help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chan, G. K.
Right arrow Articles by Karaplis, A. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chan, G. K.
Right arrow Articles by Karaplis, A. C.
Endocrinology Vol. 142, No. 11 4900-4909
Copyright © 2001 by The Endocrine Society

ARTICLES

PTHrP Inhibits Adipocyte Differentiation by Down-Regulating PPAR{gamma} Activity via a MAPK-Dependent Pathway

George K. Chan, Ron A. Deckelbaum, Isabel Bolivar, David Goltzman and Andrew C. Karaplis

Division of Endocrinology, Department of Medicine, McGill University (G.K.C., R.A.D., A.C.K.), and Lady Davis Institute for Medical Research, Montréal, Québec, Canada H3T 1E2; and Calcium Research Laboratory, Department of Medicine, McGill University Health Center and McGill University (G.K.C., R.A.D., I.B., D.G.), Montréal, Québec, Canada H3A 1A1

Address all correspondence and requests for reprints to: Andrew C. Karaplis, M.D., Ph.D., Lady Davis Institute for Medical Research, Department of Medicine, McGill University, Montréal, Québec, Canada H3T 1E2. E-mail: akarapli{at}ldi.jgh.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined the capacity of PTHrP to modulate the terminal differentiation of the preadipocytic cell line, 3T3-L1. These cells express endogenous PTHrP and its receptor, but expression levels were undetectable after differentiation into mature adipocytes. Cells stably overexpressing PTHrP failed to differentiate when induced to undergo adipogenesis and proliferated at a faster rate. MAPK activity was elevated in PTHrP-transfected 3T3-L1 cells, and treatment with the PKA inhibitor H-8 decreased this activity. Inhibition of MAPK kinase with PD098059 permitted terminal differentiation of PTHrP-transfected 3T3-L1 cells to proceed. Although PPAR{gamma} gene expression levels remained relatively constant in the PTHrP-transfected cells, PPAR{gamma} phosphorylation was enhanced. Furthermore, the capacity of PPAR{gamma} to stimulate transcription in the presence of troglitazone was diminished by PTHrP. Expression of the PPAR{gamma}-regulated adipocytespecific gene aP2 transiently rose and then fell in PTHrP-transfected cells. These results indicate that PTHrP can increase MAPK activity in 3T3-L1 cells via the PKA pathway, thereby enhancing PPAR{gamma} phosphorylation. This modification can inactivate the transcriptional enhancing activity of PPAR{gamma} and diminish the expression of adipocyte-specific genes. These studies therefore demonstrate that PTHrP may inhibit the terminal differentiation of preadipocytes and describe a molecular pathway by which this action can be achieved.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOBLASTS, THE PRINCIPLE bone-forming cells, and adipocytes are believed to originate from the same pluripotent bone marrow stromal stem cells, and their relative numbers in bone are ultimately determined by the number of stem cells that commit to each of these lineages (1, 2). Signals for cell fate determination therefore regulate whether pluripotent stem cells pursue one specific differentiation program relative to another. One hypothesis is that the increase in bone marrow adiposity observed in many forms of osteoporosis arises as a consequence of a shift in the differentiation program of the common precursor cell. Such a preferential differentiation process is detrimental, for it leads to a reduction in functional osteoblasts, decreased bone formation, and, ultimately, the osteopenic state.

Evidence also exists that the relationship between adipocytes and osteoblasts may extend beyond that of simply sharing the same precursor. Committed adipocytes and osteogenic cells, for example, exhibit a form of plasticity that allows for transdifferentiation between the two cell types. Thus, after cells have assumed an adipogenic phenotype, they are capable of reverting to a more immature state and pursue an osteogenic fate (3). Furthermore, primary osteogenic cultures can undergo adipogenic differentiation when treated with glucocorticoids or thiazolidinediones, which activate the glucocorticoid receptor and the receptor for the adipocyte master differentiation factor, PPAR{gamma}, respectively (4). Alternatively, when the same osteogenic cells are treated with 1,25-dihydroxyvitamin D3, the cells resist adipogenesis, and an increase in the expression of phenotypic markers of bone, i.e. osteocalcin, type I collagen, and alkaline phosphatase, is observed. It is therefore critical to understand the molecular switches regulating cell fate determination within the bone marrow microenvironment.

PTH and PTHrP have empirically been shown to have potent anabolic effects on bone. Although presently, there is little understanding of the factors that tend to favor these anabolic effects, by careful selection of the dose and pattern of administration, these agents stimulate bone formation in adult and aged animals of either sex, and in animals with osteopenia induced by disuse, denervation, and immobilization (for review, see Ref. 5 and references therein). On the other hand, we observed that young heterozygous mice carrying a targeted PTHrP-null allele display reduced PTHrP expression in bone and a premature form of osteoporosis characterized by decreased trabecular bone volume and increased bone marrow adiposity (6). Given that osteoblasts and adipocytes originate from the same pluripotent stem cells (7, 8), the increased number of adipocytes observed in the bone marrow of these mice could be the result of pluripotent mesenchymal cells committing to the adipocytic lineage with a concomitant decrease in osteoblastogenesis, as a consequence of PTHrP haploinsufficiency within the skeletal microenvironment.

We show here that PTHrP and the PTH/PTHrP receptor are expressed in cells of the adipocytic lineage and that PTHrP signaling by the cAMP-dependent PKA enhances MAPK activity, leading to phosphorylation of PPAR{gamma}, the master regulator of adipocyte differentiation, and thereby repression of the adipogenic differentiation program. These studies, therefore, identify inhibition of adipogenesis within the bone marrow as a novel mechanism for at least part of the anabolic action of PTHrP, PTH, and their analogs in bone and in the treatment of osteoporosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The 3T3-L1 cell line was obtained from American Type Culture Collection (Manassas, VA), and cells were maintained in DMEM containing 10% FCS, with fresh medium being applied every second day. To induce adipocytic differentiation of 3T3-L1 cells, cells were allowed to grow to confluence, followed by treatment with DMEM-10% FCS supplemented with 0.5 M isobutylmethylxanthine, 1 µM dexamethasone, and 5 µg/ml insulin. In preliminary experiments, periods of induction from 18 to 48 h were assessed. Inasmuch as no increase in differentiation was observed in our system after 18 h of induction, this time was employed in subsequent studies. After 18 h of exposure to differentiation medium, cells were subsequently cultured in DMEM-10% FCS supplemented with 5 µg/ml insulin, which was changed every second day. In some experiments, forskolin was applied at a concentration of 100 nmol/ml culture medium.

