pM triiodothyronine (Sigma, St. Louis, MO, U.S.A.), 60 ng/ml insulin, and 1 mM cysteine, with or without 30 ng/ml recombinant human (rh) BMP-2, BMP-4, or BMP-7 (Genetics Institute, Cambridge, MA, U.S.A.). Cultures were washed twice in CMF-HBSS before switching to SF conditions. Cells were harvested 48 h after switching to SF media for mRNA analysis, ALP assays, DNA analyses, and reporter assays as described below.

mRNA analysis

Cultures were rinsed with CMF-HBSS and placed in 4 M guanidine isothiocyanate solution containing 0.5% sarkosyl, 5 mM EDTA, 20 mM sodium acetate (pH 5.2), and 0.1 M β-mercaptoethanol. Cells were homogenized by repeated extrusion through a 21-gauge needle; the homogenate was then extracted with an equal volume of acid phenol chloroform (Ambion, Austin, TX, U.S.A.). The aqueous phase was removed and precipitated twice with 2.5 vol of ethanol. RNA was reprecipitated with guanine hydrochloride/ethanol as described previously.27 RNA samples (8 μg) were denatured by glyoxylation, electrophoresed on agarose gels for 2 h at 7 V/cm, and transferred onto nylon membrane (Gene Screen Plus; Du Pont, Wilmington, DE, U.S.A.). Levels of specific mRNAs were determined by hybridizing Northern blots to32P-labeled chick riboprobes. The chick type II and type X collagen probes have been described previously.27 The extent of hybridization to blots was quantitated with a Molecular Dynamics FluorImager (Sunnyvale, CA, U.S.A.).

ALP and DNA assays

Cultures were rinsed twice with CMF-HBSS and extracted with 0.15 M Tris, pH 9.0, 0.1 mM ZnCl2, 0.1 mM MgCl2, and 1% Triton X-100 for 30 minutes at 37°C. An aliquot of the solubilized cell layer extract was reacted with 7.5 mM p-nitrophenol phosphate (Sigma 104) in 1.5 M Tris (pH 9.0), 1 mM ZnCl2, and 1 mM MgCl2 at room temperature, and absorption was measured at 410 nm over a 6-minute time period. Enzyme levels are expressed as nanomoles of p-nitrophenol formed/minute/well with A410 = 64 nmol of product. The DNA content of the extracts used for ALP assays was determined as previously described.28 Briefly, DNA from 75-μl aliquots was precipitated by the addition of ethanol followed by 2-day storage at –20°C. The DNA was resuspended in alkaline EDTA, the sample neutralized, and an equal volume of Hoescht dye 33258 (Sigma) was added. Fluorescence was measured in four-sided plastic cuvettes (Fisher Scientific, Fairlawn, NJ, U.S.A.) using a spectrofluorometer (Photon Technology International, South Brunswick, NJ, U.S.A.), with an excitation wavelength of 365 nm and an emission wavelength of 460 nm.

Promoter-reporter constructs

Preparation of collagen X-chloramphenicol acetyltransferase (CAT) constructs has been previously described.29 For preparation of luciferase constructs, the B-640 fragment was excised from the B-640 CAT construct with PstI and SalI restriction enzymes and ligated into the polylinker region of PBSKSII, in which the SacI site had been deleted (PBSKSII SacI). The b1b2–640 fragment was generated from B-640 by excising the b3 region using the restriction enzymes BglII and HindIII, followed by blunt-end ligation. The b1–640 fragment was generated from B-640 using BglII and HindIII to excise the b2b3 fragment, followed by blunt-end ligation. The constructs were excised from PBSKSII SacI with SpeI and SalI, and ligated into the same sites within the polylinker region of the promoterless Renilla luciferase reporter plasmid, pRLnull (Promega, Madison, WI, U.S.A.). Additional constructs were derived using judicious restriction site selection followed by ligations using DNA rapid ligation kit (Boehringer Mannheim, Indianapolis, IN, U.S.A.). The b2 fragment representing –2649/–2007 (relative to the transcriptional start site) was created by deleting the b1 fragment (–3193/–2649) using SacI which cut 5′ to the b1 fragment within the multiple cloning site and at –2649. The 533 bp DNA fragment extending from –2866 to –2333 was isolated by digestion with SnaBI and SwaI, blunt-ended and cloned into the Nsi site 5′ to the 640 bp collagen X promoter. The 533 bp deletions in both the B (–3193/–1583) and b1b2 (–3193/–2007) fragments were created using SnaBI and SwaI restriction digest, followed by blunt-ending and religation yielding the constructs (BΔ533) and (b1b2Δ533), respectively. All constructs were sequenced to confirm their identity using an automated 373 DNA sequencer (Applied Biosystems, Foster City, CA, U.S.A.). The sequence for the 640 proximal promoter and adjacent 3.8 kb upstream region referred to as “ABC” has been deposited into GenBank (accession #AFO44678).

