Wugang Hou

N-MYC downstream-regulated gene 2, a novel estrogen-targeted gene is involved in the regulation of NA+/K+-ATPase

Na+/K+-ATPase, a plasma membrane protein abundantly expressed in epithelial tissues, has been identified and linked to numerous biological events, including ion transport and reabsorption. In Na+/K+-ATPase, the β-subunit plays a fundamental role in the structural integrity and functional maturation of holoenzyme. Estrogens are important circulating hormones that can regulate Na+/K+-ATPase abundance and activity; however, the specific molecules participating in this process are largely unknown. Here, we characterize that N-myc downstream-regulated gene 2 (NDRG2) is an estrogen up-regulated gene. 17β-Estradiol binds with estrogen receptor β but not estrogen receptor α to up-regulate NDRG2 expression via transcriptional activation. We also find that NDRG2 interacts with the β1-subunit of Na+/K+-ATPase and stabilizes the β1-subunit by inhibiting its ubiquitination and degradation. NDRG2-induced prolongation of the β1-subunit protein half-life is accompanied by a similar increase in Na+/K+-ATPase-mediated Na+ transport and Na+ current in epithelial cells. In addition, NDRG2 silencing largely attenuates the accumulation of β1-subunit regulated by 17β-estradiol. Our results demonstrate that estrogen/NDRG2/Na+/K+-ATPase β1 pathway is important in promoting Na+/K+-ATPase activity and suggest this novel pathway might have substantial roles in ion transport, fluid balance, and homeostasis.

The pleiotropic roles of sphingolipid signaling in autophagy

The autophagic process involves encompassing damaged proteins and organelles within double- or multi-membraned structures and delivering these molecules to the lytic compartments of vacuoles. Sphingolipids (SLs), which are ubiquitous membrane lipids in eukaryotes, participate in the generation of various membrane structures, including rafts, caveolae, and cytosolic vesicles. SLs are a complex family of molecules that have a growing number of members, including ceramide, sphingosine-1-phosphate, and dihydroceramide, which have been associated with the essential cellular process of autophagy. This review highlights recent studies focusing on the regulation and function of SL-associated autophagy and its role in cell fate, diseases, and therapeutic interventions.

The effects of different doses of estradiol (E2) on cerebral ischemia in an in vitro model of oxygen and glucose deprivation and reperfusion and in a rat model of middle carotid artery occlusion

BackgroundBecause neuroprotective effects of estrogen remain controversial, we aimed to investigate the effect of different doses of estradiol (E2) on cerebral ischemia using both in vivo and in vitro experiments.ResultsPC12 cells were cultured at physiological (10 nM and 20 nM) or pharmacological (10 μM and 20 μM) dosages of E2 for 24 hours (h). The results of 5-bromodeoxyuridine (Brdu) incorporation and flow cytometric analysis showed that physiological doses of E2 enhanced cell proliferation and pharmacological doses of E2 inhibited cell proliferation. After the cells were exposed to oxygen and glucose deprivation (OGD) for 4 h and reperfusion for 20 h, the results of 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release assay, flow cytometric analysis and Western blot analysis showed that physiological doses of E2 enhanced cell viability, reduced cell apoptosis and decreased the expression of pro-apoptotic protein caspase-3. In contrast, pharmacological doses of E2 decreased cell viability and induced cell apoptosis. In vivo, adult ovariectomized (OVX) female rats received continuous subcutaneous injection of different doses of E2 for 4 weeks. Transient cerebral ischemia was induced for 2 h using the middle cerebral artery occlusion (MCAO) technique, followed by 22 h of reperfusion. The results of Garcia test, 2, 3, 5-triphenyltetrazolium chloride (TTC) staining showed that 6 μg/kg and 20 μg/kg E2 replacement induced an increase in neurological deficit scores, a decrease in the infarct volume and a reduction in the expression of caspase-3 when compared to animals in the OVX group without E2 treatment. However, 50 μg/kg E2 replacement treatment decreased neurological deficit scores, increased the infarct volume and the expression of caspase-3 when compared to animals in the control group and 6 up/kg or 20 μg/kg E2 replacement group.ConclusionWe conclude that physiological levels of E2 exhibit neuroprotective effects on cerebral ischemia; whereas, pharmacological or supraphysiological doses of E2 have damaging effects on neurons after cerebral ischemia.

Spatial-temporal expression of NDRG2 in rat brain after focal cerebral ischemia and reperfusion.

