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Address correspondence to Brendan Lee, Dept. of Molecular and Human Genetics Baylor College of Medicine One Baylor Plaza, Rm 635E, Houston, TX 77030. Tel.: (713) 798-8835. Fax: (713) 798-5168. email: blee{at}bcm.tmc.edu
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Abstract |
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Key Words: CBFA1; COL1OA1; CCD; MCT cells; transgenic mice
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Introduction |
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Although several signaling molecules including Indian Hedgehog and parathyroid hormone related peptide have been shown to be required for chondrocyte hypertrophy by mouse genetic studies, few transcriptional determinants specifying hypertrophic chondrocytespecific type X collagen gene expression have been identified to date (Karaplis et al., 1994; Schipani et al., 1995, 1997; Lanske et al., 1996; Vortkamp et al., 1996; St-Jacques et al., 1999; Chung et al., 2001). One candidate gene likely important for Col10a1 gene regulation is the runt domain transcription factor Runx2/Cbfa1. Runx2 has been shown in mouse genetic studies to be required for differentiation of the mesenchymal stem cell into the osteoblast lineage (Komori et al., 1997; Otto et al., 1997). Runx2-null mice do not have bone and accordingly Runx2 has also been demonstrated to transactivate a host of genes highly expressed in osteoblasts including osteocalcin and type I collagen (Ducy et al., 1997). Runx2 has also been shown to be important for chondrocyte differentiation, i.e., during endochondral ossification. Supporting this are recent histomorphologic studies showing alteration of chondrocyte maturation in some long bones of Runx2-null mice as well as cell culture studies indicating that Runx2 is a positive regulatory factor for chondrocyte maturation (Inada et al., 1999; Kim et al., 1999; Enomoto et al., 2000). In fact, two studies mis-expressing Runx2 in proliferating chondrocytes were able to induce chondrocyte hypertrophy and partially rescue Runx2-null mice (Takeda et al., 2001; Ueta et al., 2001). Although recent studies have demonstrated several transcriptional targets for Runx2 in osteoblastic and chondrocytic cells (Jimenez et al., 1999; Zelzer et al., 2001), the direct link between Runx2 and the tissue-specific expression of type X collagen, the only known hypertrophic chondrocytespecific molecular marker, has not been identified yet.
Here, we present identification of the first murine Col10a1 promoter that can direct reporter activity selectively in hypertrophic chondrocytes in transgenic mice. We further show that Runx2 can directly transactivate this Col10a1 promoter both in vitro and in vivo via putative Runx2 binding sites found in this promoter region. Last, we discussed the altered endochondral ossification in the Runx2 mutant mice. We surmise that this is probably due to down-regulation of Runx2 targets in the hypertrophic chondrocytes including Col10a1.
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Results |
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To determine the in vivo relevance of these data, we generated four independent transgenic mouse lines harboring the 4-kb proximal Col10a1 promoter upstream of the ß-galactosidase reporter gene. Three lines of mice exhibited similar X-gal staining, whereas the fourth one did not show any staining. We performed whole embryo staining of E15.5 mouse embryos and blue staining was noted only at the ends of long bones (Fig. 3 A). No blue staining was observed in any other tissues when analyzing sagittal sections of the whole embryos featuring a variety of tissues (unpublished data). Although some background staining in craniofacial region probably due to endogenous ß-galactosidase, activity was observed both in transgenic and control postnatal day 1 (P1) mice, specific blue staining was observed only in the chondro-osseus junction of limbs, ribs and also in the nasal cartilage of transgenic mice (Fig 3 B). Indeed, histological analysis confirmed that ß-galactosidase expression was observed in the lower zone of hypertrophy of rib sections and in the long bone sections of the limbs including proximal humerus and proximal femur at P1 stage (Fig. 3, C and E, and not depicted). Although some weak staining was also present in bone marrow along trabeculae presumably in osteoblasts, no blue staining was observed in other tissues including perichondrium, resting or proliferating chondrocytes, muscle fibers, or adhering connective tissues (Fig. 3 B and not depicted). These data show that the proximal 4-kb Col10a1 promoter was able to direct hypertrophic chondrocyte expression of the ß-galactosidase reporter in vivo and, therefore, contributes to Col10a1 expression in these cells. Because a 1.7-kb mouse promoter was previously reported to be unable to direct expression of ß-galactosidase to hypertrophic chondrocytes (Eerola et al., 1996), we hypothesized that the positive regulatory elements in this 4-kb construct most likely reside in the 5' portion where the two conserved Runx2 binding sites A and B are found.
