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Endocrinology Vol. 140, No. 2 871-879
Copyright © 1999 by The Endocrine Society

ARTICLES

Insulin-Like Growth Factor I Suppresses Parathyroid Hormone (PTH)/PTH-Related Protein Receptor Expression via a Mitogen-Activated Protein Kinase Pathway in UMR-106 Osteoblast-Like Cells1

Tetsuya Kawane and Noboru Horiuchi

Department of Biochemistry, Ohu University School of Dentistry, Koriyama 963-8611, Japan

Address all correspondence and requests for reprints to: Noboru Horiuchi, D.D.S., Ph.D., Department of Biochemistry, Ohu University School of Dentistry, Koriyama 963-8611, Japan. E-mail: fwga4746{at}mb


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like growth factor I (IGF-I) is important in skeletal growth and has been implicated in the maintenance of bone integrity. PTH stimulates bone resorption through the G protein-linked PTH/PTH-related protein (PTHrP) receptor in osteoblasts. Using a heterogeneous nuclear RNA assay and Northern blot analysis, we showed that IGF-I inhibited expression of the gene for PTH/PTHrP receptor in a dose- and time-dependent fashion, but did not alter the stability of the receptor messenger RNA (mRNA) in UMR-106 osteoblast-like cells. IGF-I treatment for 48 h also caused a decrease in the receptor number to 45% of that in controls without affecting receptor affinity and in functional receptor expression to 50–60% of that in controls as measured by PTH-stimulated cAMP production. In MC3T3-E1 murine nontransformed osteoblasts, IGF suppressed receptor mRNA expression dose dependently. In UMR-106 cells, IGF-I induced the mitogen-activated protein (MAP) kinase pathway. The effect of IGF-I was blocked by PD98059, a specific inhibitor of the MAP kinase-activating kinase, but not by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. IGF-I inhibition of PTH/PTHrP receptor mRNA expression in UMR-106 cells was abrogated completely by pretreatment with cycloheximide, an inhibitor of protein synthesis. These findings indicate that IGF-I suppresses gene expression for PTH/PTHrP receptor via the MAP kinase pathway, and this inhibition is required for new protein synthesis in UMR-106 osteoblast-like cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor I (IGF-I), a 70-amino acid polypeptide, shares considerable structural homology with insulin and IGF-II. IGFs constitute a family of cellular modulators that plays an essential role in the regulation of cell cycle progression, cell proliferation, and tumor progression (1, 2). In skeletal tissue, the anabolic role of IGF-I is well documented (3, 4). Although it was thought that most of the IGF-I in the skeleton arrived through the circulation after production by the liver, there is increasing evidence that IGF-I is secreted by skeletal cells such as osteoblasts. It may act as an autocrine and paracrine regulator of osteoblastic function to stimulate bone type I collagen synthesis (5) and to decrease collagenase-mediated skeletal collagen degradation in osteoblasts (6, 7). Thus, IGF-I serves as a crucial factor in skeletal growth, integrity, and bone remodeling. The central role of IGF-I in bone integrity has prompted investigators to examine its mechanisms of action in bone cells.

Most of the effects of IGF-I are mediated by specific receptor binding, i.e. IGF-I-receptor (8, 9). Binding of IGF-I to its receptor induces receptor autophosphorylation in the intracellular kinase domain, which initiates a cascade of cellular signal transduction pathways. The predominant substrate of IGF-I receptor is insulin receptor substrate-1 (IRS-1), a docking protein (10). Phosphorylated IRS-1 or Src homology/{alpha}-collagen (Shc) not only transduces the IGF-I signal to the phosphatidylinositol 3-kinase (PI 3-kinase) pathway (11, 12, 13, 14), but also activates a wide range of signaling molecules, including the Ras-Raf-mitogen-activated protein (MAP) kinase network (15, 16, 17). The MAP kinase cascade is one of the major signaling pathways by which cells transduce extracellular stimuli into intracellular responses. Binding of phosphorylated IRS-1 and other docking proteins leads to stepwise activation of Ras, Raf (MEK-activating kinase), MEK [extracellular signal-regulated kinase (ERK)-activating kinase], and MAP kinases. Some of these kinases, such as MAP kinases, can control the activities of cellular and nuclear proteins, including transcription factors, and can regulate the gene expression of certain proteins (2).

