Results presented in part as an oral presentation at the American Society for Bone and Mineral Research meeting, Toronto, Ontario, Canada, 2000
Accelerated Up-Regulation of L-Sox5, Sox6, and Sox9 by BMP-2 Gene Transfer During Murine Fracture Healing*
Article first published online: 1 OCT 2001
Copyright © 2001 ASBMR
Journal of Bone and Mineral Research
Volume 16, Issue 10, pages 1837–1845, October 2001
How to Cite
Uusitalo, H., Hiltunen, A., Ahonen, M., Gao, T.-J., Lefebvre, V., Harley, V., Kähäri, V.-M. and Vuorio, E. (2001), Accelerated Up-Regulation of L-Sox5, Sox6, and Sox9 by BMP-2 Gene Transfer During Murine Fracture Healing. J Bone Miner Res, 16: 1837–1845. doi: 10.1359/jbmr.2001.16.10.1837
- Issue published online: 2 DEC 2009
- Article first published online: 1 OCT 2001
- Manuscript Accepted: 5 FEB 2001
- Manuscript Revised: 18 DEC 2000
- Manuscript Received: 28 JUL 2000
- transcription factor Sox;
- bone morphogenetic protein 2;
Fracture repair is the best-characterized situation in which activation of chondrogenesis takes place in an adult organism. To better understand the mechanisms that regulate chondrogenic differentiation of mesenchymal progenitor cells during fracture repair, we have investigated the participation of transcription factors L-Sox5, Sox6, and Sox9 in this process. Marked up-regulation of L-Sox5 and Sox9 messenger RNA (mRNA) and smaller changes in Sox6 mRNA levels were observed in RNAse protection assays during early stages of callus formation, followed by up-regulation of type II collagen production. During cartilage expansion, the colocalization of L-Sox5, Sox6, and Sox9 by immunohistochemistry and type II collagen transcripts by in situ hybridization confirmed a close relationship of these transcription factors with the chondrocyte phenotype and cartilage production. On chondrocyte hypertrophy, production of L-Sox5, Sox9 and type II collagen were down-regulated markedly and that of type X collagen was up-regulated. Finally, using adenovirus mediated bone morphogenetic protein 2 (BMP-2) gene transfer into fracture site we showed accelerated up-regulation of the genes for all three Sox proteins and type II collagen in fractures treated with BMP-2 when compared with control fractures. These data suggest that L-Sox5, Sox6, and Sox9 are involved in the activation and maintenance of chondrogenesis during fracture healing and that enhancement of chondrogenesis by BMP-2 is mediated via an L-Sox5/Sox6/Sox9-dependent pathway.
SOX9 IS a transcription factor belonging to the SRY-type family of high mobility group (HMG) box proteins.(1) In addition to its established function as a male sex-determining factor,(2, 3) Sox9 is involved in embryonic chondrocyte differentiation and transcriptional up-regulation of the Col2a1 gene, coding for the major cartilage collagen of type II.4-6) Expression of Sox9 is activated in prechondrogenic mesenchyme and maintained at a high level in fully differentiated chondrocytes where its direct targets are enhancer elements in the Col2a1 gene1, 4-6); in the Col11a2 gene coding for the α2-chain of type XI collagen, a minor component of cartilage collagen fibrils(7); and in the Agc gene coding for the core protein of aggrecan,(8) the major cartilage-specific proteoglycan. Using mouse chimeras of normal and Sox9−/− embryonic stem (ES) cells, the expression of Sox9 has been shown to be essential for chondrocyte differentiation and expression of the Col2a1 gene.(9) In humans, the importance of SOX9 for development is shown by the severe skeletal abnormalities and sex reversal in patients with campomelic dysplasia.10-12) At the molecular genetic level, the disease results from haploinsufficiency of SOX9, because of heterozygous mutations in the SOX9 gene, which affect, for example, DNA binding or transcriptional activation.(13, 14)
More recently, L-Sox5 (a longer form of Sox5) and Sox6 have been shown to cooperate with Sox9 in the activation of the Col2a1 gene.(15) L-Sox5 and Sox6 share a 67% overall homology and belong to a different subclass of Sox proteins than Sox9. Their homology with Sox9 is limited to the HMG domain. L-Sox5 and Sox6 form homodimers and heterodimers through their coiled coil domains and bind to adjacent HMG sites on DNA.(15) In the Col2a1 gene, Sox9 and L-Sox5/Sox6 proteins bind to a 48-base pair (bp) chondrocyte-specific enhancer, which contains four HMG-like sites necessary for its activity. During mouse development, L-Sox5/Sox6 is produced along with Sox9 in all prechondrogenic areas and cartilages.(15) Transcripts for the three Sox genes are present also in some nonchondrogenic cells, but their coexpression is always connected with a chondrogenic phenotype and Col2a1 expression.