N-myc downstream regulated gene 2 (NDRG2) was reported to be widely expressed in the nervous system. However, the expression and potential role of NDRG2 in focal cerebral ischemia brain remain unclear. Herein, we investigated spatial-temporal expression of NDRG2 in the rat brain following transient focal cerebral ischemia. Male Sprague-Dawley rats underwent a 120-min transient occlusion of middle cerebral artery. Rats were killed and brain samples were harvested at 4, 12, 24, and 72h after reperfusion. Expression of NDRG2 in the brain was determined by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot analysis and immunohistochemical staining. Cellular apoptosis was assessed by TUNEL staining. The results showed that NDRG2 was expressed on cells with an astrocytes-like morphology in ischemic penumbra. NDRG2 mRNA and protein expression began to increase at 4h after reperfusion and peaked at 24h in the ischemic penumbra. By using immunofluorescence, NDRG2 signals were co-localized with GFAP-positive astrocytes, and NDRG2 expression in astrocytes translocated from a cytoplasm to a nuclear localization at 24h after reperfusion. Double immunofluorescent staining for TUNEL and NDRG2 showed that some NDRG2 signals co-localized with TUNEL-positive cells, and that the apoptotic cells increased with enhancement of NDRG2-positive signals. In conclusion, NDRG2 expression is up-regulated in ischemic penumbra following transient focal cerebral ischemia. NDRG2 expression in astrocytes may play important pathological roles in cell apoptosis after stroke.

Upregulation and nuclear translocation of testicular ghrelin protects differentiating spermatogonia from ionizing radiation injury

Proper control of apoptotic signaling is important for maintenance of testicular homeostasis after ionizing radiation (IR). Herein, we challenged the hypothesis that ghrelin, a pleiotropic modulator, is potentially involved in IR-induced germ cell injury. Lower body exposure to 2 Gy of IR induced a notable increase of ghrelin expression in the nuclear of differentiating spermatogonia at defined stages, with an impairment in the Leydig cells (LCs)-expressing ghrelin. Unexpectedly, inhibition of the ghrelin pathway by intraperitoneal injection of a specific GHS-R1α antagonist enhanced spermatogonia elimination by apoptosis during the early recovery following IR, and thereafter resulted in impaired male fertility, suggesting that the anti-apoptotic effects of evoked ghrelin, although transient along testicular IR injury, have a profound influence on the post-injury recovery. In addition, inhibition of ghrelin signaling resulted in a significant increase in the intratesticular testosterone (T) level at the end of 21 days after IR, which should stimulate the spermatogenic recovery from surviving spermatogonia to a certain extent during the late stage. We further demonstrated that the upregulation and nuclear trafficking of ghrelin, elaborately regulated by IR-elicited antioxidant system in spermatogonia, may act through a p53-dependent mechanism. The elicitation of ghrelin expression by IR stress, the regulation of ghrelin expression by IR-induced oxidative stress and the interaction between p53 and ghrelin signaling during IR injury were confirmed in cultured spermatogonia. Hence, our results represent the first evidence in support of a radioprotective role of ghrelin in the differentiating spermatogonia. The acutely, delicate regulation of local-produced ghrelin appears to be a fine-tune mechanism modulating the balance between testicular homeostasis and early IR injury.

The loss of estrogen efficacy against cerebral ischemia in aged postmenopausal female mice.

Estrogen has been shown to have neuroprotective effects in numerous experimental studies involving young and adult animals. However, several clinical trials have found that in aged postmenopausal women who received estrogen replacement therapy, there did not appear to be a reduction in the incidence of stroke. The aim of this study was to investigate the effects of physiological dosages of estrogen on aged female mice subjected to ischemia-reperfusion injury. Adult ovariectomized (OVX) female mice and 22-month-old female mice received daily subcutaneous injections of 100 μg/kg or 300 μg/kg 17β-estradiol (E2) at the back of the neck for four weeks, and the expression levels of estrogen receptor (ER) α and β in the cerebral cortex were determined using real-time PCR and Western blotting analyses. To mimic ischemic stroke, the mice received middle cerebral artery occlusion (MCAO) treatment for 1h followed by a 24-h reperfusion period. The mice were then subjected to neurological deficit testing and infarct volume evaluation. The aged mice showed higher neurological deficit scores and larger infarct volumes compared with the adult mice. Both the lower and higher physiological dosages of E2 significantly improved the neurological test scores and decreased the infarct volume in the adult mice; however, E2 showed no neuroprotective effects in the aged mice. Furthermore, the protein expression of ERα and ERβ in the cerebral cortex was significantly decreased in the aged mice compared with the adult mice, and this decrease was not rescued by E2 treatment. These results indicate that the down-regulation of ERα and ERβ in the cerebral cortex may contribute to the loss of estrogen efficacy against ischemic injury in aged females and may point to new therapies for ischemic stroke in aged postmenopausal women.

Up-regulation of NDRG2 through nuclear factor-kappa B is required for Leydig cell apoptosis in both human and murine infertile testes

