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.

Cyclic AMP (cAMP)-dependent protein kinase (PKA) and ribosomal S6 kinase 1 (RSK1) share several cellular proteins as substrates. However, to date no other similarities between the two kinases or interactions between them have been reported. Here, we describe novel interactions between subunits of PKA and RSK1 that are dependent upon the activation state of RSK1 and determine its subcellular distribution and biological actions. Inactive RSK1 interacts with the type I regulatory subunit (RI) of PKA. Conversely, active RSK1 interacts with the catalytic subunit of PKA (PKAc). Binding of RSK1 to RI decreases the interactions between RI and PKAc, while the binding of active RSK1 to PKAc increases interactions between PKAc and RI and decreases the ability of cAMP to stimulate PKA. The RSK1/PKA subunit interactions ensure the colocalization of RSK1 with A-kinase PKA anchoring proteins (AKAPs). Disruption of the interactions between PKA and AKAPs decreases the nuclear accumulation of active RSK1 and, thus, increases its cytosolic content. This subcellular redistribution of active RSK1 is manifested by increased phosphorylation of its cytosolic substrates tuberous sclerosis complex 2 and BAD by epidermal growth factor along with decreased cellular apoptosis.Cyclic AMP (cAMP)-dependent protein kinase (PKA) regulates a wide variety of metabolic and functional processes, including cell proliferation, actin cytoskeleton rearrangements, and gene transcription (41). PKA is a tetramer that consists of two regulatory (R) and two catalytic (PKAc) subunits in its inactive form. Binding of cAMP to the R subunits of PKA results in the dissociation of the PKAc subunits, which can then phosphorylate their substrate proteins. The PKA holoenzyme is localized in the proximity of its target proteins by A-kinase PKA anchoring proteins (AKAPs) that act as scaffolds (2, 52). Moreover, by binding to additional signaling molecules, the AKAPs facilitate the coordination and integration of several signals to regulate biological events (2, 52). The two main forms of the PKA regulatory subunits, RI and RII, have different affinities for the various AKAPs. Although most of the AKAPs appear to have a higher affinity for RII subunits (2), certain AKAPs such as D-AKAP1 and D-AKAP2 preferentially bind the RI subunit (22-24).In addition to AKAPs, other well-characterized protein interactions are involved in the intracellular targeting and regulation of PKA and its subunits. For instance, RI␣ associates with cytochrome c oxidase subunit Vb and increases its activity whereas exposure to cAMP inhibits cytochrome c oxidase activity and releases cytochrome c from mitochondria and activates apoptosis (55). PAP7, a protein involved in hormonal regulation of steroid formation, also binds RI␣ at the outer mitochondrial membrane and helps in cholesterol uptake and transport to the inner mitochondrial membrane (28). Likewise, Rab32, a small GTP binding protein, can act as an AKAP for the RII subunits and target PKA to mitochondria (3). In addition, the amount of RI...

TGF-␤ 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-␤ signaling. To evaluate the in vivo function of TGF-␤ in the development of pancreatic cancers, we generated a transgenic mouse model with pancreas-specific expression of Smad7, a specific inhibitor of TGF-␤ 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-␤ 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.mouse model ͉ pancreatic cancer ͉ pancreatic intraepithelial neoplasia ͉ fibrosis ͉ Sma-and Mad-related protein 2