Study on the mechanism of glucocorticoid receptor mitochondrial translocation and glucocorticoid-induced apoptosis in macrophages.

OBJECTIVE: Our research aimed to investigate the therapeutic effects of Tubastatin-A, a glucocorticoid receptor (GR) mitochondrial translocation inhibitor, and mitoquinone (MitoQ), an antioxidant, on attenuating dexamethasone (DEX)-induced macrophage apoptosis. METHODS: We treated RAW264.7 macrophages with different combinations of DEX and either Tubastatin-A or MitoQ. Parameters such as mitochondrial GR translocation, mitochondrial reactive oxygen species levels, mitochondrial membrane potential, mitochondrial permeability transition pore opening, cytochrome C efflux to the cytosol, and apoptosis were subsequently evaluated in the different treatment groups via qRT-PCR, western blotting, and immunofluorescence assays. RESULTS: DEX intervention increased the translocation of GRs into the mitochondria, while reducing the expression of the mitochondrial gene MT-CO1 and the activity of mitochondrial respiratory chain complex IV in macrophages. In addition, DEX administration increased mtROS levels, mitochondrial permeability transition pore opening, and mitochondrial cytochrome C release in macrophages, which promoted their apoptosis. We found that Tubastatin-A inhibited mitochondrial GR translocation and reversed the DEX-induced increase in GR levels within the mitochondria. Furthermore, Tubastatin-A mitigated various mitochondrial changes induced by DEX, including reducing the efflux of mitochondrial cytochrome C and inhibiting macrophage apoptosis. Similarly, MitoQ exerted its effects on macrophage apoptosis by reducing mtROS levels through the mitochondrial pathway. CONCLUSIONS: The DEX-mediated translocation of GR into mitochondria disrupts the mitochondrial function of macrophages, which induces their apoptosis. By inhibiting mitochondrial translocation of GR and reducing mtROS levels, Tubastatin-A and MitoQ can effectively attenuate macrophage apoptosis, which has clinical implications for reducing the notable side effects associated with glucocorticoid use.

Adipsin inhibits Irak2 mitochondrial translocation and improves fatty acid ß-oxidation to alleviate diabetic cardiomyopathy.

BACKGROUND: Diabetic cardiomyopathy (DCM) causes the myocardium to rely on fatty acid ß-oxidation for energy. The accumulation of intracellular lipids and fatty acids in the myocardium usually results in lipotoxicity, which impairs myocardial function. Adipsin may play an important protective role in the pathogenesis of DCM. The aim of this study is to investigate the regulatory effect of Adipsin on DCM lipotoxicity and its molecular mechanism. METHODS: A high-fat diet (HFD)-induced type 2 diabetes mellitus model was constructed in mice with adipose tissue-specific overexpression of Adipsin (Adipsin-Tg). Liquid chromatography-tandem mass spectrometry (LC-MS/MS), glutathione-S-transferase (GST) pull-down technique, Co-immunoprecipitation (Co-IP) and immunofluorescence colocalization analyses were used to investigate the molecules which can directly interact with Adipsin. The immunocolloidal gold method was also used to detect the interaction between Adipsin and its downstream modulator. RESULTS: The expression of Adipsin was significantly downregulated in the HFD-induced DCM model (P < 0.05). Adipose tissue-specific overexpression of Adipsin significantly improved cardiac function and alleviated cardiac remodeling in DCM (P < 0.05). Adipsin overexpression also alleviated mitochondrial oxidative phosphorylation function in diabetic stress (P < 0.05). LC-MS/MS analysis, GST pull-down technique and Co-IP studies revealed that interleukin-1 receptor-associated kinase-like 2 (Irak2) was a downstream regulator of Adipsin. Immunofluorescence analysis also revealed that Adipsin was co-localized with Irak2 in cardiomyocytes. Immunocolloidal gold electron microscopy and Western blotting analysis indicated that Adipsin inhibited the mitochondrial translocation of Irak2 in DCM, thus dampening the interaction between Irak2 and prohibitin (Phb)-optic atrophy protein 1 (Opa1) on mitochondria and improving the structural integrity and function of mitochondria (P < 0.05). Interestingly, in the presence of Irak2 knockdown, Adipsin overexpression did not further alleviate myocardial mitochondrial destruction and cardiac dysfunction, suggesting a downstream role of Irak2 in Adipsin-induced responses (P < 0.05). Consistent with these findings, overexpression of Adipsin after Irak2 knockdown did not further reduce the accumulation of lipids and their metabolites in the cardiac myocardium, nor did it enhance the oxidation capacity of cardiomyocytes expose to palmitate (PA) (P < 0.05). These results indicated that Irak2 may be a downstream regulator of Adipsin. CONCLUSIONS: Adipsin improves fatty acid ß-oxidation and alleviates mitochondrial injury in DCM. The mechanism is related to Irak2 interaction and inhibition of Irak2 mitochondrial translocation.

