PTHrP and PTH/PTHrP Receptor 1 Expression in Odontogenic Cells of Normal and HHM Model Rat Incisors

Abstract

Parathyroid hormone related peptide (PTHrP) was discovered as a causative factor of humoral hypercalcemia of malignancy (HHM). We examined PTHrP and its receptor (PTHR1) expression patterns in odontogenic cells in normal and HHM model rat incisors. Nontreated nude rats serving as the normal control and HHM model rats produced by implantation of PTHrP-expressing tumor (LC-6) cells were prepared. HHM rats fractured its incisor, and histopathologically, restrict population of odontoblasts showed findings classified as “shortening of high columnar odontoblasts” and “dentin niche.” The incisors were immunostained against PTHrP and PTHR1. In normal rats, PTHrP and PTHR1 colocalized in ameloblasts, cementoblasts, and odontoblastic cells from mesenchymal cells to columnar odontoblasts. In high columnar odontoblasts, PTHrP solely expressed. In the HHM animals, although the expression patterns were identical to those of the normal rats in normal area, the shortened high columnar odontoblasts maintained PTHR1 expression and dentin niche comprising odontoblastic cells expressed both proteins. In the HHM model, the protein expression patterns changed in the odontoblastic cells with histological anomalies, and thus direct relations between the anomalies and PTHrP/PTHR1 axis are suggested.

Introduction

Parathyroid hormone related peptide (PTHrP) was initially discovered as an essential factor causing humoral hypercalcemia of malignancy (HHM) (Roskams and Desmet, 1997; Strewler, 2000). In the past decade, extensive research has elucidated PTHrP and/or PTHR1 distribution and its functions in several tissues (Strewler, 2000).

In tooth development, PTHrP is known to express in enamel epithelia (Beck et al., 1995; Lee et al., 1995; Liu et al., 1998), while its receptor (PTH/PTHrP receptor 1 or PTHR1) is expressed in cementoblasts (Tenorio and Hughes, 1996), dental papilla (Lee et al., 1995; Liu et al., 1998), and odontoblasts (Lundgren et al., 1998; Calvi et al., 2004). In addition, functional studies have revealed that PTHrP is required for tooth eruption (Schipani et al., 1997; Phibrick et al., 1998; Kitahara et al., 2002), and recently Calvi and colleagues revealed, using continuously PTHR1 expressing transgenic mice, the critical role of the receptor in early dentinogenesis in fetal and neonatal mice (Calvi et al., 2004).

For the exploration of the nature of HHM and its effective clinical treatments, several animal models have been developed that successfully manifest symptoms similar to those of HHM patients, including hypercalcemia, and HHM-related morphological and functional changes in the bones and kidneys (Kukreja et al., 1990; Haq et al., 1993; Sato et al., 1993; Liu et al., 1995; Yamato et al., 1995; Takaori-Kondo et al., 1998; Yaghoobian et al., 1998).

In the same manner, our group developed several HHM xenograft models by implantation of human cancer cell lines in rodents (Endo et al., 1998). We discovered that one of the models showed incisor fractures that appeared with other known HHM symptoms (Kato et al., 2003). Time course observation of the lesions suggested that certain populations of odontoblasts respond to an increased level of PTHrP and form 2 distinctive types of change: decrease of odontoblast cell height and dentin niche. The former is responsible for incisor fracture (Kato et al., 2005).

Based on our observations of HHM model rats, possible relations of the PTHrP/PTHR1 axis in odontogenesis, especially in dentinogenesis, are suggested. However, direct evidence with respect to the PTHrP and/or PTHR1 expressions in the odontogenic cells mainly accumulated in fetus and neonates but was quite limited in adult rodents. The primary aim of the current report is to provide further information and elucidate PTHrP and PTHR1 expressions in odontogenic cells along with the sequence of their differentiation in adult rats, immunohistochemically. The secondary aim of the study is to determine possible relations between HHM-related odontoblastic changes and expressions of PTHrP and PTHR1 from immunohistochemical detection in HHM model rats in comparison with normal adult rats. Furthermore, from the accumulated knowledge of the PTHrP/PTHR1 axis in chondrogenesis, early dentinogenesis, and tissue healing processes, both the physiological and pathological roles of the axis in dentinogenesis of mature rats are discussed.

