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Endocrinology Vol. 138, No. 5 1995-2004
Copyright © 1997 by The Endocrine Society

Articles

Expression of p27Kip1 in Osteoblast-Like Cells during Differentiation with Parathyroid Hormone1

Takehisa Onishi and Keith Hruska

Renal Division, Barnes-Jewish Hospital, Washington University School of Medicine, St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Keith Hruska, M.D., Renal Division, Barnes-Jewish Hospital North, 216 South Kingshighway Boulevard, St. Louis, Missouri 63110.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH is a major systemic regulator of bone metabolism and plays an important role in both bone formation and resorption. PTH either inhibits or stimulates osteoblastic cell proliferation depending on the model that is studied. We analyzed the cell cycle of the UMR-106 cell line, a relatively differentiated osteoblastic osteogenic sarcoma line in which PTH is known to inhibit proliferation but the mechanism of action is unknown. PTH decreased the proportion of cells in S phase and increased the number of G1 phase cells. We examined the effect of PTH on the regulators of the G1 phase cyclin-dependent kinases and found that PTH increased p27Kip1, but not p21Cip1, levels. This effect was mimicked by 8-bromo-cAMP, but not by phorbol 12-myristate 13-acetate. The protein kinase A inhibitor KT5720 abolished the effect of PTH on the increase in p27Kip1 expression. PTH increased CDK2-associated p27Kip1 without affecting the levels of CDK2. CDK2 activity was down-regulated by both PTH and 8-bromo-cAMP treatment. These data suggest that PTH blocks entry of cells into S phase and inhibits cell proliferation as the consequence of an increase in p27Kip1, which is mediated through the protein kinase A pathway. The inhibition of G1 cyclin-dependent kinases by p27Kip1 could cause a reduction of phosphorylation of key substrates and inactivation of transcription factors essential for entry into S phase. The inhibition of cell cycle progression through PKA-mediated p27Kip1 induction might play an important role in PTH-induced differentiation of osteoblasts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH ACTS ON bone and kidney as a systemic modulator for maintaining calcium and phosphate homeostasis. PTH binds to its receptor and activates two signal transduction systems: the cAMP-dependent protein kinase (PKA) (1, 2) and the phospholipase C-activated calcium/protein kinase C (Ca2+/PKC) pathways (1, 3). PTH receptor expression is a major phenotypic feature of the osteoblast lineage. The effect of PTH on osteoblast function is complex, as it affects both bone resorption and formation (4). In stimulating bone resorption, PTH activates cells in the osteoblast lineage to produce cytokines that regulate osteoclastogenesis and bone resorption. In addition, PTH may stimulate differentiated osteoblasts in a manner that favors bone resorption (4). PTH inhibition of osteoblast proliferation, type 1 collagen gene expression, and stimulation of a collagenase gene are to be considered in this context (4). The effects of PTH on osteoblast proliferation are either stimulatory or inhibitory depending upon the osteoblastic model (5, 6, 7, 8, 9, 10, 11, 12, 13).

Proliferation of the rat osteoblastic osteogenic sarcoma cell line, UMR-106, is inhibited by PTH (5, 10, 13). Activation of PKA by substances such as 8-bromo-cAMP, forskolin, and cholera toxin mimics the inhibitory actions of PTH on proliferation of osteoblastic cells (8, 10, 12, 13), and a previous report has shown that PTH-(1–34) inhibits mitogen-activated protein (MAP) kinase activity in a PKA-dependent manner in both ROS 17/2.8 and UMR-106 cells (14). As the UMR-106 cell line represents a relatively differentiated, well characterized osteoblast phenotype, it was chosen for examination of the mechanisms by which PTH produces inhibition of proliferation in differentiating osteoblasts. As it is a transformed cell, finding aberrations in the control of cell growth was expected, but the actions of PTH to inhibit proliferation are likely to be applicable to other osteoblast-like cells.

Proliferation of eukaryotic cells depends on progression through the cell cycle, and cell cycle control is achieved through the actions of a family of cyclin-dependent protein kinases (CDKs) and cyclins that initiate phosphorylation events to allow progression through checkpoints in the cell cycle. In mammalian cells, progression through the G1 phase requires the activity of the cyclin D-dependent kinases, CDK4 and/or CDK6, early in G1 and the cyclin E-dependent kinase CDK2 (G1 kinases) later in G1. G1 kinases can be regulated by changes in cyclin levels or CDK activity and by a CDK-activating kinase (CAK) (15). CDK4 and CDK6 activities are first detected in mid-G1 phase and increased as cells approach the G1/S boundary (16, 17). The retinoblastoma gene product (pRb) is a key physiological substrate of CDK4 and CDK6, and it negatively regulates transcription factors such as E2F-DP1 heterodimers by binding E2F. The functions of E2F are important for the G1/S transition. pRb phosphorylation by G1 kinases releases transcription factors from pRb and enables them to activate genes whose products are necessary for entry into the S phase (18, 19, 20). Cyclin E is expressed periodically at maximum levels near the G1/S transition, assembling with CDK2 (21, 22). Cyclin E-CDK2 complexes regulate the transition differently from that promoted by the D-type cyclin-CDK4/6 complexes. Cyclin E, but not cyclin D1, is essential for entry into S phase in mammalian cells lacking functional pRb (23, 24, 25). Cyclin E-CDK2 complexes may contribute to pRb phosphorylation late in G1 phase (26, 27) and probably phosphorylate other critical G1/S substrates (28).

