182 research outputs found
The Physiological Function of Endothelin-2 in Mice
No full textIn order to directly explore the physiological function of ET-2, we generated constitutive, tissue-specific and systemically inducible knockout mice. Global ET-2 deficient mice exhibited severe growth retardation and juvenile lethality. Despite normal milk intake, they suffered from an apparent internal starvation characterized by hypoglycemia, ketonemia, and increased expression of starvation-induced genes in liver. Based on its abundant expression in the gut, I hypothesized that intestinal function of ET-2 is essential for the growth and survival of mice. However, unexpectedly, the intestine was morphologically and functionally normal in the global mutant mice. Moreover, intestine-specific ET-2 deficient mice showed no detectable abnormalities in growth and survival. Instead, I observed that colonic ET-2 has a protective role in epithelial cell injury. Global ET-2 knockout mice were profoundly hypothermic, even at ambient temperatures. Despite the severe hypothermia, DIO2 and UCP-1 failed to increase in brown adipose tissue in ET-2 knockouts. Housing these mice in a warm environment significantly extended the median life span. As temperature regulation is controlled by the central nervous system (CNS), I examined the phenotype in neuron-specific ET-2 knockout mice. However, the mutant mice displayed normal core body temperature, suggesting that ET-2 is not playing a role in CNS-regulated body temperature. ET-2 expression is clearly detected in the lung, with a sharp and transient increase soon after birth. The emphysematous structural change, which is associated with an increase of total lung capacity, resulted in chronic hypoxemia, hypercapnia, and increased erythropoietin synthesis. Finally, to rule out effects of ET-2 during embryonic development, I used the Cre-loxP system to delete ET-2 in neonatal and adult mice, and found that these mice fully reproduced the phenotype previously observed in global knockouts. Together, these findings reveal that ET-2 is critical for growth and survival of postnatal mice by playing important roles in energy homeostasis, thermoregulation, and maintenance of lung morphology and function. My studies rule out ET-2 function in the intestine and brain as being responsible for these phenotypes. However, the dramatic effects of the lung are newly discovered as a potential candidate tissue for critical ET-2 action and lung ET-2 function deserves further investigation
Resistance of mitochondrial DNA-deficient cells to TRAIL: role of Bax in TRAIL-induced apoptosis
No full textMitochondrion is one of the master players in both apoptosis and necrosis. We studied the role of mitochondrial function in TRAIL-induced apoptosis. TRAIL killed SK-Hep1 cells with characteristic features of apoptosis such as DNA fragmentation, sub-G1 ploidy peak and cytochrome c translocation. In contrast, mitochondrial DNA-deficient SK-Hep1 rho(0) cells were resistant to TRAIL. Dissipation of mitochondrial potential or cytochrome e translocation did not occur in rho(0) cells after TRAIL treatment. TRAIL induced translocation of Bax subsequent to the cleavage of Bid in parental cells. However, Bax translocation was absent in rho(0) cells, accounting for the failure of cytochrome c release in rho(0) cells. Forced expression of Bax induced caspase-3 activity in rho(0) cells. Incubation of rho(0) cells with ADP+Pi to increase intracellular ATP restored sensitivity to TRAIL. Despite different sensitivity to TRAIL, parental cells and rho(0) cells did not show significant difference in susceptibility to agonistic anti-Fas antibody, TNF-alpha or staurosporine. Our results indicate that TRAIL-induced apoptosis is dependent on intact mitochondrial function and susceptibility of mitochondrial DNA-deficient cells to apoptosis depends on the type of apoptotic stimuli. Tumor cells with mitochondrial mutations or dysfunction might have the ability to evade tumor surveillance imposed by TRAIL in vivo
Death effectors of beta-cell apoptosis in type 1 diabetes
