Histone acetyltransferase PCAF is involved in transactivation of Bcl-6 and Pax5 genes in immature B cells.

Histone acetyltransferase p300/CBP-associated factor (PCAF) belonging to GCN5 family regulates various epigenetic events for transcriptional regulation through alterations in the chromatin structure. During normal development of B cells, gene expressions of numerous transcription factors are strictly regulated by epigenetic mechanisms including histone acetylation and deacetylation to complete their development pathways. Here, by analyzing PCAF-deficient DT40 mutants, ΔPCAF, we report that PCAF takes part in transcriptional activation of B cell lymphoma-6 (Bcl-6) and Paired box gene 5 (Pax5), which are essential transcription factors for normal development of B cells. PCAF-deficiency caused drastic decrease in mRNA levels of Bcl-6 and Pax5, and remarkable increase in that of B lymphocyte-induced maturation protein-1 (Blimp-1). In addition, chromatin immunoprecipitation assay showed that PCAF-deficiency caused remarkable decrease in acetylation levels of both H3K9 and H3K14 residues within chromatin surrounding the 5'-flanking regions of Bcl-6 and Pax5 genes in vivo, suggesting that their gene expressions may be regulated by PCAF. These results revealed that PCAF is involved in transactivation of Bcl-6 and Pax5 genes, resulting in down-regulation of Blimp-1 gene expression, and plays a key role in epigenetic regulation of B cell development.

Lack of GCN5 remarkably enhances the resistance against prolonged endoplasmic reticulum stress-induced apoptosis through up-regulation of Bcl-2 gene expression.

The endoplasmic reticulum (ER), a complex membrane structure, has important roles in all eukaryotic cells. Catastrophe of its functions would lead to ER stress that causes various diseases such as cancer, neurodegenerative diseases, diabetes and so on. Prolonged ER stress could trigger apoptosis via activation of various signal transduction pathways. To investigate physiological roles of histone acetyltransferase GCN5 in regulation of ER stress, we analyzed responses of homozygous GCN5-deficient DT40 mutants, ΔGCN5, against ER stress. GCN5-deficiency in DT40 caused drastic resistance against apoptosis induced by pharmacological ER stress agents (thapsigargin and tunicamycin). Pharmaceutical analysis using specific Bcl-2 inhibitors showed that the drastic resistance against prolonged ER stress-induced apoptosis is, in part, due to up-regulation of Bcl-2 gene expression in ΔGCN5. These data revealed that GCN5 is involved in regulation of prolonged ER stress-induced apoptosis through controlling Bcl-2 gene expression.

Paired box gene 5 isoforms A and B have different functions in transcriptional regulation of B cell development-related genes in immature B cells.

The transcription factor paired box gene 5 (Pax5) is essential for B cell development. In this study, complementation analyses in Pax5-deficient DT40 cells showed that three Pax5 isoforms Pax5A, Pax5B and Pax5BΔEx8 (another spliced isoform of Pax5B lacking exon 8) exhibit distinct roles in transcriptional regulation of six B cell development-related genes (activation-induced cytidine deaminase, Aiolos, BTB and CNC homology 2, B cell lymphoma-6, early B cell factor 1, origin binding factor-1 genes), transcriptions of which are remarkably down-regulated by Pax5-deficiency. Moreover, ectopic expression study shows that these Pax5 isoforms may regulate themselves and each other at the transcriptional level.

Histone acetyltransferase p300/CBP-associated factor is an effective suppressor of secretory immunoglobulin synthesis in immature B cells.

The histone acetyltransferase p300/CBP-associated factor (PCAF) catalyzes acetylation of core histones and plays important roles in epigenetics by altering the chromatin structure in vertebrates. In this study, PCAF-deficient DT40 mutants were analyzed and it was found that PCAF participates in regulation of secretory IgM heavy chain (H-chain) synthesis. Remarkably, PCAF-deficiency causes an increase in the amount of secretory IgM H-chain mRNA, but not in that of IgM light chain and membrane-bound IgM H-chain mRNAs, resulting in dramatic up-regulation of the amount of secretory IgM protein. These findings suggest that PCAF regulates soluble antibody production and is thus an effective suppressor of secretory IgM H-chain synthesis.

