Deregulation of Cdc2 kinase induces caspase-3 activation and apoptosis

Progression of the cell cycle and control of apoptosis are tightly linked processes. It has been reported that manifestation of apoptosis requires cdc2 kinase activity yet the mechanism(s) of which is largely unclear. In an attempt to study the role of human MDM2 (HDM2) in interphase and mitosis, we employed the Xenopus cell-free system to study HDM2 protein stability. Interestingly, HDM2 is specifically cleaved in Xenopus mitotic extracts but not in the interphase extracts. We demonstrate that HDM2 cleavage is dependent on caspase-3 and that activation of cdc2 kinase results in caspase-3 activation in the Xenopus cell-free system. Furthermore, expression of cdc2 kinase in mammalian cells leads to activation of caspase-3 and apoptosis. Taken together, these data indicate that deregulation of cdc2 kinase activity can trigger apoptotic machinery that leads to caspase-3 activation and apoptosis.     

Smurf1 facilitates myogenic differentiation and antagonizes the bone morphogenetic protein-2-induced osteoblast conversion by targeting Smad5 for degradation

Controlled proteolysis mediated by Smad ubiquitination regulatory factors (Smurfs) plays a crucial role in modulating cellular responses to signaling of the transforming growth factor-beta (TGF-beta) superfamily. However, it is not clear what influences the selectivity of Smurfs in the individual signaling pathway, nor is it clear the biological function of Smurfs in vivo. Using a mouse C2C12 myoblast cell differentiation system, which is subject to control by both TGF-beta and bone morphogenetic protein (BMP), here we examine the role of Smurf1 in myogenic differentiation. We show that increased expression of Smurf1 promotes myogenic differentiation of C2C12 cells and blocks the BMP-induced osteogenic conversion but has no effect on the TGF-beta-induced differentiation arrest. Consistent with an inhibitory role in the BMP signaling pathway, the elevated Smurf1 markedly reduces the level of endogenous Smad5, whereas it leaves unaltered that of Smad2, Smad3, and Smad7, which are components of the TGF-beta pathway. Adding back Smad5 from a different source to the Smurf1-overexpressing cells restores the BMP-mediated osteoblast conversion. Finally, by depletion of the endogenous Smurf1 through small interfering RNA-mediated RNA interference, we demonstrate that Smurf1 is required for the myogenic differentiation of C2C12 cells and plays an important regulatory role in the BMP-2-mediated osteoblast conversion.     

Regulation of cell proliferation, apoptosis, and carcinogenesis by activin

The aim of this review is to provide insight into the molecular mechanisms by which activin A modulates cell proliferation, apoptosis, and carcinogenesis in vitro and in vivo. Activin A, a member of the TGFbeta superfamily, has various effects on diverse biological systems, including cell growth inhibition in many cell types. However, the mechanism(s) by which activin exerts its inhibitory effects are not yet understood. This review highlights activin's effects on activin receptors and signaling pathway, modulation of activin signaling, and regulation of cell proliferation and apoptosis by activin. Based on the experiences of all the authors, we emphasized cell cycle inhibitors such as p16 and p21 and regulators of apoptosis such as p53 and members of the bcl-2 family. Aside from activin's inhibition of cell proliferation and enhancement of apoptosis, other newly developed methods for molecular studies of apoptosis by activin were briefly presented that support the role of activin as an inhibitor of carcinogenesis and cancer progression. These methods include subtractive hybridization based on covalent bonding, a simple and accurate means to determine molecular profile of as few as 20 cells based on an RNA-PCR approach, and a messenger RNA-antisense DNA interference phenomenon (D-RNAi), resulting in a long-term gene knockout effects.     

Isolation and characterization of a human novel RAB (RAB39B) gene

Rab proteins are small-molecular-weight GTPases that control vesicular trafficking in eukaryotic cells. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA clone encoding a novel Rab protein, which showed 74.2% identity with previously isolated Rab39A at the amino acid level. RAB39B was expressed in a variety of human tissues and located in human chromosome Xq28. It consisted of two exons spanning 3764 bp of human genomic DNA.     

