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Endocrinology Vol. 140, No. 7 3154-3162
Copyright © 1999 by The Endocrine Society

ARTICLES

3',5'-Cyclic Adenosine Monophosphate Activation in Osteoblastic Cells: Effects on Parathyroid Hormone-1 Receptors and Osteoblastic Differentiation in Vitro1

Amy J. Koh, Christopher A. Beecher, Thomas J. Rosol and Laurie K. McCauley

The University of Michigan, Department of Periodontics/Prevention/Geriatrics (A.J.K., C.A.B., L.K.M.), Ann Arbor, Michigan 48109; and The Ohio State University (T.J.R.), Department of Veterinary Biosciences, Columbus, Ohio 43210

Address all correspondence and requests for reprints to: Laurie K. McCauley, Department of Periodontics/Prevention/Geriatrics, University of Michigan, 1011 North University Avenue, Ann Arbor, Michigan 48109-1078. E-mail: mccauley{at}umich.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH has anabolic and catabolic effects in bone through activation of the PTH-1 (PTH/PTHrP) receptor and the cAMP/protein kinase A pathway. The effects of agents that regulate cAMP in nontransformed osteoblasts in relation to cell differentiation have not been described. The purpose of this study was to determine the effects of PTH fragments with differing cAMP-stimulating activity, and nonPTH cAMP regulators on PTH-1 receptor expression and activity, and osteoblast differentiation in vitro using MC3T3-E1 and primary rat calvarial cells. PTH (1–34), but not PTH (53–84), (7–34), or PTHrP (107–139) treatment (24 h) resulted in down-regulation of steady-state messenger RNA for the PTH-1 receptor. Forskolin (a stimulator of cAMP accumulation) also down regulated the PTH-1 receptor, whereas 9-(tetrahydro-2-furyl) adenine (THFA) (an inhibitor of adenylyl cyclase) had no effect. Similarly, PTH (1–34) treatment for 48 h abolished PTHrP binding to cell surface receptors; however, neither the PTH analogs nor the cAMP regulating agents altered PTH binding or numbers of binding sites on osteoblastic cells. Basal levels of cAMP were reduced in cultured cells treated for 6 days with PTH (7–34) or THFA compared with controls. In contrast, PTH-stimulated cAMP levels were significantly increased in cultures treated with PTH (7–34) and THFA for 6 days during osteoblast differentiation and were decreased in cultures treated with PTH (1–34) and forskolin compared with controls. To evaluate effects of the cAMP pathway on osteoblast differentiation, cultures were treated continuously with PTH analogs and cAMP regulators during an 18-day differentiation regime, total RNA was isolated at multiple time points, and Northern blot analysis for osteocalcin (OCN) was performed. THFA and PTH (7–34)-treated cultures had increased OCN expression; whereas, PTH (1–34) and forskolin reduced OCN expression. Interestingly, PTH (7–34) and THFA-treated cultures had increased mineralized nodule formation, in contrast to PTH (1–34) and forskolin treatment, which reduced nodule formation. Similarly, calcium accumulation in cultures was significantly increased in the PTH (7–34) and THFA-treated cultures and reduced in the PTH (1–34) and forskolin-treated cultures. These data demonstrate that agents that increase cAMP down regulate PTH-1 receptor messenger RNA and inhibit osteoblast differentiation in vitro. Agents that reduce or block adenylyl cyclase or cAMP activity do not alter PTH-1 receptor expression or binding, but have striking effects on promoting osteoblast differentiation. We conclude that many effects of PTH on osteoblasts may be mimicked or antagonized by agents that alter cAMP activity and bypass the PTH-1 receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH IS A SYSTEMIC calcium-regulating hormone with catabolic and anabolic actions in bone. Although the anabolic effects of PTH on bone have been known for decades, recent interest has focused on its utilization as a therapeutic agent to restore bone mass by means of systemic administration to patients with osteoporosis (1, 2) and local gene therapy in osteotomy sites (3). Studies have relied on empirical findings because the mechanisms of the anabolic effects are still unknown. Animal studies have provided some insight and indicate that the anabolic effects of PTH are optimal with intermittent administration, and are dependent on N-terminal PTH, and cAMP stimulation. Interestingly, prostaglandin E2 stimulates cAMP via a G protein-coupled receptor and also has anabolic effects in bone when administered intermittently.

