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Parathyroid Hormone-Related Protein Down-Regulates Bone Sialoprotein Gene Expression in Cementoblasts: Role of the Protein Kinase A Pathway¹

Departments of Periodontics/Prevention/Geriatrics (H.O., R.T.F., L.K.M., D.W., M.J.S.), and Restorative Sciences/Cardiology/Endodontics (H.O.), School of Dentistry, Departments of Biochemistry (R.T.F.), and Pharmacology (M.J.S.), School of Medicine, The University of Michigan, Ann Arbor, Michigan 48109-1078

Address all correspondence and requests for reprints to: Hongjiao Ouyang, D.D.S., Ph.D., Departments of Periodontics/Prevention/Geriatrics and Restorative Sciences/Cardiology/Endodontics, The University of Michigan, 1011 North University Avenue, Ann Arbor, Michigan 48109-1078. E-mail: hongjiao{at}umich.edu

	Abstract

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PTH-related protein (PTHrP) acts as a paracrine and/or autocrine regulator of cell proliferation, apoptosis, and differentiation and is implicated in tooth development. The current studies employed cementoblasts to determine the role(s) and mechanisms of PTHrP in regulating cementum formation. Results demonstrated that PTHrP repressed gene expression and protein synthesis of bone sialoprotein (BSP) and abolished cementoblast-mediated biomineralization in vitro. The BSP gene inhibition required protein synthesis. The PTHrP analog (1–31) and other activators of the PKA pathway (3-isobutyl-1-methylxathine (IBMX), forskolin (FSK) and Sp-Adenosine-3', 5'-cyclic monophosphorothioate (Sp-cAMPss) also down-regulated BSP gene expression and blocked cementoblast-mediated biomineralization. In contrast, the PTHrP analog (7–34), a PTHrP antagonist, and the activators of the PKC pathway [phorbol 12-myristate 13-acetate (PMA) and phorbol 12, 13-dibutyrate (PDBu)] promoted BSP gene expression. In addition, the PKA pathway inhibitor (9-(2-tetrahydrofuryl) adenine (THFA) partially, but significantly reversed the PTHrP-mediated down-regulation of BSP gene expression. Furthermore, THFA alone significantly increased BSP messenger RNA (mRNA) expression in cementoblasts. In contrast, the inhibitor of the PKC pathway (GF109203X) did not reverse the PTHrP inhibitory effect on BSP gene expression. Furthermore, GF109203X alone dramatically reduced the BSP transcript levels. These data indicate that the cAMP/PKA pathway mediates the PTHrP-mediated down-regulation of BSP mRNA expression in cementoblasts; and furthermore, this pathway may, through an intrinsic inhibition mechanism, regulate the basal level of BSP mRNA expression. In contrast, the activation of PKC promotes BSP gene expression. These data provide new insights into the molecular mechanisms involved in PTHrP regulation of cementogenesis.

	Introduction

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PTH-RELATED PROTEIN (PTHrP) was initially isolated and cloned from tumors due to its PTH-like properties and resultant humoral hypercalcemia of malignancy (1, 2). Subsequently, it was recognized that PTHrP is associated with a variety of normal tissues/organs and acts as an important autocrine and/or paracrine factor of cell proliferation, apoptosis, and differentiation (3). Overexpression of PTHrP in chondrocytes in vivo causes a delay in their differentiation and in mineralization of cartilage (4). In contrast, but consistent with these findings, disruption of either PTHrP (5) or PTH-1 receptor (6) genes leads to an accelerated differentiation of chondrocytes. These studies demonstrated that PTHrP plays a pivotal role in regulating formation of the skeletal tissues. Although the regulatory functions of PTHrP during the development of the skeletal system have been well established, the role of PTHrP in regulating other mineralized tissues, such as those associated with teeth, is poorly understood. Tooth-related mineralized tissues differ from bone in many aspects, such as histological features and physiological function (7). Therefore, understanding the role of PTHrP in controlling formation and maintenance of these tooth-related mineralized tissues will expand the knowledge of regulatory mechanisms involved in PTHrP effects on mineralized tissue formation.