Down-regulation of Fas-mediated apoptosis by plasma transglutaminase factor XIII that catalyzes fetal-specific cross-link of the Fas molecule.

The Fas antigen, also designated as APO-1 or CD95, is a member of the tumor necrosis factor receptor superfamily and can mediate apoptotic cell death in various cells. We report here that blood coagulation factor XIII (plasma transglutaminase, fibrin stabilizing factor) inhibits apoptosis induced by a cytotoxic anti-Fas monoclonal antibody in Jurkat cells. When cells were treated with the antibody in fetal calf serum-containing media, higher-molecular-weight (180K) polypeptides containing Fas molecule were detected by immunoblotting. Under conditions where the transglutaminase activity was eliminated or suppressed, the cross-link of Fas was not observed, and concurrently cell death was hastened. Moreover, an antibody against factor XIII strongly accelerated the Fas-mediated apoptosis. Furthermore, addition of partially purified factor XIII neutralized the apoptosis-promoting effect of anti-factor XIII antibody, indicating that this enzyme is involved in cross-link of Fas and down-regulates Fas-mediated apoptotic cell death. Significantly, the cross-link of Fas was seen only in fetal calf serum but not in newly-born calf serum, 1-year-old calf serum or adult bovine serum. These data suggest that plasma transglutaminase factor XIII may play a key role in fetal development of vertebrates via cross-link of Fas antigen.

Centrosomal BRCA2 is a target protein of membrane type-1 matrix metalloproteinase (MT1-MMP).

BRCA2 localizes to centrosomes between G1 and prophase and is removed from the centrosomes during mitosis, but the underlying mechanism is not clear. Here we show that BRCA2 is cleaved into two fragments by membrane type-1 matrix metalloproteinase (MT1-MMP), and that knockdown of MT1-MMP prevents the removal of BRCA2 from centrosomes during metaphase. Mass spectrometry mapping revealed that the MT1-MMP cleavage site of human BRCA2 is between Asn-2135 and Leu-2136 ((2132)LSNN/LNVEGG(2141)), and the point mutation L2136D abrogated MT1-MMP cleavage. Our data demonstrate that MT1-MMP proteolysis of BRCA2 regulates the abundance of BRCA2 on centrosomes.

GCN5 protects vertebrate cells against UV-irradiation via controlling gene expression of DNA polymerase η.

By UV-irradiation, cells are subjected to DNA damage followed by mutation, cell death and/or carcinogenesis. DNA repair systems such as nucleotide excision repair (NER) and translesion DNA synthesis (TLS) protect cells against UV-irradiation. To understand the role of histone acetyltransferase GCN5 in regulation of DNA repair, we studied the sensitivity of GCN5-deficient DT40, GCN5(-/-), to various DNA-damaging agents including UV-irradiation, and effects of GCN5-deficiency on the expression of NER- and TLS-related genes. After UV-irradiation, cell death and DNA fragmentation of GCN5(-/-) were appreciably accelerated as compared with those of DT40. Interestingly, GCN5(-/-) showed a remarkable sensitivity to only UV-irradiation but not to other DNA-damaging agents tested. Semiquantitative RT-PCR showed that transcription of DNA polymerase η (POLH) gene whose deficiency is responsible for a variant form of xeroderma pigmentosum was drastically down-regulated in GCN5(-/-) (to ∼25%). In addition, ectopic expression of human POLH in GCN5(-/-) dramatically reversed the sensitivity to UV-irradiation of GCN5(-/-) to almost the same level of wild type DT40. Moreover, chromatin immunoprecipitation assay revealed that GCN5 binds to the chicken POLH gene 5'-flanking region that contains a typical CpG island and acetylates Lys-9 of histone H3, but not Lys-14 in vivo. These data suggest that GCN5 takes part in transcription regulation of POLH gene through alterations in the chromatin structure by direct interaction with its 5'-flanking region, and protects vertebrate cells against UV-induced DNA damage via controlling POLH gene expression.

