Dietary Regulation of Adult Stem Cells.

PURPOSE OF REVIEW: Dietary intake is a critical regulator of organismal physiology and health. Tissue homeostasis and regeneration are dependent on adult tissue stem cells that self-renew and differentiate into the specialized cell types. As stem cells respond to cues from their environment, dietary signals and nutrients influence tissue biology by altering the function and activity of adult stem cells. In this review, we highlight recent studies that illustrate how diverse diets such as caloric restriction, fasting, high fat diets, and ketogenic diets impact stem cell function and their microenvironments. RECENT FINDINGS: Caloric restriction generally exerts positive effects on adult stem cells, notably increasing stem cell functionality in the intestine and skeletal muscle as well as increasing hematopoietic stem cell quiescence. Similarly, fasting confers protection of intestinal, hematopoietic, and neuronal stem cells against injury. High fat diets induce intestinal stem cell niche independence and stem-like properties in intestinal progenitors, while high fat diets impair hematopoiesis and neurogenesis. SUMMARY: Caloric restriction and fasting are generally beneficial to adult stem cell function, while high fat diets impair stem cell function or create opportunities for tumorigenesis. However, the effects of each diet on stem cell biology are complex and vary greatly between tissues. Given the recent interest in developing dietary interventions or mimetics as therapeutics, further studies, including on ketogenic diets, will be essential to understand how adult stem cells respond to diet-induced signals and physiology.

Developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters DNA methyltransferase (dnmt) expression in zebrafish (Danio rerio).

DNA methylation is one of the most important epigenetic modifications involved in the regulation of gene expression. The DNA methylation reaction is catalyzed by DNA methyltransferases (DNMTs). Recent studies have demonstrated that toxicants can affect normal development by altering DNA methylation patterns, but the mechanisms of action are poorly understood. Hence, we tested the hypothesis that developmental exposure to TCDD affects dnmt gene expression patterns. Zebrafish embryos were exposed to 5nM TCDD for 1h from 4 to 5h post-fertilization (hpf) and sampled at 12, 24, 48, 72, and 96 hpf to determine dnmt gene expression and DNA methylation patterns. We performed a detailed analysis of zebrafish dnmt gene expression during development and in adult tissues. Our results demonstrate that dnmt3b genes are highly expressed in early stages of development, and dnmt3a genes are more abundant in later stages. TCDD exposure upregulated dnmt1 and dnmt3b2 expression, whereas dnmt3a1, 3b1, and 3b4 are downregulated following exposure. We did not observe any TCDD-induced differences in global methylation or hydroxymethylation levels, but the promoter methylation of aryl hydrocarbon receptor (AHR) target genes was altered. In TCDD-exposed embryos, AHR repressor a (ahrra) and c-fos promoters were differentially methylated. To characterize the TCDD effects on DNMTs, we cloned the dnmt promoters with xenobiotic response elements and conducted AHR transactivation assays using a luciferase reporter system. Our results suggest that ahr2 can regulate dnmt3a1, dnmt3a2, and dnmt3b2 expression. Overall, we demonstrate that developmental exposure to TCDD alters dnmt expression and DNA methylation patterns.

Cigarette smoke activates NFκB-mediated Tnf-α release from mouse middle ear cells.

OBJECTIVES/HYPOTHESIS: : Cigarette smoke exposure is a significant risk factor in the development of otitis media. NF-κB is a transcription factor known to mediate cigarette smoke effects in multiple cell types. We hypothesized that stimulation of murine middle ear epithelial cells (MEEC) with cigarette smoke condensate (CSC) activates NF-κB resulting in upregulation of proinflammatory cytokines. STUDY DESIGN: : In vitro model of cultured murine middle ear epithelial cells. METHODS: : Time course CSC stimulation of MEEC was performed. Antibody microarrays were then utilized to simultaneously measure 40 inflammatory cytokines. Enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcriptase-polymerase chain reaction were performed to validate and further evaluate array results. Luciferase reporter assays were performed to evaluate NF-κB activation with CSC in MEEC. Chromatin immunoprecipitation (ChIP) assays were performed to determine whether CSC induces NF-κB interaction with the Tnf-α promoter. RESULTS: : Multiple cytokines showed significant increases with CSC exposure. ELISA studies demonstrated that Tnf-α secretion increased the most. CSC stimulation likewise increased Tnf-α mRNA abundance and induced promoter activity 4.8-fold in a Tnf-α reporter plasmid. Reporter assays demonstrated 4.84-fold activation of NF-κB with CSC. ChIP assays demonstrated NF-κB binding to canonical κB sites in the Tnf-α promoter with CSC stimulation. CONCLUSIONS: : CSC activates NF-κB in MEEC. Furthermore, this activation results in CSC induced Tnf-α promoter activation, gene expression, and levels in cell secretions. Laryngoscope, 120:2508-2515, 2010.