Cementum is a thin mineralized tissue covering the tooth root surface. It differs from bone in that it lacks innervation and vascularization (7). Furthermore, cementum has limited remodeling potential when compared with bone (7). The unique functions of cementum are related to its role as an important component of the periodontal attachment apparatus (the periodontium), anchoring teeth to the surrounding alveolar bone, and thereby contributing to the maintenance of structural stability and physiologic function of the dentition. Although cementum has these important functions, the molecular mechanisms involved in regulating the formation of cementum remain largely unknown. Accumulating evidence indicates that PTHrP may serve as a critical paracrine and/or autocrine regulator of cementogenesis. These findings include 1) during cementogenesis, PTHrP is produced by cells associated with the periodontium, such as Hertwig’s epithelial cells, osteoblasts lining the surrounding alveolar bone, and cementoblasts themselves (8); 2) cementoblasts possess PTH/PTHrP receptors (9, 10); and 3) PTHrP deficiency of certain stains of PTHrP knock-out mice results in the failure of tooth eruption and in fusion of cementum with surrounding alveolar bone (11). To further elucidate the function of PTHrP in cementogenesis, it is important to determine how PTHrP regulates cementoblast activities, such as their gene expression and biomineralization ability. The current studies focus on identifying the signal transduction pathways underlying PTHrP regulation of cementoblast gene expression in vitro.

It is well established that PTHrP and PTH interact with a G protein-coupled receptor, the PTH-1 receptor. The PTH-1 receptor-G protein complex activates two well-defined signal transduction pathways (12). Activation of adenylyl cyclase and phospholipase C leads to production of cAMP, inositol phosphates (IPs) and diacylglycerol (DAG), respectively. These intracellular mediators ultimately exert their functions through activation of various protein kinase complexes, notably, protein kinase A (PKA) by cAMP and protein kinase C (PKC) by DAG. PKA and PKC complexes can subsequently phosphorylate specific transcription factors and consequently affect transcription of specific genes. PKA mainly targets members of the cAMP response element binding protein families (CREBs and ATF members), whereas PKC mainly acts on members of the AP-1 family. The selective stimulation of PKA and/or PKC pathways and the resulting specific activation of transcription factors generates the diversity of actions by which different genes, i.e. extracellular matrix molecules, may be regulated in a tissue/cell type-specific manner in response to PTH/PTHrP.

The immortalized cementoblast cell lines used in the current study were obtained from tooth root surfaces of OC-TAg transgenic mice, which carry an osteocalcin promoter driving the SV-40 large T antigen gene to ensure the selective immortalization of OCN expressing cells (13, 14). We have shown that these cementoblast cell lines closely resemble their in vivo counterparts in many properties, such as gene expression and ability to mineralize. Relevant to the current study is that these cementoblast cell lines express PTH-1 receptor messenger RNA (mRNA), and exhibit a PTHrP-induced cAMP elevation and c-fos gene expression, thereby providing an in vitro model to study the effects of PTHrP on cementoblast activity (15). Moreover, PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) represses the expression of cementum-associated gene markers in these cells, such as BSP, OCN, and type I collagen (15). BSP was selected for the current studies because 1) the temporo-spatial gene expression pattern of BSP closely correlates with the initiation of cementum and bone formation (16, 17); and 2) BSP protein is thought to play a critical role in promoting biomineralization (18, 19, 20). The current investigation focused on determining the molecular mechanisms underlying PTHrP’s effect on BSP gene expression in cementoblasts in vitro.

	Materials and Methods

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Cell culture and experimental design
The cementoblast cell line, OC-CM 30 (15), was cultured in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FBS (Life Technologies, Inc.), 100 U/ml penicillin and 100 g/ml streptomycin (BioWhittaker, Inc., Walkersville, MD) in a humidified atmosphere of 5% CO₂ at 37 C. This clonal population was selected for the current studies based on its robust expression of PTH-1 receptor mRNA and responses to PTH/PTHrP stimulation, in vitro (15).