C3H10T1/2 cells were obtained from American Type Culture Collection and maintained in DMEM containing 10% FCS, with fresh medium applied every second day. To induce adipocytic differentiation, cells were grown to confluence, followed by treatment with {alpha}MEM-5% FCS supplemented with 100 µg/ml ascorbic acid, 5 mM ß-glycerophosphate, and 100 ng/ml bone morphogenetic protein-2 (BMP2; Genetics Institute, Cambridge, MA), with fresh medium applied every 3 d.

For the proliferation assay, 3T3-L1 cells were plated in triplicate at an initial density of 10,000 cells/well in 6-well plates and then trypsinized and counted every second day for 6 d, once again on d 10, and then again on d 14. For treatment with PD098059 (Sigma, St. Louis, MO), differentiation and postdifferentiation media were supplemented with 20 nmol PD098059/ml medium. The PKA inhibitor H8 (10 nmol/ml culture medium) and the PKC inhibitor chelerythrin chloride (5 nmol/ml culture medium) were added for a period of 24 h to subconfluent cells before lysis.

Oil Red O staining
Cells were washed twice with PBS, then fixed for 30 min with 10% formalin. Oil Red O stain (5% Oil Red 0 in 70% pyridine) was applied for 30 min, and cells were then washed three times with PBS.

Vectors and transfections
The PTHrP/pCDNA3 plasmid was constructed as previously described (9). 3T3-L1 cells were stably transfected with 5 µg of either PTHrP/pCDNA3 or pCDNA3 (Invitrogen, San Diego, CA) plasmid DNA using a Gene Pulser (Bio-Rad Laboratories, Inc., Hercules, CA; 0.2 kV and 960 µF). Cells were allowed to recover for 36 h, and selection for stable transformants was accomplished using 400 µg/ml Geneticin (Life Technologies, Inc., Grand Island, NY). For subsequent experiments, populations of stably transfected cells were used. C3H10T1/2 cells were stably transfected with 1 µg of either PTHrP/pCDNA3 or pCDNA3 plasmid DNA using FUGENE6 reagent (Roche Molecular Biochemicals, Indianapolis, IN). Stable transfected cells were selected using 400 µg/ml Geneticin. COS-7 cells were stably transfected with cDNA of the PTH/PTHrP receptor cloned into the expression plasmid pCDNA3.1 (Invitrogen), provided by G. Hendy. Transfection was performed using FUGENE6 reagent. Cells were then selected for stable transformants with 400 µg/ml Geneticin.

RT-PCR and Northern blot analysis
Total RNA was isolated by a variation of the CsCl method, as previously described (10). For RT of RNA, 100 ng total RNA in 10 µl diethylpyrocarbonate-treated H2O was used as template. RNA was denatured in the presence of 1 mM oligo(deoxythymidine) for 5 min at 80 C and then allowed to cool to room temperature. To the reaction, 6 µl 2.5 mM deoxy-NTPs, 6 µl first strand buffer, 4 µl dithiothreitol (100 mM), 0.5 µl BSA (5 µg/ml), 1.0 µl RNasin inhibitor (Promega Corp., Madison, WI), and 1.0 µl SuperScript polymerase (Roche Molecular Biochemicals) were added. The mixture was incubated at 40 C for 1 h and then amplified in Ready to Go PCR tubes (Amersham Pharmacia Biotech, Arlington Heights, IL) for 45 cycles (melting at 94 C, annealing at 60 C, and extension at 72 C, each for 30 sec, followed by an extension cycle at 72 C for 7 min). Primers for PTHrP (5'-TAC AAA GAG CAG CCA CTC-3' and 5'-GAT CCC AAT GCA TTT ACA GT-3', forward and reverse, respectively) and PTH/PTHrP receptor (5'-TGG TGA GGT GCA GGC AGA GAT TAG-3'and 5'-AAA CAC TGG CTT CTT GGT CCA TC-3', forward and reverse, respectively) were designed to span splice sites in their respective cDNAs. The amplification products for PTHrP were fractionated on agarose gel and subjected to Southern blot analysis using as probe an internal oligonucleotide (5'-GGA CTC GGT CTG CCT GGC CAG G-3') end labeled with 32P, which compliments the expected RT-PCR product.

For Northern blot analysis, 6 µg total RNA/sample were electrophoresed through a formaldehyde/agarose gel (10). The RNA was transferred to Bio-Trans nitrocellulose membrane and probed with an internal EcoRI fragment of PPAR{gamma} cDNA and the full-length cDNA of aP2, both labeled by the random priming method (11).

Determination of immunoreactive PTHrP
PTHrP in conditioned medium was determined using a two-site immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA). The detection limit of the assay is 4.51 pg/ml.

Immunoprecipitation and immunoblotting
Cells were lysed in 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 1 mM EGTA, 10 mM sodium glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1% Triton X-100, 0.1% ß-mercaptoethanol, and 1 mM sodium vanadate with the protease inhibitors leupeptin (2 µg/ml) and phenylmethylsulfonylfluoride (100 µg/ml). Twenty micrograms of total cell lysate were incubated with 2.5 µg monoclonal anti-PPAR{gamma} antibody (Research Diagnostics, Inc., Flanders, NJ) in 100 µl PBS at 4 C overnight. The sample was then incubated in 50 µl protein A-Sepharose for 4 h, washed with immunoprecipitation buffer [50 mM Tris-HCl (pH 7.5), 0.1% Triton X-100, and 150 mM NaCl], and boiled in 60 µl loading buffer, and 5-µl aliquots were used for loading. Proteins were electrophoresed through a 10% SDS-polyacrylamide gel and transferred at 45 V for 16 h to BioTrans nitrocellulose membranes (ICN Pharmaceuticals, Inc., Costa Mesa, CA). After blotting with either the anti-PPAR{gamma} antibody or a monoclonal antiphosphoserine antibody (Life Technologies, Inc.), detection was performed using the ECL chemiluminescent kit (Roche).

For phosphorylated MAPK and actin detection, 20 µg cell lysate were similarly analyzed using antiphosphorylated p42/44 (New England Biolabs, Inc., Beverley, MA) and antiactin (Amersham Pharmacia Biotech) antibodies.

PKA assay
PTHrP-transfected 3T3-L1 cells were plated at an initial density of 50,000 cells/35-mm plate and grown to 80% confluence. After overnight culture in serum-free medium, the cells were incubated with fresh serum-free DMEM with or without PTHrP 1–34 (1 x 10-7 M) for 5 min. The cells were then lysed and assayed for PKA activity using an assay kit in which 32P is incorporated into Kemptide by PKA (Upstate Biotechnology, Inc., Lake Placid, NY). Results were obtained from six independent samples, and their means and SE were calculated. Fisher’s test was performed to determine the P value.