PTH is a potent activator of osteoclastic bone resorption that elicits a wide variety of biological responses in osteoblastic cells. PTH mediates its effects on bone metabolism by binding to the G protein-linked PTH/PTH-related protein (PTHrP) receptor that is expressed in cells of osteoblasts and osteoblastic cell lines (18, 19). PTH, when it binds to this receptor, increases the levels of intracellular second messengers, including cAMP, ionized calcium, and diacylglycerol (20, 21, 22). Activation of these second messenger systems leads to changes in osteoblastic gene expression by a transcriptional mechanism. PTH is thought to mediate most of the biological effects in osteoblastic cells, primarily via cAMP-protein kinase A signaling (23). The coupling of bone resorption to bone formation is necessary for the maintenance of healthy bone. The IGF-I produced by osteoblasts stimulates bone formation and has been proposed as one of the factors that couples bone formation to bone resorption in normal skeletal homeostasis (24). In fact, IGF-I has been shown to reduce PTH/PTHrP receptor number and inhibit PTH-stimulated cAMP production in human osteoblast-like SaOS-2 cells (25). We have found that PTH/PTHrP receptor messenger RNA (mRNA) expression in bone increases markedly in starved rats with decreased levels of serum IGF-I (26). Thus, IGF-I may affect PTH-induced bone metabolism by attenuating PTH signaling in osteoblasts.

In this report, we investigated the mechanism of IGF-I-suppressed PTH/PTHrP receptor expression in UMR-106 rat osteoblast-like cells. We found that IGF-I inhibited PTH/PTHrP receptor gene expression with signal transduction of the MAP kinase pathway, but not the PI 3-kinase pathway. Its inhibition was abolished by cycloheximide pretreatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Rat osteoblast-like osteosarcoma UMR-106 cells (American Type Culture Collection, Manassas, VA; CRL 1661) were grown routinely in monolayer culture at 37 C in 5% CO2-95% air in DMEM (ICN Pharmaceuticals, Inc., Costa Mesa, CA) supplemented with 5% FCS (Filtron Pty. Ltd., Brooklyn, Australia) and were passed once per week. At 70% confluence, the culture medium was replaced with serum-free DMEM for 24 h with 0.1% BSA (Sigma Chemical Co., St. Louis, MO). Cells were exposed to a test substance such as human (h) IGF-I (Austral Biologicals, San Ramon, CA) in DMEM with 0.1% BSA. In studies of inhibitors, serum-starved cells were treated with 1 µM wortmannin (Wako Pure Chemical Industries, Ltd., Osaka, Japan), a PI 3-kinase inhibitor; 100 µM PD98059 (Alexis Corp., San Diego, CA), a MAP kinase pathway inhibitor; or 35 µM cycloheximide (Sigma Chemical Co.,), a protein synthesis inhibitor, for 1 h before the indicated duration of incubation with 100 nM IGF-I. In some experiments, cells were exposed to 75 µM 5,6-dichlorobenzimidazole riboside (DRB; Sigma Chemical Co.), a transcriptional inhibitor, dissolved in dimethylsulfoxide (Me2SO) 24 h after treatment with 100 nM IGF-I or vehicle. For some experiments, MC3T3-E1 murine calvarial osteoblasts obtained from Dr. Y. Amagai (Ohu University, Japan) were grown in {alpha}MEM (ICN Pharmaceuticals, Inc.) with 10% FCS for 6 days, and cells reached confluence. Cells were further cultured for 6 days in the absence or presence of graded doses of hIGF-I. At the end of culture, MC3T3-E1 cells had differentiated to osteoblasts, as alkaline phosphatase activity highly increased compared with that in proliferating cells (27).

Measurement of cAMP accumulation
Monolayer cells in 24-well plates were washed twice with assay buffer [135 mM NaCl, 6 mM KCl, 1 mM MgCl2, 2.8 mM glucose, 1.2 mM CaCl2, and 20 mM HEPES (Sigma Chemical Co.), pH 7.4] and then incubated in the same buffer containing 0.1% heat-inactivated BSA, 1 mM isobutylmethylxanthine (Sigma Chemical Co.), and rat (r) PTH-(1–34), supplied by Peninsula Laboratories, Inc. (Belmont, CA), or hPTH-(1–84) (a gift from Chugai Pharmaceutical Co., Tokyo, Japan) at 37 C for 15 min (28). The buffer then was rapidly aspirated, 0.3 M perchloric acid was added to the plates, and the acid extracts were used for the cAMP assay. Cellular cAMP was measured using a RIA kit, a gift from Yamasa Shoyu Co. (Choshi, Japan). Cellular protein also was measured. Results are expressed as picomoles of cAMP per mg protein generated over 1 min.