Dose-dependent effect of PTHrP
Cementoblasts were plated as described above, and upon confluence, were treated with PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (Bachem California, Inc., Torrance, CA) at concentrations ranging from 10^-13 M to 10^-6 M for 24 h. At these doses, cell viability was maintained as monitored by morphological observation and by cell proliferation assay (data not shown). To determine steady-state BSP mRNA levels, total RNA was isolated from PTHrP- and vehicle- (1 mg/ml bovine albumin/4 mM HCl) treated cultures and analyzed by Northern blotting. At the doses of PTHrP used for these studies, cell viability was maintained.

Time-course experiment
To determine the temporal effect of PTHrP on BSP mRNA expression, cementoblasts were plated at 5 x 10⁴ cells/cm² in 100-mm tissue culture plates and grown in DMEM media as described above. At confluence (designated as 0 h), cells were treated with human PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (10^-7 M) (hPTHrP, Bachem California, Inc.) and total RNA extracted at 0, 2, 6, 12, and 24 h for determination of the steady-state BSP mRNA levels by Northern blot analysis. The specific dose of PTHrP was selected due to its prominent inhibition of BSP gene expression.

Effect of cycloheximide
The effect of cycloheximide (Sigma, St. Louis, MO) (21) on PTHrP-mediated inhibition of BSP gene expression was also determined. Confluent cementoblasts were exposed to either vehicle, or 10^-7 M PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), or 10^-7 M PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) plus cycloheximide (10 µg/ml), or cycloheximide (10 µg/ml) alone for 9 h, followed by total RNA extraction and Northern blot analysis to measure steady-state BSP mRNA levels.

Effect of actinomycin D
To determine whether PTHrP affects the stability of BSP mRNA, the decay rates of BSP mRNA in vehicle- and PTHrP-pretreated groups were measured. Confluent cementoblasts were pretreated with either vehicle or PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (10^-7 M) for 6 h before transcription inhibition. Then, media were removed and cells were stimulated with vehicle or PTHrP (10^-7 M), either in the presence or in the absence of actinomycin D (Sigma, 1 µg/ml), an inhibitor of transcription (22), for designated time intervals (0, 3, 6, 9, and 12 h). Total RNA was harvested and Northern blot analysis performed to follow the decay course of BSP mRNA associated with different treatment conditions. Data are presented as logarithmic values of mean ± SD of cpm of BSP mRNA after being normalized to 18S rRNA.

Dissection of signal transduction pathway(s)
Three strategies were employed to identify the signal transduction pathway(s) involved in PTHrP regulation.

First, the effect of PTHrP analogs on BSP mRNA expression was examined. Confluent cementoblasts were treated with human PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31), or (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), or (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (Bachem California, Inc.) (23) at a concentration of 10^-7 M for 24 h and total RNA was extracted for Northern blot analysis.

In a parallel approach, the effects of various activators of PKA/PKC pathways on BSP mRNA expression in cementoblasts were analyzed. At confluence, cementoblasts were treated with various activators for 24 h, and total RNA was extracted for Northern blot analysis. Activators used included 3-isobutyl-1-methylxathine (IBMX, 10 µM) (Sigma) (24), Sp-Adenosine-3', 5'-cyclic monophosphorothioate (Sp-cAMPss) (10 µM) (BIOMOL Research Laboratories, Inc., Plymouth, PA) (25) or forskolin (FSK, 10 µM) (Sigma) (26) for activating the PKA pathway, and phorbol 12, 13-dibutyrate (PDBu 10 ng/ml) (BIOMOL Research Laboratories, Inc.) (27, 28, 29) and phorbol 12-myristate 13-acetate (PMA 10 µM) (BIOMOL Research Laboratories, Inc.) (27, 28, 29) for stimulating the PKC pathway.

Additionally, the effects of inhibitors of PKA/PKC pathways on BSP mRNA expression in the presence and absence of PTHrP were determined. Confluent cementoblasts were pretreated with an inhibitor for 12 h before treatment with either PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (10^-7 M) or vehicle in the presence of the inhibitor for an additional 12 h. Total RNA was extracted and Northern blot analysis performed to determine the level of BSP mRNA. A cell-permeable adenylyl cyclase inhibitor, 9-(2-tetrahydrofuryl) adenine (THFA) (100 µM) (Sigma) (24, 30), was used to inhibit the activity of cAMP/PKA pathway, whereas GF109203X (3 µM) (BIOMOL Research Laboratories, Inc.) (31) was used to repress/block the PKC pathway.

