Proto-Oncogene c-fos Is Transcriptionally Regulated byParathyroid Hormone (PTH) and PTH-Related Protein ina Cyclic Adenosine Monophosphate-Dependent Mannerin Osteoblastic Cells*

LAURIE K. MCCAULEY, AMY J. KOH, CHRIS A. BEECHER, AND

THOMAS J. ROSOL

Department of Periodontics/Prevention/Geriatrics (L.K.M., A.J.K., C.A.B.), University of Michigan,Ann Arbor, Michigan 48109-1078 and Department of Veterinary Biosciences (T.J.R.), The Ohio StateUniversity, Columbus, Ohio 43210

ABSTRACTPTH and PTH-related protein (PTHrP) bind to the PTH-1 (PTH/

PTHrP) receptor and produce anabolic and catabolic effects in bone.To investigate postreceptor mechanisms of action, MC3T3-E1 cellswere induced to differentiate to optimize PTH-1 receptor expression,and differentiated MC3T3-E1 cells were treated with varying dosesof PTH (1–34) for 1 h. Northern blot analysis revealed a dose-depen-dent stimulation of steady state c-fos messenger RNA (mRNA), withmeasurable expression at doses as low as 1 pM PTH. The time courseof c-fos mRNA induction was rapid, with peak levels detected at30–45 min. Increased steady state c-fos mRNA was due to increasedtranscription of the c-fos gene as demonstrated by nuclear run-onassays and was dependent on the temporal differentiation state of theMC3T3-E1 cells. Stimulation of c-fos mRNA was induced exclusivelyby N-terminal PTH and PTHrP (which is also responsible for cAMPactivation), and did not occur with PTH (7–34), (53–84), or PTHrP

(107–139). The effects of PTH (1–34) on c-fos stimulation were de-pendent on intracellular cAMP. Forskolin [a guanine-nucleotide-binding protein (Ga) agonist] stimulated c-fos mRNA, whereas 9-(tetrahydro-2-furyl) adenine (THFA) (a cAMP antagonist), 1,9dideoxyforskolin (a cAMP independent analog of forskolin), and phor-bol 12-myristate 13-acetate (a protein kinase C activator) did not.Furthermore, THFA inhibited the ability of PTH (1–34) to stimulatec-fos mRNA in a time-dependent manner. These findings indicate thatc-fos is transcriptionally regulated by PTH (1–34) in osteoblastic cells,and that cAMP is a mediator of PTH-stimulated c-fos induction. Sev-eral known bone-associated proteins contain DNA binding sites intheir promoter regions that recognize c-fos in conjunction with c-jun(AP-1 sites). Consequently, the induction of c-fos by PTH (1–34) inosteoblastic cells may be a sensitive indicator of PTH effects in vitroand in vivo, and provide valuable information regarding mechanismsof PTH action in bone. (Endocrinology 138: 5427–5433, 1997)

PTH is an 84-amino acid hormone responsible for regu-lating systemic calcium levels. PTH-related protein

(PTHrP) is a humoral factor produced by various tumorsincluding squamous cell, breast and prostate carcinoma inaddition to many normal tissues including keratinocytes andlactating mammary gland (1–3). PTH and PTHrP have bothcatabolic and anabolic actions in bone; however, the mech-anisms of these functions are poorly understood. Anabolicmechanisms of PTH have been reported to be dependent oncAMP activation (4, 5). PTH and PTHrP bind to the PTH-1receptor (the PTH/PTHrP receptor) on osteoblasts and ac-tivate protein kinase A and protein kinase C pathways (6).PTH has been found to regulate alkaline phosphatase, typeI collagen, collagenase, osteopontin, bone sialoprotein, anddihydroxyvitamin D3 (6). Recently, PTH (1–34) was found toincrease levels of c-fos in vitro and in vivo in osteoblastic cells(7–9).

