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Transient Co-localization of c-Jun N-terminal Kinase and c-Jun with Heat Shock Protein 70 in Pulp Cells during Apoptosis
1 Departments of Operative Dentistry and Endodontics, and Correspondence: *corresponding author, chi-aki-k{at}kyu-dent.ac.jp
The c-Jun N-terminal kinase (JNK) pathway and heat-shock proteins (HSPs) are involved in stress-induced apoptosis. Here we examined the association of JNK, c-Jun, and anti-apoptotic HSPs with pulp apoptosis during wound healing. In normal pulp, c-Jun was activated only in a few pulp cells, but JNK was not. HSP70 was expressed in the cytoplasm of pulp cells. One day after injury, active JNK and c-Jun were detected in apoptotic pulp cells, whereas HSP70 was detected in non-apoptotic cells. We also found the translocation of HSP70 into nuclei of pulp cells, and co-localization with active JNK and c-Jun. Four days after injury, active JNK and c-Jun disappeared in pulp cells, and HSP70 was relocalized from nuclei to the cytoplasm. These results suggest that the JNK pathway may be one of the compartments inducing apoptosis in pulp cells, and that HSP70 may have an inhibitory role in the apoptosis of pulp cells during wound healing.
Key Words: apoptosis • c-Jun • c-Jun N-terminal kinase • heat-shock protein 70 • wound healing
Pulp wound healing involves apoptosis, the death of damaged cells (Bronckers et al., 1996; Vermelin et al., 1996), as well as reactionary dentinogenesis by surviving odontoblasts (Smith et al.1995) and reparative dentinogenesis by odontoblast-like cells (Mitsiadis et al., 1999). We previously reported that c-jun and jun-B genes may play roles in surviving odontoblasts and odontoblast-like cells (Kitamura et al., 1999). We also found that apoptosis is induced both in odontoblasts and in pulp cells, as one of the cell death mechanisms, during pulp wound healing (Kitamura et al., 2001). c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinases, phosphorylates the transactivation domain of c-Jun. Recently, it has been demonstrated that the JNK pathway is a compartment of signaling pathways, leading cells into apoptosis in response to several stresses, including heat shock (Bossy-Wetzel et al., 1997; Tournier et al, 2000). Other molecules, heat-shock proteins (HSPs) 70 and 27, have also been shown to play an important role in the apoptotic signaling pathway in heat-stressed conditions (Garrido et al., 2001). In particular, HSP70 exhibits anti-apoptotic action through the inhibition of the JNK cascade (Gabai et al., 1997, 2000). Heat stress brought on by cavity preparation is recognized as one of the major causes of damage to pulp tissues. In this study, we examined whether JNK, c-Jun, HSP70, and HSP27 are associated with the induction of apoptosis during pulp wound healing after cavity preparation.
Preparation of Rat Molars Twenty Wistar specific pathogen-free rats (9 wks old), each weighing from 250 to 350 g, were cared for under barrier system conditions. The animal protocol was carried out according to the guidelines for animal care of Kyushu Dental College, and ethical approval was obtained from the institutional panel for animal care. Rats were deeply anesthetized by intraperitoneal injection of 5% pentobarbital sodium (Nembutal®, Dainippon Pharmaceutical Co., Suita, Japan) at a dose of 30 mg/kg, and cavities were prepared on mesial aspects of maxillary first molars at a half-thickness of dentin, by means of a No. 1/2 round bur. Cavities were left unrestored, and one molar from each group was without cavity. The post-operative time intervals of death were 1 hr, 1, 4, and 14 days. After each interval, rats were anesthetized by the above method, and killed by transcardial vital perfusion with 4% paraformaldehyde-phosphate buffer, pH 7.3 (PFA). First molars dissected from maxillary segments were then demineralized in 10% EDTA-phosphate-buffered saline (1xPBS), pH 7.3, at 4°C.
