ENDOPLASMIC RETICULUM INVOLVEMENT IN APOPTOSIS OF NORMAL AND TREATED GINGIVAL FIBROBLASTS Ancuţa Goriuc, Raluca Jipu, Roxana Irina Iancu, M. Costuleanu GR. T. POPA UNIVERSITY OF MEDICINE AND PHARMACY, IAŞI, ROMANIA Summary Accumulation of misfolded proteins and alterations in Ca 2+ homeostasis in the endoplasmic reticulum (ER) causes ER stress and leads to cell death. However, the signal transduction events that connect ER stress to cell death pathways are incompletely understood, especially in fibroblasts from patients with gingival hypertrophy. Gingival fibroblasts were achieved from 6 weeks old-male rats, 150-170 g body weight, from gingival explants, and grown up in specific culture medium, with and without cyclosporine A (CsA) treatment (1µg/ml), nifedipine (3mM) and phenytoin (2.5mM). The control group received no treatment. We aimed the involvement of ER in the apoptosis of normal fibroblasts and as well as of those treated with CsA, nifedipine and phenytoin. As technique we used flow cytometry (FACS) and calcein-am (C-AM) as the marker for the mitochondrial permeability transition pore (MPTP) opening. As inductor of apoptosis we used thapsigargin (THP). THP is considered one inducer of apoptosis through the RE stress. Several studies indicate that THP might produce apoptosis mainly in malignant or genetically modified cells, but not in normal cells. Facs images and statistical analysis showed differences between normal fibroblasts under thapsigargin action and those treated with CsA, nifedipine and phenytoin in culture medium under the same substance. Induction of ER stress with THP in normal fibroblasts had no statistically significant effects on MPTP opening. In fibroblasts treated with CsA, nifedipine and phenytoin, thapsigargin reduced mitochondrial calcein, suggesting the opening of mitochondrial permeability transition pore as a result of endoplasmic reticulum stress. Keywords: apoptosis, gingival fibroblasts, endoplasmic reticulum stress, thapsigargin, flow cytometry anky_ses@yahoo.com Introduction The endoplasmic reticulum (ER) is a subcellular organelle involved in the synthesis of secretory/membrane proteins and lipids. The synthesis, folding and processing of the secretory/membrane proteins by the endoplasmic reticulum (ER) requires the functioning of ER chaperones, maintenance of ER calcium pools, and an oxidative environment. To maintain homeostasis against any ER dysfunction, the ER responds through a complex and coordinated adaptive signaling mechanism called the unfolded protein response (UPR). The UPR relays ER stress to the cytosol and nucleus to counter the imbalance in protein synthesis, folding, modification, translocation and degradation. Several physiological and pathological conditions such as nutrient or glucose deprivation, elevated protein synthesis, virus infection, disturbances in Ca 2+ fluxes and redox regulation have been shown to promote ER dysfunction and elicit UPR (Zhao and Ackerman, 2006). Analysis of the UPR signaling pathways has become possible using various pharmacological agents such as tunicamycin (an inhibitor of protein glycosylation), cycloheximide (an inhibitor of translational elongation), thapsigargin (that affects ER Ca 2+ levels), redox agents such as DTT, GSH or GSSG (that influence disulphide bond formation), brefeldin A (an inhibitor of ER to Golgi protein transport) and inhibitors of protein degradation such as bortezomib. 181
The UPR plays an important role in embryonic development, maturation of secretory cell types such as antibody producing plasma cells, osteoblasts that secrete collagen, and insulin-secreting pancreatic β-cells. It is also implicated in normal biological processes like aging, liver development, sleep deprivation, and in nutrient sensing in yeast (Wu and Kaufman, 2006). Incessant ER stress beyond the limits of adaptation can trigger the proapoptotic potential of the UPR. The suicide of unhealthy cells via apoptosis represents the last resort of multicellular organisms to clear the non-functional cells. Cell death results in loss of