No full textWhile it is generally agreed that apoptosis of pancreatic beta-cells is the most important and final step in the progression of type 1 diabetes without which clinical diabetes does not develop, it has not been elucidated which molecule(s) are the real culprit(s) in type 1 diabetes. Perforin, FasL, TNFalpha, IL-1, IFNgamma, and NO have been claimed as the effector molecules; however, they, as a single agent, might explain only part of beta-cell death in type 1 diabetes. While FasL was initially considered as a strong candidate for the most important death effector, following experiments cast doubt on such a hypothesis. Combinations or synergism between IFNgamma and TNFalpha or IL-1beta are being revisited as the death effectors, and molecular mechanism explaining such a synergism was addressed in several recent papers. The role of NF-kappaB for pancreatic beta-cell death in type 1 diabetes is also controversial. While NF-kappaB plays anti-apoptotic roles in most other death models, its role in type 1 diabetes might be different probably due to the involvement of multiple cytokines at different stages of the disease progression and the peculiarity of pancreatic beta-cells. Recent papers also suggested a role for Ca2+ in cytokine-mediated pancreatic beta-cell death. Such participation of Ca2+ in beta-cell death appears to have a close relevance to the mitochondrial events or ER stress that constitutes an important part of cell death machinery recently identified.ope
Steroidogenic Factor 1 in the Ventromedial Nucleus of the Hypothalamus Regulates Age-Dependent Obesity.
No full textThe ventromedial nucleus of the hypothalamus (VMH) is important for the regulation of whole body energy homeostasis and lesions in the VMH are reported to result in massive weight gain. The nuclear receptor steroidogenic factor 1 (SF-1) is a known VMH marker as it is exclusively expressed in the VMH region of the brain. SF-1 plays a critical role not only in the development of VMH but also in its physiological functions. In this study, we generated prenatal VMH-specific SF-1 KO mice and investigated age-dependent energy homeostasis regulation by SF-1. Deletion of SF-1 in the VMH resulted in dysregulated insulin and leptin homeostasis and late onset obesity due to increased food intake under normal chow and high fat diet conditions. In addition, SF-1 ablation was accompanied by a marked reduction in energy expenditure and physical activity and this effect was significantly pronounced in the aged mice. Taken together, our data indicates that SF-1 is a key component in the VMH-mediated regulation of energy homeostasis and implies that SF-1 plays a protective role against metabolic stressors including aging and high fat diet.ope
Caspase-Mediated p65 Cleavage Promotes TRAIL-Induced Apoptosis
No full textTumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) is cytotoxic to a wide variety of transformed cells, but not to most normal cells, implying potential therapeutic value against advanced cancer. However, signal transduction in TRAIL-mediated apoptosis is not clearly understood compared with other TNF family members. Specifically, it is not yet understood how TRAIL controls nuclear factor κB (NF-κB) activation and overcomes its antiapoptotic effect. We explored the regulation of NF-κB activity by TRAIL and its role in apoptosis. TRAIL combined with IκBα-“superrepressor” induced potent apoptosis of SK-Hep1 hepatoma cells at low concentrations of TRAIL that do not independently induce apoptosis. Apoptosis by high concentrations of TRAIL was not affected by IκBα-superrepressor. Although TRAIL alone did not induce NF-κB activity, TRAIL combined with z-VAD significantly increased NF-κB activation. Analysis of the NF-κB activation pathway indicated that TRAIL unexpectedly induced cleavage of p65 at Asp97, which was blocked by z-VAD, accounting for all of these findings. p65 expression abrogated apoptosis and increased NF-κB activity in TRAIL-treated cells. Cleavage-resistant p65D97A further increased NF-κB activity in TRAIL-treated cells, whereas the COOH-terminal p65 fragment acted as a dominant-negative inhibitor. XIAP levels were increased by TRAIL in combination with z-VAD, whereas XIAP levels were decreased by TRAIL alone. Cleavage of p65 was also detected after FRO thyroid cancer cells were treated with TRAIL. These results suggest that TRAIL induces NF-κB activation, but simultaneously abrogates NF-κB activation by cleaving p65, and thereby inhibits the induction of antiapoptotic proteins such as XIAP, which contributes to the strong apoptotic activity of TRAIL compared with other TNF family members.ope