MAPK assay
MAPK activity assays were conducted using cell lysates from pCDNA3- and PTHrP-transfected 3T3-L1 cells by measuring 32P incorporation into a MAPK-specific substrate from the Biotrak p42/p44 MAPK enzyme assay system (Amersham Pharmacia Biotech). Three readings were taken per determination, and their means and SE were determined. Fisher’s test was performed to determine the P value.

PPAR{gamma} luciferase assay
The luciferase assay was performed by cloning the PPAR{gamma} response element from the aP2 promoter (12) in the luciferase vector pXP2. COS-7 cells stably transfected with the PTH/PTHrP receptor were plated at a density of 75,000 cells/35-mm plate and transiently transfected with PPAR{gamma}2 cDNA in the expression plasmid pSVSPort (13), with the aP2 luciferase reporter, and with a ß-galactosidase reporter plasmid to determine transfection efficiency. All transfections were performed using FUGENE6 reagent. Two days after transfection, the cells were serum-deprived for 4 h, then treated with 100 µM troglitazone and with or without PTHrP-(1–34) (1 x 10-6 M). Two hours after the first treatment, the cells were treated again with PTHrP-(1–34) (1 x 10-6 M) due to the high degrading activity of COS-7 cells. Fifteen minutes after the second treatment, the cells were lysed. Luciferase activity was assessed using the Promega Corp. luciferase detection assay. All luciferase assay readings were performed in triplicate and were corrected for ß-galactosidase expression levels in each cell population.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of PTHrP and its receptor in 3T3-L1 preadipocytes
First, we examined whether PTHrP and the PTH/PTHrP receptor are expressed in 3T3-L1 cells. We selected these cells because although they are committed to the adipocytic lineage, they require further stimulation to terminally differentiate into adipocytes. We induced the differentiation of these cells as described in Materials and Methods and isolated total RNA at three different time points, d 0 (uninduced cells), d 3 (induced cells), and d 14 (post differentiated cells). RNA from 3T3-L1 cells transfected with PTHrP cDNA was used as positive control. Total RNA was subjected to RT-PCR and examined for expression of PTHrP and PTH/PTHrP receptor transcripts. The RT-PCR products were verified by probing with a radiolabeled oligonucleotide specific for the PTHrP product and by sequencing the amplified receptor product.

PTHrP mRNA was detected before differentiation (d 0) by RT-PCR. Two weeks after induction (d 14), PTHrP transcript levels were undetectable by Southern blot analysis of the RT-PCR products (Fig. 1AGo). PTH/PTHrP receptor mRNA followed a similar pattern, with expression observed before induction (d 0), but not after differentiation (d 14), of 3T3-L1 preadipocytes (Fig 1BGo). Therefore, expression of both PTHrP and its receptor were coordinately decreased during progression of the adipocyte differentiation program.



View larger version (41K):
[in this window]
[in a new window]
  		Figure 1. PTHrP and PTH/PTHrP receptor expression in 3T3-L1 preadipocytes. RNA was isolated at three different time points during 3T3-L1 differentiation, reverse transcribed, and amplified by PCR for PTHrP (A) and PTH/PTHrP receptor (B) mRNA expression. The number above each lane indicates days after induction of differentiation. The negative control (-ve) corresponds to PCR amplification using oligonucleotide primers alone; the positive control (+ve) represents amplification of the RT product of 3T3-L1 cells transfected with PTHrP cDNA (A) or the cDNA for the PTH/PTHrP receptor (B) as template. The identity of the products was confirmed by probing with an internal radiolabeled oligonucleotide specific for the PTHrP product (A, lower panel) or by direct sequencing of the receptor transcript. The RT products were evaluated based on concurrent amplification of GAPDH transcripts (B, lower panel). Each experiment is representative of three triplicate experiments that produced the same results.

 
Characteristics of PTHrP transfected 3T3-L1 cells
Populations of 3T3-L1 cells stably transfected with PTHrP cDNA were generated to maintain constant delivery of intact peptide to the cells. PTHrP protein was detected in conditioned medium 14 d postinduction of differentiation (mean ± SE of triplicate determinations, 16.7 ± 0.8 pg/ml; data not shown). PTH/PTHrP receptor transcripts were also present in transfected cells (Fig. 2AGo), and in contrast to vector-transfected cells, expression of receptor mRNA was sustained throughout the culture period. Moreover, exogenous PTHrP stimulated PKA activity in these cells (Fig. 2BGo), demonstrating the functional integrity of the receptor. As shown in Fig. 2CGo, PTHrP-expressing cells also proliferated at a higher rate than control vector-transfected 3T3-L1 cells.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 2. Characteristics of PTHrP-transfected 3T3-L1 cells. A, Persistent expression of the PTH/PTHrP receptor mRNA. RT-PCR for receptor transcripts was performed on total RNA isolated from PTHrP-transfected 3T3-L1 cells on the indicated days after induction of differentiation. B, PTHrP stimulates PKA activity in PTHrP-transfected 3T3-L1 cells. Cells were serum-deprived overnight, and fresh DMEM was applied the following day without (control) and with PTHrP-(1–34) (1 x 10-7 M; PTHrP) for 5 min. Cell lysates were then assayed for PKA activity by monitoring incorporation of 32P into a PKA-specific substrate. Readings are representative of six independent samples. *, Differences between PTHrP-treated and control cultures are statistically significant (P < 0.05). C, Increased proliferative capacity of PTHrP-transfected 3T3-L1 cells. Populations of vector- and PTHrP-transfected 3T3-L1 cells were plated at an initial density of 10,000 cells. Cells were then trypsinized and counted every 2 d for 6 d, then once on d 10, and again on d 14. Cell numbers are plotted for pCDNA3-transfected ({blacksquare}) and PTHrP-transfected ({blacktriangledown}) 3T3-L1 cells. Each data point is the mean ± SE of triplicate determinations.

 
PTHrP inhibits terminal differentiation of 3T3-L1 cells
To determine whether PTHrP might also alter the differentiation program of the preadipocytic cell line, PTHrP- and vector-transfected 3T3-L1 cells were induced to differentiate. After 2 wk of treatment, differentiation was assessed by staining of lipid droplets with Oil Red O. As shown in Fig. 3AGo, the control 3T3-L1-pCDNA3 cells differentiated readily, and intense staining of fat cells that had accumulated large amounts of lipid was observed. In contrast, differentiation in 3T3-L1-PTHrP cells was hardly detectable, as demonstrated by the low intensity of staining for lipid accumulation.



View larger version (115K):
[in this window]
[in a new window]
  		Figure 3. PTHrP inhibits the adipocyte differentiation program. 3T3-L1 (A) and C3H10T1/2 (B) cells stably transfected with vector (left panels) or PTHrP cDNA (right panels) were induced to differentiate toward the adipocytic lineage, as described in Materials and Methods. Fourteen days after induction of differentiation, the cells were fixed and stained with Oil Red O. The data shown are representative of triplicate determinations.