A mitochondrial EglN1-AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress.

Mitochondria play essential roles in cancer cell adaptation to hypoxia, but the underlying mechanisms remain elusive. Through mitochondrial proteomic profiling, we here find that the prolyl hydroxylase EglN1 (PHD2) accumulates on mitochondria under hypoxia. EglN1 substrate-binding region in the ß2ß3 loop is responsible for its mitochondrial translocation and contributes to breast tumor growth. Furthermore, we identify AMP-activated protein kinase alpha (AMPKα) as an EglN1 substrate on mitochondria. The EglN1-AMPKα interaction is essential for their mutual mitochondrial translocation. After EglN1 prolyl-hydroxylates AMPKα under normoxia, they rapidly dissociate following prolyl-hydroxylation, leading to their immediate release from mitochondria. In contrast, hypoxia results in constant EglN1-AMPKα interaction and their accumulation on mitochondria, leading to the formation of a Ca2+ /calmodulin-dependent protein kinase 2 (CaMKK2)-EglN1-AMPKα complex to activate AMPKα phosphorylation, ensuring metabolic homeostasis and breast tumor growth. Our findings identify EglN1 as an oxygen-sensitive metabolic checkpoint signaling hypoxic stress to mitochondria through its ß2ß3 loop region, suggesting a potential therapeutic target for breast cancer.

Angiotensin-(1-7) promotes mitochondrial translocation of human telomerase reverse transcriptase in HUVECs through the TOM20 complex.

BACKGROUND: Angiotensin (Ang) (1-7) is a vasodilator peptide that ameliorates microcirculation dysfunction, increases telomerase activity in cells, and exerts vasodilatory, anti-inflammatory, antioxidative stress, and antiapoptotic effects. Mitochondrial human telomerase reverse transcriptase (hTERT) plays an important role in the processes of antiapoptosis, antioxidative stress, and immortalization. This study aimed to investigate the effect of Ang(1-7) on the mitochondrial translocation of hTERT. METHODS: An in vitro model of lipopolysaccharide (LPS)-induced inflammation was established in human umbilical vein endothelial cells (HUVECs). Ang(1-7) was added to cells 30 min before LPS stimulation. The Ang(1-7)/Mas receptor antagonist A779 plus Ang(1-7) were added to the cells 30 min before LPS stimulation. The translocase outer membrane (TOM)20-overexpression HUVECs (HUVEC-TOM20OE), TOM20-knockdown HUVECs (HUVEC-TOM20KD), and the corresponding negative control cell lines were constructed by lentiviral transfection of HUVECs. Cells subjected to LPS stimulation alone, LPS plus Ang(1-7), LPS plus Ang(1-7) and A779, vehicle and no treatment were termed the LPS group, LPS + A group, LPS + A + A779 group, Con group and Neg group, respectively. Immunofluorescence staining was used to detect the distribution of hTERT in the nuclei and mitochondria of HUVECs and to locate TOM20, TOM40, and translocase inner membrane (TIM)23 in the mitochondria. The protein expression levels of total hTERT, mitochondrial hTERT, TOM20, TOM40, and TIM23 were measured by Western blot. The mRNA expression levels of hTERT, TOM20, TOM40, and TIM23 were assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: hTERT colocalized with TOM40, TOM20 and TIM23 in the mitochondria. The mitochondrial hTERT protein level of the LPS + A group was significantly greater than that of the LPS group (P = 0.001), and the LPS group showed significantly increased expression of mitochondrial hTERT compared with that of the control group (P = 0.001). No significant difference in the level of total hTERT was observed between the LPS + A and LPS groups. The mitochondrial hTERT protein level of the LPS + A + A779 group was significantly lower than that of the LPS + A group (P = 0.021). The protein level of mitochondrial hTERT in HUVEC-TOM20KD treated with or without LPS alone or LPS + A was significantly decreased compared with the corresponding groups of control HUVECs (HUVEC-TOM20KD-Con vs. HUVEC-Con, P = 0.035; HUVEC-TOM20KD-LPS vs. HUVEC-LPS, P = 0.003; HUVEC-TOM20KD-LPS + A vs. HUVEC-LPS + A, P = 0.001), and treatment with Ang(1-7) did not restore the downregulation of mitochondrial hTERT in HUVEC-TOM20KD. HUVEC-TOM20OE showed a significantly increased level of mitochondrial hTERT (HUVEC-TOM20OE-Con vs. HUVEC-Con, P = 0.010), which was further elevated by Ang(1-7) stimulation (HUVEC-TOM20OE-LPS + A vs. HUVEC-TOM20OE-Con, P = 0.011). Lastly, the protein expression levels of TOM40 (HUVEC-TOM20KD-Con vs. HUVEC-Con, P = 0.007) and TIM23 (HUVEC-TOM20KD-Con vs. HUVEC-Con, P = 0.001) were significantly increased in HUVEC-TOM20KD in comparison to HUVECs. CONCLUSIONS: Ang(1-7) effectively promoted mitochondrial translocation of hTERT in HUVECs via TOM20, indicating that hTERT may be transported to the mitochondria through the TOM20 complex. In addition, A779 could block the effects of Ang(1-7) in HUVECs.