Members of the bone morphogenetic protein (BMP) subfamily of the transforming growth factor β (TGF-β) superfamily are among the best-characterized growth factors known to promote chondrogenesis and osteogenesis, that is, to induce endochondral bone formation during fracture healing as well as at ectopic sites.(16) However, the relationship between BMP and L-Sox5/Sox6/Sox9 production remains poorly understood. In chick embryos, exogenous BMP-2 has been shown to up-regulate Sox9 expression.(17) On the other hand, in a chondrocyte culture system in which recombinant BMP-2 was added, no increase in Sox9 expression was observed.(18) In mouse embryos, BMP-2 is expressed in areas of developing cartilage and bone and also in various other tissues where it has been shown to have a multitude of other effects.19-21)
Although the role of Sox9 in cartilage and endochondral bone formation has been under intensive research, L-Sox5 and Sox6 have received considerably less attention. This study was initiated to elucidate the role of these transcription factors and their hierarchy with respect to BMP-2 during fracture healing, which provides an interesting model for studies on chondrogenesis in an adult organism as it recapitulates the various steps of endochondral ossification encountered during skeletal development and growth.(22, 23) We hypothesized that activation of chondrogenesis during fracture healing involves the same pathway(s) and regulatory factors as during embryonic development. In this study we present data to support this hypothesis and show that overexpression of BMP-2 in fracture callus through gene transfer accelerates the expression of Sox5, Sox6, Sox9, and Col2a1 genes as well as subsequent chondrogenesis in vivo.
MATERIALS AND METHODS
Experimental fracture repair model
Standardized tibial fractures were produced bilaterally in 66 male C57 black/DBA mice at the age of 3 months under general anesthesia as described earlier.(24) Briefly, a 0.2-mm stainless steel rod was introduced into the intramedullary cavity of both tibias. The prenailed tibias were fractured under anesthesia using 2.5% Avertin. After recovery from anesthesia, the animals were allowed free unrestricted weight bearing in cages. Age-matched control animals underwent no surgery. The mice were killed 3, 5, 7, 9, 14, and 28 days after the operation and the fracture calluses were harvested for histological examination, in situ hybridization, immunohistochemistry, and RNA extraction. The study plan was approved by the institutional committee for animal welfare.
Gene transfer into fracture callus
To study the effects of BMP-2 gene transfer on fracture repair, an additional 30 mice were used. Adenoviral vectors RAdBMP-2 (H. Uusitalo, T.-J. Gao, V.-V. Välimäki, P. Virolainen, M. Ahonen, H. Aro, V.-M. Kähäri, E. Vuorio, unpublished observations, 2001) carrying the BMP-2 gene or RAdLacZ(25) harboring the Escherichia coli LacZ gene under the control of cytomegalovirus (CMV) promoter were injected locally (106 plaque-forming units [pfu]) into the fracture site immediately after fracturing the tibia. The mice were killed 5, 7, and 14 days after the operation and the samples were harvested for RNA extraction and histology.