cell/tissue function and may be the primary reason for the manifestation of disease in several ER stress-related disorders. Although the exact mechanism is not known, the ER stressinduced apoptosis is mediated by the mitochondria (intrinsic pathway) and/or through the activation of proapoptotic downstream kinases that are triggered typically in the death-induced receptor mediated extrinsic apoptotic pathway (Yoshida, 2007). Accumulation of misfolded proteins and alterations in Ca 2+ homeostasis in the endoplasmic reticulum (ER) causes ER stress and leads to cell death. However, the signal transducing events that connect ER stress to cell death pathways are incompletely understood. Material and methods Our purpose was to evaluate the inv olvement of endoplasmic reticulum in the apoptosis of normal gingival fibroblasts and treated gingival fibroblasts with CsA, nifedipine and phenytoin. Gingival fibroblasts were achieved from 6 weeks old-male rats, 150-170 g body weight, from gingival explants, and grown up in specific culture medium, consisting of DMEM, 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin in an atmosphere containing 5% CO 2 at 37 C. Medium was supplemented with CsA treatment (1µg/ml), nifedipine (3mM) and phenytoin (2.5mM). The control group received no treatment. We aimed the involvement of ER in the apoptosis of normal fibroblasts and as well as of those treated with CsA, nifedipine and phenytoin. As technique we used flow cytometry (FACS) and calcein- AM (C-AM) as the marker for the mitochondrial permeability transition pore (MPTP) opening. As inductor of apoptosis we used thapsigargin (THP). THP is considered one inducer of apoptosis through the RE stress. Several studies indicate that THP might produce apoptosis mainly in malignant or genetically modified cells, but not in normal cells. The grown cells were used after the third passage at least. The normal and treated fibroblasts were separated with trypsin-edta standard solution, flushed by several centrifugations at 300xg for 5 minutes, after which were resuspended in 1 ml culture medium. Cells were counted (about 1.000.000/ml) and were equally divided in tubes. A tube represented the control group for each treatment, in all other tubes we added calcein-am 5μl/ml (2 μm) and 5μl/ml of CoCl 2 (80 μm) for 20 minutes at 37 C and 5% CO 2. As apoptosis inducer we added thapsigargin 10 μl/ml (10 μm) for 24 hours at 37 C and 5% CO 2. After that we centrifuged the tubes at 300xg for 5 minutes, and then the cells were resuspended in PBS and centrifuged again, thus applying a double wash. The flow cytometry settings were as follows: 623 V for FL1, 505 V for FL2, 10,000 events and 488 nm laser. Data were processed using FlowJo 7.6.1 software. Meanwhile, the same protocol was applied for untreated fibroblasts. Statistical data were analyzed using One Way ANOVA method (completed with Student-Newman-Keuls method). Results were considered statistically significant for a p value < 0.05 and were expressed as mean ± S.E.M. (5 experiments). 182
Results and discussion Added together with CoCl 2, calcein- AM appears to be a good technique for MPT channel opening, following Ca 2+ concentration variations in the cell cytoplasm or mitochondria. We aimed the involvement of ER in the apoptosis of normal fibroblasts and as well as of those treated with CsA, nifedipine and phenytoin. Thapsigargin is considered one inducer of apoptosis through the RE stress. Several studies indicate that THP might produce apoptosis mainly in malignant or genetically modified cells, but not in normal cells. Despite the importance and possible involvement of the ER stress in various diseases, the pathway of ER stress-induced cell death has not been fully elucidated. Thus, in the present experiments we examined the role of thapsigargin in normal and treated gingival fibroblasts apoptosis. Fig.1. FACS fluorescence images of calcein-am loading, with reference to normal gingival fibroblasts (the upper image), of CsA-, nifedipine- and phenytoin-treated fibroblasts (second to fourth top to down images) under the action of THP for 24 hours. 183