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In cotransfection studies in COS7 cells, RUNX2 was able to transactivate reporter constructs containing eight copies of either of these RUNX2-binding elements upstream of a 44-bp Col10a1 minimal promoter and the luciferase reporter gene (Fig. 5 A). Promoter constructs containing the A element or B element were transactivated >20- and 40-fold above baseline, respectively (Fig. 5 A). These data show that RUNX2 can bind to the distal sequences of the 4-kb Col10a1 promoter and transactivate a Col10a1 minimal promoter via these sequences. We also transfected the Col10a1 4-kb promoter-ß-galactosidase reporter plasmid with or without the RUNX2 expression plasmid into hypertrophic MCT cells. The endogenous activity of the 4-kb promoter was 10-fold greater than that of the basal promoter (Fig. 5 B, left). In addition, when RUNX2 is over-expressed in these cells, reporter activity is further upregulated more than two-fold above the endogenous activity of this promoter (Fig. 5 B, left). In converse, when the two RUNX2 binding sites are mutated in the 4-kb promoter, reporter activity is decreased by 35% compared to the wild-type promoter (Fig. 5 B, right). Together, these data show that the 4-kb Col10a1 promoter is up-regulated when MCT chondrocytes hypertrophy in culture and that RUNX2 binding contributes to this transactivation.
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Discussion |
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This paper utilizes a unique type X collagenexpressing MCT cell line to delineate the cis-acting elements for chondrocyte hypertrophy. Compared to the bovine or chicken primary hypertrophic chondrocytes previously used in the cell culture studies, the MCT cell line has the advantage of homogeneity and phenotypic stability (Lefebvre et al., 1995). Most importantly our results from MCT cells correlate with that of transgenic mice. Therefore, MCT cells could be used to further delineate the cis elements controlling chondrocyte hypertrophy and eventually to characterize the transcription factor(s) specifying this process.
The 4-kb Col10a1 promoter described can direct reporter expression selectively to lower hypertrophic chondrocytes in transgenic mice. However, it is likely that additional positive and negative regulatory elements outside of this 4-kb Col10a1 promoter are also required for tissue-specific expression during embryogenesis. This is supported by the detection of weak ß-galactosidase expression in the bone trabeculae of our transgenic mice, as well as by the localization of reporter gene expression primarily in the lower hypertrophic zone, and less so in the upper hypertrophic zone. Together, these observations suggest the requirement of additional positive and negative regulatory elements outside this 4-kb promoter to achieve high level Col10a1 expression in hypertrophic chondrocytes in vivo. Runx2 is likely only one of several factors that are required for this coordinated process. Runx2 is essential for osteoblast differentiation and it is also important for chondrocyte maturation. It has been shown that Runx2 transcriptional regulation of collagenase-3, osteopontin, and VEGF is critical for the transition from chondrogenesis to ossification (Sato et al., 1998; Jimenez et al., 1999; Zelzer et al., 2001). Runx2 may interact with different factors within osteoblast and hypertrophic chondrocytes to regulate different downstream genes. It alone is not sufficient to specify chondrocyte hypertrophy in all bones, because loss of hypertrophic chondrocytes is observed only in some of the long bones of Runx2-null mice (Inada et al., 1999; Kim et al., 1999). Identification of other transcription factors important for chondrocyte hypertrophy and their relation with Runx2 will be crucial for our understanding of chondrogenesis.
Whether decreased Col10a1 expression might directly affect chondrocyte hypertrophy is still controversial. There is some evidence to support that haploinsufficiency of COL10A1 accounts for cases of SMCD (Chan and Jacenko, 1998; Chan et al., 2001; Wilson et al., 2002), whereas others have pointed to a possible dominant negative effect (Marks et al., 1999; Gregory et al., 2000). A very recent study showed that nonsense mutations leads to the complete degradation of mutant collagen X mRNA in cartilage in SMCD (Bateman et al., 2003). Interestingly, SMCD patients also have an altered zone of hypertrophy (Wasylenko et al., 1980; Lachman et al., 1988; Nielsen et al., 2000). However, it appears that chondrocyte hypertrophy by itself, at least in mice, does not require type X collagen (Rosati et al., 1994). Instead, defective mineralization, altered hematopoiesis, and growth plate compression were reported in Col10a1-null mice (Kwan et al., 1997; Gress and Jacenko, 2000). It may be that the structural integrity of the hypertrophic zone requires normal type X collagen expression in humans and a metaphyseal dysplasia develops in the presence of decreased expression. However, the same effect may not be seen in mice because of different biomechanical forces impinging upon metaphyseal development in a tetrapod.