In vitro effects of acid and pepsin on mouse middle ear epithelial cell viability and MUC5B gene expression.

OBJECTIVE: To examine whether in vitro exposure of mouse middle ear epithelial cells (mMEECs) to conditions that mimic physiologic reflux upregulates Muc5b gene expression and alters cell viability. DESIGN: In vitro mMEEC model. SETTING: Center for Genetic Medicine Research, Children's National Medical Center, Washington, DC. PARTICIPANTS: Cells from the immortalized mMEEC line. MAIN OUTCOME MEASURES: Cell viability, the quantity of Muc5b messenger RNA abundance, and Muc5b promoter activity. RESULTS: The 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide assays demonstrated an acidic dose-dependent decrease in cell survival, with pH less than 4 significantly decreasing viability at 1 hour. Pepsin had a mild protective effect up to 8 hours, with greater cell viability, in the pH range of 5.0 to 7.6. Reverse-transcriptase polymerase chain reaction demonstrated induction of Muc5b messenger RNA levels over controls after exposure to acidic pH levels of 5.7, and 4 with and without pepsin. Similarly, a pH of 4.0 significantly increased Muc5b promoter activation 5.4-fold. Pepsin at neutral or acidic pH values did not significantly alter Muc5b expression or promoter activity. CONCLUSIONS: Despite decreasing cell viability, acidic pH drives middle ear epithelial Muc5b gene expression in vitro, which perhaps explains how laryngopharyngeal reflux can contribute to otitis media. Pepsin at neutral or acidic pH levels had minimal effects on Muc5b gene expression; thus, although pepsin may be a useful marker for detecting the presence of reflux, our results suggest that acid itself is a more likely pathologic component of gastric juice in the middle ear.

Development of an enantioselective synthetic route to neocarzinostatin chromophore and its use for multiple radioisotopic incorporation.

A convergent, enantioselective synthetic route to the natural product neocarzinostatin chromophore (1) is described. Synthesis of the chromophore aglycon (2) was targeted initially. Chemistry previously developed for the synthesis of a neocarzinostatin core model (4) failed in the requisite 1,3-transposition of an allylic silyl ether when applied toward the preparation of 2 with use of the more highly oxygenated substrates 27 and 54. An alternative synthetic plan was therefore developed, based upon a proposed reduction of the epoxy alcohol 58 to form the aglycon 2, a transformation that was achieved in a novel manner, using a combination of the reagents triphenylphosphine, iodine, and imidazole. The successful route to 1 and 2 began with the convergent coupling of the epoxydiyne 15, obtained in 9 steps (43% overall yield) from D-glyceraldehyde acetonide, and the cyclopentenone (+)-14, prepared in one step (75-85% yield) from the prostaglandin intermediate (+)-16, affording the alcohol 22 in 80% yield and with > or =20:1 diastereoselectivity. The alcohol 22 was then converted into the epoxy alcohol 58 in 17 steps with an average yield of 92% and an overall yield of 22%. Key features of this sequence include the diastereoselective Sharpless asymmetric epoxidation of allylic alcohol 81 (98% yield); intramolecular acetylide addition within the epoxy aldehyde 82, using Masamune's lithium diphenyltetramethyldisilazide base (85% yield); selective esterification of the diol 84 with the naphthoic acid 13 followed by selective cleavage of the chloroacetate protective group in situ to furnish the naphthoic acid ester 85 in 80% yield; and elimination of the tertiary hydroxyl group within intermediate 88 using the Martin sulfurane reagent (79% yield). Reductive transposition of the product epoxy alcohol (58) then formed neocarzinostatin chromophore aglycon (2, 71% yield). Studies directed toward the glycosylation of 2 focused initially on the preparation of the N-methylamino --> hydroxyl replacement analogue 3, an alpha-D-fucose derivative of neocarzinostatin chromophore, formed in 42% yield by a two-step Schmidt glycosylation-deprotection sequence. For the synthesis of 1, an extensive search for a suitable 2'-N-methylfucosamine glycosyl donor led to the discovery that the reaction of 2 with the trichloroacetimidate 108, containing a free N-methylamino group, formed the alpha-glycoside 114 selectively in the presence of boron trifluoride diethyl etherate. Subsequent deprotection of 114 under mildly acidic conditions then furnished the labile chromophore (1). The synthetic route was readily modified for the preparation of singly and doubly (3)H- and (14)C-labeled 1, compounds unavailable by other means, for studies of the mechanism of action of neocarzinostatin in vivo.