RNA extraction and Northern analyses
The fracture calluses were dissected free of surrounding tissues and frozen. For isolation of total RNA, the calluses were pulverized under liquid nitrogen, homogenized in 4 M guanidinium isothiocyanate, and sedimented through 5.7 M cesium chloride.(26) For Northern analyses, 10-μg aliquots of total RNA were denatured by formaldehyde, electrophoresed on 1% agarose gels, and transferred to Pall Biodyne membranes for hybridization with [32P]-labeled probes under standard conditions recommended by the supplier (Pall Biodyne Europe, Portsmouth, UK). For the detection of Sox9 messenger RNA (mRNA), a 255-bp murine complementary DNA (cDNA) clone was used.(4) The mRNAs for type II and X collagens were detected using cDNA inserts of clones pMCol2a1-1(27) and pMCol10a1-1,(28) respectively. All inserts were labeled with [32P]deoxycytosine triphosphate (dCTP) using the random priming method. The bound probes were detected and quantified on a molecular imager phosphoimager and the mRNA signals were corrected for variations in the 28S ribosomal RNA (rRNA) levels determined by hybridization. For each mRNA, the means ± SD was calculated for each experimental group and analyzed for statistical significance using the unpaired two-tailed Student's t-test. A value of p < 0.05 was considered statistically significant.
RNAse protection assay
Linearized cDNA probes for L-Sox5, Sox6, and Sox9(4, 15) were used as templates in transcription reactions with T7 or T3 RNA polymerase using [32P]CTP as the labeled nucleotide. Full-length transcripts were purified on a denaturing polyacrylamide gel. Dried RNA samples were dissolved in hybridization buffer (80% [vol/vol] formamide/40 mM Pipes [pH 6.4]/400 mM NaCl/1 mM EDTA) containing all three complementary RNA (cRNA) probes at 5 × 104 counts per minute (cpm)/μl. Hybridization was performed at 55°C overnight, after which the samples were incubated for 1 h at 37°C with 300 μl of digestion mixture (300 mM sodium acetate/5 mM EDTA/12 μg/ml RNAse A/30 U/ml RNAse T1). The digestion was terminated by adding 50 μg of proteinase K and 3.5 μl of 20% (wt/vol) sodium dodecyl sulfate (SDS) followed by incubation for 15 minutes at 37°C, extraction with phenol/chloroform (1:1 [vol/vol]), and precipitation with ethanol. The precipitates were dissolved, denatured, and subjected to electrophoresis on 4.5% polyacrylamide gels in the presence of 8 M urea. Dried gels were subjected to autoradiography at −70°C for 1-5 days.
Histology, immunohistochemistry, and measurement of β-galactosidase activity
The calluses were fixed in paraformaldehyde, demineralized in 10% EDTA for 2-14 days, dehydrated, embedded in paraffin, and sectioned sagittally. For histological examination, the sections were stained with hematoxylin and eosin or toluidine blue.
For immunohistochemical analyses, the sections were deparaffinized, rehydrated, and digested for 1 h with hyaluronidase (2 mg/ml) in phosphate-buffered saline (PBS), pH 5. The distribution of L-Sox5, Sox6, and Sox9 was studied with polyclonal antibodies raised in rabbits against human Sox9 and mouse Sox5 and Sox6 peptides.(3, 15) The antibodies were applied overnight at +4°C (at dilutions 1:200, 1:100, and 1:2000, respectively). Thereafter, the sections were washed and incubated consecutively with biotinylated secondary antibody and horseradish peroxidase-conjugated streptavidin using the Histostain-Plus bulk kit (Zymed Laboratories, Inc., San Francisco, CA, USA). Visualization of peroxidase activity was performed using the 3,3′-diaminobenzidine tetrahydrochloride-plus kit (Zymed Laboratories, Inc.).
Type II collagen was detected with a monoclonal antibody prepared against chick type II collagen,(29) and type X collagen was detected with a monoclonal antibody X53 against the human recombinant protein,(30) as described earlier.(31) Briefly, the anti-type II and X primary antibodies (1:1000 and 1:20 dilutions, respectively) were applied overnight at +4°C. Thereafter, the sections were washed and incubated consecutively with biotin-labeled secondary antibody and alkaline phosphatase-conjugated streptavidin (Links and Label kit; BioGenex, San Ramon, CA, USA). Thereafter, the sections were stained with Fast Red TR (Sigma, St. Louis, MO, USA) as described earlier.(31) Finally, the sections were counterstained with hematoxylin.
