Rees C, Clemmons D R, Horvitz G D, Clarke J B, Busby W H
Department of Medicine, University of North Carolina School of Medicine, Chapel Hill 27599-7170, USA.
Endocrinology. 1998 Oct;139(10):4182-8. doi: 10.1210/endo.139.10.6266.
Smooth muscle cells (SMC) secrete a serine protease that cleaves insulin-like growth factor (IGF) binding protein (IGFBP)-4 into fragments that have low affinity for IGF-1. When IGFBP-4 is added to monolayer cultures of cell types that do not secrete this protease, IGF-1 stimulation of DNA synthesis is significantly inhibited. In contrast, if cell types that secrete this protease are used, IGFBP-4 is a much less potent inhibitor. These studies were conducted to determine whether proteolysis of IGFBP-4 accounted for its reduced capacity to inhibit IGF-1-stimulated DNA synthesis. The cleavage site in IGFBP-4 that the SMC protease uses was determined to be lysine120, histidine121. A protease-resistant mutant form of IGFBP-4 was prepared, expressed, purified, and tested for biologic activity using porcine SMC cultures. Addition of the protease-resistant mutant resulted in inhibition of DNA and cell migration responses to IGF-1. The inhibition was concentration dependent and was maximal when 500 ng/ml (20 nM) of the mutant was added with 20 ng/ml (2.8 nM) of IGF-1. When the mutant was added in the absence of IGF-1, it had no activity. The results show that cleavage of IGFBP-4 at lysine120, histidine121 results in inactivation of the ability of IGFBP-4 to bind to IGF-1. Creation of a mutant form of IGFBP-4 that was not cleaved by the protease resulted in inhibition of IGF-1-stimulated actions. The results suggest that IGFBP-4 can act as a potent inhibitor of the anabolic effects of IGF-1 and that the variables that regulate protease activity may indirectly regulate IGF-1 actions.
平滑肌细胞（SMC）分泌一种丝氨酸蛋白酶，该酶可将胰岛素样生长因子（IGF）结合蛋白（IGFBP）-4裂解成与IGF-1亲和力低的片段。当将IGFBP-4添加到不分泌这种蛋白酶的细胞单层培养物中时，IGF-1对DNA合成的刺激作用会受到显著抑制。相反，如果使用分泌这种蛋白酶的细胞类型，IGFBP-4的抑制作用则弱得多。进行这些研究是为了确定IGFBP-4的蛋白水解是否是其抑制IGF-1刺激的DNA合成能力降低的原因。已确定SMC蛋白酶作用于IGFBP-4的裂解位点为赖氨酸120、组氨酸121。制备了一种抗蛋白酶的IGFBP-4突变体形式，进行表达、纯化，并使用猪SMC培养物测试其生物活性。添加抗蛋白酶突变体导致对IGF-1的DNA和细胞迁移反应受到抑制。这种抑制作用呈浓度依赖性，当添加500 ng/ml（20 nM）的突变体与20 ng/ml（2.8 nM）的IGF-1时抑制作用最大。当在无IGF-1的情况下添加突变体时，它没有活性。结果表明，IGFBP-4在赖氨酸120、组氨酸121处的裂解导致其与IGF-1结合的能力失活。创建一种不被蛋白酶裂解的IGFBP-4突变体形式导致对IGF-1刺激作用的抑制。结果表明，IGFBP-4可作为IGF-1合成代谢作用的有效抑制剂，调节蛋白酶活性的变量可能间接调节IGF-1的作用。
