Montesano Roberto, Soulié Priscilla
Department of Morphology, University of Geneva Medical Center, CH-1211 Geneva 4, Switzerland.
J Cell Sci. 2002 Dec 1;115(Pt 23):4419-31. doi: 10.1242/jcs.00164.
Lumen formation is a fundamental step in the development of the structural and functional units of glandular organs, such as alveoli and ducts. In an attempt to elucidate the molecular signals that govern this morphogenetic event, we set up an in vitro system in which cloned mammary epithelial cells grown in collagen gels under serum-free conditions form solid, lumen-less colonies. Addition of as little as 0.1% donor calf serum (DCS) was sufficient to induce the formation of a central cavity. Among a number of serum constituents analyzed, retinol was found to mimic the effect of DCS in inducing lumen morphogenesis. Since the biological activities of retinol are largely dependent on its conversion to all-trans-retinoic acid (RA), we examined in more detail the effect of RA on lumen formation. RA induced the formation of lumen-containing colonies (cysts) in a concentration- and time-dependent manner, a half-maximal effect after 9 days of culture being observed with 100 pM RA. The pleiotropic effects of retinoids are mediated by nuclear retinoic acid receptors (RARs; alpha, beta and gamma) and retinoid X receptors (RXRs; alpha, beta and gamma). To identify the signaling pathway involved in RA-induced lumen formation, we used receptor-specific synthetic retinoids. TTNPB, a selective RAR agonist, promoted lumen morphogenesis, whereas RXR-selective ligands lacked this activity. Lumen formation was also induced at picomolar concentrations by Am-580, a synthetic retinoid that selectively binds the RARalpha receptor subtype. Moreover, co-addition of Ro 41-5253, an antagonist of RARalpha, abrogated the lumen-inducing activity of both RA and DCS, indicating that this biological response is mediated through an RARalpha-dependent signaling pathway. To gain insight into the mechanisms underlying RA-induced lumen formation, we assessed the potential role of matrix metalloproteinases (MMP). Using gelatin zymography, we observed a dose-dependent increase in latent and active forms of gelatinase B (MMP-9) upon RA treatment. In addition, lumen formation was abrogated by addition of the synthetic MMP inhibitor BB94, indicating that this morphogenetic process is likely to require MMP activity. Collectively, our results provide evidence that RA promotes lumen formation by mammary epithelial cells in vitro and suggest that it plays a similar role during mammary gland development in vivo.
管腔形成是腺器官（如肺泡和导管）结构和功能单位发育的一个基本步骤。为了阐明控制这一形态发生事件的分子信号，我们建立了一个体外系统，在该系统中，克隆的乳腺上皮细胞在无血清条件下于胶原凝胶中生长形成实心的、无管腔的集落。添加低至0.1%的供体小牛血清（DCS）就足以诱导中央腔的形成。在分析的多种血清成分中，发现视黄醇在诱导管腔形态发生方面可模拟DCS的作用。由于视黄醇的生物学活性很大程度上依赖于其转化为全反式视黄酸（RA），我们更详细地研究了RA对管腔形成的影响。RA以浓度和时间依赖性方式诱导含管腔集落（囊肿）的形成，培养9天后，100 pM RA可观察到半数最大效应。类视黄醇的多效性作用由核视黄酸受体（RARs；α、β和γ）和视黄醇X受体（RXRs；α、β和γ）介导。为了确定参与RA诱导管腔形成的信号通路，我们使用了受体特异性合成类视黄醇。TTNPB是一种选择性RAR激动剂，可促进管腔形态发生，而RXR选择性配体则缺乏这种活性。合成类视黄醇Am - 580（一种选择性结合RARα受体亚型的物质）在皮摩尔浓度下也可诱导管腔形成。此外，RARα拮抗剂Ro 41 - 5253的共同添加消除了RA和DCS的管腔诱导活性，表明这种生物学反应是通过RARα依赖性信号通路介导的。为了深入了解RA诱导管腔形成的潜在机制，我们评估了基质金属蛋白酶（MMP）的潜在作用。使用明胶酶谱法，我们观察到RA处理后明胶酶B（MMP - 9）的潜伏形式和活性形式呈剂量依赖性增加。此外，添加合成MMP抑制剂BB94可消除管腔形成，表明这种形态发生过程可能需要MMP活性。总的来说，我们的结果提供了证据表明RA在体外促进乳腺上皮细胞形成管腔，并表明它在体内乳腺发育过程中可能发挥类似作用。
