Presented in part at the 21st Annual Meeting of the American Society for Bone and Mineral Research, St. Louis, Missouri, U.S.A., 1999 and appears in J Bone Miner Res 14(Suppl 1):S211 (abstract)
Sox-4 Messenger RNA Is Expressed in the Embryonic Growth Plate and Regulated via the Parathyroid Hormone/Parathyroid Hormone-Related Protein Receptor in Osteoblast-like Cells†
Article first published online: 1 DEC 2000
Copyright © 2000 ASBMR
Journal of Bone and Mineral Research
Volume 15, Issue 12, pages 2402–2412, December 2000
How to Cite
Reppe, S., Rian, E., Jemtland, R., Olstad, O. K., Gautvik, V. T. and Gautvik, K. M. (2000), Sox-4 Messenger RNA Is Expressed in the Embryonic Growth Plate and Regulated via the Parathyroid Hormone/Parathyroid Hormone-Related Protein Receptor in Osteoblast-like Cells. J Bone Miner Res, 15: 2402–2412. doi: 10.1359/jbmr.2000.15.12.2402
- Issue published online: 2 DEC 2009
- Article first published online: 1 DEC 2000
- Manuscript Accepted: 12 JUN 2000
- Manuscript Revised: 11 MAY 2000
- Manuscript Received: 1 NOV 1999
- growth plate;
- parathyroid hormone
Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert potent and diverse effects in cells of the osteoblastic and chondrocytic lineages. However, downstream mediators of these effects are characterized inadequately. We identified a complementary DNA (cDNA) clone encoding the 5′ end of the transcription factor Sox-4, using a subtracted cDNA library enriched in PTH-stimulated genes from the human osteoblast-like cell line OHS. The SOX-4 gene is a member of a gene family (SOX and SRY) comprising transcription factors that bind to DNA through their high mobility group (HMG)-type binding domain, and previous reports have implicated Sox proteins in various developmental processes. In situ hybridization of fetal and neonatal mouse hindlimbs showed that Sox-4 messenger RNA (mRNA) was expressed most intensely in the zone of mineralizing cartilage where chondrocytes undergo hypertrophy, and by embryonic day 17 (ED17), after the primary ossification center was formed, its expression was detected only in the region of hypertrophic chondrocytes. Sox-4 mRNA was detected in osteoblast-like cells of both human and rodent origin. In OHS cells, physiological concentrations (10−10–10−9 M) of human PTH 1-84 [hPTH(1-84)] and hPTH(1-34), but not hPTH(3-84), stimulated Sox-4 mRNA expression in a time-dependent manner, indicating involvement of the PTH/PTHrP receptor. Sox-4 transcripts also were detected in various nonosteoblastic human cell lines and tissues, in a pattern similar to that previously reported in mice. The presence of Sox-4 mRNA in hypertrophic chondrocytes within the mouse epiphyseal growth plate at sites that overlap or are adjacent to target cells for PTH and PTHrP, and its strong up-regulation via activated PTH/PTHrP receptors in OHS cells, makes it a promising candidate for mediating downstream effects of PTH and PTHrP in bone.