Recently, two families of mammalian CDK inhibitors have been discovered that can bind to CDKs and inhibit their kinase activity. One includes p21Cip1 (also known as WAF1, Sdi1, and CAP20) (29, 30, 31, 32, 33), p27Kip1 (34, 35), and p57Kip2 (36, 37). The second family of CDK inhibitors includes p16Ink4A (38), p15Ink4B (39), p18Ink4C (40, 41), and p19Ink4D (41, 42). In vitro, p21Cip1, p27Kip1, and p57Kip2 inhibit a wide variety of cyclin-CDK complexes, including cyclin D-CDK4/6 and cyclin A/E-CDK2 (30, 31, 33, 35, 36, 43), and these inhibitors can prevent the activation of CDKs by binding these cyclin-CDK complexes (43) and thus prevent cells from entering S phase. Ink4 inhibitors are specific for CDK4 and CDK6 and interfere with cyclin D binding to these kinases (38, 40, 41, 42). p27Kip1 and p21Cip1 share 44% amino acid homology in their amino-terminal region, and both inhibit a wide variety of cyclin-CDK complexes (43). Overexpression of p27Kip1 blocks cell cycle progression in G1 phase in Saos-2 osteosarcoma cells or Mv1Lu cells (35, 43). Transforming growth factor-ß has been shown to repress cyclin D or CDK4 synthesis (44, 45) and to increase p15Ink4B synthesis (39); this could induce the release of p27Kip1 from cyclin D-CDK4/CDK6 complexes. In turn, the released p27Kip1 binds to the cyclin E-CDK2 complex, resulting in inhibition of its kinase activity (46). The level of p27Kip1 is increased, and more p27Kip1 accumulates in complexes with cyclin D1-CDK4 in cAMP-treated murine macrophages. Under these circumstances, CAK is blocked by CDK4-bound p27Kip1 from accessing and activating kinases, underscoring the capacity of p27Kip1 not only to inhibit fully functional cyclin-CDK complexes but also to interfere with their activation by CAK (47).

We report herein that the antiproliferative effects of PTH on UMR-106 cells involve inhibition of G1 progression, and we focused our studies on the effect of PTH on the G1 cyclin-dependent kinases. We show that PTH increases p27Kip1 expression in UMR-106 cells leading to reduced CDK2, but not CDK4, activity. PKA-activating substances such as 8-bromo-cAMP mimicked the effect of PTH, but PKC-activating substances such as phorbol 12-myristate 13-acetate (PMA) did not. We also show that UMR-106 cells have no or very little functional pRb, and the association of cyclin D with CDK4 is not seen in this cell line. This suggests that the cyclin E-CDK2 complexes that PTH inhibit are essential for entry into S phase in these cells (23, 24, 25). Our results suggest that PTH blocks entry of cells into S phase and inhibits cell proliferation as the consequence of an increase in p27Kip1 expression. The p27Kip1-mediated inhibition of cyclin E-dependent CDK2 activity presumably causes the hypophosphorylation of an unknown key substrate and inactivation of transcription factors essential for entry into S phase.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Rat PTH [rPTH-(1–34)], bovine PTH [bPTH-(3–34)], 8-bromo-cAMP (8Br-cAMP), and PMA were purchased from Sigma Chemical Co. (St. Louis, MO), and PKA inhibitor KT5720 was obtained from Kamiya Biomedical (Thousand Oaks, CA). PTH (10-5 M) and 8Br-cAMP (100 mM) were dissolved in distilled water and stored at -70 C; PMA (2 x 10-3 M) and KT5720 were dissolved in 100% dimethylsulfoxide (DMSO) and stored at -20 C until use. [3H]Thymidine and [{gamma}-32P]ATP were purchased from Amersham (Arlington Heights, IL). Antibodies against CDK2 (M2), CDK4 (c-22), and p21cip1 (c-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody against p27kip1 was purchased from Transduction Laboratory (Lexington, KY), and that against cyclin D (PMG124–259.5) was purchased from Pharmingen (San Diego, CA). Two kinds of antibodies against pRB (PMG3–245 and c-15) were obtained from Pharmingen and Santa Cruz Biotechnology, respectively.

