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ADAMTS-1: A Cellular Disintegrin and Metalloprotease with Thrombospondin Motifs Is a Target for Parathyroid Hormone in Bone

Endocrine Division (R.R.M., J.P.S., D.L.H., R.F.S., L.V.H., K.T., R.J.S.G., J.M.H., J.E.O.) and Cardiovascular Division (L.B.), Lilly Research Laboratories, Indianapolis, Indiana 46285

Address all correspondence and requests for reprints to: Dr. J. E. Onyia, Bone Metabolism Research Group, 0403, Endocrine Division, Lilly Research Laboratories, Indianapolis, Indiana 46285. E-mail: jeo{at}lilly.com

	Abstract

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PTH stimulates bone formation in animals and humans, and the expressions of a number of genes have been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon we used differential display RT-PCR and screened for genes that are selectively expressed in osteoblast- enriched femoral metaphyseal primary spongiosa of young male rats after a single sc injection of human PTH-(1–38) (8 µg/100 g). We show that one of the messenger RNAs that is up-regulated in bone is ADAMTS-1, a new member of the ADAM (A disintegrin and metalloprotease) gene family containing thrombospondin type I motifs. ADAMTS-1 consists of multiple domains common to ADAM family of proteins, including pro-, metalloprotease-like, and disintegrin-like domains. However, unlike other ADAMs, ADAMTS-1 does not possess a transmembrane or cytoplasmic domain and is a secreted protein. Northern blot analysis confirmed that ADAMTS-1 was up-regulated in both metaphyseal (14- to 35-fold) and diaphyseal (4.2-fold) bone 1 h after PTH-(1–38) injection and returned to control levels by 24 h. We also analyzed the regulation of ADAMTS-1 in response to various PTH/PTH-related peptide (PTHrP) analogs and found that PTH-(1–31) and PTHrP-(1–34), which activate the protein kinase A (PKA) pathway, induce ADAMTS-1 expression 1 h after injection, whereas PTH-(3–34) and PTH-(7–34), which do not activate the PKA pathway, did not regulate expression. To investigate the effect of other osteotropic agents, we analyzed ADAMTS-1 expression after a single dose of PGE₂ (6 mg/kg) and found that it was up-regulated 1 h after injection and returned to control levels by 6 h. In vitro ADAMTS-1 is expressed in primary osteoblasts and osteoblastic cell lines, but was not detectable in osteoclasts generated from macrophage colony-stimulating factor/receptor activator of NF-B ligand/transforming growth factor-ß1-treated bone marrow cells. Treatment of UMR 106 osteosarcoma cells with PTH, PGE₂, forskolin, or (Bu)₂cAMP increased ADAMTS-1 expression 7-, 4-, 5-, and 5-fold, respectively. Also, in vitro treatment with 1,25-dihydroxyvitamin D₃ increased ADAMTS-1 expression 3-fold. Tissue distribution analysis showed that ADAMTS-1 is expressed at high levels in many tissues, including the heart, lung, liver, skeletal muscle, and kidney. Taken together, these results demonstrate that ADAMTS-1 is specifically up-regulated in bone and osteoblasts by the osteotropic agents PTH, PTHrP, and PGE₂ possibly via the cAMP/PKA pathway. We speculate that the rapid and transient increase in ADAMTS-1 expression may contribute to some of the effects of PTH on bone turnover.

	Introduction

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PTH IS A potent modulator of bone metabolism. In experimental animals and patients with osteoporosis, intermittent administration of PTH increases net bone mass by stimulating de novo bone formation to enhance trabecular thickness, connectivity, and strength (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). On a cellular level, this increase in bone mass is associated with an increased number of osteoblasts (11, 12). We have recently demonstrated in young rats that PTH targets proliferating bone cells in the primary metaphyseal trabecular spongiosa and increases the number that differentiate into osteoblasts (11, 13, 14, 15). In this model, an increase in the trabecular bone-forming surface may be detected as early as 24 h after a single normocalcemic dose of PTH. The molecular mechanisms that mediate PTH-induced bone formation have been difficult to elucidate, primarily because this phenomenon cannot be fully recapitulated in vitro. In vivo, we and others have demonstrated that the earliest events that may lead to the effects of PTH in bone involve the expression of genes such as c-fos, c-jun, c-myc, interleukin-6, leukemia inhibitory factor, histone H4, and regulator of G protein signaling-2 (14, 16, 17, 18, 19, 20). The changes in the expression of these genes after in vivo PTH treatment have been localized predominantly to cells of the osteoblast lineage that control de novo bone formation (16, 17, 20). However, the exact contribution of this relatively few number of genes to PTH actions in bone remains to be unequivocally determined. The PTH-induced effects on bone probably involve the complex interaction of numerous gene products, many of which remain unidentified to date.

