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Journal of Dental Research
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Biological

Hormonal Regulation of Androgen Receptor Messenger Ribonucleic Acid Expression in Human Tooth Pulp

J.B. Dale1, S.L. Sarich1, T.M. Bretz2, J.F. Hatton1,2 and R.J. Zachow1,2,*

1 St. Louis University Center for Advanced Dental Education, Department of Endodontics, St. Louis, MO; and
2 Southern Illinois University School of Dental Medicine, Department of Applied Dental Medicine, 2800 College Ave., Alton, IL 62002;

Correspondence: *corresponding author, rzachow{at}siue.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tooth pulp contains steroid receptors and therefore is likely to respond to steroids. Steroids and cytokines together can alter steroid receptor content in many tissues; thus, similar mechanisms may exist in tooth pulp. In this study, reverse-transcription/polymerase chain-reaction was used to screen human pulp for the mRNAs encoding receptors for androgen (AR), estrogens (ERβ), and hepatocyte growh factor (HGF: c-Met). AR mRNA content was greater in male pulp vs. female pulp in all age groups. In both genders, AR mRNA content diminished with age. In pulp cell cultures, androstenedione, estradiol-17β, and HGF each stimulated AR mRNA accumulation. Testosterone inhibited, whereas 5{alpha}-dihydrotestosterone did not affect, AR mRNA content. ERβ was not hormonally altered in pulp cell cultures. By showing steroid- and cytokine-orchestrated regulation of AR mRNA in vitro, it is possible that age- and/or pathogen-dependent changes in available steroids and cytokines can affect any androgen-responsiveness of pulp.

Key Words: androgen receptor • androgen • hepatocyte growth factor • estrogen • tooth pulp


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dental tissue possesses several functional, developmental, and anatomical similarities to bone. For example, the maintenance of hard tissue in the tooth and bone depends upon the stimulation of morphologically and functionally related cells identified as odontoblasts and osteoblasts. Studies have demonstrated that osteoblast activity that enables hard tissue to form is promoted by gonadal steroid hormones (e.g., androgens and estrogens). Steroid hormone activity within bone is facilitated by locally produced cytokines, including hepatocyte growth factor (HGF) (Fuller et al., 1995; Grano et al., 1996; Spelsberg et al., 1999). By comparison, odontoblasts mediate hard-tissue (dentin) formation in teeth; however, the steroid hormone- and cytokine-dependence of this phenomenon has yet to be established in odontoblasts.

Although hormonal regulation of human odontoblast function has not been directly shown in vivo, fluctuations in steroid hormone and cytokine levels do affect dental health, as evidenced by (1) the apparent androgen-dependence of tooth morphogenesis during human development (Molsted et al., 1997), (2) progestin-correlated changes that occur in the oral cavity during pregnancy (Ojanotko-Harri et al., 1991), and (3) compromised dentition resulting from the menopausal-associated loss in ovarian estradiol-17β (E2) production (Krall et al., 1997). Moreover, altered patterns of dentin apposition have been reported in teeth from ovariectomized rats (Hietala and Larmas, 1992). Although the precise steroid hormone-dependent mechanisms that may mediate the aforementioned processes are debatable, the similarities between tooth and bone tissues can be used to develop a model in which steroid hormones and cytokines support tooth integrity by modulating odontoblast activity.

Steroid hormone bioactivity is controlled at the cellular level via the activation of steroid hormone receptors. Upon an elaborate activation scheme, steroid hormone receptors mediate the transcription of steroid-responsive genes. Isoforms of the estrogen receptor (ER{alpha} and ERβ) have been identified in human osteoblasts (Byers et al., 2001); and while ER{alpha} appears to mediate the anti-osteoporotic actions of E2 in human bone (Ericksen and Mosekilde, 1990), the function of ERβ is uncertain. In addition, androgens, via the activation of osteoblastic androgen receptors (AR) (Wiren et al., 1997), appear to support E2 in thwarting age-associated osteoporosis (Rosenberg et al., 1997). Of particular interest is the observation that cytokines (e.g., HGF, interleukins, tumor necrosis factor-{alpha}, and transforming growth factor-β) regulate the steroid hormone-responsiveness within bone (Spelsberg et al., 1999), perhaps by modulating the level of steroid hormone receptor expression (Jilka, 1998).

