Regulation Of Differentiation In Lung And Epidermal Kera

在早期胚胎发育过程中，气管和食道是由前肠内胚层进化而来的。这些上皮细胞经历了一系列明确的结构变化，包括祖细胞分化为几种细胞类型，最终导致成熟上皮的形成。在这项研究中，我们监测了p63在发育过程中不同阶段的食道和气管支气管上皮中的表达，并确定了p63表达缺失对p63-/-小鼠这些上皮细胞形态发生的影响。E15.5d时，食道和气管上皮含有2-3层细胞，但只有前体细胞表达p63。祖细胞首先分化为纤毛细胞(p63-/b-微管蛋白IV+)，出生时分化为基底细胞(p63+/K14+/BS-I-B4+)。在成年小鼠和人中，食道衬里成熟为(非)角化的复层上皮，而气管、支气管衬里发育为假复层的柱状上皮，内含基细胞、纤毛细胞和粘膜记忆细胞。在成熟的上皮细胞中，K14+/BS-I-B4+基底层细胞p63的表达最强。在体内或在培养的细胞中，p63的表达在鳞状细胞分化过程中被显著抑制。一般来说，人鳞状细胞癌染色强烈，而腺癌不染色p63。与野生型小鼠的食道和气管不同，新生p63-/-小鼠的食道和气管上皮主要由柱状纤毛上皮组成，似乎缺乏基底细胞。我们的研究表明，p63对于食道和气管支气管上皮的正常形态发生是至关重要的。在p63-/-小鼠中，前体细胞能够分化为纤毛细胞，但似乎不能产生基底细胞，这表明p63在调节前体细胞向基底细胞的分化或在基底细胞的存活中发挥作用。 维甲酸在气管、支气管上皮中起着重要的作用。类维甲酸可以通过核类维甲酸受体或其他机制发挥作用。合成的类维A酸类6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene羧酸既有激活RAR-γ的作用，又有非受体依赖性的作用机制。我们分析了几种维甲酸对正常人气管、支气管壁上皮(HTBE)细胞和几种肺癌细胞株中非甾体抗炎药物激活基因(NAG-1)表达的影响。维甲酸AHPN以时间和剂量依赖的方式显著增强人肺腺癌H460细胞和其他几种癌细胞株NAG-1mRNA和蛋白的表达。这种诱导对于AHPN是特异的，因为RAR-和RXR-PAN激动剂维甲酸不能诱导NAG-1，这表明这种诱导不是通过激活视黄酸受体来介导的。虽然NAG-1是P53反应基因，但AHPN诱导的NAG-1表达不需要P53。AHPN诱导NAG-1的表达至少部分是由于NAG-1mRNA的稳定性增加了8倍。与癌细胞相比，正常HTBE细胞中NAG-1的表达可被维甲酸和RAR选择性泛激动剂有效地诱导，并伴随着鳞状细胞分化的抑制和正常分化的启动。在体内，NAG-1在正常的气管、支气管上皮中有表达，而在鳞状化生的气管上皮和人肺肿瘤的切片中没有表达。我们的结果表明，维甲酸诱导正常HTBE和肺癌细胞NAG-1的表达受到不同机制的调控，并与不同的生物学过程有关。本研究揭示了AHPN治疗与NAG-1表达之间的联系，为AHPN的抗肿瘤活性提供了新的机制。

I. The trachea and esophagus have a common origin evolving from the foregut endoderm during early embryonic development. These epithelia undergo a series of well-defined structural changes involving differentiation of progenitor cells into several cell types that ultimately result in the formation of the mature epithelium. In this study, we monitor the expression of p63 in the esophageal and tracheobronchial epithelium at several stages during development and determine the effect of the lack of p63 expression on the morphogenesis of these epithelia in p63-/- mice. At day E15.5, the esophageal and tracheobronchial epithelium contain 2-3 layers of cells; however, only the progenitor cells express p63. The progenitor cells differentiate first into ciliated cells (p63-/b-tubulin IV+) and at birth into basal cells (p63+/K14+/BS-I-B4+). In adult mouse and human, the lining of the esophagus matures into a (non)keratinizing, stratified epithelium while the tracheobronchial lining develops into a pseudostratified, columnar epithelium containing basal, ciliated and mucosecretory cells. In mature epithelia, the K14+/BS-I-B4+ basal cells are the most intensely stained for p63. Expression of p63 is dramatically repressed during squamous differentiation in vivo or in cultured cells. Generally, human squamous cell carcinomas stained strongly while human adenocarcinomas did not stain for p63. In contrast to the esophagus and trachea from wild type mice, the esophageal and tracheobronchial epithelium from newborn p63-/- mice consist largely of a columnar, ciliated epithelium that appear to lack basal cells. Our study indicates that p63 is critical for normal morphogenesis of the esophageal and tracheobronchial epithelium. In p63-/- mice, progenitor cells are able to differentiate into ciliated cells but do not appear to generate basal cells suggesting a role for p63 either in the regulation of the differentiation of progenitor cells into basal cells or in the survival of basal cells. II. Retinoids play an important role in the tracheobronchial epithelium. Retinoids can act via nuclear retinoid receptors or through other mechanisms. The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) can act both activate RARgamma and act by receptor-independent mechanisms. We have analyzed the effect of several retinoids on the expression of non-steroidal anti-inflammatory drug-activated gene (NAG-1) in normal human tracheobronchial epithelial (HTBE) cells and several lung carcinoma cell lines. The retinoid AHPN greatly enhances the expression of NAG-1 mRNA and protein in a time and dose-dependent manner in human lung adenocarcinoma H460 cells and several other carcinoma cell lines. This induction was specific for AHPN since retinoic acid, an RAR- and an RXR-pan-agonist were unable to induce NAG-1 suggesting that this induction is not mediated through activation of retinoid receptors. Although NAG-1 is a p53-responsive gene, AHPN-induced NAG-1 expression does not require p53. The induction of NAG-1 expression by AHPN is at least in part due to a 8-fold increase in the stability of NAG-1 mRNA. In contrast to carcinoma cells, NAG-1 expression is effectively induced by retinoic acid and the RAR-selective pan-agonist in normal HTBE cells and accompanies the inhibition of squamous differentiation and the initiation of normal differentiation. In vivo, NAG-1 expression was observed in the normal tracheobronchial epithelium while no expression was found in either squamous metaplastic tracheal epithelium or in sections of human lung tumors. Our results suggest that the induction of NAG-1 expression by retinoids in normal HTBE and lung carcinoma cells is regulated by distinct mechanisms and is associated with different biological processes. The linkage between AHPN treatment and NAG-1 expression revealed in this study provides a new mechanism for the anti-tumorigenic activity of AHPN.