PTH binding to receptor
UMR-106 cells at confluence were preincubated with 100 nM IGF-I or vehicle in serum-free DMEM containing BSA for 48 h, and then washed twice with ice-cold binding buffer (100 mM NaCl, 50 mM Tris, 5 mM KCl, 2 mM CaCl2, 5% heat-inactivated bovine serum, and 0.5% heat-inactivated BSA, pH 7.7). Cells were incubated with 0.1 ml binding buffer containing increasing concentrations of 125I-labeled [Nle8,21,Tyr34]rPTH-(1–34) amide (SA, 81.4 TBq/mmol; New England Nuclear Life Science Products, Inc., Boston, MA) in the absence or presence of 1 µM unlabeled rPTH-(1–34) (Peninsula Laboratories, Inc.) at 15 C for 6 h. After incubation, binding buffer was aspirated, and cells were washed twice with ice-cold binding buffer without serum. Cells were solubilized in 1 N NaOH plus 0.1% SDS and transferred to fresh tubes. Cell-associated radioactivity was determined using a {gamma}-counter (25).

In vitro assays for p42/p44 MAP kinase activity
Cells treated with IGF-I at the indicated concentrations and duration were washed twice with cold PBS and lysed in an ice-cold buffer [10 mM Tris, 150 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM orthovanadate, 1 mM phenylmethylsulfonylfluoride (Sigma Chemical Co.), 10 µg/ml leupeptin (Sigma Chemical Co.), and 10 µg/ml aprotinin, pH 7.4]. Cellular debris was precipitated at 25,000 x g for 20 min, and the supernatant was retained for the MAP kinase assay. The BIOTRAK p42/p44 MAP kinase enzyme assay system (Amersham, Aylesbury, UK) was used to measure p42/p44 MAP kinase activities. The synthesized substrate peptide based on the Thr669 phosphorylation site of the EGF receptor contains one PLS/TP sequence as a phosphorylation site for the p42/p44 MAP kinase. Briefly, 30 µl reaction mixture containing 15 µl lysates or lysis buffer, substrate, Mg2+, ATP, and 1 µCi [{gamma}-32P]ATP (SA, 110 TBq/mmol; Amersham) were incubated at 30 C for 30 min, and the incubation was stopped by adding 10 µl stop reagent. Phosphorylated peptide in the 30 µl stopped reaction mixtures was separated from unincorporated label on binding paper. After washing the binding paper with 1% acetic acid and distilled water, the extent of 32P radioactivity of the substrate peptide was detected by scintillation counting.

Determination of PTH/PTHrP receptor mRNA abundance
The effect of IGF-I on the abundance of PTH/PTHrP receptor mRNA was quantified by Northern blot analysis using a complementary DNA (cDNA) probe excised from a T vector, as described previously (26). Monolayer cultures in FCS-free medium were set up in T-25 flasks, followed by treatment with IGF-I. Total RNA was isolated using 4 M guanidinium isothiocyanate followed by phenol-chloroform extraction and was quantified by absorbance at 260 nm. Twenty micrograms of total RNA were separated by electrophoresis in 1.2% agarose gel with 6% formaldehyde and transferred to Hybond-N+ membrane (Amersham) by capillary immobilization. The membrane was prehybridized and hybridized as described previously (26). The PTH/PTHrP receptor cDNA probe was radiolabeled with [{alpha}-32P]deoxy-CTP (SA, 110 TBq/mmol; ICN) by random priming and was added to the hybridization solution. Hybridization was performed at 42 C for 48 h, followed by washing with 0.1–2 x SSPE (150 mM NaCl, 10 mM sodium phosphate, and 1 mM EDTA, pH 7.4)-0.1% SDS at 65 C. Blots were visualized by autoradiography. PTH/PTHrP receptor mRNA abundance was corrected for the concentration of cyclophilin mRNA by reprobing the membrane. The integrity of the RNA was assessed by visual inspection of the 18S and 28S ribosomal RNA bands stained with ethidium bromide after agarose-formaldehyde gel electrophoresis. In MC3T3-E1 cells, PTH/PTHrP receptor mRNA abundance was determined by RT-PCR and was normalized to the amount of ß-actin mRNA expression, as described previously (29). Specific primers were 5'-ACGCGCAACTACATCCACAT-3' and 5'-CTGGAAGGAGTTGAAGAGCA-3' for PTH/PTHrP receptor, and 5'-ACCTTCTACAATGAGCTGCG-3' and 5'-TGCCAATAGTGATGACCTGG-3' for ß-actin.

