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RESEARCH REPORT |
Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803;
*corresponding author, gwise{at}vetmed.lsu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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isoform may be critical in OPG regulation because PKC- gene expression is enhanced by PMA and reduced by either CSF-1 or PTHrP.
KEY WORDS: dental follicle • PKC • PKA • osteoprotegerin • tooth eruption
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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To begin to analyze the regulation of OPG expression in the follicle cells at the signal transduction level, we incubated the cells with either phorbolmyristate acetate (PMA) or dibutyryl cyclic AMP (dbcAMP) to up-regulate either protein kinase C (PKC) or protein kinase A (PKA) activity, respectively. Up-regulation of PKC activity may enhance OPG secretion in human bone marrow stromal cells, whereas up-regulation of PKA activity decreases OPG secretion (Brändström et al., 2001). Thus, the effect of PMA or dbcAMP on the gene expression of OPG, CSF-1, and PTHrP receptor (PTHrP-R) in the dental follicle cells (DFC) was examined. The gene expression of specific isoforms of PKC was also studied.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Incubation Experiments
DFC of passage 5 or higher were grown in 10 x 2-cm Petri dishes or 25-cm2 flasks for the incubation experiments. One day prior to a given experiment, DFC were pre-treated with serum-free MEM for 12 hrs and then incubated with either PMA (Calbiochem-Novabiochem. Corp., San Diego, CA, USA) at concentrations of 0, 1, 10, 25, 50, and 100 ng/mL or dbcAMP (Calbiochem-Novabiochem. Corp.) at concentrations of 0, 1, 5, 10, 25, 50, and 100 µg/mL for 3 hrs. For the time-dependent studies, the cells were incubated with 50 ng/mL of PMA for 0, 1, 3, 6, 12, and 24 hrs or with 5 µg/mL of dbcAMP for 0, 1, 3, 6, 12, 24, and 48 hrs. All cultures were maintained in a 37°C incubator.
DFC were also incubated with CSF-1 or PTHrP. The concentrations tested for both CSF-1 and PTHrP were 0, 1, 10, 25, 50, and 100 ng/mL for 3 hrs. In the time-course study, 25 ng/mL CSF-1 or 10 ng/mL PTHrP was added, and the cells were incubated for 0, 1, 3, 6, 12, and 24 hrs. After incubation, DFC were collected for RNA isolation. Each of the incubation experiments was repeated three times.
RNA Extraction
Total RNA was isolated from DFC by means of the Tri-Reagent protocol (Molecular Research Center, Cincinnati, OH, USA) and treated with DNase I for the removal of possible contamination of DNA (Ambion Inc., Austin, TX, USA). Total RNA was quantitated by the optical density of OD260, and the ratio of OD260/OD280 was greater than 1.9.
RT-PCR
For detection of gene expression, 0.5 µg (for OPG gene) or 2.0 µg (for PKC, CSF-1, and PTHrP-R genes) of total RNA from each sample was reverse-transcribed by M-MLV Reverse Transcriptase (Life Technologies, Grand Island, NY, USA) to generate 20 µL cDNA. The reaction was incubated at 37°C for 1 hr followed by a 10-minute incubation at 70°C.
Based on the nucleotide sequences of the cDNA of rat PKCs (Knopf et al., 1986; Housey et al., 1988; Tani et al., 1993) and CSF-1 (Borycki et al., 1993), the specific primer pairs for PKC-, PKC-ß, PKC-
, and CSF-1 were chosen. For each PCR, a 2-µL quantity of cDNA generated in the above RT reaction was mixed with PCR buffer (containing MgCl2), dNTP, primers, and AmpliTaq DNA polymerase to make a total reaction volume of 25 µL. The reaction was carried out with denaturing at 94°C for 45 sec, annealing at 55 to 60°C for 1 min, and extension at 72°C for 2 min. As an internal control, primers for the ß-actin gene were used in parallel amplification with the target gene.
After PCR, a 10-µL quantity of PCR product was loaded onto an agarose gel containing ethidium bromide for electrophoresis, and then viewed under UV light. The net intensity of DNA band(s) in the gel was measured with the use of Kodak Digital Science 1D Image Analysis Software. The gene expression was described as the ratio of gene investigated/ß-actin.
