Wnt proteins form a family of highly conserved, secreted signaling molecules that regulate cell-to-cell interactions during embryogenesis. Wnt genes and Wnt signaling are also implicated in cancer. It has been shown that Wnt proteins bind to receptors of the frizzled family on the cell surface. Through several cytoplasmic relay components including DVL-1, the human counterpart of the Drosophila disheveled gene, the signal is transduced to β β β β-catenin, which then enters the nucleus and forms a complex with T-cell factor (TCF) to activate transcription of Wnt target genes. nt genes encode a family of highly conserved, secreted glycoproteins that modulate cell fate and behavior in embryos through activation of receptor-mediated signaling pathways. In the absence of a Wnt signal, β-catenin levels are kept low through interactions with the protein kinase zw3/ GSK-3β (zeste white-3, shaggy in Drosophila/glycogen synthase kinase-3β), CK1α (casein kinase 1α), APC (adenomatous polyposis coli) and axin.

Abstract-Robo4, a member of the roundabout family, is expressed exclusively in endothelial cells and has been implicated in endothelial cell migration and angiogenesis. Here we report the cloning and characterization of the human Robo4 promoter. The 3-kb 5Ј-flanking region directs endothelial cell-specific expression in vitro. Deletion and mutation analyses revealed the functional importance of two 12-bp palindromic DNA sequences at Ϫ2528 and Ϫ2941, 2 SP1 consensus motifs at Ϫ42 and Ϫ153, and an ETS consensus motif at Ϫ119. In electrophoretic mobility shift assays using supershifting antibodies, the SP1 motifs bound SP1 protein, whereas the ETS site bound a heterodimeric member of the ETS family, GA binding protein (GABP). These DNA-protein interactions were confirmed by chromatin immunoprecipitation assays. Transfection of primary human endothelial cells with small interfering RNA against GABP and SP1 resulted in a significant (Ϸ50%) reduction in endogenous Robo4 mRNA expression. The 3-kb Robo4 promoter was coupled to LacZ, and the resulting cassette was introduced into the Hprt locus of mice by homologous recombination.Reporter gene activity was observed in the vasculature of adult organs (particularly in microvessels), tumor xenografts, and embryos, where it colocalized with the endothelial cell-specific marker CD31. LacZ mRNA levels in adult tissues and tumors correlated with mRNA levels for endogenous Robo4, CD31, and vascular endothelial cadherin. Moreover, the pattern of reporter gene expression was similar to that observed in mice in which LacZ was knocked into the endogenous Robo4 locus. Together, these data suggest that 3-kb upstream promoter of human Robo4 contains information for cell type-specific expression in the intact endothelium. here is increasing evidence that migration and patterning of axons and blood vessels share similar guidance mechanisms. Among the guidance systems involved in axonal and vascular networks are the ephrin-Eph, netrin-unc5b, semaphorin-plexin, and slit-Robo molecules. 1 Robo (roundabout) is a member of the neural cell adhesion molecule family. Robo was originally isolated from Drosophila melanogaster. 2 The ligand for Robo, Slit, was first identified in Drosophila as an extracellular molecule involved in axonal branching and neural migration. 3 In vertebrates, 3 Robo receptor family members (Robo1 to -3) and 3 Slit ligands (Slit1 to -3) have been implicated in guiding axon growth via repulsive signaling. 2 A fourth Robo receptor family member, Robo4, was cloned and shown to be restricted in its tissue distribution to endothelial cells. 4,5 Robo4 is expressed in areas of in vivo angiogenesis. For example, the receptor is present in the endothelial lining of blood vessels in the developing embryo, 6 placenta, 4 and tumors. 4,7 Robo4 has also been detected in the endothelium of normal nonangiogenic tissues, including the heart and lung. 6,7 Recent studies support a role for Robo4 in endothelial cell migration, proliferation, and angiogenesis. 6,8 -10 The goal of th...

