Tumor growth inhibition by synthetic and expressed siRNA targeting focal adhesion kinase.

Focal adhesion kinase (FAK) was first identified as a viral Src substrate, and substantial experimental data have significantly correlated the elevated FAK expression in human tumor cells with an increased cell adhesion and invasion potential. However, studies investigating the role of FAK in cell proliferation have been limited. Recently, a technique known as RNA interference (RNAi) was successfully adapted to mammalian cells to decrease specifically the expression of targeted cellular genes. In this study, we investigated the role of FAK in cell proliferation, adhesion, and migration by using small interfering RNA (siRNA) technique. Firstly, we constructed a plasmid library expressing short hairpin RNAs (shRNAs) targeting FAK and selected clones substantially suppressing FAK expression in HeLa and HT1080 cells. We then studied the function of FAK in the highly invasive human prostate cancer cell line, PC3M, and mouse breast cancer cell line 4T1, by using selected shRNA clones (#40 and #42) and siRNAs chemically synthesized following the target sequences of #40 and #42. We demonstrated that the decrease of FAK protein expression by treatment with shRNA/siRNA targeting FAK inhibited cell adhesion on a fibronectin/laminin-coated plate, cell migration in a haptotactic migration assay, and cell proliferation in vitro. Furthermore, it suppressed tumor growth in vivo in heterotopic/orthotopic mice models. These results support our hypothesis that FAK plays a crucial role in tumor formation and growth in vivo by regulation of cell adhesion and proliferation by FAK-dependent signals.

Species differences between human and rat in the substrate specificity of cathepsin K.

Cathepsin K is known to play an important role in bone resorption, and it has the P2 specificity for proline. Rat cathepsin K has 88% identity with the human enzyme. However, it has been reported that its enzymatic activity for a Cbz-Leu-Arg-MCA substrate is lower than that of human cathepsin K, and that the rat enzyme is not well inhibited by human cathepsin K inhibitors. For this study, we prepared recombinant enzyme to investigate the substrate specificity of rat cathepsin K. Cleavage experiments using the fragment of type I collagen and peptidic libraries demonstrated that rat cathepsin K preferentially hydrolyses the substrates at the P2 Hyp position. Comparison of the S2 site between rat and human cathepsin K sequences indicated that two S2 residues at Ser134 and Val160 in rat are varied to Ala and Leu, respectively, in the human enzyme. Cleavage experiments using two single mutants, S134A and V160L, and one double mutant, S134A/V160L, of rat cathepsin K showed that all the rat mutants lost the P2 Hyp specificity. The information obtained from our comparative studies on rat and human cathepsin K should make a significant impact on developing specific inhibitors of human cathepsin K since rat is usually used as test species.

Group IVA phospholipase A2 is associated with the storage of lipids in adipose tissue and liver.

Prostaglandin (PG) E(2) is considered to participate in the storage of fat in adipocytes and hepatocytes, but roles of group IVA phospholipase A(2) (PLA(2)), a key PLA(2) isozyme in the arachidonic acid cascade, remain unclear. The present study examined the possible involvement of the enzyme using group IVA PLA(2)-deficient mice (C57BL/6 background, 22 weeks of age) fed a normal diet (5.3% fat). The ratio of epididymal fat pad weight to body weight was significantly reduced in group IVA PLA(2)-deficient mice compared to wild-type mice. Histological analysis revealed that in group IVA PLA(2)-deficient mice, the adipocytes were smaller, and hepatocytes bearing cytoplasmic vacuolation were scarce. Hepatic triglyceride content and the serum levels of PGE(2) in the deficient mice were also lower. However, there was no difference in the serum levels of insulin, glucose, non-esterified free fatty acid, or total cholesterol between the deficient and wild-type mice. Our findings suggest that group IVA PLA(2) is involved in the storage of lipids in the adipose tissue and liver and in determining circulating PGE(2) levels.

The receptor for advanced glycation end-products (RAGE) directly binds to ERK by a D-domain-like docking site.

The receptor for advanced glycation end-products (RAGE)-mediated cellular activation through the mitogen-activated protein kinase (MAPK) cascade, activation of NF-kappaB and Rho family small G-proteins, cdc42/Rac, is implicated in the pathogenesis of inflammatory disorders and tumor growth/metastasis. However, the precise molecular mechanisms for the initiation of cell signaling by RAGE remain to be elucidated. In this study, proteins which directly bind to the cytoplasmic C-terminus of RAGE were purified from rat lung extracts using an affinity chromatography technique and identified to be extracellular signal-regulated protein kinase-1 and -2 (ERK-1/2). Their interactions were confirmed by immunoprecipitation of ERK-1/2 from RAGE-expressing HT1080 cell extracts with anti-RAGE antibody. Furthermore, the augmentation of kinase activity of RAGE-bound ERK upon the stimulation of cells with amphoterin was demonstrated by determining the phosphorylation level of myelin basic protein, an ERK substrate. In vitro binding studies using a series of C-terminal deletion mutants of human RAGE revealed the importance of the membrane-proximal cytoplasmic region of RAGE for the direct ERK-RAGE interaction. This region contained a sequence similar to the D-domain, a ERK docking site which is conserved in some ERK substrates including MAPK-interacting kinase-1/2, mitogen- and stress-activated protein kinase-1, and ribosomal S6 kinase. These data suggest that ERK may play a role in RAGE signaling through direct interaction with RAGE.

Silencing of focal adhesion kinase by tumor direct injection of small interfering RNA decreases in vivo tumor growth.