Electroacupuncture Attenuates Ischemic Brain Injury and Cellular Apoptosis via Mitochondrial Translocation of Cofilin.

OBJECTIVE: To investigate the potential mechanisms of electroacupuncture (EA) to prevent ischemic stroke. METHODS: The method of middle cerebral artery occlusion (MCAO) was employed to establish a rat model of ischemic stroke. Seventy-eight Sprague-Dawley rats were divided into the sham group, MCAO + EA control (EC) group, and MCAO + EA (EA) group according to a random number table (n=26 per group). EA was applied to the acupoints of Baihui (DU 20) and Shenting (DU 24) 5 min and 6 h, respectively after the onset of MCAO. Rats in the sham and EC groups received only light isoflurane anesthesia for 30 min after MCAO. The neuroprotective effects of EA were evaluated by rota-rod test, neurological deficit scores and infarct volumes. Additionally, Nissl staining and immunostaining were performed to examine brain damage, rod formation, cellular apoptosis, and neuronal loss induced by ischemia. The activities of caspase-3, and expression levels of cofilin and p-cofilin in mitochondria and cytoplasm after ischemic injury were determined by Western blot. RESULTS: Compared with the EC group, EA significantly improved neuromotor function and cognitive ability after ischemic stroke (P<0.05 or P<0.01). Therapeutic use of EA also resulted in a significant decrease of cofilin rod formation and microtubule-associated protein-2 (MAP2) degradation in the cortical penumbra area compared with the EC rats (P<0.01). Furthermore, Western blot analysis showed that EA stimulation significantly inhibited mitochondrial translocation of cofilin and caspase-3 cleavage (P<0.05 or P<0.01). Additionally, brain damage (infarct volume and neuropathy), cellular apoptosis and neuronal loss induced by ischemia were remarkably suppressed by EA in the cortical penumbra of rats (P<0.05 or P<0.01). CONCLUSION: EA treatment after ischemic stroke may attenuate ischemic brain injury and cellular apoptosis through the regulation of mitochondrial translocation of cofilin, a novel mechanism of EA therapy.

Frontline Science: P2Y11 receptors support T cell activation by directing mitochondrial trafficking to the immune synapse.

