Local cell damage and increased inflammation are found to contribute to the complex range of responses occurring clinically in the dental pulp after tooth restoration with resin containing materials (Hebling et al., 1999
). Micrograms to milligrams of resin based monomers such as HEMA is released into the adjacent aqueous phase from a broad range of resin-based bonding, cementing and direct filling materials from clinically used amounts during the first days after placement (Gerzina and Hume, 1996
; Hume and Gerzia, 1996
). We have found that loss of mitochondrial membrane potential and activation of effector caspases were two major hallmarks of the intrinsic death pathway induced by HEMA (Paranjpe et al., 2008a
). Moreover, the analysis of the genes involved in apoptotic pathways by microarray technology indicated that several critical genes related to cell cycle checkpoints, signaling proteins and cell death mediators were also elevated in the presence of HEMA treatment (refer to supplemental data of Paranjpe et al., 2005
). Therefore, many of the inhibitors which have been shown to block apoptotic cell death should in practice block HEMA mediated cell death. However, inhibition of cell death should also parallel gain of functional competency of the cells if effective strategies are sought to reverse HEMA mediated adverse effects. In this regard, the cell death inhibitor NAC fulfills these criteria since both the death and loss of cellular function were inhibited by NAC.
Blockade of cell death by NAC was suggested to relate to its inhibitory effect on the generation of ROS and oxidative stress in HEMA treated cells since NAC was previously shown to have anti-oxidant effect (Chang et al., 2005
; Spagnuolo et al., 2006
). However, since well known anti-oxidants such as Trolox and Ascorbates not only did not change the course of HEMA mediated cell death but also they contributed to it (Paranjpe et al., 2007
), it is therefore believed that NAC may function via mechanisms which are distinct from those reported for Trolox and Ascorbates (Walther et al., 2004
). Indeed, we report here that NAC is an important inducer of NF-κB in oral epithelial cells. Increased activation of NF-κB in other epithelial cells by NAC was also reported previously (Das et al., 1995
). However, in contrast to the previous reports our data clearly demonstrate an NF-κB dependent and independent protective effect of NAC on the cells. It is important to note that the addition of a number of potent activators of NF-κB such as TNF-α () and PMA/Ionomycin (data not shown) to NFκB knock down HEp2 cells were unable to increase NFκB in NF-κB knockdown HEp2 cells. Thus, the NF-κB independent protection of HEp2 cells by NAC is clearly not due to an increase in NF-κB in NF-κB knockdown HEp2 cells ().
NAC may provide the protective mechanism in part by increasing the anti-apoptotic proteins regulated by NF-κB. In this respect MnSOD (Mn superoxide dismutase) may be implicated in this process since NF-κB is an important modulator of MnSOD (Murley et al., 2001
). In our experiments, the partial role of NF-κB in survival and function of the cells is evident by the following observations. First, treatment of HEp2 cells with HEMA significantly blocked nuclear NF-κB expression and function. This is in contrast to the previously published data where the addition of HEMA was reported to increase rather than decrease the nuclear NF-κB expression in fibroblasts (Spagnuolo et al. 2004
). This discrepancy could be due to the type of cells employed or the timing of the treatment. However, we did not observe any induction of NF-κB activity by HEMA from 4 to 24 h of treatment in the cells tested. Second, the addition of NAC significantly increased NF-κB expression in oral epithelial cells, and restored NF-κB activity in HEMA-treated cells, with concomitant rescue of cell viability and function. NAC had an effect similar to TNF-α in this system. Third, when nuclear NF-κB was inhibited in oral keratinocytes both HEMA () and TNF-α (Jewett et al., 2003
) exacerbated death of the cells, and the protective effect of NAC on cell death was partially lost (Paranjpe et al., 2007
). Collectively, these results are consistent with a model in which NAC blocks HEMA mediated cell death partially through the induction of NFκB.
