Eun Soo Han

The in vivo Gene Expression Signature of Oxidative Stress

How higher organisms respond to elevated oxidative stress in vivo is poorly understood. Therefore, we measured oxidative stress parameters and gene expression alterations (Affymetrix arrays) in the liver caused by elevated reactive oxygen species induced in vivo by diquat or by genetic ablation of the major antioxidant enzymes CuZn-superoxide dismutase (Sod1) and glutathione peroxidase-1 (Gpx1). Diquat (50 mg/kg) treatment resulted in a significant increase in oxidative damage within 3-6 h in wild-type mice without any lethality. In contrast, treatment of Sod1(-/-) or Gpx1(-/-) mice with a similar concentration of diquat resulted in a significant increase in oxidative damage within an hour of treatment and was lethal, i.e., these mice are extremely sensitive to the oxidative stress generated by diquat. The expression response to elevated oxidative stress in vivo does not involve an upregulation of classic antioxidant genes, although long-term oxidative stress in Sod1(-/-) mice leads to a significant upregulation of thiol antioxidants (e.g., Mt1, Srxn1, Gclc, Txnrd1), which appears to be mediated by the redox-sensitive transcription factor Nrf2. The main finding of our study is that the common response to elevated oxidative stress with diquat treatment in wild-type, Gpx1(-/-), and Sod1(-/-) mice and in untreated Sod1(-/-) mice is an upregulation of p53 target genes (p21, Gdf15, Plk3, Atf3, Trp53inp1, Ddit4, Gadd45a, Btg2, Ndrg1). A retrospective comparison with previous studies shows that induction of these p53 target genes is a conserved expression response to oxidative stress, in vivo and in vitro, in different species and different cells/organs.

cDNA expression arrays reveal incomplete reversal of age-related changes in gene expression by calorie restriction.

Calorie restriction (CR) extends life span and retards many age-related cellular and molecular changes in laboratory rodents. However, neither the breadth of its effects, its underlying mechanisms, nor the limits of its action is fully understood. Expression levels of 588 genes in livers from 3- and 24-month-old ad libitum-fed (AL), and 24-month-old CR (60% of AL intake) male C57BL/6J mice (four per group) were measured. Six genes met the statistical criteria for differential expression in old AL compared to young AL mice. Only one of these age-related changes was attenuated by CR. Four additional gene products, that did not change with age in AL mice, were differentially expressed in old CR compared to old AL mice. Northern and RT-PCR analyses confirmed differential expression of four of the six candidate genes identified by the array results. Many of the identified genes have not previously been reported to be affected by CR or aging. Some of the age-related changes in gene expression are consistent with an increased vulnerability of the aged liver to carcinogenic or other insults, with only partial protection against insult by CR. Incomplete reversal by CR of age-related changes in gene expression provides a potentially important path for probing the limits of CR action. These results also show the importance of independent confirmation in expression array profiling of age-related changes in gene expression.

Meta-analysis of gene expression in the mouse liver reveals biomarkers associated with inflammation increased early during aging☆

Aging is associated with a loss of cellular homeostasis, a decline in physiological function and an increase in various pathologies. Employing a meta-analysis, hepatic gene expression profiles from four independent mouse aging studies were interrogated. There was little overlap in the number of genes or canonical pathways perturbed, suggesting that independent study-specific factors may play a significant role in determining age-dependent gene expression. However, 43 genes were consistently altered during aging in three or four of these studies, including those that (1) exhibited progressively increased expression starting from 12 months of age, (2) exhibited similar expression changes in models of progeria at young ages and dampened or no changes in old longevity mouse models, (3) were associated with inflammatory tertiary lymphoid neogenesis (TLN) associated with formation of ectopic lymphoid structures observed in chronically inflamed tissues, and (4) overlapped with genes perturbed by aging in brain, muscle, and lung. Surprisingly, around half of the genes altered by aging in wild-type mice exhibited similar expression changes in adult long-lived mice compared to wild-type controls, including those associated with intermediary metabolism and feminization of the male-dependent gene expression pattern. Genes unique to aging in wild-type mice included those linked to TLN.

Disruption of thymopoiesis in ST6Gal I-deficient mice

Thymocyte development is accompanied by sequential changes in cell surface glycosylation. For example, medullary thymocytes have increased levels of alpha2,3-linked sialic acid and a loss of asialo core 1 O-glycans as compared to cortical thymocytes. Some of these changes have been linked to fine tuning of the T cell receptor avidity. We analyzed ST6Gal I transcript abundance and levels of alpha2,6-linked sialic acid across thymocyte subsets. We found that ST6Gal I transcript levels increased following T cell receptor beta-selection suggesting that this sialyltransferase may influence the development of early thymocyte populations. Indeed, low levels of alpha2,6-linked sialic acid were found in the earliest T lineage cells, and then increased in T cell receptor beta-selected cells. To determine whether ST6Gal I influences T cell development, we analyzed ST6Gal I-deficient mice for disruptions in thymocyte populations. We found reduced thymic cellularity in the ST6Gal I-deficient mice starting in the early thymocyte compartments.