Sorting nexin 27 interacts with multidrug resistance-associated protein 4 (MRP4) and mediates internalization of MRP4.

Multidrug resistance-associated protein 4 (MRP4/ABCC4) makes a vital contribution to the bodily distribution of drugs and endogenous compounds because of its cellular efflux abilities. However, little is known about the mechanism regulating its cell surface expression. MRP4 has a PDZ-binding motif, which is a potential sequence that modulates the membrane expression of MRP4 via interaction with PDZ adaptor proteins. To investigate this possible relationship, we performed GST pull-down assays and subsequent analysis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. This method identified sorting nexin 27 (SNX27) as the interacting PDZ adaptor protein with a PDZ-binding motif of MRP4. Its interaction was confirmed by a coimmunoprecipitation study using HEK293 cells. Knockdown of SNX27 by siRNA in HEK293 cells raised MRP4 expression on the plasma membrane, increased the extrusion of 6-[(14)C]mercaptopurine, an MRP4 substrate, and conferred resistance against 6-[(14)C]mercaptopurine. Cell surface biotinylation studies indicated that the inhibition of MRP4 internalization was responsible for these results. Immunocytochemistry and cell surface biotinylation studies using COS-1 cells showed that SNX27 localized to both the early endosome and the plasma membrane. These data suggest that SNX27 interacts with MRP4 near the plasma membrane and promotes endocytosis of MRP4 and thereby negatively regulates its cell surface expression and transport function.

Purification of N-acetyllactosamine-binding activity from the porcine sperm membrane: possible involvement of an ADAM complex in the carbohydrate-binding activity of sperm.

Although the importance of carbohydrate recognition by sperm during egg zona pellucida binding has been widely reported, the sperm molecular species that recognize the carbohydrates are poorly characterized. Our previous cytochemical study indicated that two kinds of carbohydrate-binding proteins are expressed on porcine sperm heads-one recognizes N-acetyllactosamine (Galß1-4GlcNAc-), and the other recognizes the Lewis X structure (Galß1-4(Fucα1-3)GlcNAc-). For this report, we used proteomic techniques to characterize the sperm proteins that bind N-acetyllactosamine. Porcine sperm plasma membrane was solubilized with a detergent solution and subjected to sequential chromatography with dextran sulfate agarose, affinity, and hydroxyapatite, and the binding activities in the eluates were monitored by a solid-phase binding assay. The tryptic peptides of two proteins most likely associated with the binding activities were subjected to tandem mass spectrometry sequencing. A subsequent database search identified one of the two proteins as predicted disintegrin and metalloprotease domain-containing protein 20-like (XP_003128672). The other protein was identified as disintegrin and metalloprotease domain-containing protein 5 (AB613817) by database searches for homologous amino acid sequences, cDNA cloning, nucleotide sequencing and nucleotide database searches. Furthermore, two-dimensional blue native/SDS-PAGE demonstrated that they formed a variety of non-covalent complexes. Therefore, these ADAM complexes probably are responsible for the N-acetyllactosamine-binding activity. An affinity-purified fraction containing these ADAM complexes showed zona pellucida-binding activity, though the activity was relatively weak, and the presence of another zona pellucida-binding protein that probably works in concert with these ADAM complexes was suggested. Immunofluorescence testing suggested that ADAM20-like was localized on the anterior part of the sperm plasma membrane.

GCN5 regulates the activation of PI3K/Akt survival pathway in B cells exposed to oxidative stress via controlling gene expressions of Syk and Btk.