Cell culture
UMR-106 cells (passages 11–21) were cultured in DMEM containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml) in a water-jacketed incubator with a humidified atmosphere (5% CO2-95% air) and maintained at 37 C. Experimental cultures were grown to 70–80% confluence in serum-containing medium, and then cells were serum deprived by replacing the medium with DMEM containing 0.1% bovine albumin (serum-free DMEM) for 18–24 h before the addition of test agents for all experiments.

Cell cycle analysis
Cell cycle distribution was analyzed by flow cytometry. One to 2 x 106 cells incubated in serum-free DMEM with or without PTH (10-8 M) were trypsinized, washed once with PBS, and fixed in 70% ethanol for at least 1 h at 4 C. Fixed cells were washed with PBS and incubated with PI staining solution (0.05 mg/ml propidium iodine, 0.1% sodium citrate, 20 µg/ml ribonuclease A, and 0.3% Nonidet P-40, pH 8.3) for 30 min at 4 C in the dark. The stained cells were analyzed by FACScan (Becton Dickinson, Mountain View, CA).

[3H]Thymidine incorporation assay
Cells were plated in 24-well plastic dishes and maintained in DMEM containing 10% FBS until they were approximately 70–80% confluent, washed with PBS, and refed with serum-free DMEM containing test agents for 24 h. Two hours before cell harvest, cells were labeled with [3H]thymidine (1 µCi/well). After washing with PBS twice, cells were incubated in 1 ml 10% (wt/vol) trichloroacetic acid (TCA) for 20 min on ice, washed twice with cold 5% TCA, and solubilized with 0.5 ml 0.5 N NaCl for 10 min on ice; 0.25 ml 1 N HCl and 0.25 ml 40% TCA were added to each well (final concentration of TCA, 10%) for 20 min, and [3H]thymidine incorporation was determined by scintillation counting.

Immunoblot analysis
Cells were rinsed twice with ice-cold PBS and lysed in 1 ml Nonidet P-40 lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM NaF, 5 mM EDTA, 5 mM EGTA, 2 mM sodium vanadate, 0.5% sodium deoxycholate 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonylfluoride, 2 µg/ml aprotinin, and 0.1% Nonidet P-40]. Cells were scraped after rocking for 30 min at 4 C, and lysates were cleared by centrifugation at 16,000 x g for 5 min at 4 C (total cell lysate). The protein concentration was measured at least twice using the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA). For immunoblot analysis, protein samples (50 µg) were boiled for 5 min in Laemmli buffer [62.5 mM Tris (pH 6.8), 2% SDS, 20% glycerol, 0.01% bromophenol blue, and 100 mM DTT] and separated on 7–12% SDS-PAGE. Gels were then transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) using a semidry transfer method. The transferred membranes were soaked in Ponceau-S solution (Sigma) to check that comparable amounts of proteins were loaded on the gel and the homogeneity of the transfer. After blocking with PBS-T [PBS (pH 7.4) and 0.1% Tween-20] containing 5% low fat milk, the membranes were incubated with primary antibody for 1–2 h in PBS-T, incubated with secondary antibody conjugated with horseradish peroxidase for 1 h, and developed by the chemiluminescence detection system (Pierce Chemical Co., Rockford, IL).

Immunoprecipitation
To investigate CDK2/4-p27kip1 or CDK4-cyclin D complex formation, total lysates were immunoprecipitated with rabbit polyclonal anti-CDK2 or anti-CDK4 antibodies for 2 h at 4 C, and the immunoprecipitates were brought down with protein A-Sepharose beads (1 h at 4 C), and pellets were washed four times with ice-cold Nonidet P-40 lysis buffer. The samples were separated on 10–12% SDS-PAGE as described for immunoblots.