As remodeling (proteolysis) of the extracellular matrix (ECM) plays a critical role in establishing bone tissue architecture in PTH action, it has been speculated that novel adhesive proteins, their receptors, and secreted proteases would be targets of PTH action in vivo. Many of these secreted and cell surface proteins and proteases are expressed at critical sites/locations/time to facilitate cell-cell and cell-matrix interactions and matrix remodeling that could be essential in bone turnover (21, 22, 23). One group of metalloproteases, in particular ADAMs (A disintegrin and metalloprotease), which possess both a metalloprotease (proteolytic) domain and a distingerin (adhesion) domain, has emerged as a key participant in the diverse biological and pathological processes requiring proteolytic modifications of cell surface proteins and ECM. These proteins have been implicated in a variety of important cellular processes, including neurogenesis, protein ectodomain shedding, integrin binding, and cell-cell binding and fusion in diverse systems (21, 22, 24, 25, 26, 27, 28, 29). To date, over 20 full-length ADAM complementary DNA (cDNA) species have been reported, some of which have been shown to be expressed in bone (30, 31). Recently, new members of the ADAM family, known as ADAMTSs (A disintegrin and metalloprotease with thrombospondin motifs), have been cloned and designated ADAMTS 1–11 (32, 33, 34, 35, 36, 37). ADAMTSs are novel in that they contain unique thrombospondin (TSP) type I motifs in addition to some of the multiple domains common to the ADAM family of proteins, including pro-, metalloprotease-like, and disintegrin-like domains. Unlike other ADAMs, ADAMTSs do not possess a transmembrane or cytoplasmic domain and are secreted proteins that closely associate with the ECM (38). Presently, evidence for the proteolytic activity and biological functions of some of the ADAMTSs has been demonstrated (33, 34, 35, 37, 39, 40), but information on their expression and regulation is limited.

Based on the roles of these ADAM/ADAMTS proteins in diverse processes requiring proteolysis, it seems likely that the expression and regulation of one or more of these genes might contribute to PTH actions in bone. In an effort to discover new early response genes (earliest events) involved in PTH actions in bone, we used differential display PCR (DDRT-PCR) to identify messenger RNAs (mRNAs) that are differentially expressed in the distal femur metaphysis of PTH-treated young male rats. In the present study we show that a member of the ADAMTS subfamily, ADAMTS-1, is rapidly and transiently up-regulated in bone and osteoblasts after PTH treatment [as well as other osteotropic agents, such as PTH-related peptide (PTHrP), PGE₂, and 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃)]. We speculate that ADAMTS-1 and related proteins may be involved in the cascade of events leading to bone remodeling in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
PTH
Synthetic human PTH-(1–38), PTH-(1–31), PTHrP-(1–34), and bovine PTH-(3–34) and -(7–34) (Bachem, Torrance, CA) were prepared in a vehicle of acidified saline containing 2% heat-inactivated rat serum. PGE₂ (Sigma, St. Louis, MO) was first dissolved in 100% ethanol and further diluted in vehicle to a final ethanol concentration of 10%. 1,25-(OH)₂D₃ was purchased from Sigma and solubilized in dimethylsulfoxide.

Animals
Young, virus antibody-free, male Sprague Dawley rats, 60–75 g (Harlan Sprague Dawley, Inc., Indianapolis, IN), were housed with a 12-h light-dark cycle. Animals were fed Purina chow (1% calcium and 0.61% phosphate; PMI Feeds, Inc., St. Louis, MO) and water ad libitum. Animal protocols were approved by the Lilly animal care and use committee (Eli Lilly & Co., Indianapolis, IN).

In vivo protocols
Rats were weighed and sorted into groups of comparable mean body weight (four rats per group). The rats were injected sc with the various analogs of PTH (80 µg/kg), PTHrP-(1–34) (80 µg/kg), or PGE₂ (6 mg/kg) and were killed using CO₂ at the indicated time points. Control rats received an equal amount of the vehicle sc and were killed at the same times. The doses of PTH and PGE₂ were chosen based on our previous work (14, 18, 41) and reports from other laboratories (42, 43) demonstrating an effect on bone and gene expression. After death, rat femora were resected, and all connective tissues, including periosteum, were completely removed. The distal epiphysis, including the growth plate, was removed, and a subjacent 3-mm-wide band of the metaphyseal primary spongiosa or diaphyseal middle third of the same femur was resected and frozen in liquid nitrogen until mRNA analyses were performed (14, 18, 41). For experiments involving PGE₂, the distal metaphysis (6 mm subjacent to the growth plate) was used for mRNA analysis.