 
To further solidify the inhibitory influence of PTHrP on adipocyte differentiation, the effects of the peptide were also examined in the pluripotent mesenchymal cell line C3H10T1/2. PTHrP and pCDNA3 stably transfected populations were generated and induced to differentiate toward the adipocytic lineage by addition of BMP2 (100 ng/ml) in the conditioned medium. Members of the BMP family, including BMP2, have been shown to induce adipogenesis in a variety of pluripotent mesenchymal cell lines, including the C3H10T1/2 (14, 15, 16). PTHrP expression in these cells also inhibited their capacity to differentiate into adipocytes compared with control cells (Fig. 3BGo).

MAPK activity in PTHrP/3T3-L1 cells
The increased proliferative capacity of the PTHrP-transfected 3T3-L1 cells suggested that PTHrP expression might lead to activation of MAPK in these cells. To assess the degree of MAPK activation, a monoclonal antibody recognizing dually phosphorylated MAPK (Thr202/Tyr204) was employed on whole cell lysates from both control and PTHrP-transfected cells. A 5-fold increase in activated MAPK (determined by densitometric scanning) was observed in 3T3-L1-PTHrP cell lysates compared with cell lysates from 3T3-L1-pCDNA3 cells (Fig. 4Go).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 4. Activation of MAPK in PTHrP-transfected 3T3-L1 cells. pCDNA3- and PTHrP-transfected cells were grown to confluence and lysed, and 20 µg whole cell lysate were assayed by Western blot analysis for activated forms of MAPK. Loading was verified by probing the same membrane for actin (lower panel). Each blot is representative of data obtained from triplicate experiments.

 
PTHrP activates MAPK through a PKA-dependent pathway
PTHrP binds to its G protein-coupled PTH/PTHrP receptor, thereby activating both PKA and PKC intracellular signal transduction pathways (17, 18). To determine whether there is a preferred pathway for signaling to MAPK via the PTHrP G-coupled receptor in 3T3-L1 cells, PTHrP-transfected cells were treated with either H8 or chelerythrin chloride to inhibit PKA or PKC signaling, respectively (19, 20). After treatment for 24 h with the respective inhibitors, cells were lysed and assayed for MAPK activity by measuring 32P incorporation into a MAPK-specific substrate. Inhibition of PKC by chelerythrin resulted in a very slight increase in MAPK activity. In contrast, inhibition of PKA by H8 caused a significant (39%) decrease in MAPK activity (Fig. 5AGo). These results suggest that PTHrP mediates the increase in MAPK activity in 3T3-L1 cells through a PKA-dependent pathway.



View larger version (62K):
[in this window]
[in a new window]
  		Figure 5. Signaling by PKA stimulates MAPK activity and inhibits adipocyte differentiation. A, PTHrP-transfected 3T3-L1 cells were treated with the PKA inhibitor H8, the PKC inhibitor chelerythrin chloride, or dimethylsulfoxide as vehicle for a period of 24 h. MAPK activity was determined by assaying incorporation of 32P into MAPK-specific substrate. Total counts per min were standardized against the amount of cell lysate protein per sample. Each bar represents the mean ± SE of three determinations. *, Differences between H8- and vehicle-treated cultures are statistically significant (P < 0.05). B, Forskolin inhibits the terminal differentiation of 3T3-L1 cells. 3T3-L1 cells were induced to differentiate in the absence or presence of the PKA activator forskolin (100 nmol/ml). After differentiation, cells were fixed and stained with Oil Red O. Each result is representative of those obtained from triplicate experiments.

 
To determine whether inhibition of the adipocyte differentiation program by PTHrP is attributed to PKA signaling, 3T3-L1 cells were induced to differentiate with and without addition to the culture medium of the PKA activator forskolin. Two weeks after induction, the cells were fixed and stained with Oil Red O. 3T3-L1 cells treated with forskolin resisted terminal differentiation, as assessed by the lack of Oil Red O staining (Fig. 5BGo), providing further evidence that signaling of PTHrP through the PKA pathway leads to increased MAPK activity and inhibition of terminal differentiation of 3T3-L1 preadipocytes.

Differentiation of PTHrP/3T3-L1 cells with PDO98059
If excessive MAPK signaling is responsible for the inhibition of differentiation by PTHrP, then inhibition of MAPK signaling should enhance the differentiation potential of 3T3-L1-PTHrP cells. We therefore induced adipocyte differentiation in the presence of the MAPK kinase (MEK) inhibitor PD098059 that blocks the phosphorylation and activity of p42 and p44 MAPK isoforms. When PTHrP-transfected 3T3-L1 cells were induced to differentiate in the presence of PD098059, they were able to terminally differentiate into adipocytes (Fig. 6AGo), whereas vehicle-treated (dimethylsulfoxide) control cells remained resistant to differentiation (Fig. 6BGo). This data therefore suggest that activation of the MAPK cascade is involved in the pathway leading to inhibition of adipogenesis by PTHrP.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 6. Inhibition of MAPK signaling leads to differentiation of PTHrP-transfected 3T3-L1 cells. PTHrP-expressing 3T3-L1 cells were induced to differentiate in the presence of PD098059 (A; 20 nmol/ml) or dimethylsulfoxide (B) as vehicle. Fourteen days later, the cells were fixed, then stained with Oil Red O. Each result is representative of those obtained from triplicate experiments.

 
PTHrP alters the phosphorylation status of PPAR{gamma}
PPAR{gamma}, a transcription factor acting as the master controller of the adipogenic program, has a consensus MAPK site (PASP), and its transcriptional activity is down-regulated upon phosphorylation by MAPK (21). Therefore, we next examined the phosphorylation status of PPAR{gamma} in PTHrP-transfected and control 3T3-L1 cells to determine whether PPAR{gamma} phosphorylation was altered by PTHrP expression. Whole cell lysates were isolated from both PTHrP-transfected and control 3T3-L1 cells, and PPAR{gamma} was immunoprecipitated using a monoclonal anti-PPAR{gamma} antibody. After SDS-PAGE and probing of PPAR{gamma} immunoprecipitates with an anti-PPAR{gamma} antibody, densitometric scanning showed that there was 43% more PPAR{gamma} protein in control 3T3-L1-pCDNA3 cells compared with PTHrP-transfected 3T3-L1 cells (Fig. 7AGo). However, when the same immunoprecipitates were probed with an antiphosphoserine antibody, there was a 78% increase in phosphorylated PPAR{gamma} in PTHrP-transfected cells (Fig. 7BGo). The ratio of phosphorylated to total (phosphorylated and dephosphorylated) PPAR{gamma} in PTHrP-transfected cells was greater than 2.5 times that in control pCDNA3 cells. This suggests that a substantially greater ratio of inactive to active PPAR{gamma} was present in PTHrP-transfected cells compared with control 3T3-L1 cells.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 7. Effects of PTHrP on the phosphorylation status and transcriptional activity of PPAR{gamma}. Five microliters of immunoprecipitates from lysates of pCDNA3- and PTHrP-transfected 3T3-L1 cells, which were obtained 14 d after induction of differentiation, were examined by Western blot analysis. After SDS-PAGE, immunoprecipitates were transferred onto membranes and probed with the anti-PPAR{gamma} antibody to quantify the total PPAR{gamma} protein loaded onto each lane (A) and an antiphosphoserine antibody to quantify the serine phosphorylation status of PPAR{gamma} (B). Each blot is representative of triplicate determinations. C, PTHrP inhibits PPAR{gamma} transcriptional activity. COS-7 cells expressing the PTH/PTHrP receptor were transfected with PPAR{gamma} cDNA and stimulated with the PPAR{gamma} ligand, troglitazone (100 µM), in the presence of PTHrP-(1–34) (1 x 10-6 M). Untreated cells were maintained as a control. Luciferase activity was measured and standardized against ß-galactosidase expression. Each bar represents the mean ± SE of three determinations. *, Differences between with PTHrP-treated and control cultures are statistically significant (P < 0.05).

