Proteolytic activation of latent transforming growth factor-beta from fibroblast-conditioned medium.
Proteolytic activation of latent transforming growth factor-beta from fibroblast-conditioned medium.
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DOI：
10.1083/jcb.106.5.1659
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发表时间：
1988-05
期刊：
影响因子：
--
通讯作者：
Moses HL
中科院分区：
文献类型：
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作者：
Lyons RM;Keski-Oja J;Moses HL
转化生长因子-β（TGF β）由大多数培养细胞以无活性形式产生。使用成纤维细胞（NRK-49 F和AKR-MCA）细胞条件培养基作为模型研究潜伏TGF β的潜在活化机制。通过放射受体和软琼脂测定以及抗体抑制和免疫沉淀来监测活性TGF β。在未处理的条件培养基中检测到很少或没有TGF β。用极端pH（1.5或12）处理培养基导致TGF β的显著活化，如放射受体测定所示，而弱酸处理（pH 4.5）仅产生pH 1.5所实现的竞争的20-30%。为了定义TGF β激活的更多生理手段，测试了一些蛋白酶的作用。发现纤溶酶和组织蛋白酶D产生25 kD条带，对应于TGF β的活性形式，如放射性标记的细胞条件培养基的免疫沉淀分析所示。纤溶酶处理的培养基导致的活动，定量类似于轻度酸处理的放射性受体和软琼脂测定。此外，纤溶酶产生的活性被抗TGF β抗体抑制。顺序处理AKR-MCA细胞条件培养基用弱酸，然后纤溶酶或纤溶酶，然后用弱酸得到的活化与单独处理相当。这些数据表明，条件培养基可能含有至少两种不同的潜伏TGF β池。一个样本池耐弱酸和/或纤溶酶，需要强酸或强碱处理以进行活化。第二个池通过温和的pH变化和/或纤溶酶活化。这种形式的潜伏性TGF β的活化可以通过前体分子或假设的TGF β结合蛋白复合物的解离或蛋白水解消化来发生。
Transforming growth factor-beta (TGF beta) is produced by most cultured cells in an inactive form. Potential activation mechanisms of latent TGF beta were studied using fibroblastic (NRK-49F and AKR-MCA) cell- conditioned medium as a model. Active TGF beta was monitored by radioreceptor and soft agar assays as well as by antibody inhibition and immunoprecipitation. Little or no TGF beta was detected in untreated conditioned medium. Treatment of the medium with extremes of pH (1.5 or 12) resulted in significant activation of TGF beta as shown by radioreceptor assays, while mild acid treatment (pH 4.5) yielded only 20-30% of the competition achieved by pH 1.5. In an effort to define more physiological means of TGF beta activation, the effects of some proteases were tested. Plasmin and cathepsin D were found to generate 25-kD bands corresponding to the active form of TGF beta as shown by immunoprecipitation analysis of radiolabeled cell-conditioned medium. Plasmin treatment of the medium resulted in activity that was quantitatively similar to that of mild acid treatment as measured by radioreceptor and soft agar assays. In addition, the plasmin-generated activity was inhibited by anti-TGF beta antibodies. Sequential treatments of AKR-MCA cell-conditioned medium with mild acid followed by plasmin or plasmin followed by mild acid gave activation comparable to either treatment alone. The data suggest that conditioned medium may contain at least two different pools of latent TGF beta. One pool is resistant to mild acid and/or plasmin and requires strong acid or alkali treatment for activation. A second pool is activated by mild pH change and/or plasmin. Activation of this form of latent TGF beta may take place by dissociation or proteolytic digestion from a precursor molecule or hypothetical TGF beta-binding protein complex.