Pussinen P J, Karten B, Wintersperger A, Reicher H, McLean M, Malle E, Sattler W
Institute of Medical Biochemistry and Molecular Biology, University of Graz, Harrachgasse 21, 8010 Graz, Austria.
Biochem J. 2000 Jul 15;349(Pt 2):559-66. doi: 10.1042/0264-6021:3490559.
Aberrant cell proliferation is one of the hallmarks of carcinogenesis, and cholesterol is thought to play an important role during cell proliferation and cancer progression. In the present study we examined the pathways that could contribute to enhanced proliferation rates of HBL-100 cells in the presence of apolipoprotein E-depleted high-density lipoprotein subclass 3 (HDL(3)). When HBL-100 cells were cultivated in the presence of HDL(3) (up to 200 microg/ml HDL(3) protein), the growth rates and cellular cholesterol content were directly related to the concentrations of HDL(3) in the culture medium. In principle, two pathways can contribute to cholesterol/cholesteryl ester (CE) uptake from HDL(3), (i) holoparticle- and (ii) scavenger-receptor BI (SR-BI)-mediated selective uptake of HDL(3)-associated CEs. Northern- and Western-blot analyses revealed the expression of CLA-1 (CD-36 and LIMPII analogous 1), the human homologue of the rodent HDL receptor SR-BI. In line with CLA-1 expression, selective uptake of HDL(3)-CEs exceeded HDL(3)-holoparticle uptake between 12- and 58-fold. Competition experiments demonstrated that CLA-1 ligands (oxidized HDL, oxidized and acetylated low-density lipoprotein and phosphatidylserine) inhibited selective HDL(3)-CE uptake. In line with the ligand-binding specificity of CLA-1, phosphatidylcholine did not compete for selective HDL(3)-CE uptake. Selective uptake was regulated by the availability of exogenous cholesterol and PMA, but not by adrenocorticotropic hormone. HPLC analysis revealed that a substantial part of HDL(3)-CE, which was taken up selectively, was subjected to intracellular hydrolysis. A potential candidate facilitating extralysosomal hydrolysis of HDL(3)-CE is hormone-sensitive lipase, an enzyme which was identified in HBL-100 cells by Western blots. Our findings demonstrate that HBL-100 cells are able to acquire HDL-CEs via selective uptake. Subsequent partial hydrolysis by hormone-sensitive lipase could provide 'free' cholesterol that is available for the synthesis of cellular membranes during proliferation of cancer cells.
异常细胞增殖是致癌作用的标志之一，胆固醇被认为在细胞增殖和癌症进展过程中发挥重要作用。在本研究中，我们检测了在载脂蛋白E缺失的高密度脂蛋白亚类3（HDL(3)）存在的情况下，可能导致HBL-100细胞增殖率提高的途径。当HBL-100细胞在HDL(3)（高达200μg/ml HDL(3)蛋白）存在的条件下培养时，生长速率和细胞胆固醇含量与培养基中HDL(3)的浓度直接相关。原则上，有两条途径可促进从HDL(3)摄取胆固醇/胆固醇酯（CE），（i）完整颗粒途径和（ii）清道夫受体BI（SR-BI）介导的HDL(3)相关CE的选择性摄取。Northern印迹和Western印迹分析揭示了CLA-1（CD-36和LIMPII类似物1）的表达，CLA-1是啮齿动物HDL受体SR-BI的人类同源物。与CLA-1的表达一致，HDL(3)-CEs的选择性摄取比HDL(3)完整颗粒摄取高出12至58倍。竞争实验表明，CLA-1配体（氧化HDL、氧化和乙酰化低密度脂蛋白以及磷脂酰丝氨酸）抑制HDL(3)-CE的选择性摄取。与CLA-1的配体结合特异性一致，磷脂酰胆碱不竞争HDL(3)-CE的选择性摄取。选择性摄取受外源性胆固醇和佛波酯的可用性调节，但不受促肾上腺皮质激素调节。HPLC分析表明，被选择性摄取的HDL(3)-CE的很大一部分会经历细胞内水解。促进HDL(3)-CE胞外溶酶体水解的一个潜在候选物是激素敏感性脂肪酶，通过Western印迹在HBL-100细胞中鉴定出了这种酶。我们的研究结果表明，HBL-100细胞能够通过选择性摄取获得HDL-CEs。随后由激素敏感性脂肪酶进行的部分水解可以提供“游离”胆固醇，这些胆固醇可用于癌细胞增殖过程中细胞膜的合成。
