Sakurai Hiroyuki, Bush Kevin T, Nigam Sanjay K
Division of Nephrology-Hypertension, Department of Medicine, University of California San Diego, La Jolla, California 92093, USA.
J Biol Chem. 2005 Dec 23;280(51):42181-7. doi: 10.1074/jbc.M507962200. Epub 2005 Sep 23.
We have purified a protein present in a conditioned medium derived from the metanephric mesenchyme that supports non-branching growth and epithelial differentiation of the isolated ureteric bud (UB) independent of glial cell line-derived neurotrophic growth factor (GDNF). By sequential liquid chromatography, together with protein microsequencing, the protein was identified as heregulin (HRG)alpha. The addition of recombinant HRG to the isolated UB grown in three-dimensional culture confirmed the proliferative activity of HRG. In branching UBs induced by whole metanephric mesenchyme cell-conditioned medium, proliferating cells were localized at ampullae, where a binding receptor for GDNF, GFRalpha1, was found. In HRG-induced UBs, however, the expression of GFRalpha1 was down-regulated, and proliferating cells were distributed throughout the structure. Electron microscopic examination of the HRG-induced UB revealed the presence of structurally mature and polarized epithelial cells reminiscent of the epithelial cells found in the stalk portion of the branching UB. cDNA array analysis further revealed that genes ontologically classified as developmental were down-regulated by HRG, whereas those involved in transport were up-regulated. For example, the mRNA for the GDNF receptors, GFRalpha1 and ret9, was down-regulated, whereas the mRNA for collecting duct transporters, such as urea transporter2, aquaporin3, and sodium-hydrogen exchanger2 was up-regulated in HRG-treated UBs compared with UBs grown in the presence of branch-promoting factors. Moreover, HRG promoted growth of UBs cultured in the absence of GDNF. Taken together, the data suggest that HRG supports UB epithelial cell differentiation and non-GDNF-dependent growth, raising the possibility that this kind of activity plays a role in the growth and differentiation of the stalk portion of the branching epithelial UB.
我们已经从后肾间充质来源的条件培养基中纯化出一种蛋白质，该蛋白质可支持分离的输尿管芽（UB）的非分支生长和上皮分化，且不依赖于胶质细胞系衍生的神经营养生长因子（GDNF）。通过连续液相色谱法结合蛋白质微测序，该蛋白质被鉴定为heregulin（HRG）α。将重组HRG添加到在三维培养中生长的分离的UB中，证实了HRG的增殖活性。在由整个后肾间充质细胞条件培养基诱导的分支UB中，增殖细胞位于壶腹，在那里发现了GDNF的结合受体GFRα1。然而，在HRG诱导的UB中，GFRα1的表达下调，增殖细胞分布于整个结构。对HRG诱导的UB进行电子显微镜检查发现，存在结构成熟且极化的上皮细胞，这让人联想到在分支UB的茎部发现的上皮细胞。cDNA阵列分析进一步显示，从本体论上分类为发育相关的基因被HRG下调，而参与转运的基因则被上调。例如，与在存在促进分支因子的情况下生长的UB相比，HRG处理的UB中GDNF受体GFRα1和ret9的mRNA下调，而集合管转运蛋白（如尿素转运蛋白2、水通道蛋白3和钠氢交换蛋白2）的mRNA上调。此外，HRG促进了在无GDNF条件下培养的UB的生长。综上所述，这些数据表明HRG支持UB上皮细胞分化和非GDNF依赖性生长，这增加了这种活性在分支上皮UB茎部的生长和分化中起作用的可能性。