β-galactosidase activity was determined by enzyme histochemistry using X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranosine) as the substrate. The reactions were carried out overnight at room temperature in 2 mM MgCl2, 0.2% NP-40 and 0.1% Na-deoxycholate in 0.1 M phosphate buffer, pH 7.3, supplemented with 1 mg/ml X-gal, 5 mM potassium ferrocyanide and 5 mM potassium ferricyanide. After staining, the samples were thoroughly rinsed with PBS and fixed for 24 h in 4% paraformaldehyde. Thereafter the samples were photographed, decalcified and embedded in paraffin for sectioning.
In situ hybridization
For in situ hybridization, the sections were deparaffinized and treated as described earlier.(32) Proteins were denatured in 0.2 M HCl, followed by digestion with proteinase K (5 μg/ml) in PBS for 30 minutes at 37°C and postfixation in 1% paraformaldehyde for 5 minutes. The slides were washed twice, dehydrated, and dried at room temperature. Hybridizations were performed using cRNA probes essentially as described earlier.(33) Sense and antisense cRNA probes were synthesized using linearized plasmids as templates by T7 and T3 RNA polymerases and [35S]uridine (thio)triphosphate as the radiolabeled nucleotide. The hybridizations were performed at 51–55°C for 40 h. Thereafter, the samples were washed, dehydrated, and exposed for 7-28 days with autoradiographic emulsion (Kodak NTB-3; Kodak, Rochester, NY, USA).
Detection of L-Sox5, Sox6, and Sox9 mRNA in fracture callus
Of the three Sox mRNAs analyzed, only that for Sox9 could be detected by Northern analysis. Although unfractured bone contained virtually no Sox9 mRNA, the levels increased markedly during the first week of healing, remained at this level for a few days, and thereafter declined by day 28 to a level comparable with unfractured bone (Fig. 1A). The expression profile of proα1(II) collagen mRNA resembled that of Sox9 mRNA (Fig. 1A). The changes observed in the mRNA levels of Sox9, proα1(II) collagen, and α1(X) collagen are summarized in Fig. 1B.
Because of insufficient sensitivity of Northern hybridization, an RNAse protection assay was used for detection of mRNAs for L-Sox5 and Sox6. Simultaneous hybridization of pooled RNA samples with probes for L-Sox5, Sox6, and Sox9 revealed that the mRNA levels for L-Sox5 and Sox6 were much lower than those for Sox9 (Fig. 2). The genes for Sox9 and Sox5 were coexpressed, whereas Sox6 transcripts were present already in the unfractured bone. During fracture repair, the relative amounts of all three Sox mRNAs were the highest at day 7 of healing (Fig. 2).
Localization of L-Sox5, Sox6, Sox9, and type II collagen transcripts and proteins in fracture callus
During overt chondrogenesis at day 7 of fracture healing, immunohistochemical analysis revealed the presence of L-Sox5, Sox6, and Sox9 in the nuclei of all cells exhibiting a chondrocytic phenotype (Figs. 3B, 3D, and 3E). In situ hybridization of serial sections revealed that these cells were actively transcribing the Col2a1 gene (Fig. 3C). Immunohistochemistry showed deposition of type II collagen in the extracellular matrix surrounding these chondrocytes (Fig. 3A). On chondrocyte hypertrophy, the nuclear staining for L-Sox5, Sox6, and Sox9 started to disappear (Figs. 3B, 3D, and 3E) at the same time as the transcription of the type II collagen gene was turned off (Fig. 3C). The cells became surrounded by a matrix that contained newly synthesized type X collagen (Fig. 3F) but also retained immunostaining for type II collagen (Fig. 3A).
