Role of sestrin2 in the regulation of proinflammatory signaling in macrophages.

Sestrins (Sesns) are conserved antioxidant proteins that accumulate in cells in response to various stresses. However, the regulatory roles of Sesn2 in the immune system and in inflammatory responses remain obscure. In the present study, we investigated whether Sesn2 regulates Toll like receptor (TLR)-mediated inflammatory signaling and sought to identify the molecular mechanism responsible. In cells expressing Sesn2, it was found that Sesn2 almost completely inhibited lipopolysaccharide (LPS)-induced NO release and iNOS expression. A gene knockdown experiment confirmed the role of Sesn2 in LPS-activated RAW264.7 cells. Consistently, proinflammatory cytokine (e.g., TNF-α, IL-6, and IL-1ß) release and expression were inhibited in Sesn2-expressing cells. Furthermore, Sesn2 prevented LPS-elicited cell death and ROS production via inhibition of NADPH oxidase. NF-κB and AP-1 are redox-sensitive transcription factors that regulate the expressions of diverse inflammatory genes. Surprisingly, Sesn2 specifically inhibited AP-1 luciferase activity and its DNA binding, but not those of NF-κB. AP-1 inhibition by Sesn2 was found to be due to a lack of JNK, p38, and c-Jun phosphorylation. Next, we investigated whether Sesn2 protects galactosamine (Gal)/LPS-induced liver injury in mice infected with a recombinant adenovirus Sesn2 (Ad-Sesn2). Ad-Sesn2 present less severe hepatic injury as supported by decreases in the ALT, AST, and hepatocyte degeneration. Moreover, Ad-Sesn2 attenuated Gal/LPS-induced proinflammatory gene expression in mice. The study shows that Sesn2 inhibits TLR-induced proinflammatory signaling and protects cells by inhibiting JNK- or p38-mediated c-Jun phosphorylation.

Human nuclear clusterin mediates apoptosis by interacting with Bcl-XL through C-terminal coiled coil domain.

Clusterin (CLU), a glycoprotein, is involved in apoptosis, producing two alternatively spliced isoforms in various cell types. The pro-apoptotic CLU appears to be a nuclear isoform (nuclear clusterin; nCLU), and the secretory CLU (sCLU) is thought to be anti-apoptotic. The detailed molecular mechanism of nCLU as a pro-apoptotic molecule has not yet been clear. In the current study, overexpressed nCLU induced apoptosis in human kidney cells. Biochemical studies revealed that nCLU sequestered Bcl-XL via a putative BH3 motif in the C-terminal coiled coil (CC2) domain, releasing Bax, and promoted apoptosis accompanied by activation of caspase-3 and cytochrome c release. These results suggest a novel mechanism of apoptosis mediated by nCLU as a pro-apoptotic molecule.

Temporal changes of angiopoietins and Tie2 expression in rat lungs after monocrotaline-induced pulmonary hypertension.

Angiogenic factors such as vascular endothelial growth factor (VEGF) are implicated in pulmonary hypertension (PH). However, the pathway of angiogenic factor-mediated pathologic angiogenesis in PH remains unclear. In this study, we evaluated the temporal expression of angiopoietin (Ang) 1, Ang2, and their receptor (Tie2) as well as VEGF, endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and heme oxygenase 1 (HO1) in the monocrotaline-induced PH model. Histologic evaluation showed pathologic vascular remodeling in the arteries of lung sections 1 wk after monocrotaline treatment. Protein levels of Ang1, Ang2, eNOS, iNOS, HO1, and VEGF were increased 1 wk after monocrotaline treatment but Tie2 protein levels were decreased 2 wk afterward. These results suggest that Ang2 mediates vascular remodeling in PH by decreasing Tie2 expression. Therefore, the Ang-Tie2 system may play a role in the pathophysiology of PH.

RhoGDI2 expression is associated with tumor growth and malignant progression of gastric cancer.