Focal adhesion kinase (FAK) is shown to be frequently correlated with malignancy of the tumor and poor prognosis of the diseases.Because FAK resides immediately downstream of the interaction of cell surface adhesion molecules and extracellular matricies, it is considered to be critical to regulate several cellular processes including growth, differentiation, adhesion, motility and apoptosis. However, the studies on the role of FAK related to cell proliferation have been limited even in vitro. Here, in order to validate the role of FAK in in vivo tumor formation and proliferation, we employed direct intratumoral injection of short hairpin RNA (shRNA) targeting FAK with cationic liposome. Using shRNAs targeting FAK selected from the constructed shRNA library for FAK and by optimization of in vivo delivery conditions, we demonstrated different patterns of the association of FAK inhibition with in vivo tumor formation/proliferation inhibition in two models, PC3M heterotopic xenograft and 4T1 orthotopic syngraft models. These observations indicated that the roles of FAK in tumorigenesis are different among the tumor species. In addition, we showed that ERK is the critical MAP kinase in the signaling pathway down stream of FAK in in vivo proliferation of 4T1 tumor cells.

Alleviation of high-fat diet-induced fatty liver damage in group IVA phospholipase A2-knockout mice.

Hepatic fat deposition with hepatocellular damage, a feature of non-alcoholic fatty liver disease, is mediated by several putative factors including prostaglandins. In the present study, we examined whether group IVA phospholipase A(2) (IVA-PLA(2)), which catalyzes the first step in prostanoid biosynthesis, is involved in the development of fatty liver, using IVA-PLA(2)-knockout mice. Male wild-type mice on high-fat diets (20% fat and 1.25% cholesterol) developed hepatocellular vacuolation and liver hypertrophy with an increase in the serum levels of liver damage marker aminotransferases when compared with wild-type mice fed normal diets. These high-fat diet-induced alterations were markedly decreased in IVA-PLA(2)-knockout mice. Hepatic triacylglycerol content was lower in IVA-PLA(2)-knockout mice than in wild-type mice under normal dietary conditions. Although high-fat diets increased hepatic triacylglycerol content in both genotypes, the degree was lower in IVA-PLA(2)-knockout mice than in wild-type mice. Under the high-fat dietary conditions, IVA-PLA(2)-knockout mice had lower epididymal fat pad weight and smaller adipocytes than wild-type mice. The serum level of prostaglandin E(2), which has a fat storage effect, was lower in IVA-PLA(2)-knockout mice than in wild-type mice, irrespective of the kind of diet. In both genotypes, high-fat diets increased serum leptin levels equally between the two groups, but did not affect the serum levels of adiponectin, resistin, free fatty acid, triacylglycerol, glucose, or insulin. Our findings suggest that a deficiency of IVA-PLA(2) alleviates fatty liver damage caused by high-fat diets, probably because of the lower generation of IVA-PLA(2) metabolites, such as prostaglandin E(2). IVA-PLA(2) could be a promising therapeutic target for obesity-related diseases including non-alcoholic fatty liver disease.

Sulfation of the galactose residues in the glycosaminoglycan-protein linkage region by recombinant human chondroitin 6-O-sulfotransferase-1.

6-O-Sulfated galactose residues have been demonstrated in the glycosaminoglycan-protein linkage region GlcUAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser isolated from shark cartilage chondroitin 6-sulfate (Sugahara, K., Ohi, Y., Harada, T., de Waard, P., and Vliegenthart, J. F. G. (1992) J. Biol. Chem. 267, 6027-6035). In this study, we investigated whether a recombinant human chondroitin 6-sulfotransferase-1 (C6ST-1) catalyzes the sulfation of C6 on both galactose residues in the linkage region using structurally defined acceptor substrates. The C6ST-1 was expressed as a soluble protein A chimeric form in COS-1 cells and purified using IgG-Sepharose. The purified C6ST-1 utilized the linkage tri-, tetra-, penta-, and hexasaccharide-serines and hexasaccharide alditols, including GlcUAbeta1-3GalNAc(4-O-sulfate)beta1-4GlcUAbeta1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xylbeta1-O-Ser and DeltaGlcUAbeta1-3GalNAc(6-O-sulfate)beta1-4GlcUAbeta1-3Galbeta1-3Gal(6-O-sulfate)beta1-4Xyl-ol. Identification of the reaction products obtained with the linkage tetra-, penta-, and hexasaccharide-serines revealed that the C6ST-1 catalyzed the sulfation of C6 on both galactose residues in the linkage region. Notably, the linkage tetrasaccharide-peptide GlcUAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-(Gly)Ser-(Gly-Glu) was a good acceptor substrate for the C6ST-1, suggesting that the sulfation of the galactose residues can occur before the transfer of the first N-acetylhexosamine residue to the linkage tetrasaccharide. In contrast, no incorporation was observed into DeltaGlcUAbeta1-3GalNAc(4-O-sulfate)beta1-4GlcUAbeta1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xyl-ol, indicating that an intact xylose is necessary for the transfer of a sulfate to the second sugar residue Gal from the reducing end. These findings clearly demonstrated that the recombinant C6ST-1 catalyzes the sulfation of C6 on both galactose residues in the linkage region in vitro. This is the first identification of the sulfotransferase responsible for the sulfation of galactose residues in the glycosaminoglycan-protein linkage region.

Analyses of group III secreted phospholipase A2 transgenic mice reveal potential participation of this enzyme in plasma lipoprotein modification, macrophage foam cell formation, and atherosclerosis.

Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.