The PTH-1 (PTH/PTHrP) receptor is a seven transmembrane G protein-coupled receptor that signals through the protein kinase A and C pathways. Although other receptors have been described (4, 5, 6), the PTH-1 receptor is responsible for the principal effects of PTH in bone and cartilage as evidenced by phenotypic characteristics of human and animal models of altered receptor function (7, 8, 9). Studies in osteosarcoma cells report that PTH down-regulates the PTH-1 receptor at the messenger RNA (mRNA) and posttranslational levels, but little is known of regulation in nontransformed osteoblasts during differentiation. The PTH-1 receptor is expressed at peak levels as osteoblastic cells are in the stage of active matrix mineralization in vitro (10). The role of agonist or cAMP-dependent regulation of the PTH-1 receptor in osteoblastic cells in relation to the anabolic actions of PTH in vivo is unknown. After PTH-1 receptor down-regulation, there is a period of nonresponsiveness before a return of receptor availability. Understanding this phenomenon may provide information regarding the necessity of intermittent dosing for the anabolic effects of PTH.

One of the difficulties in discovering the mechanisms of anabolic effects of PTH on bone is the contradiction between its effects in vivo and in vitro. PTH stimulates bone formation in both humans and animal models but inhibits bone formation in models of in vitro bone formation (11, 12, 13). Several other effects of PTH are disparate when comparing in vitro and in vivo studies. In vivo, PTH upregulates alkaline phosphatase whereas it inhibits alkaline phosphatase in vitro. The PTH receptor is down regulated by its ligand in vitro, whereas, contradictory results have been reported in vivo. Studies in the kidney indicate that with renal failure and secondary hyperparathyroidism the PTH-1 receptor is down-regulated (14, 15). In an animal model of hypercalcemia of malignancy with increased circulating levels of PTHrP, the mRNA for the PTH receptor was unchanged in the kidney and upregulated in the calvaria (16). In vivo, PTH up-regulates osteopontin, whereas in vitro it inhibits osteopontin (17). Despite contradictory findings, there are effects of PTH that are similar whether using an in vitro or in vivo model. Both in vitro and in vivo, PTH stimulates cAMP and c-fos (17, 18), which are early events in the PTH signaling cascade.

To understand the late events in PTH action, it is important to evaluate the relationship between cAMP activation and downstream events such as PTH-1 receptor expression and activity, and osteoblast differentiation using well-characterized in vitro model systems. In vitro models of osteoblast differentiation have been widely used to characterize the osteoblast phenotype. These models have been employed to evaluate effects of PTH on bone formation, the levels of PTHrP production during differentiation, and the temporal sequence of the PTH-1 receptor expression (10, 19, 20). Still, the subcellular actions of PTH in this model system have not been elucidated. The purpose of this study was to determine the effects of PTH fragments with differing cAMP stimulation activity and nonPTH cAMP regulators on the PTH-1 receptor and osteoblast differentiation in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and in vitro differentiation
MC3T3-E1 cells were obtained from Dr. M. Kumegawa (Meikai University, Sakado, Japan) through Dr. Renny Franceschi (University of Michigan) and maintained as previously described (21). Briefly, stock cultures were grown in {alpha}-modified Eagle’s medium ({alpha}-MEM) (Life Technologies, Inc., Grand Island, NY) and 10% FBS containing 100 U/ml of penicillin and streptomycin. Cells were passaged every 4–5 days and were not used beyond passage 15.