Transient transfections and reporter assays

One hour prior to transfection, cells were refed with fresh media containing 10% fetal bovine serum (Atlanta Biological, Atlanta, GA, U.S.A.) supplemented with 4 U/ml hyaluronidase and 100 U of penicillin/streptomycin. Chondrocytes were transiently transfected using CaPO4 precipitation with a 10:1 ratio of Collagen X promoter-PBLCAT3 and RSV β-galactosidase vector DNA or with Dual Luciferase DNA (Promega) in which the Renilla luciferase was regulated by Collagen X promoter sequences and the firefly luciferase constitutively expresses firefly luciferase under the control of an SV40 early promoter. For CAT assays, transfections were performed in 60-mm dishes in duplicate. For luciferase assays, 2.5 μg of pRL-null or equimolar amounts of experimental constructs was cotransfected with 0.25 μg Control-pGL2 per well in 12-well plates (Corning, Corning, NY, U.S.A.). Precipitates were allowed to remain on the cells for 5–6 h before washing twice with CMF-HBSS and replacement with SF media. rhBMP-2, rhBMP-4, or rhBMP-7 (kindly provided by Genetics Institute) was added to a concentration of 30 ng/ml where appropriate. Approximately 48 h later, cells were washed twice in phosphate-buffered saline in preparation for CAT or luciferase assays.

For CAT assays, cell lysates were prepared by resuspension in 0.25 M Tris-HCl (pH 7.8) and disruption by three freeze-thaw cycles from ethanol/dry ice to 37°C. Supernatants were tested for β-galactosidase activity with ONPG substrate to determine transfection efficiency. Acetyl CoA and14C-chloramphenicol were added to cell extracts containing equal amounts of β-galactosidase activity, and acetylation was carried out at 37°C for 1 h. The resulting product and substrate were extracted in ethyl acetate and separated by silica gel thin layer chromatography (TLC) in 95:5 chloroform:methanol. Relative amounts of acetylated and total14C-chloramphenicol were then assayed by densitometry scanning of X-ray film. For luciferase assays, lysis of cells was accomplished using Passive Lysis buffer (Promega). Firefly and Renilla luciferase assays were performed on cell extracts using the Dual-Luciferase Assay System according to manufacturer's recommendations and measured for 10 s/reaction using an Opticomp luminometer (MGM Instruments, Hamden, CT, U.S.A.). The effect of BMPs on various constructs were compared pairwise using an Student's t-test; values of p ≤ 0.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

BMP induction of chondrocyte hypertrophy

Previous studies have demonstrated that rhBMP-2 in SF media induces maturation of prehypertrophic chondrocytes derived from cephalic sternae of 14-day chick embryos.6 We therefore determined whether other BMPs of the same subgroup (BMP-4), or a different subgroup (BMP-7), could similarly elicit chondrocyte maturation. A comparison of type II and type X collagen mRNA synthesis in chick embryo sternal chondrocytes cultured with BMP-2, BMP-4, and BMP-7 is shown in Fig. 1. Analysis of type X collagen mRNA indicates that all three BMPs are strong inducers of chondrocyte maturation in chondrocytes destined for hypertrophy (15-day upper sternal chondrocytes [USC]), but BMP-4 was consistently the most potent inducer. As anticipated from previous studies,6 chondrocytes derived from the caudal sterna (LSC) showed no induction of type X collagen synthesis, either with BMP-2 or with BMP-4 and BMP-7 (data not shown) and also maintained high levels of type II collagen. A summary of BMP effects on collagen mRNAs is presented in Table 1.

