In vivo disruption of TGF-beta signaling by Smad7 leads to premalignant ductal lesions in the pancreas.

TGF-beta has been postulated to play an important role in the development of pancreatic cancers. More than 50% of human pancreatic cancers bear mutations of Sma- and Mad-related protein (Smad) 4, a critical protein required for TGF-beta signaling. To evaluate the in vivo function of TGF-beta in the development of pancreatic cancers, we generated a transgenic mouse model with pancreas-specific expression of Smad7, a specific inhibitor of TGF-beta signaling. Through the use of elastase I promoter, we directed the tissue specific expression of exogenous Smad7. Consistently, the exogenous Smad7 was detected only in the pancreas in the transgenic mice, and, furthermore, phosphorylation of Smad2 was blocked in the pancreatic tissues. At 6 months of age, most transgenic animals developed premalignant ductal lesions in the pancreas, with characteristics of pancreatic intraepithelial neoplasia (PanIN), a precursor to invasive pancreatic cancers. The premalignant lesions of the pancreas were accompanied by accelerated proliferation of the ductal epithelium and acinar cells, as well as increased fibrosis around the ductal lesions. This study not only demonstrated that in vivo inactivation of TGF-beta signaling is implicated in the development of early stage of pancreatic cancers, but also provided a promising animal model useful for the investigation and intervention of pancreatic cancers in humans.

Arsenite induces a cell stress-response gene, RTP801, through reactive oxygen species and transcription factors Elk-1 and CCAAT/enhancer-binding protein.

RTP801 is a newly discovered stress-response gene that is induced by hypoxia and other cell stress signals. Arsenic is a heavy metal that is linked to carcinogenesis in humans. Here, we investigated the mechanism by which arsenic induces RTP801 transcription. In HaCaT human keratinocytes, arsenite was able to induce a rapid rise in the RTP801 mRNA level. Correspondingly, arsenite treatment was capable of stimulating a 2.5 kb human RTP801 promoter. Such a stimulatory effect was inhibited by co-expression of superoxide dismutase or glutathione peroxidase, and was abrogated by N-acetylcysteine, implying that ROS (reactive oxygen species) were involved in transcriptional regulation of the RTP801 gene. A series of deletion studies with the promoter revealed a critical arsenic-responsive region between -1057 and -981 bp of the promoter. Point mutations of the putative Elk-1 site and the C/EBP (CCAAT/enhancer-binding protein) site within this region were able to reduce the stimulatory effect of arsenite, indicating that Elk-1 and C/EBP are involved in transcriptional regulation of the RTP801 gene by arsenite. Furthermore, a gel mobility-shift assay demonstrated that arsenite was able to mount the rapid formation of a protein complex that bound the arsenic-responsive region as well as the C/EBP-containing sequence. The arsenite stimulation on RTP801 transcription was partly mediated by the ERK (extracellular-signal-regulated kinase) pathway, since the effect of RTP801 was inhibited by a selective ERK inhibitor. In addition, overexpression of Elk-1 and C/EBPbeta was able to elevate the promoter activity. Therefore these studies indicate that RTP801 is a transcriptional target of arsenic in human keratinocytes, and that arsenic and ROS production are linked to Elk-1 and C/EBP in the transcriptional control.

Modulation of cord blood CD8+ T-cell effector differentiation by TGF-beta1 and 4-1BB costimulation.

Transforming growth factor-beta1 (TGF-beta1), an immunosuppressive cytokine, inhibits cytotoxic T cell (CTL) immune responses. In contrast, 4-1BB (CD137), a costimulatory molecule in the tumor necrosis factor (TNF) receptor family, amplifies CTL-mediated antitumor immune responses. We investigated whether TGF-beta1 responses could be reversed by 4-1BB costimulation during in vitro differentiation of naive CD8+ T cells into effector CTL cells. TGF-beta1 potently suppressed CTL differentiation of human cord blood naive CD8+ T cells as determined by reduced induction of characteristic phenotypes of effector cells and cytotoxic activity. TGF-beta1-mediated suppression of CTL differentiation was abrogated by 4-1BB costimulation but not by CD28 or another member in the TNF receptor family, CD30. 4-1BB costimulation suppressed Smad2 phosphorylation induced by TGF-beta1, suggesting that 4-1BB effects were at the level of TGF-beta1 signaling. 4-1BB effects on the TGF-beta1-mediated suppression were enhanced by interleukin 12 (IL-12) but counteracted by IL-4; 4-1BB expression was up- or down-regulated, respectively, by IL-12 and IL-4. IL-4 was more dominant than IL-12 when both cytokines were present during 4-1BB costimulation in the presence of TGF-beta1. This indicates critical roles for IL-4 and IL-12 in regulating 4-1BB effects on TGF-beta1-mediated suppression.

Repression of Smad2 and Smad3 transactivating activity by association with a novel splice variant of CCAAT-binding factor C subunit.

Activation by transforming growth factor-beta (TGF-beta)/activin receptors leads to phosphorylation of Smad2 (Sma- and Mad-related protein 2) and Smad3, which function as transcription factors to regulate gene expression. Using the MH2 domain (Mad homologue domain of Smad proteins 2) of Smad3 in a yeast two-hybrid screening, we isolated a novel splice variant of CAATT-binding factor subunit C (CBF-C), designated CBF-Cb, that associated with Smad3. CBF-C is one of the subunits that form a heterotrimeric CBF complex capable of binding and activating the CAATT motif found in the promoters of many eukaryotic genes. CBF-Cb is 62 amino acids shorter than the wild-type CBF-C in the N-terminal region. In addition, CBF-Cb is expressed ubiquitously in various mouse tissues. By an immunoprecipitation assay, we detected an in vivo association of CBF-Cb with Smad2 and Smad3, independent of signalling by activated TGF-beta type I receptors. In transient transfection experiments, overexpression of CBF-Cb was able to repress the transactivating activity of Smad2 and Smad3, mediated either by direct binding to the Smad-responsive element or through their association with the Smad-interacting transcription factor FAST-2 (forkhead activin signal transducer-2). The Smad-mediated transcriptional response after TGF-beta receptor activation was also inhibited by overexpression of unspliced CBF-C. In addition, the repressive activity of CBF-Cb on Smad2- and Smad3-mediated transcriptional regulation was abrogated by co-expression of the general transcription activator p300. The association of CBF-Cb with Smad2 was competitively inhibited by overexpression of p300. These data indicate a novel mechanism for modulation of the transcriptional activity of Smad proteins, whereby the interaction of CBF-Cb, as well as canonical CBF-C, with the MH2 domain of Smads may prevent the association of Smads with transcriptional co-activators.