Immunohistochemical studies were made of the distribution of various cytokeratins (CK), Clara cell secretory protein (CC10), surfactant protein A (SP-A) and type VII collagen in normal human airways. Electron microscopic studies were made to identify hemidesmosomes and anchoring fibrils on the basal surfaces of the epithelial cells. CK19 was detected in all epithelial cells, and CK17 in all basal cells. CK14 was coexpressed in a few basal cells, and this coexpression was decreased in the distal airways. Two types of basal cells were recognized. One type, found mainly in large airways, was characterized by abundant intermediate filaments and well-developed hemidesmosomes and anchoring fibrils. The second type contained few intermediate filaments and poorly developed hemidesmosomes and anchoring fibrils. Reactivity for type VII collagen was found along the basement membrane throughout the airways, but not in the alveoli. Clara cells were reactive for CC10 and CK17, but not for CK14 and SP-A. The bronchiolar cuboidal cells in the respiratory bronchioles were positive only for CK19. Surfactant protein A was present only in type II alveolar epithelial cells. Thus, two types of basal cells are present in airways, and the bronchiolar cuboidal cells appear distinct from these basal cells, Clara cells and type II alveolar epithelial cells.

We recently demonstrated inactivation in hepatocellular carcinomas (HCCs) of the gene encoding SOCS1/JAB1/SSI-1, a JAK-binding protein that regulates the JAK/STAT signal-transduction pathway. In a follow-up immunochemical investigation of expression of SOCS-1 in hepatoblastomas (HBLs), the protein was markedly reduced in half of the HBL tumors we examined. CpG-rich regions upstream of the SOCS-1 gene were hypermehylated in 7 of the 15 HBL cases. The results suggest that hypermethylation may play an important role in silencing the SOCS-1 gene, not only in adult HCCs, but also in liver tumors arising in childhood.

Human airway trypsin-like protease (HAT), a novel serine protease in the airways, enhances cell growth and IL-8 production. The expression and role of HAT in the skin however, is unknown. Immunofluorescence staining and reverse transcription (RT)-PCR were done to know HAT production in normal and psoriatic tissues and keratinocyte cell lines. Cell growth and/or IL-8 release analyses were made by bromo-deoxyuridine (BrdU) uptake and ELISA. Psoriatic epidermis showed more extensive immunofluorescence expression of HAT, and less extensive expression of protease-activated receptor (PAR)-2. RT-PCR demonstrated a higher HAT and a lesser PAR-2 mRNA expressions in psoriatic epidermis. Normal keratinocyte and epidermoid carcinoma cell lines expressed HAT and PAR-2 mRNA, and immortalized keratinocytes (HaCaT) expressed PAR-2, but not HAT mRNA. PAR-2 was detected along the keratinocyte surface in culture and became invisible upon HAT stimulation, suggesting a process of its internalization. HAT or PAR-2 activating peptide did not enhance BrdU uptake, but induced an IL-8 release. Treatment with HAT and IL-1beta synergistically increased the effect of IL-8 release. Inhibition of PAR-2 resulted in a decreased HAT-induced IL-8 release. Thus, HAT might promote PAR-2-mediated IL-8 production to accumulate inflammatory cells in the epidermal layer of psoriasis.

No abstract

We recently demonstrated that the 3-kb 5'-flanking region of the human ROBO4 gene directs endothelial cell-specific expression in vitro and in vivo. Moreover, a GA-binding protein (GABP)-binding motif at -119 was necessary for mediating promoter activity in vitro. The goal of the present study was to confirm the functional relevance of the -119 GABP-binding site in vivo. To that end, the Hprt locus of mice was targeted with a Robo4-LacZ transgenic cassette in which the GABP site was mutated. In other studies, the GABP mutation was introduced into the endogenous mouse Robo4 locus in which LacZ was knocked-in. Compared with their respective controls, the mutant promoters displayed a significant reduction in activity in embryoid bodies, embryos, and adult animals. Together, these data provide strong support for the role of the GABP-binding motif in mediating Robo4 expression in the intact endothelium.

The Ras-CRK-Rap1 cellular signal-transduction system is regulated by guanine nucleotide exchange factors (GEFs). Transcription of C3G on chromosome 9q34 and a key member of the GEF gene family is activated by the CRK-adaptor protein; the C3G product is a CRK SH3 domain-binding guanine nucleotide-releasing factor. We document here the amplification of C3G in five of 18 primary non-small cell lung cancers examined and its increased expression in 18 of 28 tumors in comparison to corresponding non-cancerous lung tissues. Immunohistochemical staining revealed prominent C3G protein in the cytoplasm of cancer cells, associated with faint staining at the nucleolar membrane, but C3G was not detectable in normal bronchial mucoepithelial cells or in broncholoalveolar cells of the bronchial/bronchiolar ducts or alveoli. These data indicate that amplification and increased expression of the C3G gene may play some role in human lung carcinogenesis through derangement of the CRK-Rap1 signaling pathway.