At day 9 and day 14, L-Sox5, Sox6, and Sox9 also were found in the nuclei of chondrocytes expressing type II collagen, whereas hypertrophic chondrocytes rapidly lost nuclear Sox9 staining and cytoplasmic type II collagen mRNA (data not shown). By day 28 of fracture healing, cells containing L-Sox5, Sox6, or Sox9 or expressing Col2a1 mRNA had disappeared virtually from the callus, which consisted of new bone undergoing remodeling (not shown).
We next turned to the earlier time points of fracture healing. At day 5, the number of chondrocytic cells was still small but they all exhibited strong nuclear staining for L-Sox5, Sox6, and Sox9 (Figs. 4D, 4F, and 4H). Sometimes staining also was seen in the cytoplasm (Figs. 4F and 4H). Sox-expressing cells were located consistently close to cortical bone in areas of thickened periosteum. Immunohistochemistry showed that such cells had started to secrete type II collagen into the surrounding matrix (Fig. 4B).
At day 3 of fracture healing, the callus tissue consisted predominantly of loose granulation tissue with undifferentiated mesenchymal cells (Fig. 4C). The periosteum was considerably thickened and consisted mostly of fibroblastic cells. Occasionally, these cells were beginning to acquire a roundish chondrocytic phenotype and showed nuclear staining for Sox9 (Fig. 4C). The exact location of the faint immunostaining for L-Sox5 and Sox6 could not be determined at this point (Figs. 4E and 4G). In bone marrow, unidentified cells stained positive for Sox6, which probably accounts for the presence of the mRNA in unfractured bone.
BMP-2 gene transfer
Using adenovirus RAdLacZ (106 pfu), we first showed that adenovirus is able to infect effectively cells through much of the fracture callus (Fig. 5). Reliable calculation of the number of cells infected could not be performed, because processing of callus tissue for paraffin sectioning dissolves much of the blue X-gal staining. However, large areas of β-galactosidase-positive cells frequently were observed also in histological sections (Fig. 5B). The expression of β-galactosidase persisted at least for 2 weeks although the level started to decline after the first week.
The effects of BMP-2 on L-Sox5, Sox6, and Sox9, as well as type II and X collagens in fracture callus, then were examined using adenovirus-mediated gene transfer. At day 5 after fracture, Northern analysis (Fig. 6A) showed that the expression of Sox9 and type II collagen mRNAs was significantly higher in fractures injected with RAdBMP-2 than in control fractures injected with RAdLacZ or without viral injection. After RAdBMP-2 injection, the Sox9 mRNA level reached its maximum already at day 5 whereas in the controls the maximum was reached at day 7 (Figs. 6A and 6C). The expression of Sox9 and Col2a1 genes also was increased in the RAdBMP-2 group at day 7, whereas at day 14 the expression of both genes essentially had ceased in all study groups. Although calluses injected with RAdLacZ adenoviruses did not show any difference from noninjected controls at day 5 of healing, the mRNA levels for type II collagen and Sox9 increased by day 7 of repair to the level seen in the RAdBMP-2 group (Figs. 6A and 6C). The latter change did not reach statistical significance.
RNAse protection assay of the same RNAs showed that the BMP-2 gene transfer caused also the mRNA levels of L-Sox5 mRNAs to peak earlier than in the controls, that is, at day 5 of fracture healing (Figs. 6B and 6C). The effect of RAdBMP-2 injection on the kinetics of Sox6 gene expression was similar, except that the highest relative Sox6 mRNAs levels were seen in nonfractured bone.