Materials and Methods

Animal Models

Twenty-five 5-week-old male nude rats were purchased from Clea Japan Inc. (Tokyo, Japan), housed in sterilized cages in an animal room maintained at a temperature of 24 ± 2°C and a humidity of 55 ± 10%, with 14 to 16 air changes per hour and a 14-hour light and 10-hour dark cycle. They were allowed free access to standard rodent chow (CE-2, Clea Japan Inc.) and sterilized water throughout the study.

The animals were divided into 3 groups designated as: Nontreated (NT), HHM, and Antibody (AB) group. For the NT group, 5 animals remained without treatment throughout the study period, were sacrificed at 14 weeks of age, and served to show normal structure and PTHrP and PTHR1 expression patterns in the incisors of adult rats. Additionally, they served as age-matched controls for immunohistochemical distribution patterns for both HHM and AB animals.

For the HHM and AB groups, the cancer cell line LC-6 established from human large-cell lung cancer at the Central Institute for Experimental Animal, Kanagawa, Japan (Tashiro et al., 1989), and known to express a large amount of PTHrP (Endo et al., 1998; Kato et al., 2003, 2005), was prepared. After 1 week of acclimatization, 20 animals received subcutaneous implants of 2-mm³ fragments of LC-6 in the right flank.

At 5 weeks after implantation (11 weeks of age), 200 μL of blood was collected through the retroobital plexus of each animal of all groups under ether anesthesia, and blood ionized calcium (iCa) levels were measured by the electrode method using an autoanalyzer (M-634, Chiba Corning Diagnostics Co. Ltd., Tokyo Japan) Based on the iCa levels, implanted animals were divided into 2 groups (HHM and AB) with similar mean iCa levels. Once a week from 5 to 8 weeks after tumor implantation, the animals of the HHM group received 1 ml/kg of phosphate buffered saline, while the animals of the AB group received 3 mg/kg of anti-human PTHrP antibody (Chugai Pharmaceutical, Tokyo, Japan), known to neutralize the effect of the PTHrP (Ogata, 2000; Iguchi et al., 2001; Onuma, 2004).

All animals were monitored for the occurrence of incisor fracture twice a week, and at 8 weeks after implantation (14 weeks of age), blood iCa levels were measured with same process as the measurement done at week 5 in all animals of all groups. Under deep ether anesthesia, plasma samples were collected from the abdominal artery from both NT and HHM animals and human intact PTHrP concentrations were measured with a 2-site immunoradiometric assay kit using recombinant PTHrP (1-87) as the standard (detectable limit: 1.1 pmol/mL; YUKA-MEDIAS Co. Ltd., Tokyo, Japan). Considering possible interference from treated anti-human PTHrP antibodies to the assay system, measurements of PTHrP concentration in the AB group were not carried out.

The experimental protocols were approved by the Ethics Committee for the Treatment of Laboratory Animals at Chugai Pharmaceutical Co., Ltd.

Histopathology

At 8 weeks after tumor implantation (14 weeks of age), and after the aforementioned plasma sampling in the NT and HHM groups, all animals were exsanguinated through bleeding from the abdominal artery. Both mandibular incisors of all animals and both tibias of the NT group were removed; the left side samples were fixed with 20% neutral buffered formalin and decalcified with ethylenediaminetetraacetic acid · 4Na (EDTA · 4Na) for 2 weeks at room temperature. After transversal trimming at apical, middle, and incisal regions of the incisors and longitudinal trimming of the tibias, the samples were embedded in paraffin using the conventional method and stained with hematoxylin and eosin (HE). The right-side samples were fixed with periodate-lysine-paraformaldehyde fixative (PLP fixative, containing 4% paraformaldehyde) (McLean and Nakane, 1974) at 4°C overnight and decalcified with EDTA•4Na for 1 month at −5°C. After the same trimming that was applied to left side, the samples were embedded in paraffin with AMeX method (Sato et al., 1986) and stained with PTHrP or PTHR1 immunohistochemically.