For real-time PCR, cDNA was mixed with 2x SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), primers, and Milliq H2O to make a reaction volume of 25 µL. The reactions were performed in an ABI Prism 7700 Sequence Detection System (Applied Biosystems) with 10 min of initial stage at 95°C to activate the DNA polymerase, followed by PCR cycles of 95°C for 15 sec and 60°C for 1 min. The ß-actin was used as endogenous control. A 
CT method was used to obtain relative gene expression (RGE).
Immunocytochemistry
The DFC were grown on coverslips for 1-2 days and then transferred into serum-free medium overnight. The cells were treated with or without PMA (100 ng/mL) for 12 hrs before being fixed in methanol. The coverslips were treated with a pre-incubation solution of 5% normal donkey serum and 0.3% Triton X-100 in PBS for 1 hr at room temperature. They were washed with 0.01 M PBS containing 0.03% (v/v) Triton X-100, and then incubated for 24 hrs at 4°C with 0.5 µg/mL goat polyclonal anti-human OPG (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) followed by biotin-conjugated donkey anti-goat IgG (1:500) for 2 hrs. Next, the sections were rinsed with PBS and incubated in DAB for 5 min before being counterstained. In control experiments, either the primary antibody was eliminated or the sections were incubated with pre-immune serum.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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PMA stimulated OPG gene expression in the cells in a concentration-dependent manner, with maximal OPG expression at a PMA concentration of 50 ng/mL (RT #50-100ng) (Fig. 1A
). Conversely, PMA inhibited PTHrP-R and CSF-1 expression, with a PMA concentration of 10-50 ng/mL (RT #25-100 ng) being optimal (Fig. 1A). Time-course studies showed that PMA stimulated gene expression of OPG in the cultured DFC up to 24 hrs, with a maximal enhancement after 6 hrs of incubation with PMA at 50 ng/mL (Fig. 1B). PTHrP-R and CSF-1 gene expressions in rat DFC were down-regulated when the cells were incubated in PMA, with maximal inhibition of expression at 3-6 hrs (RT #3-12 hrs) (Fig. 1B).
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). Conversely, dbcAMP up-regulated the expression of PTHrP-R and CSF-1, with maximal enhancement at 5 µg/mL for PTHrP and 5-25 µg/mL for CSF-1 (RT #10-50 µg) (Fig. 2A). In time-course studies, dbcAMP at 5 µg/mL maximally down-regulated OPG gene expression at 3-6 hrs (Fig. 2B). Again, dbcAMP enhanced PTHrP-R and CSF-1 expression in a time-dependent fashion, with maximal up-regulation of PTHrP-R at 12-24 hrs and maximal up-regulation of CSF-1 at 12 hrs (Fig. 2B).
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, which shows that PMA maximally up-regulates PKC- expression at a concentration of 25 ng/mL (RT #50-100 ng), and, timewise, maximal enhancement of PKC- expression is at 6 hrs (RT #3 hr). Another PKC isoform, PKC-ß, is also expressed in vitro, but PMA has no effect on its expression (Fig. not shown). PKC- is not expressed in vitro.
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in the follicle cells is down-regulated by either PTHrP or CSF-1. In concentration-dependent studies, maximal inhibition of PKC- was at a concentration of 25 ng/mL (RT #25-50 ng) of PTHrP (Fig. 3B
). In time-course studies, maximal inhibition of PKC- was at 6 hrs (RT #6-12 hrs) of incubation with PTHrP (Fig. 3B). For CSF-1, maximal inhibition of PKC- was also at a concentration of 25 ng CSF-1/mL (Fig. 3C). Timewise, maximal inhibition of PKC- was at 3 hrs (RT #6-12 hrs) of incubation with CSF-1 (Fig. 3C). In vivo, both PKC- and PKC-ß were expressed in the dental follicle, with PKC- being slightly expressed (Fig. not shown).
Immunostaining to detect OPG in the cultured DFC showed that incubating the cells in PMA enhanced immunostaining (Fig. 4). Controls do not stain (Fig. not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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isoform. Treatment of the cells with PMA up-regulates both OPG expression (Fig. 1) and PKC- expression (Fig. 3A) but not PKC-ß. Moreover, treating the cells either with CSF-1 or PTHrP, molecules previously shown to down-regulate OPG expression (Wise et al., 2000a), reduces PKC- expression (Figs. 3B, 3C). Changes in the gene expression of PKC- to regulate gene expression of another molecule are not without precedent. Enhanced gene expression of PKC- in an adrenocortical cell line impairs their ability to synthesize steroids (Reyland et al., 1998). Antisense treatment to inhibit PKC- expression in human lung carcinoma cells will inhibit some phorbol ester-mediated responses (Dean et al., 1994).