Immunohistochemistry For double-immunofluorescence staining, sections were reacted with a primary antibody against either p-JNK or p-c-Jun. Goat anti-mouse IgG-Alexa Fluor 488 (Molecular Probes, Inc., Eugene, OR, USA) was used as a secondary antibody at a dilution of 1:200 in 1xPBS for 1 hr at room temperature. Sections were subsequently reacted with a primary antibody against either HSP70 or HSP27, and labeled with donkey anti-goat IgG-Alexa Fluor 546 (Molecular Probes, Inc., Eugene, OR, USA) at a dilution of 1:200 in 1xPBS. Signals were observed under the fluorescence microscope, and analyzed by Adobe Photoshop software (Adobe Systems Inc., San Jose, CA, USA).
Double-staining with Terminal Deoxynucleotidyl Transferase-mediated Labeling (TUNEL) and Immunohistochemistry
We first examined the temporospatial patterns of p-JNK, p-c-Jun, HSP70, and HSP27 in dental pulp. Fig. 1  shows representative data of non-treated and one-hour-treated sections. The upper panels in each group show the odontoblast layer and pulp cells close to odontoblasts, and the lower panels show pulp cells distant from the odontoblast layer. In non-treated molars, there were no signals of either p-JNK or p-c-Jun in the odontoblast layer (Figs. 1a, 1c). Signals of p-c-Jun were detected in the nuclei of a few pulp cells close to odontoblasts (Figs. 1c, 1d), whereas signals of p-JNK were not detected in any cells (Figs. 1a, 1b). HSP70 and HSP27 were observed in the cytoplasm of most odontoblasts and some pulp cells (Figs. 1e-1h). Expression of HSPs in odontoblasts was sustained during pulp wound healing. One hour after injury, primary apoptosis was induced in odontoblasts, and the arrangement of the odontoblast layer under the cavities was disrupted. In this phase, signals of p-JNK and p-c-Jun were detected in a few pulp cells in the subodontoblastic area, but not in odontoblasts (Figs. 1i-1l). There were no changes in levels of p-c-Jun signals in 1 hr, compared with those in the non-treated group. Localization of HSP70 and HSP27 was the same as that of non-treated molars (Figs. 1m-1p).
Fig. 2 shows distribution patterns of these molecules one day after injury. In this phase, secondary apoptosis was induced in pulp cells. Signals of both p-JNK and p-c-Jun were more frequently detected in pulp cell nuclei at one day than in non-treated and one-hour groups (Figs. 2a-2f). HSP70 signals translocated from the cytoplasm to the nuclei of pulp cells in the subodontoblastic area (Figs. 2h, 2i). Intracellular translocation of HSP70 was not observed in odontoblasts (Fig. 2g). The distribution pattern of HSP27 in the one-day group was the same as that of the one-hour group (Figs. 2j-2l).
Fig. 3 shows representative data 4 and 14 days after injury. At 4 days, signals of p-JNK and p-c-Jun were detected only in the nuclei of some pulp cells close to functioning odontoblasts, but not in other pulp cells far from the odontoblast layer (Figs. 3a-3d). Pulp cells expressing p-JNK and p-c-Jun appeared to move to the front of the reparative dentin formation. HSP70 also appeared to relocalize from nuclei to the cytoplasm of pulp cells (Fig. 3f). Intense signals of HSP27 were detected in the cytoplasm of subodontoblastic pulp cells (Fig. 3h). Fourteen days after injury, signals of p-JNK and p-c-Jun were maintained only in nuclei of some pulp cells close to functioning odontoblasts, not in other pulp cells (Figs. 3j-3m). Distribution patterns and levels of HSP70 and HSP27 returned to those in non-treated molars (Figs. 3n-3q).
To clarify the relationship between the intracellular localization of p-JNK, p-c-Jun, and HSP70, and secondary apoptosis in pulp cells one day after injury, we next examined whether these molecules were detected in apoptotic cells, and whether translocated HSP70 co-localized with p-JNK and p-c-Jun in the nuclei of pulp cells (Fig. 4 ). Signals of p-JNK and p-c-Jun were detected in TUNEL-positive cells (Figs. 4a, 4b), whereas signals of HSP70 were not noted in any TUNEL-positive cells (Fig. 4c). HSP70 appeared to co-localize with p-JNK and p-c-Jun in nuclei of some pulp cells (yellow signals in Figs. 4d, 4e). Pulp cells predominantly expressing HSP70 (red signals in Figs. 4d, 4e) were also observed.