T cells form an immune synapse (IS) with antigen-presenting cells (APCs) to detect antigens that match their TCR. Mitochondria, pannexin-1 (panx1) channels, and P2X4 receptors congregate at the IS where mitochondria produce the ATP that panx1 channels release in order to stimulate P2X4 receptors. P2X4 receptor stimulation causes cellular Ca2+ influx that up-regulates mitochondrial metabolism and localized ATP production at the IS. Here we show that P2Y11 receptors are essential players that sustain these T cell activation mechanisms. We found that P2Y11 receptors retract from the IS toward the back of cells where their stimulation by extracellular ATP induces cAMP/PKA signaling that redirects mitochondrial trafficking to the IS. P2Y11 receptors thus reinforce IS signaling by promoting the aggregation of mitochondria with panx1 ATP release channels and P2X4 receptors at the IS. This dual purinergic signaling mechanism involving P2X4 and P2Y11 receptors focuses mitochondrial metabolism to the IS where localized ATP production sustains synaptic activity in order to allow successful completion of T cell activation responses. Our findings have practical implications because rodents lack P2Y11 receptors, raising concerns as to the validity of rodent models to study treatment of infections and inflammatory conditions.

Electroacupuncture Attenuates Ischemic Brain Injury and Cellular Apoptosis via Mitochondrial Translocation of Cofilin / 中国结合医学杂志

OBJECTIVE@#To investigate the potential mechanisms of electroacupuncture (EA) to prevent ischemic stroke.@*METHODS@#The method of middle cerebral artery occlusion (MCAO) was employed to establish a rat model of ischemic stroke. Seventy-eight Sprague-Dawley rats were divided into the sham group, MCAO + EA control (EC) group, and MCAO + EA (EA) group according to a random number table (n=26 per group). EA was applied to the acupoints of Baihui (DU 20) and Shenting (DU 24) 5 min and 6 h, respectively after the onset of MCAO. Rats in the sham and EC groups received only light isoflurane anesthesia for 30 min after MCAO. The neuroprotective effects of EA were evaluated by rota-rod test, neurological deficit scores and infarct volumes. Additionally, Nissl staining and immunostaining were performed to examine brain damage, rod formation, cellular apoptosis, and neuronal loss induced by ischemia. The activities of caspase-3, and expression levels of cofilin and p-cofilin in mitochondria and cytoplasm after ischemic injury were determined by Western blot.@*RESULTS@#Compared with the EC group, EA significantly improved neuromotor function and cognitive ability after ischemic stroke (P<0.05 or P<0.01). Therapeutic use of EA also resulted in a significant decrease of cofilin rod formation and microtubule-associated protein-2 (MAP2) degradation in the cortical penumbra area compared with the EC rats (P<0.01). Furthermore, Western blot analysis showed that EA stimulation significantly inhibited mitochondrial translocation of cofilin and caspase-3 cleavage (P<0.05 or P<0.01). Additionally, brain damage (infarct volume and neuropathy), cellular apoptosis and neuronal loss induced by ischemia were remarkably suppressed by EA in the cortical penumbra of rats (P<0.05 or P<0.01).@*CONCLUSION@#EA treatment after ischemic stroke may attenuate ischemic brain injury and cellular apoptosis through the regulation of mitochondrial translocation of cofilin, a novel mechanism of EA therapy.

STAT3 inhibition induced temozolomide-resistant glioblastoma apoptosis via triggering mitochondrial STAT3 translocation and respiratory chain dysfunction.

Recent evidence has demonstrated that the signal transducer and activator of transcription 3 (STAT3) gene are abnormally active in glioblastoma multiforme (GBM), and this change is crucial for the tumor survival and chemotherapy-resistant. Certain preclinical pharmacology studies have focused on STAT3 phosphorylation and homodimerization, and have developed a class of salicylic acid-based inhibitors, which blocks the nuclear translocation-dependent canonical STAT3 signaling. In the present study, we demonstrated that the salicylic acid-based compound SH-4-54 was quite toxic to temozolomide (TMZ)-resistant GBM cells and could trigger apoptosis in these cells via enhancing mitochondrial translocation-dependent non-canonical STAT3 pathway. We demonstrated that incubation of TMZ-resistant GBM cells with SH-4-54 led to mitochondrial STAT3 (mitoSTAT3) activation and respiratory dysfunction reflected by disrupted (or suppressed) activities of oxidative phosphorylation complexes and oxygen consumption rate. Mechanistically, we proved that SH-4-54 could increase mitoSTAT3 transmembrane import via GRIM-19 and reinforce the association between mitoSTAT3 and mitochondrial transcription factor A (TFAM), indicating that SH-4-54 could facilitate the binding of mitoSTAT3 to mitochondria DNA (mtDNA) and negatively regulate mitochondrial-encoded genes, thus leading to the abnormal oxidation respiratory. Lastly, using GRIM-19 knockout cell line and subcutaneous xenotransplanted tumor model, we elaborately showed the enrichment of SH-4-54 in mitochondria by LC-MS/MS analysis. In conclusion, our data demonstrate thatthe salicylic acid-based compound SH-4-54 is quite effective in killing TMZ-resistant GBM cells and this cytotoxicity is attributed to mitoSTAT3 activation.

