The Ink4a/Arf locus encodes 2 tumor suppressor molecules, p16INK4a and Arf, which are principal mediators of cellular senescence. To study the links between senescence and aging in vivo, we examined Ink4a/Arf expression in rodent models of aging. We show that expression of p16INK4a and Arf markedly increases in almost all rodent tissues with advancing age, while there is little or no change in the expression of other related cell cycle inhibitors. The increase in expression is restricted to well-defined compartments within each organ studied and occurs in both epithelial and stromal cells of diverse lineages. The age-associated increase in expression of p16INK4a and Arf is attenuated in the kidney, ovary, and heart by caloric restriction, and this decrease correlates with diminished expression of an in vivo marker of senescence, as well as decreased pathology of those organs. Last, the age-related increase in Ink4a/Arf expression can be independently attributed to the expression of Ets-1, a known p16INK4a transcriptional activator, as well as unknown Ink4a/Arf coregulatory molecules. These data suggest that expression of the Ink4a/Arf tumor suppressor locus is a robust biomarker, and possible effector, of mammalian aging.
Janakiraman Krishnamurthy, Chad Torrice, Matthew R. Ramsey, Grigoriy I. Kovalev, Khalid Al-Regaiey, Lishan Su, Norman E. Sharpless
The enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) is selectively expressed in aldosterone target tissues, where it confers aldosterone selectivity for the mineralocorticoid receptor by inactivating 11β-hydroxyglucocorticoids. Variable activity of 11βHSD2 is relevant for blood pressure control and hypertension. The present investigation aimed to elucidate whether an epigenetic mechanism, DNA methylation, accounts for the rigorous control of expression of the gene encoding 11βHSD2, HSD11B2. CpG islands covering the promoter and exon 1 of HSD11B2 were found to be densely methylated in tissues and cell lines with low expression but not those with high expression of HSD11B2. Demethylation induced by 5-aza-2′-deoxycytidine and procainamide enhanced the transcription and activity of the 11βHSD2 enzyme in human cells in vitro and in rats in vivo. Methylation of HSD11B2 promoter–luciferase constructs decreased transcriptional activity. Methylation of recognition sequences of transcription factors, including those for Sp1/Sp3, Arnt, and nuclear factor 1 (NF1) diminished their DNA-binding activity. Herein NF1 was identified as a strong HSD11B2 stimulatory factor. The effect of NF1 was dependent on the position of CpGs and the combination of CpGs methylated. A methylated-CpG–binding protein complex 1 transcriptional repression interacted directly with the methylated HSD11B2 promoter. These results indicate a role for DNA methylation in HSD11B2 gene repression and suggest an epigenetic mechanism affecting this gene causally linked with hypertension.
Rasoul Alikhani-Koopaei, Fatemeh Fouladkou, Felix J. Frey, Brigitte M. Frey
How ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) lower plasma lipid levels is incompletely understood. We previously showed that marine ω-3 PUFAs (docosahexaenoic acid [DHA] and eicosapentaenoic acid) stimulate a novel pathway, post-ER presecretory proteolysis (PERPP), that degrades apolipoprotein B100 (ApoB100), thereby reducing lipoprotein secretion from liver cells. To identify signals stimulating PERPP, we examined known actions of ω-3 PUFA. In rat hepatoma or primary rodent hepatocytes incubated with ω-3 PUFA, cotreatment with the iron chelator desferrioxamine, an inhibitor of iron-dependent lipid peroxidation, or vitamin E, a lipid antioxidant, suppressed increases in thiobarbituric acid–reactive substances (TBARSs; a measure of lipid peroxidation products) and restored ApoB100 recovery and VLDL secretion. Moreover, ω-6 and nonmarine ω-3 PUFA, also prone to peroxidation, increased ApoB100 degradation via intracellular induction of TBARSs. Even without added fatty acids, degradation of ApoB100 in primary hepatocytes was blocked by desferrioxamine or antioxidant cotreatment. To extend these results in vivo, mice were infused with DHA, which increased hepatic TBARSs and reduced VLDL-ApoB100 secretion. These results establish a novel link between lipid peroxidation and oxidant stress with ApoB100 degradation via PERPP, and may be relevant to the hypolipidemic actions of dietary PUFAs, the basal regulation of ApoB100 secretion, and hyperlipidemias arising from ApoB100 overproduction.