 
PTHrP inhibits the capacity of PPAR{gamma} to enhance transcription
We next determined whether PTHrP signaling reduces the capacity of PPAR{gamma} to regulate gene transcription. Due to the low transfection efficiency of 3T3-L1 cells and the low levels of PPAR{gamma} expression before induction, we tested this hypothesis in COS-7 cells stably transfected with the cDNA encoding PTH/PTHrP receptor. These cells were transiently transfected with PPAR{gamma} cDNA and a luciferase reporter plasmid whose expression was driven by the PPAR{gamma} response element from the adipocytespecific gene aP2 (13). A ß-galactosidase reporter plasmid was used as a transfection control. After PPAR{gamma} activation by addition of the PPAR{gamma} ligand troglitazone, the cells were incubated with or without PTHrP (1 x 10-6 M). As shown in Fig. 7CGo, cells treated with PTHrP displayed a marked reduction in luciferase activity, indicating that exogenous PTHrP, acting via its receptor, is able to directly inhibit the transcriptional activity of PPAR{gamma}.

Effects of PTHrP on PPAR{gamma} and aP2 expression
We next examined mRNA levels of the fat-specific gene aP2 in 3T3-L1 cells. As tissue-specific expression of this fatty acid-binding protein is directly regulated by the transcriptional activity of PPAR{gamma}, any changes in aP2 expression levels will be the result of changes in PPAR{gamma} activity (12). Total RNA was isolated from 3T3-L1-pCDNA3 and 3T3-L1-PTHrP cells at various time points after culture in the differentiation medium and assessed by Northern blot analysis using labeled probes for PPAR{gamma} and aP2 transcripts. We observed that upon induction of differentiation, PPAR{gamma} mRNA levels increased and remained relatively constant in both pCDNA- and PTHrP-transfected 3T3-L1 cells (Fig. 8Go). In contrast, aP2 levels initially increased faster in PTHrPexpressing cells. However, the levels plateaued earlier and thereafter fell and remained at a lower level than in the control 3T3-L1-pCDNA3 cells, consistent with the decreased capacity of these cells to undergo adipocytic differentiation.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 8. PPAR{gamma} and aP2 gene expression in pCDNA3- and PTHrP-transfected 3T3-L1 cells. Total RNA was isolated at four different time points during the differentiation program of pCDNA3- and PTHrP-transfected 3T3-L1 cells. A, Six micrograms of RNA were loaded per sample and probed with a radiolabeled probe for PPAR{gamma} and aP2, concurrently. Number above lanes represent days following induction of differentiation. B, The membrane was reprobed for 18S rRNA as a loading control. C, Graph depicting relative levels of aP2 vs. 18S expression, as determined by densitometric scanning. Results shown are representative of triplicate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
An inverse relationship generally exists between the total number of osteoblasts and adipocytes within bone marrow (2). This inverse relationship is reflected in virtually every form of osteopenia, such that a decrease in bone mass is always accompanied by an increase in adiposity within the bone marrow. It has been postulated that this accumulation may be a consequence of the inappropriate differentiation of pluripotent stem cells that are diverted from other lineages, such as osteoblasts (22). Although the molecular signals that are ultimately involved in this process are poorly understood, our studies of the bone alterations arising in mice heterozygous for targeted disruption of the PTHrP gene have suggested the possibility that PTHrP expressed within the skeletal microenvironment functions as one such signal in regulating cell fate determination (6). We show here that cells of the adipocytic lineage, the 3T3-L1 preadipocytic cell line, express PTHrP and the PTH/PTHrP receptor. Under conditions that promote terminal differentiation of 3T3-L1 cells, expression levels for both PTHrP and its receptor decrease until they are not detectable in the terminally differentiated cells. This sequence of PTHrP expression in adipocytes is reversed in osteoblasts where PTHrP transcripts are detected after commitment to the osteoblast lineage, and their levels increase as the cells proceed through the differentiation program (23).

When PTHrP levels are induced by stable transfection to remain constant throughout differentiation of the preadipocytic 3T3-L1 cells, this appears sufficient to perturb the progress of the cells in acquiring an adipocytic phenotype. If PTHrP limits adipocyte differentiation by signaling via the PTH/PTHrP receptor, then it is likely that inhibition of adipogenesis within the bone marrow could result not only from PTHrP derived from preadipocytes, but also from neighboring developing osteoblasts secreting PTHrP (23, 24, 25), thereby diminishing their adipogenic potential. Systemic PTH that also acts on the PTH/PTHrP receptor might contribute to the inhibition of bone marrow adiposity.