For immunohistochemistry, anti-human PTHrP (10 μg/ mL) or PTHR1 (8 μg/mL) goat antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were applied as primary antibodies and positive reactions were visualized by the LSAB method with a diaminobenzidine reaction. Anti-PTHrP and PTHR1 antibody were raised against a peptide mapping at the amino terminus of PTH-rP and near the carboxy terminus of PTHR1 of human origin, respectively. As a negative control, normal goat serum (NGS) was applied as a substitute for the primary antibodies for all specimens. To confirm the specificity of the immunohistochemistry, additional negative staining was applied to 1 of the incisors of each group and 1 of the tibias of control group with pre-absorbed antibodies incubated with corresponding antigen proteins (PTHrP: 50 μg/mL or PTHR1: 40 μg/mL, at 4°C overnight) (Santa Cruz Biotechnology, Santa Cruz, CA). For the positive control, tibial epiphyseal chondrocytes of the NT group were stained with anti-PTHrP and anti-PTHR1 antibodies and compared to a previously described distribution pattern in the rat growth plate.

The characteristics and distribution of odontoblastic changes in HE sections and expression patterns of odontogenic cells for both proteins in the immunostained sections were recorded for the lingual, mesial, lateral, and labial areas of each apical, middle, and incisal section (Figure 1). The nomenclature for the differentiation of odontoblastic lineage cells was based on previous reports (Dahl, 1983; Bishop and Boyde, 1986; Ohshima and Yoshida, 1992; Tabata et al., 1993; Sakaki and Garant, 1996; Yoshida and Ohshima, 1996), and classified as mesenchymal cells, pre-odontoblasts, columnar odontoblasts, high columnar odontoblasts or post-odontoblasts (Figure 1).

Statistics

Statistical analysis was conducted on the iCa concentrations. The means and standard deviations of the measured values were calculated for the NT, HHM, and AB groups, and the significant difference was calculated for the HHM or AB groups against the NT group at each sampling time point (at 5 and 8 weeks). Data was first examined by the F-test for homogeneity of the variance among groups. The Student’s t-test was applied to test the significant difference between the group means if the variance was equal; otherwise, Aspin-Welch’s t-test was applied. The difference was considered significant for those with statistical test results of 5%.

Results

PTHrP and PTHR1 Expressions in NT Group

In the apical sections of the PTHrP immunostain, the mesenchymal cells (lingual) were weakly positive, while pre-odontoblasts (lateral and mesial) and columnar odontoblasts (labial) were positive. In the middle section, PTHrP positive reactions were detected in columnar odontoblasts (lingual, lateral, and mesial) and high columnar odontoblasts (labial). In the incisor sections, no PTHrP positive reactions were detected in the post-odontoblasts of any area (Table 1, Figure 2). Throughout all 3 transversal sections, inner and outer ameloblasts and cementoblasts showed positive PTHrP reactions (Table 1, Figure 2).

In the PTHR1 immunostained apical sections, the mesenchymal cells (lingual) were weakly positive, while pre-odontoblasts (lateral and mesial) and columnar odontoblasts (labial) were positive. In the middle sections, PTHR1 positive reactions were found in columnar odontoblasts (lingual, lateral, and mesial) but, in high columnar odontoblasts (labial), both intensity of positive stains and incidence of positive cells were dramatically less. In these sections, an intense positive area was observed in the juxtanuclear compartment of columnar odontoblasts and ameloblasts (Figure 2, i and k). In the incisal sections, no PTHR1 positive reactions were detected in post-odontoblasts of any area; however, PTHR1 was expressed in inner and outer ameloblasts and cementoblasts of all 3 transversal sections (Table 1, Figure 2).