Meihui Pan, Arthur I. Cederbaum, Yuan-Li Zhang, Henry N. Ginsberg, Kevin Jon Williams, Edward A. Fisher
Here we describe the effect of immunization with dendritic cells loaded with syngeneic tumor cells (DC/Ts) by polyethylene glycol treatment, on tumor development in adenomatous polyposis coli (APC) gene mutant mouse models, APC1309 and APCMin–/+, in which adenomatous polyps of the gastrointestinal tracts develop with a high incidence. Treatment with DC/Ts prevented the development of gastrointestinal tumors, and coadministration of DC/Ts and IL-12 caused a further reduction in tumor incidence. Splenocytes from APC1309 mice treated with DC/Ts and IL-12 showed no cytotoxic activity toward the tumor cells, but serum antibody specific to them was detected. IgG from the treated mice exhibited cytotoxic activity against the tumor cells in vitro. Predominance of Th2 cell response over Th1 response was also suggested by ELISPOT assays in the treated mice. Depletion in vivo of CD4+ T cells, not CD8+ T cells, by the intraperitoneal administration of corresponding mAb’s decreased the antitumor effect of DC/T inoculation. Immunofluorescence microscopic studies showed that Ig was attached to tumor cells in mice treated with DC/Ts and IL-12. These findings indicate that DC/T vaccination prevents tumor development through APC gene mutation and that its preventive effects are mediated by humoral antitumor immunity.
Toshio Iinuma, Sadamu Homma, Tetsuo Noda, Donald Kufe, Tsuneya Ohno, Gotaro Toda
Recent reports of tumor regression following delivery of autologous tumor antigen–pulsed DCs suggest that defective antigen presentation may play a key role in tumor escape. Here we show in two different murine tumor models, CT26 (colon adenocarcinoma) and B16 (melanoma), that the number and activation state of intratumoral DCs are critical factors in the host response to tumors. We used CCL20/macrophage inflammatory protein-3α (MIP-3α) chemokine to increase the number of tumoral DCs and intratumoral injections of CG-rich motifs (CpGs) to activate such cells. Expression of CCL20 in the tumor site attracted large numbers of circulating DCs into the tumor mass and, in the case of CT26 tumors, led to complete tumor regression. Intratumoral CpG injections, in addition to CCL20, were required to induce therapeutic immunity against B16 tumors. In this model CpG overcame tumor-mediated inhibition of DC activation and enabled tumoral DCs to cross-present tumor antigens to naive CD8 T cells. CpG activation of tumoral DCs alone was not sufficient to induce tumor regression in either tumor model, nor was systemic delivery of the DC growth factor, Flt3 ligand, which dramatically increased the number of circulating DCs but not the number of tumoral DCs. These results indicate that the number of tumoral DCs as well as the tumor milieu determines the ability of tumor-bearing hosts to mount an effective antitumor immune response. Our results also suggest that DCs can be manipulated in vivo without delivery of defined tumor antigens to induce a specific T cell–mediated antitumor response and provide the basis for the use of chemokines in DC-targeted clinical strategies.