Table TABLE 1. RELATIVE COLLAGEN MRNA LEVELS IN BMP-TREATED CEPHALIC STERNAL CHONDROCYTES
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Figure FIG. 1. Representative Northern blot of RNA from upper sternal chondrocyte (USC) cultures in SF medium without BMPs (SF) or supplemented with 30 ng/ml BMP-2 (B2), BMP-4 (B4), or BMP-7 (B7). Total cellular RNA (8 μg) from each sample was denatured with glyoxyl and loaded on an agarose gel as described in the Materials and Methods. Blots were hybridized to32P-labeled riboprobe for chick type X collagen. The blot was then rehybridized to labeled riboprobe for chick type II collagen. Methylene blue staining of rRNA was used to confirm that each lane contained similar amounts of RNA.

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Another hallmark of chondrocyte maturation is the induction of high levels of ALP activity. We have previously reported that 30–40 ng/ml rhBMP-2 added to serum-free media was effective in stimulating ALP, and that this elevation was seen as early as 24 h after adding rhBMP-2.6 A comparison of the ability of BMP-2, BMP-4, and BMP-7 to induce ALP activity in 15-day USCs cultured under SF conditions is presented in Fig. 2. Again, all three BMPs are capable of increasing ALP levels, but as with the collagen X data presented in Fig. 1, BMP-4 was significantly more effective at inducing ALP levels than either BMP-2 or BMP-7 (p < 0.05). Measurement of DNA content per well (Fig. 2) confirmed our previous observation that BMPs do not increase proliferation of prehypertrophic chondrocytes and that increased cell number is not contributing to the higher ALP activity seen in response to BMP treatment.

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Figure FIG. 2. ALP levels of USC cultures in SF medium with and without rhBMPs. Chondrocytes were initially plated in 12-well plates with DMEM containing 10% NuSerum supplemented with hyaluronidase to promote attachment. Twenty-four hours later, cells were washed twice with CMF-HBSS and switched to DMEM supplemented with triiodothyronine, insulin, cysteine (SF media) with or without 30 ng/ml BMP-2, BMP-4, or BMP-7. ALP levels were measured 48 h later. Values represent the mean ± SEM for four independent experiments with triplicate samples.

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To examine the hypothesis that BMP regulation of hypertrophy-related genes occurs at the transcriptional level, promoter-CAT constructs containing portions of the 3.8 kb region 5′ to the chick type X collagen gene29 were tested in transient transfection studies. The initial survey for BMP-responsive sites was limited to studies with rhBMP-2, since this was the most readily available of the recombinant BMPs. These constructs contained the 640 bp promoter-containing region including the transcriptional start site (–558 to +82), along with A, B, and C subfragments of the 3.8-kb upstream region (Fig. 3). The 3.8-kb region has previously been reported to contain multiple negative elements which act to restrict transcription of the collagen X gene to hypertrophic chondrocytes, while the 640 bp region contains basal promoter activity permitting expression of a reporter gene.29 Prehypertrophic sternal chondrocytes transfected with constructs containing the 1610 bp “B” fragment upstream of the 640 bp promoter showed a marked BMP response (Fig. 3). Addition of the “A” and “C” fragments did not augment the BMP response, suggesting that the “B” fragment was sufficient.

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Figure FIG. 3. Collagen X-CAT promoter constructs and their relative BMP responsiveness. The upstream promoter sequence of the chicken type X collagen gene is schematically represented in the left panel. The 640 bp fragment, which contains the transcriptional start site and 558 bp of 5′ flanking sequence, has been previously reported to contain a strong promoter. This fragment, plus subfragments (A, B, and C) representing 3.8 kb of 5′ flanking type X collagen sequence were subcloned upstream of the CAT reporter gene. As seen in the righthand panel, addition of the B fragment to the 640 bp is sufficient for BMP response and addition of A and C fragments do not significantly augment this response. Data presented is from a representative experiment performed with duplicate samples.