PTH and PTHrP bind to the common PTH/PTHrP receptor leading to the activation of two signal transduction systems: a Gs{alpha}-mediated increase in cAMP and activation of PKA, and a Gq{alpha}-mediated increase in intracellular calcium and IP3 levels and activation of PKC. Although the MAPK cascade represents the basic mechanism used by many growth factors to transduce mitogenic and differentiation signals by receptors with intrinsic tyrosine kinase activity, this pathway is also subject to regulation or cross-talk by G protein-coupled receptor signaling. PKA has been reported, for example, to directly activate the small G protein Rap1 that, in turn, activates B-Raf, leading to the sequential activations of MEK and MAPK in a Ras-independent pathway (26, 27). Although activation of PKC also stimulates increased MAPK activity, the signaling pathways involved are less well defined. All three groups of PKCs (conventional, novel, and atypical) are able to activate MAPK and MEK, but only conventional and novel PKCs are potent activators of c-Raf1 (26, 28). Here we show that MAPK activity in 3T3-L1 cells is under the regulation of PTH/PTHrP receptor signaling as a consequence of increasing PKA activity. PTH/PTHrP signaling has previously been reported to either trigger or inhibit MAPK activity depending on the cell type examined. Thus, PTH inhibits growth factor-induced MAPK activation in UMR 106 and ROS 17/2.8 osteosarcoma cells (29) and MAPK in F9 embryonal carcinoma cells (30) through activation of PKA, whereas it enhances activation of MAPK in Chinese hamster ovary R15 and parietal yolk-sac carcinoma cells (31). Activation of MAPK by PTH in these cell lines was also mediated by cAMP and was independent of Ras. This argues either for a cAMP site of action downstream of Ras in the Ras-Raf-MEK-MAPK cascade or a parallel Ras-independent pathway, such as Rap1 and B-Raf. In 3T3-L1 preadipocytes, PTHrP signaling via cAMP is also a positive regulator of the MAPK pathway at a level upstream of MEK, as indicated by our findings. Whether Raf1 or B-Raf kinase is involved in the activation of MAPK by the cAMP-dependent PKA in these cells remains to be determined. Nevertheless, activation of MAPK, regardless of the pathway used, had profound effects on the differentiation program of 3T3-L1 preadipocytes by increasing phosphorylation of PPAR{gamma}, the master controller of the adipogenic program. Conversely, inhibition of MEK activity by PD098059 was sufficient to restore the adipogenic differentiation program. This observation is consistent with a recent report showing that MAPK inhibition in pluripotent cells enhanced adipogenesis (32).

It has been reported that activation of PPAR{gamma} is able to induce adipocyte differentiation in fibroblasts and myoblasts as well as in bone marrow stromal cells (13, 21, 33). This appears to occur by enhancing transcription of a variety of adipocyte-specific genes, such as aP2 and phosphoenolpyruvate carboxykinase (34). PTHrP was shown in our studies to diminish the capacity of ligand-bound PPAR{gamma} to act as a transcriptional enhancer at an aP2 promoter site. Consequently, it also decreased gene expression of aP2. Another mechanism by which PPAR{gamma} functions, although less well understood, is by acting synergistically with other fat regulatory factors, such as CAAT/enhancer-binding protein-{alpha}, to drive forward the adipogenic program (13). Therefore, inactivation of PPAR{gamma} could also reduce the response of these cells to other fat-determining factors, such as CAAT/enhancer-binding protein-{alpha}. PPAR{gamma}, therefore, is a potent target by which PTHrP signaling can promote its inhibitory effects on the development of the adipocyte phenotype.

Our findings illustrate that although PTHrP does not affect the transcription of PPAR{gamma}, it does, however, down-regulate its activity. A striking feature of our study was the rapid initial increase in aP2 expression observed in PTHrP-transfected cells compared with control cells after induction of differentiation. This rapid early increase in aP2 expression could be the result of activated MAPK in PTHrP-transfected cells, as these cells are more likely to proceed at an accelerated rate through the mitotic divisions required to take place before adipocyte differentiation, thereby allowing the differentiation program to begin earlier (35). This conclusion is further substantiated by the increased proliferative capacity of PTHrP-transfected 3T3-L1 cells, as illustrated by the proliferation assay. Despite this early increase in aP2, however, PTHrP overall down-regulates the transcript levels of this marker. These observations are consistent with previous reports that cAMP has dual effects on the differentiation of preadipocytes in that it potentiates early events but exerts potent inhibitory effects on terminal differentiation (36). Therefore, early aP2 expression followed by its down-regulation in PTHrP/3T3-L1 cells would be consistent with cells being initially directed toward an adipogenic fate but subsequently prevented from further differentiation. This ability to withdraw from the adipogenic differentiation program has been observed even with mature adipocytes derived from primary bone marrow cultures, as they are capable of reverting to a more proliferative state and undertake an osteogenic fate (3), indicative of the plasticity that allows for transdifferentiation between adipocytes and osteoblasts.

PTH and PTHrP analogs are considered one of the most effective forms of anabolic treatment of osteoporosis. When PTH and PTHrP are administered continuously, their effect on bone is catabolic. However, when PTH or PTHrP is administered intermittently, an increase in bone formation follows (5, 37, 38, 39). Current theories regarding the anabolic action of PTH and PTHrP action in bone suggest that these agents act primarily on cells of the osteoblast lineage. PTHrP and the PTH/PTHrP receptor are expressed in osteoblast precursors and differentiated osteoblasts (25, 40, 41, 42). Consequently, PTH and PTHrP are also likely to influence the biology of cells of the osteoblast lineage. Prevention of osteoblast apoptosis was reported recently to be a mechanism of increased bone formation observed after PTH administration in vivo (43). Here we present evidence for another possible mode of action for PTH and PTHrP analogs, specifically altering the commitment and differentiation of pluripotent bone marrow stromal cells. Although PTHrP was shown here to inhibit the adipogenic differentiation program, the question remains whether PTHrP reciprocally influences the commitment of stem cells to the osteoblast lineage. Recent findings demonstrate that PTHrP is able to stimulate the osteoblast differentiation factor CBFA1 by PKA signaling (44). These findings taken together with our observations suggest that PTHrP is able to inhibit adipogenesis and at the same time enhance osteogenesis by PKA signaling.

In summary, our findings here provide a molecular mechanism to explain the increased bone marrow adiposity observed with PTHrP haploinsufficiency and perhaps with the osteopenic state in general. The capacity of PTHrP to inhibit adipocyte differentiation and ultimately enhance osteogenesis may provide novel targets and strategies for the treatment of osteoporosis.


   Acknowledgments
 
We thank Dr. J. E. Silva for his invaluable advice.


   Footnotes
 
This work was supported by the Medical Research Council of Canada, the National Cancer Institute of Canada, and a Scientist Award from the Medical Research Council of Canada (to A.C.K.).

Abbreviations: BMP2, Bone morphogenetic protein-2; MEK, MAPK kinase.

Received March 20, 2001.