Osteoblast cell cultures
The rat osteosarcoma cell line UMR 106 was maintained in DMEM/Ham’s F-12 (3:1; Life Technologies, Inc., Grand Island, NY) containing 10% FBS (HyClone Laboratories, Inc., Logan, UT) plus 2 mM glutamine (Life Technologies, Inc.). ROS 17/2.8 osteosarcoma cells were maintained in the growth medium-Ham’s F-12 nutrient mixture (Life Technologies, Inc.) containing 10% FBS plus 2 mM glutamine (Life Technologies, Inc.). BALC cells, a mouse calveria-derived stromal/osteoblastic cell line (44), were grown in RPMI 1640 supplemented with 5% FBS and 2 mM glutamine. Primary osteoblast cultures were derived from the rat femur metaphysis and diaphysis as previously described (15, 45, 46). All cultures were maintained in a humidified 5% CO₂ atmosphere at 37 C_. For mRNA analysis, cultures (4-T150 flasks/group) of cells were grown (as described above) to 80–90% confluence, and for PTH treatment experiments, cells were then switched to medium containing 0.1% FBS overnight. The cells (UMR106 cells) were treated with human (h) PTH-(1–38) (5 x 10^-8 M) or 1,25-(OH)₂D₃ (1 x 10^-8 M) for 0, 1, 6, 24, or 48 h. In additional experiments cells were treated with the indicated concentrations of PGE₂, forskolin, or (Bu)₂cAMP for 1 h.

Bone marrow-derived osteoclast cell cultures
Osteoclasts were generated in vitro from bone marrow as previously described (47). In brief, bone marrow was flushed from the femurs of male BALB/c mice (aged 6–12 weeks) with culture medium (RPMI with 5% FBS and 1% antibiotic/antimycotic). The bone marrow cells were seeded at a density of 2.5 x 10⁵ mononuclear cells/cm² in 150-mm cluster dishes (Falcon, Becton Dickinson and Co., Franklin Lakes, NJ). The cultures were treated with the following factors: recombinant murine macrophage colony-stimulating factor (M-CSF; 50 ng/ml; R and D Systems, Minneapolis, MN), recombinant human soluble receptor activator of NF-B ligand (RANKL; 50 ng/ml; Chemicon, Temecula, CA), and recombinant human transforming growth factor-ß1 (TGFß1; 100 ng/ml). The media and factors were replaced on day 3 and polyadenylated [poly(A)⁺] RNA was isolated on day 6. In this culture system, theformation of osteoclasts is totally dependent on the presence of RANKL and M-CSF, whereas TGFß1 was added to the cultures to enhance osteoclast formation. As we recently reported, these osteoclasts display the distinguishing characteristics of authentic osteoclasts, including the ability to resorb bone, and the expression of lineage-specific markers, including tartrate-resistant acid phosphatase and calcitonin receptor (47). These cultures consist of osteoclasts and progenitor cells with very few contaminating stromal or osteoblastic cells (47).

Isolation of RNA and cDNA synthesis
To ensure reproducibility and to reduce the false positives, total RNA from three independent experiments was used in the cDNA synthesis and differential display. For each experiment, RNA was extracted from the metaphyseal primary spongiosa of vehicle- or PTH-treated rats at 1 and 24 h as previously described (14, 41). With each experiment, samples were pooled into treated or control groups (four animals per group) for each indicated time point after treatment. Samples were removed from the animals, snap-frozen, and pooled for isolation of RNA. Total RNA was extracted by homogenization in Ultraspec-II (Biotecx, Houston, TX) using an LS 10–35 Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY) as recommended by the manufacturer. Isolated RNA was quantitated using spectrophotometry by measuring the absorbance at 260 nm, and the 260/280 nm ratio was calculated to ensure the absence of protein contamination. To remove contaminating DNA from the RNA preparation, samples were incubated with ribonuclease-free deoxyribonuclease I (Roche, Indianapolis, IN) for 15 min at room temperature and then extracted with phenol/chloroform. First strand cDNA was synthesized from 4 µg total RNA by oligo(deoxythymidine) priming, using the Superscript preamplification kit (Life Technologies, Inc.) in a final volume of 40 µl.

