Ku P T, D'Amore P A
Program in Cell and Developmental Biology, Harvard Medical School, Boston, Massachusetts, USA.
J Cell Biochem. 1995 Jul;58(3):328-43. doi: 10.1002/jcb.240580307.
Basic fibroblast growth factor (bFGF; FGF-2) lacks a signal sequence and thus is not secreted by classical pathways. It has been speculated that one mode of bFGF release may be injury, either sublethal or lethal; and, transient disruption of the plasma membrane has been shown to release bFGF [Muthukrihnan et al. (1991): J Cell Physiol 148:1-16]. This observation has led to the concept of bFGF as a "wound hormone," involved in tissue integrity and repair. Findings of elevated bFGF following injury in vivo support this concept. Using an in vitro model, we have examined the regulation of bFGF gene expression following its release by sublethal injury. Analysis of bFGF protein by ELISA revealed that scraping subconfluent bovine aortic EC (BAE) released up to 80% of their bFGF. Following scraping, there was a 4- to 10-fold increase in the steady state level of bFGF mRNA, which reached a maximum at 2-3 h. There was a parallel increase in protein so that by 6 h after the scrape-induced release, bFGF levels were restored to those measured prior to scraping. Since bFGF has been reported to induce its own expression, we hypothesized that the released bFGF might be responsible for the increase in bFGF mRNA. However, inclusion of neutralizing antibodies against bFGF had a negligible effect on the scrape-induced increase in bFGF mRNA levels. Because of the important role of transforming growth factor type-beta 1 (TGF-beta 1), the plasminogen/plasminogen activator system, and thrombin in wound healing, we investigated their potential contributions to the increase in bFGF expression. Addition of anti-TGF-beta 1 antibodies, plasminogen activator inhibitor-1 (PAI-1), or the thrombin inhibitory combination of heparin and anti-thrombin III (AT III) to the cells at the time of scraping blocked about 50% of the increase in bFGF mRNA; the effects of these agents were not additive. The suppression of bFGF mRNA was associated with a proportional reduction in bFGF protein. Inclusion of the antagonists for 2 h at the time of scraping led to reduced cell proliferation, suggesting that cell-associated bFGF may be required for recovery and growth. Finally, studies to characterize the molecular mechanisms underlying the increased bFGF mRNA following sublethal injury revealed an increase in the transcriptional activation of bFGF gene. These results indicate that in spite of the fact that bFGF is not a secreted protein, levels of bFGF in the cell are tightly regulated. Furthermore, these findings suggest a role for bFGF in recovery from cell injury.
碱性成纤维细胞生长因子（bFGF；FGF - 2）缺乏信号序列，因此不会通过经典途径分泌。据推测，bFGF释放的一种方式可能是损伤，无论是亚致死性损伤还是致死性损伤；并且，已经证明质膜的短暂破坏会释放bFGF[穆图克里南等人（1991年）：《细胞生理学杂志》148:1 - 16]。这一观察结果引出了bFGF作为“伤口激素”的概念，它参与组织完整性和修复。体内损伤后bFGF升高的研究结果支持了这一概念。我们使用体外模型研究了亚致死性损伤释放bFGF后其基因表达的调控。通过酶联免疫吸附测定（ELISA）分析bFGF蛋白发现，刮擦亚汇合的牛主动脉内皮细胞（BAE）可释放高达80%的bFGF。刮擦后，bFGF mRNA的稳态水平增加了4至10倍，在2 - 3小时达到最大值。蛋白质水平也有相应增加，以至于在刮擦诱导释放后6小时，bFGF水平恢复到刮擦前测量的水平。由于据报道bFGF可诱导其自身表达，我们推测释放的bFGF可能是bFGF mRNA增加的原因。然而，加入抗bFGF中和抗体对刮擦诱导的bFGF mRNA水平增加的影响可忽略不计。鉴于转化生长因子β1（TGF - β1）、纤溶酶原/纤溶酶原激活剂系统和凝血酶在伤口愈合中的重要作用，我们研究了它们对bFGF表达增加的潜在贡献。在刮擦时向细胞中加入抗TGF - β1抗体、纤溶酶原激活剂抑制剂 - 1（PAI - 1）或肝素与抗凝血酶III（AT III）的凝血酶抑制组合可阻断约50%的bFGF mRNA增加；这些试剂的作用不是相加的。bFGF mRNA的抑制与bFGF蛋白的相应减少相关。在刮擦时加入拮抗剂2小时导致细胞增殖减少，这表明细胞相关的bFGF可能是恢复和生长所必需的。最后，表征亚致死性损伤后bFGF mRNA增加背后分子机制的研究表明bFGF基因的转录激活增加。这些结果表明，尽管bFGF不是分泌蛋白，但细胞内bFGF的水平受到严格调控。此外，这些发现表明bFGF在细胞损伤恢复中发挥作用。