Quantification of PTH/PTHrP receptor heterogeneous nuclear RNA (hnRNA)
PTH/PTHrP receptor hnRNA was analyzed by RT-PCR using specific primers designed to amplify DNA from an intron between exons M7 and T of the rat PTH/PTHrP receptor gene (30). The nucleotide sequences of the introns and parts of exon M6/7 and T were determined and submitted to the databases. A sense primer (5'-CGTCTTTGGGGCATTTGAGT-3'), spanning nucleotides 590–609 of the intron between exons M7 and T of the rat PTH/PTHrP receptor gene, and an antisense primer (5'-AAACACTGGCTTCTTGGTCC-3'), spanning nucleotides 1199–1218 of exon T, were synthesized. Total RNA from control and test samples was prepared as described for the Northern blot analysis and treated with ribonuclease-free grade deoxyribonuclease I (Boehringer Mannheim, Indianapolis, IN) to remove potentially contaminating DNA (31). One microgram of RNA was copied into cDNA using Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer, Norwalk, CT) and random hexanucleotide primers. A 20-µl aliquot of the newly synthesized cDNA was amplified by PCR with 21 cycles at 93 C for 1 min, 59 C for 1 min, and 72 C for 2 min in the presence of Taq polymerase (Perkin-Elmer) and 0.15 µM sense and antisense primers. PCR products were loaded onto a 1.2% agarose gel and transferred to a Hybond-N+ membrane by capillary immobilization. The membrane was prehybridized and hybridized in hybridization solution (5 x Denhart’s solution, 5 x SSPE, 0.5% SDS, and 20 µg/ml calf thymus DNA) in the presence of a PTH/PTHrP receptor cDNA probe in T vector, encompassing the region from positions 776-1218 of the rat PTH/PTHrP receptor mRNA, radiolabeled with [{alpha}-32P]deoxy-CTP by random priming. Hybridization was performed at 65 C for 24 h, followed by washing with 0.1–2 x SSPE-0.1% SDS at 65 C. Amplified RNA was corrected for the level of cyclophilin mRNA by RT-PCR using the 20-µl aliquot of the same synthesized cDNA. PCR was performed for 11 cycles at 93 C for 1 min, 55 C for 1 min, and 72 C for 2 min in the presence of Taq polymerase, 0.15 µM sense primer (5'-CAAAGTTCCAAAGACAGCAG-3'), and 0.15 µM antisense primer (5'-TGAGCTACAGAAGGA- ATGGT-3').

Statistical analysis
The data are described as the mean ± SEM. Statistical analysis was performed using ANOVA followed by Fisher’s protected least significant difference (StatView 4.02, Abacus Concepts, Inc., Berkeley, CA). P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Action of IGF-I on PTH/PTHrP receptor expression
To determine whether IGF-I inhibited PTH-sensitive adenylyl cyclase activity by UMR-106 osteoblast-like cells, we examined the effect of IGF-I pretreatment on PTH-stimulated accumulation of cAMP in the cells (Fig. 1Go). When the cells were pretreated with increasing concentrations of IGF-I for 24 h, dose-dependent inhibition of the cAMP accumulation was observed with 10-8 M hPTH-(1–84) and rPTH-(1–34) treatment. Maximal inhibition occurred after pretreatment with 100 nM IGF-I for 24 h and 1–10 nM for 48 h. Heterologous desensitization of the adenylyl cyclase response induced by IGF-I was greater with 48-h pretreatment than with 24-h pretreatment. At a concentration of 100 nM, IGF-I treatment for 48 h maximally inhibited PTH-stimulated cAMP accumulation by 50–60%. The effect of IGF-I on PTH-sensitive adenylyl cyclase activity was also examined in MC3T3-E1 nontransformed osteoblasts. When MC3T3-E1 cells with osteoblastic phenotype were treated with 100 nM IGF-I in the serum-free medium for 48 h, IGF-I significantly decreased rPTH-stimulated cAMP accumulation (20.1 ± 0.6 pmol/min·mg protein in vehicle-treated cells vs. 16.4 ± 0.1 pmol/min·mg protein in IGF-I-treated cells; P < 0.01). The presence of PTH/PTHrP receptor in UMR-106 cells was examined by receptor binding study. Figure 2Go depicts the binding of increasing concentrations of 125I-labeled [Nle8,21,Tyr34]rPTH-(1–34)amide to UMR-106 cells 48 h after treatment with either vehicle alone or 100 nM IGF-I. Scatchard analysis showed that IGF-I reduced the number of cell-surface PTH-binding sites to 45% of the control value without affecting the affinity (Kd) of the receptor. Binding capacity values were 24,000 ± 5,000 and 11,000 ± 3,000 sites/cells in vehicle- and IGF-I-treated UMR-106 cells, respectively.



