Regulation of nuclear DNA damage response by mitochondrial morphofunctional pathway.

Cells are constantly challenged by genotoxic stresses that can lead to genome instability. The integrity of the nuclear genome is preserved by the DNA damage response (DDR) and repair. Additionally, these stresses can induce mitochondria to transiently hyperfuse; however, it remains unclear whether canonical DDR is linked to these mitochondrial morphological changes. Here, we report that the abolition of mitochondrial fusion causes a substantial defect in the ATM-mediated DDR signaling. This deficiency is overcome by the restoration of mitochondria fusion. In cells with fragmented mitochondria, genotoxic stress-induced activation of JNK and its translocation to DNA lesion are lost. Importantly, the mitochondrial fusion machinery of MFN1/MFN2 associates with Sab (SH3BP5) and JNK, and these interactions are indispensable for the Sab-mediated activation of JNK and the ATM-mediated DDR signaling. Accordingly, the formation of BRCA1 and 53BP1 foci, as well as homology and end-joining repair are impaired in cells with fragmented mitochondria. Together, these data show that mitochondrial fusion-dependent JNK signaling is essential for the DDR, providing vital insight into the integration of nuclear and cytoplasmic stress signals.

Mitochondrial fission and fusion mediators, hFis1 and OPA1, modulate cellular senescence.

The number and morphology of mitochondria within a cell are precisely regulated by the mitochondrial fission and fusion machinery. The human protein, hFis1, participates in mitochondrial fission by recruiting the Drp1 into the mitochondria. Using short hairpin RNA, we reduced the expression levels of hFis1 in mammalian cells. Cells lacking hFis1 showed sustained elongation of mitochondria and underwent significant cellular morphological changes, including enlargement, flattening, and increased cellular granularity. In these cells, staining for acidic senescence-associated beta-galactosidase activity was elevated, and the rate of cell proliferation was greatly reduced, indicating that cells lacking hFis1 undergo senescence-associated phenotypic changes. Reintroduction of the hFis1 gene into hFis1-depleted cells restored mitochondrial fragmentation and suppressed senescence-associated beta-galactosidase activity. Moreover, depletion of both hFis1 and OPA1, a critical component of mitochondrial fusion, resulted in extensive mitochondrial fragmentation and markedly rescued cells from senescence-associated phenotypic changes. Intriguingly, sustained elongation of mitochondria was associated with decreased mitochondrial membrane potential, increased reactive oxygen species production, and DNA damage. The data indicate that sustained mitochondrial elongation induces senescence-associated phenotypic changes that can be neutralized by mitochondrial fragmentation. Thus, one of the key functions of mitochondrial fission might be prevention of the sustained extensive mitochondrial elongation that triggers cellular senescence.

Raf-1 and protein kinase B regulate cell survival through the activation of NF-kappaB in hepatitis B virus X-expressing cells.

We previously demonstrated that activation of NF-kappaB by the hepatitis B virus X (HBx) gene plays an important role in cell survival. In the present study, we explored the upstream mediators of NF-kappaB activation and their correlations with cell survival. XTT assays and colony generation assays revealed that inhibition of NF-kappaB activation indeed increased cell death in HBx-expressing cells. Utilizing inactivating mutants of signal transducers, we showed that dominant negative mutants of stress-activated protein kinase/extracellular signal-regulated kinase (SEK1) or PKCalpha significantly diminished the HBx-mediated NF-kappaB activation. However, neither of these mutants significantly affected the cell survival in colony generation assays. In contrast, inactivating mutants of Raf-1 or PKB (protein kinase B)/Akt abrogated the HBx-mediated NF-kappaB activation and also suppressed the cell survival. Our results suggest that the Raf-1 or PKB-mediated NF-kappaB activation promotes cell survival in HBx-expressing cells.

Hypertonic sodium chloride induction of cyclooxygenase-2 occurs independently of NF-kappaB and is inhibited by the glucocorticoid receptor in A549 cells.

Cellular response to a hypertonic environment is important for fluid clearance in the lung. Hypertonicity modulates prostaglandin synthesis by influencing cyclooxygenase-2 (COX-2) expression in tissues such as liver and kidney via a mitogen-activated protein kinase (MAPK)-dependent pathway. However, little is known about COX-2 expression in response to hypertonicity in the lung. COX-2 mRNA accumulation induced by hypertonic NaCl was detected after 1 h of treatment, and COX-2 mRNA continued to accumulate until 18 h, the longest time point examined, in human alveolar epithelial A549 cells. This induction was a transcriptional event that occurred in the absence of the protein synthesis inhibitor cycloheximide and was the result of enhanced promoter activity, as examined with the use of full-length COX-2 promoter-driven reporter plasmids. The induction of COX-2 expression by hypertonic NaCl did not require the activation of NF-kappaB. The p38 MAPK inhibitor, SB203580, or MEK1/2 inhibitor, U0126, inhibited hypertonic induction of COX-2 expression. We examined whether the hypertonic induction of COX-2 was under the influence of glucocorticoid; we found that COX-2 promoter activity and mRNA and protein levels were depressed by dexamethasone and antagonized by the glucocorticoid receptor (GR) antagonist RU486. Our data demonstrate that the induction of COX-2 expression by hypertonic NaCl occurs independently of NF-kappaB and is inhibited by the GR in A549 cells.

