Differential roles of JNK and Smad2 signaling pathways in the inhibition of c-Myc-induced cell death by TGF-beta

The transforming growth factor beta (TGF-beta) plays an important role in constraining cellular proliferation, but it is also a potent inducer of programmed cell death or apoptosis. Here, we demonstrate that TGF-beta can have an opposite effect, acting as a survival factor to prevent c-Myc-induced cell death in Rat-1 fibroblasts. However, in marked contrast to TGF-beta, Smad2, which is a critical intracellular mediator of the TGF-beta signaling pathway, functions as an antagonist to induce increased cell death. The protective activity of TGF-beta was associated with the activation of c-Jun N-terminal Kinase (JNK) and was not linked to the ability of TGF-beta to promote cell cycle progression. Expression of dominant-interfering forms of various components of the JNK signaling pathway, including Rac1, Cdc42, mitogen-activated protein kinase kinase 4 (MKK4), and c-Jun, abolished TGF-beta-mediated cell survival. Furthermore, overexpression of the constitutively activated mutant RacL61F37A, which selectively stimulates JNK cascade but not G1 cell cycle progression or actin polymerization, was sufficient to prevent apoptosis induced by c-Myc. These findings describe a differential effect of two separated signaling pathways of TGF-beta and indicate for the first time that Smad2 can act as antagonist to suppress TGF-beta-dependent cell survival. Oncogene (2000) 19, 1277 - 1287.     

Mutational analysis of TGF-beta type II receptor,Smad2, Smad3, Smad4, Smad6 and Smad7 genes in colorectal cancer

Transforming growth factor-beta(TGF-beta) is known to play an important role in controlling embryonal development, cell proliferation and homeostasis. The purpose of this study is to elucidate the involvement of the TGF-beta pathway in colorectal carcinogenesis. DNA was extracted from 100 patients with colorectal cancer. Then, all coding regions of the TGF-beta type II receptor (TRII) and the genes for Smad2, Smad3, Smad4, Smad6, and Smad7 were analyzed by PCR-SSCP and direct sequencing. Also, a LOH analysis of 18q21, where the Smad2 and Smad4 genes are located, was performed. We detected 11 cases of frameshift mutation in the TRII gene (11%) and 5 cases of point mutations in the Smad4 gene (5.0%); LOH at 18q21 was detected with 33% frequency. No abnormalities were found in the genes for Smad2, Smad3, Smad6, and Smad7. These results suggest that the abnormalities of TRII and Smad4 play an important role inhibiting TGF-beta signaling in colorectal carcinogenesis.     

Mammary development and tumorigenesis in mice expressing a truncated human Notch4/Int3 intracellular domain (h-Int3sh)

Recently, we have identified a novel 1.8 kb human Notch4/Int3 RNA species (designated h-Int3sh). The h-Int3sh RNA encodes a protein that is missing the CBF1-binding region (RAM23) of the Notch 4/Int3 intracellular domain (ICD). Expression of h-Int3sh in the MCF10A 'normal' human mammary epithelial cell line has been previously shown to induce changes characteristic of oncogenic transformation, including anchorage-independent growth in soft agar. To study the consequences of h-Int3sh expression in vivo on mammary gland development and tumorigenesis, three transgenic mouse lines were established, in which the transgene is the Whey acidic protein (WAP) promoter linked to h-Int3sh. Expression of WAP-Int3sh was detectable in the mammary gland at day 15 of pregnancy in each transgenic line. Mammary gland development in all founder lines is normal and the females can lactate. WAP-h-Int3sh females from each of the founder lines develop mammary tumors, but with a long latency (average age of 18 months). Tumor development was associated with activation of Notch pathway, as evidenced by upregulation of Hes-1. The long latency of mammary tumors in WAP-h-Int3sh mice could be due in part to the subcellular localization of h-Int3sh. Immunofluorescence analysis of transfected COS-1 cells showed that h-Int3sh is localized in the cytoplasm and nucleus, while Int3-ICD is detected only in the nucleus. We speculate that the Notch4/Int3 ICD-induced block to mammary gland development and tumorigenesis are consequences of an increasing gradient of CBF1-dependent Notch4/Int3 signaling.     