The complement of steroid hormone receptors in human tooth pulp is uncertain, because contradictory data have been presented thus far. For example, immunoreactive ER was shown in pulp tissue (Hietala et al., 1998); whereas, in a separate study, immunoreactive progesterone receptor was identified in pulp tissue, but neither AR nor ER was detected (Whitaker et al., 1998). Thus, it appears that human tooth pulp contains steroid hormone receptors, and pulp tissue would therefore be steroid-responsive. The present study extends this concept by identifying AR and ERβ mRNAs in human tooth pulp. In addition, the effects of androgens, E2, and HGF on AR mRNA content in pulp cells were investigated in vitro.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
All procedures for the usage of human pulp tissue were approved by the St. Louis University and Southern Illinois University Institutional Review Boards for Human Subjects. Subjects were grouped according to gender and in three age groups: 18-24, 25-40, and 41-55 yrs old. Dental pulp tissue was obtained from permanent, impacted, or partially erupted non-carious third molars that were extracted for prophylactic purposes. Upon extraction, the teeth were cleaned then submerged in ice-cold Medium 199 (M199; Gibco, Grand Island, NY, USA) that was supplemented with the following: 100 U/mL penicillin, 100 mg/mL streptomycin, 100 mg/mL neomycin, 100 mg/mL nystatin, 1% fetal calf serum (FCS), and 1 mg/mL bovine serum albumin (BSA). Within 1 hr following extraction, the teeth were split, and the pulp tissue was removed and either snap-frozen pending RNA extraction, or processed for cell culture. Only 18- to 24-year-old pulp was used for cell culture.

For cell culture, pulp was placed in M199 and minced. The minced pulp tissue was enzymatically dissociated in a collagenase/deoxyribonuclease cocktail as previously described (Magoffin and Erickson, 1988). Enzymatic dispersal was conducted for up to 60 min in a 37°C Dubnoff shaking H2O bath. The enzymatic dissociation was terminated by the addition of an excess volume of ice-cold McCoy's 5A medium (M5A, Gibco) with added antibiotics and 1% FCS. The pulpal cell suspension was centrifuged for 5 min at 450 x g (4°C), and the pellet was re-suspended in a known volume of M5A. Cell viability and number were determined by means of a hemacytometer following a challenge with trypan blue.

Cells (1-2 x 105 viable cells/well/500 µL M5A) were pipetted into 24-well culture plates. Cultures were incubated at 37°C in a H2O-saturated atmosphere containing 5% CO2 in air. Following an overnight acclimation period, cell-conditioned media were discarded, and cells were given fresh, pre-warmed (37°C) M5A. The cells were then challenged with androstenedione (0.1, 1, 10 µM), E2 (0.01, 0.1, 1 µM), 5{alpha}-dihydrotestosterone (DHT; 0.1, 1, 10 µM), testosterone (0.1, 1, 10 µM), or recombinant human HGF (1, 10, 30 ng/ml; R&D Systems, Minneapolis, MN, USA). We chose steroid hormone concentrations to bracket the serum levels of these hormones in reproductive-age men and women (Williams, 1981). We chose the HGF doses to bracket the reported dissociation constant Kd (24-32 pM) for HGF binding to c-Met (Higuchi and Nakamura, 1991).

RT-PCR
Total RNA was extracted from the pulp cells by means of TriReagent and Microcarrier (Molecular Research Center, Inc., Cincinnati, OH, USA), according to the manufacturer's protocol. Upon purification, RNA was re-suspended in RNase-free H2O, and RNA concentration was measured at A260.

