Crosstalking Of TGF-β1/Smad4 And Ras-MAPK(ERK2) Pathway In Rhabdomyosarcoma

Transforming growth factor-β(TGF-β) is a member of a large superfamily of cytokines that plays an essential role in the regulation of normal physiologic processes such as cell proliferation, differentiation, apoptosis, extracellular matrix(ECM) production, migration, adhesion and embryogenesis. In addition, it is of key biological importance in the pathogenesis of fibrosis and tumorigenesis. Many of the mechanisms defined for TGF-β-induced postreceptor signaling have focused on early immediate signaling molecules such as Smads, members of the Rac-Ras family of GTPases as well as members of the Src family of kinases. Among the several signaling components, Smads have been well studied. So, at its simplest, the basic TGF-β signaling engine consists of a receptor complex that activates Smads and a Smad complex that controls transcription. Mitogen-activated protein kinases(MAPK) compose a family of protein kinases whose function and regulation have been conserved during evolution from unicellular organisms to complex organisms including humans. Extracellular stimulationcan be transduced to nucleus to regulate cellular activaties. Moreover, the extracellular-signal regulated protein kinase(ERKs) was found to play a vital role in the responses of many cell types to growth factors and other mitogens, which would provoke both proliferation and malignant differentiation when persistent activated. In epithelial cells, TGF-β have been shown to regulate several MAP kinase pathways, but the mode of activation appears to be highly variable and cell type-specific. However, the effects of TGF-β on soft tissue sarcoma were seldom reported.Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence, which is a class of myoblast-derived solid tumors expressing some muscle-specific markers, with a 10-20% morbidity and incidence lower than leukemia. It has been shown that various autocrine growth factor, such as IGF, bFGF, EGF, can be expressed by RMS cells abnormally and are likely to be involved in the growth of cancer cells in vitro. Better understanding of the molecular mechanism about how RMS cells is developed and differentiated may ultimately have highly diagnostic and therapeutic implications.In our previous study, we have identified that TGF-β1 and down-stream molecules-Smads were highly expressed in both RMS and human embryonal rhabdomyosarcoma cell RD. RD also autocrined TGF-β1 and expressed TβRs and Smads. It has been identified that TGF-β1/Smad signaling dose function in RD. More importantly, the obstruction of endogenous TFG-β1/Smad signaling can inhibit growth of RD and induce apoptosis of RD. It was suggested physiological TGF-β1/Smad signal is indispensable to normal growth of RD, or there should be some other signal transduction pathway besides Smads which is important to RD growth. In thepresent study, We examined the expression and functions of ERK2 and Smad4 in human RMS tissue and RMS cell line RD. The results were summarized as follows:1. RD cell and myoblast(Mb) were studied to establish the profile of expression of ERK2 at the mRNA and protein levels using RT-PCR and immunofluorescent. ERK2 protein and ERK2, Smad4 mRNA were shown expressed at higher level in RD cell than in Mb, indicating that TGF-/31/Smad pathway and Ras-MAPK(ERK2) pathway may participate in development and progression of RMS.2. The signal transduction stimulated by TGF-/31 involved in growth regulation of RD cells was investigated by RT-PCR and immunofluorescent. Exogenous TGF-/?l(5ng/ml) showed a growth inhibitory effect in RD after supplying for 24 hours. And immunofluorescent staining exhibited a stronger staining pattern of ERK2 in nucleus than unsettled RD, indicating the nuclear translocation of ERK2. With RT-PCR, the ERK2 mRNA expression was definitely increased after 24h, then declined gradually after 48h and 72h. All this further suggested that, in RD, there exist the activation of Ras-MAPK(ERK2) pathway by TGF-jSl, and it may be unrelated to Smads pathway. Then, RD was supplied with exogenous TGF-/31(10ng/ml). It was showed that ERK2 mRNA ascended after 4h, and became the peak value when 6h, then descended after 8h, ascended again when 12h, then declined after 24h, and came to near normal while 72h. This mode of MAPK activation differs from other reported cells.3. Immunohistochemistry was used to detect the expression of ERK2 and Smad4 in 50 cases of RMS and 23 normal skeletal muscles. Increased expression of ERK2 and Smad4 were found in RMS compared with normalskeletal muscle with significant difference, localized in cytoplasm and nucleus. It was further indicated that TGF-/31/Smad pathway and Ras-MAPK(ERK2) pathway may participate in development and progression of RMS simultaneously. There was no correlation found between ERK2 and Smad4, suggesting that the activation by TGF-/31 of Ras-MAPK(ERK2) may be Smad4 independent. Clinically, the overexpression of ERK2 and Smad4 has no indicative effect in histological subtypes, histological grading, gender, ages and prognosis.In summary, our data shows that in RMS, signalling of TGF-/31 from the cell surface to the nucleus can also through Ras-MAPK(ERK2) pathway besides TGF-/31/Smads pathway. However, the activation of Ras-MAPK(ERK2) by TGF-/31 may be Smad4 independent and the two pathways may take part in the development of RMS respectively. And physiological Ras-MAPK(ERK2) signal may also be indispensable to normal growth of RD. Moreover, there may present some tanglesome relationships between the pathways after TGF-/31, which involved in the growth and progression of RMS. The experiments lay the good foundations of continuously studying the precise relationship of the two pathways and supplies a new insight for the anticipative RMS gene therapy based on the bidirectional effect of TGF-/31 on the growth of tumor cells.