Michael Satre

Characterization of a Novel Airway Epithelial Cell-specific Short Chain Alcohol Dehydrogenase/Reductase Gene Whose Expression Is Up-regulated by Retinoids and Is Involved in the Metabolism of Retinol

Multiple retinoic acid responsive cDNAs were isolated from a high density cDNA microarray membrane, which was developed from a cDNA library of human tracheobronchial epithelial cells. Five selected cDNA clones encoded the sequence of the same novel gene. The predicted open reading frame of the novel gene encoded a protein of 319 amino acids. The deduced amino acid sequence contains four motifs that are conserved in the short-chain alcohol dehydrogenase/reductase (SDR) family of proteins. The novel gene shows the greatest homology to a group of dehydrogenases that can oxidize retinol (retinol dehydrogenases). The mRNA of the novel gene was found in trachea, colon, tongue, and esophagus. In situhybridization of airway tissue sections demonstrated epithelial cell-specific gene expression, especially in the ciliated cell type. Both all-trans-retinoic acid and 9-cis-retinoic acid were able to elevate the expression of the novel gene in primary human tracheobronchial epithelial cells in vitro. This elevation coincided with an enhanced retinol metabolism in these cultures. COS cells transfected with an expression construct of the novel gene were also elevated in the metabolism of retinol. The results suggested that the novel gene represents a new member of the SDR family that may play a critical role in retinol metabolism in airway epithelia as well as in other epithelia of colon, tongue, and esophagus.

Identification of transcriptional networks responding to pyrroloquinoline quinone dietary supplementation and their influence on thioredoxin expression, and the JAK/STAT and MAPK pathways.

PQQ (pyrroloquinoline quinone) improves energy utilization and reproductive performance when added to rodent diets devoid of PQQ. In the present paper we describe changes in gene expression patterns and transcriptional networks that respond to dietary PQQ restriction or pharmacological administration. Rats were fed diets either deficient in PQQ (PQQ−) or supplemented with PQQ (approx. 6 nmol of PQQ/g of food; PQQ+). In addition, groups of rats were either repleted by administering PQQ to PQQ− rats (1.5 mg of PQQ intraperitoneal/kg of body weight at 12 h intervals for 36 h; PQQ−/+) or partially depleted by feeding the PQQ− diet to PQQ+ rats for 48 h (PQQ+/−). RNA extracted from liver and a Codelink® UniSet Rat I Bioarray system were used to assess gene transcript expression. Of the approx. 10000 rat sequences and control probes analysed, 238 were altered at the P<0.01 level by feeding on the PQQ− diet for 10 weeks. Short-term PQQ depletion resulted in changes in 438 transcripts (P<0.01). PQQ repletion reversed the changes in transcript expression caused by PQQ deficiency and resulted in an alteration of 847 of the total transcripts examined (P<0.01). Genes important for cellular stress (e.g. thioredoxin), mitochondriogenesis, cell signalling [JAK (Janus kinase)/STAT (signal transducer and activator of transcription) and MAPK (mitogen-activated protein kinase) pathways] and transport were most affected. qRT-PCR (quantitative real-time PCR) and functional assays aided in validating such processes as principal targets. Collectively, the results provide a mechanistic basis for previous functional observations associated with PQQ deficiency or PQQ administered in pharmacological amounts.

Alterations in cellular retinol metabolism contribute to differential retinoid responsiveness in normal human mammary epithelial cells versus breast cancer cells

The present study was undertaken to compare ROH growth responsiveness between normal human mammary epithelial cells (HMECs), estrogen receptor positive (MCF-7) and negative (MDA-MB-231) breast cancer cells, and assess whether this responsiveness is correlated with differences in ROH metabolism, particularly RA synthesis. HMECs were markedly more growth sensitive to a physiological dose of ROH than breast cancer cells, exhibiting a significant decrease in cell number by 48 h and >70% decrease by 144 h. In comparison, numbers of MCF-7s were only decreased 32% by 144 h. MDA-MB-231 cells were not affected. However, HMECs and MCF-7 cells displayed similar growth responsiveness to 1 μM RA, while MDA-MB-231 cells were minimally affected. Although the initial rates and extent of ROH uptake were comparable among cell types, ROH levels in HMECs progressively decreased to 20% of the peak by 24 h and ≤10% by 72 h. In contrast, ROH levels in the cancer cells remained relatively constant through 48 h. The decrease in HMEC ROH was attributable to greater metabolism as evidenced by rapid and predominant retinyl ester formation. HMECs also produced ∼5 times more RA from ROH than MCF-7s and ∼10 times more than MDA MB-231 cells. Our results demonstrate that normal HMECs are markedly more responsive to the growth inhibitory effects of ROH than breast cancer cells, and that this responsiveness is associated with greater ROH metabolism including greater RA synthesis. These data suggest that altered ROH metabolism may be a factor in breast cancer progression.