Genetic inhibition of PKCε attenuates neurodegeneration after global cerebral ischemia in male mice.

Global cerebral ischemia that accompanies cardiac arrest is a major cause of morbidity and mortality. Protein Kinase C epsilon (PKCε) is a member of the novel PKC subfamily and plays a vital role in ischemic preconditioning. Pharmacological activation of PKCε before cerebral ischemia confers neuroprotection. The role of endogenous PKCε after cerebral ischemia remains elusive. Here we used male PKCε-null mice to assess the effects of PKCε deficiency on neurodegeneration after transient global cerebral ischemia (tGCI). We found that the cerebral vasculature, blood flow, and the expression of other PKC isozymes were not altered in the PKCε-null mice. Spatial learning and memory was impaired after tGCI, but the impairment was attenuated in male PKCε-null mice as compared to male wild-type controls. A significant reduction in Fluoro-Jade C labeling and mitochondrial release of cytochrome C in the hippocampus was found in male PKCε-null mice after tGCI. Male PKCε-null mice expressed increased levels of PKCδ in the mitochondria, which may prevent the translocation of PKCδ from the cytosol to the mitochondria after tGCI. Our results demonstrate the neuroprotective effects of PKCε deficiency on neurodegeneration after tGCI, and suggest that reduced mitochondrial translocation of PKCδ may contribute to the neuroprotective action in male PKCε-null mice.

Differential protein acetylation assists import of excess SOD2 into mitochondria and mediates SOD2 aggregation associated with cardiac hypertrophy in the murine SOD2-tg heart.

SOD2 is the primary antioxidant enzyme neutralizing •O2- in mitochondria. Cardiac-specific SOD2 overexpression (SOD2-tg) induces supernormal function and cardiac hypertrophy in the mouse heart. However, the reductive stress imposed by SOD2 overexpression results in protein aggregation of SOD2 pentamers and differential hyperacetylation of SOD2 in the mitochondria and cytosol. Here, we studied SOD2 acetylation in SOD2-tg and wild-type mouse hearts. LC-MS/MS analysis indicated the presence of four acetylated lysines in matrix SOD2 and nine acetylated lysines in cytosolic SOD2 from the SOD2-tg heart. However, only one specific acetylated lysine residue was detected in the mitochondria of the wild-type heart, which was consistent with Sirt3 downregulation in the SOD2-tg heart. LC-MS/MS further detected hyperacetylated SOD2 with a signaling peptide in the mitochondrial inner membrane and matrix of the SOD2-tg heart, indicating partial arrest of the SOD2 precursor in the membrane during translocation into the mitochondria. Upregulation of HSP 70 and cytosolic HSP 60 enabled the translocation of excess SOD2 into mitochondria. In vitro acetylation of matrix SOD2 with Ac2O deaggregated pentameric SOD2, restored the profile of cytosolic SOD2 hyperacetylation, and decreased matrix SOD2 activity. As revealed by 3D structure, acetylation of K89, K134, and K154 of cytosolic SOD2 induces unfolding of the tertiary structure and breaking of the salt bridges that are important for the quaternary structure, suggesting that hyperacetylation and HSP 70 upregulation maintain the unfolded status of SOD2 in the cytosol and mediate the import of SOD2 across the membrane. Downregulation of Sirt3, HSP 60, and presequence protease in the mitochondria of the SOD2-tg heart promoted protein misfolding that led to pentameric aggregation.