In vitro protein kinase assays
For CDK2 assay, cell lysates were immunoprecipitated with anti-CDK2 antibody as described above. After a final wash with Nonidet P-40 lysis buffer, immunoprecipitates were washed twice with H1 kinase buffer [50 mM HEPES (pH 7.5), 1 mM DTT, 1 mM EDTA, 10 mM MgCl2, 0.1 mM sodium orthovanadate, 1 mM NaF, 15 mM ß-glycerophosphate, and 1 mM phenylmethylsulfonylfluoride] and then resuspended in 40 µl H1 kinase buffer containing 200 µM ATP, 5 µCi [{gamma}-32P]ATP (6000 Ci/mmol; Amersham), and 5 µg histone H1. After incubation for 30 min at 30 C, the reactions were stopped by the addition of 25 µl 2 x Laemmli sample buffer. The samples were boiled for 5 min and separated on 12% SDS-PAGE; the gel was dried and visualized by autoradiography. For CDK4 assays, immunoprecipitates of cell lysates were prepared using the anti-cdk4 antibody. After the final wash with Nonidet P-40 lysis buffer, immunoprecipitates were washed three times with Rb kinase buffer [50 mM HEPES (pH 7.5), 1 mM DTT, 10 mM MgCl2, 10 mM ß-glycerophosphate, 1 mM NaF, and 0.1 mM sodium orthovanadate]; resuspended in 50 µl Rb kinase buffer containing 20 µM ATP, 5 µCi [{gamma}-32P]ATP, and 1.5 µg gluthathione-S-transferase (GST)-Rb fusion protein as a substrate; and incubated for 30 min at 30 C. The reactions were stopped as described above. The samples were separated on 8% SDS-PAGE and visualized by autoradiography as described for the histone H1 kinase assay. All assays were performed at least twice.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of G1 progression by PTH
Initial experiments were performed to investigate the effect of PTH on DNA synthesis and progression of the cell cycle. Consistent with previous reports (5, 12), the incorporation of [3H]thymidine into DNA decreased in PTH-treated UMR-106 cells compared to that in vehicle-treated cells (7807 ± 943 vs. 18767 ± 1990 cpm/well; P < 0.001; n = 4). This result indicated that the population of cells entering into S phase was reduced by PTH. Analysis by fluorescence-activated cell sorting revealed that PTH treatment resulted in a higher proportion of cells in G1 (Fig. 1Go). Seventy-three percent of cells treated with PTH for 12 h, and 76% of cells treated with PTH for 24 h were in G1 compared to 56% of control cells. As a consequence, fewer cells were in S phase (19% of cells treated with PTH for 12 h, 10% of cells treated with PTH for 24 h) compared to control cells (31%; Fig. 1Go). Repeated experiments revealed a mean reduction of 22 ± 3% (n = 4; P < 0.01) of cells in S phase and a coordinate increase in G1 phase cells. There was no effect of PTH on G2, except that the proportion of cells in S phase decreased, and the proportion of G2 phase cells increased from 12–24 h as expected with a G1 phase block.



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  		Figure 1. The effect of PTH on cell cycle progression. UMR-106 cells were incubated in serum-free DMEM for 24 h (0 h) and then treated with rPTH (10-8 M) for 12 and 24 h or with vehicle for 24 h (control). The distribution of cells in the cell cycle was analyzed by flow cytometry (fluorescence-activated cell sorting) analysis of DNA content.

 
Effects of PTH on the expression of G1 CDKs inhibitors
As PTH inhibited progression from G1 phase in UMR-106 cells, we focused on the inhibitors of G1 CDKs, p27Kip1 and p21Cip1. To investigate the effect of PTH on p27Kip1 and p21Cip1 expression, the levels of p27Kip1 and p21Cip1 were detected by immunoblotting with specific antibodies. p27Kip1 levels increased approximately 2.7-fold in cells treated with PTH for 12 h and 3.5-fold in cells treated with PTH for 24 h relative to those in control cells (Fig. 2Go, A and B), whereas p21Cip1 levels were unaffected by PTH treatment (Fig. 2CGo). Densitometric scans of the experiment portrayed in Fig. 2BGo and another identical experiment revealed the fold change in p21Cip1 levels to be 1.01 and 1.15 at 12 and 24 h, respectively. These results suggest that PTH-induced G1 inhibition in UMR-106 cells is associated with an increase in p27Kip1 levels.



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  		Figure 2. PTH increases p27Kip1, but not p21Cip1 expression. UMR-106 cells were treated as described in Fig. 1Go, and cell lysates (50 µg) were prepared as described in Materials and Methods. The lysates were separated on 12% SDS-PAGE, and immunoblots were prepared using anti-p27Kip1 (A) or anti-p21Cip1 (C) antibodies. Densitometric analyses were performed on the anti-p27Kip1 immunoblots from three experiments (B). The bar graphs represent the mean ± SD. *, P < 0.01 compared to the corresponding control (-) value.