Primary rat calvarial cells were isolated as previously described (10). Briefly, calvaria of fetal rats at 21 days gestation were dissected, isolated from periosteum, and subjected to sequential digestions in collagenase A (2 mg/ml Roche Molecular Biomedicals, Indianapolis, IN) and 0.25% trypsin (Life Technologies, Inc.) for 20, 40, and 90 min. Cells from the third digest were washed, counted, and plated in {alpha}-MEM with 10% FBS containing 100 U/ml of penicillin and streptomycin. Primary cultures were used without passage.

MC3T3-E1 or primary calvarial cells were plated at 50,000/cm2 and induced to differentiate and form mineralized matrix with the addition of ascorbic acid (50 µg/ml) and ß-glycerophosphate (10 mM). After 5–7 days, in culture, the cells displayed maximal PTH-1 receptor expression as previously described (10) and were used for single time point experiments. For experiments where effects of treatments on osteoblast differentiation were evaluated, cells were differentiated as above with the addition of treatment or vehicle control agents during media replacement (every 2 days) for the indicated time course.

Northern blot analysis
Total RNA was isolated from MC3T3-E1 or primary osteoblastic cells and Northern blot analysis performed as described (21). Briefly, total RNA was isolated from 60-mm dishes using guanidinium isothiocyanate (22) and quantitated by spectrophotometry. Total RNA (10 µg) was electrophoresed on 1.2% agarose-formaldehyde gels. The RNA was transferred to nylon membranes (Duralon U.V.; Stratagene, La Jolla, CA) and cross-linked with UV light (Stratalinker, Stratagene). The nylon membranes were hybridized with a complementary DNA (cDNA) probe for osteocalcin (OCN) (23) labeled with {alpha}-[32P] deoxycytidine triphosphate (Amersham Pharmacia Biotech, Arlington Heights, IL) using random primer labeling (Amersham Pharmacia Biotech). After hybridization and washing, radioactivity (cpm) was measured using an Instant Imager (Packard Instrument Co., San Diego, CA) and blots were exposed to Kodak X-OMAT or Biomax film (Eastman Kodak Co., Rochester, NY) at -70 C for 24 to 72 h. Blots were stripped and reprobed with a cDNA probe for 18S ribosomal RNA (rRNA) to control for RNA loading (10).

von Kossa staining and calcium content of cultures
At the indicated time points, plates from each treatment group were fixed with 95% ethanol and stained with AgNO3 by the von Kossa method to detect phosphate deposits in bone nodules as previously described (21).

Calcium accumulation in cell layers was determined by extraction with 15% trichloroacetic acid followed by colorimetric determination with cresolphthalein complexone (Sigma, St. Louis, MO) (21).

Adenylyl cyclase stimulation assay
The adenylyl cyclase stimulation assay and cAMP binding assay were performed as previously described (24). Briefly, following the indicated treatment regimes, cell cultures were stimulated for 10 min with hPTH (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 vehicle control at 37 C in calcium- and magnesium-free HBSS containing 0.1% BSA and 1 mM isobutylmethylxanthine (IBMX). The medium was aspirated and 250 µl ice-cold 5% perchloric acid was added to each well. The plates were incubated at -20 C overnight to extract the cAMP. After thawing, the extract was transferred to tubes, the pH was adjusted to 7.5 with 4N KOH, and the extract was centrifuged to remove the precipitate. The neutralized extract was then assayed for cAMP content using a cAMP binding protein assay. Parallel wells were analyzed for DNA content by fluorometric analysis as previously described (10). In selected assays, the potential for residual ligand to alter PTH stimulation was evaluated by treating cells immediately before stimulation with an acid rinse treatment (HBSS with HCl and 0.1% BSA, pH 4.5 vs. vehicle control). Because there was no difference between the acid or vehicle rinse vs. nonrinsed cells (data not shown), it was concluded that residual occupation of receptors by ligands used for treatments during differentiation did not alter the ability of PTH to stimulate cell surface receptors.