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  		Figure 1. Dose-dependent effect of IGF-I on PTH-stimulated cAMP production by UMR-106 cells. Cells were preincubated with vehicle alone or with the indicated concentrations of IGF-I for 24 h ({blacksquare} and {square}) or 48 h (• and {circ}) before incubation with 10 nM hPTH-(1–84) ({blacksquare} and •) or rPTH-(1–34) ({square} and {circ}) for 15 min in assay buffer containing 100 µM isobutylmethylxanthine. Cellular cAMP is expressed as a percentage of cAMP accumulation observed in cells pretreated with vehicle alone. In the experiment shown, basal cAMP accumulation in vehicle-treated cells was 2.6 ± 0.1 pmol/min·mg protein, whereas maximal stimulation was 186 ± 2 and 194 ± 8 pmol/min·mg protein for 10 nM hPTH-(1–84) and rPTH-(1–34), respectively. Each point represents the mean ± SEM of triplicate determinations. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with vehicle control).

 


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  		Figure 2. Saturation curves (A) and Scatchard analysis (B) of PTH binding to UMR-106 cells. Cells were cultured with 100 nM IGF-I (•) or vehicle ({circ}) for 48 h. Specific binding of PTH was determined using increasing concentrations of 125I-labeled [Nle8,21,Tyr34]rPTH-(1–34)amide (1.25 x 10-10 to 4 x 10-9 M). Nonspecific binding, determined in the presence of 1 µM rPTH-(1–34), was subtracted from the total bound counts to give specific binding. The calculated Kd values were 1.1 ± 0.2 and 1.0 ± 0.3 nM in control and IGF-I groups, respectively, and maximal binding (Bmax) values of vehicle and IGF-I-treated cells were 4.1 and 1.8 fmol/105 cells, respectively.

 
IGF-I inhibition of PTH/PTHrP receptor mRNA
The time course of the effect of IGF-I on the amount of PTH/PTHrP receptor mRNA was determined in UMR-106 cells treated with 100 nM IGF-I (Fig. 3Go). There was a significant decrease in PTH/PTHrP receptor mRNA as early as 3 h after initiation of treatment with IGF-I. The inhibition was already maximal at 6 h and was maintained for up to 48 h. Figure 4Go shows the concentration-response curve for PTH/PTHrP receptor mRNA measured after IGF-I treatment for 24 h in osteoblastic cell lines. The reduction in PTH/PTHrP receptor mRNA in response to IGF-I was significant at a concentration of 10-9 M and was maximal at 10-7 M in rat osteoblast-like osteosarcoma UMR-106 cells (Fig. 4AGo). To demonstrate that IGF-I significantly reduces PTH/PTHrP receptor mRNA expression in nontransformed osteoblasts, MC3T3-E1 murine calvarial osteoblasts were grown to confluence and further cultured for 6 days to allow differentiation to mature osteoblast-like cells (27). Treatment of MC3T3-E1 cells with IGF-I decreased PTH/PTHrP receptor mRNA abundance in a dose-dependent manner (Fig. 4BGo).



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  		Figure 3. Time course of the inhibition of PTH/PTHrP receptor mRNA expression by IGF-I in UMR-106 cells. After various periods of incubation (1–48 h) with vehicle or 100 nM IGF-I in serum-free DMEM containing 0.1% BSA, cells were harvested, and total RNA was extracted. A, Northern blots of total RNA (20 µg) for PTH/PTHrP receptor mRNA (PTHR) and cyclophilin mRNA (Cyclo.). The estimated sizes of PTH/PTHrP receptor and cyclophilin mRNAs were 1.5 and 0.9 kb, respectively. B, Determination of PTH/PTHrP receptor mRNA abundance in cells treated with vehicle ({circ}) or 100 nM IGF-I (•). The PTH/PTHrP receptor mRNA concentration was determined densitometrically and normalized to that of cyclophilin mRNA. The data are expressed as the mean ± SEM of triplicate determinations. **, P < 0.01; ***, P < 0.001 (compared with vehicle control at each time point).

 


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  		Figure 4. Effect of IGF-I on PTH/PTHrP receptor (PTHR) mRNA abundance in UMR-106 cells and MC3T3-E1 murine calvarial osteoblasts. A, At 70% confluence of UMR-106 cells, the medium was changed to serum-free DMEM for 24 h, and cells were treated with the indicated amounts of IGF-I for 24 h. Total RNA (20 µg) from cells was subjected to Northern blot analysis. The results of quantitative Northern blot analysis of the receptor mRNA abundance were normalized to those of the cyclophilin mRNA. B, MC3T3-E1 cells were cultured in {alpha}MEM for 6 days after reaching confluence and treated with vehicle or the indicated amounts of IGF-I for 6 days. Total RNA (0.5 µg) was subjected to RT-PCR. PCR products were measured by Southern blot analysis. The receptor mRNA abundance was corrected for the ß-actin mRNA. The data are expressed as the mean ± SEM of triplicate determinations. **, P < 0.01; ***, P < 0.001 (compared with vehicle control).