The c-fos proto-oncogene is an immediate-early responsegene that undergoes rapid transcriptional activation by mi-

togens and growth factors (10, 11). The c-fos protein forms aheterodimer with members of the c-jun family and binds tothe promoter of various target genes to regulate transcrip-tion. Numerous lines of evidence suggest that c-fos may beinvolved in the regulation of osteoblast proliferation anddifferentiation and ultimately bone formation. In vitro, c-fosmRNA expression is stimulated by PTH (1–34) (9). c-fos isexpressed in a temporal pattern during osteoblastic differ-entiation (12). The expression of c-fos is greatest during pro-liferation and decreases as the cells enter into the phase ofextracellular matrix synthesis and maturation. Early inmouse development, c-fos is expressed primarily in thegrowth regions of developing cartilage and bone (13, 14).Transgenic mice that overexpress c-fos develop chondroblas-tic osteosarcomas, and knock-out mice develop osteopetrosisand lack osteoclasts (14, 15). In vivo, administration of PTH(1–34) to normal mice induces c-fos expression in osteoblastswithin minutes followed by expression in osteoclasts 2 h later(8). Because PTH up-regulates c-fos and also stimulates boneformation when administered intermittently in vivo, it is pos-sible that the anabolic effects of PTH may be mediatedthrough c-fos. The purpose of this study was to determine theeffects of PTH on steady state and transcriptional regulationof the c-fos gene and its dependence on cAMP in activematrix-producing osteoblastic cells.

Received January 23, 1997.Address all correspondence and requests for reprints to: Laurie K.

McCauley, Department of Periodontics/Prevention/Geriatrics, Univer-sity of Michigan, 1011 North University Avenue, Ann Arbor, Michigan48109-1078. E-mail: mccauley@umich.edu.

* These studies were supported by National Institutes of HealthGrants DK-46919 and SPORE in prostate cancer P50-CA-69568.

0013-7227/97/$03.00/0 Vol. 138, No. 12Endocrinology Printed in U.S.A.Copyright © 1997 by The Endocrine Society

5427

Materials and MethodsCell culture

MC3T3-E1 cells were obtained from Dr. M. Kumegawa (Meikai Uni-versity, Sakado, Japan) via Dr. Renny Franceschi, and maintained aspreviously described (16). Briefly, stock cultures were grown in a-mod-ified Eagle’s medium (a-MEM) (Gibco BRL, Gaithersburg, MD) and 10%FBS (Hyclone, Logan, UT) containing 100 U/ml penicillin and strepto-mycin. Cells were passaged every 4–5 days and were not used beyondpassage 15. MC3T3-E1 cells were plated at initial densities of 50,000/cm2

and induced to differentiate and form a mineralized matrix with theaddition of ascorbic acid (50 mg/ml) and b-glycerophosphate (100 mm).After 5–7 days in culture, the cells display maximal PTH-1 receptorexpression as previously described (17) and were then used forexperiments.

Northern blot analysis

Total RNA was isolated from cultured MC3T3-E1 cells and Northernblot analysis performed as described (18). Briefly, total RNA was isolatedfrom one 60-mm dish by the guanidinium isothiocyanate method (19)and quantitated by spectrophotometry. Total RNA (20 mg) was electro-phoresed on 1.2% agarose-formaldehyde gels. The RNA was transferredto nylon membranes (Duralon U.V.; Stratagene, La Jolla, CA) and cross-linked with UV light. The nylon membranes were hybridized with acomplementary DNA (cDNA) probe for c-fos (American Type TissueCollection, Rockville, MD) labeled with a-[32P]deoxycytidine triphos-phate (NEN Dupont, Boston, MA) using random primer labeling (Strat-agene). After hybridization and washing, blots were exposed to KodakX-OMAT film (Eastman Kodak, Rochester, NY) at 270 C for 24–72 h.Blots were stripped and reprobed with a cDNA probe for 18S ribosomalRNA (rRNA) (20) to control for RNA loading.

Nuclear run-on assays

Nuclear run-on assays were performed as described (21) with thefollowing modifications. MC3T3-E1 cells were induced to differentiatefor 6 days and were treated with 0.1 mm hPTH (1–34) for 1 h, and intactnuclei isolated by incubating cells with hypotonic lysis buffer (0.5%NP-40). The nuclei were induced to transcribe, incorporate the labeledprecursor nucleotide a-[32P]uridine triphosphate (NEN Dupont), andthe nascent transcripts were isolated by the guanidinium-isothiocyanatemethod (19). The labeled RNA was hybridized for 48 h to c-fos cDNA,18S rRNA cDNA (loading control), and pcDNA plasmid (negative con-trol) previously immobilized by slot blot onto a Duralon-UV membrane(Stratagene). Blots were washed three times with 0.1% SDS and 23 SSCsolution for 30 min. Counts per minute were obtained from an InstantImager (Packard Instrument Co., San Diego, CT), and blots were ex-posed to Kodak BIOMAX film (Eastman Kodak) at 270 C forautoradiography.