The cAMP binding assay was performed with triplicate samples of standards or unknowns incubated with [3H]-cAMP (10,000 cpm) (ICN Radiochemicals, Irvine, CA) and cAMP binding protein sufficient to bind 40–60% of the radioactivity for 90 min on ice. Dextran-coated charcoal was added to each tube and centrifuged to remove the unbound from bound cAMP-binding protein-[3H]-cAMP complexes. The radioactivity in the supernatants was counted with a liquid scintillation spectrophotometer and cAMP concentration calculated by the log-logit method using Graph Pad Prism.

Receptor binding assays
PTH-1 receptor binding assays were performed as described (10). Briefly, cells were plated in 24-well plates and binding assays were performed in triplicate with the addition of 20,000 cpm of high performance liquid chromotography-purified monoiodinated-125I-[Tyr36]-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, 35, 36) amide (24), in addition to varying concentrations of nonradioactive 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) amide (Bachem, Inc., Torrance, CA). The cells were incubated for 90 min at 4C with gentle shaking. The unbound peptides were washed twice from the cell monolayer with HBSS, the cells were lysed with 0.5 M NaOH for 30 min, and the suspension counted in a scintillation counter. Three wells from each plate were used to determine DNA levels using fluorometric analysis.

Statistical analysis
All experiments were performed a minimum of three times with similar results. Results of experiments were analyzed using ANOVA followed by a Tukey-Kramer multiple comparison test or Student’s t test, with the Instat 2.1 biostatistics program (GraphPad Software, Inc., San Diego, CA). For Northern analyses, a representative assay is shown along with a plot of data from multiple assays including their statistical evaluation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of peptide fragments and cAMP regulators on the PTH-1 receptor mRNA, binding and biologic activity
We have previously shown that the PTH-1 receptor expression follows a similar course of expression for both MC3T3-E1 cells and primary rat calvarial cells during differentiation with peak receptor expression during the phase of active matrix-producing (10). Consequently, for current studies of PTH-1 receptor activity, cells were maintained in culture for 5–7 days to maximize receptor expression. All experiments for the current study were performed with both primary rat calvarial cells and MC3T3-E1 cells. In our laboratory, these cells follow a similar pattern of differentiation, with subtle differences in the magnitude of mineralization, as the primary cells tend to have slightly higher numbers of nodules and calcium accumulation than the MC3T3-E1 cells. Despite small differences in differentiation magnitude, the effects of the treatment agents vs. control for the experiments reported here were the same for both cell lines.

Fragments of PTH and PTHrP have varied effects in bone but have not been evaluated for their direct effects on PTH-1 receptor expression. One goal of the current study was to evaluate these fragments in relation to their effects on receptor regulation. PTH (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) decreased steady state PTH-1 receptor mRNA after 24 h of treatment whereas 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), PTH (53–84), and PTHrP (107–139) did not alter PTH-1 receptor mRNA (Fig. 1Go). The down-regulation of PTH-1 receptor mRNA by PTH (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) resulted in diminished PTH-1 receptor binding. Figure 2Go demonstrates that pretreatment with PTH (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 the ability of PTHrP to bind to the PTH-1 receptor on MC3T3-E1 preosteoblasts. Cultures treated with vehicle only (control), 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), PTH (53–84) or PTHrP (107–139) all displayed similar competition binding curves.



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  		Figure 1. Effects of PTH fragments on PTH-1 receptor mRNA. A, Representative autoradiograph of Northern blot analysis of PTH-1 receptor (PTH-1R) mRNA and 18S rRNA isolated from MC3T3-E1 cells induced to differentiate for 5 days and treated for 24 h with the PTH/PTHrP analogs [Control (C), PTH (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 ), 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 ), PTH (53–84), and PTHrP (107–139)] at 0.1 µM. B, Plot of mean values of relative expression of PTH-1 receptor mRNA vs. 18S rRNA from three separate experiments. PTH (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 the steady state expression of the PTH-1 receptor. Data are expressed as mean ± SEM.

