Autocrine growth inhibitors of keratinocytes and their significance in carcinogenesis

(1)TGF-β通路限制性SMAD的功能：我们鉴定了一个缺失外显子3的Smad 2剪接变异体。当p3 TP-lux被用作转录因子时，Smad 2DELTAexon 3具有比野生型Smad 2更强的转录活性，并且与Smad 3相似。Smad 2DELTAexon 3具有与p3 TP启动子中的Smad结合元件结合的活性，这些结果表明Smad 2外显子3编码的多肽干扰了Smad 2与DNA的直接结合。我们还发现TGF-β在HaCaT细胞中同时激活了Smad 2和Smad 3，激活素对Smad 3的激活作用与TGF-β相同，但激活素对Smad 2的激活作用弱于TGF-β。(2)Smad 3突变体对TGF-β信号的抑制作用：在一例结肠癌中发现的Smad 2突变体被改造为Smad 3的相应残基，该突变体Smad 3D 407 E对TGF-β信号具有显性负效应，Smad 3D 407 E的表达赋予HaCaT细胞侵袭性生长潜能，并对TGF-β的生长抑制作用产生抵抗。(3)癌细胞中TGF-β信号的破坏：原发灶中TGF-β染色阳性的乳头状甲状腺微腺癌患者的远处转移发生率高于阴性病例。(4)TGF-β信号的靶基因：c-Myc是SMADs抑制角质形成细胞TGF-β生长的重要靶基因，我们还发现了一个新的TGF-β早期反应基因，该基因编码一个分子量为19 kDa的核蛋白，等电点为10.9。(5)TGF-β信号的生理功能：TGF-β被认为在角膜伤口愈合过程中参与上皮细胞迁移。

(1) Functions of TGF-beta pathway-restricted SMADs : We identified a splice variant of Smad2 which omitted exon 3.Smad2DELTAexon3 had a transcriptional activity more potent than wild-type Smad2 and similar to Smad3, when p3TP-lux was used as a reporter.Smad2DELTAexon3 had an activity to bind to a Smad binding element in the p3TP promoter, which Smad3 can bind to but Smad2 can not.These results suggested that the peptide coded by exon 3 of Smad2 interfered with the direct DNA binding of Smad2.We also found that TGF-beta activated both Smad2 and Smad3 in HaCaT cells, and activin activated Smad3 as potent as TGF-beta did, but the activation of Smad2 by activin was less potent than that by TGF-beta.(2) Inhibition of TGF-beta signals by a mutant Smad3 : A mutation of Smad2 identified in a case of colon cancer was reconstructed on the corresponding residue of Smad3.This mutant Smad3D407E had a dominant negative effect on TGF-beta signals.Expression of Smad3D407E conferred invasive growth potential on HaCaT cells as well as resistance to the growth inhibitory effects of TGF-beta.(3) Disruption of TGF-beta signals in cancer cells : Patients of papillary thyroid microadenocarcinoma with a positive staining for TGF-beta in the primary lesions had higher incidence of distant metastasis than negative cases.(4) Target genes of TGF-beta signals : c-Myc was identified to be an essential target of SMADs in growth inhibitory signals of TGF-beta in keratinocytes.We also identified a new early responsive gene to TGF-beta.This gene coded a nuclear protein of 19 kDa with an isoelectric point of 10.9.(5) Physiological function of TGF-beta signals : TGF-beta was suggested to be involved in epithelial cell migration during the corneal wound healing process.