Mechanisms of TGFbeta-mediated fibroblastic transition

转化生长因子β (tgfβ)家族分泌因子在生物系统的发育、稳态和功能中起着重要作用。许多人类疾病都与tgf - β信号网络的异常运作有关。tgf β表现出肿瘤抑制活性，其信号通路成分在癌症中经常发生突变或沉默。然而，TGFbeta通过刺激上皮到间质转化（EMT）和细胞迁移来促进肿瘤的进展和侵袭性。EMT是一个复杂的过程，与上皮细胞接触的改变、细胞形态的改变、细胞骨架的重组、成纤维细胞标记物（纤连蛋白、波形蛋白）的表达和细胞迁移的增强有关。tgf - β的这些作用也涉及到各种组织的伤口修复和纤维化。tgf - β诱导EMT和细胞迁移的机制尚不清楚。我们做了重要的观察，tgf β介导的EMT和细胞迁移被p38丝裂原活化蛋白激酶（p38Mapk）抑制剂破坏，而不抑制tgf β的抗增殖活性。因此，p38Mapk通路可能在TGFbeta的促肿瘤活性中发挥关键作用。我们还观察到p38Mapk的激活是由rho家族GTPases介导的。tgf β向rho样GTPases和p38Mapk发出信号的机制尚未明确。我们假设，由rho家族GTPases介导的激酶活性tgf β受体激活p38Mapk通路是建立细胞表型变化程序的必要条件，从而导致tgf β诱导的成纤维细胞转化和细胞迁移。这一假设将通过以下具体目标进行检验。目的1：研究tgfβ受体激酶功能在rho样gtpase激活和p38Mapk通路中的作用。目的2：在体外检测p38Mapk激活激酶MKK3和MKK6显性阴性和组成活性突变体对tgf β诱导的EMT和细胞迁移的影响。目的3：在小鼠模型中评价p38Mapk通路在肿瘤细胞运动、侵袭性和转移中的作用。本研究将有助于明确TGFbeta信号网络，并有助于设计新的选择性抑制TGFbeta促肿瘤活性的方法。

The transforming growth factor beta (TGFbeta) family of secreted factors play important role(s) in development, homeostasis and functioning of biological systems. A number of human diseases are associated with abnormal operation of the TGFbeta signaling network. TGFbeta exhibits a tumor suppressor activity and components of its signaling pathway are frequently mutated or silenced in cancers. However, TGFbeta contributes to the progression and invasiveness of tumors by stimulating an epithelial to mesenchymal transition (EMT) and cell migration. EMT is a complex process associated with alterations in epithelial cell contacts, changes in cell morphology, reorganization of the cell cytoskeleton, expression of fibroblastic markers (fibronectin, vimentin) and enhancement of cell migration. These effects of TGFbeta are also involved in wound repair and fibrosis of various tissues. The mechanism(s) of TGFbeta-induced EMT and cell migration are not well understood. We made the important observation that TGFbeta- mediated EMT and cell migration are impaired by inhibitors of p38 mitogen activated protein kinase (p38Mapk), without inhibition of the antiproliferative activity of TGFbeta. Thus, the p38Mapk pathway may play a critical role in TGFbeta's tumor promoting activity. We also observed that p38Mapk activation is mediated by Rho-family GTPases. The mechanism by which TGFbeta signals to Rho-like GTPases and p38Mapk has not yet been defined. We hypothesize that activation of the p38Mapk pathway by kinase- active TGFbeta receptors mediated by Rho-family GTPases is required for setting up a program of changes in the cell phenotype leading to TGFbeta-induced fibroblastic transition and cell migration. This hypothesis will be tested by the following specific aims. Aim 1: To examine the role of kinase function of TGFbeta receptors in activation of Rho-like GTPases and the p38Mapk pathway. Aim 2: To examine in vitro the effect of dominant negative and constitutively active mutants of MKK3 and MKK6, p38Mapk activating kinases, on the TGFbeta-induced EMT and cell migration. Aim 3: To evaluate the role of the p38Mapk pathway in tumor cell motility, invasiveness and metastasis in mouse models. The proposed research will help to define the TGFbeta signaling network and will facilitate the design of novel selective inhibition of TGFbeta's tumor promoting activity.