 


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  		Figure 2. Effects of PTH fragments on PTH-1 receptor binding. Competition binding curves for MC3T3-E1 cells induced to differentiate for 5 days followed by 48-h treatment with; vehicle (control), PTH (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 ), 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 ), PTH (53–84), or PTHrP (107–139) at 0.1 µM each. Competition binding curves were performed using 20,000 cpm 125I-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 35 36 ) and the indicated concentrations of noniodinated 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 ). Data are expressed as mean ± SEM for triplicate samples from one of three experiments with similar results. Derived values for EC50 and number of binding sites are inset. n.d., Not determined.

 
PTH (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) was effective at homologous down-regulation of the PTH-1 receptor. Because PTH stimulates cAMP activity, other agents that mediate cAMP levels in osteoblasts were evaluated for their ability to alter PTH-1 receptor activity. We previously reported the effects of these agents on c-fos proto-oncogene expression in osteoblastic cells and used these studies to select time points and doses for evaluating their effects on the PTH-1 receptor and osteoblastic differentiation (18). Interestingly, the stimulator of cAMP accumulation, forskolin, down-regulated the PTH-1 receptor after 24 h as did PTH (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). In contrast, 9-(tetrahydro-2-furyl) adenine (THFA) did not alter steady state PTH-1 receptor mRNA (Fig. 3Go). THFA inhibits adenylyl cyclase and also inhibits the PTH-stimulated increase in c-fos mRNA expression and PTH-stimulated bone resorption (18, 25). The competition binding curves for PTH after treatment with cAMP regulating agents indicated that they did not alter binding significantly from controls (Fig. 4Go). There was a trend toward higher binding affinity and lower numbers of binding sites for PTH with THFA and forskolin-treatment compared with controls; however, data from three independent assays did not reach statistical significance. When MC3T3-E1 cells were induced to differentiate for 6 days with agents that alter cAMP (medium and treatments were changed every 48 h) basal and stimulated cAMP levels were significantly altered (Fig. 5Go). Basal levels of cAMP were significantly reduced compared with controls for 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) and THFA-treated cultures, whereas forskolin significantly increased basal cAMP levels compared with controls (Fig. 5aGo). The reduction in cAMP levels followed a dose response with maximal reduction for primary rat calvarial cells with 1 µM 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) incubation (Table 1Go). After PTH stimulation for 10 min, cAMP levels increased only slightly in the PTH (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) and forskolin-treated groups, and were significantly reduced compared with controls (Fig. 5bGo). In contrast, the 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) and THFA-pretreated groups had a significant increase in PTH-stimulation of cAMP compared with controls. Treatment with PTH (53–84) and PTHrP (107–139) had no effect on basal or PTH-stimulated cAMP levels (data not shown).



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  		Figure 3. Effects of cAMP regulators on PTH-1 receptor mRNA. A, Representative autoradiograph of Northern blot analysis of the PTH-1 receptor (PTH-1R) mRNA and 18S rRNA isolated from MC3T3-E1 cells induced to differentiate for 5 days then treated for 24 h with PTH (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 ) (0.1 µM), forskolin (Forsk) (10 µM), THFA (100 µM) or vehicle control. B, Plot of mean values of relative expression of PTH-1 receptor mRNA vs. 18S rRNA from two separate experiments. Data are expressed as mean ± SEM. PTH (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 ) and forskolin significantly reduced the steady-state expression of the PTH-1 receptor vs. control.

 


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  		Figure 4. Effects of cAMP regulators on PTH-1 receptor binding. Competition binding curves for MC3T3-E1 cells induced to differentiate for 5 days followed by a 48 h treatment with; vehicle (control), PTH (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 ) (0.1 µM), THFA (100 µM), or forskolin (10 µM). Competition binding curves were performed using 20,000 cpm 125I-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 35 36 ) and the indicated concentrations of noniodinated 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 ). Data are expressed as mean ± SEM for triplicate samples from one of three experiments with similar results. Derived values for EC50 and number of binding sites are inset. n.d., Not determined.

