Autocrine growth inhibitors of keratinocytes and their significance in carcinogenesis

(1) TGF-β途径限制性SMAD的功能：我们鉴定了Smad2的剪接变体，其省略了外显子3。当使用p3TP-lux作为报告基因时，Smad2DELTAexon3的转录活性比野生型Smad2更有效，并且与Smad3相似。Smad2DELTAexon3具有与p3TP启动子中的Smad结合元件结合的活性，Smad3可以结合该元件，但Smad2不能。这些结果表明Smad2外显子3编码的肽干扰了Smad2与DNA的直接结合。我们还发现HaCaT细胞中TGF-β同时激活Smad2和Smad3，激活素激活Smad3的效果与TGF-β一样强，但激活素对Smad2的激活不如TGF-β激活。(2)突变型Smad3对TGF-β信号的抑制：在结肠癌病例中发现的 Smad2 突变是在 Smad3 的相应残基上重建的。该突变体 Smad3D407E 对 TGF-β 信号具有显性负作用。Smad3D407E 的表达赋予 HaCaT 细胞侵袭性生长潜力以及对 TGF-β 生长抑制作用的抵抗力。(3) 癌细胞中 TGF-β 信号的破坏：甲状腺乳头状微腺癌患者原发灶中TGF-β染色阳性的患者远处转移发生率高于阴性病例。(4)TGF-β信号的靶基因：c-Myc被鉴定为角化细胞中TGF-β生长抑制信号中SMADs的重要靶标。我们还鉴定了一个新的TGF-β早期反应基因。该基因编码19 kDa的核蛋白，等电点为10.9。(5) TGF-β的生理功能TGF-β信号：TGF-β被认为参与角膜伤口愈合过程中的上皮细胞迁移。

(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.