Analysis of angiotensin II mediated COX-2 downregulation in angiotensin II- or aldosterone-infused hypertensive rat.

The renin-angiotensin cascade plays an important role in blood pressure control and sodium homeostasis. This study investigated whether cyclooxygenase-2 expression is regulated in the kidney, in an angiotensin II- and aldosterone-induced hypertension model. For this purpose, we treated male Sprague-Dawley rats (n=8 per group) with angiotensin II (9 mg/h, subcutaneously) for 14 d and aldosterone (0.75 mg/h, subcutaneously) for 42 d. Systolic blood pressure was significantly increased by angiotensin II (p<0.001) and by aldosterone (p<0.001). We found that angiotensin II downregulated cyclooxygenase-2 protein in the kidney cortex, whereas aldosterone showed no effect. These results indicate that angiotensin II may act directly to inhibit kidney cortex cyclooxygenase-2 protein expression, rather than acting via stimulation of aldosterone.

Ginsenoside Rc and Re stimulate c-fos expression in MCF-7 human breast carcinoma cells.

We have found that ginsenoside Rc and Re induce c-fos in MCF-7 human breast carcinoma cells at both the mRNA and protein levels. However, neither ginsenoside activated the expression of reporter gene under the control of AP-1/TPA response elements. We have also examined the possibility that ginsenoside Rc and Re act by binding to intracellular steroid hormone receptors that act as transcriptional factors in the nucleus in inducing c-fos mRNA in MCF7 human breast carcinoma cells. However, ginsenoside Rc and Re did not bind to glucocorticoid, androgen, estrogen, or retinoic acid receptors as examined by the transcription activation of the luciferase reporter genes in CV-1 cells that were transiently transfected with the corresponding steroid hormone receptors and hormone responsive luciferase reporter plasmids. These data demonstrate that ginsenoside Rc and Re act via other transcription factors and not via estrogen receptor in c-Fos expression.

Ginsenoside-Rb1 acts as a weak phytoestrogen in MCF-7 human breast cancer cells.

Ginseng has been recommended to alleviate the menopausal symptoms, which indicates that components of ginseng very likely contain estrogenic activity. We have examined the possibility that a component of Panax ginseng, ginsenoside-Rb1, acts by binding to estrogen receptor. We have investigated the estrogenic activity of ginsenoside-Rb1 in a transient transfection system using estrogen-responsive luciferase plasmids in MCF-7 cells. Ginsenoside-Rb1 activated the transcription of the estrogen-responsive luciferase reporter gene in MCF-7 breast cancer cells at a concentration of 50 microM. Activation was inhibited by the specific estrogen receptor antagonist ICI 182,780, indicating that the estrogenic effect of ginsenoside-Rb1 is estrogen receptor dependent. Next, we evaluated the ability of ginsenoside-Rb1 to induce the estrogen-responsive gene c-fos by semi-quantitative RT-PCR assays and Western analyses. Ginsenoside-Rb1 increased c-fos both at mRNA and protein levels. However, ginsenoside-Rb1 failed to activate the glucocorticoid receptor, the retinoic acid receptor, or the androgen receptor in CV-1 cells transiently transfected with the corresponding steroid hormone receptors and hormone responsive reporter plasmids. These data support our hypothesis that ginsenoside-Rb1 acts a weak phytoestrogen, presumably by binding and activating the estrogen receptor.

Effects of estrogen receptor and estrogen on the chromatin structure in estrogen receptor stable transfectants.

The estrogen receptor (ER), a member of the nuclear hormone receptor superfamily recruits coactivators that modify local chromatin structure. Here we investigated the effect of the estrogen receptor and estrogen on the global chromatin structure and the local chromatin structure of the progesterone receptor gene during the process of transcriptional activation using Rat1+ER cells stably expressing the estrogen receptor. The total chromatin was more accessible to DNasel in Rat1+ER cells than in the parental estrogen receptor-negative Rat1 cells. After 18 h of estrogen treatment, total chromatin was more dispersed in Rat1+ER cells than in Rat1 cells. The chromatin structure of the progesterone receptor gene was more sensitive to DNasel in Rat1+ER cells than in Rat1 cells. However, the chromatin structure of the progesterone receptor gene did not change further on estrogen treatment. Our results suggest that under certain circumstances unoccupied estrogen receptors may play some role in reorganizing the repressive chromatin structure to induce gene activation.