 


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  		Figure 5. Effects of cAMP regulators on PTH-stimulated cAMP. MC3T3-E1 preosteoblastic cells were induced to differentiate with ascorbic acid and ß-glycerophosphate for 6 days with the following treatments; vehicle (control), PTH (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 ) (0.1 µM), 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 ) (0.1 µM), THFA (100 µM), or forskolin (Forsk) (10 µM). Treatments and media were changed every 2 days. Cultures then were stimulated for 10 min with vehicle (control) to determine basal cAMP levels, or PTH (0.1 µM) to determine PTH responsiveness. A) basal cAMP levels, B) PTH-stimulated cAMP levels (PTH-stimulated minus basal). Note different scale for each figure. Data are expressed as mean ± SEM for triplicate samples from one of three experiments with similar results.

 

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  		Table 1. Basal cAMP levels in rat primary calvarial cells

 
Effects of peptide fragments and cAMP regulators on osteoblast differentiation: mRNA, calcium accumulation, and mineralized nodules
PTH fragments and agents that alter the cAMP pathway were evaluated to determine their effects on osteoblast differentiation. Northern blot analysis for osteocalcin (OCN) mRNA was performed because OCN expression characterizes the osteoblast phenotype and is expressed in the latter stage of osteoblast differentiation (26). The stimulator of cAMP accumulation, forskolin, dramatically reduced the steady-state mRNA expression of osteocalcin in vitro (Fig. 6Go). THFA and 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) treatment increased OCN expression compared with forskolin-treated cultures in MC3T3-E1 cells and compared with control in rat primary calvarial cells. PTH (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)-treated cultures had reduced OCN expression compared with THFA-treated cultures. The PTH fragments (53–84) and PTHrP (107–139) did not significantly alter osteoblast differentiation (data not shown). Effects of cAMP regulators on differentiation also were evaluated qualitatively in the in vitro experiments with von Kossa staining to identify mineralized nodule formation. PTH (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) and forskolin reduced mineralized nodule formation in primary rat calvarial cells; whereas, interestingly, 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) and THFA increased mineralized nodule formation (Fig. 7Go). Quantitatively, calcium accumulation in the cell layers and nodules was also altered by cAMP regulation (Fig. 8Go). PTH (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) and forskolin-treatment resulted in a significant reduction in calcium accumulation; whereas 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) and THFA-treatment resulted in a significant increase in calcium accumulation compared with controls. The effects of PTH fragments and regulators of the cAMP pathway on gene expression, mineralized nodule formation, and calcium levels were not associated with an alteration in cell numbers since DNA concentrations for the treatment groups were not significantly different (data not shown, but were used to standardize calcium and cAMP data).



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  		Figure 6. Effects of cAMP regulators on osteoblast differentiation: Northern blot analysis of osteocalcin (OCN). A, Representative autoradiograph of OCN mRNA isolated from MC3T3-E1 cells that were induced to differentiate for 18 days with vehicle (control), PTH (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 ) (0.1 µM), 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 ) (0.1 µM), THFA (100 µM), or forskolin (Forsk) (10 µM). B, Plot of mean cpm values (T/C) of OCN mRNA compared with 18S rRNA from two separate experiments. C, Representative autoradiograph of OCN mRNA isolated from primary rat calvarial cells that were induced to differentiate for 10 d with vehicle (control), 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 ) (0.1 µM), or THFA (100 µM). D, Plot of mean cpm values (T/C) of OCN mRNA compared with 18S rRNA from four separate experiments.

 


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  		Figure 7. Effects of cAMP regulators on osteoblast differentiation: von Kossa staining. Representative 35-mm culture plates stained for mineralized nodules with the von Kossa method. A, MC3T3-E1 cells treated with vehicle (control) or 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 ) (0.1 µM) for 21 days. B, Primary rat calvarial cells treated with vehicle (control), THFA (100 µM), PTH (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 ) (0.1 µM), or forskolin (10 µM) for 26 days.