Many pro-apoptotic factors, such as nuclear factor-kappa B (NF-κB) and Fas, play crucial roles in the process of Leydig cell apoptosis, ultimately leading to male sterility, such as in Sertoli cell only syndrome (SCO) and hypospermatogenesis. However, the molecular mechanism of such apoptosis is unclear. Recent reports on N-myc downstream-regulated gene 2 (ndrg2) have suggested that it is involved in cellular differentiation, development, and apoptosis. The unique expression of NDRG2 in SCO and hypospermatogenic testis suggests its pivotal role in those diseases. In this study, we analyzed NDRG2 expression profiles in the testes of normal spermatogenesis patients, hypospermatogenesis patients, and SCO patients, as well as in vivo and in vitro models, which were Sprague-Dawley rats and the Leydig cell line TM3 treated with the Leydig cell-specific toxicant ethane-dimethanesulfonate (EDS). Our data confirm that NDRG2 is normally exclusively located in the cytoplasm of Leydig cells and is up-regulated and translocates into the nucleus under apoptotic stimulations in human and murine testis. Meanwhile, transcription factor NF-κB was activated by EDS administration, bound to the ndrg2 promoter, and further increased in expression, effects that were abolished by NF-κB inhibitor Pyrrolidine dithiocarbamate (PDTC). Furthermore, siRNA knock-down of ndrg2 led to increased proliferative or decreased apoptotic TM3 cells, while over-expression of ndrg2 had the reverse effect. This study reveals that ndrg2 is a novel gene that participates in Leydig cell apoptosis, with essential functions in testicular cells, and suggests its possible role in apoptotic Leydig cells and male fertility.

Anti-depressive mechanism of repetitive transcranial magnetic stimulation in rat: the role of the endocannabinoid system.

Repetitive transcranial magnetic stimulation (rTMS) to treat depression has been thoroughly investigated in recent years. However, the underlying mechanisms are not fully understood. In this study, a chronic unpredictable mild stress (CUMS) paradigm was applied to male Sprague Dawley rats. Then rTMS was performed for 7 consecutive days, and the anti-depressive effects were evaluated by the sucrose preference test (SPT), the forced swimming test (FST), and the open-field test (OFT). Hippocampal cannabinoid type I receptor (CB1) expression was measured, and the expression levels of brain-derived neurotrophic factor (BDNF), Bcl-2, and Bax and the number of bromodeoxyuridine (BrdU)-positive cells were also investigated. These parameters were also observed after the selective CB1 receptor antagonist AM251 was used as a blocking agent. The results showed that CUMS induced a significant decrease in sucrose preference, a significant increase in immobility time in the FST, and a significantly decreased horizontal distance in the OFT. In addition, reduced hippocampal CB1 receptor, BDNF, and Bcl-2/Bax protein expression levels in CUMS rats, as well as decreased cell proliferation were also observed in the dentate gyrus. Meanwhile, rTMS treatment up-regulated cell proliferation; elevated CB1 receptor, BDNF, and Bcl-2/Bax expression levels in the hippocampus; and ameliorated depressive-like behaviors. All of these beneficial effects were abolished by AM251. These results indicate that rTMS increases BDNF production and hippocampal cell proliferation to protect against CUMS-induced changes through its effect on CB1 receptors.

Rosmarinic acid ameliorates PTSD-like symptoms in a rat model and promotes cell proliferation in the hippocampus.

Rosmarinic acid (RA) is an important component of Chinese herbal medicine treatments and has been demonstrated to exert therapeutic effects in mood disorders. The present study was designed to assess the effects of RA on post-traumatic stress disorder (PTSD)-like symptoms, hippocampal cell proliferation and phosphorylation extracellular regulated protein kinases (pERK1/2) expression. We found that administration of RA (10mg/kg) alleviated PTSD-like symptoms in rats exposed to an enhanced single prolonged stress (ESPS) paradigm and restored hippocampal proliferation and pERK1/2 expression. Interestingly, the effects of RA were inhibited by the blockage of the ERK signaling. These data support the use of RA for treating PTSD and indicate that the ERK1/2 signaling cascade may play a critical role in the therapeutic efficacy of RA in treating such conditions.

NDRG2 Is a Novel p53-Associated Regulator of Apoptosis in C6-Originated Astrocytes Exposed to Oxygen-Glucose Deprivation

N-myc downstream-regulated gene 2 (NDRG2) has been documented to be a pro-differentiative and anti-proliferative gene in cancer research. Our previous study found a significant NDRG2 up-regulation in reactive astrocytes of penumbra after transient focal cerebral ischemia, which was parallel to the enhancement of TUNEL-positive signals. However, it is still uncertain whether NDRG2 participates in cellular apoptosis induced by ischemia-reperfusion injury in brain. In this study, we investigated the role of NDRG2 in cellular apoptosis induced by oxygen-glucose deprivation (OGD) in IL-6-differentiated C6 glioma cells. The results showed that NDRG2 was up-regulated and translocated from the cytoplasm to the nucleus after OGD exposure. NDRG2 over-expression exhibited an anti-proliferative effect and increased the Bax/Bcl-2 ratio after OGD exposure, while NDRG2 silencing promoted the cellular proliferation and attenuated the up-regulation of Bax/Bcl-2 ratio. The pro-apoptotic effect of p53 was verified by the results in which p53 silencing greatly reduced the percentage of OGD-induced apoptotic cells. p53 silencing also reduced the OGD-induced NDRG2 up-regulation. However, over-expression of p53 did not further improve the NDRG2 up-regulation. In conclusion, NDRG2 is a p53-associated regulator of apoptosis in C6-originated astrocytes after OGD exposure. These findings bring insight to the roles of NDRG2 in ischemic-hypoxic injury and provide potential targets for future clinical therapies on stroke.