PURPOSE: Rho GDP dissociation inhibitor 2 (RhoGDI2) has been identified as a regulator of Rho family GTPase. However, there is currently no direct evidence suggesting whether RhoGDI2 activates or inhibits Rho family GTPase in vivo (and which type), and the role of RhoGDI2 in tumor remains controversial. Here, we assessed the effects of RhoGDI2 expression on gastric tumor growth and metastasis progression. EXPERIMENTAL DESIGN: Proteomic analysis was done to investigate the tumor-specific protein expression in gastric cancer and RhoGDI2 was selected for further study. Immunohistochemistry was used to detect RhoGDI2 expression in clinical samples of primary gastric tumor tissues which have different pathologic stages. Gain-of-function and loss-of-function approaches were done to examine the malignant phenotypes of the RhoGDI2-expressing or RhoGDI2-depleting cells. RESULTS: RhoGDI2 expression was correlated positively with tumor progression and metastasis potential in human gastric tumor tissues, as well as cell lines. The forced expression of RhoGDI2 caused a significant increase in gastric cancer cell invasion in vitro, and tumor growth, angiogenesis, and metastasis in vivo, whereas RhoGDI2 depletion evidenced opposite effects. CONCLUSION: Our findings indicate that RhoGDI2 is involved in gastric tumor growth and metastasis, and that RhoGDI2 may be a useful marker for tumor progression of human gastric cancer.

Temporal expression of AMP-activated protein kinase activation during the kainic acid-induced hippocampal cell death.

Kainic acid (KA)-induced seizure induces the hippocampal cell death. There are reports that AMP-activated protein kinase (AMPK), which is an important regulator of energy homeostasis of cells, has been proposed as apoptotic molecule. In this study, we investigated the altered expression of AMPK cascade in the hippocampus of mice during KA-induced hippocampal cell death. Mice were killed at 2, 6, 24 or 48 h after KA (30 mg/kg) injection. Histological evaluation of KA-treated hippocampus revealed hippocampal cell death first at 6 h and appearing prominently by 48 h after KA injection. Immunoreactivity of Ca(2+)/calmodulin-dependent protein kinase kinasebeta (CaMKKbeta) was increased after KA treatment. In Western blot analysis, AMPK activation was increased 2 h after KA treatment. The proteins of downstream AMPK, including those of glucose transporter1 (GLUT1) and phosphorylation of Acetyl CoA Carboxylase (ACC) were increased in the hippocampus after KA treatment. These results indicate that sustained AMPK activation might be a mechanism by which KA-induced seizure causes hippocampal cell death of mice.

C-jun N-terminal kinase regulates the interaction between 14-3-3 and Bad in ethanol-induced cell death.

Activation of the c-jun N-terminal kinase (JNK) is known to be an important step during ethanol-induced cell death, but it has yet to be identified how JNK regulates apoptosis. Therefore, we investigated the mechanism by which JNK induces cell death following ethanol treatment. Ethanol (6 g/kg, 20% in saline) was administered subcutaneously to postnatal 7 day rat pups. Twelve hours after the first ethanol administration, rat pups were decapitated, and extracts of total protein from cerebral cortices were prepared. Ethanol exposure induced phosphorylation of JNK but did not affect the expression levels of pro- and antiapoptotic proteins. Furthermore, interactions of phospho-JNK (p-JNK) with 14-3-3 as well as with Bad were enhanced in the cerebral cortices of ethanol-treated rats. Pretreatment with JNK inhibitor (SP600125) of SH-SY5Y cells inhibited JNK phosphorylation and interaction between p-JNK and 14-3-3 resulting from ethanol. Furthermore, 14-3-3 interaction with Bad was diminished in the cerebral cortices of ethanol-treated rats. These findings suggest that JNK induces Bad release from 14-3-3 by inhibiting their interaction. After this event, Bad binds to Bcl-xL, releasing Bax from Bcl-xL and leading to cell death. We hypothesize that JNK may play an important role during ethanol-induced cell death via the inhibition of antiapoptotic function of 14-3-3 as well as activation of proapoptotic function of Bad.

Ethanol-induced oxidative stress is mediated by p38 MAPK pathway in mouse hippocampal cells.