 
Effects of wortmannin and PD98059
The involvement of the MAP kinases and PI 3-kinase in the inhibition of PTH/PTHrP receptor expression by IGF-I was examined by pretreating the cells with their inhibitors for 1 h. The incubation period of the cells with IGF-I was 6 h because maximal suppression of the mRNA was observed at that point in the time-course study (Fig. 3Go). Pretreatment with 1 µM wortmannin, a specific inhibitor of PI 3-kinase, did not affect the IGF-I-induced suppression of PTH/PTHrP receptor mRNA expression (Fig. 5Go). Next, to determine whether MAP kinases are responsible for IGF-I-induced inhibition of PTH/PTHrP receptor mRNA synthesis, PD98059, a highly selective inhibitor of the MAP kinase pathway, was used. PD98059 completely blocked the decrease in receptor mRNA induced by IGF-I in UMR-106 cells (Fig. 5Go).



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  		Figure 5. Effects of the MEK inhibitor PD98059 (PD) and PI 3-kinase inhibitor wortmannin (W) on IGF-I-induced suppression of PTH/PTHrP receptor (PTHR) mRNA expression in UMR-106 cells. Serum-starved cells were treated with 1 µM wortmannin or 100 µM PD98059 for 1 h before incubation for 6 h with 100 nM IGF-I. A, Northern blot analysis. B, Densitometric determination of the Northern blots. PTH/PTHrP receptor mRNA abundance normalized relative to cyclophilin mRNA was expressed as a percentage of the vehicle control value. Each bar represents the mean ± SEM of triplicate determinations. ***, P < 0.001 (compared with the vehicle control); {dagger}{dagger}{dagger}, P < 0.001 (between wortmannin-treated groups). NS, Not significantly different between PD98059-treated groups.

 
MAP kinases are a family of serine/threonine kinases that are activated by threonine and tyrosine phosphorylation by MAP kinase kinase upon activation of various growth factor receptors (32, 33). Prominent proteins among them are the p42/p44 MAP kinases, which are involved in the regulation of transcription factor activity by phosphorylation (34). To determine whether IGF-I induces p42/p44 MAP kinases in UMR-106 osteoblast-like cells, changes in p42/p44 MAP kinase activities were analyzed by their ability to phosphorylate a peptide substrate containing the PLS/TP as a site for phosphorylation (35). IGF-I treatment of UMR-106 cells increased significantly for the p42/p44 MAP kinase activities for 5–10 min and declined after 30 min to the control level (Fig. 6AGo). The stimulating effect of IGF-I on the p42/p44 MAP kinase activities was abolished by the addition of the MEK inhibitor, PD98059, although PD98059 itself decreased basal activities of MAP kinases (Fig. 6BGo). Subsequently, we studied the effect of PD98059 on PTH/PTHrP receptor number. IGF-I-induced suppression of PTH/PTHrP receptor expression was abrogated completely by treatment with PD98059 (Fig. 7).



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  		Figure 6. Effects of IGF-I and PD98059 on p42/p44 MAP kinase activities in UMR-106 osteoblast-like cells. A, Time course of changes in IGF-I-induced p42/p44 MAP kinase activities. Cells were cultured in serum-free DMEM for 24 h and then treated with 100 nM IGF-I for the indicated periods, and p42/p44 MAP kinase activities were measured. B, Serum-starved cells were treated with 100 nM IGF-I in the presence or absence of 100 µM PD98059. Control cells were treated with 0.1% Me2SO alone. After 5 min, p42/p44 MAP kinase activities were measured. Data are the mean ± SEM of triplicate determinations. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with control; time zero for A and vehicle control for B). {dagger}{dagger}{dagger}, P < 0.001 (between PD98059-treated groups).

 
Transcriptional regulation of the PTH/PTHrP receptor gene by IGF-I
To determine whether IGF-I caused a change in PTH/PTHrP receptor mRNA stability, the rate of receptor mRNA degradation was tested in control and IGF-I-treated cultures. UMR-106 cells were exposed to vehicle and 100 nM IGF-I-containing medium for 24 h, and then 75 µM DRB, a RNA polymerase II inhibitor (36), was added to the cultures. The decay of PTH/PTHrP receptor mRNA was determined by Northern blot analysis (Fig. 8Go). The receptor mRNA decay occurred at similar rates in both control and IGF-I-treated cultures, indicating that IGF-I did not alter the stability of this transcript. Because transcriptional regulation frequently leads to changes in hnRNA synthesis, concentrations of hnRNA were examined to assess the transcriptional rate of the PTH/PTHrP receptor gene during treatment with IGF-I. Addition of IGF-I at 100 nM for 3–6 h in UMR-106 cells caused a significant decrease in PTH/PTHrP receptor hnRNA (Fig. 9Go). To determine whether this event required protein synthesis in UMR-106 cells, the reduction of PTH/PTHrP receptor mRNA by IGF-I was assessed in the presence of the protein synthesis inhibitor, cycloheximide (10 µg/ml). Cycloheximide completely abolished IGF-I-induced inhibition of PTH/PTHrP receptor mRNA expression, demonstrating that de novo protein synthesis was required for such inhibition (Fig. 10Go).