Statistical analysis

Northern blot analyses and nuclear run-on assays were performedtwo to four times each. The results of multiple experiments were ana-lyzed using a Student’s t test (nuclear run-on assays) or ANOVA fol-lowed by a Tukey-Kramer multiple comparison test with the Instat 2.1biostatistics program (GraphPad Software). The figures are presentedwith a representative assay, in addition to a plot of data from multipleassays including their statistical evaluation.

Results

The experiments described were performed withMC3T3-E1 cells that had been induced to differentiate for 5–7days. We have previously reported that PTH-1 receptor ex-pression, binding, and biological activity peaks during thisphase of differentiation. This optimization of receptor ex-pression likely contributed the sensitive PTH-stimulated c-fos response detected in this study. PTH (1–34) treatment for1 h stimulated steady state c-fos mRNA expression in a dose-

dependent manner in MC3T3-E1 cells (Fig. 1). There was adetectable increase in c-fos expression with a dose as low as1 pm PTH (1–34). Maximal expression was detected at 10 nm,which is approximately 10-fold higher than the dissociationconstant (Kd) for the PTH-1 receptor in these cells. There wasno further increase in PTH-stimulated steady state c-fosmRNA expression at doses above 10 nm. An investigation ofthe time response of c-fos mRNA induction by PTH (1–34)indicated that gene expression was rapid with detectableincreases noted at 20 min (Fig. 2). This up-regulation wassustained for up to 1 h and decreased thereafter to unde-tectable levels. To determine whether this increase was dueto alterations in c-fos gene transcription, nuclear-run on tran-scription assays were performed. MC3T3-E1 cells weretreated with PTH (1–34) for 30 min before isolation of nucleiand labeling of nascent transcripts. Nuclear run-on assaysrevealed an increase in c-fos transcript expression followingPTH (1–34) stimulation in MC3T3-E1 cells (Fig. 3).

The PTH-stimulated increase in steady-state c-fos mRNAwas regulated temporally as indicated in Fig. 4. Basal levelsof c-fos were minimal throughout the differentiation period.

FIG. 1. Effects of PTH (1–34) (0–1 mM) for (1 h) on steady state c-fosmRNA levels in MC3T3-E1 cells. A, Autoradiograph of representativenorthern blot of c-fos mRNA and 18S rRNA. B, plot of counts perminute (mean values expressed as treatment vs. control 6 SEM) forc-fos vs. 18S rRNA from two separate experiments. PTH at all dosestested significantly stimulated steady state c-fos mRNA levels vs.control, P , 0.05.

5428 PTH REGULATES c-fos IN OSTEOBLASTS Endo • 1997Vol 138 • No 12

PTH-stimulated c-fos levels increased early during differen-tiation, peaked at day 6, and decreased thereafter. This trendis similar to the effects of PTH-stimulated cAMP duringdifferentiation and corresponds to peak PTH-1 receptor ex-pression and binding as we have previously reported (17).The PTH-stimulated up-regulation in c-fos mRNA gene ex-pression was not detected with PTH (7–34), PTH (53–84), orPTHrP (107–139) (Fig. 5). Although other fragments of PTHand PTHrP have been found to have biological activity inosteoblastic cells, the N-terminal fragments of PTH andPTHrP are the ones responsible for cAMP stimulation (6). Tofurther characterize the cAMP dependence on c-fos stimu-lation, agents that stimulate and inhibit cAMP were evalu-ated. Forskolin, a potent cAMP agonist, was effective at 10mm in stimulating c-fos expression in MC3T3-E1 cells (Fig. 6).In contrast, 9-(tetrahydro-2-furyl) adenine (THFA), which isa cAMP inhibitor, did not stimulate c-fos expression. PTH(1–34) and PTHrP (1–34) both stimulated a 5- to 6-fold in-crease in steady state c-fos mRNA compared with control