 
Effects of PTH on the association of G1 CDKs with p27Kip1
p27Kip1 is one of a family of CDK inhibitory proteins that control cell cycle progression by binding to and inactivating cyclin-CDK complexes, including cyclin D-CDK4/6 and cyclin A/E-CDK2 complexes (46). In addition, p27Kip1 may directly inhibit CDK activity (47). To demonstrate that PTH affects the association of G1 CDKs with p27Kip1, immunoprecipitations of total cell extracts from PTH-treated or untreated cells were performed using an anti-Cdk2 antibody or an anti-Cdk4 antibody. Immunoprecipitates were separated on 12% SDS-PAGE, and immunoblots were prepared using an anti-p27 antibody. As shown in Fig. 3AGo, the levels of p27Kip1 bound to CDK2 were increased by PTH treatment. Densitometric scans of the experiment shown in Fig. 3AGo and one other experiment demonstrated the stimulation by PTH to be 2.7-fold in cells treated for 12 h and 4.4-fold in cells treated for 24 h. CDK2 levels were not altered by PTH (Fig. 3BGo). Induction of CDK2 by PTH was 1.21- and 0.88-fold at 12 and 24 h, respectively. These results indicate that only association of p27Kip1 with CDK2 is increased by PTH. However, an interaction of CDK4 with p27Kip1 was not seen in UMR cells (Fig. 3CGo). Previous reports have shown that in cells that lack functional pRb, the D-type cyclins are not found associated with either CDK4 or CDK6 (24, 48, 49). We considered that UMR cells might not express pRb or, if present, it would not be functional. To confirm this, we investigated the association of CDK4 with cyclin D and pRb expression in UMR-106 cells. Immunoprecipitates of total cell lysates from UMR-106 cells and NIH 3T3 cells were prepared using an anti-CDK4 antibody. The immunoprecipitates were separated on 12% SDS-PAGE followed by immunoblotting with an anti-cyclin D antibody that recognizes cyclin D1, D2, and D3. In NIH 3T3 cells, cyclin D formed detectable complexes with CDK4, but not in UMR cells (Fig. 4AGo). Next, to investigate whether pRb is expressed in UMR cells, total cell lysates from UMR cells and another osteoblastic cell line, TE-85, were examined by immunoblotting with two kinds of anti-Rb antibodies. As shown in Fig. 4BGo, pRb expression was detected in growing TE-85 cells (lane 1 in TE-85), and faster and slower migrating bands (unphosphorylated and phosphorylated pRb) were seen in serum-deprived TE-85 cells (lane 2), whereas they were not detectable in UMR cells.



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  		Figure 3. PTH increases the association of p27Kip1 with CDK2. Cells were treated as described in Fig. 1Go. The effect of PTH on the levels of CDK2-associated p27Kip1 (A), CDK2 (B), and CDK4-associated p27Kip1 (C) were examined by immunoprecipitation with respective antibodies and subsequent immunoblotting with anti-p27Kip1 or Cdk2 antibody. The same membrane as that in A was stripped and reprobed with anti-CDK2 antibody in B. Rabbit nonimmune serum (NIS) was used instead of anti-Cdk2 antibody to show the specificity of the immunoprecipitation. In A and B, 3 mg whole cell lysates from PTH-treated or untreated cells were used for the immunoprecipitations. In C, 5 mg total cell lysates from cells treated with (lane 1) or without (lane 2) PTH for 24 h and from cells cultured in DMEM containing 10% FBS (lane 3) were used for the immunoprecipitation. NIH-3T3 cells were used as a positive control.

 


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  		Figure 4. Cyclin D-CDK4 complex and pRb expression are not observed in UMR-106 cells. Total cell lysates (4 mg) from cells treated with (lane 1) or without (lane 2) PTH for 24 h and cells cultured in DMEM containing 10% FBS (lane 3) were immunoprecipitated with anti-CDK4 antibody and then probed with anti-cyclin D antibody (A). The expression of pRb in UMR-106 and TE-85 cells was examined by immunoblots of whole cell extracts [100 µg for UMR cells, 50 µg for TE-85 cells cultured in serum containing medium (lane 1), or serum-free medium (lane 2)] separated on 7% SDS-PAGE (B). NIH-3T3 cells and TE-85 cells were used as positive controls. In pRb immunoblotting, similar results were obtained from different anti-pRb antibodies [only the immunoblots with antibody against pRb (PMG3–245) are shown].

 
Effects of second messenger analogs on p27Kip1 expression
PTH is known to activate various signal transduction pathways in osteoblastic cells (1, 2, 3, 4). To further determine the signal transduction pathway(s) involved in increasing p27Kip1 expression, we examined the abilities of different second messenger analogs to mimic the action of PTH. The PKA activator 8Br-cAMP (1 mM) and the PKC activator PMA (10-6 and 10-7 M) significantly decreased the incorporation of [3H]thymidine into DNA as well as PTH (10-8 M) (Fig. 5Go). Analysis of immunoblots demonstrated that p27Kip1 expression was increased in the cells treated with 8Br-cAMP, but not in those treated with 10-6 M PMA (Fig. 6AGo). Densitometric scans of the experiment in Fig. 6AGo and two additional experiments revealed that p27Kip1 expression was induced 3.5-fold by PTH-(1–34), 3.8-fold by 8Br-cAMP, and 1.1-fold by PMA. To confirm the idea that activation of PKA is necessary for the induction of p27Kip1, we next used PTH-(3–34), a PTH analog that does not activate PKA but can activate PKC in these cells (1, 50, 51), and the PKA inhibitor KT5720. PTH-(3–34) had no effect on p27Kip1 expression. The significant elevation of p27Kip1 expression induced by PTH-(1–34) was not observed in the cells treated with PTH plus KT5720 (Fig. 6BGo; 3.2- vs. 1.4-fold, respectively, by densitometry of two experiments). These data suggest that the effect of PTH to inhibit DNA synthesis may be mediated by a mechanism that involves both PKA and PKC activation. However, the increase in p27Kip1 expression by PTH is mediated by the cAMP-PKA-dependent pathway.