SOX-4 IS a member of a family of transcription factors consisting of the mammalian testis-determining factor SRY and the Sox proteins of which about 30 have been found so far. (1–3) Sox proteins are defined by the presence of a high mobility group (HMG) box of about 80 amino acids that also are found in the chromatin-associated proteins HMG-1 and HMG-2, suggesting that they represent architectural components of chromatin in addition to their function as transcription factors.(4)
Evidence that Sox proteins play important roles during embryonic development and cellular differentiation recently has been presented. Disruption of the Sox-4 gene in mice leads to circulatory failure in embryos homozygous for the mutation at embryonic day 14 (ED14). This is related to impaired development of the endocardial ridges, consistent with Sox-4 messenger RNA (mRNA) expression in the endocardial anlagen of wild-type embryos.(5) Furthermore, a role for Sox-4 in lymphocyte development is implicated from studies showing that the gene is expressed in cell lines representing early pre-B and T lymphocyte lineages(6) and that differentiation of B lymphocytes is arrested at the pro-B cell stage in SOX-4 (−/−) mice.(5) Recent studies have shown that Sox-4 mRNA is stimulated by the progesterone analogue progestin in breast cancer cells(7) and by prostaglandin (PG)A2 and Δ12-PGJ2 in Hep3B human hepatocellular carcinoma cells.(8) Several reports also suggest important roles of Sox proteins in chondrogenesis: Sox-4 is expressed in embryonic cartilage of the respiratory tract, the orthosympatic trunk, and the spinal cord(5); defects in the transcription factor Sox-9 lead to camptomelic dysplasia, which is characterized by skeletal malformation(9,10); and L-Sox-5, Sox-6, and Sox-9 cooperatively regulate chondrocyte-associated genes such as type II and type XI collagen.(11,12) Postnatally, although Sox-4 is expressed most abundantly in the heart, testis, ovary, and thymus,(6,13) gross histological examination of SOX-4 (−/−) embryos at ED14 did not reveal manifestations of any obvious abnormalities at these sites, except dysplasia of the semilunar valves.(5)
Parathyroid hormone (PTH) is produced in the parathyroid glands and is a main systemic regulator of calcium homeostasis and bone remodeling. Full-length PTH contains 84 amino acid residues, and although circulating forms of the hormone include distinct N-terminal, midregion, and C-terminal fragments, the latter forms predominate.(14) By comparison, the PTH-related protein (PTHrP) is produced locally in a wide variety of cell types and tissues, including the skeleton, and various proteolytic fragments of PTHrP also are known to exist. PTH and PTHrP share structural homology in the 34 amino acids N-terminal region, enabling the two proteins to bind and activate a common cloned G protein-coupled PTH/PTHrP receptor present on the surface of chondrocytes, osteoblasts, and renal epithelial cells(15,16) with equal potency. This then leads to stimulation of multiple effector systems, including adenylate cyclase and phospholipase C, and increases of intracellular calcium. (17–19) N- and C-terminal fragments of PTH and PTHrP also show distinct actions in bone-derived cells that are not mediated by the “classical” PTH/PTHrP receptor and thus are likely to be mediated by unique PTH and PTHrP receptors. (20–23) For example, a novel PTH receptor (C-PTH receptor) that is expressed by parathyroid and osteoblastic cells specifically binds C-terminal fragments of PTH but not PTHrP.(24,25) Although the biochemical properties and biological significance of these other receptors remain to be elucidated, the PTH/PTHrP receptor system has been shown to play crucial roles in embryonic skeletal development. Mice lacking functional copies of the respective genes show a similar phenotype, including severe defects in the epiphyseal growth plates associated with increased rate of chondrocyte differentiation.(26) The more severe skeletal abnormalities observed in mouse embryos, where the PTH/PTHrP receptor gene has been ablated as compared with those lacking one of the ligands (i.e., PTHrP (−/−) mutants), can be taken as evidence for a compensatory role of circulating PTH in the latter knockout model.(27)
The PTH and PTHrP signals in bone cells are transduced via activation of receptor-coupled secondary messenger systems that lead to transcriptional regulation of downstream target genes. We have used subtractive cloning to identify genes that are expressed differentially in response to PTH (or PTHrP) in the human osteoblast-like OHS osteosarcoma cell line. From a subtracted complementary DNA (cDNA) library, we have identified a clone encoding a part of the human Sox-4 gene. Our data showing for the first time that Sox-4 gene expression is stimulated by human PTH 1-84 [hPTH(1-84)]and PTH(1-34) in osteoblastic cells, implicate a member of the HMG-transcription factors as a novel component of the PTH signal transduction pathway in osteoblastic cells. Furthermore, expression of Sox-4 within the embryonic growth plate indicates that Sox-4 has an important function in bone formation.