Enhanced Phosphorylation of Bax and Its Translocation into Mitochondria in the Brains of Individuals Affiliated with Alzheimer's Disease.

BACKGROUND: Despite increased neuronal death, senile plaques, and neurofibrillary tangles observed in patients suffering from Alzheimer's disease (AD), the detailed mechanism of cell death in AD is still poorly understood. METHOD: We hypothesized that p38 kinase activates and then phosphorylates Bax, leading to its translocation to mitochondria in AD brains compared to controls. The aim of this study was to investigate the role of p38 kinase in phosphorylation and sub-cellular localization of pro-apoptotic Bax in the frontal cortex of the brains from AD and control subjects. Increased oxidative stress in AD individuals compared to control was evaluated by measuring the levels of carbonylated proteins and oxidized peroxiredoxin, an antioxidant enzyme. The relative amounts of p38 kinase and phospho-Bax in mitochondria in AD brains and controls were determined by immunoblot analysis using the respective antibody against each protein following immunoprecipitation. RESULTS: Our results showed that the levels of oxidized peroxiredoxin-SO3 and carbonylated proteins are significantly elevated in AD brains compared to controls, demonstrating the increased oxidative stress. CONCLUSION: The amount of phospho-p38 kinase is increased in AD brains and the activated p38 kinase appears to phosphorylate Thr residue(s) of Bax, which leads to its mitochondrial translocation, contributing to apoptosis and ultimately, neurodegeneration.

TRAF6 Restricts p53 Mitochondrial Translocation, Apoptosis, and Tumor Suppression.

Mitochondrial p53 is involved in apoptosis and tumor suppression. However, its regulation is not well studied. Here, we show that TRAF6 E3 ligase is a crucial factor to restrict mitochondrial translocation of p53 and spontaneous apoptosis by promoting K63-linked ubiquitination of p53 at K24 in cytosol, and such ubiquitination limits the interaction between p53 and MCL-1/BAK. Genotoxic stress reduces this ubiquitination in cytosol by S13/T330 phosphorylation-dependent translocation of TRAF6 from cytosol to nucleus, where TRAF6 also facilitates the K63-linked ubiquitination of nuclear p53 and its transactivation by recruiting p300 for p53 acetylation. Functionally, K63-linked ubiquitination of p53 compromised p53-mediated apoptosis and tumor suppression. Colorectal cancer samples with WT p53 reveal that TRAF6 overexpression negatively correlates with apoptosis and predicts poor response to chemotherapy and radiotherapy. Together, our study identifies TRAF6 as a critical gatekeeper to restrict p53 mitochondrial translocation, and such mechanism may contribute to tumor development and drug resistance.

HPOB, an HDAC6 inhibitor, attenuates corticosterone-induced injury in rat adrenal pheochromocytoma PC12 cells by inhibiting mitochondrial GR translocation and the intrinsic apoptosis pathway.

High levels of glucocorticoids (GCs) have been reported to damage normal hippocampal neurons, and such damage has been positively correlated with major depression (MD) and chronic stress. Our previous study showed that HDAC6 might be a potential target to regulate GC-induced glucocorticoid receptor (GR) translocation to the mitochondria and subsequent apoptosis. In the present study, we investigated the effect of HPOB, a selective HDAC6 inhibitor, on corticosterone (Cort)-induced apoptosis and explored the possible mechanism of action of HPOB in rat adrenal pheochromocytoma (PC12) cells, which possesses typical neuron features and expresses high levels of glucocorticoid receptors. We demonstrated that pre-treatment with HPOB remarkably reduced Cort-induced cytotoxicity and confirmed the anti-apoptotic effect of HPOB via the caspase-3 activity assay and H33342/PI and TUNEL double staining. Mechanistically, we demonstrated that HPOB reversed the Cort-induced elevation of GR levels in the mitochondria and blocked concomitant mitochondrial dysfunction and the intrinsic apoptosis pathway. Furthermore, HPOB was shown to attenuate expression of the multi-chaperone machinery (Hsp90-Hop-Hsp70) and cooperate with mitochondrial translocase of the outer/inner membrane (TOM/TIM) complex recruitment by triggering hyperacetylation of Hsps through HDAC6 inhibition. Considering all of these findings, the neuroprotective effect of HPOB demonstrated the crucial role of HDAC6 inhibition in reducing Cort-induced apoptosis in PC12 cells. The data further suggested that the anti-apoptotic activity of HDAC6 inhibition against the mitochondria-mediated impairment pathway might be mechanistically linked to the hyperacetylation of Hsps and consequent suppression of GR translocation to the mitochondria.