Katsuyoshi Furumoto, Luis Soares, Edgar G. Engleman, Miriam Merad
Cellular acquisition of folate is mediated by folate receptors (FRs) in many malignant and normal human cells. Although FRs are upregulated in folate deficiency and downregulated following folate repletion, the mechanistic basis for this relationship is unclear. Previously we demonstrated that interaction of an 18-base cis-element in the 5′-untranslated region of FR mRNA and a cystolic trans-factor (heterogeneous nuclear ribonucleoprotein E1 [hnRNP E1]) is critical for FR synthesis. However, the molecular mechanisms controlling this interaction, especially within the context of FR regulation and folate status, have remained obscure. Human cervical carcinoma cells exhibited progressively increasing upregulation of FRs after shifting of folate-replete cells to low-folate media, without a proportionate rise in FR mRNA or rise in hnRNP E1. Translational FR upregulation was accompanied by a progressive accumulation of the metabolite homocysteine within cultured cells, which stimulated interaction of the FR mRNA cis-element and hnRNP E1 as well as FR biosynthesis in a dose-dependent manner. Abrupt reversal of folate deficiency also led to a rapid parallel reduction in homocysteine and FR biosynthesis to levels observed in folate-replete cells. Collectively, these results suggest that homocysteine is the key modulator of translational upregulation of FRs and establishes the linkage between perturbed folate metabolism and coordinated upregulation of FRs.
Aśok C. Antony, Ying-Sheng Tang, Rehana A. Khan, Mangatt P. Biju, Xiangli Xiao, Qing-Jun Li, Xin-Lai Sun, Hiremagalur N. Jayaram, Sally P. Stabler
Viruses can cause but can also prevent autoimmune disease. This dualism has certainly hampered attempts to establish a causal relationship between viral infections and type 1 diabetes (T1D). To develop a better mechanistic understanding of how viruses can influence the development of autoimmune disease, we exposed prediabetic mice to various viral infections. We used the well-established NOD and transgenic RIP-LCMV models of autoimmune diabetes. In both cases, infection with the lymphocytic choriomeningitis virus (LCMV) completely abrogated the diabetic process. Interestingly, such therapeutic viral infections resulted in a rapid recruitment of T lymphocytes from the islet infiltrate to the pancreatic draining lymph node, where increased apoptosis was occurring. In both models this was associated with a selective and extensive expression of the chemokine IP-10 (CXCL10), which predominantly attracts activated T lymphocytes, in the pancreatic draining lymph node, and in RIP-LCMV mice it depended on the viral antigenic load. In RIP-LCMV mice, blockade of TNF-α or IFN-γ in vivo abolished the prevention of T1D. Thus, virally induced proinflammatory cytokines and chemokines can influence the ongoing autoaggressive process beneficially at the preclinical stage, if produced at the correct location, time, and levels.
Urs Christen, Dirk Benke, Tom Wolfe, Evelyn Rodrigo, Antje Rhode, Anna C. Hughes, Michael B.A. Oldstone, Matthias G. von Herrath
Liver IGF-1–deficient (LID) mice have a 75% reduction in circulating IGF-1 levels and, as a result, a fourfold increase in growth hormone (GH) secretion. To block GH action, LID mice were crossed with GH antagonist (GHa) transgenic mice. Inactivation of GH action in the resulting LID + GHa mice led to decreased blood glucose and insulin levels and improved peripheral insulin sensitivity. Hyperinsulinemic-euglycemic clamp studies showed that LID mice exhibit severe insulin resistance. In contrast, expression of the GH antagonist transgene in LID + GHa mice led to enhanced insulin sensitivity and increased insulin-stimulated glucose uptake in muscle and white adipose tissue. Interestingly, LID + GHa mice exhibit a twofold increase in white adipose tissue mass, as well as increased levels of serum-free fatty acids and triglycerides, but no increase in the triglyceride content of liver and muscle. In conclusion, these results show that despite low levels of circulating IGF-1, insulin sensitivity in LID mice could be improved by inactivating GH action, suggesting that chronic elevation of GH levels plays a major role in insulin resistance. These results suggest that IGF-1 plays a role in maintaining a fine balance between GH and insulin to promote normal carbohydrate and lipid metabolism.