PCR and differential display
Differential display was carried out using arbitrary primer sets as previously described (18, 41, 48). The upstream (arbitrary primer) and downstream (anchored) primers that detected ADAMTS-1 were 5'-AGGTGACCGT-3' and 5'-TTTTTTTTTTTTC-3', respectively. Using cDNA (diluted 1:25) or a no cDNA template control (negative control) duplicate PCR reactions were assembled robotically (Tecan Genesis, Reading, UK) to final concentrations of 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 50 mM KCl, 2.0 mM deoxy (d)-NTPs, 15 nM [³³P]dATP (Amersham Pharmacia Biotech, Arlington Heights, IL), and 1 U AmpliTaq polymerase (Perkin-Elmer Corp., Foster City, CA) in a final volume of 20 µl. Reactions were then subjected to the following PCR conditions on a DNA Engine PTC-225 thermocycler (MJ Research, Inc., Watertown, MA): 1 cycle of 92 C for 2 min; 40 cycles of 92 C for 15 sec, 40 C for 2 min, and 72 C for 1 min; and 1 cycle of 72 C for 5 min. Subsequently, PCR products were separated on a 6% Tris-borate/EDTA/urea sequencing gel (Sequagel, National Diagnostics, Atlanta, GA) for 3 h at 1700 V. Gels were dried and exposed to Biomax x-ray film (Eastman Kodak Co., Rochester, NY). The negative controls with no cDNA template yielded no PCR products.

Reamplification, cloning, and sequencing of cDNA
Bands of interest representing differentially expressed genes were excised from the gel, boiled for 5 min in H₂O, and purified over a Centricon 50 column (Amicon, Beverly, MA). Samples were then reamplified to confirm the size and specificity of the primer sets used in the display. Reamplified products were ligated into pCR2.1 TA cloning vector (Invitrogen, San Diego, CA) and transformed into DH10B cells (Life Technologies, Inc.). For each clone, 10 colonies were picked and amplified in Luria Bertoni broth, and the plasmids were isolated (Wizard Plus, Promega Corp., Madison, WI). Clones that contained inserts were submitted for automated cycle sequencing (Lilly DNA Technology Group, Indianapolis, IN). All sequences were analyzed using BLAST2 against GenBank and EMBL databases to determine sequence identity.

Cloning ADAMTS-1 cDNA
Poly(A)⁺ RNA (1.0 µg) isolated from femur metaphyseal primary spongiosa of 1-h PTH-treated rats was used for Marathon ready cDNA synthesis using the Marathon cDNA Amplification Kit according to the manufacturer’s protocol (CLONTECH Laboratories, Inc., Palo Alto, CA). The ADAMTS-1 gene was amplified from Marathon ready cDNA by PCR using nested primers corresponding to differential display PCR product (1006HS2, 5'-AATGAGAGAATGTGACAACCCGGTCCCA-AAGAACG-3'; and 1006HS3, 5'-AAGTACTGCGAAGGCAAACGAG-TCCGCTACAGGTC-3') and AP1 and AP2 primers (CLONTECH Laboratories, Inc.). Hot start PCR was performed using a 1:1 mixture of anti-Taq antibody (CLONTECH Laboratories, Inc.) and TaKaRa LA Taq using buffers suggested by the manufacturer for the polymerase (Pan Vera Corp., Madison, WI). Cycling conditions for both the first round and the nested PCR were 94 C for 2 min (1 cycle); 5 cycles of 94 C for 30 sec and 72 C for 5 min; 5 cycles of 94 C for 30 sec and 70 C 5 min; and 25 cycles of 94 C for 30 sec and 68 C for 5 min. The resulting 2.9-kb fragment was isolated from an agarose gel using QIAEX II gel extraction kit (QIAGEN, Santa Clarita, CA) and cloned into the TOPO-TA cloning vector (Invitrogen, San Diego, CA).

Generation of radiolabeled probes for Northern analysis
To generate radioactive probes for Northern analysis, the inserts containing ADAMTS-1 cDNA were released from the plasmid by restriction digest. Rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes were cloned using PCR with specific primer pairs as previously described (45). Twenty-five nanograms of cDNA were labeled by the random primer method (Life Technologies, Inc.) using [-³²P]dCTP (Amersham Pharmacia Biotech). Free nucleotides were removed by centrifugation through a Centricon-50 column (Amicon).