Fig. 2. Statistical analysis of calcein-am loaded and treated fibroblasts with CsA, nifedipine and phenytoin under thapsigargin action, as compared to normal fibroblasts. Since mitochondria are juxtaposed to the ER, in many cases ER stress is communicated to the mitochondria, and ER stress-induced apoptosis is mediated through a dysfunction in the mitochondria. Recent work suggests that BCL2 protein family regulates the ER-Ca 2+ release and communication of ER stress signal to the mitochondria. Persistent ER stress can also induce a switch in the UPR signalling from prosurvival to proapoptotic pathways, like the induction of CHOP, a proapoptotic transcriptional factor and GADD34 (a cofactor of eif2α phosphatase), through the PERK-eIF2α pathway, and activation of proapoptotic kinases such as ASK1 (apoptosis signal regulating kinase) and JNK (c-jun-n-terminal kinase) through the IRE-1 pathway. The major players involved in ER stress-induced apoptosis and their roles are described below (Hussain and Ramaiah, 2007). Thapsigargin induces acute response s in a large variety of cell types, but thapsigargin-induced cellular activation appears, in all cases, to be initiated by a single common event: a rapid and pronounced increase in the concentration of cytosolic free Ca 2+ that occurs via a direct discharge of intracellular stored Ca 2+ without hydrolysis of inositolphospholipids These results imply that thapsigargin might act directly on a recognition site associated with the intracellular Ca 2+ store (Thastrupt, 1990). Our experiments showed that THP has no effect on normal gingival fibroblasts. But showed significant effects of thapsigargin on fibroblasts treated with CsA, nifedipine and less phenytoin as mitochondrial permeability transition pore opening, evidenced by calcein_am release from mitochondria. Any alteration in the environment surrounding the endoplasmic reticulum, such as disturbance in Ca 2+ homeostasis changes in secretory protein synthesis, deprivation of glucose or other sugar, altered glycosylation and accumulation of unfolded proteins in the endoplasmic reticulum stress can cause cell death (Park et al., 1999, Bullon et al., 2007). Conclusions All the elements involved in apoptosis are interconnected both physically and functionally, making possible the existence of a variety ways to initiate the response to very different stimuli and also the means of adjusting them. Mitochondrial permeability transition pore opening, as evidenced by reduction of 184
calcein-am fluorescence in the presence of cobalt chloride represents, according to current data, an initial step in the development of apoptosis. Induction of endoplasmic reticulum stress in normal fibroblasts with thapsigargin had no statistically significant effects on the opening of mitochondria permeability transition pore. In contrast, in fibroblasts treated with CsA, thapsigargin reduced mitochondrial calcein, suggesting the opening of permeability transition pore as a result of endoplasmic reticulum stress. Thapsigargin caused significant reduction of mitochondrial calcein loading even in fibroblasts treated with nifedipine and phenytoin References Bullon, P., Gallardo, I., Goteri, G., Rubini, G.: Nifedipine and cyclosporin affect fibroblast calcium and gingival growth. J. Dent. Res., 86, 4, 357-362, 2007. Hussain, S.G., Ramaiah, K.V.A.: Endoplasmic reticulum: Stress, signalling and apoptosis. Curr. Sci., 93, 12-19, 2007. Park, K.S., Kim, T.K., Kim, D.H.: Cyclosporin A treatment alter characteristic of Ca 2+ - release channel in sarcoplasmic reticulum. Am. J. Periodontol., 4, 12, 154-159, 1999. Thastrupt, O., Cullent, P.J.: Proc. Natl. Acad. Sci., 87, 2466-2470, 1990. Wu, J., Kaufman, R.J.: From acute ER stress to physiological roles of the unfolded protein response. Cell Death Differ., 13, 374-384, 2006. Yoshida, H.: ER stress and diseases. FEBS J., 274, 630-658, 2007. Zhao, L., Ackerman, S.L.: Endoplasmic reticulum stress in health and disease. Curr. Opin. Cell. Biol., 18, 444-452, 2006. 185