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Elucidating the mechanism of regulation of transforming growth factor ? Type II receptor expression in human lung cancer cell lines.

ABSTRACT: Lung carcinogenesis in humans involves an accumulation of genetic and epigenetic changes that lead to alterations in normal lung epithelium, to in situ carcinoma, and finally to invasive and metastatic cancers. The loss of transforming growth factor ? (TGF-?)-induced tumor suppressor function in tumors plays a pivotal role in this process, and our previous studies have shown that resistance to TGF-? in lung cancers occurs mostly through the loss of TGF-? type II receptor expression (T?RII). However, little is known about the mechanism of down-regulation of T?RII and how histone deacetylase (HDAC) inhibitors (HDIs) can restore TGF-?-induced tumor suppressor function. Here we show that HDIs restore T?RII expression and that DNA hypermethylation has no effect on T?RII promoter activity in lung cancer cell lines. TGF-?-induced tumor suppressor function is restored by HDIs in lung cancer cell lines that lack T?RII expression. Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by either activated Ras or epidermal growth factor signaling is involved in the down-regulation of T?RII through histone deacetylation. We have immunoprecipitated the protein complexes by biotinylated oligonucleotides corresponding to the HDI-responsive element in the T?RII promoter (-127/-75) and identified the proteins/factors using proteomics studies. The transcriptional repressor Meis1/2 is involved in repressing the T?RII promoter activity, possibly through its recruitment by Sp1 and NF-YA to the promoter. These results suggest a mechanism for the downregulation of T?RII in lung cancer and that TGF-? tumor suppressor functions may be restored by HDIs in lung cancer patients with the loss of T?RII expression.

SUBMITTER: Halder SK

PROVIDER: S-EPMC3201568 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Elucidating the mechanism of regulation of transforming growth factor β Type II receptor expression in human lung cancer cell lines.

Halder Sunil K SK Cho Yong-Jig YJ Datta Arunima A Anumanthan Govindaraj G Ham Amy-Joan L AJ Carbone David P DP Datta Pran K PK

Neoplasia (New York, N.Y.) 20111001 10

Lung carcinogenesis in humans involves an accumulation of genetic and epigenetic changes that lead to alterations in normal lung epithelium, to in situ carcinoma, and finally to invasive and metastatic cancers. The loss of transforming growth factor β (TGF-β)-induced tumor suppressor function in tumors plays a pivotal role in this process, and our previous studies have shown that resistance to TGF-β in lung cancers occurs mostly through the loss of TGF-β type II receptor expression (TβRII). Howe ...[more]

PMID: 22028617

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Project description:TGF-beta (transforming growth factor-beta) is implicated in the pathogenesis of diabetic nephropathy. We previously demonstrated that up-regulation of type II TGF-beta receptor (TbetaRII) induced by high glucose might contribute to distal tubular hypertrophy [Yang, Guh, Yang, Lai, Tsai, Hung, Chang and Chuang (1998) J. Am. Soc. Nephrol. 9, 182-193]. We have elucidated the mechanism by using cultured Madin-Darby canine kidney cells. Enhancer assay and electrophoretic-mobility-shift assay were used to estimate the involvement of transcription factors. Western blotting and an in vitro kinase assay were used to evaluate the level and activity of protein kinase. We showed that glucose (100-900 mg/dl) induced an increase in mRNA level and promoter activity of TbetaRII (note: 'mg/dl' are the units commonly used in diabetes studies). The promoter region -209 to -177 appeared to contribute to positive transactivation of TbetaRII promoter by comparing five TbetaRII-promoter-CAT (chloramphenicol acetyl-transferase) plasmids. Moreover, the transcription factor AP-1 (activator protein 1) was significantly activated and specifically binds to TbetaRII promoter (-209 to -177). More importantly, we found that atypical PKC iota might be pivotal for high glucose-induced increase in both AP-1 binding and TbetaRII promoter activity. First, high glucose induced cytosolic translocation, activation and autophosphorylation of PKC iota. Secondly, antisense PKC iota expression plasmids attenuated high-glucose-induced increase in AP-1 binding and TbetaRII promoter activity; moreover, sense PKC iota expression plasmids enhanced these instead. Finally, we showed that antisense PKC iota expression plasmids might partly attenuate a high-glucose/TGF-beta1-induced increase in fibronectin. We conclude that PKC iota might mediate high-glucose-induced increase in TbetaRII promoter activity. In addition, antisense PKC iota expression plasmid effectively suppressed up-regulation of TbetaRII and fibronectin in hyperglycaemic distal-tubule cells.

Project description:The response to transforming growth factor-? (TGF-?) depends on cellular context. This context is changed in mitosis through selective inhibition of vesicle trafficking, reduction in cell volume and the activation of mitotic kinases. We hypothesized that these alterations in cell context may induce a differential regulation of Smads and TGF-? receptors. We tested this hypothesis in mesenchymal-like ovarian cancer cells, arrested (or not) in mitosis with 2-methoxyestradiol (2ME2). In mitosis, without TGF-? stimulation, Smad3 was phosphorylated at the C-terminus and linker regions and localized to the mitotic spindle. Phosphorylated Smad3 interacted with the negative regulators of Smad signaling, Smurf2 and Ski, and failed to induce a transcriptional response. Moreover, in cells arrested in mitosis, Smad3 levels were progressively reduced. These phosphorylations and reduction in the levels of Smad3 depended on ERK activation and Mps1 kinase activity, and were abrogated by increasing the volume of cells arrested in mitosis with hypotonic medium. Furthermore, an Mps1-dependent phosphorylation of GFP-Smad3 was also observed upon its over-expression in interphase cells, suggesting a mechanism of negative regulation which counters increases in Smad3 concentration. Arrest in mitosis also induced a block in the clathrin-mediated endocytosis of the type II TGF-? receptor (T?RII). Moreover, following the stimulation of mitotic cells with TGF-?, the proteasome-mediated attenuation of TGF-? receptor activity, the degradation and clearance of T?RII from the plasma membrane, and the clearance of the TGF-? ligand from the medium were compromised, and the C-terminus phosphorylation of Smad3 was prolonged. We propose that the reduction in Smad3 levels, its linker phosphorylation, and its association with negative regulators (observed in mitosis prior to ligand stimulation) represent a signal attenuating mechanism. This mechanism is balanced by the retention of active TGF-? receptors at the plasma membrane. Together, both mechanisms allow for a regulated cellular response to TGF-? stimuli in mitosis.

Dataset Information

Elucidating the mechanism of regulation of transforming growth factor ? Type II receptor expression in human lung cancer cell lines.

Publications

Elucidating the mechanism of regulation of transforming growth factor β Type II receptor expression in human lung cancer cell lines.

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