Isolation of poly(A)⁺ RNA and Northern blotting
ADAMTS-1 mRNA expression was analyzed by Northern blot. Bone, various tissues, and cell culture samples were pooled into treated or control groups for each indicated time point after treatment. Total RNA was extracted from bone and various tissues by homogenization in Ultraspec-II (Biotecx) using an LS 10–35 Polytron homogenizer (Brinkmann Instruments, Inc.) as recommended by the manufacturer. Total RNA was extracted from the osteoblast or osteoclast cultures by adding Ultraspec-II directly to the culture flasks. The resulting cell lysates were passed several times through a 10-ml pipette before collection. Poly(A)⁺ RNA was isolated from total RNA using Oligotex (QIAGEN) according to the manufacturer’s protocol and was quantitated by spectrophotometry. The absorbance at 260 nm was determined, and the 260/280 nm absorbance ratio was calculated to ensure the absence of protein contamination. Samples of poly(A)⁺ RNA (2 µg) were denatured in 0.04 M 3-(N-morpholino)propanesulfonic acid (pH 7.0), 10 mM sodium acetate, 1 mM EDTA, 2.2 M formaldehyde, and 50% formamide at 60 C for 10 min; size-fractionated by electrophoresis through 1% agarose gels in 2.1 M formaldehyde and 1 x 3-(N-morpholino)propanesulfonic acid); and transferred to nylon membranes (Brightstar-Plus, Ambion, Inc., Austin, TX). The nylon membranes were air-dried, and the RNA samples were cross-linked to the membranes by UV irradiation in a Stratalinker (Stratagene, La Jolla, CA). Migration of 28S and 18S ribosomal RNA was determined by ethidium bromide staining. DNA probes were labeled by the random primer method (Life Technologies, Inc.) using [-³²P]dCTP. Prehybridization and hybridization were carried out at 48 C in NorthernMax buffers (Ambion, Inc.). After hybridization, membranes were washed for 30 min at room temperature in buffer containing 2 x SSC and 0.1% SDS, then for 30 min at 48 C in 0.2 x SSC (standard saline citrate) and exposed to Biomax MS x-ray film (Eastman Kodak Co.) at -70 C. Autoradiograms were quantitated by scanning laser densitometry (2400 Gel Scan XL, LKB, Piscataway, NJ). Labeled bands were quantitated as densitometric units and normalized to that of the GAPDH signals to correct for variations in RNA transfer and gel loading. The data were expressed as fold change vs. untreated control samples. The experiments were repeated two to four times for each time point to confirm findings.

Multitissue RNA analysis
To determine the distribution of the ADAMTS-1 transcript, we probed poly(A)⁺ RNA from rat tissues using multiple tissue Northern blots (CLONTECH Laboratories, Inc.). The multiple tissue Northern blot contained 2 µg/lane poly(A)⁺ RNA from heart, brain, kidney, spleen, lung, liver, skeletal muscle, and testis.

	Results

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Identification of ADAMTS-1 as a PTH-regulated gene in rat metaphyseal bone
We screened for genes that are differentially regulated by PTH in rat metaphyseal bone using DDRT-PCR (48). cDNA derived from RNA isolated from hPTH-(1–38)-treated and control femoral metaphyses at 1 and 24 h after treatment was differentially displayed using primer sets as described in Materials and Methods. To enhance reproducibility and reduce the occurrence of false positives, as reported previously (49, 50, 51, 52, 53), the DDRT-PCR was conducted on cDNA prepared from total RNA samples derived from three independent experiments. A parallel display of duplicate samples from control and treated bones showed a 498-bp band that was rapidly up-regulated in 1 h, but returned to control levels by 24 h (Fig. 1). This band was excised from the gel and reamplified by PCR, and the resulting PCR product was then cloned and sequenced. PCR primers designed to this sequence were used to extend the length and clone the cDNA corresponding to this gene. The largest cDNA fragment amplified (2.9 kb) was cloned and sequenced. Sequence analysis revealed that cDNA from this band encodes a partial open reading frame of 372 amino acids and is 100%, 96%, and 84% identical with rat, mouse, and human ADAMTS-1 proteins, respectively (data not shown; GenBank no. g4929478, D67076, and g5725505). Based on sequence similarity, we designated this clone rat ADAMTS-1.

View larger version (71K): [in this window] [in a new window]   Figure 1. DDRT-PCR identification of ADAMTS-1 as a PTH-regulated gene in rat metaphyseal bone. DDRT-PCR products amplified from cDNA derived from vehicle- and hPTH-(1–38)-treated femurs (pooled; n = 4/group) were resolved on a 6% TBE/urea sequencing gel. To eliminate false positives, cDNAs derived from total RNA prepared from three independent experiments were analyzed simultaneously. A negative control omitting the cDNA template (no cDNA control) was also analyzed, and it yielded no bands (data not shown). Samples were run in duplicate for each time point examined. The band representing the candidate PTH-regulated gene is indicated by the arrow. This band was excised from the gel, reamplified by PCR, and cloned for sequence analysis.

