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J Dent Res 85(9):824-828, 2006
© 2006 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Re-oxygenation Improves Hypoxia-induced Pulp Cell Arrest

Y. Ueno1, C. Kitamura1, M. Terashita1, and T. Nishihara2,*

1 Division of Pulp Biology, Operative Dentistry, and Endodontics, Department of Cariology and Periodontology, Science of Oral Functions, and
2 Division of Infections and Molecular Biology, Department of Health Promotion, Science of Health Improvement, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita, Kitakyushu 803-8580, Japan

* corresponding author, tatsujin{at}kyu-dent.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dental pulp cells can be exposed to hypoxia during severe inflammation or restorative procedures, though their response to hypoxia is not well-understood. We hypothesized that hypoxia has effects on the growth of pulp cells in vitro. When the cells were exposed to hypoxia for 48 hr, cell growth was suppressed, and cell death was detected by Hoechst staining. Western blot analysis revealed that phosphorylation of retinoblastoma protein was inhibited in cells exposed to hypoxia. Analyses of the molecules involved in retinoblastoma protein phosphorylation revealed that hypoxia suppressed cyclin D2 and activated p21CIP1/WAF1. Further, hypoxia-exposed pulp cells showed improvement of cell viability, cell-cycle progression, and expression of cyclin D2 with re-oxygenation. These findings indicate that hypoxia-induced cell cycle arrest in pulp cells is reversible, while cyclin D2 may play an essential role in the improvement of cell proliferation with re-oxygenation.

KEY WORDS: hypoxia • cell-cycle arrest • re-oxygenation • dental pulp cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypoxia induces cell-cycle arrest in the G1 phase by inactivation of the molecules responsible for DNA synthesis, and inhibits progression of the cell cycle (Thelander et al., 1983; Loffler, 1989; Gardner et al., 2001). Cell death induced by hypoxia is recognized as apoptosis and is known to play a role in diseases that exhibit a hypoxic microenvironment, such as atherosclerosis. Numerous molecules—including p53, retinoblastoma protein (Rb), cyclins, cyclin-dependent kinase (CDKs), CDK inhibitors, and hypoxia-inducible factor 1{alpha}—play essential roles in the cell fate decision that leads to either cell proliferation or apoptosis during G1 arrest caused by hypoxia (Krtolica et al., 1998; Goda et al., 2003).

Following carious infection of teeth, pathological changes such as inflammation occur in dental pulp, and give rise to painful symptoms in teeth (Mjör, 2002). Restorative procedures to alleviate painful symptoms can produce a variety of types of stress, such as hypoxic stress, which is known to be one of the major adverse responses encountered during restorative treatment of the dental pulp. Administration of local anesthetic that contains a vasoconstrictor, which is often given during restorative procedures, results in a reduction of pulpal blood flow and produces a hypoxia in the dental pulp (Kim et al., 1984, 1992). Further, abolition of the vasculature after pulp exposure may also induce hypoxia during pulp wound-healing.

With proper treatment, the hypoxic condition produced in dental pulp is transient, and pulp viability is generally improved by re-oxygenation after an increase of pulpal blood flow. We previously demonstrated that heat stress induces pulp apoptosis during wound-healing and regeneration (Kitamura et al., 2001, 2003), and that pulp cells show thermo-resistance following heat stress (Kitamura et al., 2005). However, there are few reports regarding the responses of pulp cells to hypoxia, or the molecular mechanisms responsible for resistance to hypoxia by pulp cells (Amemiya et al., 2003). In the present study, we hypothesized that hypoxia has effects on the growth of dental pulp cells in vitro, and we further examined pulp cell response to re-oxygenation after hypoxia.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
We cloned RPC-C2A-K4 cells twice from rat clonal dental pulp cells (RPC-C2A cells) by limiting dilution (Kasugai et al., 1988) and used them as clonal dental pulp cells. The RPC-C2A-K4 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Corp., Carlsbad, CA, USA) containing 10% heat-inactivated fetal calf serum (FCS), 100 µg/mL of streptomycin, and 100 U/mL of penicillin, and then incubated in a humidified atmosphere of 5.0% CO2 in air at 37°C. Thirty hours after the subculture began, the cells were exposed to hypoxia for 3 to 60 hrs. A hypoxic condition was accomplished by the exchange of air in the chamber to a gas mixture containing 5.0% CO2, 1.0% O2, and 94% N2. During culture in the hypoxic condition, we monitored the oxygen tension in the gas mixture inside the chamber by means of a gas analyzer (Astec Co., LTD., Fukuoka, Japan). Further, the cells exposed to hypoxia for 24 hrs were subsequently re-oxygenated by exposure to a normoxic condition (20% O2).