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Based on these results, the BMP response region was further localized using smaller fragments of the “B” fragment cloned into a luciferase reporter vector. The constructs, and results of transfecting them into prehypertrophic sternal chondrocytes, are summarized in Fig. 4. Neither the pRL-null (promoterless) vector alone nor the pRL vector driven by the 640 bp fragment containing the collagen X promoter was responsive to any of the BMPs tested in either USCs or LSCs (data not shown). When the “B” fragment was divided into three regions (b1, b2, b3), deleting the b1 or b3 regions did not significantly affect the magnitude of the BMP response seen with the entire “B” fragment. Thus, the BMP response region was within the b2 region containing the central portion of “B” (–2649 to –2007); this region showed 15–20× stimulation by BMP-4. Interestingly, the b1 region consistently showed modest BMP responsiveness; however, this BMP response was an order of magnitude lower than that seen with the b2 region.

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Figure FIG. 4. Collagen X promoter-luciferase constructs and their relative BMP responsiveness in prehypertrophic chick sternal chondrocytes (15-day USC). The 5′ flanking region of the chick type X collagen gene is schematically represented at the top. Once a BMP response was localized to the B fragment, further subdivision of this region by restriction fragment deletion/subcloning was performed as described in the Materials and Methods. Relative BMP response was based upon data from at least six independent assays. The pattern of response was similar for BMP-2, BMP-4, and BMP-7.

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The relative luciferase activity in sternal chondrocytes cultured under SF conditions with BMP-4 following transfection with portions of the “B” fragment is presented in Fig. 5. For any given construct, normalized luciferase activity (experimental pRL/control pGL) values were not statistically different between USC and LSC. Removing a 533 bp segment from the entire B fragment (BΔ533) or from the b1b2 fragment (b1b2Δ533) obliterated the BMP response and therefore implicates the 533 bp as a BMP-responsive region. All constructs containing this 533 bp, which includes 217 bp of the 3′ region of b1 and 316 bp of the 5′ region of b2, showed similar BMP responsiveness. Furthermore, the 533 bp fragment alone was capable of responding to BMP. Caudal sternal chondrocytes transfected with plasmid constructs containing the 533 bp region showed no BMP response; therefore, this region within the upstream promoter region of the type X collagen gene contains a BMP-responsive site which functions in cephalic sternal chondrocytes undergoing hypertrophy, but not in nonhypertrophic caudal sternal chondrocytes.

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Figure FIG. 5. Renilla luciferase activity in sternal chondrocytes transfected with constructs containing various upstream regions of the chick Collagen X promoter and cultured in the presence of 30 ng/ml BMP-4 for 48 h. Renilla luciferase values were normalized for transfection efficiency based on levels of SV40-firefly luciferase activity in the same sample. BMP responsiveness was then calculated as the ratio of Renilla luciferase in BMP-treated versus untreated cultures. Data represents the mean ± SEM of at least four independent experiments with triplicate samples. Probabilities were calculated using a Student's t-test. **Significantly different from untreated culture (p < 0.01). *Significantly different from untreated culture (p < 0.05).

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The fact that BMP response is seen with the the b2 fragment (–2649 to –2007), but not with b1b2Δ533 which lacks –2649 to –2333, implies that the BMP response element(s) must be within the 316 bp region spanning –2649 to –2333. The sequence of the 316 bp region containing the putative BMP response element for the chick type X gene is presented in Fig. 6. Comparison of this sequence with consensus regulatory elements, identified using the tfsites program of the Genetics Computer Group (Madison, WI, U.S.A.), indicate previously reported transcription factor binding sites.

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Figure FIG. 6. DNA sequence of the 316 bp region at the 5′ end of the type X collagen “b2” region. Putative enhancer elements are underlined and numbered at their 5′ residue.

