Ziehr Jacqueline, Sheibani Nader, Sorenson Christine M
Department of Pediatrics, University of Wisconsin Medical School, Madison, WI 53792, USA.
Am J Physiol Renal Physiol. 2004 Dec;287(6):F1154-63. doi: 10.1152/ajprenal.00129.2004. Epub 2004 Aug 3.
Bcl-2 protects cells from apoptosis initiated by a variety of stimuli including loss of cell adhesion. Bcl-2 -/- mice develop renal hypoplastic/cystic dysplasia with renal cyst formation coinciding with renal maturation in normal mice. To gain a better understanding of the role cell-adhesive mechanisms play during renal maturation, we generated proximal tubule and collecting duct cell lines from postnatal day 10 (P10) and P20 bcl-2 +/+ and bcl-2 -/- mice. Very little is known about the role cell-adhesive and migratory mechanisms play during renal maturation. We observed that modulation of cell-adhesive properties, which normally occur in a nephron segment-specific manner during renal maturation, and cell migration were altered in cells from bcl-2 -/- mice. Enhanced migration of bcl-2 -/- proximal tubule cells in a scratch wound assay was completely inhibited by incubation with PP1 (Src inhibitor) and moderately affected by incubation with SB-203580 (p38 inhibitor). These cells expressed increased levels of fibronectin and had numerous central focal adhesions. P20 bcl-2 -/- proximal tubule cells adhered to fibronectin but adhered poorly to collagen, vitronectin, or laminin. Collecting duct cells, similar to proximal tubule cells from bcl-2 -/- mice, demonstrated enhanced migration in a scratch wound assay that was inhibited by incubation with PP1. Migration of these cells was moderately affected by incubation with PD-98059 (MEK inhibitor) or LY-294002 (PI3 kinase inhibitor), whereas incubation with SB-203580 had no effect. P10 bcl-2 -/- collecting duct cells also expressed increased levels of fibronectin but decreased levels of thrombospondin-1 and demonstrated precocious binding to fibronectin and vitronectin compared with bcl-2 +/+ cells. The ability of P20 bcl-2 +/+ collecting duct cells to adhere to fibronectin and vitronectin corresponded with a decline in thrombospondin-1 expression. Therefore, alterations in cell-adhesive and migratory characteristics may be an early indicator of aberrant renal epithelial cell differentiation.
Bcl-2可保护细胞免受包括细胞黏附丧失在内的多种刺激所引发的细胞凋亡。Bcl-2基因敲除小鼠会出现肾发育不全/囊性发育异常，肾囊肿形成与正常小鼠的肾成熟过程同时发生。为了更好地理解细胞黏附机制在肾成熟过程中所起的作用，我们从出生后第10天（P10）和第20天（P20）的bcl-2基因野生型和基因敲除小鼠中生成了近端小管和集合管细胞系。关于细胞黏附及迁移机制在肾成熟过程中所起的作用，目前所知甚少。我们观察到，在肾成熟过程中通常以肾单位节段特异性方式发生的细胞黏附特性的调节以及细胞迁移，在bcl-2基因敲除小鼠的细胞中发生了改变。在划痕试验中，bcl-2基因敲除近端小管细胞的迁移增强，与PP1（Src抑制剂）孵育可完全抑制这种增强，与SB-203580（p38抑制剂）孵育则有中度影响。这些细胞中纤连蛋白表达水平升高，并有大量中央黏着斑。P20的bcl-2基因敲除近端小管细胞可黏附于纤连蛋白，但对胶原蛋白、玻连蛋白或层粘连蛋白的黏附较差。集合管细胞与bcl-2基因敲除小鼠的近端小管细胞类似，在划痕试验中迁移增强，与PP1孵育可抑制这种增强。与PD-98059（MEK抑制剂）或LY-294002（PI3激酶抑制剂）孵育对这些细胞的迁移有中度影响，而与SB-203580孵育则无作用。与bcl-2基因野生型细胞相比，P10的bcl-2基因敲除集合管细胞中纤连蛋白表达水平也升高，但血小板反应蛋白-1水平降低，且对纤连蛋白和玻连蛋白的结合早熟。P20的bcl-2基因野生型集合管细胞对纤连蛋白和玻连蛋白的黏附能力与血小板反应蛋白-1表达的下降相对应。因此，细胞黏附及迁移特性的改变可能是肾上皮细胞异常分化的早期指标。
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