Northern blot analysis
Total RNA was isolated from cells and quantitated by spectrophotometry as described (32). Ten micrograms of total RNA was electrophoresed on 1.2% agarose-formaldehyde gels, blotted to Duralon-UV membranes Stratagene, La Jolla, CA), and immobilized by UV light, using Stratagene’s Stratalinker. The membranes were hybridized with a labeled cDNA probe. The probes were labeled with -³²P-dCTP (Amersham Pharmacia Biotech, Arlington Heights, IL) using a Rediprime labeling kit (Amersham Pharmacia Biotech). After hybridization and washing, blots were exposed to Kodak X-OMAT film at -70 C with intensifying screens for 24–48 h. Blots were subsequently stripped and reprobed with either GAPDH and/or 18S rRNA cDNA probe to standardize RNA loading and hybridization efficiency. Blots were quantitatively scanned by a phosphoImager, Packard A2024 InstantImager (Downers Grove, IL); and the signals were normalized by calculating the ratio of signal intensities of the assayed markers vs. either the internal control GAPDH mRNA or 18S rRNA. For most of the studies, GAPDH and/or 18S rRNA were used for normalization and to rule out possible nonspecific effects of agents used. No differences were noted for using GAPDH vs. 18S rRNA. Therefore, in some experiments, GAPDH data are presented, whereas in other situations 18S rRNA was presented. Probes used included: 1) a mouse BSP cDNA probe cloned into PCR II vector (33); 2) a 1100 bp chicken GAPDH cDNA cloned into pGEM3 (34); and 3) an 18S rRNA cDNA in PBR 322 (35).

Western immunoblot analysis
The procedures were described previously (36). Briefly, cells were plated at a density of 5 x 10⁴ cells/cm² in 60-mm culture plates and incubated in DMEM containing 10% FBS. On reaching confluence, media were removed and cells were exposed to either vehicle or 10^-7 M hPTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) for 24 h in serum-free DMEM. Conditioned media were collected, lyophilized, and subsequently dissolved in 1x SDS-PAGE loading buffer. A 10 µg aliquot of protein was fractionated by SDS-PAGE. LF-6 anti-BSP antibody (37), diluted at a ratio of 1:1000, was used to detect BSP protein. A secondary antibody (horseradish peroxidase-conjugated goat antirabbit immunoglobin G) was used at a dilution of 1: 10,000. Hybridization with antibodies was performed as previously reported (38). Immunoreactivity was determined by an enhanced chemiluminescence reaction assay (Amersham Pharmacia Biotech).

Adenylyl cyclase stimulation assay
Adenylyl cyclase stimulation assays were performed as described previously (39). Briefly, the OC-CM 30 cells were plated in DMEM media in 24-well tissue culture plates in triplicate, at 5 x 10⁴ cells/cm². At confluence, cells were stimulated with either vehicle, or PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31), or (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) or (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (Bachem California, Inc.) for 15 min at 37 C in calcium- and magnesium-free HBSS (Life Technologies, Inc.) containing 0.1% BSA and 1 mM IBMX (Sigma), a phosphodiesterase inhibitor. Intracellular cAMP was extracted and measured, using a cAMP binding protein assay (39). The cAMP levels were calculated by a log-logit method (Graph Pad Prism, normalized to cell number, and expressed as pmol/10⁵ cells.

In vitro mineralization assay
The in vitro mineralization assay was performed as reported previously (40). Cells were plated at 5 x 10⁴ cells/cm² in 24-well plates with DMEM containing 10% FBS and supplemented with 50 µg/ml ascorbic acid. When cell reached confluence, designated day 0, medium was removed and cells were incubated in mineralization media (ascorbic acid-free -MEM) (Life Technologies, Inc.) containing 10% FBS and supplemented with 50 µg/ml ascorbic acid) for 7 days, with inorganic phosphate (3 mM) added on day 6, to induce mineral nodule formation. Samples were processed on day 7 and von Kossa staining used to detect mineral nodule formation. To determine the effect of various agents on cementoblast-mediated mineralization, either vehicle or test agent was added to cultures on day 6. On day 7, samples were assayed for mineral deposition.