The relative abundances of target gene mRNAs were determined by semi-quantitative RT-PCR based upon the method previously described (Orly et al., 1994). Approximately 1 µg RNA/reaction were used as the template for the synthesis of cDNA in a cocktail containing 1 x PCR buffer II (PE Applied Biosystems, Hercules, CA, USA), 0.5 mM dNTP, 100 ng oligo dT16, 1 U/µL recombinant placental RNase inhibitor, 200 U MuLV reverse transcriptase, and 4 mM MgCl2. After incubation at 37°C for 30 min, the reaction was heat-inactivated at 95°C for 5 min, and then cooled to 22°C. The cDNA products were then divided for the subsequent amplification of AR, ERβ, ER{alpha}, c-Met, and GAPDH in separate PCR procedures. Specific cDNA primers (10-50 pmol) for human AR, ERβ, ER{alpha}, and c-Met were used during PCR as previously described (Takeuchi et al., 1994; Hillier et al., 1998; Osuga et al., 1999). Each reaction also contained 1 µCi of {alpha}-32P-dCTP and 0.5 U AmpliTaq Gold polymerase in 1 x PCR Buffer II. Gene-specific cycles of PCR were run as follows for AR, ERβ, ER{alpha}, and c-Met: 95°C for 12 min, followed by 35 cycles at 95°C (1 min), 54°C (1 min), and 72°C (1 min). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was amplified during 23 cycles at the above temperatures and times. All PCR reactions were terminated with a 10-minute extension phase at 72°C. The gene-specific PCR cycles were chosen based upon preliminary experiments in which the linear phase of amplification was determined (data not shown). All RT-PCR reactions included a negative control in which no RNA was added, and the absence of DNA contamination was determined in PCR reactions that were conducted in the absence of reverse transcriptase.

Amplification of a specific 414-bp region of cDNA for GAPDH (Wiren et al., 1997) was conducted as an internal control for differences in total RNA concentration among samples, and variability among individual PCR amplifications.

All PCR products were visualized in 2% agarose gels stained with ethidium bromide. Scanned gel images were digitally analyzed with the use of the Kodak 1D Image Analysis software program (version 3.0). The relative mRNA abundance is presented in this study as the ratio of target gene/GAPDH.

Statistical Analysis
For in vivo studies (freshly isolated pulp), data from pulp samples were grouped according to gender and age. We analyzed the data by comparing age groups (16-20, 21-35, and 36-45) within and among men and women. In all experiments, mean values from independent experiments were analyzed by unpaired t test, and multiple comparisons were performed by one-way Analysis of Variance followed by Tukey's analysis. Values were determined to be significant when P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gender- and Age-dependent Differences in AR mRNA Expression in Human Tooth Pulp
In the 18- to 24-year-old age group, AR mRNA content in freshly isolated pulp tissue was greater in males when compared with females (Fig. 1Go). No significant differences in AR mRNA levels were measured in the other age groups evaluated in this study. The relative abundance of AR mRNA declined as age increased in both sexes. ERβ mRNA was not detectable in freshly isolated pulp tissue but was present in pulp cell cultures at 48 hrs (Fig. 2Go). The procedures used in this study failed to demonstrate significantly different changes in ERβ mRNA content in response to the hormones that were tested (data not shown). Estrogen-R{alpha} was not detected in freshly isolated pulp tissue or in pulp cell cultures.


Figure 1
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  		Figure 1. Gender- and age-correlated differences in AR mRNA expression in human tooth pulp. RNA was extracted from pulp tissue that was harvested from non-carious third molars (age, 18-55 yrs), and semi-quantitative RT-PCR was conducted to determine the relative abundance of AR mRNA. Values shown represent the ratio of AR:GAPDH in relative units. Bars represent the mean ± SEM of three independent experiments, and significant differences (P ≤ 0.05) in AR mRNA content among the subject groups are indicated by different letters.

 

Figure 2
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  		Figure 2. ERβ expression in tooth pulp. Cultures of pulp cells were prepared and incubated for 48 hrs. RT-PCR was used to screen for ERβ mRNA. The arrow indicates the predicted 439-bp ERβ amplicon. Lane 1, PhiX 174 RF HaeIII DNA standard; lane 2, negative control; lanes 3-4, RNA from independent pulp cell cultures.

 
Regulation of AR mRNA Content by E2 and Androgens
Neither E2 nor androstenedione altered AR mRNA content at 24 hrs. However, at 48 hrs, E2 and androstenedione each induced a significant increase in AR mRNA content (Figs. 2a and 2bGo, respectively) when compared with control (untreated) cultures.