Neither immunostaining with NGS nor pre-absorbed antibodies produced specific positive reactions in any of the sections (Figure 2). The distribution patterns of the PTHrP or PTHR1 positive chondrocytes in the tibial epiphyseal growth plate in the NT group were identical to those previously described (Stevens et al., 2000, Van Der Eerden et al., 2000, 2003). From these results, it was concluded that the immunostains for both PTHrP and PTHR1 were valid.

Pathophysiology of the HHM Model

In the HHM and AB groups, blood iCa levels were significantly higher at 5 weeks after tumor implantation than that of the NT group. At 8 weeks, the blood iCa levels for the HHM animals remained higher but in the AB group the levels were not significantly different from the NT group, statistically (Figure 3). The mean plasma PTHrP levels of the HHM group were 42.42 ± 18.54 pmol/mL, whereas the mean of the NT group was below the detectable limit.

Live phase monitoring of the incisors revealed that mandibular and maxillary incisor fractures occurred on both sides at 7 weeks after tumor implantation in the HHM group. On the contrary, fracture did not occur in any animals of the NT or AB groups.

Histopathology of the HE stained sections showed 2 distinct odontoblastic changes, classified as decrease of odontoblastic cell height and dentin thickness and dentin niche with osteodentin, in the HHM group.

A decrease of odontoblastic cell height was observed in the labial area of the middle sections and decreased dentin thickness was distributed in the labial area of both middle and incisal sections in all animals of the HHM group. These changes were not observed in any animals of the NT or AB groups (Table 2). Histopathologically, cell height of the odontoblasts in the affected area decreased, while those of the corresponding area of the NT and AB groups were of a high columnar phenotype (Figure 4).

Dentin niche, characterized by a sharply demarcated recessed area of the dentin, with osteodentin was observed in 1 out of 10 animals in the HHM group and was localized in the labial area of the apical section, but not found in any area of the NT or AB groups (Table 2). Histopathologically, odontoblasts lost their polarized columnar shape and incorporated within the dentin (osteodentin). Toward the center of the changed area, adjacent pulpal satellite cells showed odontoblastic morphology and produced dentin matrix (Figure 5).

PTHrP and PTHR1 Expression in HHM and AB Groups

Except for the areas with histopathological odontoblastic changes, PTHrP and PTHR1 expression patterns of odontoblastic cells, ameloblasts, and cementoblasts were the same for all groups (Table 1).

In the labial area of the middle sections of the HHM group, odontoblasts with decreased cell height expressed PTHR1, whereas expression significantly decreased in high columnar odontoblasts of the corresponding area in the NT and AB groups (Table 1, Figure 4). At the site of dentin niche with osteodentin, which was observed in 1 animal of the HHM group, incorporated odontoblasts as well as the surrounding pulpal satellite cells expressed PTHrP and PTHR1 (Figure 5).

Discussion

The incisors of rodents are known to grow throughout life, are composed of a dentin core covered by enamel and cementum at the labial and lingual surfaces, respectively, and enclose incisor pulp in the center. Within the pulp, mesenchymal cells around the apical end of the incisor differentiate into odontoblasts, which start secreting dentin and moving toward the incisal direction from differentiation (Kuijpers et al., 1996).

Extensive research in the past decade has revealed PTHrP and/or PTHR1 expressions and their contribution to tooth development in both rodent and human (Beck et al., 1995; Lee et al., 1995; Tenorio and Hughes, 1996; Liu et al., 1998; Lundgren et al., 1998; Philbrick et al., 1998; Kitahara et al., 2002; Wysolmersky et al., 2001; Comier et al., 2003). Recently, Calvi and colleagues showed PTHR1 expression and suggest its possible function in the odontoblasts of fetus and neonates of continuously PTHR1 expressing transgenic mice (Calvi et al., 2004). It is of interest to understand the expression of PTHrP and PTHR1 in adult rodent incisors, along with the differentiational morphological changes of odontogenic cells which are spatially and temporally sequenced.