Statistical analysis
All experiments were performed a minimum of two times with comparable findings obtained. For cAMP stimulation assays, a Student’s t test was conducted to analyze the results (Instat 2.0, GraphPad Software, Inc., San Diego, CA). For mRNA decay rate measurements, the data were analyzed by either ANOVA followed by a Tukey-Kramer multiple comparison test and a Student’s t test as needed. For Northern blot analyses, representative autoradiographs are shown with a graphical analysis demonstrating statistical evaluation of multiple assays.

	Results

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Dose-dependent inhibitory effect of PTHrP on BSP gene expression
Northern blot analysis demonstrated that PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) exhibited a dose-dependent inhibition of BSP mRNA expression in cementoblasts; a significant inhibitory effect was first detected at 10^-9 M (P < 0.001) with a greater magnitude of inhibition associated with higher concentrations (10^-8, 10^-7, and 10^-6 M) of PTHrP (Fig. 1, A and B). Given that BSP protein has been implicated in initiating biomineralization (18, 20, 41), the ability of PTHrP at these same concentrations to regulate cementoblast-mediated biomineralization also was determined. As revealed by a von Kossa assay, PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) abolished cementoblast-induced formation of mineralized nodules in a dose-dependent fashion with the first detectable inhibition of mineralization noted at 10^-9 M (Fig. 1C). Furthermore, the dose-dependent profile of PTHrP effects on both BSP gene expression and biomineralization were consistent with the dose-dependent effect of PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) on elevating cAMP synthesis in cementoblasts (Fig. 1D and Ref. 15).

View larger version (39K): [in this window] [in a new window]   Figure 1. Dose-dependent effects of PTHrP on cementoblast activities. A, A representative Northern blot autoradiograph of BSP mRNA and 18S rRNA levels associated with 24 h PTHrP treatment at concentrations ranging from 10-13 M to 10-6 M. BSP mRNA of murine cementoblasts runs slightly above 18S rRNA with sizes approximating 2.0 kb. B, Graphical analysis of cpm (mean values expressed as BSP mRNA normalized to 18S rRNA ± SD). PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) exhibited a dose-dependent inhibitory effect on BSP gene expression by cementoblasts; and compared with vehicle (V), PTHrP at doses from 10-9 M to 10-6 M, significantly down-regulated BSP mRNA levels (* P < 0.001 vs. vehicle). The experiments were performed twice in duplicate with similar results noted. C, Dose-dependent effect of PTHrP on cementoblast-mediated biomineralization, in vitro. When cultured for 7 days in the presence of ascorbic acid (50 µg/ml) and incubated for the last 24 h with 3.0 mM inorganic phosphate (Pi), vehicle-treated cementoblasts promoted mineral nodule formation. However, when present for the last 24 h, PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) blocked mineralization in a dose-dependent manner with a dose as low as 10-9 M having an inhibitory effect. This experiment was performed twice in duplicate with comparable results obtained. D, PTHrP dose-dependent cAMP response: cementoblasts were exposed to PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) at increasing concentrations ranging from 10-11 M to 10-6 M for 15 min. cAMP levels were measured by a cAMP stimulation assay and the values were expressed as the mean ± SEM pmol cAMP/105 cells. PTHrP significantly stimulated cAMP elevation in cementoblasts in a dose-dependent manner (* P < 0.001 significance vs. basal level of cAMP). Experiments were performed three times with similar results noted.

View larger version (61K): [in this window] [in a new window]   Figure 2. Time-dependent effect of PTHrP on BSP mRNA expression by cementoblasts. Cementoblasts were plated at an initial density of 5 x 104 cells/cm2. At confluence, designated as 0 h, either vehicle (V) or PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (10-7 M) (P) was added followed by isolation of total RNA at designated time points (0, 2, 6, 12, 24 h). A, A representative Northern blot autoradiograph of BSP mRNA and 18S rRNA levels. 18S rRNA hybridization is shown to demonstrate relative loading and blotting efficiency. B, Graphical analysis of BSP mRNA levels. Plot of cpm (mean values expressed as BSP mRNA normalized to 18S rRNA ± SD). Compared with vehicle, PTHrP significantly down regulated BSP mRNA levels at 6, 12, and 24 h (* P < 0.001). The experiments were performed four times with similar results observed.