MATERIALS AND METHODS
Commercial premade blots were from Clontech (Palo Alto, CA, U.S.A.). Recombinant hPTH(1-84) and hPTH(3-84) were prepared in our laboratory as described.(28,29) hPTH(1-34) was purchased from Sigma (St. Louis, MO, U.S.A.); ExpressHyb and Marathon-Ready cDNA were from Clontech; the OHS, KPDXM, and KRIB cell lines were obtained from Dr. Ø. Bruland (The Norwegian Radium Hospital, Oslo, Norway). MNNG-HOS, SaOS-2, ROS 17/2.8, UMR-106, and MDA-MB-231 cell lines, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were from the American Type Culture Collection (ATCC; Manassas, VA, U.S.A.); genomic DNA for mouse type X collagen was obtained from Dr. B. R. Olsen (Harvard Medical School, Boston, MA, U.S.A.); and the rat PTH/PTHrP receptor cDNA and C29 cell line were obtained from Dr. H. Jüppner (Massachusetts General Hospital, Boston, MA, U.S.A.).
Construction of cDNA libraries and directional tag polymerase chain reaction subtraction
Cytoplasmic RNA was isolated from OHS cells treated with 10−7 M hPTH(1-84) or vehicle for 24 h and enriched for poly(A+) RNA using oligo (dT)25 Dynabeads(DYNAL, Oslo, Norway). A target cDNA library was prepared from the PTH-treated material as described(30) except that the Sal I linker (Gibco BRL, Stafford, TX, U.S.A.), rather than EcoRI linker, was ligated to the double-stranded cDNA, and pSPORT-Sal I/Not I (Gibco BRL), rather than pT7T3D, was used as vector. The cDNA library was transformed into Epicurian coli SURE electroporation-competent cells according to vendor of the cells (Stratagene, La Jolla, CA, U.S.A.). The driver library was prepared as described(30) from poly(A)+ RNA from untreated OHS cells, except that pT7T3D-EcoRI/Not I/bacterial alkaline phosphatase (BAP) was used rather than the pGEM11Zf(−) vector. The subtractive hybridization was performed once using the previously described procedures.(30,31) Briefly, both libraries were linearized with Not I, followed by in vitro transcription with T7 RNA polymerase using the megascript kit (Ambion, Austin, TX, U.S.A.). Thirty micrograms of the sense strand OHS-purified complementary RNA (cRNA) were used directly in the subtractive hybridization procedure as driver, whereas the target OHS-PTH had to go through an additional [32P]-labeled antisense cDNA synthesis prepared as described in the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech; Uppsala, Sweden), using 3 μg of the sense cRNA, 0.75 μg of Not I-d(T)18 primer, and 150 μCi of α[32P]deoxycytosine triphosphate (dCTP) to allow later quantification of the target cDNA. The product was treated with alkali to hydrolyze the RNA template and purified before annealing for 24 h at 68°C in 15 μl of hybridization buffer(30) with the 30 μg of the driver sense cRNA (ratio 1:10). After chromatography on hydroxyapatite, the single-stranded target cDNA fraction corresponded to 3% of the input material, as judged by tracer quantitation. The sample was then purified and an aliquot (1/10) of the single-stranded material was used as template in a 30-cycle polymerase chain reaction (PCR) (94°C for 15 s, 60°C for 30 s, and 72°C for 1 minute) using primers corresponding to the tag sequences in the target (5′-AACTGGAAGAATTCGCGG-3′ and 5′-ACCGGTCCGGAATTCCCG-3′) and Taq polymerase (Perkin Elmer, Branchburg, NJ, U.S.A.). The amplification product was cleaved sequentially with Not I and EcoRI and then separated from primers and the flanking sequences with a Sephacryl S 400 spin column, before it was ligated into the pT7T3D EcoRI/Not I/BAP vector. An aliquot (1/10) of this subtracted library was electroporated into Epicurian coli cells. The subtracted library was named ΔOHS-PTH and represented 1.5 × 106 clones.
