Stimulation of the proliferation of the Madin-Darby canine kidney (MDCK) epithelial cell line by high-density lipoproteins and their induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.
Stimulation of the proliferation of the Madin-Darby canine kidney (MDCK) epithelial cell line by high-density lipoproteins and their induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.
复制标题
高密度脂蛋白刺激 Madin-Darby 犬肾 (MDCK) 上皮细胞系的增殖及其诱导 3-羟基-3-甲基戊二酰辅酶 A 还原酶活性。
DOI：
10.1002/jcp.1041170112
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发表时间：
1983
影响因子：
5.6
通讯作者：
Massoglia,SL
中科院分区：
文献类型：
--
作者：
Gospodarowicz,D;Cohen,DC;Massoglia,SL
MDCK 细胞接种在细胞外基质 (ECM) 包被的培养皿上，并暴露于补充有高密度脂蛋白（HDL，750 μg 蛋白质/ml）和转铁蛋白（10 μg/ml）的培养基中，其增殖率、最终细胞密度和形态外观与在补充血清的培养基中生长的细胞相似。 HDL 提供的促有丝分裂刺激不受接种培养物的初始细胞密度的限制，也不受时间的限制，因为在补充有转铁蛋白和 HDL 的培养基中生长的细胞至少生长 50 代。细胞存活需要培养基中存在 HDL，因为在补充有 HDL 和转铁蛋白的培养基中活跃增殖的细胞在转移至仅补充转铁蛋白的培养基后 2 天内开始死亡。低密度脂蛋白 (LDL) 在低浓度（2.5 至 100 μg 蛋白/ml）时对 MDCK 细胞具有促有丝分裂作用。超过 100 μg 蛋白质/ml 时，LDL 具有细胞毒性，因此不能支持细胞以最佳速率增殖。当细胞保存在纤连蛋白包被的培养皿上时，也可以观察到 HDL 的促有丝分裂作用。然而，细胞的增殖率不是最理想的，并且培养物不能无限期地在这种底物上传代，因为它们可以在 ECM 包被的培养皿上传代。观察到 HDL 支持细胞增殖的能力与其诱导 3-羟基-3-甲基戊二酰辅酶 A (HMG CoA) 还原酶活性的能力之间存在密切关联。增殖细胞中的 HMG CoA 还原酶活性（70 pmoles/min/106 个细胞）比融合的非分裂细胞（4 pmoles/min/106 个细胞）高 18 倍。稀疏细胞的 HMG CoA 还原酶活性对 HDL 的诱导更敏感（比对照细胞高八倍），比汇合细胞的酶活性（比对照水平高两倍）更敏感。 HDL 支持增殖和诱导 HMG CoA 还原酶活性的能力之间的剂量反应关系相似。暴露于 HDL 后稀疏静止细胞的增殖刺激和酶活性增加的时间过程是平行的。暴露于 Compactin（HMG CoA 还原酶的竞争性抑制剂）后，稀疏 MDCK 细胞的 HMG CoA 还原酶活性被诱导六倍。 HMG CoA 还原酶的这种诱导受到甲羟戊酸的阻止，不受 LDL 的影响，并且通过同时暴露于 HDL 来协同增强。高密度脂蛋白（HDL）可以挽救康帕素的细胞毒性作用，而低密度脂蛋白则不能。更具体地说，在 HDL 存在下增殖的细胞对康帕素毒性作用的抵抗力比暴露于 LDL 的细胞高 100 倍。这些结果共同表明 HDL 诱导 HMG CoA 还原酶活性可能在介导 HDL 增殖作用中发挥作用。通过较高水平的 HMG CoA 还原酶而增加的甲羟戊酸的重要性似乎不在于大量提供细胞胆固醇，而在于提供特定的内源合成甾醇库，或甲羟戊酸的一种或多种非甾醇产物。在响应 HDL 增殖的细胞中，HMG CoA 还原酶活性的诱导似乎是对 HDL 的可能多型代谢反应的一致且基本的特征。
MDCK Cells seeded on extracellular matrix‐ (ECM‐) coated dishes and exposed to medium supplemented with high density lipoproteins (HDLs, 750 μg protein/ml) and transferrin (10 μg/ml) have a proliferative rate, final cell density, and morphological appearance similar to those of cells grown in serum‐supplemented medium. The mitogenic stimulus provided by HDLs is not limited by the initial cell density at which cultures are seeded, nor is it limited in time, since cells grown in medium supplemented with transferrin and HDLs grew for at least 50 generations. The presence of HDLs in the medium is required in order for cells to survive, since cells actively proliferating in the presence of medium supplemented with HDLs and transferrin begin to die within 2 days after being transferred to medium supplemented only with transferrin. Low‐density lipoprotein (LDL) is mitogenic for MDCK cells when present at low concentrations (from 2.5 to 100 μg protein/ml). Above 100 μg protein/ml, LDL is cytotoxic and therefore cannot support cell proliferation at an optimal rate. The mitogenic effect of HDLs is also observed when cells are maintained on fibronectin‐coated dishes. However, the proliferative rate of the cells is suboptimal and cultures cannot be passaged on this substrate indefinitely, as they can be on ECM‐coated dishes.A close association between the ability of HDLs to support cell proliferation and their ability to induce the activity of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase is observed. HMG CoA reductase activity is 18 times higher (70 pmoles/min /106cells) in proliferating cells than in confluent, nondividing cells (4 pmoles/min /106cells). The HMG CoA reductase activity of sparse cells is more sensitive to induction by HDLs (eight‐fold higher than control cells) than is the enzyme activity of confluent cells (twofold higher than control levels). The dose‐response relationships between the abilities of HDLs to support proliferation and to induce HMG CoA reductase activity are similar. The time course of the stimulation of proliferation and the increase in enzyme activity of sparse, quiescent cells after exposure to HDLs are parallel.The HMG CoA reductase activity of sparse MDCK cells is induced six‐fold by exposure to compactin, a competitive inhibitor of HMG CoA reductase. This induction of HMG CoA reductase is prevented by mevalonic acid, not affected by LDL, and synergistically enhanced by simultaneous exposure to HDLs. HDLs effect a rescue from the cytotoxic effect of compactin, whereas LDL does not. More specifically, cells proliferating in the presence of HDLs are 100 times more resistant to the toxic effects of compactin than are cells exposed to LDL. These results taken together suggest that the induction of HMG CoA reductase activity by HDLs may play a role in mediating the proliferative effect of HDLs. The significance of the increased mevalonate made available by higher levels of HMG CoA reductase appears not to lie in the bulk provision of cellular cholesterol, but rather in the provision of a specific pool of endogenously synthesized sterol, or in one or more of the nonsterol products of mevalonate. In cells that proliferate in response to HDLs, the induction of HMG CoA reductase activity appears to be a consistent and essential feature of a possibly pleiotypic metabolic response to HDLs.