miRNA-708 Control of CD44(+) Prostate Cancer-Initiating Cells
No full textTumor recurrence in prostate cancer has been attributed to the presence of CD44-expressing tumor-initiating cells. In this study, we report that miR-708 is a key negative regulator of this CD44+ subpopulation of prostate cancer cells, with important implications for diagnosis and prognosis of this disease. miR-708 was underexpressed in CD44+ cells from prostate cancer xenografts. Reconstitution of miR-708 in prostate cancer cell lines or CD44+ prostate cancer cells led to decreased tumorigenicity in vitro. Intratumoral delivery of synthetic miR-708 oligonucleotides triggered regression of established tumors in a murine xenograft model of human prostate cancer. Conversely, miR-708 silencing in a purified CD44− population of prostate cancer cells promoted tumor growth. Functional studies validated CD44 to be a direct target of miR-708 and also identified the serine/threonine kinase AKT2 as an additional target. Clinically, low miR-708 expression was associated significantly with poor survival outcome, tumor progression, and recurrence in patients with prostate cancer. Together, our findings suggest that reduced miR-708 expression leads to prostate cancer initiation, progression, and development by regulating the expression of CD44 as well as AKT2. miR-708 therefore may represent a novel therapeutic target or diagnostic and prognostic biomarker in prostate cancer.ope
IFN-gamma/TNF-alpha synergism as the final effector in autoimmune diabetes: a key role for STAT1/IFN regulatory factor-1 pathway in pancreatic beta cell death
No full textFas ligand (FasL), perforin, TNF-alpha, IL-1, and NO have been considered as effector molecule(s) leading to beta cell death in autoimmune diabetes. However, the real culprit(s) in beta cell destruction have long been elusive, despite intense investigation. We and others have demonstrated that FasL is not a major effector molecule in autoimmune diabetes, and previous inability to transfer diabetes to Fas-deficient nonobese diabetic (NOD)-lpr mice was due to constitutive FasL expression on lymphocytes from these mice. Here, we identified IFN-gamma/TNF-alpha synergism as the final effector molecules in autoimmune diabetes of NOD mice. A combination of IFN-gamma and TNF-alpha, but neither cytokine alone, induced classical caspase-dependent apoptosis in insulinoma and pancreatic islet cells. IFN-gamma treatment conferred susceptibility to TNF-alpha-induced apoptosis on otherwise resistant insulinoma cells by STAT1 activation followed by IFN regulatory factor (IRF)-1 induction. IRF-1 played a central role in IFN-gamma/TNF-alpha-induced cytotoxicity because inhibition of IRF-1 induction by antisense oligonucleotides blocked IFN-gamma/TNF-alpha-induced cytotoxicity, and transfection of IRF-1 rendered insulinoma cells susceptible to TNF-alpha-induced cytotoxicity. STAT1 and IRF-1 were expressed in pancreatic islets of diabetic NOD mice and colocalized with apoptotic cells. Moreover, anti-TNF-alpha Ab inhibited the development of diabetes after adoptive transfer. Taken together, our results indicate that IFN-gamma/TNF-alpha synergism is responsible for autoimmune diabetes in vivo as well as beta cell apoptosis in vitro and suggest a novel signal transduction in IFN-gamma/TNF-alpha synergism that may have relevance in other autoimmune diseases and synergistic anti-tumor effects of the two cytokines.ope
Nuclear factor kappaB protects pancreatic beta-cells from tumor necrosis factor-alpha-mediated apoptosis