Differential screening of the subtracted cDNA library
The single-stranded OHS-PTH cDNA target library was used to prepare a cDNA probe. This subtracted cDNA (ΔOHS-PTH) was labeled using PCR (same conditions and primers as described previously for construction of the cDNA subtraction library, except for a reduced dCTP concentration and the use of α[32P]dCTP(30) and was used as probe for the subtractive library, ΔOHS-PTH probe. To prepare a representative probe for the target and driver libraries, 2 μg from the OHS-PTH sense cRNA and 2 μg from OHS sense cRNA, respectively, were used to synthesize the corresponding antisense cDNAs. These probes were prepared in the same way as the single-stranded cDNA that was used as target in the subtractive hybridization. An aliquot (1/100) each of the single-stranded cDNA (OHS-PTH and OHS) was labeled by PCR using the same conditions as for the subtracted probe. The OHS probe was synthesized by PCR using the same primers as for the subtracted probe. To synthesize the OHS probe (driver), the primers were 5′-ACTGGAAGAATTCGCGG-3′ and 5′-AGGCCAAGAATTCGGCACGA-3′. To screen the subtracted cDNA library, 960 single colonies were picked and grown up in microtiter plates and three identical nylon replica filters were prepared from each plate. These identical filters were hybridized to each of the three different probes, target probe (OHS-PTH), driver probe (OHS), and subtraction probe (ΔOHS-PTH), using equal amounts of32P radioactivity (>106 cpm/ml) for each filter. Colonies that gave strong signals with the subtractive probe (ΔOHS-PTH) and with the target probe (OHS-PTH) but no signals with the driver probe (OHS) were picked for further analysis. The inserts were sequenced, using the ThermoSequenase kit (Amersham, Arlington Heights, IL, U.S.A.). The inserts were then isolated from these clones and used as probes in Northern blot analysis.
Cloning of the 5′ untranslated region by rapid amplification of cDNA ends
For 5′ rapid amplification of cDNA ends (RACE), the AP1 and AP2 anchoring primers in the Marathon-Ready cDNA kit (Clontech) were used, in addition to the following downstream primers: internal (5′AGCAAACTGCAGCGCGGTGAGAGAG) and external (5′TCGTTTGACGTCGCGCCACTCTCTC). Marathon-Ready cDNA from the MOLT4 human T lymphocyte cell line was used as template as these cells contained large amounts of the transcript. PCR was performed with four cycles of annealing at 68°C and then four cycles at 65°C and finally 30 cycles at 63°C. Extension time was 60 s at 70°C, and denaturation time was 15 s at 94°C for all cycles. The GeneAmp XL PCR kit (Perkin Elmer, Foster City, CA, U.S.A.) was used according to the manufacturer's instructions, and the product was cloned into pCRII-TOPO (Invitrogen, Carlsbad, CA, U.S.A.) and sequenced using Thermo Sequenase (Amersham). These sequence data are available from GenBank/EMBL/DDBJ under the accession number AF124147.
Tissue preparation and in situ hybridization
Animals were maintained in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Pregnant Swiss-Webster mice were killed by CO2, and hindlimbs of fetuses and neonatal pups were removed and dipped into freshly prepared 4% paraformaldehyde (PFA)-phosphate-buffered saline (PBS; pH 7.4) for at least 1 h. Bones were dehydrated and embedded in paraffin wax. Bone sections of 5 μm were prepared by standard histological procedures and mounted on Superfrost/Plus slides (Fisher, Pittsburgh, PA, U.S.A.).
Antisense or sense [35S]-labeled RNAs were synthesized from linearized plasmids containing the genomic DNA for mouse type X collagen DNA (nucleotides 903-2215; no. X65121), the 350-base pair (bp) Sox-4 human cDNA, or the rat PTH/PTHrP receptor cDNA (nucleotides 258-1671; no. M77184) using the Gemini Transcription kit (Promega, Madison, WI, U.S.A.). The specificity of in situ hybridization was indicated by the absence of hybridization signal when adjacent tissue sections were subjected to identical conditions with radiolabeled sense RNA probes (data not shown).