When compared with control cells at 24 and 48 hrs, testosterone, at all concentrations tested, caused a significant decline in the control levels of AR mRNA (Fig. 2cGo). In contrast, DHT did not significantly alter the relative abundance of AR mRNA at 24 hrs (not shown) and 48 hrs (Fig. 2dGo).

Cytokine Regulation of Pulpal AR Expression: HGF and c-Met
At 48 hrs, pulp cells were screened by RT-PCR for the presence of c-Met mRNA. A single band representing the predicted 222-bp c-Met amplicon was visualized, and a representative gel is shown in Fig. 4aGo.


Figure 4
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  		Figure 4. The effect of HGF on AR mRNA in tooth pulp. (Panel a) At 48 hrs in vitro, RT-PCR was used to screen pulp cells for c-Met mRNA. A representative gel shows the presence of c-Met mRNA. The arrow indicates the position of the predicted 222-bp c-Met amplicon. Lane 1, 100-bp DNA ladder standard; lanes 1-3, RNA from three independent pulp cell cultures. (Panel b) Pulp cells were incubated in the presence and absence of HGF for 24 and 48 hrs. Semi-quantitative RT-PCR was used to measure relative differences in AR mRNA content. Values shown represent the ratio of AR:GAPDH in relative units. Bars represent the mean ± SEM of three independent experiments. Significant differences (P ≤ 3; 0.05) in AR mRNA levels as a result of time and treatment are indicated by different letters.

 
HGF did not alter pulpal AR mRNA content from basal levels at 24 hrs (Fig. 4bGo). At 48 hrs, HGF (1 and 10 ng/mL) induced a dose-dependent increase in AR mRNA content when compared with control cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrated age- and gender-correlated differences in AR mRNA content in human tooth pulp. Furthermore, the expression of AR could be hormonally manipulated in vitro. Although AR mRNA levels diminished with age in freshly isolated male and female pulp tissue, the relative abundance of AR mRNA in pulp tissue was consistently greater in males when compared with females. This difference was especially apparent in the youngest age group that was screened. Because pulp cells expressed AR, it is likely that this tissue, like bone (Kasperk et al., 1990), is androgen-responsive. At present, there are no data showing that androgens regulate pulpal function in vivo. However, we suggest that androgens support odontoblast-directed dentinogenesis during development and/or in response to injury. This activity would parallel the androgen-orchestrated events within osteoblasts that enable bone mineralization to occur (Khosla et al., 1999). If this is the case, then tooth viability, like bone, would be supported in response to the cyclically rising levels of gonadal androgen production that occur in the second though fifth decades of life; whereas, in response to advanced age, diminished gonadal androgen production in men and women, combined with reduced AR mRNA content in pulp tissue, may result in an uncoupling of an androgen support mechanism that promotes pulpal viability. Such a mechanism could support an in vivo model to explain why younger pulp tissue possesses a higher reparative potential than older pulp tissue (Trowbridge and Kim, 1998). It is important to consider that the aforementioned androgen-dependent pulpal mechanism remains speculative pending further research.

Interestingly, both the non-anabolic androgen, androstenedione, as well as the predominant estrogen, E2, stimulated an increase in AR mRNA expression in vitro. In contrast, the anabolic gonadal androgen, testosterone, caused a significant reduction in AR content in pulp cells; whereas the more potent androgen, DHT, did not alter AR expression. The regulation of AR expression by androgens and E2 has not been previously reported in dental tissues; however, this regulatory mechanism is not without precedent, as demonstrated in osteoblasts, human prostatic cells, and adipocytes in vitro (Wiren et al., 1997; Blanchere et al., 1998; Dieudonne et al., 1998). Analysis of the present data indicates that fluctuations in the predominant gonadal androgens (androstenedione and testosterone) and E2 can affect pulpal responsiveness to androgens via changes in the expression of AR. Unlike the effects of androstenedione and testosterone, DHT did not alter the relative level of AR mRNA in vitro. To conceptualize this apparent paradox, one must consider that the steroid pathway enzymes, 17β-hydroxysteroid dehydrogenase (17β-HSD) and cytochrome P450 aromatase (P450arom), can convert (i.e., aromatize) androstenedione and testosterone into estrogens; whereas DHT is a non-aromatizable androgen. The presence of pulpal P450arom and 17β-HSD has not been reported to our knowledge; however, if these enzymes are present, the observed effects of androstenedione and testosterone could result from AR- and/or ER-mediated events; whereas any steroid hormone-dependent actions of DHT would be mediated by AR. This is probably a simplistic interpretation, because numerous signaling mechanisms, including cross-talk among steroid hormone receptors, can influence steroid hormone receptor function (Beato and Klug, 2000). Therefore, it is plausible that complex signaling arrays mediate any androgen- and estrogen-dependent changes in AR expression in pulp cells.