View larger version (52K): [in this window] [in a new window]   Figure 2. Effect of hPTH-(1–38) on ADAMTS-1 mRNA expression in rat femur metaphysis and diaphysis. A, A representative autoradiograph showing the time course of hPTH-(1–38) treatment on ADAMTS-1 mRNA expression in rat femur metaphysis. B, Comparison of basal and PTH effects on ADAMTS-1 in metaphyseal and diaphyseal bone. RNA was isolated from the femur metaphyseal and diaphyseal bones of young male rats (pooled; n = 4/group) at the indicated times after a single PTH injection (80 µg/kg, sc). Two micrograms of poly(A)+ RNA were loaded per lane and analyzed for ADAMTS-1 expression by Northern blot hybridization. GAPDH was rehybridized as a control for RNA integrity and quantification. After normalization to GAPDH, the values are shown as the fold induction over the vehicle (V)-treated control value (which is set at 1).

View larger version (61K): [in this window] [in a new window]   Figure 3. Effects of PTH analogs and PTHrP on ADAMTS-1 mRNA expression in rat femur metaphysis. Poly(A)+ RNA, isolated from the distal femur metaphysis of rats (pooled: n = 4/group) 1 h after injection (sc) with the indicated PTH analogs (80 µg/kg), PTHrP (80 µg/kg), or vehicle equivalent, were analyzed for ADAMTS-1 expression by Northern blot hybridization. GAPDH was rehybridized as a control for RNA integrity and quantification. ADAMTS-1 mRNA levels normalized to GAPDH signals are expressed as fold induction over the vehicle-treated control value (which is set at 1).

View larger version (63K): [in this window] [in a new window]   Figure 4. Effect of PGE2 on ADAMTS-1 mRNA expression in rat femur metaphysis. Poly(A)+ RNA isolated 1, 6, or 24 h after injection of PGE2 (6 mg/kg) or vehicle equivalent was analyzed for ADAMTS-1 mRNA expression. Two micrograms per lane of poly(A)+ RNA were used for Northern blot hybridization. GAPDH was rehybridized as a control for RNA integrity and quantification. ADAMTS-1 mRNA levels normalized to GAPDH signals are expressed as fold induction over the vehicle-treated control value (which is set at 1).

View larger version (48K): [in this window] [in a new window]   Figure 5. Expression and regulation of ADAMTS-1 in UMR106 osteosarcoma cells. A, Time course of the effects of hPTH-(1–38) and 1,25-(OH)2D3 on ADAMTS-1 mRNA expression. UMR106 osteosarcoma cells were treated with hPTH-(1–38) (5 x 10-8 M) or 1,25-(OH)2D3 (10-8 M) for 0, 1, 6, 24, or 48 h. B, The effects of forskolin, (Bu)2cAMP, and PGE2 on ADAMTS-1 mRNA expression in UMR106 osteosarcoma cells. Cells were treated with the indicated concentration of forskolin (FSK) or (Bu)2cAMP (db cAMP) or PGE2 for 1 h. Poly(A)+ RNA was isolated at the end of the treatments, and ADAMTS-1 mRNA levels were determined by Northern analysis [2 µg/lane poly(A)+ RNA]. GAPDH was rehybridized as a control for RNA integrity and quantification. ADAMTS-1 mRNA levels normalized to GAPDH signals are expressed as fold induction over the vehicle-treated control value (which is set at 1).

View larger version (46K): [in this window] [in a new window]   Figure 6. Expression of ADAMTS-1 mRNA in bone cells. Two micrograms of poly(A)+ RNA from the various bone cells (primary cultures of osteoblasts derived from rat diaphyseal and metaphyseal bone, ROS 17/2.8 osteosarcoma cells, BALC stromal/osteoblastic cells, and osteoclast-like cells derived from M-CSF/RANKL/TGFß1-treated mouse bone marrow cells) were analyzed for ADAMTS-1 and GAPDH mRNA. Autoradiograms showing ADAMTS-1 expression in the osteoblastic cells are of comparable exposure (20 h), whereas the exposure time for the osteoclast-like cell was 2 weeks. Autoradiograms showing GAPDH expression were exposed for 1 h.

View larger version (60K): [in this window] [in a new window]   Figure 7. Tissue distribution of ADAMTS-1 mRNA expression. Expression of ADAMTS-1 mRNA was examined using poly(A)+ RNA from nonosseous tissues as indicated. GAPDH was rehybridized as a control for RNA integrity and quantification.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