Assignment of tyrosine-specific T-cell phosphatase to conserved syntenic groups on human chromosome 18 and mouse chromosome 18

Phosphorylation of proteins on tyrosine is crucially involved in signal transduction and mitogenesis and is regulated by both kinases and phosphatases. Recently, a number of soluble and transmembrane receptor-linked protein tyrosine phosphatases (PTPase) have been characterized. Among these is a 48.4-kDa PTPase encoded by a cDNA isolated from a T-lymphocyte library by low-stringency screening with probes derived from placental PTPase 1B. A human T-cell PTPase (PTPT) cDNA and somatic cell hybrids were used to assign a PTPT gene to conserved syntentic groups on human chromosome 18 and on mouse chromosome 18. Two unlinked sequences, one on human chromosome 1, were also detected.

Food Restriction Differentially Affects mRNAs Encoding the Major Anterior Pituitary Tropic Hormones

Abstract Chronic food restriction (FR) leads to adaptive cellular changes, some of which retard aging. Moreover, some of these changes occur within weeks after onset of FR. Because neuroendocrine mechanisms may mediate these effects, we measured the effect of FR on the messenger ribonucleicacids (mRNAs) encoding all of the tropic hormones of the anterior pituitary (AP). Slot blot and solution hybridization were conducted on AP ribonucleicacid (RNA) samples obtained at 0500 h (AM) and 1500 h (PM) from 3-month-old male Fischer 344 rats fed ad libitum (AL) or FR (60% of AL calories) since 6 weeks of age. Poly A RNA/μg total RNA was similar in AL and FR rats, indicating that there was no overall effect of FR on mRNA levels. The level of proopiomelanocortin (POMC) mRNA was not reduced by FR when expressed per fig of RNA or as total AP content. By contrast, the total AP content of the mRNAs encoding LHβ, FSHβ TSHβ, GH, and PRL was markedly reduced by FR. When expressed per fig of RNA, however, only GH (AM and PM), FSHβ (AM), TSHbeta; (PM), and PRL (PM) were reduced by FR. These results reveal that FR differentially affects pituitary tropic hormone mRNA levels within weeks after onset of FR, and are consistent with a role for neuroendocrine alterations in the initiation of adaptive cellular responses to FR.

Mouse melanoma growth stimulatory activity gene (Mgsa) is polymorphic and syntenic with the W, patch, Rumpwhite, and recessive spotting loci on chromosome 5

Melanoma growth stimulatory activity (Mgsa) is a polypeptide growth factor originally detected in culture medium of the human malignant melanoma cell line Hs294T and may have an autocrine role in neoplastic growth. Mgsa is a member of the small inducible gene (SIG) family and shares homology with beta-thromboglobulin and platelet factor 4. Mgsa was localized to chromosome 5 using a cDNA probe for mouse Mgsa and somatic cell hybrids and is thus syntenic with Kit (W), Ph, Rw, and rs loci. The results eliminate Mgsa as the product of the Steel locus on chromosome 10, but raise the possibility that Mgsa might be synonymous with a chromosome 5 locus affecting skin pigmentation.

Sigje, a member of the small inducible gene family that includes platelet factor 4 and melanoma growth stimulatory activity, is on mouse Chromosome 11

Stiles and coworkers originally identified a gene they termed JE that is transcriptionally activated in mouse fibroblasts early after treatment with platelet derived growth factor or serum. This gene, now named Sigje, can encode a 148-amino acid secreted, basic polypeptide that belongs to the small inducible gene (SIG) family whose members include, for example, platelet factor 4, melanoma growth stimulatory activity (Mgsa), and interferon inducible protein 10. SIG family members share a conserved array of cysteine and proline residues and a similar predicted secondary structure, and may have evolved from a common ancestral gene. Several members of the SIG family have been assigned to the proximal long arm of human chromosome 4, to a region that has genetic homoeology with a portion of mouse Chromosome 5. We report here that mouse Sigje, in contrast to Mgsa, is on Chromosome 11. Sigje restriction fragment length polymorphisms in Mus spretus DNA were identified with the enzymes TaqI, MspI, BclI, and XbaI, and will be useful in mapping by meiotic recombination.