View larger version (44K): [in this window] [in a new window]   Figure 3. Effect of PTHrP on BSP protein level in cementoblasts. Confluent cementoblasts were treated with either vehicle or 10-7 M PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) for 24 h in serum-free DMEM media. Media were collected and BSP protein levels were measured by Western blot analysis as described in Materials and Methods section. MC3T3E1 cell conditioned medium served as a positive control. The size of the BSP protein is approximately 70 kDa. Experiments were done twice with one performed in duplicate.

View larger version (44K): [in this window] [in a new window]   Figure 4. Effect of cycloheximide on PTHrP-mediated BSP mRNA down-regulation. Confluent cementoblasts were treated for 9 h with either vehicle (V), or PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (10-7 M), or PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (10-7 M) plus cycloheximide (cyclo. 10 µg/ml), or cycloheximide (10 µg/ml) alone, followed by total RNA extraction and Northern blot analysis. A, A representative Northern blot autoradiograph of BSP mRNA and 18S rRNA levels. B, Graphic analysis of normalized BSP mRNA levels. Values are expressed as mean values of the normalized BSP mRNA vs. 18S rRNA ± SD. The BSP mRNA levels were significantly reduced in PTHrP- and PTHrP plus cycloheximide-treated groups (* P < 0.001 for PTHrP vs. vehicle control and ** P < 0.05 for PTHrP plus cycloheximide vs. vehicle control). Nevertheless, cycloheximide significantly reversed the PTHrP-mediated repression of BSP gene expression (*** P < 0.01 for PTHrP plus cycloheximide vs. PTHrP). Experiments were performed in duplicate on two separate occasions with similar results observed.

View larger version (23K): [in this window] [in a new window]   Figure 5. Effect of actinomycin D on PTHrP-mediated inhibition of BSP gene expression. BSP mRNA stability was analyzed in the presence and absence of PTHrP. Cementoblasts were pretreated with either vehicle or PTHrP (10-7 M) for 6 h. Media were then changed (designated as 0 h) and the cementoblasts were treated with either vehicle or PTHrP (10-7 M) in either the presence or the absence of actinomycin D for designated time intervals. Total RNA was extracted at 0, 3, 6, 9, and 12 h and Northern analyses performed to detect BSP gene expression. y-axis, Log of mean values of BSP mRNA vs. 18S rRNA ± SD. Line V, The basal level BSP gene expression in the absence of transcription inhibitor actinomycin D; line V+Act. D, the decay curve of BSP mRNA in vehicle-treated group with a half-life (t1/2) of 6.5 h in the presence of actinomycin D; line PTHrP, PTHrP-mediated down-regulation of BSP gene expression in the absence of actinomycin D; line PTHrP+Act. D, the decay curve of BSP mRNA in PTHrP-treated group with a half-life (t1/2) of 7.2 h in the presence of actinomycin D. Experiments were performed in duplicate on two separate occasions with similar results observed. Statistical analyses revealed no significant difference in BSP mRNA decay rate between vehicle- and PTHrP-treated group (P > 0.5).

