Savla U, Waters C M
Department of Biomedical Engineering, Northwestern University, Chicago, Illinois 60611, USA.
Radiat Res. 1998 Aug;150(2):195-203.
In patients undergoing radiation therapy in the thoracic region, ionizing radiation causes immediate damage to pulmonary endothelial and epithelial cells. We have recently shown that keratinocyte growth factor (KGF) protects against increases in permeability induced by hydrogen peroxide in human airway epithelial cells. Since radiation injury involves the production of oxygen free radicals, we tested the hypothesis that KGF would protect against radiation-induced increases in permeability. Two lines of human airway epithelial cells (Calu-3 and 16HBE14o-) were grown on collagen-coated polyester membranes (Transwell, Costar) and the permeability of the monolayers was determined by measuring the flux of tracers from the top chamber to the bottom chamber as a function of time. Changes in permeability were apparent 4 h after exposure. Increasing doses of radiation (2-10 Gy) stimulated significant increases in permeability compared with control monolayers (P < 0. 05, n=5-10) in Calu-3 and 16HBE14o- cells. KGF (50 ng/ml) alone reduced permeability significantly compared with controls, protected against increases in permeability with low doses of radiation and provided partial protection at higher doses. KGF also provided a significant effect in cells irradiated with 10 Gy (n=5, P < 0. 05) when given for the 4 h immediately after irradiation. The effects of KGF were sustained. After a full 24-h pretreatment with KGF, cells were incubated in medium without KGF for 8 or 12 h prior to 10 Gy irradiation. Both of these treatments significantly reduced permeability to albumin in sham-irradiated and irradiated cells (n=3, P < 0.05). To investigate the signal transduction pathways through which KGF mediates protection, permeability was measured in the presence of the protein kinase C (PKC) inhibitor, calphostin C, or the tyrosine kinase inhibitor, genistein. Inhibition of PKC blocked the decrease in basal tracer flux caused by KGF treatment in both cell types and removed the KGF-mediated protection against radiation. Incubation with genistein completely blocked the KGF-mediated decrease in the baseline tracer flux, as well as the ameliorating effect observed after irradiation. Rhodamine-phalloidin staining of the F-actin cytoskeleton showed disruption of the cytoskeleton with radiation exposure, increased density of F-actin filaments with KGF treatment, and resistance to disruption when cells were pretreated with KGF and exposed to radiation. Our results suggest that KGF regulates permeability in airway epithelium through a pathway mediated by PKC and tyrosine kinase that stabilizes the F-actin cytoskeleton.
在接受胸部放疗的患者中，电离辐射会立即对肺内皮细胞和上皮细胞造成损伤。我们最近发现，角质形成细胞生长因子（KGF）可防止过氧化氢诱导的人气道上皮细胞通透性增加。由于辐射损伤涉及氧自由基的产生，我们测试了KGF可防止辐射诱导的通透性增加这一假设。将两株人气道上皮细胞系（Calu-3和16HBE14o-）培养在胶原包被的聚酯膜（Transwell，Costar）上，通过测量示踪剂从顶部腔室到底部腔室的通量随时间的变化来确定单层细胞的通透性。暴露4小时后通透性发生明显变化。与对照单层细胞相比，增加辐射剂量（2-10 Gy）可刺激Calu-3和16HBE14o-细胞的通透性显著增加（P<0.05，n=5-10）。单独使用KGF（50 ng/ml）与对照相比可显著降低通透性，可防止低剂量辐射引起的通透性增加，并在高剂量时提供部分保护。当在照射后立即给予4小时时，KGF对接受10 Gy照射的细胞也有显著作用（n=5，P<0.05）。KGF的作用是持续的。在用KGF进行24小时的充分预处理后，在10 Gy照射前，将细胞在不含KGF的培养基中孵育8或12小时。这两种处理均显著降低了假照射和照射细胞中白蛋白的通透性（n=3，P<0.05）。为了研究KGF介导保护作用的信号转导途径，在存在蛋白激酶C（PKC）抑制剂钙泊三醇C或酪氨酸激酶抑制剂染料木黄酮的情况下测量通透性。抑制PKC可阻断KGF处理引起两种细胞类型中基础示踪剂通量的降低，并消除KGF介导的辐射防护作用。用染料木黄酮孵育可完全阻断KGF介导的基线示踪剂通量降低以及照射后观察到的改善作用。用罗丹明-鬼笔环肽对F-肌动蛋白细胞骨架进行染色显示，辐射暴露会破坏细胞骨架，KGF处理会增加F-肌动蛋白丝的密度，并且当细胞用KGF预处理并暴露于辐射时可抵抗破坏。我们的结果表明，KGF通过由PKC和酪氨酸激酶介导的途径调节气道上皮的通透性，该途径可稳定F-肌动蛋白细胞骨架。