 


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  		Figure 8. Effects of PTH analogs and cAMP regulators on calcium accumulation. Primary rat calvarial cells were induced to differentiate with continuous treatment with PTH analogs and cAMP regulators for 13 days. Mean calcium concentrations in trichloroacetic acid precipitates were expressed (± SEM) for triplicate samples. The groups treated with adenylyl cyclase stimulators (PTH 1–34 and forskolin) had significantly less calcium accumulation than controls, whereas the 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 ) antagonist and adenylyl cyclase inhibitor (THFA) significantly increased calcium accumulation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrated that agents that increase, decrease, or block endogenous adenylyl cyclase or cAMP production have prominent effects on PTH-1 receptor expression and osteoblast differentiation. Several studies have evaluated ligand-mediated PTH receptor regulation in osteoblastic cells; however, these studies have used transformed osteosarcoma cells. Receptor regulation in primary or nontransformed cells has not been reported. These are important studies because transformed cells often display different characteristics when compared with nontransformed cells and do not undergo normal differentiation in vitro (27). Furthermore, in vitro models of osteoblast differentiation with primary osteoblast-like cells are being used extensively as model systems to examine the anabolic mechanisms of PTH action in bone, yet factors regulating the receptor in these models are not well characterized. In the current study, new data demonstrated that agents that inhibit or block adenylyl cyclase alter osteoblastic differentiation.

We found that agents that stimulate cAMP also down-regulate steady-state PTH-1 receptor mRNA. In contrast, Abou-Samra et al. used the rat osteosarcoma (ROS 17/2.8) cell line and reported that agonist-dependent PTH-1 receptor downregulation was cAMP independent (28). Pretreatment of ROS 17/2.8 cells with PTH (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) reduced the PTH-stimulated increase in cAMP, whereas a pretreatment with 8-Br-cAMP or forskolin (stimulators of intracellular cAMP accumulation) did not alter PTH-stimulated cAMP levels. In the current study, PTH (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) and forskolin treatment reduced steady-state mRNA for the PTH-1 receptor in addition to the PTH-stimulated increase in cAMP. The current data are similar to that reported in a study by Fukayama et al. using SaOS-2 human osteosarcoma cells in which agents that activated cAMP (PTH 1–34, 8-Br-cAMP and forskolin) reduced steady-state levels of PTH-1 receptor (29). Similar results were reported by Mitchell and Goltzman with the UMR-106 rat osteosarcoma cells (30). They showed down-regulation of PTH-stimulated cAMP with PTH (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), prostaglandin E2, and (Bu)2-cAMP, consistent with homologous and heterologous protein kinase A-dependent regulation of PTH receptors in osteosarcoma cells.

In the present study, fragments of PTH and PTHrP that have been reported to have biologic activity in bone were evaluated for their ability to alter PTH-1 receptor expression and binding. PTH (53–84) does not activate cAMP-dependent protein kinase or protein kinase C but does stimulate calcium flux and increases alkaline phosphatase in osteoblastic cells (31). PTH (53–84) did not alter PTH-1 receptor expression or binding. This is similar to a previous study with SaOS-2 cells in which neither PTH fragments (39–84) or (53–84) altered PTH-1 receptor mRNA expression (29). PTHrP (107–139) has been reported to stimulate membrane-associated PKC activity and cAMP in osteoblastic cells (32, 33). We have not found PTHrP (107–139) to stimulate cAMP accumulation in MC3T3-E1 or primary rat calvarial cells and did not see alteration of steady state PTH-1 receptor mRNA. These data suggest that down-regulation of the PTH-1 receptor with PTH (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) in nontransformed osteoblastic cells is dependent on cAMP stimulation. Our data supports this concept because forskolin down-regulated the PTH-1 receptor, whereas the adenylyl cyclase inhibitor THFA had no effect.