In situ hybridization was performed as described.(32) In brief, sections were dewaxed with xylene, dehydrated with increasing concentrations of ethanol, and postfixed with 4% PFA-PBS for 15 minutes. After washing with PBS and another treatment with 4% PFA-PBS for 10 minutes, sections then were washed sequentially with PBS, incubated with 0.2N HCl (10 minutes), again washed with PBS, acetylated with 0.25% acetic anhydride in the presence of triethanolamine (0.1 M; 10 minutes), dehydrated with increasing concentrations of ethanol, and air-dried. Hybridizations with [35S]-labeled cRNAs (5 × 107 cpm/ml) were performed in a humidified chamber in a solution containing 50% formamide, 10% dextran sulfate, 1× Denhardt's solution (0.02% Ficoll 400, 0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin [BSA]), 600 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, 50 mM dithiothreitol (DTT), 0.25% sodium dodecyl sulfate (SDS), and 200 μg/ml transfer RNA (18 h; 55°C). After hybridization, sections were washed briefly with 5× SSC at 50°C, with 50% formamide in 2× SSC at 50°C for 30 minutes, and then with 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA (TNE) at 37°C for 30 minutes. Sections were treated with 10 μg/ml RNase A in TNE (37°C; 30 minutes). After being washed with TNE, sections were incubated once with 2× SSC (50°C; 20 minutes) and twice with 0.2× SSC (50°C; 20 minutes). Sections were dehydrated with increasing concentrations of ethanol and air-dried. Slides were then placed on X-ray films (hyperfilm β-max; Amersham) and film autoradiographs were obtained after overnight exposure. Slides were dipped into NTB-2 (Eastman Kodak, New Haven, CT, U.S.A.) and stored at 4°C for the times estimated from the intensity of expression on X-ray film (1-7 days). After development, sections were counterstained with hematoxylin and eosin and mounted.
Northern blot analysis
Total RNA was prepared using the TRIZOL reagent (Life Technologies, Rockville, MD, U.S.A.). Poly(A+) RNA was isolated from total RNA preparations using Dynabeads according to the manufacturer's instructions. Northern blots were prepared as described previously(33) using Hybond N+ filters (Amersham). The filters were prehybridized, hybridized with a radiolabeled Sox-4 cDNA fragment, washed, and exposed to X-ray films according to the manufacturer's recommendation. Quantification of the Northern blot signals on X-ray film was performed by scanning densitometry using an XRS 3sc scanner and the BioImage System (Millipore Corp., Ann Arbor, MI, U.S.A.). Values for Sox-4 mRNA signals were normalized to those of the GAPDH or β-actin to correct for variations in loading and are presented as fold stimulation in PTH-treated cultures relative to untreated control.
Normal osteoblasts (NOs) were prepared from bone biopsy specimens of patients undergoing hip surgery and cultured as described.(34) OHS, KPDXM, and SaOS-2 cells were cultured in RPMI-1640 medium with 10% fetal calf serum (FCS), L-glutamine (0.3 mg/ml), penicillin (5 × 105 IU/liter), and streptomycin (50 mg/liter). The MNNG-HOS, SaOS-2, ROS 17/2.8, UMR-106, and MDA-MB-231 cell lines were cultured according to instructions from ATCC and subcultured weekly. The medium was changed every 3-4 days, and the cells always received fresh medium containing 5% FCS 24 h before the experiments were started. C29 cells were cultured as described.(35) Full-length PTH or truncated PTH peptides of concentrations or vehicle (acetic acid, 1.7 × 10−5 M final concentration) were added to the culture medium of cells that had just reached confluence, and the cells were harvested at the given time points, as described in the figure legends.
Cloning and sequencing of a highly conserved region within the 5′ untranslated region of Sox-4
The osteogenic human OHS cell line exhibits characteristics of a relatively well-differentiated osteoblastic phenotype, including high expression of alkaline phosphatase and PTH-stimulated adenylyl cyclase activities, (36–38) and thus represents a valuable model system to investigate downstream effectors of PTH and PTHrP in bone. Immediate and early response genes such as c-fos are activated within minutes in osteoblast-like cells on PTH stimulation without the need of foregoing protein synthesis.(39,40) Secondary target genes are then transcriptionally activated, and in order to enrich for their mRNAs, OHS cells were stimulated with PTH over a period of 24 h. Subtractive cloning was used to identify the target genes for PTH signaling. A cDNA clone of 350 bp isolated from the subtracted library (see Introduction) showed 86% identity to the 5′ untranslated region of mouse Sox-4 but no homology to the published human sequence(13) (Fig. 1). To map the 5′ end of the Sox-4 transcript, 5′ RACE was performed (see Materials and Methods). We identified a putative transcription start site at position −767 relative to the translational start codon, in agreement with the 5′ end of a number of expressed sequence tags (EST) clones with identical or very similar sequences (e.g., the clones with GenBank accession numbers W25179, W16493, and AA534016).