No full textRecent studies incriminating tumor necrosis factor (TNF)-α as the final effector in pancreatic β-cell death in type 1 diabetes underscore the potential role of TNF-α-dependent NF-κB activation as an important modulator of pancreatic β-cell death in autoimmune diabetes. Although nuclear factor (NF)-κB activation has been implicated in the protection of target cells against apoptosis by a variety of death effectors, its role in pancreatic islet cell death is not clear. We studied the role of NF-κB activation in pancreatic islet cell death by using a γ-interferon (IFN-γ)/TNF-α synergism model we had previously reported. TNF-α induced inhibitor of κB (IκB) degradation and p65 translocation from cytoplasm to nuclei in MIN6N8 insulinoma cells. The NF-κB DNA-binding nuclear complex activated by TNF-α contained both the p65 and p50 subunit. IFN-γ pretreatment did not affect TNF-α-induced NF-κB activation. Treatment with a proteasome inhibitor blocked p65 translocation and induced susceptibility to TNF-α in otherwise resistant insulinoma cells or primary pancreatic islet cells. Specific inhibition of NF-κB activation by adenoviral transduction of IκB “superrepressor” also sensitized insulinoma cells and primary islet β-cells to TNF-α-induced apoptosis. These results suggest the protective role of NF-κB activation against cytokine-mediated pancreatic β-cell death, contrary to previous reports implicating NF-κB as a mediator of pancreatic islet cell death.
Although apoptosis of pancreatic β-cells is a critical step in the development of type 1 diabetes (1,2), it has not yet been clearly elucidated which molecules are the real effectors of pancreatic β-cell death. We have recently published in vitro and in vivo data suggesting that γ-interferon (IFN-γ) and tumor necrosis factor (TNF)-α synergism is responsible for apoptosis of pancreatic β-cells (3). IFN-γ seems to sensitize otherwise resistant pancreatic islet cells to TNF-α-mediated apoptosis, and TNF-α is thought to exert the final apoptosis on pancreatic islet cells. The role of TNF-α as the final death effector molecule is consistent with other studies that use genetic ablation models (4,5). However, other data showing the opposite effect of TNF-α in autoimmune diabetes have been published, reflecting the complexity of the pathogenesis and probably different role of cytokines in the different stages of the disease progression (6,7).
Although TNF-α is one of the most important death effector molecules, most primary or immortalized cells are not susceptible to apoptosis by TNF-α alone because of the concomitant activation of the antiapoptotic process by TNF-α (8–10). Many studies have implicated nuclear factor (NF)-κB as an important player in the protection of target cells against TNF-α-induced apoptosis (11–13). However, other studies have reported increased cell death by NF-κB activation using neuronal cells, pancreatic islet cells/insulinoma cells, or others (14–20), reflecting a complex interplay of cytokines and transcriptional factors that could be different according to the cell types or modes of cell death.
This investigation was carried out to determine the role of NF-κB activation in cytokine-induced pancreatic β-cell death. We found evidence supporting the role of NF-κB in the protection of pancreatic β-cells against TNF-α-induced apoptosis.ope
MicroRNA-34a suppresses malignant transformation by targeting c-Myc transcriptional complexes in human renal cell carcinoma
No full textWe investigated the functional effects of microRNA-34a (miR-34a) on c-Myc transcriptional complexes in renal cell carcinoma. miR-34a down-regulated expression of multiple oncogenes including c-Myc by targeting its 3′ untranslated region, which was revealed by luciferase reporter assays. miR-34a was also found to repress RhoA expression by suppressing the c-Myc–Skp2–Miz1 transcriptional complex that activates RhoA. Overexpression of c-Myc reversed miR-34a suppression of RhoA expression and inhibition of cell invasion, suggesting that miR-34a inhibits invasion by suppressing RhoA through c-Myc. miR-34a was also found to repress the c-Myc–P-TEFb transcription elongation complex, indicating one of the mechanisms by which miR-34a has profound effects on cellular functions. Our results demonstrate that miR-34a suppresses assembly and function of the c-Myc complex that activates or elongates transcription, indicating a novel role of miR-34a in the regulation of transcription by c-Myc.ope
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