(Fig. 7). Phorbol 12-myristate 13-acetate (PMA), a proteinkinase C stimulator, and the forskolin analog (1, 9-dideoxy-forskolin) did not stimulate c-fos mRNA levels (Fig. 7). Theforskolin analog, 1,9-dideoxyforskolin is a naturally occur-ring analog of forskolin that does not activate adenylyl cy-clase but has cAMP-independent forskolin effects including

FIG. 2. Effects of PTH (1–34) (0.1 mM) for 0–24 h on stimulation ofc-fos mRNA levels in MC3T3-E1 cells. A, Autoradiograph of repre-sentative northern blot of c-fos mRNA and 18S rRNA. B, plot of countsper minute (mean values expressed as treatment vs. control 6 SEM)for c-fos vs. 18S rRNA from two separate experiments. PTH signifi-cantly stimulated steady state c-fos mRNA levels at 30 and 45 min(P , 0.01) and 1 h (P , 0.05).

FIG. 3. Effects of PTH (1–34) on c-fos transcription. MC3T3-E1 cellswere treated with 0.1 mM PTH (1–34) for 30 min. Nuclei were isolated,labeled with [32P]uridine triphosphate, and hybridized to immobilizedcDNA for c-fos, 18S rRNA (standardization control) and pcDNA plas-mid (negative control). A, Autoradiograph of representative nuclearrun-on assay. B, plot of signal counts per minute hybridized to run-ontranscripts (c-fos vs. 18S rRNA). PTH significantly stimulated c-fostranscript levels vs. control, P , 0.05.

PTH REGULATES c-fos IN OSTEOBLASTS 5429

inhibition of glucose transport and effects on nicotinic ace-tylcholine receptors (22).

To provide further evidence of the cAMP dependence,THFA (0.1 mm) reduced PTH-stimulated c-fos expression. Inexperiments shown in Fig. 8, MC3T3-E1 cells were treatedwith THFA from 0–24 h before PTH treatment for 1 h. Therewas a 1.6-fold reduction in c-fos expression when MC3T3-E1cells were treated with THFA and PTH (1–34) concomitantly.However, when MC3T3-E1 cells were pretreated for 6–12 hwith THFA, the reduction in c-fos stimulation was over 6-foldcompared with PTH (1–34) treatment and no THFA.

Discussion

The findings of effects of PTH (1–34) on c-fos mRNA ex-pression in MC3T3-E1 cells support a cAMP-mediated mech-anism in osteoblastic cells. Interestingly, in the present studyPTH (1–34) at doses as low as 1 pm was capable of stimulatinga detectable increase in c-fos expression. The Kd for PTH-stimulation of cAMP in osteoblastic cells is in the range of0.1–1 nm with assay sensitivity to 10 pm (17, 23). The finding

that PTH (1–34) stimulates c-fos at such low doses couldprovide valuable information regarding the physiologicalrole of PTH in bone, because normal circulating values arein this range. Furthermore, low doses of PTH have anaboliceffects in bone, and patients with mild primary hyperpara-thyroidism have increased trabecular bone volume (24). Nor-mal serum PTH levels are in the range of 1–5 pmol/L,whereas for patients with primary hyperparathyroidism,PTH levels range up to 30 pmol/L (25).

The time response of c-fos induction by PTH and its roleas an early response gene provide insight into the earlydownstream effects of PTH. PTH-stimulated cAMP accu-mulation is rapid and can be detected within minutes afterexposure (18, 23). In the present study, c-fos mRNA expres-sion peaked at 30–45 min and decreased thereafter. c-fos is

FIG. 4. Effects of differentiation on PTH-stimulated c-fos mRNA lev-els. MC3T3-E1 cells were plated at 50,000 cells/cm2 and induced todifferentiate with addition of ascorbic acid (50 mg/ml) and b-glycer-ophosphate (100 mM). At designated time points, cells were stimu-lated with 0.1 mM PTH (1–34) for 45 min. Total RNA was isolated, andnorthern blot analysis performed. A, Autoradiograph of representa-tive northern blot analysis of c-fos mRNA and 18S rRNA levels. B, Plotof counts per minute (mean values for replicate experiments 6 SEM)from two separate experiments. Open circles represent controls; solidcircles represent PTH-treated samples. PTH-stimulated c-fos valueswere significantly elevated on day 6 vs. day 0, P , 0.05.

FIG. 5. Effects of PTH and PTHrP analogs on steady state c-fosmRNA levels. MC3T3–1 cells were treated with 0.1 mM PTH (1–34),PTH (7–34), PTH (53–84), or PTHrP (107–139) for 1 h followed bytotal RNA isolation and northern blot analysis. A, Autoradiograph ofrepresentative northern blot analysis of c-fos mRNA and 18S rRNAlevels. B, Plot of counts per minute (mean values expressed as treat-ment vs. control 6 SEM) for c-fos vs. 18S rRNA from two separateexperiments. PTH (1–34) was the only analog to significantly stim-ulate steady state c-fos mRNA vs. control, P , 0.001.

5430 PTH REGULATES c-fos IN OSTEOBLASTS Endo • 1997Vol 138 • No 12

an early response gene and a transcription factor that formsheterodimers with c-jun protein family members, binds DNAat activating protein-1 (AP-1) sites, and transregulates theexpression of many genes (10, 11). Examples pertinent tobone are osteocalcin and the collagenase gene, which havebeen reported to have AP-1 sites that may be the target ofPTH-stimulated c-fos induction (26, 27). The expression ofc-fos is associated with a variety of cellular processes includ-ing cell cycle progression and cell differentiation (10). In thepresent study during osteoblast differentiation, PTH-stimu-

lation of c-fos was greatest during the phase of extracellularmatrix synthesis coincident with the time of maximal PTH-1receptor binding and cAMP stimulation (17). The temporalresponse of c-fos induction by PTH (1–34) coupled with thewidespread presence of AP-1 sites in genes that have im-portant regulatory features in bone makes it an attractivecandidate for mediating the PTH effects in bone.

There are several lines of evidence that indicate that c-fosplays a prominent role in bone formation. Ablation of ex-pression of the c-fos gene in vivo leads to osteopetrosis char-acterized by foreshortening of the long bones and ossification

FIG. 6. Effects of cAMP stimulation or inhibition on c-fos mRNAlevels. MC3T3-E1 cells were treated with 0.1 mM PTH (1–34), 10 mMforskolin, or 100 mM THFA for 1 h followed by total RNA isolation andnorthern blot analysis. A, Autoradiograph of representative northernblot of c-fos and 18s rRNA mRNA levels. B, Plot of counts per minute(mean values expressed as treatment vs. control 6 SEM) for c-fos vs.18S rRNA from three separate experiments. PTH (1–34) and forskolinsignificantly stimulated steady state c-fos mRNA levels vs. control,P , 0.05.

FIG. 7. Effects of protein kinases A and C modulators on steady statec-fos mRNA levels. MC3T3-E1 cells were treated with PTHrP (1–34)(0.1 mM), PTH (1–34) (0.1 mM), PMA (10 nM or 0.1 nM), forskolin (10mM), or forskolin analog 1,9-dideoxyforskolin (Forsk 1,9; 10 mM) for 1 hfollowed by total RNA isolation and northern blot analysis. A, Auto-radiograph of representative northern blot of c-fos and 18s rRNAmRNA levels. B, Plot of counts per minute (mean values expressed astreatment vs. control 6 SEM) for c-fos vs. 18S rRNA from two separateexperiments. PTH (1–34), PTHrP (1–34), and forskolin significantlystimulated steady state c-fos mRNA levels vs. control, P , 0.05.

PTH REGULATES c-fos IN OSTEOBLASTS 5431

of the marrow space (15). These animals have reduced num-bers of osteoblasts lining the endosteum and periosteum. Incontrast, overexpression of c-fos in transgenic mice leads toosseous hyperplasia and formation of large calcified tumorsin all areas of the skeleton (14). The tumors are chondro-blastic osteosarcomas containing large amounts of bone linedby cuboidal osteoblasts with some chondrocytes. Duringnormal development, c-fos is expressed in the growth regionsof developing cartilage and bones, which have been found tobe target tissues for PTHrP action, because ablation of thePTHrP gene or the PTH-1 receptor leads to severe cartilagedysgenesis (28, 29). In the adult, c-fos expression is main-tained in bone and has been detected in high levels in os-teosarcomas. Interestingly, in patients with fibrous dysplasiadue to activating mutations in the a-subunit of the guanine-nucleotide-binding protein (Gsa) linked to adenylyl cyclase,there is increased expression of c-fos (30). These patientsmanifest lesions characterized by bone marrow fibrosis andenhanced formation of woven bone. The physiological ac-tivity of PTH and PTHrP occurs via stimulation of Gsa,consequently these data support the concept that G protein-mediated stimulation of c-fos is an intermediary for PTH andPTHrP effects in bone.

The PTH-1 receptor is a Gsa-linked receptor that is coupledto adenylyl cyclase. Gsa proteins enhance transcription ofc-fos via a cAMP response element binding (CREB)-mediatedpathway (31). Gsa-mediated c-fos transcription is abolishedby overexpression of regulatory units of protein kinase Alacking cAMP-binding sites in pituitary cells (31). SeveralcAMP response elements have been reported in the c-fospromoter, and Evans et al. (32) reported that PTH (1–34)stimulated c-fos transcription led to the transient phosphor-ylation of the transcription factor CREB, which binds to CREat positions 264 to 257 in the c-fos promoter in osteosarcomacells. In these studies the protein kinase A pathway wasimplicated as the route for PTH (1–34) effects on c-fos, and theprotein kinase C pathway was excluded.

In the present study, PTH (1–34) was responsible for thetranscriptional regulation of c-fos expression. Partridge et al.(9, 32) and other investigators have reported a CREB-likeresponse element in the c-fos promoter. The present studysupports the regulation of c-fos transcription by PTH in acAMP-dependent manner. Fragments of PTH and PTHrPthat do not activate cAMP and additionally have not beenfound to be anabolic in vivo were unable to stimulate c-fosexpression in MC3T3-E1 cells. Forskolin, a potent stimulatorof cAMP, activated c-fos in a similar manner as PTH, whereasthe forskolin analog 1,9-dideoxyforskolin did not. The for-skolin analog has properties of forskolin but does not stim-ulate cAMP, and rules out effects of forskolin that are notrelated to its ability to stimulate cAMP. The findings thatPMA did not stimulate c-fos mRNA levels further corrobo-rate the cAMP dependence, because PMA is a protein kinaseC activator. THFA, a cAMP inhibitor did not activate c-fos.Finally, THFA was effective in blocking the PTH-stimulatedinduction of c-fos when the cells had been treated for 6–12 h.This time course is similar to that reported for THFA inhi-bition of PTH-stimulated bone resorption, which is a cAMP-dependent process (33). It is clear that stimulation of cAMPresults in activation of c-fos, and if the adenylyl cyclase path-

FIG. 8. Effects of inhibition of adenylyl cyclase on steady state c-fosmRNA levels. MC3T3-E1 cells were preincubated with 100 mM THFAfor 0–24 h followed by treatment with PTH (1–34) (0.1 mM) for 1 h(hatched bars). Control group (open bar) received PTH (1–34) for 1 hand no THFA. A, Outline of experimental design. B, Autoradiographof representative northern blot analysis of c-fos and 18s rRNA mRNAlevels. C, Plot of counts per minute (mean values expressed as treat-ment vs. control 6 SEM) for c-fos vs. 18S rRNA from two separateexperiments. All THFA treatment times were significantly differentfrom control, P , 0.05; whereas, only THFA preincubation times of 6,12, and 24 h were significantly different from THFA 0 (THFA notpreincubated but included during 1-h treatment period). P , 0.01, P ,0.001, P , 0.05, respectively.

5432 PTH REGULATES c-fos IN OSTEOBLASTS Endo • 1997Vol 138 • No 12

way is blocked, PTH will not be effective to activate c-fostranscription.

The c-fos gene expression findings are of particular interestin light of in vivo findings of PTH effects on c-fos expression.PTH (1–34) administration in vivo results in expression ofc-fos mRNA within 15–60 min (detected by in situ hybrid-ization) in osteoblasts, chondrocytes, and to a lesser degreein stromal cells, followed by transient c-fos expression in themajority of stromal cells and osteoclasts at 1–2 h (8). Stromalcells in the bone microenvironment have been suggested tobe osteoblastic precursors, but the factors responsible fortheir differentiation are unknown. PTH-1 receptors are as-sociated with active matrix-producing osteoblastic cells bothin vitro and in vivo and not with less differentiated cells (8,17). Interestingly, the anabolic effects of PTH (1–34) in vivohave been attributed to an increase in numbers or recruit-ment of cells of the osteoblastic lineage. These findings sug-gest that PTH and PTHrP likely bind to receptors on osteo-blastic cells that activate cAMP and subsequently stimulatec-fos transcription. The osteoblastic cells may respond bysecreting a factor(s) that stimulates differentiation of adjacentstromal cells into active osteoblasts with a subsequent in-crease in bone formation. Understanding the role of c-fos inthe downstream events of PTH-1 receptor activation willprovide critical information regarding the mechanisms ofaction of PTH and PTHrP in stimulating bone formationduring physiological bone remodeling and in metabolic andmetastatic bone disease.

Acknowledgments

We gratefully acknowledge Dr. Renny Franceschi for providing theMC3T3-E1 cells, and Drs. Victoria Shalhoub and Jane Lian for nuclearrun-on assay protocols.

References

1. Rosol TJ, Capen CC 1992 Biology of disease: mechanisms of cancer-inducedhypercalcemia. Lab Invest 67:680–702

2. Philbrick WM, Wysolmerski JJ, Galbraith S, Holt E, Orloff JJ, Yang KH,Vasavada RC, Weir EC, Broadus AE, Stewart AF 1996 Defining the roles ofparathyroid hormone-related protein in normal physiology. Physiol Rev76:127–173

3. Iwamura M, Abrahamsson PA, Foss KA, Wu G, Cockett ATK, Deftos LJ 1994Parathyroid hormone-related protein: a potential autocrine growth regulatorin human prostate cancer lines. Urology 43:675–679

4. Rixon RH, Whitfield JF, Gagnon L, Isaacs RJ, Maclean S, Chakravarthy B,Durkin JP, Neugebauer W, Ross V, Sung W, Willick GE 1994 Parathyroidhormone fragments may stimulate bone growth in ovariectomized rats byactivating adenylyl cyclase. J Bone Miner Res 9:1179–1189

5. Dempster DW, Cosman F, Parisien M, Shen V, Lindsay R 1993 Anabolicactions of parathyroid hormone on bone. Endocr Rev 14:690–709

6. Fitzpatrick LA, Bilezikian JP 1996 Actions of parathyroid hormone. In: Bilez-ikian JP, Raisz LG, Rodan GA (eds) Principles of Bone Biology. Academic Press,San Diego, pp 339–346

7. Scott DK, Brakenhoff KD, Clohisy JC, Quinn CO, Partridge NC 1992 Para-thyroid hormone induces transcription of collagenase in rat osteoblastic cellsby a mechanism using cyclic adenosine 39, 59-monophosphate and requiringprotein synthesis. Mol Endocrinol 6:2153–2159

8. Lee K, Deeds JD, Chiba S, Un-No M, Bond AT, Segre GV 1994 Parathyroidhormone induces sequential c-fos expression in bone cells in vivo: in situ lo-

calization of its receptor and c-fos messenger ribonucleic acids. Endocrinology134:441–450

9. Pearman AT, Chou WY, Bergman KD, Pulumati MR, Partridge NC 1996Parathyroid hormone induces c-fos promoter activity in osteoblastic cellsthrough phosphorylated cAMP response element (CRE)-binding protein bind-ing to the major CRE. J Biol Chem 271:25715–25721

10. Angel P, Karin M 1991 The role of Jun, Fos, and the AP-1 complex in cellproliferation and transformation. Biochim Biophys Acta 1072:129–157

11. Curran T, Franza BR 1988 Fos and Jun: the AP-1 connection. Cell 55:395–39712. McCabe LR, Kockz M, Lian J, Stein J, Stein G 1995 Selective expression of

fos- and jun-related genes during osteoblast proliferation and differentiation.Exp Cell Res 218:255–262

13. Machwate M, Jullienne A, Moukhtar M, Marie PJ 1995 Temporal variationof c-fos proto-oncogene expression during osteoblast differentiation and os-teogeneis in developing rat bone. J Cell Biochem 57:62–70

14. Grigoriadis AE, Wang ZQ, Wagner EF 1995 Fos and bone cell development:lessons from a nuclear oncogene. Trends Genet 11:436–441

15. Johnson RS, Spiegelman BM, Papaioannou V 1992 Pleiotropic effects of a nullmutation in the c-fos proto-oncogene. Cell 71:577–586

16. Franceschi RT, Iyer BS 1992 Relationship between collagen synthesis andexpression of the osteoblast phenotype in MC3T3–E1 cells. J Bone Miner Res7:235–246

17. McCauley LK, Koh AJ, Beecher CA, Cui Y, Rosol TJ, Franceschi RT 1996PTH/PTHrP receptor is temporally regulated during osteoblast differentiationand is associated with collagen synthesis. J Cell Biochem 61:638–647

18. McCauley LK, Beecher CA, Melton ME, Werkmeister JR, Juppner H, Abou-Samra AB, Segre GV, Rosol TJ 1994 Transforming growth factorb regulatessteady state PTH/PTHrP receptor mRNA levels and PTHrP binding in ROS17/2.8 osteosarcoma cells. Mol Cell Endocrinol 101:331–336

19. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acidguanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem162:156–159

20. Renkawitz R, Gerbi SA 1979 Ribosomal DNA of the fly Sciara coprophila hasa very small and homogenous repeat unit. Mol Gen Genet 173:1–13

21. Shalhoub V, Bortell R, Jackson ME, Marks SC, Stein JL, Lian JB, Stein GS1994 Transcriptionally active nuclei isolated from intact bone reflect modifiedlevels of gene expression in skeletal development and pathology. J Cell Bio-chem 55:182–189

22. Laurenza A, McHugh Sutkowski E, Seamon KB 1989 Forskolin: a specificstimulator of adenylyl cyclase or a diterpene with multiple sites of action?Trends Pharmacol Sci 10:442–447

23. Segre GV 1996 Receptors for parathyroid hormone and parathyroid hormone-related protein. In: Bilezikian JP, Raisz LG, Rodan GA (eds) Principles of BoneBiology. Academic Press, San Diego, pp 377–403

24. Vogel M, Hahn M, Delling G 1995 Trabecular bone structure in patients withprimary hyperparathyroidism. Virchows Arch 426:127–134

25. Kao PC, Grant CS, Klee GG, Khosla S 1992 Clinical performance of para-thyroid hormone immunometric assays. Mayo Clin Proc 67:637–645

26. Stein GS, Lian JB, Van Wijnen AJ, Montecino M 1996 Transcriptional controlof osteoblast growth and differentiation. Physiol Rev 76:593–629

27. Rajakumas RA, Quinn CO 1996 Parathyroid hormone induction of rat in-terstitial collagenase mRNA in osteosarcoma cells is mediated through an AP-1binding site. Mol Endocrinol 10:867–878

28. Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, Karperien M,Defize LHK, Ho C, Mulligan RC, Abou-Samra AB, Jueppner H, Segre GV,Kronenberg HM 1996 PTH/PTHrP receptor in early development and Indianhedgehog-regulated bone growth. Science 273:663–666

29. Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ 1996Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273:613–622

30. Candeliere GA, Glorieux FH, Prud’homme J, St.-Arnaud R 1995 Increasedexpression of the c-fos proto-oncogene in bone from patients with fibrousdysplasia. N Engl J Med 332:1546–1551

31. Gaiddon C, Boutillier AL, Monnier D, Mercken L, Loeffler JP 1994 Genomiceffects of the putative oncogene Gas. J Biol Chem 269:22663–22671

32. Evans DB, Hipskind RA, Bilbe G 1996 Analysis of signaling pathways usedby parathyroid hormone to activate the c-fos gene in human SaOS2 osteoblast-like cells. J Bone Miner Res 11:1066–1074

33. Herrmann-Erlee MPM, van der Meer JM, Lowik CWGM, Van LeeuwenJPTM, Boonekamp PM 1988 Different roles for calcium and cyclic AMP in theaction of PTH: studies in bone explants and isolated bone cells. Bone 9:93–100

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