Expression of Sox-4 mRNA in fetal and newborn mouse hindlimbs
The spatial and temporal expression of Sox-4 mRNA in developing mouse tibias from ED15.5 throughout postnatal day 1 (P1) was studied using in situ hybridization. By ED15.5, Sox-4 mRNA was most highly expressed in the zone of mineralizing cartilage where chondrocytes characteristically express type X collagen (Kaechong Lee, personal communication, 1999), undergo hypertrophy, and the future bone marrow cavity is formed (Figs. 2A–2D and 2G). Furthermore, although expression of the PTH/PTHrP receptor is greatest in the zones flanking the central portion of the cartilage core, lower levels of the transcript are expressed throughout the central portion, overlapping with the Sox-4 expressing domain ( Figs. 2C–2F). At ED17.0, the primary ossification center has been formed in the tibias, giving rise to areas consisting of epiphyseal cartilage at the ends of the developing bones that are separated by a central marrow cavity (Figs. 2H and 2I). At this stage, expression of Sox-4 mRNA is evident in the zone of hypertrophic chondrocytes, although the levels of Sox-4 transcripts are lower than at ED15.5. By ED18.5, although mRNA for Sox-4 colocalizes with that of type X collagen and thereby identifies the Sox-4 expressing cells as hypertrophic chondrocytes, its expression is adjacent to or partly overlapping with the zone of PTH/PTHrP receptor mRNA expressing cells (Figs. 2J–2O). Postnatally (P1), expression of Sox-4 is reduced compared with earlier time points (Figs. 2P–2S).
Regulation of Sox-4 mRNA by PTH in osteoblast-like cells
Dose and time response studies were carried out in OHS cell cultures using the full-length PTH(1-84) form. Northern analysis revealed a single Sox-4 mRNA species of about 5 kilobases (kb), as previously reported in humans and mice.(6,13) The Sox-4 mRNA levels increased dose dependently from 10−10 M PTH(1-84), and maximum response (∼10-fold stimulation) was observed at a dose of 10−9 M (Fig. 3A). Sox-4 mRNA levels peaked after 12-24 h of treatment with PTH(1-84) (10−7 M; Fig. 3B), and elevated levels relative to the untreated controls were observed also at 48 h and 5 days (not shown). Similar effects of PTH on Sox-4 mRNA expression were found in the rat UMR-106 osteosarcoma cell line (not shown).
To examine whether Sox-4 mRNA was regulated by PTH also in NOs, calvarial osteoblasts (released from calvaria by collagenase digestion) were grown in the presence of 10% fetal bovine serum supplemented with β-glycerophosphate and vitamin C to induce osteogenic differentiation.(41,42) Northern analysis of total RNA from prolonged cultures of these cells showed that levels of Sox-4 mRNA increased about 2-fold in response to treatment with 2 × 10−9 M hPTH(1-84) for 16 h (not shown). Our data show that PTH regulation of Sox-4 mRNA takes place also in normal primary osteoblasts and not only in clonal osteoblast-like cells.
An intact N terminus of PTH is required for its stimulatory effect on Sox-4 mRNA expression in OHS cells as shown by the equipotent ability of PTH(1-84) and PTH(1-34) to increase levels of Sox-4, whereas the N-terminally truncated form PTH(3-84) had no effect. PTH(3-84) is able to bind to the receptor but unable to stimulate cyclic monophosphate (cAMP) production.(28) Furthermore, Sox-4 mRNA expression in OHS cells increased 3-fold when the OHS cells were treated with the cAMP analog 8-Br-cAMP, but not with phorbol-12-myristate-13-acetate (TPA; not shown). These results indicate that increased Sox-4 expression on PTH stimulation is mediated by the PTH/PTHrP receptor via increases in cAMP levels.
Sox-4 mRNA expression in human tissues and cell lines
Northern blot analysis was performed in order to define the pattern of Sox-4 mRNA expression in various cell lines. We found that Sox-4 mRNA is abundantly expressed in human MNNG-HOS, KRIB, OHS, SaOS-2, KPDXM, and in rat ROS 17/2.8 osteosarcoma cell lines (Fig. 4A), while lower levels of the transcript were detected in human osteoblast cultures derived from normal trabecular bone. The osteoblastic nature of the osteosarcoma cell lines was verified by positive hybridization signals using cDNA probes for the following osteoblastic markers: type I collagen, osteonectin, PTH/PTHrP receptor, and alkaline phosphatase (data not shown). No or very little expression of Sox-4 mRNA was found in the breast cancer cell line MDA-MB-231 (Fig. 4A).