PEDF attenuates hypoxia-induced apoptosis and necrosis in H9c2 cells by inhibiting p53 mitochondrial translocation via PEDF-R.

Pigment epithelial-derived factor (PEDF) is a multifunctional secreted glycoprotein, which could protect against hypoxia-induced cell death related to its anti-oxidative effect in cultured cardiomyocytes. However, the pathway mediating this cytoprotective process has not been fully established. Here we confirmed that PEDF bound to pigment epithelial-derived factor receptor (PEDF-R) expressed on the membrane of H9c2 cells. Under hypoxic condition, PEDF increased the ratio of MDM2:p53, so as to inhibited p53 mitochondrial translocation via PEDF-R. As a result, mitochondrial outer membrane permeabilization (MOMP) and mitochondrial permeability transition pore (MPTP) opening were inhibited, meanwhile cleaved caspase-3, PARP and the release of HMGB1 were reduced. Accordingly, apoptosis and necrosis were attenuated simultaneously. We conclude that PEDF-R mediates PEDF attenuates hypoxia-induced apoptosis and necrosis in H9c2 cells by inhibiting p53 mitochondrial translocation.

Involvement of mortalin/GRP75/mthsp70 in the mitochondrial impairments induced by A53T mutant α-synuclein.

Mutations and excessive accumulation of α-synuclein (α-syn) can lead to the degeneration of dopaminergic neurons, indicating a pivotal role of α-syn in the pathogenesis of Parkinson's disease (PD). Although how α-syn contributes to PD is still elusive, mitochondrial impairments have been reported to be implicated in. Mortalin, a molecular chaperone mainly located in mitochondria, has been linked to the pathogenesis of PD in recent studies. Moreover, some proteomics studies indicate that mortalin is associated with PD-related proteins, including α-syn. Therefore it is of interest to understand the function of mortalin in the mitochondrial disruption induced by A53T α-syn overexpression. The present study modulated the expression of mortalin and detected the effect of mortalin on the mitochondrial impairments induced by A53T α-syn in SH-SY5Y cells. Our data revealed that A53T α-syn could disrupt mitochondrial dynamics and increase the neuronal susceptibility to neurotoxin rotenone. The expression of mortalin decreased significantly in dopaminergic cells overexpressing A53T α-syn; furthermore, the down-regulation of mortalin could attenuate the disrupted mitochondrial dynamics by reducing α-syn translocation to mitochondria, suggesting that a compensatory mechanism of mortalin might be implicated in the pathogenesis of PD.

Extramitochondrial OPA1 and adrenocortical function.

We have previously described that silencing of the mitochondrial protein OPA1 enhances mitochondrial Ca(2+) signaling and aldosterone production in H295R adrenocortical cells. Since extramitochondrial OPA1 (emOPA1) was reported to facilitate cAMP-induced lipolysis, we hypothesized that emOPA1, via the enhanced hydrolysis of cholesterol esters, augments aldosterone production in H295R cells. A few OPA1 immunopositive spots were detected in ∼40% of the cells. In cell fractionation studies OPA1/COX IV (mitochondrial marker) ratio in the post-mitochondrial fractions was an order of magnitude higher than that in the mitochondrial fraction. The ratio of long to short OPA1 isoforms was lower in post-mitochondrial than in mitochondrial fractions. Knockdown of OPA1 failed to reduce db-cAMP-induced phosphorylation of hormone-sensitive lipase (HSL), Ca(2+) signaling and aldosterone secretion. In conclusion, OPA1 could be detected in the post-mitochondrial fractions, nevertheless, OPA1 did not interfere with the cAMP - PKA - HSL mediated activation of aldosterone secretion.