Accepted for publication August 7, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Meunier P, Aaron J, Edouard C, Vignon G 1971 Osteoporosis and the replacement of cell populations of the marrow by adipose tissue. A quantitative study of 84 iliac bone biopsies. Clin Orthop 80:147–154[Medline]
  2. Beresford JN, Bennett JH, Devlin C, Leboy PS, Owen ME 1992 Evidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J Cell Sci 102:341–351[Abstract/Free Full Text]
  3. Bennett JH, Joyner CJ, Triffitt JT, Owen ME 1991 Adipocytic cells cultured from marrow have osteogenic potential. J Cell Sci 99:131–139[Abstract/Free Full Text]
  4. Nuttall ME, Patton AJ, Olivera DL, Nadeau DP, Gowen M 1998 Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype: implications for osteopenic disorders. J Bone Miner Res 13:371–382[CrossRef][Medline]
  5. Reeve J 1996 PTH: a future role in the management of osteoporosis? J Bone Miner Res 11:440–445[Medline]
  6. Amizuka N, Karaplis AC, Henderson JE, Warshawsky H, Lipman ML, Matsuki Y, Ejiri S, Tanaka M, Izumi N, Ozawa H, Goltzman D 1996 Haploinsufficiency of parathyroid hormone-related peptide (PTHrP) results in abnormal postnatal bone development. Dev Biol 175:166–176[CrossRef][Medline]
  7. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR 1999 Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147[Abstract/Free Full Text]
  8. Grigoriadis AE, Heersche JN, Aubin JE 1988 Differentiation of muscle, fat, cartilage, and bone from progenitor cells present in a bone-derived clonal cell population: effect of dexamethasone. J Cell Biol 106:2139–2151[Abstract/Free Full Text]
  9. Henderson JE, Amizuka N, Warshawsky H, Biasotto D, Lanske BM, Goltzman D, Karaplis AC 1995 Nucleolar localization of parathyroid hormone-related peptide enhances survival of chondrocytes under conditions that promote apoptotic cell death. Mol Cell Biol 15:4064–4075[Abstract]
  10. Maniatis T, Sambrook J, Fritsch E 1989 Molecular cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press
  11. Feinberg AP, Vogelstein B 1984 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137:266–267[CrossRef][Medline]
  12. Tontonoz P, Graves RA, Budavari AI, Erdjument-Bromage H, Lui M, Hu E, Tempst P, Spiegelman BM 1994 Adipocyte-specific transcription factor ARF6 is a heterodimeric complex of two nuclear hormone receptors, PPAR{gamma} and RXR{alpha}. Nucleic Acids Res 22:5628–5634[Abstract/Free Full Text]
  13. Tontonoz P, Hu E, Spiegelman BM 1994 Stimulation of adipogenesis in fibroblasts by PPAR{gamma}2, a lipid-activated transcription factor. Cell 79:1147–1156[CrossRef][Medline]
  14. Ahrens M, Ankenbauer T, Schroder D, Hollnagel A, Mayer H, Gross G 1993 Expression of human bone morphogenetic proteins-2 or -4 in murine mesenchymal progenitor C3H10T1/2 cells induces differentiation into distinct mesenchymal cell lineages. DNA Cell Biol 12:871–880[Medline]
  15. Asahina I, Sampath TK, Hauschka PV 1996 Human osteogenic protein-1 induces chondroblastic, osteoblastic, and/or adipocytic differentiation of clonal murine target cells. Exp Cell Res 222:38–47[CrossRef][Medline]
  16. Wang EA, Israel DI, Kelly S, Luxenberg DP 1993 Bone morphogenetic protein-2 causes commitment and differentiation in C3H10T1/2 and 3T3 cells. Growth Factors 9:57–71[Medline]
  17. Abou-Samra AB, Juppner H, Force T, Freeman MW, Kong XF, Schipani E, Urena P, Richards J, Bonventre JV, Potts Jr JT, Kronenberg HM, Segre GV 1992 Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium. Proc Natl Acad Sci USA 89:2732–2736[Abstract/Free Full Text]
  18. Bringhurst FR, Juppner H, Guo J, Urena P, Potts Jr JT, Kronenberg HM, Abou-Samra AB, Segre GV 1993 Cloned, stably expressed parathyroid hormone (PTH)/PTH-related peptide receptors activate multiple messenger signals and biological responses in LLC-PK1 kidney cells. Endocrinology 132:2090–2098[Abstract/Free Full Text]
  19. Hidaka H, Inagaki M, Kawamoto S, Sasaki Y 1984 Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23:5036–5041[CrossRef][Medline]
  20. Herbert JM, Augereau JM, Gleye J, Maffrand JP 1990 Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 172:993–999[CrossRef][Medline]
  21. Hu E, Kim JB, Sarraf P, Spiegelman BM 1996 Inhibition of adipogenesis through MAP kinase-mediated phosphorylation of PPAR{gamma}. Science 274:2100–2103[Abstract/Free Full Text]
  22. Gimble JM, Robinson CE, Wu X, Kelly KA 1996 The function of adipocytes in the bone marrow stroma: an update. Bone 19:421–428[Medline]
  23. Kartsogiannis V, Moseley J, McKelvie B, Chou ST, Hards DK, Ng KW, Martin TJ, Zhou H 1997 Temporal expression of PTHrP during endochondral bone formation in mouse and intramembranous bone formation in an in vivo rabbit model. Bone 21:385–392[Medline]
  24. Suda N, Gillespie MT, Traianedes K, Zhou H, Ho PW, Hards DK, Allan EH, Martin TJ, Moseley JM 1996 Expression of parathyroid hormone-related protein in cells of osteoblast lineage. J Cell Physiol 166:94–104[CrossRef][Medline]
  25. Amizuka N, Warshawsky H, Henderson JE, Goltzman D, Karaplis AC 1994 Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation. J Cell Biol 126:1611–1623[Abstract/Free Full Text]
  26. Vossler MR, Yao H, York RD, Pan MG, Rim CS, Stork PJ 1997 cAmp activates Map kinase and Elk-1 through a B-Raf- and Rap1-dependent pathway. Cell 89:73–82[CrossRef][Medline]
  27. Wan Y, Huang XY 1998 Analysis of the Gs/mitogen-activated protein kinase pathway in mutant S49 cells. J Biol Chem 273:14533–14537[Abstract/Free Full Text]
  28. Schonwasser DC, Marais RM, Marshall CJ, Parker PJ 1998 Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes. Mol Cell Biol 18:790–798[Abstract/Free Full Text]
  29. Verheijen MH, Defize LH 1995 Parathyroid hormone inhibits mitogen-activated protein kinase activation in osteosarcoma cells via a protein kinase A-dependent pathway. Endocrinology 136:3331–3337[Abstract]
  30. Verheijen MH, Wolthuis RM, Bos JL, Defize LH 1999 The Ras/Erk pathway induces primitive endoderm but prevents parietal endoderm differentiation of F9 embryonal carcinoma cells. J Biol Chem 274:1487–1494[Abstract/Free Full Text]