Histone acetyltransferase(s) (HATs) are involved in the acetylation of core histones, which is an important event for transcription regulation through alterations in the chromatin structure in eukaryotes. General control non-depressible 5 (GCN5) was first identified as a global coactivator and transcription-related HAT. Here we report that GCN5 regulates the activation of phosphatidylinositol 3-kinase (PI3K)/acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt) survival pathway in B cells exposed to oxidative stress via controlling gene expressions of spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk). The GCN5-deficiency remarkably caused apoptotic cell death by treatment with exogenous hydrogen peroxide (H(2)O(2)) in chicken DT40 cells. In GCN5-deficient DT40 cells, gene expressions of Syk and Btk, which are involved in activation of PI3K/Akt survival pathway in DT40 cells exposed to exogenous H(2)O(2), were remarkably decreased compared with those in wild type DT40 cells. In addition, phosphorylation of Akt in H(2)O(2)-treated GCN5-deficient cells was remarkably suppressed as compared to that of DT40. Chromatin immunoprecipitation assay revealed that GCN5 binds to proximal 5'-upstream regions of Syk and Btk genes in vivo. These results suggest that GCN5 takes part in transcriptional regulations of the Syk and Btk genes, and plays a key role in epigenetic regulation of PI3K/Akt survival pathway in B cells exposed to reactive oxygen species such as H(2)O(2).

BRCA2 interacts with the cytoskeletal linker protein plectin to form a complex controlling centrosome localization.

The breast cancer susceptibility gene (BRCA2) is localized mainly in the nucleus where it plays an important role in DNA damage repair. Some BRCA2 protein is also present in the centrosome. Here, we demonstrate that BRCA2 interacts with plectin, a cytoskeletal cross-linker protein, and that this interaction controls the position of the centrosome. Phosphorylation of plectin by cyclin-dependent kinase 1/cyclin B (CDK1/CycB) kinase has been reported to abolish its cross-linking function during mitosis. Here, we induced phosphorylation of plectin in prepared fractions of HeLa cells by adding activated CDK1/CycB kinase. Consequently, there was significant dissociation of the centrosome from the nuclear membrane. Plectin has six homologous ankyrin-like repeat domains (termed PLEC M1-M6). Using a pull-down assay, we found that GST-PLEC M1 and a GST-C-terminal region fusion protein (which comprised PLEC M6, along with an adjacent vimentin site) interacted with BRCA2. Since each PLEC module exhibits high homology to the others, the possibility of all six domains participating in this interaction was indicated. Moreover, when PLEC M1 was overexpressed in HeLa cells, it competed with endogenous plectin and inhibited the BRCA2-plectin interaction. This inhibitory effect resulted in dissociation of the centrosomes from the nucleus and increased the rate of micronuclei formation which may lead to carcinogenesis. In addition, when either BRCA2 or plectin was suppressed by the appropriate siRNA, a similar change in centrosomal positioning was observed. We suggest that the BRCA2-plectin interaction plays an important role in the regulation of centrosome localization and also that displacement of the centrosome may result in genomic instability and cancer development.

Cell adhesion markedly increases lucigenin-enhanced chemiluminescence of the phagocyte NADPH oxidase.

Lucigenin-enhanced chemiluminescence (LECL) is widely used for the detection of reactive oxygen species released from various cells and mitochondria. However, the LECL response varies depending on cell species and assay conditions at least in part by unknown factors. Here we report that cell adhesion is an important factor for increasing LECL of tetradecanoylphorbol acetate (TPA)-stimulated human neutrophils. More than 90% LECL remained even after complete removal of the cell suspension 10 min after TPA stimulation, and approximately 22.5% of neutrophils were adhered to the reaction tube. These results indicate that LECL by an adhering neutrophil is approximately 45x higher than that by a non-adhering neutrophil. LECL by leukocyte adhesion deficiency neutrophils was one-fifth of that by normal neutrophils and completely disappeared when the cell suspension was removed, confirming that LECL depends highly on cell adhesion. The oxidase activity of adhering neutrophils measured after permeabilization with Renex 30 together with NADPH addition was similar to that of non-adhering neutrophils, indicating that lucigenin and cell adhesion do not enhance the oxidase activity. Based on these findings, we propose that a mixture of adhering and non-adhering neutrophils can be used for simultaneous screenings of adhering activity and the oxidase activity of neutrophils.