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  		Figure 5. Effects of second messenger analogs on DNA synthesis. Serum-deprived UMR-106 cells were incubated in the serum-free DMEM for 24 h in the absence (control for 8Br-cAMP and PTH, C) or presence of test agents (1 mM 8Br-cAMP, 10-8 M PTH, and 10-8, 10-7, and 10-6 M PMA). DMSO (0.1%) was used as a vehicle for PMA. [3H]Thymidine (0.5 µCi/well) was present during the last 2 h of incubation. The data represent the mean ± SE of six wells. *, P < 0.001 (vs. C). **, P < 0.01; ***, P < 0.001 (vs. DMSO). Similar results were obtained in two other independent experiments.

 


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  		Figure 6. Effects of second messenger analogs on p27Kip1 expression. A, Serum-deprived cells were cultured in serum-free DMEM for 24 h in the absence (control) or presence of test agents (1 mM 8Br-cAMP, 10-6 M PMA, and 10-8 M PTH). DMSO (0.1%) was used as a vehicle for PMA. B, Cells were treated with test agents [10-8 M PTH-(1–34) and 10-8 M PTH-(3–34)] for 24 h. For inactivation of PKA, cells were pretreated with KT5720 (10-6 M) for 1 h, then 10-8 M PTH-(1–34) was added in the culture medium, and cells were incubated for 24 h. Total cell lysates (50 µg) were analyzed by immunoblotting with anti-p27Kip1 antibody.

 
Effects of PTH and cAMP on the activities of G1 CDKs
We postulated that the PTH-mediated increase in p27Kip1 expression and its binding to CDK2/cyclin E complexes would reduce CDK2 activity. Immunoprecipitates of 0.5 mg total UMR-106 cell lysates were prepared using an antibody against CDK2. The immunoprecipitates were used in protein kinase assays in vitro. The activity of CDK2, as determined by the phosphorylation of histone H1 in vitro, was decreased approximately 50% by the immunoprecipitates from cells treated with PTH for 12 h compared to the effect of immunoprecipitates prepared from control cells. After 24-h treatment of PTH, CDK2 activity was only 3–6% that of control cells (Fig. 7AGo). Similar results were observed in cells treated with 8Br-cAMP (Fig. 7BGo). The data in Fig. 7Go are representative of four experiments with similar results. We next measured CDK4 activity by assessing the phosphorylation of its natural substrate, Rb, in vitro. Immunoprecipitations of 1 mg total cell lysates, prepared using an anti-CDK4 antibody, were used for protein kinase assays, and kinase activities were visualized by autoradiography at -70 C for 48 h. Judging from the exposure time, the kinase activity of CDK4 was very low compared with CDK2 activity, and CDK4 activity was unaffected by PTH (Fig. 7CGo). The results of the CDK4 kinase assays were consistent with the findings that there is no or very little interaction of CDK4 with cyclin D in UMR cells, and its activity is constitutive and unregulated.



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  		Figure 7. PTH decreases CDK2 activity, but not CDK4 activity. Anti-CDK2 immunoprecipitates from PTH-treated or untreated cells (A) or from 8Br-cAMP-treated or untreated cells (B) were used for protein kinase assays in vitro with histone H1 as a substrate. Anti-CDK4 immunoprecipitates from cells treated with or without PTH for 12, 18, or 24 h were also used for protein kinase assays in vitro with GST-pRb (90KD) as a substrate (C). The amounts of total cell lysates used for the anti-CDK2 immunoprecipitations were 0.5 mg (A) and 1.5 mg (B). One milligram of anti-CDK4 immunoprecipitate was used (C). The exposure times for immunoblots were 1 h (A) and 45 min (B) at room temperature and 48 h (C) at -70 C.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Many reports have shown that PTH has either proliferative or antiproliferative effects on osteoblastic cells (5, 6, 7, 8, 9, 10, 11, 12, 13), and the effect of PTH on osteoblast differentiation is either stimulatory or inhibitory depending on the osteoblastic model, the method of PTH administration, and the stage of differentiation (52, 53, 54). We hypothesized that PTH could stimulate proliferation of early osteoprogenitors while inhibiting proliferation and further stimulating differentiation in more mature cells of the lineage. To test the hypothesis and investigate the mechanisms of PTH-mediated proliferative actions, we used the human osteosarcoma cell line TE-85, in which PTH is known to stimulate proliferation (9). We have shown that PTH stimulates progression of the cell cycle and increases CDC2 (cyclin-dependent kinase 1) expression and its activity through the actions of the E2F transcription factor (unpublished data). As a next step and as reported here, we examined the mechanisms of the antiproliferative effects of PTH on osteoblastic cells by using the UMR-106 cell line, in which PTH is known to inhibit proliferation (5, 10, 12, 13). UMR-106 cells are more differentiated in the osteoblast lineage than the TE-85 cell line, in agreement with our original hypothesis. PTH inhibited G1 progression to S phase in UMR-106 cells, and as a result, we analyzed the actions of PTH on the G1 phase cyclin-CDK inhibitors.

