Effect Of Nicotine On Special Genes Expression Of Mouse Embryonic Stem Cells

It is well known that maternal cigarette smoking during pregnancy isassociated with many adverse outcomes, including low birth weight,premature delivery, spontaneous abortion, placental abruption, prenatalmortality and ectopic pregnancy. Whereas there is little disagreementregarding the adverse effects of prenatal tobacco smokes exposure, it is lessclear whether these effects are produced by exposure to nicotine, the majoraddictive component, or other substances present in tobacco smoke. Theissue is of clinical significance, because nicotine replacement for pregnantwomen is often regarded as an alternative in smoking cessation programs,though, it is controversial. Moreover, due to small size, complexity andinaccessibility of the mouse embryo within uterine environment, whether nicotine affects early embryo development and the possible mechanisms are poorly characterized.Since isolation and maintenance of mouse pluripotent cells, these cells, embryonic stem (ES) cells, can be maintained indefinitely as a pluripotent cell population in vitro in the presence cytokines of the IL-6 family, and, when reintroduced into a host blastocyst, can contribute to all adult tissues including germ cells. ES cells, therefore, retain the ability to respond to all signals that regulate normal development, and potentially represent a powerful model system for investigation of the mechanisms underlying pluripotent cell biology and differentiation within the early embryo. In the meantime, in vitro, homogeneous lineage-specific differentiation of ES cells to early primitive ectoderm-like (EPL) cells has been achieved in response to medium conditioned by the human hepatocellular carcinoma cell line HepG2 (MEDII). EPL cells have been demonstrated to be primitive ectoderm-like by morphology, gene expression, cytokine responsiveness and differentiation potential both in vivo and in vitro. The homogeneous differentiation of ES cells would further provide an excellent way to study the early embryo development.Meanwhile, it has been demonstrated the FUU tamiiy transcription iaciorOct-4 (also termed Oct-3 or Oct-3/4, encoded by Pou5fl) is regarded as acandidate master regulator for initiation, maintenance, and differentiation ofpluripotent cells. It is a critical level of Oct-4 that ES cells require tomaintain their stem cell character and pluripotency. Besides, it has found thatthe target genes of Oct-4: Rex-1 and Utf-1, and Gbx-2 are all expressedspecially in undifferentiated ES cells, and their expressions are related withthe differentiation of ES cells to EPL cells. Therefore, some factors that leadto the change of these specific genes expression would have influence on thedevelopment and differentiation of ES and EPL cells.In this paper, we first examined the effect of nicotine on the specific genes expression of ES cells and the possible mechanism; subsequently, we employed the model of homogeneous lineage-specific differentiation of ES to EPL cells, and interrogated whether nicotine had effect on the specific genes expression in the conversion. Considering the expression of glyceraldehydes-3 phosphate dehydrogenase (GAPDH) is unaffected by nicotine treatment, and GAPDH was used as an internal reference for relative quantification. Furthermore, based on the data that nicotine may reorganize cytoskeleton, induce actin disassembly, and has effect on theexpression of (3-actin, so the effect of nicotine on P-actin expression was alsoinvestigated together. The purpose of the present study was to provideexperimental evidence for clarifying the effect of nicotine on early embryodevelopment.The first part: Effect of nicotine on Oct-4, Rex-1, and P-actin expressionof ES cellsES cells were cultured on feeder cells and were treated with 1-1000 nM nicotine, which are comparable to the plasma nicotine level in moderate smokers. Tubocurarine, a usual nicotinic acetylcholine receptors blocker, was added to plates at the concentration of 10 yM concurrent with nicotine for 24 h. ES cells were purified and total RNA was isolated. Reverse transcription (RT) was performed, subsequently the conditions of amplification were optimized, and the results of RT-PCR should be a single product with the desired length and without non-specific band. The optimum conditions were transferred to the following real time PCR protocols.RT-PCR result showed: (1) The amplification of Fgf-5 was not detected in purified ES cells, that is, they were qualified with next experiments. (2) The amplification of Oct-4, Rex-1, P-actin and GAPDH all resulted in a single product with the desired length and without non-specific band, and theoptimum conditions were transferred to the following real time PCR.Real time RT-PCR analysis further showed, nicotine (10 nM-1 uM) had significantly enhanced the expression of Oct-4. Meanwhile, the effect of nicotine on Rex-1 expression is similar to the