Even though a portion of NAC's protective effect on HEMA induced cell death is regulated by NFκB, a significant portion of its effect is NF-κB independent. In this regard we demonstrate that NAC prevents HEMA mediated induction of JNK. Strong and sustained activation of JNK by a variety of stresses such as UV light and hydroxy peroxides results in an induction of significant apoptosis in the cells (Butterfield et al., 1997
; Chen et al., 1996
). As determined by a cDNA array analysis (data not shown) expression of JNK1 in NF-κB knockdown HEp2 cells augmented MAPKK1/MKK1 gene expression in HEp2 cells (data not shown). Thus, increased activation of MAPKK1 could in turn increase the levels of JNK expression in NF-κB knockdown HEp2 cells resulting in a sustained increase in the levels and function of JNK (Reuther-Madrid et al., 2002
). JNK is also an important regulator of p53 tumor suppressor protein (Fuchs et al., 1998
). Activation of p53 in many different tumor cells induces growth arrest and apoptosis (Eliyahu et al., 1984
; Lane, 1984
). JNK prolongs the half-life of p53 (Fuchs et al., 1998
). In accordance, we have observed significant induction of p53 associated protein, proline dehydrogenase (proline oxidase 1) gene when JNK was expressed in NF-κB knock down HEp2 cells (manuscript submitted). HEp2 cells do not express p53 tumor suppressor protein and thus loss of p53 expression is believed to be one of the contributory mechanisms to their transformation and survival. Thus, increased JNK activity may serve to increase the levels of p53 and its target protein proline dehydrogenase (proline oxidase 1) gene resulting in the augmentation of cell death seen in this study. These possibilities are currently under investigation in our laboratory.
We have also found that the inverse increase in JNK and NF-κB pathways in HEp2 cells is responsible for the synergistic upregulation of IgFbp6 as determined by the gene and protein analysis (Cacalano et al., 2008
). Both the IgFbp6 and proline dehydrogenase gene (proline oxidase 1) were previously shown to play significant roles in programming for death of a variety of cells (Hu et al., 2007
; Pandhare et al., 2006
). Whether synergistic induction of abovementioned genes in combination by HEMA are responsible for cell death of HEp2 cells should await future investigations.
Although the pharmacologic JNK inhibitor SP600125 was used to selectively target JNK1, 2, and 3 in previous studies, in some recent studies the specificity of this inhibitor has been challenged, pointing to perhaps the limitation of such an approach in delineating the mechanisms involving the JNK pathway (Bain et al., 2003
). Therefore, although it is likely that JNK is one of the important mediators of HEMA induced cell death in HEp2 cells, further genetic as well as pharmacological studies are warranted and will be performed in our future studies to establish the extent and the level of the contribution of this pathway in HEMA mediated cell death.
To determine the levels of functional competency under different experimental conditions we opted to use the status of VEGF and bFGF release by the cells. These two growth factors exhibit different release profiles depending on the levels of cell viability and can be used to determine the functional competency of the cells under different experimental conditions. Whereas viable HEp2 cells are capable of secreting high levels of VEGF, cells which are signaled to undergo cell death loose this ability and decrease the levels of VEGF secretion ( and ). In contrast, those that are signaled to undergo cell death release high levels of bFGF ( and ). Therefore, by using these two parameters we were able to demonstrate that JNK inhibitor was able to prevent cell death and maintain the functional competency of the HEp2 cell transfectants. Since blocking NF-κB in HEp2 cells abolishes the ability of the cells to release bFGF, we could only determine bFGF release in vector-alone transfected HEp2 cells (). Thus, NAC mediated decrease in bFGF release from DPSCs or HEp2 cells is another piece of supporting evidence regarding the role of NAC in increase functional activation of NF-κB (data not shown and ).
The results reported in this paper indicated that the protective effect of NAC on HEMA mediated cell death maybe induced via mechanisms other than or in addition to an anti-oxidant effect which was reported previously (Schweikl et al., 2006
). We have reported that protection of NAC is partly due to its capacity to induce differentiation of the cells (Paranjpe et al., 2007
) due to the increased induction of important transcription factors such as NF-κB and an increase in a number of important differentiation genes reported previously (Paranjpe et al., 2007
). The full spectrum of signals responsible for the induction of differentiation in the cells by NAC is not established yet, but it clearly involves both the NF-κB and JNK signaling pathways ( and ). Overall, studies reported in this paper and those which were performed in vivo
(Paranjpe et al., 2008b
) indicated that NAC is an important protective agent which could be used in dental restorations to protect the pulp cells from toxic effects of restorative materials. In addition, they indicated that there may be mutually inclusive and exclusive effects of NF-κB and JNK in NAC mediated protection from HEMA mediated cell death.