Cytokines manifest their function through alteration of gene expression. However, target genes for signals from cytokine receptors are largely unknown. We therefore searched for immediate‐early cytokine‐responsive genes and isolated a novel gene, CIS (cytokine inducible SH2‐containing protein) which is induced in hematopoietic cells by a subset of cytokines including interleukin 2 (IL2), IL3, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and erythropoietin (EPO), but not by stem cell factor, granulocyte colony‐stimulating factor and IL6. The CIS message encodes a polypeptide of 257 amino acids that contains an SH2 domain of 96 amino acids in the middle. To clarify the function of CIS in cytokine signal transduction, we expressed CIS in IL3‐dependent hematopoietic cell lines under the control of a steroid‐inducible promoter. The CIS product stably associated with the tyrosine‐phosphorylated beta chain of the IL3 receptor as well as the tyrosine‐phosphorylated EPO receptor. Forced expression of CIS by steroid reduced the growth rate of these transformants, suggesting a negative role of CIS in signal transduction. CIS induction requires the membrane‐proximal region of the cytoplasmic domain of the EPO receptor as well as that of the common beta chain of the IL3, IL5 and GM‐CSF receptor, whereas CIS binds to the receptor that is tyrosine phosphorylated by cytokine stimulation. Thus CIS appears to be a unique regulatory molecule for cytokine signal transduction.

Snail, a transcriptional repressor of E-cadherin expression, is involved in epithelial-mesenchymal transitions during development. We demonstrate that Snail activity is not restricted to E-cadherin downregulation. Expression of tight junction proteins, including claudin-1, occludin and ZO-1, was downregulated in MDCK cells exogenously expressing Snail protein. Although occludin mRNA levels were downregulated by Snail expression, the transcription of claudin-1 and ZO-1 were unaffected. Reporter assays using the claudin-1 promoter region revealed that promoter activity was not affected by Snail overexpression. Decreased synthesis of claudin-1 protein was observed, however, suggesting that Snail may act in translation initiation. Snail expression also altered the splicing pattern of p120. The levels of mRNA encoding the epithelial variant decreased, while the fibroblastic mRNA form increased. Although ectopic E-cadherin expression resulted in a downregulation of Snail-induced fibronectin expression, fibroblastic morphology was affected only minimally; the expression of tight junctional proteins remained at low levels. These results indicate that Snail is involved in both the direct transcriptional repression of genes, such as E-cadherin and occludin, and post-transcriptional events, including downregulation of claudin-1. These data support the idea that Snail is a transcription factor possessing pleiotropic activities.

Cadherins are transmembrane glycoproteins involved in Ca2؉ -dependent cell-cell adhesion. Previously, we showed that the conserved membrane-proximal region of the E-cadherin cytoplasmic domain negatively regulates adhesion activity. In this report, we provide several lines of evidence that p120 ctn is involved in this negative regulation. p120 ctn binds to the membraneproximal region of the nonfunctional carboxyl-terminally deleted E-cadherin protein. An additional internal deletion in this region prevented the association with p120 ctn and activated the protein, as seen in an aggregation assay. Furthermore, the nonfunctional E-cadherin can be activated through coexpression of p120 ctn proteins with amino-terminal deletions, which eliminate several potential serine/threonine phosphorylation sites but do not affect the ability to bind to cadherins. Finally, we show that staurosporine, a kinase inhibitor, induces an increased electrophoretic mobility of p120 ctn bound to E-cadherin polypeptides, activates the nonfunctional E-cadherin protein, and converts the wildtype E-cadherin and an E-cadherin-␣-catenin chimeric protein from a cytochalasin D-sensitive to a cytochalasin D-insensitive state. Together, these results indicate that p120 ctn is a modulator of E-cadherin-mediated cell adhesion.

A major functional component of the blood-brain barrier is P-glycoprotein. In principle, inhibition of this efflux transporter would permit greater distribution of its substrates into the brain and increased central effects. Tariquidar and elacridar, potent and selective P-glycoprotein inhibitors, were investigated in this regard using the opioid loperamide as an in vivo probe in mice. Pretreatment with both inhibitors converted intravenous loperamide from a drug without central effects to one producing antinociception. Radiolabeled loperamide tissue distribution studies indicated that inhibition was associated with increased uptake into brain and testes in the absence of changes in plasma levels, along with enhanced efflux of rhodamine 123 from CD3e ϩ T-lymphocytes. However, with tariquidar, the loperamide dose-response curves for testes/plasma and brain/ plasma concentration ratios were shifted 6-(p ϭ 0.07) and 25-fold (p Ͻ 0.01) to the right, respectively (ED 50 ϭ 1.48 and 5.65 mg/kg), compared with the rhodamine 123 efflux curve (ED 50 ϭ 0.25 mg/kg). Less pronounced shifts were noted with elacridar where the brain/plasma ratio was shifted only 2-fold relative to the rhodamine 123 efflux data (ED 50 ϭ 2.36 versus 1.34 mg/kg, respectively; p Ͻ 0.01). These results indicate that the P-glycoprotein localized in the blood-brain barrier and, to a lesser extent, the testes-blood barrier is more resistant to inhibition than at other tissue sites such as the lymphocyte; moreover, the extent of this effect depends on the inhibitor. Such resistance can be overcome by a sufficiently high dose of an inhibitor; however, whether this is safely attainable in the clinical situation remains to be determined.

Snail, a DNA-binding zinc finger protein, functions as a transcriptional repressor for genes including E-cadherin during development and the acquisition of tumor cell invasiveness. Human Snail is a 264-amino acid nuclear protein with an amino-terminal basic amino acid-rich domain (SNAG domain) and a carboxyl-terminal DNA-binding domain (zinc finger domain). A series of fusion proteins composed of green fluorescent protein (GFP) and portions of the Snail protein were generated, and their subcellular localization was examined. Fusion of the four zinc fingers to GFP led to the targeting of GFP to the nucleus, demonstrating that the zinc finger domain is sufficient for nuclear localization. Using an in vitro transport system, the nuclear import of Snail was reconstituted by importin (karyopherin) β β β β in the presence of Ran and NTF2. We further demonstrated that Snail binds directly to importin β β β β in a zinc finger domain-dependent manner. These results indicate that zinc finger domain of Snail functions as a nuclear localization signal and Snail can be transported into the nucleus in an importin β β β β -mediated manner.