In mammalian cells, the activities of cyclin D-dependent CDK4 and/or CDK6 and cyclin E-dependent CDK2 are implicated in the regulation of G1/S progression. One of the CDK inhibitor families, the Cip/Kip family (p21Cip1, p27Kip1, and p57Kip2), blocks the kinase activity of G1 cyclin-CDK complexes (reviewed in Ref.46). p21Cip1 expression is increased in a p53-dependent manner in cells containing damaged DNA and independently of p53 in postmitotic, terminally differentiated cells (55, 56, 57, 58, 59). p27Kip1 expression, on the other hand, primarily increases in response to extracellular antimitogenic signals (60, 61, 62, 63, 64). Thus, p27Kip1 seems to be essential for the regulation of cell cycle events at the restriction point in G1. Our data demonstrate that the level of p27Kip1 increased, whereas that of p21Cip1 was unaffected by PTH in UMR cells. These results suggest that the antimitotic effect of PTH on G1 arrest in this cell line may be mediated predominantly by p27Kip1, although p57Kip2 was not investigated. The latter is not expressed widely, and it is not known to be in bone cells.

Correlated with the elevation of p27Kip1 expression, the binding of p27Kip1 with CDK2 is increased by PTH. As the binding of p27Kip1 with CDK2 increased, CDK2 activity was decreased to 50% of control levels at 12 h and was strongly reduced at 24 h by PTH treatment. The effect of p27Kip1 to inhibit CDK2 by binding cyclin E-CDK2 complexes was probably critical for the block in the cell cycle, because no effect on PTH was seen in the CDK4 activity.

In fact, an association of p27Kip1 with CDK4 was not seen in UMR-106 cells, and binding of cyclin D with CDK4 was not observed. The retinoblastoma gene product (pRB) is one of the putative substrates for cyclin D-CDK4 or CDK6 and cyclin E-CDK2 complexes. Phosphorylation of pRb appears to release transcription factors whose functions are important for G1/S transition, such as E2F-DP1 heterodimers (18, 19, 20). However, in cells lacking functional pRb, CDK4 and CDK6 are not associated with D-type cyclins. Instead, both kinases form complexes with p16INK4A, which interfere with binding of D-type cyclins to CDK4 and CDK6 and prevent formation of active complexes (38, 40, 48, 49). This evidence and our results suggest that there is no functional pRb in UMR-106 cells. Two different anti-pRb antibodies, which recognize the epitopes between amino acids 300–380 (Pharmingen) and 914–928 (Santa Cruz) of the human pRb, were used for immunoblotting, but pRb expression was not detectable in UMR-106 cells. CDK4 activity appeared to be very low, because in the CDK2 assays, 0.5 mg total lysates was immunoprecipitated, and only 1-h exposure at room temperature was required to visualize the activities by autoradiography, whereas in CDK4 assays, larger amounts of total cell lysates (1 mg) were immunoprecipitated and longer exposure times (48 h at -70 C) were used. An inhibitory effect of PTH on CDK4 activity was not found. These results suggest that UMR cells have no functional pRb, which results in a lack of association of cyclin D with CDK4 and very low CDK4 activity, although we have not examined p16INK4A levels in this cell line.

Several lines of evidence suggest that cyclin D and cyclin E are rate limiting for the G1/S transition (65, 66, 67, 68, 69), and it has been reported that expression of either cyclin D1 or E alone advances the G1/S transition to a similar extent. Expression of cyclin D1 leads to the immediate appearance of hyperphosphorylated pRb, whereas expression of cyclin E does not. Furthermore, expression of both cyclins has an additive effect on shortening of the G1 interval, whereas the effect on pRb phosphorylation is similar to that of cyclin D1 alone in rat fibroblasts engineered to express inducible cyclin D1 and cyclin E (28). These results suggest that cyclin D1 and cyclin E control two different events, both partly rate limiting for the G1/S transition. pRb phosphorylation is likely to be controlled by cyclin D-CDK4, whereas other unknown key substrates critical for G1/S progression would be the target of cyclin E-CDK2 (Fig. 8Go). This could be the explanation for the events that occur in the G1/S progression in UMR-106 cells.



