Xie Leike, Overbeek Paul A, Reneker Lixing W
Department of Ophthalmology, School of Medicine, University of Missouri, Columbia, MO 65212, USA.
Dev Biol. 2006 Oct 15;298(2):403-14. doi: 10.1016/j.ydbio.2006.06.045. Epub 2006 Jul 4.
The vertebrate ocular lens is a simple and continuously growing tissue. Growth factor-mediated receptor tyrosine kinases (RTKs) are believed to be required for lens cell proliferation, differentiation and survival. The signaling pathways downstream of the RTKs remain to be elucidated. Here, we demonstrate the important role of Ras in lens development by expressing a dominant-negative form of Ras (dn-Ras) in the lens of transgenic mice. We show that lens in the transgenic mice was smaller and lens growth was severely inhibited as compared to the wild-type lens. However, the lens shape, polarity and transparency appeared normal in the transgenic mice. Further analysis showed that cell proliferation is inhibited in the dn-Ras lens. For example, the percentage of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in epithelial layer was about 2- to 3-fold lower in the transgenic lens than in the wild-type lens, implying that Ras activity is required for normal cell proliferation during lens development. We also found a small number of apoptotic cells in both epithelial and fiber compartment of the transgenic lens, suggesting that Ras also plays a role in cell survival. Interestingly, although there was a delay in primary fiber cell differentiation, secondary fiber cell differentiation was not significantly affected in the transgenic mice. For example, the expression of beta- and gamma-crystallins, the marker proteins for fiber differentiation, was not changed in the transgenic mice. Biochemical analysis indicated that ERK activity, but not Akt activity, was significantly reduced in the dn-Ras transgenic lenses. Overall, our data imply that the RTK-Ras-ERK signaling pathway is essential for cell proliferation and, to a lesser extent, for cell survival, but not for crystallin gene expression during fiber differentiation. Thus, some of the fiber differentiation processes are likely mediated by RTK-dependent but Ras-independent pathways.
脊椎动物的眼球晶状体是一种简单且持续生长的组织。生长因子介导的受体酪氨酸激酶(RTK)被认为是晶状体细胞增殖、分化和存活所必需的。RTK下游的信号通路仍有待阐明。在此,我们通过在转基因小鼠的晶状体中表达一种显性负性形式的Ras(dn-Ras),证明了Ras在晶状体发育中的重要作用。我们发现,与野生型晶状体相比,转基因小鼠的晶状体较小,晶状体生长受到严重抑制。然而,转基因小鼠的晶状体形状、极性和透明度看起来正常。进一步分析表明,dn-Ras晶状体中的细胞增殖受到抑制。例如,转基因晶状体上皮层中5-溴-2'-脱氧尿苷(BrdU)标记细胞的百分比比野生型晶状体低约2至3倍,这意味着Ras活性是晶状体发育过程中正常细胞增殖所必需的。我们还在转基因晶状体的上皮和纤维部分发现了少量凋亡细胞,这表明Ras在细胞存活中也发挥作用。有趣的是,尽管初级纤维细胞分化存在延迟,但转基因小鼠的次级纤维细胞分化并未受到显著影响。例如,作为纤维分化标记蛋白的β-和γ-晶状体蛋白的表达在转基因小鼠中没有变化。生化分析表明,dn-Ras转基因晶状体中的ERK活性显著降低,但Akt活性未受影响。总体而言,我们的数据表明,RTK-Ras-ERK信号通路对于细胞增殖至关重要,在较小程度上对细胞存活也很重要,但对于纤维分化过程中的晶状体蛋白基因表达并非必需。因此,一些纤维分化过程可能由RTK依赖但Ras独立的信号通路介导。