View larger version (29K): [in this window] [in a new window]   Figure 6. Effects of PTHrP analogs on cementoblast activities. A and B, Role of PTHrP analogs in regulating BSP gene expression. Confluent cementoblasts were treated with either vehicle (V) or different PTHrP analogs at 10-7 M for 24 h and total RNA was extracted to measure the steady-state BSP mRNA levels using Northern blot analysis. A, A representative Northern blot analysis autoradiograph of BSP mRNA and GAPDH mRNA levels. B, Plot of cpm (mean values expressed as BSP mRNA vs. GAPDH mRNA ± SD). PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ) and (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) significantly reduced BSP mRNA level (* P < 0.001 vs. vehicle), whereas PTHrP (7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) promoted BSP mRNA expression (** P < 0.01 vs. vehicle). C, Effect of PTHrP analogs on cAMP synthesis: Cementoblasts were stimulated with either 10-7 M of either PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ), or (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ), or (7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) for 15 min. cAMP levels were measured as described in the Materials and Methods section. PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ) and (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) significantly stimulated cAMP levels in cementoblasts (* P < 0.001 vs. vehicle), whereas PTHrP (7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) did not increase cAMP levels. Experiments were performed three times with similar results noted. D, Effect of PTHrP analogs on cementoblast-mediated biomineralization, in vitro. When cultured for 7 days in the presence of ascorbic acid and incubated for the last 24 h with inorganic phosphate (Pi; 3 mM), cementoblasts promoted mineral nodule formation. PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ) and (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) at 10-7 M, when present for the last day, blocked mineralization. In contrast, PTHrP (7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) at 10-7 M did not prevent mineral nodule formation under similar treatment conditions. The experiment was performed three times. C, negative control without A. A, Induction; V, vehicle plus A. A; 1–31, PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ); 1–34, PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ); 7–34, PTHrP (7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ).

View larger version (45K): [in this window] [in a new window]   Figure 7. Effect of PKA/PKC pathway activators on cementoblast activities. A and B, Roles of PKA/PKC pathway activators in regulating steady-state BSP mRNA levels. A, Representative autoradiographs of Northern blot analysis: GAPDH was used to standardize RNA loading. B, A plot of mean values of relative expression of BSP mRNA vs. GAPDH mRNA ± SD. The activators of the PKA pathway, IBMX (10 mM), Sp-cAMPss (10 µM) and forskolin (10 µM) significantly decreased BSP mRNA expression. In contrast, the activators of the PKC pathway, PDBu (100 ng/ml) and PMA (100 nM), promoted BSP gene expression by approximately 1.8- and 1.9-fold, respectively (** P < 0.01 vs. vehicle). IBMX, 3-isobutyl-1-methylxanthine; PDBu, phorbol 12, 13-dibutyrate; PMA, phorbol 12-myristate 13-acetate. C, Effects of activators on cementoblast-mediated biomineralization, in vitro. Twenty-four-hour treatment with forskolin (10-5 M) abolished formation of mineral nodules in cementoblast cultures. In contrast, PDBu (100 ng/ml) did not inhibit mineralization under the similar treatment condition. V, Vehicle. Experiments were performed three times with similar results observed.

View larger version (30K): [in this window] [in a new window]   Figure 8. Effect of the PKA/PKC pathway inhibitors on PTHrP-mediated repression of BSP mRNA expression. Cementoblasts were pretreated with THFA, an adenylate cyclase inhibitor, or GF109203X, a PKC pathway inhibitor, for 12 h in before the stimulation with PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (10-7 M) plus the inhibitors for an additional 12 h. A and C, Representative Northern blot autoradiographs of BSP and 18S rRNA of the experiments using inhibitors of PKA and PKC pathways, respectively. B and D, Graphic analysis of cpm (mean values expressed as BSP mRNA normalized to 18S rRNA level ± SD). PTHrP significantly inhibited BSP mRNA gene expression (* P < 0.001 vs. vehicle). THFA, partially but significantly, rescued PTHrP-induced down-regulation of BSP mRNA level (** P < 0.01 for PTHrP plus THFA vs. PTHrP only). Twenty-four-hour treatment with THFA alone significantly stimulated BSP approximately 1.7-fold (*** P < 0.01 vs. vehicle). In contrast, GF109203X failed to prevent PTHrP-induced BSP down-regulation. Furthermore, the inhibitor alone significantly reduced BSP mRNA level (* P < 0.001 vs. vehicle).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The time course experiment revealed that a minimum of 6 h exposure of cells to PTHrP was required to observe an inhibitory effect on BSP mRNA expression. This pattern indicates that inhibition of BSP mRNA expression in cementoblasts is a secondary response requiring protein synthesis, as confirmed by the experiments using cycloheximide.