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  		Figure 8. Effect of IGF-I on PTH/PTHrP receptor mRNA half-life in transcription-arrested UMR-106 cells. Serum-starved cells were exposed to 100 nM IGF-I (•) or to vehicle alone ({circ}) for 24 h before the addition of 75 µM DRB, a transcriptional inhibitor. At the indicated time points after the addition of DRB, total RNA from cultures was subjected to Northern blot analysis. PTH/PTHrP receptor mRNA normalized relative to cyclophilin mRNA was quantified by densitometry. Values are the mean ± SEM from three culture flasks.

 


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  		Figure 9. Time course of the inhibition of PTH/PTHrP receptor hnRNA concentration by IGF-I in UMR-106 cells. Serum-starved cells were treated with 100 nM IGF-I for the indicated periods. Total RNA from control and IGF-I-treated cultures was reverse transcribed and amplified by PCR. A, Schematic representation of the structure of the rat PTH/PTHrP receptor gene. The gene between exons M6/7 and T (30 ) was expanded, and these exons are shown with boxes. The nucleotide sequence of region I was determined and submitted to nucleotide sequence databases (accession no. AB01294). Region II (nucleotide 590-1218) was amplified by PCR for determination of PTH/PTHrP receptor hnRNA concentration. B, Total RNA (1 µg) was subjected to RT-PCR, and products were analyzed by Southern blots as described in Materials and Methods. The Southern blots of cyclophilin hnRNA are also shown in the lower panels. C, Quantitative determination of PTH/PTHrP receptor hnRNA in IGF-I-treated UMR-106 cells. The results were normalized relative to cyclophilin expression and are expressed as the mean ± SEM of triplicate determinations. **, P < 0.01; ***, P < 0.001 (compared with vehicle control at each time point).

 


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  		Figure 10. Effect of IGF-I at 100 nM, in the presence or absence of cycloheximide (CHX) at 35 µM, on PTH/PTHrP receptor (PTHR) mRNA expression in UMR-106 cells. Serum-starved cells were treated with cycloheximide, a protein synthesis inhibitor, for 1 h before incubation with IGF-I for 6 h. A, Northern blot analysis. B, Densitometric determination of the Northern blots. PTH/PTHrP receptor mRNA abundance normalized relative to cyclophilin mRNA was expressed as a percentage of the vehicle control value. Each bar represents the mean ± SEM of triplicate determinations. ***, P < 0.001 (compared with vehicle control).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studies have shown that IGF-I and its cognate receptors are expressed abundantly in osteoblasts (37, 38) and are essential for maintaining bone integrity. IGF-I also reduces the number of cell surface PTH-binding sites and inhibits the production of cAMP stimulated by PTH (25). However, the mechanism by which IGF-I suppresses PTH/PTHrP receptor expression is unknown. We found a pathway through which the IGF-I signal is transduced from the plasma membrane leading to PTH/PTHrP receptor expression and function in UMR-106 osteoblastic cells.

We and other investigators have shown previously that PTH/PTHrP receptor mRNA expression in bone and kidney was markedly increased in starved rats with decreased concentrations of plasma IGF-I and hepatic IGF-I transcripts (26, 39, 40). It is likely that IGF-I affects the PTH signaling pathway in osteoblasts. Bone-resorbing hormones such as PTH act through receptors, such as the PTH/PTHrP receptor found on osteoblasts, which communicate with osteoclasts, stimulating them to increase bone resorption (41). Thus, osteoblasts can participate in bone resorption in response to PTH. In contrast, IGF-I and IGF-II are the most abundant growth factors present in bone and increase bone formation with the enhancement of the differentiated functions of osteoblasts. However, the effects of IGF-I on bone resorption are not clear. MC3T3-E1 cells were used as the model of nontransformed osteoblasts for our experiments; they are preosteoblastic cells derived from newborn mouse calvaria and express the osteoblast phenotype, such as increased synthesis of collagen, osteocalcin, and alkaline phosphatase after differentiation (27). As we demonstrated the suppression of PTH/PTHrP receptor mRNA expression by IGF-I not only in UMR-106 osteoblast-like osteosarcoma cells but also in MC3T3-E1 nontransformed osteoblastic cells, the effects of IGF-I appear to be physiological. The present results suggested that IGF-I at least in part maintains bone integrity with decreased PTH signaling by attenuating the expression of the PTH/PTHrP receptor in osteoblasts.

