Takemura T, Kondo S, Homma T, Sakai M, Harris R C
Department of Medicine, Vanderbilt University School of Medicine and the Department of Veterans Affairs Medical Center, Nashville, Tennessee 37232, USA.
J Biol Chem. 1997 Dec 5;272(49):31036-42. doi: 10.1074/jbc.272.49.31036.
To understand whether expression of membrane-anchored heparin binding epidermal growth factor (proHB-EGF) is involved in renal epithelial cell survival, rat membrane-bound HB-EGF precursor was stably transfected into a renal epithelial cell line, NRK 52E cells (NRKproHB-EGF). When exposed to 10% fetal calf serum (FCS), there were no differences in growth rates among wild-type (WT), vector-transfected (NRKvector), and NRKproHB-EGF. However, when cells were grown in the presence of 1% FCS, the growth rate of NRKproHB-EGF was 65% faster. When confluent cell monolayers were exposed to H2O2 or etoposide, WT or NRKvector exhibited significant apoptotic bodies and DNA laddering; in contrast, NRKproHB-EGF were resistant to both stimuli, as indicated by increased cell viability and marked decrease of apoptotic bodies and DNA laddering. When plated at high density onto plastic dishes without FCS, WT and NRKvector formed few attachments, did not proliferate, and underwent apoptosis. By day 3, no cells survived. Addition of exogenous recombinant HB-EGF (10(-8) M) to WT or NRKvector increased cell survival by <10% and incubation with conditioned media of NRKproHB-EGF had no effect. In contrast, NRKproHB-EGF attached and formed epithelial colonies, although they did not proliferate. After 3 days, cell viability was 84% of the initial cell number plated, and no evidence of apoptosis was present. When plated in 10% FCS, NRKproHB-EGF attachment to plastic substratum at 1, 2, and 3 h was 250% greater than that of WT or NRKvector. Addition of exogenous recombinant human HB-EGF to WT or NRKvector increased attachment by <50%. When grown on poly(2-hydroxyethyl methacrylate) or in the presence of the integrin receptor-blocking peptide GRGDTP, neither WT nor NRKvector attached to the substratum or formed cell-cell attachments. Compared with WT or NRKvector, NRKproHB-EGF exhibited 300% greater cell viability on either poly(2-hydroxyethyl methacrylate)-coated dishes or in the presence of GRGDTP and formed cell clusters. When plated at low density (1 x 10(3) cells/1.5-cm dish) or at high density in the presence of an anti-HB-EGF blocking antibody, NRKproHB-EGF failed to form epithelial colonies. Addition of formalin fixed NRKproHB-EGF promoted EGF receptor tyrosine phosphorylation in quiescent A431 cells and stimulated DNA synthesis and prevented H2O2-induced apoptosis in renal epithelial cells. These results indicate that membrane-bound HB-EGF promotes renal epithelial cell survival, possibly by promoting cell-matrix and cell-cell interactions. The failure of either conditioned media or exogenous HB-EGF to reproduce these findings suggests that juxtacrine or tightly coupled paracrine interactions underlie this cytoprotection.
为了解膜锚定型肝素结合表皮生长因子（proHB - EGF）的表达是否参与肾上皮细胞存活，将大鼠膜结合型HB - EGF前体稳定转染至肾上皮细胞系NRK 52E细胞（NRKproHB - EGF）。当暴露于10%胎牛血清（FCS）时，野生型（WT）、载体转染（NRKvector）和NRKproHB - EGF细胞的生长速率无差异。然而，当细胞在1% FCS存在下生长时，NRKproHB - EGF的生长速率快65%。当汇合的细胞单层暴露于过氧化氢或依托泊苷时，WT或NRKvector出现明显的凋亡小体和DNA梯状条带；相反，NRKproHB - EGF对这两种刺激均有抗性，表现为细胞活力增加以及凋亡小体和DNA梯状条带明显减少。当高密度接种于无FCS的塑料培养皿时，WT和NRKvector几乎没有附着，不增殖并发生凋亡。到第3天，无细胞存活。向WT或NRKvector中添加外源性重组HB - EGF（10⁻⁸ M）可使细胞存活率提高不到10%，且与NRKproHB - EGF的条件培养基孵育无作用。相比之下，NRKproHB - EGF虽不增殖但能附着并形成上皮细胞集落。3天后，细胞活力为接种初始细胞数的84%，且无凋亡迹象。当在10% FCS中接种时，NRKproHB - EGF在1、2和3小时对塑料基质的附着比WT或NRKvector大250%。向WT或NRKvector中添加外源性重组人HB - EGF可使附着增加不到50%。当在聚（甲基丙烯酸2 - 羟乙酯）上生长或存在整合素受体阻断肽GRGDTP时，WT和NRKvector均不附着于基质或形成细胞 - 细胞连接。与WT或NRKvector相比，NRKproHB - EGF在聚（甲基丙烯酸2 - 羟乙酯）包被的培养皿上或存在GRGDTP时细胞活力高300%，并形成细胞簇。当低密度（1×10³细胞/1.5 cm培养皿）接种或在存在抗HB - EGF阻断抗体的情况下高密度接种时，NRKproHB - EGF无法形成上皮细胞集落。添加福尔马林固定的NRKproHB - EGF可促进静止A431细胞中表皮生长因子受体酪氨酸磷酸化，刺激DNA合成，并防止肾上皮细胞中过氧化氢诱导的凋亡。这些结果表明，膜结合型HB - EGF可能通过促进细胞 - 基质和细胞 - 细胞相互作用来促进肾上皮细胞存活。条件培养基或外源性HB - EGF均无法重现这些结果，提示这种细胞保护作用的基础是近分泌或紧密偶联的旁分泌相互作用。