From the point of view of skeleton pathogenesis, haploinsufficiency of RUNX2 causes cleidocranial dysplasia (CCD), a dominantly inherited skeletal dysplasia in humans (Mundlos et al., 1995, 1996, 1997; Lee et al., 1997). The pathognomonic features of CCD include both defective intramembranous ossification and defective endochondral ossification (Cooper et al., 2001). Interestingly, it has been reported that Runx2+/- mice exhibit some of the features of CCD including delayed closure of the fontanel and hypoplastic clavicles (Otto et al., 1997). Our findings of decreased Col10a1 expression and altered chondrocyte hypertrophy in Runx2 heterozygote mice suggest that they could also serve as a model to study the pathogenesis of long bone defects of CCD.
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Materials and methods |
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For nuclear extracts, MCT cells were grown at 32°C until subconfluency. They were further incubated at 37°C for 2 d before nuclear extract preparation. Nuclear extracts were then prepared as described previously with 10 µg/ml of leupeptin and pepstatin in all buffers (Dyer and Herzog, 1995). 3 µg of hypertrophic MCT cell nuclear extracts were used for each binding assay. 50- to 100-fold cold competitor probe, or Runx2 antibody (Ducy et al., 1997), or preimmune serum were incubated with nuclear extracts 10 min on ice before addition of probe in parallel experiments. The MCT cells were provided by B. de Crombrugghe (University of Texas, Houston, TX). The anti-Runx2 antibody was provided by G. Karsenty (Baylor College of Medicine, Houston, TX).
Chromatin immunoprecipitation assay
Hypertrophic MCT cells were incubated at 37°C for 3 d before formaldehyde fixation. Cold PBS (with Protein Inhibitor Cocktail Tablets; Roche) washing, cell harvesting, and sonication to shear DNA to 5001,000 bp was performed according to the manufacturer's protocol (Upstate Biotechnology). Precleared chromatin was incubated with 2 µg of the anti-Runx2 antibody (Santa Cruz Biotechnology, Inc.), preimmune antiserum or no antibody and rotated at 4°C for 12 h. Immunoprecipitation, washing, and elution of immune complex were carried out as described previously (Boyd and Farnham, 1999). The specific primers for A element, B element, and the control primers within Col10a1 intron II region were used for PCR amplification as described previously (Thomas et al., 2001; Weinmann and Farnham, 2002).
Transfection studies
The Col10a1 minimal promoter (+7 bp to -37 bp) was inserted into the pLuc4 luciferase reporter plasmid (Min-Col10a1-pA; Zhou et al., 1999). Each of the Runx2 binding elements A or B were concatamerized to form eight copy fragments, which were inserted upstream of the Col10a1 minimal promoter (8xA/B-Min-Col10a1-pA). Transfections using an expression plasmid (control pcDNA3.1 or pcDNA3.1/RUNX2), a reporter plasmid (Min-Col10a1-pA or 8xA/B-Min-Col10a1-pA), and a normalizing plasmid (pSV2ßgal) were performed with the Lipofectamine-plus (GIBCO BRL) reagent in COS7 cells, and luciferase and ß-galactosidase activities were assayed 24 h after transfection as described previously (Zhou et al., 1999). Transfections were performed in triplicate at three different doses (0.2, 0.5, and 1.0 µg/well, respectively) to ensure a linear-dose response.
MCT cells were grown at 32°C in standard DME media with 8% FBS (GIBCO BRL) and 8% CO2 as per published protocol (Lefebvre et al., 1995). Transfection of MCT cells were conducted at both 32°C and 37°C using reporter plasmids containing a 221-bp basal Col10a1 promoter upstream of the ßgeo reporter (basCol10a1-SAßgeobpA), the wild-type Col10a1 4-kb proximal promoter (Col10a1-SAßgeobpA), or the same promoter with mutated RUNX2 binding sites A and B (mutCol10a1-SAßgeobpA). The mutCol10a1-SAßgeobpA reporter plasmid was generated by replacing a Xho/BlpI wild-type fragment with a mutant one within the 4-kb Col10a1 promoter and it contained the same mutations within the RUNX2 binding A and B sites as described in the previous paragraph on the EMSA experiments.
MCT cells were transfected for 6 h using Lipofectamine-plus (GIBCO BRL), incubated for an additional 48 h at either 32°C or 37°C, and harvested for ß-galactosidase activity assay. A luciferase expression plasmid pRSVluc was added to all transfections and used as internal control for normalizing the cell transfection efficiency. Transfections were also performed in triplicate at three doses to ensure a linear-dose response.