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A comparison of the ability of BMP-2, BMP-4, and BMP-7 to stimulate the response by the b1b2 fragment (–3193/–2007) is presented in Fig. 7. BMP-2 and BMP-7 were equally effective at inducing a 5- to 10-fold stimulation in reporter activity while BMP-4 was significantly (p < 0.01) more effective than the other BMPs. Similar results were seen with all of the BMP-responsive constructs (data not shown). These data are consistent with results presented in Figs. 1 and 2, demonstrating that BMP-4 is a more potent inducer of chick chondrocyte maturation. Dose response analyses with 15–150 ng/ml BMP suggested that all three of the tested BMPs reached maximal stimulation by 30 ng/ml. In contrast to results obtained with the BMPs, 1–10 ng/ml activated TGF-β2 had no effect on transcription driven by the b1b2 region (data not shown).

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Figure FIG. 7. Comparison of BMP-2, BMP-4, and BMP-7 effect on reporter activity with the b1b2/640-Luc construct transfected into 15-day sternal chondrocytes. Student's t-test was used to determine statistical significance of data from at least five independent experiments with triplicate samples. All BMPs are capable of significant (p < 0.05) stimulation of renilla luciferase activity as compared with SF, untreated values. BMP-4 is significantly more effective than either BMP-2 or BMP-7 (p < 0.01). None of the three BMPs are capable of increasing luciferase activity when the b1b2/640-luciferase constructs are transfected into LSCs.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Results presented here demonstrate that BMP-4 and BMP-7, like BMP-2, are potent inducers of maturation of prehypertrophic chick sternal chondrocytes cultured under SF conditions. All three BMPs are capable of inducing type X collagen mRNA production, high levels of ALP and diminished levels of type II collagen mRNA which are hallmarks of maturation in the process of endochondral ossification. Furthermore, we have identified a region in the upstream promoter of the chick collagen X gene which shows transcriptional regulation by BMPs. Deletion of 533 bp from either the B or b1b2 constructs abolishes BMP responsiveness, while addition of the 533 bp sequence to the 640 basal promoter permits BMP response. Thus, in the presence of the 640 bp basal promoter, the 533 bp region appears both necessary and sufficient for BMP-induced transcriptional activity in cells destined for hypertrophy (USCs) but is inactive in cells derived from permanent cartilage (LSCs).

Naturally occurring and experimentally induced mutations in BMPs have provided us with information about the requirement for individual BMPs in skeletal formation. Loss of function mutation of BMP-5 results in short ears, defects in the sternum and ribs with relatively minor alterations in the appendicular skeleton,30 whereas mutation of the BMP family member, GDF-5, results in a shortened appendicular skeleton and relatively unaffected axial skeleton.31 BMP-7 null mice have severe kidney and eye defects but relatively minor skeletal pattern abnormalities of the rib cage, skull, and hindlimbs.14,32 Homozygous null mutations in BMP-4 and loss of function mutations in BMP-2 mice have also been created, but since embryos die in utero before the process of skeletogenesis occurs, the impact of BMP-2 and BMP-4 on this process cannot be examined using these techniques.10,33 Analysis of these mutations suggests that different subsets of skeletal elements may require different BMP family members for normal development. Nonetheless, analyses of BMP expression patterns indicate that multiple BMPs, including BMP-2, BMP-4, and BMP-7, are produced by chondrocytes undergoing endochondral ossification.15,34–38 Similarly, our results indicate that, in vitro, BMP-2, BMP-4, and BMP-7 are all capable of inducing maturation in prehypertrophic chick sternal chondrocytes cultured under SF conditions. Although BMP-2 and BMP-7 share less than 60% amino acid homology,11 they are equally effective at inducing type X collagen mRNA (Fig. 1) and ALP activity (Fig. 2). In contrast, BMP-4 was consistently a more potent inducer of maturation events than BMP-2, with which it shares 92% homology. Similar results have been reported for cultured osteoblasts treated with BMPs; BMP-4 is more effective than BMP-2 at promoting differentiation.39 It has been suggested that the N-terminus of BMP-2 may be responsible for its reduced potency in vitro and may not reflect differences in vivo.40