This study thus shows that an agent (PMA) which enhances PKC activity also acts to enhance gene expression of a specific isoform, PKC-, as well as to enhance OPG expression while decreasing CSF-1 and PTHrP-R gene expression. Conversely, dbcAMP, which enhances PKA activity, down-regulates OPG expression while enhancing CSF-1 and PTHrP-R gene expression. The common theme is that whatever enhances CSF-1 and PTHrP-R expression in the follicle down-regulates OPG expression, whereas any agent that down-regulates their expression up-regulates OPG. Because PTHrP itself is produced only in the stellate reticulum adjacent to the dental follicle (Philbrick et al., 1998), only the expression of its receptor could be monitored in the DF cells. Previous studies have shown that PTHrP-R is expressed in the DF cells (Philbrick et al., 1998; Wise et al., 2000b).
These studies of the regulation of gene expression of OPG in the DF cells parallel the studies of Brändström et al. (2001) in which they demonstrated, in bone marrow stromal cells, that activation of the PKC pathway stimulated the secretion of OPG, whereas stimulation of the PKA pathway decreased OPG secretion. Although our study focused on gene expression and not activation and secretion, other reports support our gene expression findings. For example, activators of PKA, such as forskolin or dbcAMP, inhibit expression of OPG mRNA in primary osteoblasts (Takami et al., 2000), as well as reducing CSF-1 mRNA induced by IL-1 in a pancreatic carcinoma cell line (Kamthong et al., 2000). Inhibiting PKA activity also can inhibit the effect of PTHrP on enhancing gene expression, as seen in inhibition of the PTHrP-induced increase in IL-6 mRNA (Valin et al., 2001) and inhibition of the PTHrP-induced increase of BMP-4 mRNA (Ito et al., 2000). Activators of the PKC pathway such as PMA stimulate the expression of OPG mRNA in primary osteoblasts (Takami et al., 2000).
The significance of this work in relation to tooth eruption pertains to the chronology of OPG expression in the dental follicle of the first mandibular molar of the rat. Tooth eruption requires the presence of the dental follicle (Cahill and Marks, 1980; Marks and Cahill, 1984) and osteoclastogenesis/alveolar bone resorption (e.g., see Marks et al., 1994; Grier and Wise, 1998). Molecules such as CSF-1 (e.g., see Felix et al., 1990; Wiktor-Jedrzejczak et al., 1990; Yoshida et al., 1990) and PTHrP (Philbrick et al., 1998) appear to be required for eruption. OPG is constitutively expressed in the dental follicle of the rat first molar but is down-regulated at day 3 post-natally, the time of maximal numbers of osteoclasts on the surrounding alveolar bone (Wise and Fan, 1989; Cielinski et al., 1994), as well as the time of maximal expression of CSF-1 in the dental follicle (Wise et al., 1995). Thus, we have hypothesized that the down-regulation of OPG at this time allows osteoclast formation to occur, and this study would suggest that CSF-1 may cause this via its down-regulating the gene expression of PKC- (Fig. 3C).
The in vitro presence of PKC in the dental follicle cells is corroborated by in vivo analysis demonstrating its presence in the dental follicle. Both PKC- and ß are seen in vivo, as well as PKC- to a much lesser extent. However, the in vitro studies show that only and ß are expressed in the DF cells and that only PKC- is the one that is affected by PMA treatment. Consequently, future in vivo studies will focus on the chronological expression of PKC-.
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ACKNOWLEDGMENTS |
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Received February 19, 2002; Last revision December 12, 2002; Accepted January 10, 2003
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REFERENCES |
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0.05). (B) RT-PCR products for OPG, PTHrP-R, and CSF-1 after incubation of DFC with 50 ng/mL PMA over different time periods. Again, PMA enhances OPG expression (six-hour maximal effect) and decreases PTHrP-R and CSF-1 (six-hour maximum effect).