Cell Viability Assay
Inhibition of cell proliferation after exposure to hypoxia was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay (Mosmann, 1983). At each time-point during hypoxia, MTT solution (20 µL/well) was added to each well, and the dishes were incubated at 37°C for 4 hrs. After acid-isopropanol (100 µL/well) was added and mixed thoroughly, we analyzed cell viability at each time-point by measuring optical density (OD) using a test wavelength of 540 nm and a reference wavelength of 620 nm with a multiscan biochromatic microplate reader (Multiscan JX) (Thermo Electron Co., Yokohama, Japan).

Cell-cycle Analysis
Hypoxia-exposed RPC-C2A-K4 cells were suspended in a hypotonic solution (0.1% Triton X-100, 1 mmol/L Tris/HCl, pH 8.0, 3.4 mmol/L sodium citrate, 0.1 mmol/L EDTA) and stained with 5 µg/mL of propidium iodide, after which cell-cycle distribution was analyzed with a FACScalibur flow cytometer EPICS XL (Beckman Coulter, Fullerton, CA, USA).

Detection of Cell Death by Hoechst Staining
RPC-C2A-K4 cells were cultured on coverslips and exposed to hypoxia. Those cells attached to the coverslips were fixed with 1.0% glutaraldehyde for 30 min, washed with 1x phosphate buffer (PBS), stained with 5 µg/mL of Hoechst dye 33342, and mounted on slides. Cells detached from the coverslips into the medium were recovered by centrifugation and stained with Hoechst dye 33342. Nuclei were observed under a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70) (Olympus Co., Tokyo, Japan).

Immunoblot Analysis
Hypoxia (0–24 hrs)-exposed RPC-C2A-K4 cells underwent lysis in a buffer containing 3.0% sodium dodecyl sulfate (SDS) in 50 mmol/L of Tris/HCl (pH 6.0). Proteins from whole-cell lysates were separated on SDS-polyacrylamide gels and electroblotted onto Immobilon-P Transfer membranes (Millipore Corp., Bedford, MA, USA). Blotted membranes were blocked in PBS containing 5.0% skim milk and incubated with the following primary antibodies: mouse anti-p21CIP1/WAF1 (F-5), mouse anti-p53 (Ab-3), rabbit anti-cyclin D2 (M-20) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit anti-phosphorylated p53 (Ser-15) (Cell Signaling Technology, Beverly, MA, USA), mouse anti-RB (BD Biosciences, San Jose, CA, USA), and mouse anti-actin (Santa Cruz Biotechnology Inc.) as a control. Subsequently, the membranes were incubated with the following secondary antibodies: donkey anti-rabbit IgG and sheep anti-mouse IgG, which were both peroxidase-linked secondary antibodies (Amersham Biosciences Corp.). Immunodetection was performed with the use of the ECL Western blotting detection system (Amersham Pharmacia Biotech, UK), according to the manufacturer’s instructions.

Statistical Analysis
We performed two-way analyses of variance to compare the means of the different conditions. Protected Fisher least-significant differences were used for pairwise comparisons. All data are expressed as the mean ± SD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell Viability of RPC-C2A-K4 Cells Exposed to Hypoxia
We first examined the viability of RPC-C2A-K4 cells exposed to normoxic or hypoxic conditions (Fig. 1Go). There were no differences in viability after 6 and 12 hrs; however, after 18 hrs of normoxia, the cells showed continuous growth, and cell numbers were gradually increased. In contrast, growth inhibition of RPC-C2A-K4 cells exposed to hypoxia was observed, and significant growth inhibition was found, from 24 to 60 hrs of hypoxia. In the long-term hypoxic condition, the viability of the cells showed no significant difference between 48 and 60 hrs of hypoxia.


Figure 1
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  		Figure 1. The viability of RPC-C2A-K4 cells exposed to hypoxia for 0 to 60 hrs was monitored by MTT assay. White bar = cells under normoxic condition; grey bar = cells under hypoxic condition. Data are expressed as the mean ± SD of triplicate cultures. The experiment was performed 3 times, with similar results obtained in each experiment. Statistical differences were determined between the cells in normoxia and the cells in hypoxia at each time-point. *p < 0.01.