Lateral inhibition of Notch signaling in neoplastic cells

During normal development, heterogeneous expression of Notch ligands can result in pathway suppression in the signal-sending cell, a process known as lateral inhibition. It is unclear if an analogous phenomenon occurs in malignant cells. We observed significant induction of Notch ligands in glioblastoma neurospheres and pancreatic carcinoma cells cultured in low oxygen, suggesting that this phenomenon could occur around hypoxic regions. To model lateral inhibition in these tumors, the ligand Jagged1 was overexpressed in glioblastoma and pancreatic carcinoma cells, resulting in overall induction of pathway targets. However, when ligand high and ligand low cells from a single line were co-cultured and then separated, we noted suppression of Notch pathway targets in the former and induction in the latter, suggesting that neoplastic lateral inhibition can occur. We also found that repression of Notch pathway targets in signal-sending cells may occur through the activity of a Notch ligand intracellular domain, which translocates into the nucleus. Understanding how this neoplastic lateral inhibition process functions in cancer cells may be important in targeting ligand driven Notch signaling in solid tumors.     

The type III TGF-beta receptor signals through both Smad3 and the p38 MAP kinase pathways to contribute to inhibition of cell proliferation

Transforming growth factor beta (TGFbeta) has an important role as a negative regulator of cellular proliferation. The type III transforming growth factor beta receptor (TbetaRIII) has an emerging role as both a TGFbeta superfamily co-receptor and in mediating signaling through its cytoplasmic domain. In L6 myoblasts, TbetaRIII expression enhanced TGFbeta1-mediated growth inhibition, with this effect mediated, in part, by the TbetaRIII cytoplasmic domain. The effects of TbetaRIII were not due to altered ligand presentation or to differences in Smad2 phosphorylation. Instead, TbetaRIII specifically increased Smad3 phosphorylation, both basal and TGFbeta-stimulated Smad3 nuclear localization and Smad3-dependent activation of reporter genes independent of its cytoplasmic domain. Conversely, SB431542, a type I transforming growth factor beta receptor (TbetaRI) inhibitor, as well as dominant-negative Smad3 specifically and significantly abrogated the effects of TbetaRIII on TGFbeta1-mediated inhibition of proliferation. TbetaRIII also specifically increased p38 phosphorylation, and SB203580, a p38 kinase inhibitor, specifically and significantly abrogated the effects of TbetaRIII/TGFbeta1-mediated inhibition of proliferation in L6 myoblasts and in primary human epithelial cells. Importantly, treatment with the TbetaRI and p38 inhibitors together had additive effects on abrogating TbetaRIII/TGFbeta1-mediated inhibition of proliferation. In a reciprocal manner, short hairpin RNA-mediated knockdown of endogenous TbetaRIII in various human epithelial cells attenuated TGFbeta1-mediated inhibition of proliferation. Taken together, these data demonstrate that TbetaRIII contributes to and enhances TGFbeta-mediated growth inhibition through both TbetaRI/Smad3-dependent and p38 mitogen-activated protein kinase pathways.     

Modulation of the functional binding sites for TGF-beta on the type II receptor leads to suppression of TGF-beta signaling

Transforming growth factor-beta (TGF-beta) binds to two different types of serine/threonine kinase receptors termed type II (TbetaR-II) and type I (TbetaR-I). TGF-beta is unable to bind to TbetaR-I in the absence of TbetaR-II, and initiates receptor assembly by binding with high affinity to TbetaR-II. Previous structural analysis of the TGF-beta3-TbetaR-II complex has suggested that two charged amino acid residues, D55 and E142 of TbetaR-II, are binding sites of TGF-beta. In the present study, we have shown that mutations of the amino-acid residues, D55 and E142 of TbetaR-II, resulted in loss of TGF-beta binding and downstream signaling activity. Moreover, we found that 3,5,7,2',4'-pentahydroxyflavone (Morin) inhibits TGF-beta binding to TbetaR-II, and suppresses phosphorylation of Smad2 and expression of a TGF-beta target gene Smad7 induced by TGF-beta. Our findings may thus provide useful information for designing therapeutic agents for various diseases induced by TGF-beta, including advanced cancers.     