Protein kinase C epsilon phosphorylates keratin 8 at Ser8 and Ser23 in GH4C1 cells stimulated by thyrotropin-releasing hormone.

Protein kinase C epsilon (PKCepsilon) is activated by thyrotropin-releasing hormone (TRH), a regulator of pituitary function in rat pituitary GH(4)C(1) cells. We analyzed the downstream mechanism after PKCepsilon activation. Exposure of GH(4)C(1) cells to TRH or a phorbol ester increased the phosphorylation of three p52 proteins (p52a, p52b and p52c) and decreased the phosphorylation of destrin and cofilin. GF109203X, an inhibitor of protein kinases including PKC, inhibited phosphorylation of the p52 proteins by TRH stimulation. Peptide mapping, amino-acid sequencing, and immunochemical studies indicated that p52a, p52b, and p52c are the differentially phosphorylated isoforms of keratin 8 (K8), an intermediate filament protein. The unphosphorylated K8 (p52n) localized exclusively to the cytoskeleton, whereas the phosphorylated forms (especially p52c), which are increased in TRH-stimulated cells, localized mainly to the cytosol. K8 phosphorylation was enhanced in PKCepsilon-overexpressing clones, and purified recombinant PKCepsilon directly phosphorylated K8 with a profile similar to that observed in TRH-stimulated cells. PKCepsilon and K8 colocalized near the nucleus under basal conditions and were concentrated in the cell periphery and cell-cell contact area after TRH stimulation. MS analyses of phospho-K8 and K8-synthesized peptide (amino acids 1-53) showed that PKCepsilon phosphorylates Ser8 and Ser23 of K8. Phosphorylation of these sites is enhanced in TRH-stimulated cells and PKCepsilon-overexpressing cells, as assessed by immunoblotting using antibodies to phospho-K8. These results suggest that K8 is a physiological substrate for PKCepsilon, and the phosphorylation at Ser8 and Ser23 transduces, at least in part, TRH-PKCepsilon signaling in pituitary cells.

Autoantibodies against glial fibrillary acidic protein (GFAP) in cerebrospinal fluids from Pug dogs with necrotizing meningoencephalitis.

Cerebrospinal fluids (CSFs) from 9 Pug dogs with necrotizing meningoencephalitis (NME: Pug dog encephalitis) were examined to identify the antigens for anti-astrocyte autoantibodies. Each CSF exhibited a positive reaction to the cytoplasm of cultured canine astrocytes by an indirect fluorescent antibody test. In an immunoblotting analysis on normal canine brain proteins, eight of 9 CSFs showed a common band of 52 kDa, corresponding to glial fibrillary acidic protein (GFAP), and all of 9 CSFs reacted with purified bovine GFAP. From these results, GFAP is one of the common autoantigens in Pug dogs with NME. On the other hand, the reactivity of CSFs to chymotrypsin-digested bovine GFAP fragments were variable among dogs, indicating that the antibodies in the CSFs recognized different epitopes on GFAP.

Carbonic anhydrase II is a tumor vessel endothelium-associated antigen targeted by dendritic cell therapy.

Tumor-associated antigens are promising candidates as target molecules for immunotherapy and a wide variety of tumor-associated antigens have been discovered through the presence of serum antibodies in cancer patients. We previously conducted dendritic cell therapy on 10 malignant melanoma patients and shrinkage or disappearance of metastatic tumors with massive necrosis occurred in two patients. In this study, we found a 29-kDa protein against which antibody was elicited by dendritic cell therapy in one of the two patients. Matrix-assisted laser desorption ionization-time of flight/mass spectrometry analysis of the protein isolated by two-dimensional electrophoresis combined with Western blots revealed that the 29-kDa protein was carbonic anhydrase II (CA-II). Immunohistochemistry of the tumors and normal tissues showed that CA-II was expressed in the tumor vessel but not in normal vessel endothelium. CA-II expression in tumor endothelium was observed as well in other cancers including esophageal, renal, and lung cancers. In an in vitro angiogenesis model, CA-II expression of normal human vein endothelial cells was significantly up-regulated when cells were cultured in the acidic and hypoxic conditions indicative of a tumor environment. These findings suggest that CA-II is a tumor vessel endothelium-associated antigen in melanoma and other cancers, and elicitation of serum anti-CA-II antibody by dendritic cell therapy may be associated with good clinical outcome including tumor reduction.

