A potential role of alternative splicing in the regulation of the transcriptional activity of human GLI2 in gonadal tissues

BACKGROUND: Mammalian Gli proteins are important transcription factors involved in the regulation of Sonic hedgehog signal transduction pathway. Association of Gli2 with mammalian development and human disease led us to study the structure and expression of the human GLI2. RESULTS: We show that the region encoding GLI2 repressor domain is subject to alternative splicing in the gonadal tissues and different cell lines. Two major alternatively spliced forms of GLI2 mRNA arise from skipping exon 3 (GLI2Δ3) or exons 4 and 5 (GLI2Δ4–5). Both forms contain premature translational stop codons in the GLI2 open reading frame (ORF) starting from exon 2. Translation of GLI2Δ3 and GLI2Δ4–5 in vitro, initiated from downstream AUG codons, produced N-terminally truncated proteins. In Gli-dependent transactivation assay, expression of GLI2Δ3 induced activation of the reporter gene similar to that of the full-length construct (GLI2fl) containing complete ORF. However, expression of the GLI2Δ4–5 resulted in about 10-fold increase in activation, suggesting that deletion of the major part of repressor domain was responsible for the enhanced activation of GLI2 protein. CONCLUSION: Our data suggest that in addition to proteolytic processing, alternative splicing may be another important regulatory mechanism for the modulation of repressor and activator properties of GLI2 protein

The Tumor Suppressor CYLD Interacts with TRIP and Regulates Negatively Nuclear Factor κB Activation by Tumor Necrosis Factor

Cylindromas are benign adnexal skin tumors caused by germline mutations in the CYLD gene. In most cases the second wild-type allele is lost in tumor tissue, suggesting that CYLD functions as tumor suppressor. CYLD is a protein of 956 amino acids harboring a functional deubiquitinating domain at the COOH-terminal end. To shed more light on the function of CYLD, we have performed a yeast two hybrid screen using an HaCaT cDNA library that identified the RING finger protein TRIP (TRAF-interacting protein) as interactor with full-length CYLD. Mapping of the interacting domains revealed that the central domain of CYLD binds to the COOH-terminal end of TRIP. Far Western analysis and coimmunoprecipitations in mammalian cells confirmed that full-length CYLD binds to the COOH-terminal domain of TRIP. Because TRIP is an inhibitor of nuclear factor (NF)-κB activation by tumor necrosis factor (TNF), the effect of CYLD on NF-κB activation was investigated in HeLa cells. The results established that CYLD down-regulates NF-κB activation by TNF-α. The inhibition by CYLD depends on the presence of the central domain interacting with TRIP and its deubiquitinating activity. These findings indicate that cylindromas arise through constitutive NF-κB activation leading to hyperproliferation and tumor growth

Soluble CD44 Interacts with Intermediate Filament Protein Vimentin on Endothelial Cell Surface

CD44 is a cell surface glycoprotein that functions as hyaluronan receptor. Mouse and human serum contain substantial amounts of soluble CD44, generated either by shedding or alternative splicing. During inflammation and in cancer patients serum levels of soluble CD44 are significantly increased. Experimentally, soluble CD44 overexpression blocks cancer cell adhesion to HA. We have previously found that recombinant CD44 hyaluronan binding domain (CD44HABD) and its non-HA-binding mutant inhibited tumor xenograft growth, angiogenesis, and endothelial cell proliferation. These data suggested an additional target other than HA for CD44HABD. By using non-HA-binding CD44HABD Arg41Ala, Arg78Ser, and Tyr79Ser-triple mutant (CD443MUT) we have identified intermediate filament protein vimentin as a novel interaction partner of CD44. We found that vimentin is expressed on the cell surface of human umbilical vein endothelial cells (HUVEC). Endogenous CD44 and vimentin coprecipitate from HUVECs, and when overexpressed in vimentin-negative MCF-7 cells. By using deletion mutants, we found that CD44HABD and CD443MUT bind vimentin N-terminal head domain. CD443MUT binds vimentin in solution with a Kd in range of 12–37 nM, and immobilised vimentin with Kd of 74 nM. CD443MUT binds to HUVEC and recombinant vimentin displaces CD443MUT from its binding sites. CD44HABD and CD443MUT were internalized by wild-type endothelial cells, but not by lung endothelial cells isolated from vimentin knock-out mice. Together, these data suggest that vimentin provides a specific binding site for soluble CD44 on endothelial cells

A potential role of alternative splicing in the regulation of the transcriptional activity of human GLI2 in gonadal tissues-1