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  		Figure 8. Proposed model for signal transduction pathway of PTH-mediated G1 arrest through p27Kip1 in UMR-106. PTH increases p27Kip1 levels through activation of PKA. The increase in p27Kip1 inhibits CDK2 activity by binding to cyclin E-CDK2 complexes and inhibiting CAK activity. The inhibition of CDK2 activity decreases phosphorylation of the key substrate(s) and maintains transcription factors (TF) in inactive modes. Thus, cells fail to enter into S phase. One such key substrate of CDK2 is the retinoblastoma gene product, pRb. When Rb is not phosphorylated, it binds the E2F transcription factor, which is the key for G1/S progression. pRb phosphorylation releases active E2F dimers, such as E2F/DP1.

 
PTH and its receptor result in activation of at least two signal transduction pathways: 1) cAMP-dependent PKA, and 2) phospholipase C-activated Ca/PKC (1, 2, 3). To determine the signal transduction pathway(s) involved in the PTH stimulation of p27Kip1 expression, second messenger analogs were tested for the ability to mimic the action of PTH. Both the PKA activator 8Br-cAMP and the PKC activator PMA significantly decreased DNA synthesis, as was seen with PTH. Consistent with the previous report that the levels of p27Kip1 increase in cAMP-treated macrophages (47), p27Kip1 expression was elevated, and CDK2 activity was inhibited by 8Br-cAMP. However, PMA had no effect on p27Kip1 expression. Interestingly, in vascular smooth muscle cells, PMA inhibited pRb phosphorylation, but it was not mediated by suppressing CDK4 activity, but involved the suppression of CDK2 activity that resulted from the reduced expression of cyclin E and A (70). Although the mechanism by which PKA and PKC inhibit G1/S progression remains to be investigated, and various cells could have different mechanisms, our data suggest that the cAMP-dependent PKA pathway may play an important role in PTH-mediated p27Kip1 expression. The mechanism of cell growth inhibition mediated by PKA appears to be related to p27Kip1 expression, but that of the PKC pathway must be different.

There have been conflicting reports on the effects of PTH on UMR-106 cells. PTH has been shown to increase the expression of protooncogenes such as c-fos and c-jun, which are believed to be associated with cell proliferation, through the PKA pathway (71). On the other hand, PTH has been reported to inhibit MAP kinase activity in UMR cells (14), and the effect of PTH is mimicked by PKA-activating substances such as 8Br-cAMP and forskolin. PTH-(3–34), a PTH analog that does not activate PKA in these cells, is unable to inhibit the activation of MAP kinase (14). Thus, the regulation of osteoblastic cell growth by PTH is complex and perhaps involves the interaction of several second messenger pathways. Although the signal transduction pathway(s) that links the antiproliferative signal of PTH to the G1 phase events in UMR cell cycle are unknown, the inhibition of MAP kinase by PTH might be one of the candidates up-stream of elevation of p27Kip1 through the PTH-mediated PKA-dependent pathway.

Disruption of p27Kip1 enhances the growth of mice without gross morphological abnormalities and increases the proliferation of hematopoietic progenitor cells. Furthermore, p27Kip1 deletion leads to nodular hyperplasia in the intermediate lobe of the pituitary (72, 73, 74). These data indicate the importance of p27Kip1 as a regulator of cell growth. Antimitogenic signals are essential for the maintenance of balanced tissue growth and cell differentiation. As several lines of evidence have shown, antiproliferative signals lead to the blockade of cell cycle progression by inhibiting various CDKs, through CDK inhibitors (63, 64, 75, 76).

In conclusion, we have shown that the growth inhibitory response to PTH in UMR cells includes the induction of p27Kip1 expression and its inhibition of CDK2 activity. The induction of p27Kip1 is mediated by PTH activating the PKA pathway. This inhibition of cell cycle progression by PTH might play an important role in the actions of the hormone on osteoblast-like cells at specific stages of differentiation. The analysis of the mechanism of PTH action on the cell cycle of normal cells in the human osteoblast lineage is in progress.


   Acknowledgments
 
We thank Paveen Chand (Flow Cytometry Core Laboratory, ashington University School of Medicine) for flow cytometry analysis, and Dr. Douglas Dean (Washington University School of Medicine) for providing GST-pRb.


   Footnotes
 
1 This work was supported by NIH Grants AR-39561 (to K.A.H.) and DK-09976 (to K.A.H.). Back

Received October 28, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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