An important finding of this study is that the IGF-I signal is transmitted to the nucleus to regulate PTH/PTHrP receptor gene expression. IGF-I decreases steady state levels of PTH/PTHrP receptor mRNA, but does not modify the half-life of the mRNA. The abundance of hnRNA also is reduced by IGF-I in UMR-106 osteoblastic cells. Although hnRNA may reflect alterations in RNA processing, hnRNA abundance correlates well with transcription rates measured by nuclear run-off assays (42, 43). Our results indicate that exogenously added IGF-I down-regulates PTH/PTHrP receptor transcription in UMR-106 osteoblastic cells. Cycloheximide, a protein synthesis inhibitor, completely blocks the decrease in receptor mRNA expression, indicating that de novo protein synthesis is required for IGF-I-induced repression of PTH/PTHrP receptor gene transcription.

Although IGF-I signaling has been studied extensively using various cell lines and primary culture cells, this is the study to define its signal transduction in an osteoblastic cell line. Our results indicate that IGF-I rapidly induces p42/p44 MAP kinase activities and suppresses PTH/PTHrP receptor gene transcription through de novo protein synthesis in UMR-106 osteoblast-like cells. The role of IGF-I in the MAP kinase pathway in UMR-106 cells is confirmed by the use of an inhibitor of MEK, i.e. PD98059. This compound selectively inhibits the activity of MEK and the subsequent activation of ERKs (44, 45). In this study, PD98059 significantly suppressed IGF-I-induced MAP kinase activation in UMR-106 cells. Furthermore, this compound completely abolished the IGF-I-induced suppression of PTH/PTHrP receptor mRNA and receptor expression in osteoblast-like cells.

In contrast, PI 3-kinase activation plays an important role in IGF-I signal transduction, because this enzyme mediates the activation of other protein kinases, such as protein kinase C, S6 kinase, and serine-threonine kinase Akt (46). A recent study has shown that the PI 3-kinase pathway, but not the MAP kinase pathway, is an essential step in the regulation of gene expression, such as the mitogenic action of IGF-I in human breast cancer-derived MCF-7 cells (47), although the PI 3-kinase pathway is associated primarily with metabolic properties of IGF-I, such as stimulation of glucose uptake by cells and stimulation of protein turnover (48, 49). The involvement of PI 3-kinase as a mediator of IGF-I action on PTH/PTHrP receptor gene expression was evaluated by using the specific inhibitor of the enzyme, wortmannin. We showed that wortmannin does not blunt the IGF-I action on PTH/PTHrP receptor gene transcription in UMR-106 osteoblast-like cells. These findings demonstrate that MAP kinase activation is essential in the suppression of PTH/PTHrP receptor gene expression caused by IGF-I. Furthermore, identification of the repressor will be of interest because our results demonstrated that IGF-I signaling inhibits transcription of the PTH/PTHrP receptor gene through newly synthesized repressors.

In summary, we have shown that IGF-I suppresses the expression of the PTH/PTHrP receptor in UMR-106 osteoblast-like cells by transcriptional mechanisms. Inhibition of the MAP kinase cascade with the MEK inhibitor PD98059 blocks these effects. The protein synthesis inhibitor cycloheximide abolishes the IGF-I-induced decrease in PTH/PTHrP receptor mRNA expression. Collectively, these results indicate that inhibition of the PTH/PTHrP receptor expression induced by IGF-I is transduced through MAP kinase pathways, and IGF-I regulates the receptor gene transcript through new protein synthesis.



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  		Figure 7. Effects of PD98059 on PTH/PTHrP receptor number (Bmax) in IGF-I-treated UMR-106 cells. At 70% confluence the medium was changed to serum-free DMEM for 24 h, and cells were treated with 100 nM IGF-I in the presence or absence of 100 µM PD98059 for 24 h. Control cells were treated with vehicle (0.1% Me2SO) alone. These cells were incubated with increasing concentrations of labeled hormone in the absence or presence of 1 µM unlabeled hormone. Bmax values were calculated by Scatchard analysis. Values are expressed as a percentage of the maximum (vehicle control) and are the mean ± SEM of triplicate determinations. *, P < 0.05 (compared with vehicle control).

 

   Acknowledgments
 
We thank Mr. K. Saitoh (Yamasa Shoyu Co., Choshi, Japan) for the cAMP assay kits.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan and in part by a grant from Chugai Pharmaceutical Co. The nucleotide sequence reported in this paper has been submitted to the DDBJ, EMBL, and Genbank under accession no. AB01294. Back

Received May 12, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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