Daher Ahmad, de Boer Willem I, El-Marjou Ahmed, van der Kwast Theodorus, Abbou Claude C, Thiery Jean-Paul, Radvanyi François, Chopin Dominique K
INSERM EMI 03.37, Centre de Recherches Chirurgicales, Faculté de Médecine, Université de Paris 12, Créteil, France.
Lab Invest. 2003 Sep;83(9):1333-41. doi: 10.1097/01.lab.0000086380.23263.52.
Members of the epidermal growth factor (EGF) family and their receptors are involved in many cellular processes, including proliferation, migration, and differentiation. We have previously reported that these growth factors are expressed and have specific regulatory functions in an organ-like culture model of normal human urothelial cells. Here, we used this model to investigate the involvement of EGF receptor (EGFR) in human urothelial regeneration. Three 4-mm-diameter damaged areas were made in confluent normal human urothelial cell cultures with a biopsy punch. Regeneration was measured, on fixed stained cultures, with an image analyzer, at 4, 24, and 48 hours after injury. Cell proliferation was assessed by 5-bromo-2-deoxyuridine incorporation. To identify EGF family factors potentially involved in the healing process, we studied the effect of these factors on damaged confluent cultures and the level of expression of mRNAs extracted from these cultures. EGFR inhibition of the proliferation and migration of urothelial cells was tested with (1). a specific tyrosine kinase inhibitor (AG1478) and (2). a blocking anti-EGFR antibody (LA22). Exogenously added amphiregulin, EGF, transforming growth factor-alpha and heparin-binding EGF (HB-EGF) stimulated urothelial regeneration. The damaged areas were repaired by regrowth within 48 hours. Both AG1478 and LA22 inhibited the repair (by 50% and 30%, respectively), as well as proliferation and migration. This regeneration was accompanied by increased HB-EGF mRNA expression in cultures of cells from four of six subjects, but no corresponding change in EGFR protein level was observed. These results indicate that the EGFR signaling pathway is involved in urothelial regeneration. Our data support an autocrine role of HB-EGF in this process and suggest that the EGFR pathway is a potential therapeutic target for modulating urothelial cell proliferation.
表皮生长因子（EGF）家族成员及其受体参与许多细胞过程，包括增殖、迁移和分化。我们之前报道过这些生长因子在正常人尿路上皮细胞的类器官培养模型中表达并具有特定的调节功能。在此，我们使用该模型研究表皮生长因子受体（EGFR）在人尿路上皮再生中的作用。用活检打孔器在汇合的正常人尿路上皮细胞培养物中制造三个直径4毫米的损伤区域。在损伤后4小时、24小时和48小时，使用图像分析仪在固定染色的培养物上测量再生情况。通过5-溴-2-脱氧尿苷掺入评估细胞增殖。为了确定可能参与愈合过程的EGF家族因子，我们研究了这些因子对受损汇合培养物的影响以及从这些培养物中提取的mRNA的表达水平。用（1）一种特异性酪氨酸激酶抑制剂（AG1478）和（2）一种阻断性抗EGFR抗体（LA22）测试EGFR对尿路上皮细胞增殖和迁移的抑制作用。外源性添加的双调蛋白、EGF、转化生长因子-α和肝素结合EGF（HB-EGF）刺激尿路上皮再生。损伤区域在48小时内通过再生长得以修复。AG1478和LA22均抑制修复（分别抑制50%和30%）以及增殖和迁移。这种再生伴随着六名受试者中四名受试者的细胞培养物中HB-EGF mRNA表达增加，但未观察到EGFR蛋白水平的相应变化。这些结果表明EGFR信号通路参与尿路上皮再生。我们的数据支持HB-EGF在此过程中的自分泌作用，并表明EGFR通路是调节尿路上皮细胞增殖的潜在治疗靶点。
