Transforming growth factor-beta-induced growth inhibition in a Smad4 mutant colon adenoma cell line.

Transforming growth factor-beta (TGF-beta) inhibits growth and induces apoptosis of colon epithelial cells. Binding of TGF-beta to its receptor induces phosphorylation of the Smad proteins Smad2 and Smad3, which then form heteromeric complexes with Smad4, translocate to the nucleus, and activate gene transcription. Smad4 function has been considered an obligate requirement for TGF-beta signaling, and Smad4 mutations present in some cancers have been considered sufficient to inactivate TGF-beta signaling. In this work, we describe studies with a nontransformed human colon epithelial cell line that is mutant for Smad4 but remains growth-inhibited by TGF-beta. The colon cell line VACO-235 has lost one of its Smad4 alleles via a chromosome 18q deletion. The remaining allele bears two missense point mutations located in regions important for Smad4 trimer formation, which is thought necessary for Smad4 function. As expected, pSBE4-BV/Luc, a Smad4-activated transcriptional reporter, was inactive in VACO-235. Nonetheless, VACO-235 demonstrated 80% growth inhibition in response to TGF-beta, as well as retention of some TGF-beta-mediated activation of the p3TP-Lux transcriptional reporter. Transient transfection of the VACO-235 Smad4 mutant allele into a Smad4-null cell line confirmed that this allele is functionally inactive as assayed by both the pSBE4-BV and p3TP-Lux reporters. The simplest explanation of these results is that there is a non-Smad4-dependent pathway for TGF-beta-mediated signaling and growth inhibition in VACO-235 cells.

转化生长因子 -β（TGF -β）抑制结肠上皮细胞生长并诱导其凋亡。TGF -β与其受体结合会诱导Smad蛋白Smad2和Smad3磷酸化，它们随后与Smad4形成异聚体复合物，转移至细胞核并激活基因转录。Smad4的功能一直被认为是TGF -β信号传导的必要条件，并且一些癌症中存在的Smad4突变被认为足以使TGF -β信号传导失活。在这项工作中，我们描述了对一种未转化的人结肠上皮细胞系的研究，该细胞系存在Smad4突变，但仍受TGF -β生长抑制。结肠细胞系VACO - 235通过18号染色体q缺失丢失了一个Smad4等位基因。剩余的等位基因带有两个错义点突变，位于对Smad4三聚体形成很重要的区域，而Smad4三聚体形成被认为是Smad4功能所必需的。正如预期的那样，一种由Smad4激活的转录报告基因pSBE4 - BV/Luc在VACO - 235中无活性。然而，VACO - 235在TGF -β作用下表现出80%的生长抑制，并且保留了一些由TGF -β介导的p3TP - Lux转录报告基因的激活。将VACO - 235的Smad4突变等位基因瞬时转染到Smad4缺失的细胞系中，证实了该等位基因在pSBE4 - BV和p3TP - Lux报告基因检测中均无功能活性。对这些结果最简单的解释是，在VACO - 235细胞中存在一种不依赖Smad4的TGF -β介导的信号传导和生长抑制途径。