Analysis of the function of TOB (transducer of Erb) on proliferation and differentiation of keratinocytes

已发现TOB（Transducer of Erb）是与EGF受体家族成员ErbB 2结合的因子。TOB家族由6个同源成员组成。已有研究表明，TOB在细胞停滞期具有活性，其表达受细胞周期调控，EGF受体磷酸化诱导TOB表达，组成型活性TOB过表达降低cyclin D1表达。本研究的目的是探讨TOB是否参与表皮生长因子家族相关的正常人角质形成细胞的自诱导和交叉诱导机制。使用针对每个TOB家族成员的特异性引物的RT-PCR分析显示角质形成细胞表达TOB家族mRNA。EGF刺激以剂量依赖性方式上调TOB mRNA表达。提示TOB对EGF诱导角质形成细胞增殖可能存在负反馈机制。为了进一步阐明TOB在EGF诱导的角质形成细胞生长中的作用，我们构建了Ax-TOB、AxTOB 3A和AxTOB 3SE等腺病毒载体。AxTOB 3A是作为组成型活性TOB的突变形式。AxTOB 3SE是TOB的显性-阴性形式。用腺病毒载体转染角质形成细胞，从RNA和蛋白水平分析细胞周期和分化标志物。组成型活性形式的TOB阻滞细胞周期并诱导角质形成细胞分化。另一方面，显性阴性TOG增加S期细胞，抑制分化标志物的表达。我们还建立了新生TOB基因敲除小鼠的角质形成细胞。我们得出结论，TOB是角质形成细胞生长抑制的重要因素。

TOB (Transducer of Erb) has been discovered as a factor that binds to ErbB2, which is a member of EGF receptor family. TOB family consists of 6 homologous members. It has been reported that TOB is active at cell arrest stage, its expression is regulated by cell cycle, EGF receptor phosphorylation induces TOB expression, constitutive active TOB over-expression decreases cyclin D1 expression. The purpose of this study is to investigate whether TOB is involved in EGF family-related auto- and cross- induction mechanism in normal human keratinocytes. RT-PCR analysis using specific primers for each TOB family member showed that keratinocytes express TOB family mRNA. EGF stimulation upregulated TOB mRNA expression in a dose dependent manner. These results indicate that there might be a negative feedback mechanism by TOB about EGF induced proliferation in keratinocytes. To further clarify the role of TOB on EGF-induced keratinocyte growth, we constructed adenovirus vector such as Ax-TOB, AxTOB3A and AxTOB3SE. AxTOB3A is a mutant form which acts as constitutive active TOB. AxTOB3SE is a dominant -negative form of TOB. Keratinocytes were transfected with these adenovirus vectors, cell cycle and differentiation markers were analyzed at RNA level as well as protein level. Constitutive active form of TOB arrested cell cycle and induced keratinocyte differentiation. On the other hand, dominant-negative form of TOG increased S-phase cells and inhibited the expression of differentiation markers. We also established the keratinocytes from new born TOB knockout mice. We conclude that TOB is an important factor for keratinocyte-growth inhibition.