 
Effects of Hypoxia on Cell Cycle of RPC-C2A-K4 Cells
Next, we examined the effects of hypoxia on the cell-cycle progression of RPC-C2A-K4 cells (Figs. 2A, 2BGo). Exposure to hypoxia (24 and 48 hrs) increased the numbers of cells in the G1 phase, with a concomitant reduction of those in the S and G2/M phases. An increase of RPC-C2A-K4 cells in the sub-G1 population, implying cell death such as apoptosis, was also observed. The population of the cells in the G1 phase gradually increased in a time-dependent manner, from 51.3% (0 hr) to 71.6% (48 hrs), with a reduction in cells in the S and G2/M phases from 48.5% (0 hr) to 17.6% (48 hrs). The population of RPC-C2A-K4 cells in the sub-G1 increased from 0.2% (0 hr) to 10.8% (48 hrs) during exposure to hypoxia (Fig. 2BGo).


Figure 2
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  		Figure 2. Cell-cycle analysis of RPC-C2A-K4 cells exposed to hypoxia. (A) Representative results of cell-cycle distribution of the cells under normoxic (non-treated) and hypoxic conditions for 24 and 48 hrs. (B) Changes that occurred during each phase of cell-cycle distribution during hypoxia are indicated at the times noted. (C) Representative morphologies of non-treated and hypoxia-exposed cells. Scale bar = 10.0 µm.

 
Detection of Cell Death in RPC-C2A-K4 Cells Exposed to Hypoxia
Fluorescent microscopic analysis showed that adherent RPC-C2A-K4 cells exhibited normal structures, whereas those cells that became detached into the medium during hypoxia showed cell death, with typical characteristics of apoptotic cells, such as nuclear fragmentation and apoptotic corpuscles (Fig. 2CGo).

Expression and Phosphorylation of Cell-cycle-related Molecules
We investigated the expression and phosphorylation of p21CIP/WAF1, Rb, cyclin D2, and p53 to clarify the molecular mechanisms involved with G1 arrest and cell death in RPC-C2A-K4 cells exposed to hypoxia (Fig. 3Go). The expression of p21CIP/WAF1 was strongly induced after 3 hrs of exposure to hypoxia, after which it gradually decreased, and was completely suppressed after 24 hrs of exposure to hypoxia. Hyperphosphorylated Rb was expressed throughout the period of hypoxia, whereas hypophosphorylated Rb was detected from 12 to 24 hrs of hypoxia, after the strong induction of p21CIP/WAF1 at 3 hrs of hypoxia. The expression of cyclin D2 was detected in the period of 0 to 6 hrs of hypoxia, with a peak at 3 hrs, after which it was decreased in a time-dependent manner. Unphosphorylated p53 was stably expressed in the cells throughout the period of hypoxia, while phosphorylated p53 was faintly expressed at 0 hrs (normoxic condition), and then its expression gradually increased during hypoxia and peaked at 18 hrs.


Figure 3
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  		Figure 3. Expression of cell-cycle-related molecules in hypoxia-exposed RPC-C2A-K4 cells. The immunoblot analysis was carried out as described in MATERIALS & METHODS. pRb, hypophosphorylated Rb; ppRb, hyperphosphorylated Rb; pp53, phosphorylated p53.

 
Effects of Re-oxygenation on Growth of RPC-C2A-K4 Cells Exposed to Hypoxia
After 24 hrs of exposure to hypoxia, RPC-C2A-K4 cells were subsequently incubated under normoxic conditions. With re-oxygenation, the viability of the cells gradually increased (Fig. 4AGo), G1 arrest and sub-G1 induction caused by hypoxia gradually disappeared, and the cell-cycle distribution pattern of hypoxia-insulted cells returned to that prior to exposure to hypoxia (Fig. 4BGo). Interestingly, cyclin D2, which was suppressed by hypoxia, was induced in the cells by re-oxygenation, and then returned to the level observed in the normoxic condition (Fig. 4CGo).


Figure 4
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  		Figure 4. Improvement in intracellular functions of hypoxia-exposed RPC-C2A-K4 cells by re-oxygenation. After the cells were exposed to hypoxia for 24 hrs, cells were incubated under a normoxic condition (re-oxygenation). (A) Cell viability was monitored by MTT assay. Data are expressed as the mean ± SD of triplicate cultures. The experiment was performed 3 times, with similar results obtained in each experiment. Statistical differences were determined between the cells exposed to 24 hrs of hypoxia (0 hr after re-oxygenation) and the cells exposed to normoxia after 24 hrs of hypoxia (a, 12 hrs of re-oxygenation; b, 24 hrs of re-oxygenation; c, 36 hrs of re-oxygenation). *p < 0.01. (B) DNA content was analyzed by flow cytometry. Non-treated, cells in normoxia. (C) Expression of cyclin D2 in re-oxygenated RPC-C2A-K4 cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we found that short-term exposure to hypoxia did not have an effect on the viability of pulp cells; however, long-term exposure reduced their viability, and the population of cells in the S phase decreased after long-term exposure to hypoxia, suggesting that hypoxia suppresses the proliferation of pulp cells in vitro in a time-dependent manner. We also found that long-term exposure to hypoxia in vitro (from 24 to 48 hrs of hypoxia) induced G1 arrest and cell death. It is known that cell-cycle arrest is essential for the maintenance of viability under environmental conditions preventing normal regulation of cell growth (Weinert and Hartwell, 1988). Our results suggest that hypoxic stress is a critical factor for growth arrest of pulp cells, and that the hypoxia-time-dependent reduction in pulp cell proliferation seen in the present study was the result of G1 arrest and the induction of cell death.