Effects of PTH/PTHrP on gene markers associated with mineralized tissues have been linked to transcriptional (24, 26, 44), and/or posttranscriptional (45) events. Data from the current studies suggest that the inhibitory effect of PTHrP on BSP mRNA expression in cementoblasts is at the transcription level. The mRNA decay curves revealed that when the transcription inhibitor actinomycin D was included, BSP mRNA in the vehicle-treated group decayed with a half-life of 6.5 h approximately, and the transcripts in PTHrP-pretreated cells decayed with a half-life of 7.2 h. In three different experiments, a slightly upward trend was noted for the BSP mRNA decay curve of PTHrP-treated group, compared with that of vehicle-treated group; however, no statistical difference in BSP mRNA decay rate was detected between the groups, suggesting that PTHrP did not dramatically affect the stability of BSP mRNA. Thus, the PTHrP-mediated down-regulation of BSP mRNA must result from transcriptional inhibition.

PTHrP stimulates both cAMP/PKA and PKC pathways (12, 46). The data presented in the current study suggested that the inhibitory effect of PTHrP on BSP mRNA expression in cementoblasts was mediated through the cAMP/PKA pathway, but not through the PKC pathway. First, the dose-dependent profile of PTHrP-mediated inhibition of BSP gene expression and biomineralization resembles the dose-dependent effect of PTHrP on promotion of cAMP levels in cementoblasts. Second, only the PTHrP analog capable of stimulating a cAMP response [PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31)] (47) inhibited BSP gene expression and biomineralization. In contrast, PTHrP (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), an antagonist for PTHrP (23, 48) failed to inhibit gene expression and biomineralization in cementoblast cultures. Third, other activators of the cAMP/PKA pathway, IBMX (a cAMP phosphodiesterase inhibitor) (24), forskolin (an adenylyl cyclase stimulator) (26), and Sp-cAMPs (a potent and specific activator for cAMP-dependent protein kinase A with excellent cell permeability and resistance to cyclic nucleotide phosphodiesterase) (25), mimicked the inhibitory effects of PTHrP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) on BSP gene expression and biomineralization, whereas two PKC activators, PMA and PDBU, which stimulate PKC function by binding to PKC and increasing its affinity to calcium (27, 28, 29), failed to do so. Finally, THFA, an adenylate cyclase inhibitor (24), partially but significantly blocked the effect of PTHrP on BSP gene expression, and THFA alone stimulated basal level BSP mRNA expression. In contrast, the PKC inhibitor, GF109203X (31), did not reverse PTHrP-mediated down-regulation of BSP mRNA expression, but, in fact, dramatically lowered the basal BSP mRNA level. Collectively, these results suggest that the cAMP/PKA pathway, but not the PKC pathway, mediates the inhibitory effect of PTHrP on BSP mRNA expression. Furthermore, it appears that this pathway may also play an inhibitory role in regulating the basal BSP gene expression. In support to this conclusion, Wang et al. reported that treatment of osteoblasts with forskolin blocked apatite deposition and decreased BSP mRNA expression (49).

In contrast to the inhibitory effects of the cAMP/PKA, the activation of the PKC pathway resulted in an increase of BSP gene expression in cementoblasts. PTHrP (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) is widely accepted as an effective antagonist for PTH/PTHrP functions (23, 30). Thus, the up-regulation of BSP mRNA expression might result form the PTHrP (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)-mediated inhibition of endogenous PTHrP. However, endogenous PTHrP mRNA and protein were not detected in the cementoblasts used for these studies (data not shown). Moreover, PTH (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) did not further decrease the basal cAMP level in cementoblasts (Fig. 6C). Therefore, this is not a likely explanation. Instead, stimulation of the PKC pathway may directly target a PKC-responsive element in the murine cementoblast BSP gene. This hypothesis is supported by the finding that the avian BSP gene contains a PKC-responsive cis-element in the promoter region (26) and the strong homology between the murine and avian promoter. Although this PKC-responsive element did not mediate the up-regulation of avian BSP mRNA expression associated with short-term exposure to PTH (26), it provides a potential mechanism by which cells regulate BSP gene expre