Inhibition of Connexin 26/43 and Extracellular-Regulated Kinase Protein Plays a Critical Role in Melatonin Facilitated Gap Junctional Intercellular Communication in Hydrogen Peroxide-Treated HaCaT Keratinocyte Cells

Though melatonin was known to regulate gap junctional intercellular communication (GJIC) in chick astrocytes and mouse hepatocytes, the underlying mechanism by melatonin was not elucidated in hydrogen peroxide- (H2O2-) treated HaCaT keratinocyte cells until now. In the current study, though melatonin at 2 mM and hydrogen peroxide (H2O2) at 300 μM showed weak cytotoxicity in HaCaT keratinocyte cells, melatonin significantly suppressed the formation of reactive oxygen species (ROS) in H2O2-treated HaCaT cells compared to untreated controls. Also, the scrape-loading dye-transfer assay revealed that melatonin enhances the intercellular communication by introducing Lucifer Yellow into H2O2-treated cells. Furthermore, melatonin significantly enhanced the expression of connexin 26 (Cx26) and connexin 43 (Cx43) at mRNA and protein levels, but not that of connexin 30 (Cx30) in H2O2-treated HaCaT cells. Of note, melatonin attenuated the phosphorylation of extracellular signal-regulated protein kinases (ERKs) more than p38 MAPK or JNK in H2O2-treated HaCaT cells. Conversely, ERK inhibitor PD98059 promoted the intercellular communication in H2O2-treated HaCaT cells. Furthermore, combined treatment of melatonin (200 μM) and vitamin C (10 μg/mL) significantly reduced ROS production in H2O2-treated HaCaT cells. Overall, these findings support the scientific evidences that melatonin facilitates gap junctional intercellular communication in H2O2-treated HaCaT keratinocyte cells via inhibition of connexin 26/43 and ERK as a potent chemopreventive agent.


Introduction
Gap junctional intercellular communication (GJIC) is an important biological mechanism to maintain homeostasis, growth, differentiation, and development of cells and tissues [1]. Gap junctions are made of two hemichannels, called connexons, and each in turn is composed of six molecules of the membrane-spanning connexin (Cx) protein [2,3].
The gap junctions of human keratinocytes include primarily Cx43, which is abundantly expressed within interfollicular epidermis, and Cx26, which is codistributed with Cx43 in skin [4]. Several studies showed that the downregulation of Cxs and phosphorylation of Cxs are involved in the carcinogenesis of the skin [4,5]. Cx43 is phosphorylated by several protein kinases, such as protein kinase C (PKC), casein kinase 1, and mitogen-activated protein kinase (MAPK) [3,[6][7][8]. Recent evidence suggests that the carcinogenicity of oxidative stress induced by H 2 O 2 is attributable to the inhibition of GJIC [8][9][10].
Lucifer Yellow was added to the washed cells, and three scrapes were made with a surgical steel-bladed scalpel at lowlight intensities. Three scrapes were performed to ensure that the scrape traversed a large group of confluent cells. After 3 min incubation, the cells were washed with 10 mL of PBS and then fixed with 2 mL of a 4% formalin solution. The distance traveled by the dye in a direction perpendicular to the scrape was observed with an inverted Axio Axiovert S 100 fluorescent microscope (Carl Zeiss).

Statistical
Analyses. All data were expressed as means ± SD. The statistically significant differences between control and melatonin-treated groups were calculated by ANOVA test followed by a post hoc analysis (Tukey or Dunnett's multiple-comparison test) using Prism software 5 (Graph-Pad Software, Inc., San Diego, CA, USA). is well known to produce free radicals to inhibit gap junctional intercellular communication [26]. As shown in Figure 2(a), melatonin reduced ROS production to 5 (Figures 3(d) and 3(e)). We also observed that melatonin suppressed the phosphorylation of Cx43 in H 2 O 2treated HaCaT cells (Figure 3(c)).

Combined Treatment of Melatonin and Vitamin C at Low Concentrations Exerted the Synergy in Reducing ROS Production in H 2 O 2 -Treated HaCaT Cells.
In order to evaluate the synergistic effect of melatonin with other antioxidant, we used vitamin C. As shown in Figure 5  ROS production, but combination of melatonin and vitamin C significantly reduced ROS production to 16.15% compared to H 2 O 2 -treated control (23.56%).

Discussion
H 2 O 2 plays an important role in the multistep process of carcinogenesis and directly promotes transformation in many in vivo and in vitro model systems [28][29][30]. In the present study, melatonin suppressed ROS production and facilitated H 2 O 2 -mediated inhibition of GJIC in HaCaT cells, implying the antioxidant and anti-carcinogenic potential of melatonin, which was supported by previous studies that the carcinogenicity of H 2 O 2 is attributable to the inhibition of GJIC [31]. Likewise, antioxidants such as vitamin C and quercetin protect against the disruption of GJIC induced by H 2 O 2 [32].
There are several lines of evidences that malignant lesions reveal abnormal expression of connexins and decreased GJIC [33][34][35]. The function of GJIC can be modulated at the    MAPKs are considered to play important roles in GJIC [36]. Also, ROS-activated MAPK cascades phosphorylate the various proteins involved in cell growth and development [37]. Previous studies revealed that H 2 O 2 -dependent ERK and p38 kinase activation lead to depressed GJIC and enhanced connexin degradation [36]. However, in the current study, melatonin significantly decreased the phosphorylation of ERK alone, but not p38 MAPK or JNK. Furthermore, ERK inhibitor PD98059 effectively recovered the lowered activity of GJIC in H 2 O 2 -treated HaCaT cells, suggesting the critical role of ERK in recovering the decreased GJIC activity by H 2 O 2 . Interestingly, combined treatment of melatonin (200 μM) and vitamin C (10 μg/mL) that do not affect ROS production significantly reduced ROS production in H 2 O 2 -treated HaCaT cells, implying the synergistic effect of melatonin and vitamin C at low concentrations. However, it is also required to confirm this synergistic effect in small animals or humans in the near future.
In summary, melatonin showed weak cytotoxicity in HaCaT cells, reduced ROS production, recovered the disturbed GJIC, enhanced the expression of Cx26 and Cx43 at mRNA and protein levels, suppressed the phosphorylation of ERK, and enhanced synergy with vitamin C in H 2 O 2 -treated HaCaT cells ( Figure 6). Overall, our findings suggest that melatonin recovers decreased GJIC via enhancement of Cx26 and Cx43 and inhibition of ROS production and ERK phosphorylation.