Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that is activated by cellular stresses, such as oxidative compounds. After activation, Nrf2 induces transcription of its target genes, many of which have cytoprotective functions. Previously, we have shown that activation of Nrf2 by tert-butylhydroquinone (tBHQ) skews murine CD4⁺ T-cell differentiation. Although the role of Nrf2 in murine T cells is somewhat characterized, it is largely uncharacterized in human T cells. Therefore, the aim of the current studies was to characterize the effects of the Nrf2 activator, tBHQ, on the early events of human CD4⁺ T-cell activation. Pretreatment of Jurkat T cells with tBHQ, prior to activation with anti-CD3/anti-CD28, diminished the production of interleukin-2 (IL-2) at both the transcript and protein levels. Similarly, the expression of CD25 also diminished, albeit to a lesser degree than IL-2, after pretreatment with tBHQ. The decrease in IL-2 production was not due to decreased nuclear translocation of c-fos or c-jun. Although tBHQ caused both a delay and a decrease in Ca²⁺ influx in activated Jurkat cells, no decrease in nuclear factor of activated T cells (NFAT) DNA binding or transcriptional activity was observed. In contrast to NFAT, tBHQ significantly decreased NFκB transcriptional activity. Collectively, our studies show that the Nrf2 activator, tBHQ, inhibits IL-2 and CD25 expression, which correlates with decreased NFκB transcriptional activity in activated Jurkat cells. Overall, our studies suggest that Nrf2 represents a novel mechanism for the regulation of both human and mouse T cell function.
During the dry period between successive lactations, the mammary gland of dairy cows undergoes extensive remodeling that is marked by phases of involution and mammogenesis. Changes in the mammary epithelium during the dry period have been well characterized; however, few studies have examined the changes that occur in stromal tissue. The objective of this study was to characterize changes that occur in mammary stroma during the dry period. Mammary biopsies were taken from 9 multigravid Holstein cows in late lactation, at 1 wk after dry-off, 3 wk before expected calving date, and 1 wk before expected calving date. Tissue was fixed in formalin, embedded in paraffin, and cut into 5-mum sections. Sections were stained with hematoxylin and eosin or with immunohistochemistry for expression of smooth muscle alpha actin (SMA), fibronectin, stromelysin-1 (MMP-3), transforming growth factor-beta1 (TGF-beta1), and TGF-beta receptor 2 (TGF-betaR2). Images of tissues were captured with light microscopy, and imaging software was used to measure intralobular stromal area, number of activated fibroblasts, as identified by expression of SMA, and percentage of intralobular stromal area expressing fibronectin, MMP3, TGF-beta1, and TGF-betaR2. Analyses of variance were conducted and statistical differences were based on the least squares means of biopsy stage. Number of activated fibroblasts was greater at 1 wk dry than at 1 wk before calving (2,720 vs. 1,800 cells/mm(2)), percentage intralobular stromal area was greater at 1 wk dry (32%) and 3 wk before calving (37%) than at 1 wk before calving (25%), and TGF-beta1 expression decreased 15% from late lactation to the dry period. The percentages of stromal area expressing fibronectin, MMP-3, and TGF-betaR2 and the percentage of myofibroblasts were not different across biopsy stages. These results support the concept that stromal expression of transforming growth factor-beta1 and fibroblast proliferation may be important for remodeling during the dry period.
Tissue-specific shifts in a dam's metabolism to support fetal and neonatal growth during pregnancy and lactation are controlled by differential expression of regulatory genes. The goal of this study was to identify a more detailed cohort of genes in mammary, liver, and adipose tissue that are transcriptionally controlled during the pregnancy to lactation evolution and explore the relationship of these genes to core clock genes. Total RNA was isolated from mammary, liver and adipose tissues collected from rat dams on day 20 of pregnancy (P20) and day 1 of lactation (L1) and gene expression was measured using Rat 230 2.0 Affymetrix GeneChips. Gene functional analysis revealed that pathway associated metabolism (carbohydrate, amino acid, lipid, cholesterol, protein) were enriched (P < 0.001) in the mammary gland during P20 to L1 transition. Approximately 50% of the genes associated with solute transport, as well as lipogenesis were up-regulated in the mammary gland during P20 to L1 transition compared to 10% in liver and 15% in adipose tissue. Genes engaged in conveying glucose (INSR, GLUT1, GLUT4, SGLT1, and SGLT2), bicarbonate (SLC4), sodium (SLC9), zinc (SLC30), copper (SLC31), iron (SLC40) in tandem with rate-limiting lipogenic genes (ACACA, FASN, PRLR, SREBP2, THRSP) were specifically enriched in the mammary gland during the P20 to L1 evolution. Our results provide insight into a cross-tissue transcriptional repertoire that is associated with homeorhetic adaptation needed to support lactation, and at the onset of lactation the mammary gland becomes a factory for macromolecular biosynthesis through inducing genes participating in nutrient transfer and lipid biosynthesis.