Notch1 胞内段荧光表达质粒的构建及鉴定

目的：构建能够在鼻咽癌细胞株中高效表达人Notchl信号胞内段（NIC）的荧光质粒。方法：通过RT—PCR获得人NIC的cDNA;利用基因重组技术插入到pEGFP—N1中;在脂质体介导下转染人低分化鼻咽癌细胞CNE2后,经荧光显微镜、RT—PCR和Westernblot检测NIC的表达情况。结果：RT—PCR方法得到一条2424bp的特异性扩增产物,酶切和基因测序结果表明重组质粒构建成功。转染48h后,检测到绿色荧光表达,RT～PCR和Westernblot的结果显示NIC的表达量升高。结论：正确构建了人NIC荧光表达质粒。该质粒能够在鼻咽癌细胞株中高效表达,为研究Notch信号通路在鼻咽癌中的作用奠定了实验基础。     

Yes-associated protein (YAP65) interacts with Smad7 and potentiates its inhibitory activity against TGF-beta/Smad signaling

Members of the TGF-beta family of growth factors signal from the cell surface through serine/threonine kinase receptors. Intracellular propagation of the signal occurs by phosphorylation of intracellular proteins of the Smad family. Smad7 belongs to the subclass of inhibitory Smads that function as antagonists of TGF-beta signaling. A yeast two-hybrid screen of a human placental cDNA expression library using full-length mouse Smad7 as bait identified Yes-Associated Protein (YAP65) as a novel Smad7-interacting protein. The association of Smad7 with YAP65 was confirmed using co-expressed tagged proteins in COS-7 cells. Deletion of the PY motif of Smad7 reduced but did not abolish YAP65-Smad7 association, suggesting the existence of several interacting domains. We demonstrate that YAP65 potentiates the inhibitory activity of Smad7 against TGF-beta-induced, Smad3/4-dependent, gene transactivation. Furthermore, YAP65 augments the association of Smad7 to activated TGF-beta receptor type I (TbetaRI), whereas YAP65(1-301), which exerts a dominant-negative effect against Smad7-driven inhibition of TGF-beta signaling, reduces these interactions. Together, these data provide the first evidence that YAP65 is a Smad7 partner that facilitates the recruitment of the latter to activated TbetaRI, and enhances the inhibitory activity of Smad7 against TGF-beta signaling.     

Smad3 deficiency inhibits v-ras-induced transformation by suppression of JNK MAPK signaling and increased farnesyl transferase inhibition

The ability of transforming growth factor-beta (TGF-beta) to modulate various effects on distinct cell lineages has been a central feature of its multi-faceted nature. The purpose of this study was to access the effects of deletion of a key TGF-beta signal transducer, Smad3, on MAPK activation and v-Ras(Ha)-transformation of primary mouse embryonic fibroblasts (MEFs). We observe reduced TGF-beta1 and v-ras(Ha) mediated activation of the JNK and ERK MAPK pathway upon ablation of Smad3. Further, Smad3-deficient MEFs demonstrate resistance to v-ras(Ha)-induced transformation while the absence of Smad3 results in increased inhibition of farnesyl transferase activity. Taken together, these observations demonstrate that the absence of Smad3 protects fibroblasts from oncogenic transformation by (i) augmenting farnesyl transferase inhibition and (ii) suppressing the Ras-JNK MAPK pathway. These results provide new insights into the molecular mechanisms involved in v-Ras(Ha) oncogene-induced mesenchymal phenotypic transformation.     

CDK4 inhibition and doxorubicin mediate breast cancer cell apoptosis through Smad3 and survivin

Cyclin D1/CDK4 activity is upregulated in up to 50% of breast cancers and CDK4-mediated phosphorylation negatively regulates the TGFβ superfamily member Smad3. We sought to determine if CDK4 inhibition and doxorubicin chemotherapy could impact Smad3-mediated cell/colony growth and apoptosis in breast cancer cells. Parental and cyclin D1-overexpressing MCF7 cells were treated with CDK4 inhibitor, doxorubicin, or combination therapy and cell proliferation, apoptosis, colony formation, and expression of apoptotic proteins were evaluated using an MTS assay, TUNEL staining, 3D Matrigel assay, and apoptosis array/immunoblotting. Study cells were also transduced with WT Smad3 or a Smad3 construct resistant to CDK4 phosphorylation (5M) and colony formation and expression of apoptotic proteins were assessed. Treatment with CDK4 inhibitor/doxorubicin combination therapy, or transduction with 5M Smad3, resulted in a similar decrease in colony formation. Treating cyclin D overexpressing breast cancer cells with combination therapy also resulted in the greatest increase in apoptosis, resulted in decreased expression of anti-apoptotic proteins survivin and XIAP, and impacted subcellular localization of pro-apoptotic Smac/DIABLO. Additionally, transduction of 5M Smad3 and doxorubicin treatment resulted in the greatest change in apoptotic protein expression. Collectively, this work showed the impact of CDK4 inhibitor-mediated, Smad3-regulated tumor suppression, which was augmented in doxorubicin-treated cyclin D-overexpressing study cells.