Although E2 increased AR mRNA content in pulp cells, and ERβ mRNA was detected in these cells, the hormones that were tested failed to induce measurable changes in ERβ content in vitro. These data do, however, lend further support to a mechanism in which gonadal steroidogenesis is coupled to pulp function at two levels: (1) direct effects of androgens and E2 via activation of AR and/or ERβ, and (2) a feedback loop in which androgens and E2 control AR expression in pulp cells, thereby regulating the (proposed) androgen-responsiveness of this tissue.

While ERβ mRNA was detected in human pulp tissue, ER{alpha} expression was not established. At this time, the presence of ER{alpha} in pulp cells cannot be excluded; however, we suggest that the RT-PCR procedure is an extremely sensitive technique for the detection of mRNA. Thus, if pulp tissue indeed expresses the ER{alpha} isoform, it is possible that functionally significant levels of ER{alpha} may be expressed in response to exquisite temporal and/or hormonal mechanisms that were not tested in this study.

It is important to consider that cytokine and steroid hormone interactions have been documented with regard to bone development and maintenance (Spelsberg et al., 1999). Since HGF has morphogenic actions in dental tissues (Kajihara et al., 1999) and is secreted by pulp (Ohnishi et al., 2000), the effect of HGF on AR expression in pulp cells was tested. This report showed that (1) the HGF receptor, c-Met, is expressed in pulp cells, and (2) HGF up-regulated the relative abundance of AR mRNA in pulp cell cultures. By promoting AR expression, we suggest that HGF, via c-Met-directed signaling, increases pulpal sensitivity to androgens. Thus, any effect of androgens in pulp tissue would be indirectly supported by HGF. Although osteoblastic HGF can regulate certain aspects of bone remodeling and repair (Grano et al., 1996), the parallel effects of odontoblastic HGF in dental tissue (e.g., stimulating dentinogenesis) remain speculative pending further research.


Figure 3
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  		Figure 3. Steroid hormone-dependent regulation AR mRNA content in tooth pulp. Semi-quantitative RT-PCR was used to determine relative differences in AR mRNA content following treatment with E2 (panel a), androstenedione (panel b), testosterone (panel c), and DHT (panel d). Values shown represent the ratio of AR:GAPDH in relative units. Bars represent the mean ± SEM of three independent experiments. Significant differences (P ≤ 3; 0.05) in AR mRNA levels as a result of time in vitro and treatment are indicated by different letters.

 

   ACKNOWLEDGMENTS
 
This work was supported through funds that were provided by the St. Louis University Center for Advanced Dental Education and the SIU School of Dental Medicine. We gratefully acknowledge Dr. George Helden (Alton, IL) for supplying the third molars. We thank Stacy Weitsman (Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Los Angeles, CA) for technical advice regarding the RT-PCR procedures. We also acknowledge the secretarial and technical support of Donna Young and Sandy Sawyer (SIU School of Dental Medicine).

Received for publication June 15, 2001. Revision received January 22, 2002. Accepted for publication February 28, 2002.


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 MATERIALS & METHODS
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 DISCUSSION
 REFERENCES
 

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Journal of Dental Research, Vol. 81, No. 5, 360-365 (2002)
DOI: 10.1177/154405910208100514
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