Mouse retinol binding protein gene: Cloning, expression and regulation by retinoic acid

A full-length cDNA clone encoding the retinol binding protein (RBP) was isolated from a mouse liver cDNA library by hybridization screening. The nucleotide sequence of murine RBP is 85 and 95% homologous to that of human and rat RBP, respectively, with a deduced amino acid sequence ≥ 83% homologous to both species. Analysis of the tissue expression pattern of RBP mRNA in the female mouse indicated relatively abundant expression in the liver, with lesser amounts in extrahepatic tissues including adipose, kidney, spleen and uterus, suggesting that these tissues may have a significant role in retinol homeostasis. Mouse liver cell RBP regulation by retinoids was also investigated. Both all-trans retinoic acid (AT-RA) and 9-cis retinoic acid (9c-RA) induced RBP mRNA expression in a dose- and time-dependent manner. Maximal levels (up to 4-fold above controls) were observed at ≥ 48h following treatment of both mouse hepatoma cells in vitro and in vivo in mice receiving a single, oral dose of either retinoid. Interestingly, 9c-RA was more potent at RBP induction in both in vivo and in vitro systems. Given the extent and temporal pattern of RBP induction, we suggest that the RA-mediated increase in liver RBP is part of a cellular protection mechanism. Increased levels of RBP would facilitate sequestration and possibly cellular export of RA in cells receiving prolonged exposure to high levels of RA, thus minimizing toxicity.

Metabolic conversion of retinol to retinoic acid mediates the biological responsiveness of human mammary epithelial cells to retinol

The biological effects of vitamin A are mediated in part by retinoic acid (RA) modulation of gene transcription. In this study, we examined whether normal human mammary epithelial cells (HMECs) are biologically responsive to retinol (ROH), the metabolic precursor of RA. While both ROH and tRA resulted in time‐ and dose‐dependent decreases in total cell number, tRA was markedly more potent. Metabolically, treatment of HMECs with physiological doses of ROH resulted in rapid uptake and subsequent production of both retinyl esters and tRA. Although a comparatively minor metabolite, tRA levels peaked at 6 h and remained above endogenous levels for up to 72 h in proportion to cellular ROH concentrations. In HMECs transfected with an RA‐responsive luciferase reporter gene, treatment with 3 μM ROH resulted in an increase in luciferase activity to a level intermediate between that observed with 0.001 and 0.01 μM tRA. Citral, an RA‐synthesis inhibitor, was also used to examine the biological activity of ROH. Compared to ROH alone, ROH plus citral treatment resulted in three‐fold less tRA synthesis and a > 65% attentuation of RA‐responsive reporter gene activity which persisted through 72 h. Citral also significantly attenuated the extent of ROH‐mediated reductions in total HMEC number. Thus, treatment with physiological concentrations of ROH results in fewer total numbers of HMECs and this response is a consequence of cellular tRA synthesis which can induce RA‐responsive gene expression.

Retinol binding protein expression is induced in HepG2 cells by zinc deficiency

Zinc (Zn) deficiency is often associated with low plasma vitamin A (retinol) concentrations. It has been suggested that the reduction in plasma retinol is secondary to reduced liver retinol binding protein (RBP) synthesis. In the present study, RBP expression was determined in HepG2 cells cultured in either Zn adequate media or chelated media containing varying concentrations of Zn. Levels of RBP mRNA increased in a time‐ and Zn concentration‐dependent manner such that 0.5 μM Zn‐treated cells exhibited a >7.5‐fold increase while cells treated with 15 μM Zn were increased 2.9‐fold at 72 h compared to controls. RBP protein also progressively increased by 72 h to levels >8‐fold and 3‐fold higher than controls, in 0.5 μM and 15 μM Zn‐treated cells, respectively. The increase in RBP occurred without any change in DNA concentration between groups through 72 h. The Zn deficiency‐induced elevations in RBP transcript levels could be reversed within 24–48 h of repletion in Zn adequate media. Thus, the reductions in plasma retinol observed in Zn deficiency are in part a direct consequence of the deficiency.