Among other nonosteoblastic cell lines, high levels of Sox-4 mRNA expression were found in the lymphoblastic leukemia cell line MOLT4, moderate expression was found in the lung carcinoma cell line A549, and low expression was found in the colorectal adenocarcinoma cell line SW480 as revealed by hybridization of the Sox-4 probe to a commercial human cancer cell-line multiple tissues Northern blot (MTN; Clontech). No expression was detected in the promyelocytic leukemia cell line HL60, HeLa, and S3; in the Burkitt's lymphoma cell line Raji; or in the melanoma cell line G363 (Fig. 4B). These results are in agreement with previous reports showing Sox-4 expression in early B and T cell lineages.(6)
The expression pattern of Sox-4 mRNA in human tissues was analyzed by hybridization to a human RNA Master Blot (Clontech; Fig. 5). Sox-4 mRNA levels were found to be most abundant in fetal brain, but high levels also were detected in fetal heart, kidney, liver, spleen, and lung. With the exception of thymus, expression was higher in fetal tissues than in adult tissues. In adult tissue, although Sox-4 mRNA was most abundantly expressed in the thymus and ovary, signals also were detected in the frontal lobe, uterus, testis, prostate, stomach, lung trachea, placenta, small intestine, kidney, mammary gland, and appendix. Thus, the expression pattern is reminiscent of what has been found in mice.(6,5)
Using subtractive cloning, we have identified the HMG transcription factor Sox-4 as a novel putative regulatory component of the PTH signal transduction pathway in osteoblastic cells. In situ hybridization showed that Sox-4 transcripts are predominantly localized to the zone of hypertrophic chondrocytes in the growth plates of developing fetal mouse hindlimbs. We found that the Sox-4 gene is expressed by human and rat osteoblast-like clonal cell lines and primary osteoblastic cell cultures, and that the levels of Sox-4 mRNA in human OHS and rat UMR-106 osteosarcoma cell lines are time- and dose-dependently increased in response to treatment with full-length PTH(1-84) for 24 h.
DNA sequencing revealed high homology of the isolated clone to the 5′ untranslated region of mouse Sox-4. Mapping of the Sox-4 mRNA by 5′ RACE identified the end at position −767 relative to the translational start codon. Northern analysis further showed the presence of a single transcript of about 5 kb in various cell lines and tissues. Thus, our data indicate that the Sox-4 cDNA clone represents a hitherto unknown 5′ end of the human Sox-4 transcript. Interestingly, the interspecies conservation is nearly as high for this newly discovered 5′ region as for the HMG box and the serine-rich regions (94% and 91% sequence identity, respectively, whereas the identity between the 5′ ends is 86%). By comparison, the DNA sequence of the translated coding region of mouse and Sox-4 shows only 64% identity between the HMG box and the serine-rich region (Fig. 1). Because the Sox-4 protein is encoded by a single exon in mice(43) and is caused by stop codons downstream of the conserved sequence that are in frame with the protein coding sequence, this region is not likely to be part of a translated region. We therefore propose that this region has an important regulatory function.
Sox-4 mRNA is predominantly expressed in the growth plate of hypertrophic chondrocytes of fetal mouse hindlimbs as detected using in situ hybridization. Furthermore, Sox-4 transcripts are most abundant at ED15.5 and gradually decrease to undetectable levels at P1. Its expression overlaps the same area as that of the central and less intense part of the PTH/PTHrP receptor distribution. At ED 18.5 the common area of expression of Sox-4 mRNAs and the PTH/PTHrP receptor appears to be reduced to overlapping zones near the distal ends. We have found Sox-4 mRNA expression in cultured human articular chondrocytes (not shown), indicating a function for Sox-4 also in chondrocytes outside the embryonic growth plate.