<p><b>Copyright information:</b></p><p>Taken from "A potential role of alternative splicing in the regulation of the transcriptional activity of human GLI2 in gonadal tissues"</p><p>BMC Molecular Biology 2006;7():13-13.</p><p>Published online 23 Mar 2006</p><p>PMCID:PMC1435915.</p><p></p>own). In the alignment, zebrafish yot protein (encoded by ) is shown. Su(fu) binding site SYGH is indicated by asterisks. The initiator methionine of human GLI2 defined by Tanimura et al. [32] is marked by arrowhead

A potential role of alternative splicing in the regulation of the transcriptional activity of human GLI2 in gonadal tissues-4

<p><b>Copyright information:</b></p><p>Taken from "A potential role of alternative splicing in the regulation of the transcriptional activity of human GLI2 in gonadal tissues"</p><p>BMC Molecular Biology 2006;7():13-13.</p><p>Published online 23 Mar 2006</p><p>PMCID:PMC1435915.</p><p></p>ferent cell lines (indicated on top of the panel) using -specific primers derived from exons 2 and 7. The products were hybridized with a 918 nt-riboprobe encompassing exons 2–7. Bars on the right indicate sizes of PCR products corresponding to mRNA and its alternatively spliced forms Δ3 and Δ4–5 (0.9, 0.8 and 0.5 kb, respectively). A shorter and longer exposure were used to determine the presence of minor transcripts corresponding to Δ3 (0.8 kb) in ovary and testis (panel A). Note the complex pattern of alternatively spliced products observed for cell lines (panel B) that is most likely due to promiscuous splicing (not analyzed in detail). Control, no cDNA

A potential role of alternative splicing in the regulation of the transcriptional activity of human GLI2 in gonadal tissues-0

<p><b>Copyright information:</b></p><p>Taken from "A potential role of alternative splicing in the regulation of the transcriptional activity of human GLI2 in gonadal tissues"</p><p>BMC Molecular Biology 2006;7():13-13.</p><p>Published online 23 Mar 2006</p><p>PMCID:PMC1435915.</p><p></p>erence accession numbers shown on the right). Exons (numbered) and introns are shown by vertical and horizontal lines, respectively. Alternative 5' noncoding exons in human gene are designated as 1a and 1b. Asterisks indicate exons 3–6, predicted from the comparison of mouse and human Gli2 mRNAs and genomic structures, identified by Roessler et al. [33]

A potential role of alternative splicing in the regulation of the transcriptional activity of human GLI2 in gonadal tissues-5

<p><b>Copyright information:</b></p><p>Taken from "A potential role of alternative splicing in the regulation of the transcriptional activity of human GLI2 in gonadal tissues"</p><p>BMC Molecular Biology 2006;7():13-13.</p><p>Published online 23 Mar 2006</p><p>PMCID:PMC1435915.</p><p></p>uences are designated by upper and lower case letters, respectively. Alternatively spliced forms I-III arising via exon skipping and different exon 6–7 junction usage (described in this study) are compared with the previously published sequence (Type IV) [32]. For each alternatively spliced form, GenBank accession number is shown on the right. Translation of these sequences initiated from ATG codon located in exon 2 (METSA) terminates with premature termination codons (PTC). Location of initiator codon described previously [32] is shown in exon 7 (M). Location of the SUFU binding site (SYGH) in exon 6 is shown

Regulation of Gli1 transcriptional activity in the nucleus by Dyrk1

To investigate the cellular role of dual specificity Yak1-related kinase (Dyrk) 1, a nuclear localized dual specificity protein kinase, we examined its effect on transcriptional regulation using reporter gene assays. We found that Dyrk1 can substantially enhance Gli1-dependent, but not LEF-1-, c-Jun-, or Elk-dependent, gene transcription. In part, Dyrk1 does this through retaining Gli1 in the nucleus. However, we also demonstrate that Dyrk1 can enhance the transcriptional activity of Gli1-AHA, a nuclear export mutant, suggesting that Dyrk1 may be more directly involved in regulating the transcriptional activity of Gli1. In addition, Dyrk1 acted synergistically with Sonic hedgehog (Shh) to induce gene transcription and differentiation in mouse C3H10T1/2 cells. The failure of Shh to stimulate Dyrk1 kinase activity suggests that Dyrk1 may not be directly regulated by the Shh signaling pathway but functionally interacts with it. Thus, Gli1 transcriptional activity may be subjected to further regulation in the cell nucleus by a pathway distinct from Shh signaling, one mediated by Dyrk1.This work was supported by grants from the National Institutes of Health and American Cancer Society and by American Heart Association Established Investigator Award (to D.W.).Peer reviewe