It has been known that ethanol causes neuronal cell death through oxidative stress. Ethanol itself and reactive oxygen species (ROS) produced by ethanol modulate intracellular signaling pathways including mitogen-activated protein kinase (MAPK) cascades. This study was conducted to examine the impact of ethanol on MAPK signaling in HT22 cells. Ethanol (100 and 400mM) caused activation of ERK, p38 MAPK, and JNK. ERK activation occurred in early time and p38 MAPK activation was evident when ERK activation was diminished. Specific inhibitor of p38 MAPK (SB203580) protected HT22 cells against ethanol, which was accompanied by an inhibition of ROS accumulation. However, inhibitors of ERK (U0126) and JNK (SP600125) had no effects on ethanol-induced neuronal cell death when they are treated with ethanol for 24h. These results suggest that p38 MAPK may have important roles in ROS accumulation during ethanol-induced oxidative stress in HT22 cells.

Altered gene expression of caspase-10, death receptor-3 and IGFBP-3 in preeclamptic placentas.

Enhanced apoptosis has been observed in the placentas of women with preeclampsia, but few studies have examined changes at the molecular level. This study was designed to detect genes specifically expressed in full-term preeclamptic placentas. Tissue samples were collected immediately after cesarean delivery from 11 normal and 8 preeclamptic placentas at 35-40 weeks of gestation. Total RNAs were extracted and hybridized to a cDNA microarray. Results were confirmed by reverse-transcription polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. Hematoxylin and eosin and TUNEL staining were also performed to confirm apoptosis in preeclamptic placentas. Among 205 genes, three were up- or down-regulated in preeclamptic placentas. The expression of caspase-10 and death receptor 3 (DR-3) was significantly increased, whereas insulin-like growth factor binding protein-3 (IGFBP-3) was strongly down-regulated. RT-PCR analysis and Western blotting confirmed these effects. Immunohistochemical analysis showed that the DR-3, caspase-10 and IGFBP-3 proteins were localized in the syncytial membrane. Apoptosis in the trophoblast was also increased in term placentas from women with pregnancies complicated by preeclampsia. These results suggest that caspase-10, DR-3 and IGFBP-3 are involved in apoptosis in the preeclamptic placenta.

Ketogenic diet protects the hippocampus from kainic acid toxicity by inhibiting the dissociation of bad from 14-3-3.

The ketogenic diet (KD) is often effective for intractable epilepsy, but its antiepileptic mechanisms remain largely unknown. Within the cell death/survival pathway, Akt and its downstream protein Bad play an important role in kainic acid (KA)-induced cell death. Therefore, we investigated the effects of a KD on KA-induced changes in the Akt/Bad/14-3-3 signaling pathway by evaluating Akt, Bad, 14-3-3, and cleaved caspase-3 expression levels as well as their relative interactions. Our results showed that a KD did not affect the expression levels of Akt, Bad, Bcl-xL, Bax, and 14-3-3 but increased phospho-Akt [serine 473; p-Akt (Ser473)] and phospho-Bad [serine 136; p-Bad (Ser136)] expression levels as well as decreased cleaved caspase-3 levels following a KA-induced seizure in the hippocampus. Furthermore, we found that a KD increased the protein-protein interaction between 14-3-3 and p-Bad (Ser136), which might be phosphorylated by p-Akt (Ser473), and decreased interaction of Bad and Bcl-xL. These results suggest that a KD might protect, at least partially, the hippocampus from KA-induced cell death via inhibiting the dissociation of Bad from 14-3-3.

Acute ethanol administration decreases GAP-43 and phosphorylated-GAP-43 in the rat hippocampus.