This study clearly supports the notion of the involvement of L-Sox5, Sox6, and Sox9 in the activation of the chondrogenic differentiation program in an adult organism during fracture healing. Although the intensity of immunostaining in Fig. 3. was stronger for L-Sox5 and Sox6, the mRNA analyses indicated that Sox9 mRNA is considerably more abundant than L-Sox5 and Sox6 mRNAs. This appearance of the three Sox mRNAs and proteins is consistent with their suggested role in chondrogenesis and the production of cartilaginous extracellular matrix during embryonic development.(1, 4, 5, 15, 34) The appearance of Sox9 in murine fracture callus was reported recently also by Sakano et al.(35) In the healing fractures, elevated L-Sox5, Sox6, and Sox9 mRNA levels slightly preceded the up-regulation of the Col2a1 gene (Figs. 1 and 2). Immunohistochemical localization of these three transcription factors in the nuclei of chondrocytes that were rich in Col2a1 transcripts confirmed their temporospatial relationship. On chondrocyte hypertrophy, when the transcription of the Col2a1 gene was down-regulated and that of Col10a1 was up-regulated, the three Sox proteins rapidly disappeared from the nuclei. These findings are in agreement with the suggested function of Sox9 in the induction of the chondrocytic phenotype and cooperative action of the three Sox proteins in the activation of type II collagen production.(15, 34) The strict temporospatial coexpression of the L-Sox5, Sox6, Sox9, and type II collagen in 7-day and 9-day callus suggests that they also function as maintenance factors for the chondrocytic phenotype and for the production of the specialized cartilage matrix. The disappearance of all three Sox proteins on chondrocyte hypertrophy also is in agreement with observations made in embryonic epiphyseal growth plates.(5, 15)
In this study, adenovirus-mediated overexpression of BMP-2 in fracture callus resulted in accelerated up-regulation of Sox9 and Col2a1 genes when compared with untreated or RAdLacZ-treated fractures. Although the increases in L-Sox5 and Sox6 mRNAs were smaller, BMP-2 also enhanced the transcription of these genes at an earlier stage of fracture healing. Thus, in this fracture model, the predominant effect of BMP-2 appears to be acceleration of the kinetics of Sox activation and subsequent chondrogenic response. A similar observation is seen when RAdBMP-2 is injected into intact periosteum.(36) Treatment of unstable fractures in the rabbit by injection of recombinant BMP-2 at day 3 of healing also has been shown to accelerate healing but without induction of chondrogenesis.(37) Recently, adenoviral transfer of BMP-2 into rabbit bone defects also was shown to enhance healing, but the involvement of chondrogenesis was not examined.(38) Our findings are in agreement with previous studies on chick embryos in which up-regulation of the Sox9 gene by exogenous BMP-2 and down-regulation by ectopic Noggin, a BMP antagonist, have been observed.(17) However, in chondrocyte cultures recombinant BMP-2 did not affect the expression of Sox9, suggesting that this effect is indirect and involves other effector molecules.(18) Recent studies have shown that Sox9 is a target of cyclic adenosine monophosphate (cAMP) signaling, possibly elicited by parathyroid hormone-related protein (PTHrP).(39) The presence of PTHrP as well as indian hedgehog (ihh), its receptor patched (Ptc), and core binding factor α1 (cbfa-1) at different stages of fracture healing have been shown.40-42) BMP signaling through BMP receptors and the ihh and PTHrP pathways(43) all appear to act upstream of Sox9, although their exact relationship to Sox9-dependent chondrocytic differentiation remains poorly understood. The fact that bone is a major reservoir of BMPs and TGF-βs,(19, 44) which can be liberated from fractured bone by acid hydrolysis and proteolytic degradation during osteoclastic bone resorption, and the findings of this study bring additional support for the hierarchy of these regulators in vivo.
The data suggest that L-Sox5, Sox6, and Sox9 are involved in the activation and maintenance of chondrogenesis during fracture healing and that acceleration of chondrogenesis by BMP-2 is mediated via L-Sox5-, Sox6-, and Sox9-dependent pathway(s). The data also show that adenoviruses efficiently infect cells in the fracture callus. This should allow secreted growth factors such as BMP-2, to act in a noncell-autonomous manner. Because retarded fracture healing continues to be an important orthopedic problem and the incidence of fractures is increasing because of raising incidence of osteoporosis,(45) identification of molecules capable of accelerated fracture healing potentially are of great medical importance.
We thank Päivi Auho, Merja Lakkisto, and Tuula Oivanen for excellent technical assistance. This study was supported by the Academy of Finland (projects 29496 and 37311), the Sigrid Juselius Foundation, and Turku University Central Hospital (project 13304 and 13336). H.U. is a recipient of a training grant from TULES Graduate School.
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