In these experiments, negative controls omitting the primary antibody or with non-specific IgG showed no signals, and no positive cells were observed in negative control sections after incubation in TdT mixture without TdT(AQ) (data not shown).
During wound healing after cavity preparation, the pulp shows primary apoptosis immediately induced in odontoblasts 1 hr after injury, and secondary apoptosis induced in pulp cells one day after injury (Kitamura et al., 2001). Here we examined the association of JNK, c-Jun, and HSPs with pulp apoptosis. One hour after injury, JNK and c-Jun were not activated in odontoblasts, suggesting that the activation of the JNK pathway is not essential for apoptosis in odontoblasts. Other factors stimulated by direct mechanical stresses, such as the damage to odontoblastic processes and the aspiration of odontoblastic bodies into dentinal tubules, may be associated with apoptosis of odontoblasts. In contrast, we observed the appearance of pulp cells with active JNK. This result suggests that gap junctional signaling may exist between injured odontoblasts and pulp cells, and that secondary apoptosis may initiate in pulp cells through the JNK pathway in the early healing process. Pulp cells with active c-Jun 1 hr post-injury were similar in number to untreated cells, suggesting that the activation of c-Jun in pulp cells during this early phase is not associated with apoptosis. One day after injury, JNK and c-Jun were frequently activated in pulp cells, and localized in some apoptotic pulp cells, whereas HSP70 localized only in non-apoptotic pulp cells. Furthermore, we found that HSP70 transiently translocated from the cytoplasm to nuclei of pulp cells, and co-localized with active JNK and c-Jun. HSP70 is known to translocate from the cytoplasm to the nucleus following heat-shock stress (Lepock et al., 2001). Although we need direct evidence from cell culture experiments, these results suggest that heat-shock stress from cavity preparation can induce apoptosis in pulp cells, and that JNK and c-Jun contribute to secondary apoptosis in pulp cells. It is also suggested that HSP70 plays an anti-apoptotic role by inhibiting the JNK pathway, and may contribute to the thermotolerance of pulp tissues. Pulp cells showing co-localization of these molecules may be rescued from apoptosis, and pulp cells without co-localization may enter apoptosis. We also examined the expression of HSP27, one of the survival molecules in heat-shock stress (Garrido et al., 1999). In this study, HSP27 neither translocated nor co-localized with active JNK and c-Jun in the nuclei of pulp cells, suggesting that the expression of HSP27 is independent of the regulation of pulp apoptosis. We found persistent expression of HSPs in odontoblasts throughout the pulp healing process. It is well-known that HSPs function as molecular chaperones in a variety of cells (Bukau and Horwich, 1998). Sustained expression suggests that HSPs have roles in the survival of odontoblasts for reactionary dentinogenesis. From 4 to 14 days after injury, apoptotic cells were eliminated, and reparative dentin formation was strongly activated. In these phases, JNK and c-Jun were activated and maintained only in the nuclei of some subodontoblastic pulp cells that had migrated to the front of reparative dentin formation. The JNK pathway is also known to regulate the cell differentiation process (Ip and Davis, 1998). The activation of JNK and c-Jun in specific pulp cells close to odontoblasts suggests that these cells with active JNK and c-Jun are odontoblastic progenitor cells, and that the JNK pathway is associated with the recruitment of odontoblast-like cells from pulp cells for reparative dentinogenesis. Taken together, the results indicate that the JNK pathway contributes to both apoptosis and the differentiation of pulp cells throughout wound healing after cavity preparation. Recently, cross-talk has been suggested to occur between the JNK cascade and the Smad cascade responding through the TGF-ß family of receptors (Mazars et al., 2001; Péron et al., 2001). To clear the crossroad of survival and apoptosis of pulp cells during the wound-healing process, we are now continuing to research the cooperation of JNK pathway members with other molecules, including Smads.
This research was supported by a Grant in Aid for Scientific Research, 13771136,14207082 (Kitamura), from The Ministry of Education, Science, and Culture of Japan, Tokyo, Japan. Received for publication April 23, 2002. Revision received September 23, 2002. Accepted for publication November 6, 2002.
Journal of Dental Research, Vol. 82, No. 2,
91-95 (2003)
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