Acute alcohol ingestion is well known to have deleterious effects on memory and also known to inhibit long-term potentiation, a putative cellular substrate of memory. In this study, we for the first time revealed that growth-associated protein 43 (GAP-43), which is well known as a presynaptic substrate of protein kinase C and one of the major synaptic plasticity-related genes, was down regulated by single ethanol administration (2.5 g/kg, 15% in saline, i.p.) in the rat hippocampus. Using real-time PCR, we confirmed that GAP-43 mRNA level is significantly decreased 2 h after ethanol administration. GAP-43 and p-GAP-43 (Ser41) immunoreactivities in the hippocampus were also reduced 4 h after ethanol administration. Immunohistochemical study showed that the reduction of GAP-43 and p-GAP-43 expression was associated with the perforant and mossy fibers pathways. These results suggest that the reduction of GAP-43 in the hippocampus might be, at least in part, a cause of memory impairment after acute ethanol ingestion.

Suppression of survival kinases and activation of JNK mediate ethanol-induced cell death in the developing rat brain.

Administration of ethanol to immature rat pups during the period in which synaptogenesis occurs triggers extensive apoptotic cell death in the brain. This ethanol-induced cell death is known to be mediated by Bax activation, which is caused by mitochondrial dysfunction. However, little data is available regarding the regulation of survival signaling pathways and their downstream events that lead to Bax activation. Thus, in the present study, we aimed to investigate the effect of ethanol on survival signaling pathways and their downstream events that lead to cell death in the rat brain during the brain developmental period. Ethanol (3 g/kg, 20% in saline) was administered subcutaneously to post-natal 7-day-old rat pups twice at 2-h intervals and the pups were sacrificed at 4 h following the first ethanol injection. Ethanol treatment suppressed the activation of survival kinases, particularly Akt, Erk1/2 and PKAalpha, whereas it increased the activation of JNK. Moreover, dissociation of dephosphorylated Bad from 14-3-3 and the interaction of activated JNK with Bcl-2 were elevated by ethanol treatment. The present study demonstrated that ethanol treatment during the brain developmental period induced mitochondrial dysfunction, which led to cell death by the suppression of survival kinases, Bad release from 14-3-3 and inactivation of Bcl-2 by activated JNK.

Ethanol induces cell death by activating caspase-3 in the rat cerebral cortex.

Ethanol has long been implicated in triggering apoptotic neurodegeneration. We examined the effects of ethanol on the rat brain during synaptogenesis when a spurt in brain growth occurs. This period corresponds to the first 2 postnatal weeks in rats and is very sensitive to ethanol exposure. Ethanol was administered subcutaneously to 7-day- postnatal rat pups by a dosing regimen of 3 g/kg at 0 h and again at 2 h. Blood ethanol levels peaked (677+/-16.4 mg/dl) at 4 h after the first ethanol administration. The cerebral cortexes of the ethanol-treated group showed several typical symptoms of apoptosis such as chromosome condensation and disintegration of cell bodies. Activated caspase-3 positive cells were found in the cortex within 2 h of the first injection, and reached a peak at 12 h. In addition, TUNEL staining revealed DNA fragmentation in the same regions. These results demonstrate that acute ethanol administration causes neuronal cell death via a caspase-3-dependent pathway within 24 h, suggesting that activation of caspase-3 is a marker of the developmental neurotoxicity of ethanol.

Ketogenic diet increases calbindin-D28k in the hippocampi of male ICR mice with kainic acid seizures.

The ketogenic diet (KD) increased the expression of calbindin-D(28k) (CB) in the interneurons of the hippocampus compared with the normal diet (ND)-fed mice. Also, 2 days after kainic acid (KA) administration, numerous CB-expressing astrocytes were found in the KD-fed mice compared with those of the ND-fed mice. These results suggest that the neuroprotective effect of the KD on the KA-induced toxicity may be, in part, mediated via an increased expression of CB.

Prepubertal chronic ethanol administration alters TTF-1 and Oct-2 expression in the hypothalamus of female rats.

We found that prolonged administration of ethanol (3 g/kg i.p. at 08:00, once per day) to young female rats starting on postnatal day 24 caused delayed puberty. We further found that prolonged ethanol administration changed the typical hypothalamic expression patterns of TTF-1 and Oct-2 and reduced GnRH mRNA expression. We suggest that these changes may cause the ethanol-induced disturbances in the regulation of GnRH in the hypothalamus and may be responsible for the ethanol-induced reduction in GnRH and LH associated with delayed puberty.