We previously demonstrated that activation of the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2) promotes CD4+ Th2 differentiation. In the current study, we assessed the role of Nrf2 in early events following T cell activation. The Nrf2 activators, tBHQ (tert-butylhydroquinone) and CDDO-Im (the imidazolide derivative of the triterpenoid CDDO), were used in conjunction with splenocytes derived from wild-type and Nrf2-null mice to distinguish between Nrf2-specific and off-target effects. CDDO-Im inhibited early IFNγ production in a largely Nrf2-dependent manner. In contrast, tBHQ and CDDO-Im had little effect on expression of CD25 or CD69. Furthermore, tBHQ inhibited GM-CSF and IL-2 production in both wild-type and Nrf2-null T cells, suggesting this effect is Nrf2-independent. Conversely, CDDO-Im caused a concentration-dependent increase in IL-2 secretion in wild-type, but not Nrf2-null, splenocytes, suggesting that Nrf2 promotes IL-2 production. Interestingly, both compounds inhibit NFκB DNA binding, where the suppression by tBHQ is Nrf2-independent and CDDO-Im is Nrf2-dependent. Surprisingly, as compared to wild-type splenocytes, Nrf2-null splenocytes showed lower nuclear accumulation of c-Jun, a member of the AP-1 family of transcription factors, which have been shown to drive multiple immune genes, including IL-2. Both Nrf2 activators caused a Nrf2-dependent trend toward increased nuclear accumulation of c-Jun. These data suggest that modulation of cytokine secretion by tBHQ likely involves multiple pathways, including AP-1, NFκB, and Nrf2. Overall, the data suggest that Nrf2 activation inhibits secretion of the Th1 cytokine IFNγ, and increases early production of IL-2, which has been shown to promote Th2 differentiation, and may support the later occurrence of Th2 polarization.
Genes associated with lactation evolved more slowly than other genes in the mammalian genome. Higher conservation of milk and mammary genes suggest that species variation in milk composition is due in part to the environment and that we must look deeper into the genome for regulation of lactation. At the onset of lactation, metabolic changes are coordinated among multiple tissues through the endocrine system to accommodate the increased demand for nutrients and energy while allowing the animal to remain in homeostasis. This process is known as homeorhesis. Homeorhetic adaptation to lactation has been extensively described; however how these adaptations are orchestrated among multiple tissues remains elusive. To develop a clearer picture of how gene expression is coordinated across multiple tissues during the pregnancy to lactation transition, total RNA was isolated from mammary, liver and adipose tissues collected from rat dams (n = 5) on day 20 of pregnancy and day 1 of lactation, and gene expression was measured using Affymetrix GeneChips. Two types of gene expression analysis were performed. Genes that were differentially expressed between days within a tissue were identified with linear regression, and univariate regression was used to identify genes commonly up-regulated and down-regulated across all tissues. Gene set enrichment analysis showed genes commonly up regulated among the three tissues enriched gene ontologies primary metabolic processes, macromolecular complex assembly and negative regulation of apoptosis ontologies. Genes enriched in transcription regulator activity showed the common up regulation of 2 core molecular clock genes, ARNTL and CLOCK. Commonly down regulated genes enriched Rhythmic process and included: NR1D1, DBP, BHLHB2, OPN4, and HTR7, which regulate intracellular circadian rhythms. Changes in mammary, liver and adipose transcriptomes at the onset of lactation illustrate the complexity of homeorhetic adaptations and suggest that these changes are coordinated through molecular clocks.
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