Takemura T, Hino S, Murata Y, Yanagida H, Okada M, Yoshioka K, Harris R C
Department of Pediatrics, Kinki University, Osaka, Japan, and Department of Medicine, Vanderbilt University, Nashville, Tennessee, USA.
Kidney Int. 1999 Jan;55(1):71-81. doi: 10.1046/j.1523-1755.1999.00259.x.
Transfection of renal epithelial cells (NRK 52E) with membrane-associated heparin-binding epidermal growth factor-like growth factor (proHB-EGF) increased renal epithelial cell survival by promoting cell-cell and cell-extracellular matrix interactions. ProHB-EGF has been shown to form a complex in the plasma membrane with the tetraspanin CD9, an interaction that significantly increases the effectiveness of proHB-EGF as a juxtacrine mitogenic agent.
We examined whether the coexpression of proHB-EGF and CD9 would increase renal epithelial cell survival. CD9 was stably transfected into NRK 52E cells, either alone (NRKCD9) or together with proHB-EGF (NRKboth).
Juxtacrine mitogenic activity of NRKCD9 was no different than in cells transfected with vector alone (NRKvector), but was increased by NRKboth; juxtacrine mitogenic activity by NRKboth was twofold greater than when proHB-EGF was transfected alone (NRKproHB-EGF). When grown in 10% fetal calf serum, growth rates were similar among all transfectants. However, in 1% fetal calf serum, NRKproHB-EGF grew 50% faster than NRKvector or NRKCD9, and NRKboth grew 20% to 50% faster than NRKproHB-EGF at one, two, and three days of culture. NRKproHB-EGF attachment to plastic substratum at one, two, and three hours was 250% greater than that of NRKvector, and NRKboth was 20% to 30% greater than that of NRKproHB-EGF. Coating plates with either poly 2-hydroxyethyl methacrylate or the GRGDTP peptide prevented normal cell-extracellular matrix attachment, and NRKvector or NRKCD9 failed to attach or form cell-cell attachments. NRKproHB-EGF exhibited 300% and NRKboth exhibited 600% greater cell viability under these conditions. Expression of type I and type III collagen mRNA was enhanced similarly in NRKproHB-EGF and NRKboth, but the expression of beta1 integrin was up-regulated only in NRKboth.
Coexpression of proHB-EGF and CD9 may render the renal epithelial cells more resistant to disruption of cell-cell and cell-matrix interactions and could accelerate the re-establishment of these attachments.
用膜相关肝素结合表皮生长因子样生长因子（proHB - EGF）转染肾上皮细胞（NRK 52E）可通过促进细胞间及细胞与细胞外基质的相互作用来提高肾上皮细胞的存活率。已表明proHB - EGF在质膜上与四跨膜蛋白CD9形成复合物，这种相互作用显著提高了proHB - EGF作为旁分泌有丝分裂原的有效性。
我们检测了proHB - EGF和CD9的共表达是否会提高肾上皮细胞的存活率。将CD9稳定转染到NRK 52E细胞中，单独转染（NRKCD9）或与proHB - EGF一起转染（NRKboth）。
NRKCD9的旁分泌有丝分裂活性与仅用载体转染的细胞（NRKvector）无差异，但NRKboth使其增加；NRKboth的旁分泌有丝分裂活性比单独转染proHB - EGF（NRKproHB - EGF）时高两倍。在10%胎牛血清中培养时，所有转染细胞的生长速率相似。然而，在1%胎牛血清中，NRKproHB - EGF比NRKvector或NRKCD9生长快50%，在培养1天、2天和3天时，NRKboth比NRKproHB - EGF生长快20%至50%。NRKproHB - EGF在1小时、2小时和3小时时附着于塑料基质的能力比NRKvector高250%，NRKboth比NRKproHB - EGF高20%至30%。用聚甲基丙烯酸2 - 羟乙酯或GRGDTP肽包被培养板可阻止正常的细胞与细胞外基质附着，NRKvector或NRKCD9无法附着或形成细胞间附着。在这些条件下，NRKproHB - EGF的细胞活力提高了300%，NRKboth提高了600%。NRKproHB - EGF和NRKboth中I型和III型胶原mRNA的表达同样增强，但β1整合素的表达仅在NRKboth中上调。
proHB - EGF和CD9的共表达可能使肾上皮细胞对细胞间及细胞与基质相互作用的破坏更具抗性，并可能加速这些附着的重新建立。