Rb is known to control cell-cycle progression during the G1/S transition in response to extracellular signals for growth inhibition (Weinberg, 1995). Hypophosphorylated Rb binds to E2F transcription factor and cancels its ability to activate the genes required for entry into the S phase, while hyperphosphorylation of Rb abrogates its binding to E2F, allowing E2F to activate the genes (Nevins, 1992). Rb is phosphorylated by CDKs, and forms complexes with specific regulatory subunits of cyclins. Among cyclins, cyclin D is one of the key modulators to control cell-cycle progression. When the cell cycle progresses toward the G1 to S transition, cyclin D-CDK4 kinase activity increases and participates in the regulation of Rb phosphorylation (Ewen et al., 1993). The Rb kinase activity of CDK4 is positively regulated by cyclin D and negatively regulated by CDK inhibitors, including p16INK4a, p15INK4b, p21CIP/WAF1, and p27KIP1 (Sherr and Roberts, 1995; Weinberg, 1995; Yamato et al., 1997). It has also been shown that p21CIP/WAF1 is induced through a p53-dependent or independent pathway by several environmental stress factors (Brugarolas et al., 1995; Aguero et al., 2005). p21CIP/WAF1 mediates the blockade of cyclin D-CDK4 kinase activity by directly binding to this catalytically active kinase complex, and reduces Rb phosphorylation, which results in an inhibition of cell-cycle progression (El-Deiry et al., 1994; Slebos et al., 1994; Sherr and Roberts, 1995).

Our findings indicate that p21CIP/WAF1 was transiently induced in the early phase of pulp cell exposure to hypoxia, cyclin D2 was expressed in the early phase of hypoxia, and expression of these molecules decreased throughout hypoxia. After the appreciable induction of p21CIP/WAF1 and the reduction of cyclin D2, hypophosphorylated Rb was induced from 6 to 24 hrs of hypoxia. We also indicate, in the present study, that G1 arrest appeared after 24 and 48 hrs of hypoxia after the change of p21CIP/WAF1/cyclin D2/Rb expression, and that the suppression of cell proliferation continued during long-term hypoxia (48 and 60 hrs) after the induction of G1 arrest. Taken together, these results suggest that hypoxia-induced G1 arrest of pulp cells resulted from the suppression of Rb phosphorylation through a combined modulation of cyclin D2 and p21CIP/WAF1 in pulp cells, and that G1 arrest of pulp cells induced by hypoxia resulted in the reduction of cell proliferation during long-term hypoxia. Also, p53 was phosphorylated after 12 hrs of hypoxia. The suppression of cyclin D2 by hypoxia may be independent of p21, and phosphorylated p53 may contribute to the suppression of cyclin D2 during long-term hypoxia.

It has been reported that re-oxygenated cells exhibit prolongation of the G0/G1 phase, and then show senescence or apoptosis (Zhang et al., 2005). To clarify the effect of re-oxygenation on pulp cells exposed to hypoxia, we analyzed cell viability, cell-cycle distribution, and the expression of cyclin D2 in pulp cells after re-oxygenation. Following exposure of hypoxia-insulted pulp cells to a normoxic condition, cell viability, cell-cycle distribution, and the expression of cyclin D2 returned to levels observed in non-treated pulp cells, and the induction of cell death was prevented. These results indicate that pulp cells exhibit hypoxia-resistance, and their viability and cell proliferation can be improved by re-oxygenation. The low increasing rate of viability of pulp cells after re-oxygenation may be the result of hypoxic damage to pulp cells, and of the time lag to release pulp cells from signals for hypoxia-induced cell-cycle arrest by re-oxygenation. Taken together, our findings provide important insight into the in vivo pulpal functions that occur during wound-healing and regeneration.


   ACKNOWLEDGMENTS
 
This study was supported by a Grant in Aid for Scientific Research, 15592025 (Kitamura), from the Japan Society for the Promotion of Science, Tokyo, Japan.

Received August 26, 2005; Last revision April 22, 2006; Accepted May 5, 2006


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 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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