Current evaluations of PTHrP and PTHR1 in adult rat incisors revealed both proteins express in ameloblasts, odontoblasts, and cementoblasts. To these authors’ knowledge, the present paper is the first report on immunohistochemically detected PTHrP and PTHR1 expressions in odontoblasts and ameloblasts in mature rodents, made possible by the PLP fixation and AMeX embedding applied in this study and known to have excellent ability to preserve antigens of tissue (McLean and Nakane, 1974; Sato et al., 1986).

Positive reactions of PTHrP/PTHR1 expression in ameloblasts and cementoblasts were observed in all cells; there were no transient expressional changes accompanying the cell differentiation. However, transient expressions of both proteins along with zonal and morphological differentiations were observed in the odontoblasts (Figure 6). In cells which differentiate similar morphological and special succession steps such as chondrocytes of growth plate or keratinocytes of skin, it is well documented that PTHrP and PTHR1 are expressed transitionally in those cells and modulate their differentiation in both fetus and adult of many species (Vortkamp et al., 1998; St.-Jacques et al., 1999; Strewler, 2000; Van Der Eerden et al., 2000; Nakase et al., 2001; Comier et al., 2003). From our observations of odontoblasts of normal adult rats and known information of the odontoblasts of fetal and neonatal mice, as well as of other organs, it is suggested that the PTHrP/PTHR1 axis may modulate dentinogenesis in normal adult rodents.

In a previous report, our group showed that incisor fractures occurred in HHM model rats (Kato et al., 2003) and, from the time-course observations of the changes, suggested that a decrease of the cell height in high columnar odontoblasts was the initial onset of change followed by thinning of the dentin leading to fracture (Kato et al., 2005). In addition, because the restricted localization patterns of the shortened odontoblasts coincided with the detection of PTHrP in systemic circulation and the treatment of antibody that antagonizes PTHrP activity eliminated the findings, it was concluded that PTHrP, which acts as an auto- and/or a paracrine local factor, is a direct causative factor. However, a lack of information on PTHrP and/or PTHR1 expression in odontoblasts of mature rats prevented conclusive discussion about the possible relation between the lesions and the proteins. In the current report, incisor fractures and decrease of odontoblastic cell height were observed with identical localization as the previous report. Interestingly, in the NT group, high columnar odontoblasts showed decreased expression of PTHR1, while in the HHM animals, the odontoblasts were shortened and PTHR1 expressions sustained in the corresponding labial area of the middle sections. With treatment of anti-PTHrP antibody to the HHM animals, both morphological and PTHR1 expressional changes were eliminated. From these results, a possible direct correlation between the incisal fracture of the HHM model and the PTHrP/PTHR1 axis was elucidated. Namely, increased PTHrP levels in the HHM model cause decrease of cell height of high columnar odontoblasts and maintain their PTHR1 expressions, and these changes are followed by thinning of the dentin, causing incisal fracture.

Reports regarding PTHrP or its receptor in knockout mice have revealed that the mice manifest chondrodysplasia due to premature hypertrophic differentiation (Amizuka et al., 1996; Chung et al., 1998, 2001; Karp et al., 2000, Kobayashi et al., 2002). On the other hand, PTHrP oversignaling conditions, such as in animal models with PTHrP over-expression, lead the differentiational delay in chondrocytes (Schipani et al., 1995, 1997; Weir et al., 1996), hair follicles (Wysolmerski et al., 1994), and mammary glands (Wysolmerski et al., 1995). Recently, in fetus and neonates of continuously PTHR1 expressing transgenic mice, considered to be PTHrP oversignaling, differentiational delay of odontoblasts and a decreased production of dentin were reported (Calvi, 2004). Therefore, it was plausible to conclude that the decrease of the odontoblastic cell height was not mere atrophy but a differentiating delay from columnar to high-columnar odontoblast (Kato et al., 2005). The following results from the present study allow us to further hypothesize.