Cleavage at the carboxyl-terminus of Ku80 during apoptosis in human Jurkat T cells.

We have previously reported that the amount of Apg-2, an Hsp110 family protein, decreases during apoptosis in Jurkat T cells. Since we hypothesized that Apg-2 would be cleaved by caspase-3 during apoptosis, a cleavage-site-directed antibody was raised against the carboxyl-terminus of the Apg-2 fragment that appears after the cleavage by caspase-3. Although this antibody could not detect the Apg-2 fragment in apoptotic cells, three additional fragments were unexpectedly detected. Based on the results of microsequencing, one of these fragments was identified as Ku80. Ku80 is a nuclear protein and a component of DNA-dependent protein kinase (DNA-PK). In this study, we observed that Ku80 is cleaved at Asp-730 residue during apoptosis, and this cleavage occurs in the nucleus in the early apoptotic phase. Furthermore, Ku80 is distributed in the cytoplasm of nuclear fragmented apoptotic cells, although the cleaved fragment contains the nuclear-localization signal (NLS). Our study clearly shows that Ku80 is cleaved in the nucleus, and distributes in the cytoplasm during apoptosis.

Cleavage of nonmuscle myosin heavy chain-A during apoptosis in human Jurkat T cells.

We have previously reported that calpastatin, an endogenous inhibitory protein of calpain, is cleaved by a caspase-3-like protease during apoptosis in human Jurkat T cells [Kato, M. et al. (2000) J. Biochem. 127, 297-305]. In this study, we found that nonmuscle myosin heavy chain-A (NMHC-A) is cleaved during apoptosis in Jurkat cells by using a cleavage-site-directed antibody for calpastatin. The cleavage-site-directed antibody was raised against the amino-terminal fragment of calpastatin, and this antibody detected the in vitro cleaved calpastatin fragment. Although cleaved calpastatin was not detected, a 95-kDa polypeptide (p95) was detected in apoptotic cells by this antibody. This p95 was identified as the carboxyl-terminal fragment of NMHC-A based on the results of peptide mass spectrometry fingerprinting and amino-terminal sequencing. Furthermore, two cleavage sites on NMHC-A, Asp-1153 and Asp-1948, were determined, and three cleaved fragments of NMHC-A, one cleaved at Asp-1153 and the other two cleaved at Asp-1948, were detected by cleavage-site-directed antibodies against each cleavage site. The results of confocal immunofluorescence microscopic analysis show that the cleavage at Asp-1948 occurs faster than that at Asp-1153 during apoptosis. In addition, the Asp-1153 cleaved fragment was distributed diffusely in the cytoplasm of apoptotic cells, whereas the Asp-1948 cleaved fragments were detected as condensed dots. In conclusion, our findings can be summarized as follows: (i) NMHC-A is cleaved at two sites during apoptosis, (ii) the timing of cleavage is different between these two cleavage sites, and (iii) the distribution of cleaved fragments is different in apoptotic cells.

Superoxide generation in directional coronary atherectomy specimens of patients with angina pectoris: important role of NAD(P)H oxidase.