Type X collagen expression has been shown to be transcriptionally regulated during development,41–43 but relatively little is known about the upstream regions governing transcriptional activity. Promoter studies with type X collagen from several species have suggested both silencer and enhancer regulatory elements which restrict type X collagen expression to hypertrophic chondrocytes. Chicken promoter-CAT transfection studies using three subfragments of 3.8 kb from the 5′ flanking region suggest that multiple upstream negative regulatory elements act in an additive manner, restricting type X collagen transcription to hypertrophic chondrocytes.29 In vivo footprinting using collagen X-expressing chick chondrocytes implicated a sequence overlapping an AP-2 site and a 9 bp region containing the sequence CACACA as potential important regulatory regions.44 An AP-2 site is present in the 316 bp region described in the present study (Fig. 6). However, preliminary mobility shift studies with an oligonucleotide including this AP-2 site indicate no BMP-specific gel shift (unpublished results). Within the human collagen X gene, both enhancer and repressor elements appear to play key roles in the regulation of collagen X. The region between 2400 bp and 900 bp upstream from the transcriptional start site showed enhancer activity in hypertrophic chondrocytes,45 and Beier et al.46 recently reported a second, tissue nonspecific enhancer within the first intron. Beier et al. also reported that a 400 bp region 2.4–2.8 kb upstream from the transcriptional start site contained tissue-specific repressor activity.46

Recently, a BMP-7 (Op-1) responsive element was defined in the mouse collagen X promoter.47 A luciferase construct containing a SV-40 promoter +33 bp from the type X promoter showed 1.5-fold stimulation by BMP-7; the 33 bp region contains both AP1-like and Mef-2–like sequences. The 533 bp region of the chicken collagen X gene is an order of magnitude more responsive to BMP and contains neither AP1 nor Mef-2–like sequences. An AP1-like sequence is present in the 640 bp proximal promoter for the chick gene, but this region does not respond to BMP (Figs. 3 and 4).

Given the strong evidence for Smads as downstream mediators of BMP signaling, it will be important to investigate if Smads play a role in activating elements within the chicken type X collagen 533 bp region. Although Smad2, a mediator of TGF-β signaling, has been found to associate with the winged-helix DNA binding protein member FAST-1,26 no DNA binding partner has been found for Smad1 or Smad5, which have been implicated in BMP signaling. In Drosophila, Mad has been shown to bind DNA within the quadrant enhancer of vestigial to direct dpp-induced vestigial expression within the developing wing.48 The mouse and chicken type X collagen upstream regions contain neither the consensus binding region for this MAD binding site nor sequence similar to the Smad2/FAST-1 binding site.

Both 533 and b2 constructs show BMP responsiveness. We would therefore expect the BMP response to be localized to the 316 bp region which is common to both b2 and 533, located –2649/–2333 upstream of the transcription start. This region includes the AP-2 site discussed above, as well as several other binding sites for transcription factors implicated in development and differentiation (Fig. 6). If the BMP signaling mechanism is mediated by Smad1 or Smad5 binding to a known DNA binding protein, an already reported response element identified in Fig. 6 may be responsible. Alternatively, Smad1/Smad5 may bind directly to a previously unidentified response element within the 316 bp region. Studies of TGF-β–responsive regions in several genes have implicated several different response elements. This raises the possibility that signaling downstream of Smads utilizes complexes with multiple tissue-specific transcription factors which have unique, tissue-specific DNA binding sites. Alternatively, a Smad complex may be formed that uses more than one DNA binding site.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors are grateful to the Genetics Institute for generously providing rhBMP-2, rhBMP-4, and rhBMP-7. We would like to thank Ms. Rachel Venezian and Dr. Zhi Yan for technical assistance and Mr. Tom Tucker and Dr. Bill Abrams for their expertise in sequencing and assistance with figure preparation. This work was supported by National Institutes of Health grants AR40075, AR41630, and GM07170 and grants from the Medical Research Council of Canada and the Arthritis Society.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  • 1
    Hoyland JA, Thomas JT, Donn R, Marriott A, Ayad S, Boot-Handford RP, Grant ME, Freemont AJ 1991 Distribution of type X collagen mRNA in normal and osteoarthritic human cartilage Bone Miner 15:151164.
  • 2
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