Northern analysis, real time RT-PCR, and in situ hybridization
For Northern hybridization analysis of Col10a1 mRNA derived from MCT cells grown at either 32°C or 37°C, total RNA was prepared from MCT cells using TRIzol reagent according to the manufacturer's protocol (GIBCO BRL). 10 µg RNA was fractionated, transferred to Hybond NH2 nylon membrane (Amersham Biosciences), and hybridized with murine Col10a1 cDNA (a 1.2-kb BamHI fragment) and Gapdh cDNA (a 300-bp fragment from the 3' untranslated region) probes as described previously (Lefebvre et al., 1995). The filter was then autoradiographed for 3 d.
For MCT cells, the same total RNAs for Northern analysis were also used for cDNA synthesis. Total mouse limb RNAs from littermates with wild-type, Runx2+/-, or Runx2-/- background at P1 stage was extracted by liquid nitrogen frozen, grinded on ice, and followed by TRIzol reagents extraction (GIBCO BRL). cDNA synthesis was performed by using the Superscript first-strand synthesis system RT-PCR kit (Invitrogen). The primer sequences used for PCR amplification for Runx2, Col10a1, transgene (the bovine growth hormone poly A region in the transgene construct) and Gapdh was performed using specific primers. The Gapdh gene was used as an internal control of the quantity and quality of the cDNAs. Real time PCR amplification was performed on LightCycler (Roche) according to the manufacturer's protocol and published procedures with modifications (Pfaffl, 2001). Analysis of the real time PCR results, i.e., the relative gene expression level, was achieved by using the 2- CT method for fold induction, and CT (the threshold cycle) for the fractional cycle number at which the amount of amplified target reaches a fixed threshold (Livak and Schmittgen, 2001; Pfaffl, 2001).
For Col10a1 in situ hybridization, newborn mouse hind limbs were fixed in 4% PFA in PBS, dehydrated, and embedded in paraffin. Sectioning and in situ hybridization were carried out as described previously (Albrecht, 1998) with the same Col10a1 probe used in the Northern analysis. Nine distal serial femur sections from each of two Runx2+/- and one Runx2+/+ littermates were analyzed. Representative data for comparable sections are shown.
Generation and histochemical analysis of transgenic mice
The DNA fragment containing the 4-kb Col10a1 promoter followed by SAßgeobpA reporter was released from the above-described Col10a1-SAßgeobpA construct by ApaI digestion. Purified DNA's were redissolved and microinjected into the pronuclei of one-cell ICR X B6C3F1 mouse embryos and implanted into ICR pseudopregnant foster mothers (Hogan et al., 1986). Transgenic founder mice were analyzed by genomic southern analysis of tail DNA's with a 3-kb lacZ-specific probe (Zhou et al., 1995). Runx2 heterozygote mice were obtained from M. Owen (Lincoln's Inn Fields, London, UK) and genotyped by PCR amplification as reported previously (Otto et al., 1997).
In brief, P1 mice were collected, skinned, and eviscerated. They were fixed and stained with X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) as described previously (Zhou et al., 1995). After staining, mice were paraffin embedded, sectioned, and counterstained with nuclear fast red (Poly Scientific R&D Corp.). Sections of all long bones were analyzed and comparisons were made only among littermates at the same magnifications. At least 30 sections of each growth plate were analyzed.
For densitometric analysis of transgene expression in Tg/Runx2+/+ and Tg/Runx2+/- mouse limb sections, we calculated the gray value of blue staining cells corresponding to ß-galactosidase activity using a microscope (Axioplan 2; Carl Zeiss MicroImaging, Inc.) and AxioVision 3.1 software (Carl Zeiss Vision GmbH). Analyses were made by two independent observers blinded to genotype of the sections. 200 blue staining cells in the hypertrophic zone were randomly chosen from 20 serial limb sections. A gray value with standard deviation was generated for each cell. This value inversely related to the intensity of the blue staining. Average gray values were calculated for the 200 cells of each genotype. Statistical analysis was performed with t test.
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Acknowledgments |
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This work was supported by the National Institutes of Health grants AR44738, ES 11253, and HD22657 (all to B. Lee), March of Dimes Birth Defects Foundation (to B. Lee), the Arthritis Foundation (to Q. Zheng and B. Lee), the Baylor College of Medicine Child Health Research Center (to B. Lee), the Baylor College of Medicine Mental Retardation Research Center (to B. Lee), and the Howard Hughes Medical Institute (to B. Lee and X. Garcia-Rojas).
Submitted: 20 November 2002
Accepted: 10 July 2003
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