ADAMTS-1 belongs to a subfamily of ADAM proteases referred to as ADAMTS with an additional distinct feature not present in other ADAM proteins (32, 35). Like the ADAM genes, amino-terminal regions of ADAMTS-1 consist of a proprotein, a metalloprotease, and a disintegrin-like domain. In contrast, the ADAMTS-1 protein is secreted from the cells in a catalytically active form and does not contain a transmembrane domain, but possesses three TSP type I motifs, which are the conserved motifs in both thrombospondin-1 and -2. These TSP type I motifs of ADAMTS-1 are functional for binding to heparin and anchoring it to the ECM (38, 39), a putative mechanism for targeting to specific sites of action. The disintegrin-like adhesion domain of this protein might interact with integrin-like receptors on cells to promote cell-matrix attachment or disrupt interaction between integrin receptors and the extracellular matrix. Like some of the other ADAM genes, ADAMTS-1 has a potential zinc-binding consensus motif in its metalloprotease domain. In recent studies using the proteinase trapping mechanism of ₂-macroglobulin, Kuno et al. (39) demonstrated that ADAMTS-1 is an active metalloprotease associated with the extracellular matrix; however, an in vivo substrate(s) has yet to be identified. Since the initial cloning and description of ADAMTS-1, 10 additional mammalian members and 1 Caenorhahditis elegans member of the ADAMTS family have been described (23, 33, 34, 35, 36, 65). Notable among these are 1) the recent purification and cloning of aggrecanase-1 and -2 (ADAMTS-4 and -11) (33, 34), proteases responsible for the cleavage of aggrecan; 2) procollagen-1 N-proteinase (ADAMTS-2), which cleaves collagen (35, 37, 40); and 3) Gon-1, an active metalloprotease essential for gonadal morphogenesis by remodeling basement membranes in C. elegans (23). These studies suggest that ADAMTS-1, like ADAMTS-4 and procollagen-1 N-proteinase (ADAMTS-2), may play a role in the processing of proteins that are present in the extracellular matrix.

Although the precise function of ADAMTS-1 remains unclear, predictions can be made based on its functional domains, homology to other ADAM-ADAMTS family genes, phenotype of the recently reported ADAMTS-1 null mice, and pattern of expression and regulation. The recent characterization of ADAMTS-1 null mice demonstrates that ADMATS-1 is necessary for normal growth, fertility, and organ morphology and function. The phenotype revealed a marked reduction in size, remarkable changes in kidney structures, and abnormal adrenal medullary architecture without capillary formation. In the ADAMTS-1 null females, fertilization was impaired, and phenotypic abnormalities were seen in the uterus and ovaries. Although the mechanisms responsible for the pleiotropic developmental defects in these mice are not known, it is tempting to speculate that these developmental abnormalities may be explained by changes in ECM remodeling, cell adhesion, or growth factor signaling in the absence of ADAMTS-1. The protease domain may mediate aspects of cellular degradation and dissolution of the ECM, whereas the TSP type I and disintegrin domains may control its localization and aspects of cell adhesion. Although the knockout animals provided valuable information on the role of ADAMTS-1 in development/morpho-genesis of organs, they do not suggest potential roles for ADAMTS-1 in long-term maintenance and physiology after development and in pathological states. In relation to bone, at present it would be premature to speculate on the absence of a bone phenotype in the ADAMTS-1 null mice, as it is not indicated whether the bones of these animals have been carefully studied in development and in long-term maintenance and physiology after development.

To date, evidence for ADAMTS-1 function based on its pattern of expression, and regulation after development is very limited. Initial studies suggest that expression of ADAMTS-1 can be associated with inflammation (32). ADAMTS-1 was first identified by differential display as a gene highly expressed in the transplanted colon 26 cachexigenic tumor cell line in vivo. ADAMTS-1 mRNA is induced by the inflammatory cytokine interleukin-1 and by iv administration of lipopolysaccharide, suggesting an association with certain inflammatory responses (32). Our results, demonstrating a constitutive expression of ADAMTS-1 in all tissues examined, including bone and bone cells (osteoblasts), are consistent with a more fundamental role for ADAMTS-1 in normal tissue physiology. The differences observed in levels of ADAMTS-1 expression in the various tissues may reflect the functional output needed by the tissues to exert the desired effects in the different types of cells, including osteoblasts. In bone, a low level of ADAMTS-1 was detected in the uninduced state (in vivo and in vitro), but increased levels were observed after osteotropic treatments, predicating a role in bone metabolism. The potential function in bone metabolism is emphasized by our finding that in addition to PTH, ADAMTS-1 expression is elevated by bone-active agents, PTHrP, PGE₂, and 1,25-(OH)₂D₃, in vivo and/or in vitro. Because these agents can trigger both de novo bone formation and resorption in vivo (via effects on the osteoblast/stromal cell) (3, 4, 11, 42, 43), we speculate that the up-regulated ADAMTS-1 expression may be one of the earliest events contributing to bone turnover. The in vitro findings that ADAMTS-1 is very well expressed in osteoblasts and is not detectable in osteoclasts support the idea that the osteoblast is the major cell type expressing the ADAMTS-1 gene in bone. However, because the osteoclast cultures used are enriched for differentiated osteoclasts, we cannot exclude the possibility that ADAMTS-1 is expressed in early osteoclast precursors and other hemopoietic cells. The higher levels of ADAMTS-1 mRNA expression detected in the metaphysis vs. diaphysis after PTH treatment are reproducible and consistent with the presence of more PTH-responding cells (osteoblasts) or greater responsiveness per cell in the metaphysis than in the diaphysis. This differential responsiveness in the two regions of bone is similar to results we obtained with several other PTH-responsive genes, including c-fos, c-jun, interleukin-6 (Onyia, J. E., unpublished results) and regulator of G protein signaling-2 (18). Future studies in ADAMTS-1 null mice will be required to elucidate the role of ADAMTS-1 in bone metabolism.

