An actin-binding protein Girdin regulates the motility of breast cancer cells.

Girdin (girders of actin filaments) is a novel actin-binding Akt substrate that plays an important role in actin organization and Akt-dependent cell motility in fibroblasts. Here, we find that Girdin is expressed in a variety of cancer cell lines, including the breast cancer cell line MDA-MB-231, and is phosphorylated by the stimulation of insulin-like growth factor (IGF-I). In vitro migration and invasion assays showed that Girdin is required for the IGF-I-dependent cell movement of MDA-MB-231 cells. Short hairpin interfering RNA directed against Girdin markedly inhibited the metastasis of s.c. transplanted MDA-MB-231 cells in nude mice. In addition, Girdin is highly expressed in a variety of human malignant tissues, including breast, colon, lung, and uterine cervical carcinomas. These findings highlight the important role of Girdin in tumor progression in which the Akt signaling pathway is aberrantly activated.

Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin.

The serine/threonine protein kinase Akt is involved in a variety of cellular processes including cell proliferation, survival, metabolism and gene expression. It is essential in vascular endothelial growth factor (VEGF)-mediated angiogenesis; however, it is not known how Akt regulates the migration of endothelial cells, a crucial process for vessel sprouting, branching and the formation of networks during angiogenesis. Here we report that Akt-mediated phosphorylation of Girdin, an actin-binding protein, promotes VEGF-dependent migration of endothelial cells and tube formation by these cells. We found that exogenously delivered adenovirus harbouring Girdin short interfering RNA in Matrigel embedded in mice, markedly inhibited VEGF-mediated angiogenesis. Targeted disruption of the Girdin gene in mice impaired vessel remodelling in the retina and angiogenesis from aortic rings, whereas Girdin was dispensable for embryonic vasculogenesis. These findings demonstrate that the Akt/Girdin signalling pathway is essential in VEGF-mediated postneonatal angiogenesis.

Thermodynamic instability of siRNA duplex is a prerequisite for dependable prediction of siRNA activities.

We developed a simple algorithm, i-Score (inhibitory-Score), to predict active siRNAs by applying a linear regression model to 2431 siRNAs. Our algorithm is exclusively comprised of nucleotide (nt) preferences at each position, and no other parameters are taken into account. Using a validation dataset comprised of 419 siRNAs, we found that the prediction accuracy of i-Score is as good as those of s-Biopredsi, ThermoComposition21 and DSIR, which employ a neural network model or more parameters in a linear regression model. Reynolds and Katoh also predict active siRNAs efficiently, but the numbers of siRNAs predicted to be active are less than one-eighth of that of i-Score. We additionally found that exclusion of thermostable siRNAs, whose whole stacking energy (DeltaG) is less than -34.6 kcal/mol, improves the prediction accuracy in i-Score, s-Biopredsi, ThermoComposition21 and DSIR. We also developed a universal target vector, pSELL, with which we can assay an siRNA activity of any sequence in either the sense or antisense direction. We assayed 86 siRNAs in HEK293 cells using pSELL, and validated applicability of i-Score and the whole DeltaG value in designing siRNAs.

Sprouty2 regulates growth and differentiation of human neuroblastoma cells through RET tyrosine kinase.

The Sprouty (SPRY) family of proteins includes important regulators of downstream signaling initiated by receptor tyrosine kinases. In the present study, we investigated the role of SPRY proteins in intracellular signaling via the RET receptor tyrosine kinase activated by glial cell line-derived neurotrophic factor (GDNF). Expression of SPRY1, SPRY2, SPRY3 and SPRY4 in HEK293T cells transfected with RET and GDNF receptor family alpha1 (GFRalpha1) genes significantly reduced sustained ERK activation as well as ELK-1 activation. Because expression of SPRY2 was efficiently induced by GDNF in TGW human neuroblastoma cells expressing RET and GFRalpha1, we further investigated the role of SPRY2 in the growth and differentiation of TGW cells. Expression of wild-type SPRY2 (WT-SPRY2) decreased the growth of TGW cells. In contrast, expression of a dominant negative form of SPRY2 (MT-SPRY2, with a mutated tyrosine residue) enhanced cell proliferation. In addition, expression of WT-SPRY2 reduced GDNF-dependent neurite outgrowth of TGW cells, whereas expression of MT-SPRY2 enhanced it. Taken together, our results suggest that SPRY2 regulates GDNF-dependent proliferation and differentiation of TGW neuroblastoma cells mediated by RET tyrosine kinase.