Interesting results were found with the 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) fragment that displays antagonist activity at the PTH-1 receptor. Although 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 alter steady-state PTH-1 receptor mRNA levels, pretreatment reduced basal cAMP levels and PTH-stimulated cAMP levels were increased. One possibility for the increase in PTH-stimulated cAMP is that a G protein that tonically inhibits PTH receptor availability is sensitive to down-regulation by the (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) fragment. A similar scenario was proposed for rat osteosarcoma cells when a pertussis toxin-sensitive G protein was used to explain an increase in PTH-1 receptor responsiveness (28). Alternatively, in light of the findings that 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) stimulates osteoblastic differentiation, these data suggest that 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) may block an autocrine phenomenon in osteoblasts. 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) may reduce physiologic tonic inhibition of the PTH-1 receptor by endogenous secretion of PTHrP. Osteoblastic cells have been reported to produce and secrete PTHrP at low levels (34, 35). PTHrP levels are greatest when the cells are proliferating and are less differentiated. PTH (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) and PTHrP inhibit osteoblastic differentiation in models of bone formation in vitro (11, 19, 36); consequently, 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) may occupy receptors and block the action of early, endogenously produced PTHrP, which would inhibit mineralization if allowed to activate the receptor. Finally, a third possibility is that the PTH-1 receptor, similar to other G protein-linked receptors, displays some level of constitutive activity and 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) acts as an inverse agonist in this osteoblast differentiation model system (37).

There is a lack of studies that measure the effects of cAMP-activating agents (other than PTH or PGE2) on osteoblast differentiation. One recent study evaluated short-term, continuous exposure of primary rat calvarial cells to forskolin (19). Continuous exposure for 48 h resulted in inhibition of alkaline phosphatase activity and expression of osteocalcin mRNA that mimicked continuous exposure to PTH. Turksen et al. also used fetal rat calvarial cells and reported a significant inhibition of mineralized nodule formation with high doses of forskolin (38). Interestingly, opposite results were reported for calcifying vascular cells. Treatment of cells derived from primary aortic medial cell cultures with dibutyrl-cAMP or forskolin for 3 days induced alkaline phosphatase, type I procollagen production and matrix GLA protein, and treatment with a protein kinase A-specific inhibitor, KT5720, inhibited alkaline phosphatase activity and mineralization (39). These differences likely reflect the nature of the cell culture system and its variability from osteoblast differentiation models.

The effects of adenylyl cyclase inhibitory agents, such as THFA, in osteoblast differentiation have not been explored. Our data suggests that osteoblast differentiation in vitro is under tonic inhibitory regulation by autocrine or exogenous agents in the cell culture medium that stimulate cAMP. One such agent may be PTHrP. Indeed, this may mimic the effects of PTHrP during cartilage differentiation in vivo where PTHrP plays a role in inhibiting mineralization. Mice with targeted disruption of the PTHrP gene have developmental abnormalities attributed to the loss of PTHrP inhibition of mineralization (40). In addition, overexpression of PTHrP in chondrocytes in vivo results in inhibition of the differentiation process and delayed osteogenesis (41). It is unknown whether cAMP regulatory agents act as direct mediators or counteract endogenous factors such as PTHrP that alter osteoblast differentiation.

In summary, this study reports the effects of agents that alter cAMP on PTH-1 receptor activity and osteoblast differentiation in vitro. PTH (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) and forskolin have prominent effects on down-regulation of the PTH-1 receptor and inhibition of mineralized nodule and osteoblast phenotypic characteristics during differentiation. 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) and THFA do not alter PTH-1 receptor mRNA expression, but reduce basal cAMP levels via different mechanisms and stimulate the differentiation of nontransformed osteoblastic cells in vitro. These results suggest that PTHrP may be involved in the differentiation of osteoblastic cells in vitro and also raise the question whether effects of PTH on bone in vivo may be attributed to a more general phenomena of cAMP activation. Although these studies do not discount the possibility that the PKC pathway may be involved in osteoblast differentiation, they strongly support a role for the PKA pathway. Future studies using PKA and PKC regulating agents and in vivo model systems will provide a better understanding of this intriguing area.


   Acknowledgments
 
We gratefully acknowledge Dr. Renny Franceschi for providing MC3T3-E1 cells and the OCN cDNA.


   Footnotes
 
1 These studies were supported by the National Institutes of Health Grants DK-46919 and DK-53904. Back

Received September 3, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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