Oct-4, and nicotine (10 nM-1 uM) had enhanced the expression of Rex-1, while the nicotine-induced increase of Rex-1 expression is more weaker compared with Oct-4. In contrast, nicotine produced no effect on the expression of p-actin. That is, the effect of nicotine on Oct-4 and Rex-1 expression was not general but special. Moreover, the influence was in a dose-dependent manner; nicotine (1 nM) had no evident effect on the Oct-4 and Rex-1 expression compared with control, while their expressions were both enhanced significantly with increment of nicotine (10-1000 nM).Real-time RT-PCR analysis also revealed tubocurarine (10 uM) itself could evidently enhance Oct-4 and Rex-1 expression of ES cells without nicotine treatment; however, the action was reduced concurrent with nicotine, and compared with nicotine (100 nM and 1000 nM) treatment alone, tubocurarine significantly inhibited nicotine-induced enhancement of Oct-4 and Rex-1 expression.The results demonstrated that nicotine could impinge on Oct-4 and Rex-1expression of ES cells through nAChRs.The second part: Effect of nicotine on special genes expression in theconversion of ES to EPL cells.Prior to the formation of EPL cells, ES cells were cultured on gelatin-treated flask, in the absence of a feeder layer. EPL cells were formed by the culture of ES cells in ES DMEM containing 50 % MEDII for 8 days. At the same time, the cells were treated with nicotine for 24 h at the concentrations of 10 nM, 100 nM, 1000 nM or 10 uM respectively, which are comparable to the plasma nicotine level in moderate smokers. The cell suspension was purified to discard feeder cells and got more purified EPL cells. Purified EPL cells isolated total RNA and amplified Fgf-5, Oct-4, Rex-1, Gbx-2, Utf-1, p-actin and GAPDH. RT-PCR products were subjected to electrophoresis on 2 % agarose gel and stained with ethidium bromide. Results were analyzed with semiquantitative estimation by comparing the mRNA expression of Oct-4, Rex-1, Utf-1, Gbx-2 and P-actin to that of GAPDH.RT-PCR results showed the amplification of Fgf-5 was not detected in purified EPL cells, that is, they were qualified with next experiments. During the formation of EPL cells, nicotine (10 nM-10 uM) had pronounced effecton the expression of Oct-4, Rex-1, Gbx-2 and Utf-1 expressions; in contrast, it produced no effect on the expression of 0-actin, that is, the effect of nicotine on the genes expression was not general but special.At the first two days in the conversation of ES cells to EPL cells, nicotine (10 nM- 1000 nM) produced no effect on the expression of Oct-4, Rex-1 and Utf-1; however, nicotine had evident influence on the expression of Oct-4, Rex-1 and Utf-1 at the later days (from 4 to 8 day), and the effect was the most pronounced at the fourth day. In contrast, nicotine significantly influenced the expression of Gbx-2 at the second and eighth day.In more detail, nicotine (100 nM) enhanced the expression of Oct-4,Rex-1 Utf-1 and Gbx-2; nicotine (10 uM) inhibited the Oct-4, Rex-1 andUtf-1 expression except at the fourth day, and nicotine (10 uM) increasedGbx-2 expression (2 day) while reduced its expression (8 day); nicotine (10nM) enhanced the expression of Oct-4, Gbx-2 and Utf-1 (8 day), whileinhibited Rex-1 and Utf-1 (6 day) expression; nicotine (1000 nM) increasedthe Oct-4 (8 day) and Gbx-2 expression, while reduced the expression ofUtf-1 (8 day).RT-PCR results further illustrated that nicotine had pronounced effect on the specific genes expression (Oct-4, Rex-1, Gbx-2, and Utf-1) during theformation of EPL cells.Altogether, the study revealed that nicotine specially influenced the genes (Oct-4, Rex-1, Gbx-2, and Utf-1) expression of ES cells. Among the specific genes of ES cells, the POU family transcription factor Oct-4, as a candidate master regulator for initiation, maintenance, and differentiation of pluripotent cells, is known better. Oct-4 is a candidate master regulator for initiation, maintenance, and differentiation of ES cells. ES cells require a critical level of Oct-4 to maintain their stem cell character and pluripotency. A less than twofold increase causes differentiation into endoderm and mesoderm, whereas reduction to less than 50 % of the normal expression level triggers dedifferentiation into trophectoderm. Our data demonstrated that nicotine (10-1000 nM) enhanced the expression of Oct-4, and at the concentration of nicotine (1000 nM), the Oct-4 expression was increased by more than twofold, which could cause unnatural differentiation into endoderm and mesoderm of ES cells. Meanwhile, it has been demonstrated that both Rex-1 and Utf-1 is the target gene of Oct-4, so that nicotine affects the expression of Rex-1 and Utf-1 would further state the effect of Oct-4 on the development and differentiation of ES and EPL cells.In this regard, we do not favor the suggestion that nicotine replacementfor pregnant women is regarded as a safe alternative in smoking cessationprogram.