BRANCHING TUBULOGENESIS INDUCED BY HEPARIN-BINDING EGF-LIKE GROWTH FACTOR

可溶性HB-EGF（s-HB-EGF）可诱导培养在I型胶原凝胶中的输尿管芽细胞（UBC）形成小管。s-HB-EGF诱导的微管样结构具有丰富的树枝状分支。从SV-40抗原转基因小鼠的胚胎输尿管芽中分离UBC。在本研究中使用用小鼠proHB-EGF（UBC）稳定转染的UBC<proHB-EGF>。proHB-EGF在质膜细胞外结构域中含有序列PVENPLYTYDHT，s-HB-EGF在P（脯氨酸）处被切割。然后，我们构建了proHB-EGF的截短突变体，其在发生切割的位点（UBC^）处具有9个氨基酸缺失（PVENPLYTY）<del9aa>。在<del9aa>用TPA处理后，与野生型proHB-EGF转染的UBC（UBC-1）相比<proHB-EGF>，在UBC-1中几乎没有s-HB-EGF 13 UBC-1<proHB-EGF>和UBC-2<del9aa>都诱导表达高水平EGF受体的A431细胞中EGF受体的酪氨酸磷酸化。UBC^<proHB-EGF>和UBC^<del9aa>均显示长直的管状结构，具有很少的分支，模拟体内肾小管。来自UBC^和UBC^的免疫沉淀物<proHB-EGF><del9aa>显示磷酸肌醇-3激酶（PI-3激酶）活性的上调，伴随着分支索伸长的形态学变化。UBC 1<proHB-EGF>和UBC 2<del9aa>显示顶部-基底侧极化和水通道水通道蛋白-2的表达。结论：proHB-EGF可诱导UBC中的集合管形成，其通过EGF受体激活PI-3激酶介导。用proHB-EGF激活Juxtacrine可能对集合管系统的形态发生平衡很重要。

Soluble form of HB-EGF (s-HB-EGF) induce tubulogenesis in ureteric bud cells (UBC) cultured in type I collagen gel. s-HB-EGF induced tubule-like structures with abundant arborized branches. UBC were isolated from the embryonic ureteric buds of mice transgenic for SV-40 antigen. UBC that were stably transfected with mouse proHB-EGF (UBC^<proHB-EGF>) were used in this study. proHB-EGF contains the sequence PVENPLYTYDHT in the juxtamembrane extracellular domain and s-HB-EGF is cleaved at P (proline). Then, we constructed a truncated mutant of proHB-EGF that has a 9 amino acid deletion (PVENPLYTY) at the site where cleavage occur (UBC^<del9aa>). There was almost no cleavage of s-HB-EGF in UBC^<del9aa> compared to wild type proHB-EGF transfected UBC (UBC^<proHB-EGF>) after treatment with TPA.Both UBC^<proHB-EGF> and UBC^<del9aa> induced tyrosine phosphorylation of EGF-receptors in A431 cells that express high levels of EGF-receptor. Both UBC^<proHB-EGF> and UBC^<del9aa> showed long straight tubule-like structures with few branches mimicking renal tubules in vivo. Immunoprecipitates from the UBC^<proHB-EGF> and UBC^<del9aa> showed upregulation of phosphoinositide-3 kinase (PI-3 kinase) activity, concomitantly with morphological changes in elongation of the branching cords. UBC^<proHB-EGF> and UBC^<del9aa> showed apical-basolateral polarization and expression of the water channel aquaporin-2. In conclusion, proHB-EGF can induce collecting duct formation in UBC, which was mediated by activating PI-3 kinase via the EGF-receptor. Juxtacrine activation with proHB-EGF could be important for well-balanced morphogenesis of the collecting duct system.