Pressure-induced endothelial Ca(2+) oscillations in lung capillaries

Endothelial second messenger responses may contribute to the pathology of high vascular pressure but remain poorly understood because of the lack of direct in situ quantification. In lung venular capillaries, we determined endothelial cytosolic Ca(2+) concentration [Ca(2+)](i) by the fura 2 ratioing method. Pressure elevation increased mean endothelial [Ca(2+)](i) by Ca(2+) influx through gadolinium-inhibitable channels and amplified [Ca(2+)](i) oscillations by Ca(2+) release from intracellular stores. Endothelial [Ca(2+)](i) transients were induced by pressure elevations of as little as 5 cmH(2)O and increased linearly with higher pressures. Heptanol inhibition of [Ca(2+)](i) oscillations in a subset of endothelial cells indicated that oscillations originated from pacemaker endothelial cells and were propagated to adjacent nonpacemaker cells by gap junctional communication. Our findings indicate the presence of a sensitive, active endothelial response to pressure challenge in lung venular capillaries that may be relevant in the pathogenesis of pressure-induced lung microvascular injury.     

Molecular cloning and characterization of a novel human protein phosphatase,LMW-DSP3

Reversible phosphorylation is recognized to be a major mechanism for the control of intracellular events in eukaryotic cells. From a human fetal brain cDNA library, we isolated a cDNA clone encoding a novel dual specificity protein phosphatase, which showed 88% identity with previously reported mouse LMW-DSP3 at the amino acid level. The deduced protein had a single dual-specificity phosphatase catalytic domain, and lacked a cdc25 homology domain. LMW-DSP3 was expressed in the heart, lung, liver, and pancreas, and the expression level in the pancreas was highest. The LMW-DSP3 gene was located in human chromosome 2q32, and consisted of five exons spanning 21kb of human genomic DNA. LMW-DSP3 fused to GST showed phosphatase activity towards p-nitrophenyl phosphate which was optimal at pH 7.0 and 40 degrees C, and the activity was enhanced by Ca(2+) and Mn(2+). The phosphatase activity of LMW-DSP3 was inhibited by orthovanate. LMW-DSP3 showed phosphatase activity toward oligopeptides containing pSer/Thr and pTyr, indicating that LMW-DSP3 is a protein phosphatase with dual substrate specificity.     

Cholesterol loading induces a block in the exit of VSVG from the TGN

Recent work from our laboratory demonstrated that increased cellular cholesterol content affects the structure of the Golgi apparatus. We have now investigated the functional consequences of the cholesterol-induced vesiculation of the Golgi apparatus and the role of actin for these changes. The results showed that cholesterol-induced vesiculation and dispersion of the Golgi apparatus is a reversible process and that reversal can be inhibited by cytochalasin D, an actin-disrupting reagent. Furthermore, electron microscopy revealed that jasplakinolide, which stabilizes actin filaments, prevented the dispersion, but not the vesiculation of the Golgi cisternae. Importantly, the different Golgi markers seemed to be separated even after vesiculation. To investigate whether transport through the different steps of the exocytic pathway was affected in cholesterol-treated cells, we visualized ER to plasma membrane transport by using ts045-VSVG-GFP. In COS-1 cells expressing ts045-VSVG-GFP increased cholesterol levels did not affect transport of VSVG into the vesiculated Golgi apparatus. However, increased levels of cholesterol resulted in retention of the nascent G protein in vesicles with the TGN-marker TGN46. Biotinylation of cell surface molecules to quantify arrival of VSVG at the plasma membrane confirmed that cholesterol treatment inhibited export of the VSVG protein. In conclusion, the data show that transport of VSVG into/through a vesiculated Golgi is feasible, but that cholesterol loading inhibits exit of VSVG from the vesicles containing TGN markers. Furthermore, the data illustrate the importance of actin filaments for Golgi structure.     

Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis

Very little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis-associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis-related function of candidate protein interleukin-18 (IL-18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach was used to compare the protein expression profiles between PLA801C and PLA801D sublines. Eleven proteins were identified and further verified by one-dimensional Western blotting, Northern blot and/or semiquantitative reverse transciptase polymerase chain reaction analysis. Compared with those in poorly metastatic PLA801C subline, cytokeratin 18, tissue transglutaminase, Rho GDP-dissociation inhibitor 1, tropomyosin, fibroblast type, IL-18 and annexin I were significantly up-regulated, while protein disulfide isomerase, heat shock protein 60, peroxiredoxin 1, chlorine intracellular channel protein 1 (CLI1) and creatine kinase, B chain were significantly down-regulated in the highly metastatic PLA801D subline. Intriguingly, all the identified candidate proteins except for CLI1 have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was present in highly metastatic PLA801D but absent in poorly metastatic PLA801C, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense/IL-18 antisense into PLA801C/PLA801D sublines simultaneously. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro and down-regulated E-cadherin expression of PLA801C transfectants, while IL-18 antisense remarkably decreased cell invasion potency in vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play a role in metastasis by inhibiting E-cadherin expression.