Shoshana Yakar, Jennifer Setser, Hong Zhao, Bethel Stannard, Martin Haluzik, Vaida Glatt, Mary L Bouxsein, John J. Kopchick, Derek LeRoith
In our previous genome-wide scan of Finnish nuclear families, obesity was linked to chromosome Xq24. Here we analyzed this 15-Mb region by genotyping 9 microsatellite markers and 36 single nucleotide polymorphisms (SNPs) for 11 positional and functional candidate genes in an extended sample of 218 obese Finnish sibling pairs (sibpairs) (BMI > 30 kg/m2). Evidence of linkage emerged mainly from the obese male sibpairs, suggesting a gender-specific effect for the underlying gene. By constructing haplotypes among the obese male sibpairs, we restricted the region from 15 Mb to 4 Mb, between markers DXS8088 and DXS8067. Regional functional candidate genes were tested for association in an initial sample of 117 cases and 182 controls. Significant evidence was observed for association for an SNP in the 3′-untranslated region of the solute carrier family 6 member 14 (SLC6A14) gene (P = 0.0002) and for SNP haplotypes of the SLC6A14 gene (P = 0.0007–0.006). Furthermore, an independent replication study sample of 837 cases and 968 controls from Finland and Sweden also showed significant differences in allele frequencies between obese and non-obese individuals (P = 0.003). The SLC6A14 gene is an interesting novel candidate for obesity because it encodes an amino acid transporter, which potentially regulates tryptophan availability for serotonin synthesis and thus possibly affects appetite control.
Elina Suviolahti, Laura J. Oksanen, Miina Öhman, Rita M. Cantor, Martin Ridderstråle, Tiinamaija Tuomi, Jaakko Kaprio, Aila Rissanen, Pertti Mustajoki, Pekka Jousilahti, Erkki Vartiainen, Kaisa Silander, Riika Kilpikari, Veikko Salomaa, Leif Groop, Kimmo Kontula, Leena Peltonen, Päivi Pajukanta
We have previously described the only reported case of human proprotein convertase 1 (PC1) deficiency, in a female (Subject A) with obesity, hypogonadism, hypoadrenalism, and reactive hypoglycemia. We now report the second case of human PC1 deficiency (Subject B), also due to compound heterozygosity for novel missense and nonsense mutations. While both subjects shared the phenotypes of obesity, hypoadrenalism, reactive hypoglycemia, and elevated circulating levels of certain prohormones, the clinical presentation of Subject B was dominated by severe refractory neonatal diarrhea, malabsorptive in type. Subsequent investigation of Subject A revealed marked small-intestinal absorptive dysfunction, which was not previously clinically suspected. We postulate that PC1, presumably in the enteroendocrine cells, is essential for the normal absorptive function of the human small intestine. The differences in the nature and severity of presentation between the two cases cannot readily be explained on the basis of allelic heterogeneity, as the nonsense and missense mutations from both subjects had comparably severe effects on the catalytic activity of PC1. Despite Subject A’s negligible PC1 activity, some mature ACTH and glucagon-like peptide 17-36amide were detectable in her plasma, suggesting that the production of these hormones, at least in humans, does not have an absolute dependence on PC1. The presence of severe obesity and the absence of growth retardation in both subjects contrast markedly with the phenotype of mice lacking PC1 and suggest that the precise physiological repertoire of this enzyme may vary between mammalian species.
Robert S. Jackson, John W.M. Creemers, I. Sadaf Farooqi, Marie-Laure Raffin-Sanson, Andrea Varro, Graham J. Dockray, Jens J. Holst, Patricia L. Brubaker, Pierre Corvol, Kenneth S. Polonsky, Diane Ostrega, Kenneth L. Becker, Xavier Bertagna, John C. Hutton, Anne White, Mehul T. Dattani, Khalid Hussain, Stephen J. Middleton, Thomasina M. Nicole, Peter J. Milla, Keith J. Lindley, Stephen O’Rahilly
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