In the NT group, PTHR1 expression decreased along with differentiation from columnar to high-columnar odontoblasts.

In the HHM group, PTHR1 expression maintained and coincided with delayed high-columnar differentiation of the columnar odontoblasts.

In the AB group, the findings of the HHM group recovered.

This group of results suggests that the high PTHrP levels of the HHM group maintained the expression levels of PTHR1 in columnar odontoblasts, which normally is reduced along with its high-columnar differentiation. This sustained expression of PTHR1 maintained signal inputs of the PTHrP, which caused differentiation delay from columnar to high-columnar odontoblasts. On the other hand, it can be considered that, in normal adult rats, reduction of PTHR1 decreases the input of PTHrP signals and causes high-columnar differentiation. Furthermore, since in HHM rats suppression of high-columnar differentiation caused incisor fracture, physiologically, the differentiation from columnar to high-columnar odontoblasts is the mechanism by which the strength of the incisor to bear mechanical stress in rats is maintained (Figure 7).

Dentin niche was observed in the HHM model rats of this study with low incidence (1 out of 10 animals). By immunostaining PTHrP and PTHR1, expression of the proteins was elucidated in both incorporated odontoblasts and the surrounding pulpal satellite cells that constituted the lesion. Dentin niche is known to induce by administration of cytotoxic agents with different modes of actions and is considered to be a reparative response by odontoblastic cells to the cytotoxicity (Adkins, 1972; Koppang, 1973; Adatia, 1975; Stent and Koppang, 1976, 1980; Mikkelsen, 1978; Stene, 1978, 1979; Nogueira et al., 1981; Dahl, 1984, 1985; Karim and Eddy, 1984; Dahl and Koppang, 1985; Moule et al., 1993; Long et al., 2004). In combination with the fact that PTHrP is known to contribute to the process of bone, cartilage, and skin repair (Ferguson et al., 1998; Vortkamp et al., 1998; Blomme et al., 1999; Kudo et al., 2000; Nakase et al., 2001; Okazaki et al., 2003; Van Der Eerden et al., 2003), but not known to have cytotoxicity, we previously suggested that exposure to long and sustained high PTHrP concentrations may induce the odontoblastic reparative mechanisms without the preceding cytotoxicity (Kato et al., 2005). Although low incidence of the lesion prevented a conclusive discussion, the fact that the expressions of both PTHrP and PTHR1 in the lesion comprising cells further supports the concept in which the PTHrP/PTHR1 axis is related to the regulation of the reparative response of odontoblasts. To further evaluate this relationship, PTHrP and PTHR1 expression in odontoblasts in the reparative phase after the administration of cytotoxic agents is currently under investigation.

Although knowledge about the role of PTHrP/PTHR1 in fetus and neonate has been clarified by intensive past research, it is still obscure in the mature phase of life. And, to some extent, current protein expression patterns of incisors of adult rats differed from those of previously reported neonates/fetus (i.e., PTHR1 expression in ameloblasts). Considering these differences, it is possible to speculate that among the life stages, some of the roles of the PTHrP/PTHR1 axis are different. In addition, recently a meticulous in vitro study elucidated PTHR1 subcellular trafficking after its internalization, and has shown the Golgi apparatus to be the final destination of this trafficking system (Tawfeek and Abou-Samra, 2004). Here, PTHR1 intensive positive reactions were observed at the juxtanuclear compartment of columnar odontoblasts and ameloblasts known as the Golgi area (Bishop and Boyde, 1986; Dahl, 1993; Ohshima, and Yoshida, 1992; Sakaki and Garant, 1996; Tabata et al., 1993; Yoshida and Ohshima, 1996). This accumulation of the protein in the Golgi apparatus was not described in the recent in vivo studies. Thus it was considered that the relative long succession of special and temporal differentiation of the rat odontoblasts are a suitable model to investigate the possible role (including the subcellular trafficking of the proteins) of the PTHrP/PTHR1 axis in the mature phase of life.