Regulation of the expression of intracellular beta-carbonic anhydrase in response to CO2 and light in the marine diatom Phaeodactylum tricornutum.

Cells of the marine diatom Phaeodactylum tricornutum Bohlin (UTEX 642) grown in 5% CO(2) were transferred to air-level CO(2) in the light or dark and allowed to acclimate to air. No accumulation of the transcript of the P. tricornutum beta-carbonic anhydrase 1 (ptca1) was detected in 5% CO(2)-grown cells, but ptca1 mRNA accumulated and reached a peak after 6 h acclimation to air but decreased over the next 18 h. A similar accumulation time course was observed in cells air-acclimated in the dark, except that levels of mRNA were <50% those in the light. These results suggest that air-level [CO(2)] is required to trigger the transcription of ptca1 and that light affects the extent of acclimation. During acclimation to air for 120 h in the light, levels of ptca1 mRNA exhibited a periodic oscillation with a cycle of about 24 h, which, however, was not reflected in protein accumulation levels. A 5'-upstream region from the transcription-start site toward -1,292 bp of ptca1 was cloned by inverse polymerase chain reaction, and 5'-truncations were carried out on this fragment. The truncated promoter regions were fused with the beta-glucuronidase gene (uidA) and introduced into P. tricornutum. The promoter fragments, truncated at positions -1,292, -824, -484, -225, and -70 bp, conferred on transformants clear CO(2)-responsive beta-glucuronidase expressions. In contrast, the CO(2)-responsive regulation was severely impaired or completely abolished by truncations, respectively, at position -50 or -30 bp. These results indicate that critical cis-elements required for CO(2)-responsive transcription of ptca1 may be located between -70 and -30 bp relative to the transcription start site.

Localization of soluble beta-carbonic anhydrase in the marine diatom Phaeodactylum tricornutum. Sorting to the chloroplast and cluster formation on the girdle lamellae.

A beta-carbonic anhydrase (CA) in the marine diatom Phaeodactylum tricornutum (PtCA1) is encoded by the nuclear genome. This enzyme was previously found to be important for the operation of photosynthesis with a high affinity for dissolved inorganic carbon. A cDNA sequence that encodes PtCA1 (ptca1) was shown to possess a presequence of 138 bp (pre138), which encodes an N-terminal sequence of 46 amino acids (Pre46AA) that does not exist in the mature PtCA1. In this study, pre138 was ligated with the enhanced green fluorescent protein (GFP) gene (egfp), and introduced into P. tricornutum by microprojectile bombardment. Subsequently, the expressed Pre46AA-GFP fusion was shown to be localized in the chloroplast stroma, whereas the expressed GFP without Pre46AA was localized in the cytoplasm. Insertion of the DNA sequence, encoding a mature region of ptca1 (mptca1) between pre138 and egfp, resulted in the formation of particles with concentrated GFP fluorescence in the stroma of P. tricornutum. These particles, 0.3 to 3.0 mum in size, were shown to be distinct from the mitochondria and localized on the surface of the putative girdle lamella. The attachment of the initial one-half of the pre138 to the mptca1-egfp fusion caused the expressed GFP fusion to accumulate in areas surrounding the chloroplast, presumably due to the presence of the endoplasmic reticulum signal encoded by the initial half-sequence and to the absence of the chloroplast transit sequence. These results indicate that PtCA1 is targeted to the stroma by the bipartite sequences of Pre46AA and that the observed GFP particles are formed specifically in the stroma due to the function of the mptca1.