OBJECTIVE: NADH/NADPH oxidase is an important source of reactive oxygen species (ROS) in the vasculature. Recently, we demonstrated that p22(phox), an essential component of this oxidase, was expressed in human coronary arteries and that its expression was enhanced with the progression of atherosclerosis. The present study was undertaken to investigate its functional importance in the pathogenesis of coronary artery disease. For this aim, the expression of p22(phox), the distribution of oxidized low density lipoprotein (LDL), and the generation of ROS in directional coronary atherectomy (DCA) specimens were examined. METHODS AND RESULTS: DCA specimens were obtained from patients with stable or unstable angina pectoris. The distribution of p22(phox) and of oxidized LDL was examined by immunohistochemistry. The generation of superoxide in DCA specimens was assessed by the dihydroethidium method and lucigenin-enhanced chemiluminescence. ROS were closely associated with the distribution of p22(phox) and oxidized LDL. Not only inflammatory cells but also smooth muscle cells and fibroblasts generated ROS. There was a correlation between ROS and the expression of p22(phox) or oxidized LDL. The generation of ROS was significantly higher in unstable angina pectoris compared with stable angina pectoris. CONCLUSIONS: ROS generated by p22(phox)-based NADH/NADPH oxidase likely mediate the oxidative modification of LDL and might play a major role in pathogenesis of atherosclerotic coronary artery disease.

Oxidative stress in the pathogenesis of thoracic aortic aneurysm: protective role of statin and angiotensin II type 1 receptor blocker.

OBJECTIVE: The pathogenesis of thoracic aortic aneurysms (TAA) is still unclear. A recent investigation indicated that angiotensin II, a potent activator of NADH/NADPH oxidase, plays an important role in aneurysmal formation. We investigated the potential role of p22phox-based NADH/NADPH oxidase in the pathogenesis of TAA. METHODS: Human thoracic aneurysmal (n=40) and non-aneurysmal (control, n=39) aortic sections were examined, and the localization of p22phox, an essential component of the oxidase, and its expressional differences were investigated by immunohistochemistry and Western blot. In situ reactive oxygen species (ROS) generation was examined by the dihydroethidium method, and the impact of medical treatment on p22phox expression was investigated by multiple regression analysis. RESULTS: In situ production of ROS and the expression of p22phox increased markedly in TAA throughout the wall, and Western blot confirmed the enhanced expression of p22phox. The expression was more intense in the regions where monocytes/macrophages accumulated. In these inflammatory regions, numerous chymase-positive mast cells and angiotensin converting enzyme-positive macrophages were present. Their localization closely overlapped the in situ activity of matrix metalloproteinase and the expression of p22phox. Multiple regression analysis revealed that medical treatment with statin and angiotensin II type 1 receptor blocker (ARB) suppressed p22phox expression in TAA. CONCLUSION: Our findings indicate the role of p22phox-based NADH/NADPH oxidase and the local renin-angiotensin system in the pathogenesis of TAA. Statin and ARB might have inhibitory effects on the formation of aneurysms via the suppression of NADH/NADPH oxidase.

Efficient inhibition of tumor angiogenesis and growth by a synthetic peptide blocking S100A4-methionine aminopeptidase 2 interaction.

The prometastatic calcium-binding protein, S100A4, is expressed in endothelial cells, and its downregulation markedly suppresses tumor angiogenesis in a xenograft cancer model. Given that endothelial S100A4 can be a molecular target for inhibiting tumor angiogenesis, we addressed here whether synthetic peptide capable of blocking S100A4-effector protein interaction could be a novel antiangiogenic agent. To examine this hypothesis, we focused on the S100A4-binding domain of methionine aminopeptidase 2, an effector protein, which plays a role in endothelial cell growth. Overexpression of the domain in mouse endothelial MSS31 cells reduced DNA synthesis, and the corresponding synthetic peptide (named NBD) indeed interacted with S100A4 and inhibited capillary formation in vitro and new blood vessel formation in vivo. Intriguingly, a single intra-tumor administration of the NBD peptide in human prostate cancer xenografts significantly reduced vascularity, resulting in tumor regression. Mechanistically, the NBD peptide enhanced assembly of nonmuscle myosin IIA filaments along with Ser1943 phosphorylation, stimulated formation of focal adhesions without phosphorylation of focal adhesion kinase, and provoked G1/S arrest of the cell cycle. Altogether, the NBD peptide is a potent inhibitor for tumor angiogenesis, and is the first example of an anticancer peptide drug developed on the basis of an endothelial S100A4-targeted strategy.