Molecular identification of canine podocalyxin-like protein 1 as a renal tubulogenic regulator
Molecular identification of canine podocalyxin-like protein 1 as a renal tubulogenic regulator
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DOI：
10.1681/asn.2004121145
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发表时间：
2005-06-01
影响因子：
13.6
通讯作者：
Jou, TS
中科院分区：
文献类型：
--
作者：
Cheng, HY;Lin, YY;Jou, TS
GP 135是在极化的MDCK上皮细胞中表达的顶端膜蛋白。当在三维胶原凝胶中培养时，MDCK细胞以模拟胚胎肾发育的方式响应于肝细胞生长因子刺激而形成分支小管。在此过程中，GP 135显示短暂的膜定位丢失，但当新生管腔从发育中的小管中出现时，GP 135重新出现在细胞表面。尽管几十年来一直被用作根尖表面的典型标志，但GP 135在小管形成过程中动态表达的分子身份和意义仍然难以捉摸。为了进一步研究GP 135的功能，获得了GP 135的全长cDNA。序列比对和特征分析证实GP 135是足糖萼蛋白的犬同源物，证实了早期独立研究的发现。犬肾切片的免疫组织化学测定确定了GP 135沿着肾单位的肾小球和肾小管分布。表达靶向GP 135两个区域的siRNA的突变MDCK细胞显示肝细胞生长因子诱导的小管形成缺陷。GP 135的全长和O-连接的糖基化缩短的构建体的再表达可以重现siRNA敲低细胞中缺乏的小管形成过程;然而，缺乏胞质结构域的缺失构建体未能挽救表型。总之，这些数据将MDCK顶端结构域标记物GP 135鉴定为足糖萼蛋白的管状形式，并为其在肾小管发生中的重要性提供了证据。
GP135 is an apical membrane protein expressed in polarized MDCK epithelial cells. When cultured in three-dimensional collagen gel, MDCK cells form branching tubules in response to hepatocyte growth factor stimulation in a manner that simulates the embryonic renal development. During this process, GP135 displays transient loss of membranous localization but reappears at the cell surface when nascent lumen emerges from the developing tubules. Despite being used for decades as the canonical hallmark of apical surface, the molecular identity and the significance of the dynamic expression of GP135 during the tubulogenic process remain elusive. For exploring the function of GP135, the full-length cDNA encoding GP135 was obtained. Sequence alignments and features analysis confirm GP135 as a canine homolog of podocalyxin, confirming the finding of an earlier independent study. Immunohistochemical assays on canine kidney sections identified both glomerular and tubular distribution of GP135 along the nephron. Mutant MDCK cells expressing siRNA targeted at two regions of GP135 show defects in hepatocyte growth factor-induced tubulogenesis. Re-expression of full-length and an O-linked glycosylation abbreviated construct of GP135 could recapitulate the tubulogenesis process lacking in siRNA knockdown cells; however, a deletion construct devoid of the cytoplasmic domain failed to rescue the phenotype. In summary, the data identify the MDCK apical domain marker GP135 as a tubular form of podocalyxin and provide evidence for its importance in renal tubulogenesis.