Although PTH activates multiple signal transduction pathways, our studies suggest that the increase in ADAMTS-1 expression by PTH treatment is mediated primarily by the cAMP/PKA pathway. This conclusion is supported by combined findings in the following in vivo and in vitro experiments showing that 1) both forskolin, a direct activator of adenylate cyclase that enhances cAMP accumulation, and (Bu)₂cAMP, a cell-permeable analog of cAMP, stimulated ADAMTS-1 mRNA expression in vitro; 2) agents that activate the cAMP/PKA pathway [PTH-(1–38), PTH-(1–31), PTHrP, and PGE₂] were able to increase ADAMTS-1 expression in vivo and/or in vitro. Unlike PTH-(1–38), which has a full spectrum of activity, PTH-(1–31) activates only cAMP/PKA, with no demonstrable effects on PKC or PLC (57, 58). This specificity of PTH-(1–31) to the PKA pathway has been substantiated in target cells of bone expressing endogenous PTH-1 receptor, but not in transfected cells overexpressing PTH-1 receptor (57, 66). In contrast, N-terminally truncated PTH analogs PTH-(3–34) and PTH-(7–34) were ineffective in increasing ADAMTS-1 expression. These analogs activate PKC, but not adenylate cyclase (54, 55, 56). Collectively, these results demonstrate that the responsiveness of ADAMTS-1 to PTH depends on the cAMP/PKA signaling pathway in bone. This is consistent with many previous studies showing that PTH regulates many osteoblastic genes, including c-fos, c-jun, interleukin-6, leukemia inhibitory factor, osteocalcin, osteopontin, 1(I) collagen, collagenase, prostaglandin G/H synthase 2 (PGHS-2), regulator of G protein signaling-2, and osteoprotegerin, mainly through the cAMP/PKA signaling pathway (18, 64, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76). As activation of the cAMP pathway is required for the effect of PTH on both bone formation and resorption (58, 77, 78, 79, 80), our finding showing a dramatic increase in ADAMTS-1 expression could be interpreted to suggest a particular significance in bone remodeling. To better understand the significance of ADAMTS-1 in PTH control of bone remodeling, future studies employing the different regimens of PTH will be needed.

Lastly, it is important to note that although our results demonstrate that PTH responses depend on cAMP, they do not exclude a role for other agents and signaling pathways in the regulation of ADAMTS-1 in bone. To fully explore the regulation of ADAMTS-1 in bone, extensive studies employing other agents that induce or block bone/osteoblast function, such as steroids and cytokines, will be needed. Recent studies by Robker et al. (81) and Espey et al. (82) indicate that ADAMTS-1 is regulated by progesterone in ovarian tissues. Our results with 1,25-(OH)₂D₃ further confirm that agents or signaling pathways other than cAMP can regulate ADAMTS-1 in the osteoblast. 1,25-(OH)₂D₃ plays a central role in regulating calcium homeostasis and bone cell biology through the vitamin D receptor, a member of the nuclear receptor transcription factor superfamily. The rapid induction of ADAMTS-1 mRNA by 1,25-(OH)₂D₃ suggests a direct and immediate effect on gene transcription and could be mediated by the vitamin D receptor via direct binding to vitamin D response elements in the ADAMTS-1 promoter or indirectly through interaction with another transcription factor. Clearly, further studies directed at the cloning and characterization of ADAMTS-1 promoter will be necessary to clarify the transcriptional mechanisms by which 1,25-(OH)₂D₃ regulates ADAMTS-1 gene expression.

In summary, we have cloned the cDNA for rat ADAMTS-1 and demonstrated that it is regulated by PTH both in vivo in bone and in vitro in osteoblasts. The finding that ADAMTS-1 is also regulated by the potent osteotropic agents, PTHrP, PGE₂, and 1,25-(OH)₂D₃, suggests that this protein may play a role in bone metabolism. We conclude that the rapid, but transient, increase in ADAMTS-1 expression may represent one of the initiating events involved in eliciting PTH actions in bone.

Received April 24, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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