  31. Verheijen MH, Defize LH 1997 Parathyroid hormone activates mitogen-activated protein kinase via a cAMP-mediated pathway independent of Ras. J Biol Chem 272:3423–3429[Abstract/Free Full Text]
  32. Jaiswal RK, Jaiswal N, Bruder SP, Mbalaviele G, Marshak DR, Pittenger MF 2000 Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J Biol Chem 275:9645–9652[Abstract/Free Full Text]
  33. Gimble JM, Robinson CE, Wu X, Kelly KA, Rodriguez BR, Kliewer SA, Lehmann JM, Morris DC 1996 Peroxisome proliferator-activated receptor-gamma activation by thiazolidinediones induces adipogenesis in bone marrow stromal cells. Mol Pharmacol 50:1087–1094[Abstract]
  34. Tontonoz P, Hu E, Spiegelman BM 1995 Regulation of adipocyte gene expression and differentiation by peroxisome proliferator activated receptor gamma. Curr Opin Genet Dev 5:571–576[CrossRef][Medline]
  35. Cook JR, Kozak LP 1982 sn-Glycerol-3-phosphate dehydrogenase gene expression during mouse adipocyte development in vivo. Dev Biol 92:440–448[CrossRef][Medline]
  36. Yarwood SJ, Anderson NG, Kilgour E 1995 Cyclic AMP modulates adipogenesis in 3T3–F442A cells. Biochem Soc Trans 23:175S[Medline]
  37. Stewart AF 1996 PTHrP(1–36) as a skeletal anabolic agent for the treatment of osteoporosis. Bone 19:303–306[Medline]
  38. Canalis E, Hock JM, Raisz LG 1994 Parathyroid hormone anabolic and catabolic effects on bone and interactions with growth factors. In: Bilezikian JP, Marcus R, Levine MA, eds. The parathyroids. New York: Raven Press; 65–82
  39. Frolik CA, Cain RL, Sato M, Harvey AK, Chandrasekhar S, Black EC, Tashjian Jr AH, Hock JM 1999 Comparison of recombinant human PTH(1–34) (LY333334) with a C-terminally substituted analog of human PTH-related protein(1–34) (RS-66271): in vitro activity and in vivo pharmacological effects in rats. J Bone Miner Res 14:163–172[CrossRef][Medline]
  40. Rouleau MF, Mitchell J, Goltzman D 1988 In vivo distribution of parathyroid hormone receptors in bone: evidence that a predominant osseous target cell is not the mature osteoblast. Endocrinology 123:187–191[Abstract/Free Full Text]
  41. Lee K, Deeds JD, Bond AT, Juppner H, Abou-Samra AB, Segre GV 1993 In situ localization of PTH/PTHrP receptor mRNA in the bone of fetal and young rats. Bone 14:341–345[Medline]
  42. Urena P, Kong XF, Abou-Samra AB, Juppner H, Kronenberg HM, Potts Jr JT, Segre GV 1993 Parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acids are widely distributed in rat tissues. Endocrinology 133:617–623[Abstract/Free Full Text]
  43. Jilka RL, Weinstein RS, Bellido T, Roberson P, Parfitt AM, Manolagas SC 1999 Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. J Clin Invest 104:439–446[Medline]
  44. Selvamurugan N, Pulumati MR, Tyson DR, Partridge NC 2000 Parathyroid hormone regulation of the rat collagenase-3 promoter by protein kinase A-dependent transactivation of core binding factor alpha1. J Biol Chem 275:5037–5042[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
N. Bhattacharya, J. M. Dufour, M.-N. Vo, J. Okita, R. Okita, and K. H. Kim
Differential Effects of Phthalates on the Testis and the Liver
Biol Reprod, March 1, 2005; 72(3): 745 - 754.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Chen, A. J. Koh, N. S. Datta, J. Zhang, E. T. Keller, G. Xiao, R. T. Franceschi, N. J. D'Silva, and L. K. McCauley
Impact of the Mitogen-activated Protein Kinase Pathway on Parathyroid Hormone-related Protein Actions in Osteoblasts
J. Biol. Chem., July 9, 2004; 279(28): 29121 - 29129.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
B. Zingarelli, M. Sheehan, P. W. Hake, M. O'Connor, A. Denenberg, and J. A. Cook
Peroxisome Proliferator Activator Receptor-{gamma} Ligands, 15-Deoxy-{Delta}12,14-Prostaglandin J2 and Ciglitazone, Reduce Systemic Inflammation in Polymicrobial Sepsis by Modulation of Signal Transduction Pathways
J. Immunol., December 15, 2003; 171(12): 6827 - 6837.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
R. K. Petersen, C. Jorgensen, A. C. Rustan, L. Froyland, K. Muller-Decker, G. Furstenberger, R. K. Berge, K. Kristiansen, and L. Madsen
Arachidonic acid-dependent inhibition of adipocyte differentiation requires PKA activity and is associated with sustained expression of cyclooxygenases
J. Lipid Res., December 1, 2003; 44(12): 2320 - 2330.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
G. K. Chan, D. Miao, R. Deckelbaum, I. Bolivar, A. Karaplis, and D. Goltzman
Parathyroid Hormone-Related Peptide Interacts with Bone Morphogenetic Protein 2 to Increase Osteoblastogenesis and Decrease Adipogenesis in Pluripotent C3H10T1/2 Mesenchymal Cells
Endocrinology, December 1, 2003; 144(12): 5511 - 5520.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. J. Entingh, C. M. Taniguchi, and C. R. Kahn
Bi-directional Regulation of Brown Fat Adipogenesis by the Insulin Receptor
J. Biol. Chem., August 29, 2003; 278(35): 33377 - 33383.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
K. M. Jung, K. S. Park, J. H. Oh, S. Y. Jung, K. H. Yang, Y. S. Song, D. J. Son, Y. H. Park, Y. P. Yun, M. K. Lee, et al.
Activation of p38 Mitogen-Activated Protein Kinase and Activator Protein-1 during the Promotion of Neurite Extension of PC-12 Cells by 15-Deoxy-Delta 12,14-prostaglandin J2
Mol. Pharmacol., March 1, 2003; 63(3): 607 - 616.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Z.-C. Dang, V. Audinot, S. E. Papapoulos, J. A. Boutin, and C. W. G. M. Lowik
Peroxisome Proliferator-activated Receptor gamma (PPARgamma ) as a Molecular Target for the Soy Phytoestrogen Genistein
J. Biol. Chem., January 3, 2003; 278(2): 962 - 967.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. Prusty, B.-H. Park, K. E. Davis, and S. R. Farmer
Activation of MEK/ERK Signaling Promotes Adipogenesis by Enhancing Peroxisome Proliferator-activated Receptor gamma (PPARgamma ) and C/EBPalpha Gene Expression during the Differentiation of 3T3-L1 Preadipocytes
J. Biol. Chem., November 22, 2002; 277(48): 46226 - 46232.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chan, G. K.
Right arrow Articles by Karaplis, A. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chan, G. K.
Right arrow Articles by Karaplis, A. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals