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Endocrinology Vol. 138, No. 12 5427-5433
Copyright © 1997 by The Endocrine Society

ARTICLES

Proto-Oncogene c-fos Is Transcriptionally Regulated by Parathyroid Hormone (PTH) and PTH-Related Protein in a Cyclic Adenosine Monophosphate-Dependent Manner in Osteoblastic Cells1

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 State University, Columbus, Ohio 43210

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH 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 cells were induced to differentiate to optimize PTH-1 receptor expression, and differentiated MC3T3-E1 cells were treated with varying doses of PTH (1–34) for 1 h. Northern blot analysis revealed a dose-dependent stimulation of steady state c-fos messenger RNA (mRNA), with measurable expression at doses as low as 1 pM PTH. The time course of c-fos mRNA induction was rapid, with peak levels detected at 30–45 min. Increased steady state c-fos mRNA was due to increased transcription of the c-fos gene as demonstrated by nuclear run-on assays and was dependent on the temporal differentiation state of the MC3T3-E1 cells. Stimulation of c-fos mRNA was induced exclusively by N-terminal PTH and PTHrP (which is also responsible for cAMP activation), 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 dependent on intracellular cAMP. Forskolin [a guanine-nucleotide-binding protein (G{alpha}) agonist] stimulated c-fos mRNA, whereas 9-(tetrahydro-2-furyl) adenine (THFA) (a cAMP antagonist), 1,9 dideoxyforskolin (a cAMP independent analog of forskolin), and phorbol 12-myristate 13-acetate (a protein kinase C activator) did not. Furthermore, THFA inhibited the ability of PTH (1–34) to stimulate c-fos mRNA in a time-dependent manner. These findings indicate that c-fos is transcriptionally regulated by PTH (1–34) in osteoblastic cells, and that cAMP is a mediator of PTH-stimulated c-fos induction. Several known bone-associated proteins contain DNA binding sites in their promoter regions that recognize c-fos in conjunction with c-jun (AP-1 sites). Consequently, the induction of c-fos by PTH (1–34) in osteoblastic cells may be a sensitive indicator of PTH effects in vitro and in vivo, and provide valuable information regarding mechanisms of PTH action in bone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH is an 84-amino acid hormone responsible for regulating systemic calcium levels. PTH-related protein (PTHrP) is a humoral factor produced by various tumors including squamous cell, breast and prostate carcinoma in addition to many normal tissues including keratinocytes and lactating mammary gland (1, 2, 3). PTH and PTHrP have both catabolic and anabolic actions in bone; however, the mechanisms of these functions are poorly understood. Anabolic mechanisms of PTH have been reported to be dependent on cAMP activation (4, 5). PTH and PTHrP bind to the PTH-1 receptor (the PTH/PTHrP receptor) on osteoblasts and activate protein kinase A and protein kinase C pathways (6). PTH has been found to regulate alkaline phosphatase, type I collagen, collagenase, osteopontin, bone sialoprotein, and dihydroxyvitamin D3 (6). Recently, PTH (1–34) was found to increase levels of c-fos in vitro and in vivo in osteoblastic cells (7, 8, 9).

The c-fos proto-oncogene is an immediate-early response gene that undergoes rapid transcriptional activation by mitogens and growth factors (10, 11). The c-fos protein forms a heterodimer with members of the c-jun family and binds to the promoter of various target genes to regulate transcription. Numerous lines of evidence suggest that c-fos may be involved in the regulation of osteoblast proliferation and differentiation and ultimately bone formation. In vitro, c-fos mRNA expression is stimulated by PTH (1–34) (9). c-fos is expressed in a temporal pattern during osteoblastic differentiation (12). The expression of c-fos is greatest during proliferation and decreases as the cells enter into the phase of extracellular matrix synthesis and maturation. Early in mouse development, c-fos is expressed primarily in the growth regions of developing cartilage and bone (13, 14). Transgenic mice that overexpress c-fos develop chondroblastic osteosarcomas, and knock-out mice develop osteopetrosis and lack osteoclasts (14, 15). In vivo, administration of PTH (1–34) to normal mice induces c-fos expression in osteoblasts within minutes followed by expression in osteoclasts 2 h later (8). Because PTH up-regulates c-fos and also stimulates bone formation when administered intermittently in vivo, it is possible that the anabolic effects of PTH may be mediated through c-fos. The purpose of this study was to determine the effects of PTH on steady state and transcriptional regulation of the c-fos gene and its dependence on cAMP in active matrix-producing osteoblastic cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
MC3T3-E1 cells were obtained from Dr. M. Kumegawa (Meikai University, Sakado, Japan) via Dr. Renny Franceschi, and maintained as previously described (16). Briefly, stock cultures were grown in {alpha}-modified Eagle’s medium ({alpha}-MEM) (Gibco BRL, Gaithersburg, MD) and 10% FBS (Hyclone, Logan, UT) containing 100 U/ml penicillin and streptomycin. Cells were passaged every 4–5 days and were not used beyond passage 15. MC3T3-E1 cells were plated at initial densities of 50,000/cm2 and induced to differentiate and form a mineralized matrix with the addition of ascorbic acid (50 µg/ml) and ß-glycerophosphate (100 mM). After 5–7 days in culture, the cells display maximal PTH-1 receptor expression as previously described (17) and were then used for experiments.

Northern blot analysis
Total RNA was isolated from cultured MC3T3-E1 cells and Northern blot analysis performed as described (18). Briefly, total RNA was isolated from one 60-mm dish by the guanidinium isothiocyanate method (19) and quantitated by spectrophotometry. Total RNA (20 µg) was electrophoresed on 1.2% agarose-formaldehyde gels. The RNA was transferred to nylon membranes (Duralon U.V.; Stratagene, La Jolla, CA) and cross-linked with UV light. The nylon membranes were hybridized with a complementary DNA (cDNA) probe for c-fos (American Type Tissue Collection, Rockville, MD) labeled with {alpha}-[32P]deoxycytidine triphosphate (NEN Dupont, Boston, MA) using random primer labeling (Stratagene). After hybridization and washing, blots were exposed to Kodak X-OMAT film (Eastman Kodak, Rochester, NY) at -70 C for 24–72 h. Blots were stripped and reprobed with a cDNA probe for 18S ribosomal RNA (rRNA) (20) to control for RNA loading.

Nuclear run-on assays
Nuclear run-on assays were performed as described (21) with the following modifications. MC3T3-E1 cells were induced to differentiate for 6 days and were treated with 0.1 µM hPTH (1–34) for 1 h, and intact nuclei isolated by incubating cells with hypotonic lysis buffer (0.5% NP-40). The nuclei were induced to transcribe, incorporate the labeled precursor nucleotide {alpha}-[32P]uridine triphosphate (NEN Dupont), and the nascent transcripts were isolated by the guanidinium-isothiocyanate method (19). The labeled RNA was hybridized for 48 h to c-fos cDNA, 18S rRNA cDNA (loading control), and pcDNA plasmid (negative control) previously immobilized by slot blot onto a Duralon-UV membrane (Stratagene). Blots were washed three times with 0.1% SDS and 2x SSC solution for 30 min. Counts per minute were obtained from an Instant Imager (Packard Instrument Co., San Diego, CT), and blots were exposed to Kodak BIOMAX film (Eastman Kodak) at -70 C for autoradiography.

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The experiments described were performed with MC3T3-E1 cells that had been induced to differentiate for 5–7 days. We have previously reported that PTH-1 receptor expression, binding, and biological activity peaks during this phase of differentiation. This optimization of receptor expression likely contributed the sensitive PTH-stimulated c-fos response detected in this study. PTH (1–34) treatment for 1 h stimulated steady state c-fos mRNA expression in a dose-dependent manner in MC3T3-E1 cells (Fig. 1Go). There was a detectable increase in c-fos expression with a dose as low as 1 pM PTH (1–34). Maximal expression was detected at 10 nM, which is approximately 10-fold higher than the dissociation constant (Kd) for the PTH-1 receptor in these cells. There was no further increase in PTH-stimulated steady state c-fos mRNA expression at doses above 10 nM. An investigation of the time response of c-fos mRNA induction by PTH (1–34) indicated that gene expression was rapid with detectable increases noted at 20 min (Fig. 2Go). This up-regulation was sustained for up to 1 h and decreased thereafter to undetectable levels. To determine whether this increase was due to alterations in c-fos gene transcription, nuclear-run on transcription assays were performed. MC3T3-E1 cells were treated with PTH (1–34) for 30 min before isolation of nuclei and labeling of nascent transcripts. Nuclear run-on assays revealed an increase in c-fos transcript expression following PTH (1–34) stimulation in MC3T3-E1 cells (Fig. 3Go).



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  		Figure 1. Effects of PTH (1–34) (0–1 µM) for (1 h) on steady state c-fos mRNA levels in MC3T3-E1 cells. A, Autoradiograph of representative northern blot of c-fos mRNA and 18S rRNA. B, plot of counts per minute (mean values expressed as treatment vs. control ± SEM) for c-fos vs. 18S rRNA from two separate experiments. PTH at all doses tested significantly stimulated steady state c-fos mRNA levels vs. control, P < 0.05.

 


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  		Figure 2. Effects of PTH (1–34) (0.1 µM) for 0–24 h on stimulation of c-fos mRNA levels in MC3T3-E1 cells. A, Autoradiograph of representative northern blot of c-fos mRNA and 18S rRNA. B, plot of counts per minute (mean values expressed as treatment vs. control ± SEM) for c-fos vs. 18S rRNA from two separate experiments. PTH significantly stimulated steady state c-fos mRNA levels at 30 and 45 min (P < 0.01) and 1 h (P < 0.05).

 


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  		Figure 3. Effects of PTH (1–34) on c-fos transcription. MC3T3-E1 cells were treated with 0.1 µM PTH (1–34) for 30 min. Nuclei were isolated, labeled with [32P]uridine triphosphate, and hybridized to immobilized cDNA for c-fos, 18S rRNA (standardization control) and pcDNA plasmid (negative control). A, Autoradiograph of representative nuclear run-on assay. B, plot of signal counts per minute hybridized to run-on transcripts (c-fos vs. 18S rRNA). PTH significantly stimulated c-fos transcript levels vs. control, P < 0.05.

 
The PTH-stimulated increase in steady-state c-fos mRNA was regulated temporally as indicated in Fig. 4Go. Basal levels of c-fos were minimal throughout the differentiation period. PTH-stimulated c-fos levels increased early during differentiation, peaked at day 6, and decreased thereafter. This trend is similar to the effects of PTH-stimulated cAMP during differentiation and corresponds to peak PTH-1 receptor expression and binding as we have previously reported (17). The PTH-stimulated up-regulation in c-fos mRNA gene expression was not detected with PTH (7–34), PTH (53–84), or PTHrP (107–139) (Fig. 5Go). Although other fragments of PTH and PTHrP have been found to have biological activity in osteoblastic cells, the N-terminal fragments of PTH and PTHrP are the ones responsible for cAMP stimulation (6). To further characterize the cAMP dependence on c-fos stimulation, agents that stimulate and inhibit cAMP were evaluated. Forskolin, a potent cAMP agonist, was effective at 10 µM in stimulating c-fos expression in MC3T3-E1 cells (Fig. 6Go). In contrast, 9-(tetrahydro-2-furyl) adenine (THFA), which is a cAMP inhibitor, did not stimulate c-fos expression. PTH (1–34) and PTHrP (1–34) both stimulated a 5- to 6-fold increase in steady state c-fos mRNA compared with control (Fig. 7Go). Phorbol 12-myristate 13-acetate (PMA), a protein kinase C stimulator, and the forskolin analog (1, 9-dideoxyforskolin) did not stimulate c-fos mRNA levels (Fig. 7Go). The forskolin analog, 1,9-dideoxyforskolin is a naturally occurring analog of forskolin that does not activate adenylyl cyclase but has cAMP-independent forskolin effects including inhibition of glucose transport and effects on nicotinic acetylcholine receptors (22).



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  		Figure 4. Effects of differentiation on PTH-stimulated c-fos mRNA levels. MC3T3-E1 cells were plated at 50,000 cells/cm2 and induced to differentiate with addition of ascorbic acid (50 µg/ml) and ß-glycerophosphate (100 mM). At designated time points, cells were stimulated with 0.1 µM PTH (1–34) for 45 min. Total RNA was isolated, and northern blot analysis performed. A, Autoradiograph of representative northern blot analysis of c-fos mRNA and 18S rRNA levels. B, Plot of counts per minute (mean values for replicate experiments ± SEM) from two separate experiments. Open circles represent controls; solid circles represent PTH-treated samples. PTH-stimulated c-fos values were significantly elevated on day 6 vs. day 0, P < 0.05.

 


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  		Figure 5. Effects of PTH and PTHrP analogs on steady state c-fos mRNA levels. MC3T3–1 cells were treated with 0.1 µM PTH (1–34), PTH (7–34), PTH (53–84), or PTHrP (107–139) for 1 h followed by total RNA isolation and northern blot analysis. A, Autoradiograph of representative northern blot analysis of c-fos mRNA and 18S rRNA levels. B, Plot of counts per minute (mean values expressed as treatment vs. control ± SEM) for c-fos vs. 18S rRNA from two separate experiments. PTH (1–34) was the only analog to significantly stimulate steady state c-fos mRNA vs. control, P < 0.001.

 


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

 


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  		Figure 7. Effects of protein kinases A and C modulators on steady state c-fos mRNA levels. MC3T3-E1 cells were treated with PTHrP (1–34) (0.1 µM), PTH (1–34) (0.1 µM), PMA (10 nM or 0.1 nM), forskolin (10 µM), or forskolin analog 1,9-dideoxyforskolin (Forsk 1,9; 10 µM) for 1 h followed by total RNA isolation and northern blot analysis. A, Autoradiograph of representative northern blot of c-fos and 18s rRNA mRNA levels. B, Plot of counts per minute (mean values expressed as treatment vs. control ± SEM) for c-fos vs. 18S rRNA from two separate experiments. PTH (1–34), PTHrP (1–34), and forskolin significantly stimulated steady state c-fos mRNA levels vs. control, P < 0.05.

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



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  		Figure 8. Effects of inhibition of adenylyl cyclase on steady state c-fos mRNA levels. MC3T3-E1 cells were preincubated with 100 µM THFA for 0–24 h followed by treatment with PTH (1–34) (0.1 µM) for 1 h (hatched bars). Control group (open bar) received PTH (1–34) for 1 h and no THFA. A, Outline of experimental design. B, Autoradiograph of representative northern blot analysis of c-fos and 18s rRNA mRNA levels. C, Plot of counts per minute (mean values expressed as treatment vs. control ± SEM) for c-fos vs. 18S rRNA from two separate experiments. All THFA treatment times were significantly different from control, P < 0.05; whereas, only THFA preincubation times of 6, 12, and 24 h were significantly different from THFA 0 (THFA not preincubated but included during 1-h treatment period). P < 0.01, P < 0.001, P < 0.05, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The findings of effects of PTH (1–34) on c-fos mRNA expression in MC3T3-E1 cells support a cAMP-mediated mechanism in osteoblastic cells. Interestingly, in the present study PTH (1–34) at doses as low as 1 pM was capable of stimulating a detectable increase in c-fos expression. The Kd for PTH-stimulation of cAMP in osteoblastic cells is in the range of 0.1–1 nM with assay sensitivity to 10 pM (17, 23). The finding that PTH (1–34) stimulates c-fos at such low doses could provide valuable information regarding the physiological role of PTH in bone, because normal circulating values are in this range. Furthermore, low doses of PTH have anabolic effects in bone, and patients with mild primary hyperparathyroidism have increased trabecular bone volume (24). Normal 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 role as an early response gene provide insight into the early downstream effects of PTH. PTH-stimulated cAMP accumulation is rapid and can be detected within minutes after exposure (18, 23). In the present study, c-fos mRNA expression peaked at 30–45 min and decreased thereafter. c-fos is an early response gene and a transcription factor that forms heterodimers with c-jun protein family members, binds DNA at activating protein-1 (AP-1) sites, and transregulates the expression of many genes (10, 11). Examples pertinent to bone are osteocalcin and the collagenase gene, which have been reported to have AP-1 sites that may be the target of PTH-stimulated c-fos induction (26, 27). The expression of c-fos is associated with a variety of cellular processes including cell cycle progression and cell differentiation (10). In the present study during osteoblast differentiation, PTH-stimulation of c-fos was greatest during the phase of extracellular matrix synthesis coincident with the time of maximal PTH-1 receptor binding and cAMP stimulation (17). The temporal response of c-fos induction by PTH (1–34) coupled with the widespread presence of AP-1 sites in genes that have important regulatory features in bone makes it an attractive candidate for mediating the PTH effects in bone.

There are several lines of evidence that indicate that c-fos plays a prominent role in bone formation. Ablation of expression of the c-fos gene in vivo leads to osteopetrosis characterized by foreshortening of the long bones and ossification of the marrow space (15). These animals have reduced numbers of osteoblasts lining the endosteum and periosteum. In contrast, overexpression of c-fos in transgenic mice leads to osseous hyperplasia and formation of large calcified tumors in all areas of the skeleton (14). The tumors are chondroblastic osteosarcomas containing large amounts of bone lined by cuboidal osteoblasts with some chondrocytes. During normal development, c-fos is expressed in the growth regions of developing cartilage and bones, which have been found to be target tissues for PTHrP action, because ablation of the PTHrP gene or the PTH-1 receptor leads to severe cartilage dysgenesis (28, 29). In the adult, c-fos expression is maintained in bone and has been detected in high levels in osteosarcomas. Interestingly, in patients with fibrous dysplasia due to activating mutations in the {alpha}-subunit of the guanine-nucleotide-binding protein (Gs{alpha}) linked to adenylyl cyclase, there is increased expression of c-fos (30). These patients manifest lesions characterized by bone marrow fibrosis and enhanced formation of woven bone. The physiological activity of PTH and PTHrP occurs via stimulation of Gs{alpha}, consequently these data support the concept that G protein-mediated stimulation of c-fos is an intermediary for PTH and PTHrP effects in bone.

The PTH-1 receptor is a Gs{alpha}-linked receptor that is coupled to adenylyl cyclase. Gs{alpha} proteins enhance transcription of c-fos via a cAMP response element binding (CREB)-mediated pathway (31). Gs{alpha}-mediated c-fos transcription is abolished by overexpression of regulatory units of protein kinase A lacking cAMP-binding sites in pituitary cells (31). Several cAMP response elements have been reported in the c-fos promoter, and Evans et al. (32) reported that PTH (1–34) stimulated c-fos transcription led to the transient phosphorylation of the transcription factor CREB, which binds to CRE at positions -64 to -57 in the c-fos promoter in osteosarcoma cells. In these studies the protein kinase A pathway was implicated as the route for PTH (1–34) effects on c-fos, and the protein kinase C pathway was excluded.

In the present study, PTH (1–34) was responsible for the transcriptional regulation of c-fos expression. Partridge et al. (9, 32) and other investigators have reported a CREB-like response element in the c-fos promoter. The present study supports the regulation of c-fos transcription by PTH in a cAMP-dependent manner. Fragments of PTH and PTHrP that do not activate cAMP and additionally have not been found to be anabolic in vivo were unable to stimulate c-fos expression in MC3T3-E1 cells. Forskolin, a potent stimulator of cAMP, activated c-fos in a similar manner as PTH, whereas the forskolin analog 1,9-dideoxyforskolin did not. The forskolin analog has properties of forskolin but does not stimulate cAMP, and rules out effects of forskolin that are not related to its ability to stimulate cAMP. The findings that PMA did not stimulate c-fos mRNA levels further corroborate the cAMP dependence, because PMA is a protein kinase C activator. THFA, a cAMP inhibitor did not activate c-fos. Finally, THFA was effective in blocking the PTH-stimulated induction of c-fos when the cells had been treated for 6–12 h. This time course is similar to that reported for THFA inhibition of PTH-stimulated bone resorption, which is a cAMP-dependent process (33). It is clear that stimulation of cAMP results in activation of c-fos, and if the adenylyl cyclase pathway is blocked, PTH will not be effective to activate c-fos transcription.

The c-fos gene expression findings are of particular interest in light of in vivo findings of PTH effects on c-fos expression. PTH (1–34) administration in vivo results in expression of c-fos mRNA within 15–60 min (detected by in situ hybridization) in osteoblasts, chondrocytes, and to a lesser degree in stromal cells, followed by transient c-fos expression in the majority of stromal cells and osteoclasts at 1–2 h (8). Stromal cells in the bone microenvironment have been suggested to be osteoblastic precursors, but the factors responsible for their differentiation are unknown. PTH-1 receptors are associated with active matrix-producing osteoblastic cells both in vitro and in vivo and not with less differentiated cells (8, 17). Interestingly, the anabolic effects of PTH (1–34) in vivo have been attributed to an increase in numbers or recruitment of cells of the osteoblastic lineage. These findings suggest that PTH and PTHrP likely bind to receptors on osteoblastic cells that activate cAMP and subsequently stimulate c-fos transcription. The osteoblastic cells may respond by secreting a factor(s) that stimulates differentiation of adjacent stromal cells into active osteoblasts with a subsequent increase in bone formation. Understanding the role of c-fos in the downstream events of PTH-1 receptor activation will provide critical information regarding the mechanisms of action of PTH and PTHrP in stimulating bone formation during physiological bone remodeling and in metabolic and metastatic bone disease.


   Acknowledgments
 
We gratefully acknowledge Dr. Renny Franceschi for providing the MC3T3-E1 cells, and Drs. Victoria Shalhoub and Jane Lian for nuclear run-on assay protocols.


   Footnotes
 
1 These studies were supported by National Institutes of Health Grants DK-46919 and SPORE in prostate cancer P50-CA-69568. Back

Received January 23, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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