Other members of the Sox family and various collagens are candidates for being involved with the regulative functions of Sox-4. L-Sox-5, Sox-6, and Sox-9 are all expressed in proliferating chondrocytes,(11) whereas Sox-4 mRNA is located in hypertrophic chondrocytes (Fig. 2). Sox-4 is able to bind to the same DNA binding site as other Sox proteins. Hence, it would be interesting to study whether Sox-4 acts as an inducer for type X collagen expression, which is used as a marker for hypertrophic chondrocytes, and participates in regulation of expression of other collagens. The type X collagen promoter has a Sox-9 responsive region that probably also will bind Sox-4,(44) and type II and type XI collagen as well as the cartilage-derived retinoic acid-sensitive protein (CD-RAP) can all be regulated by Sox-9 in chondrocytes through their HMG type protein binding site.(12, 45, 46)
Sox-4 mRNA was not detected in the osteoblasts of the perichondrium or the bone collar by in situ hybridization, perhaps because of the scattered distribution of osteoblasts in this tissue and/or lower levels of expression existing at various differentiation stages.
The effects of PTH on Sox-4 mRNA expression in osteoblastic cells are coupled via specific receptors. The dose-dependent increase of Sox-4 mRNA in OHS cells in response to PTH parallels the PTH-stimulated cAMP production in human OHS cells and LLC-PK1 cells stably transfected with the cloned human PTH/PTHrP receptor, with effective concentrations with half maximal effect (EC50) values of 2 × 10−9 M (unpublished results) and 5 × 10−10 M, respectively.(28,47) These results, together with the observation that PTH(3-84) was unable to stimulate cAMP production, make the classical PTH/PTHrP receptor a strong candidate for mediating the stimulatory effect of PTH on Sox-4 mRNA expression. Activated PTH/PTHrP receptors are capable of stimulating multiple intracellular effector systems, including adenylate cyclase and phospholipase C. (17–19) Stimulation of Sox-4 mRNA expression by the cAMP mimic 8-Br-cAMP but not TPA indicates that the effect of PTH is mediated through adenylate cyclase. The functional significance of Sox-4 in osteoblasts is unknown. PTH stimulation of human bone cells after several weeks of culture gave no significant Sox-4 mRNA stimulation. This may be caused by phenotypic alterations during in vitro conditions.
In addition to PTH, PTHrP, and their receptor(s), the transforming growth factor (TGF) superfamily including the bone morphogenetic proteins (BMPs) with receptors and Indian hedgehog are important players in the regulation of chondrogenesis and bone formation.(48) Recently, it was found that BMP-6 is negatively regulated by PTHrP, resulting in accelerated maturation of cultured chicken sternal chondrocytes.(49,50) Being downstream in the signaling pathway of PTH and PTHrP and being expressed in the same cells as BMP-6, Sox-4 is a candidate transcription factor for taking part in mediation of these effects.
We found Sox-4 mRNA to have similar expression pattern in human tissues and cell lines as has been found in the mouse, indicating similar roles in the two species.
High expression of Sox-4 mRNA in osteosarcoma cells and hypertrophic chondrocytes may suggest a role for Sox-4 in maturation of osteoblasts and chondrocytes, analogous to what has been found for B cell and T cell development, where lack of Sox-4 blocks further differentiation at the pro-B cell stage(5) and restricts thymocyte differentiation.(51) Interestingly, Sox-4 is highly expressed in the thymus, in which PTHrP and its receptor also is expressed.(52)
Being expressed within the embryonic growth plate and regulated via the common PTH/PTHrP receptor in osteoblast-like cells, Sox-4 appears to be an important regulator of bone development and can be added to the list of transcription factors regulated by PTH and PTHrP.
We thank Zeremariam Yohannes and Åse Karine Fjellheim for technical help, Sigurd Ørstavik for computational assistance, and Øyvind S. Bruland and Harald Jüppner for cell lines. This work was supported by The Norwegian Osteoporosis Society, The Norwegian Cancer Society, The Norwegian Research Council, Anders Jahre's Foundation for Promotion of Science, Rachel and Otto Bruuns legate, The Novo Nordisk Foundation, The Norwegian Society for Endocrinology, and Bristol-Myers Squibb. S.R. was financed by The Norwegian Foundation for Health and Rehabilitation through the Norwegian Women's Health Association.
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