Biomedical subjects

Kazufumi Katayama

Publications and source records attributed to Kazufumi Katayama.

18 recordsLinked to original sources

PubMed2006

Peroxisome proliferator-activated receptor gamma-mediated regulation of neural stem cell proliferation and differentiation.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays an important role in insulin sensitivity, tissue homeostasis, and regulating cellular functions. We found high-level expression of PPARgamma in embryo mouse brain and neural stem cells (NSCs), in contrast to extremely low levels in adult mouse brain. Here, we show that PPARgamma mediates the proliferation and differentiation of murine NSCs via up-regulation of the epidermal growth factor receptor and activation of the ERK pathway. Cell growth rates of NSCs prepared from heterozygous PPARgamma-deficient mouse brains, PPARgamma-RNA-silenced NSCs, and PPARgamma dominant-negative NSCs were significantly decreased compared with those of wild-type NSCs. Physiological concentrations of PPARgamma agonists, rosiglitazone and pioglitazone, stimulated NSC growth, whereas antagonists caused cell death in a concentration-dependent manner via activation of the caspase cascade. The stimulation of cell growth by PPARgamma was associated with a rapid activation of the ERK pathway by phosphorylation and up-regulation of epidermal growth factor receptor and cyclin B protein levels. In contrast, activation of PPARgamma by agonists inhibited the differentiation of NSCs into neurons. The inhibition of differentiation was associated with an activation of STAT3. These data indicate that PPARgamma regulates the development of the central nervous system during early embryogenesis via control of NSC proliferation.

Animals↗

PubMed2006

Leukotriene B4 and lipoxin A4 are regulatory signals for neural stem cell proliferation and differentiation.

Leukotrienes (LTs) and lipoxins (LXs) are lipid mediators that play a key role in regulating acute inflammatory responses. Their roles in neural stem cell (NSC) functions are of interest. We showed here that LTB(4) and LXA(4) regulated proliferation and differentiation of murine NSCs that were isolated from embryo brains. Proliferation of NSCs was stimulated by LTB(4) (3 to 100 nM) and blocked by receptor antagonist (IC(50)=2.7 microM). In contrast, LXA(4), and its aspirin-triggered-15-epi-LXA(4) stable analog attenuated growth of NSCs at as little as 1 nM. Both lipoxygenase (LOX) inhibitors and LTB(4) receptor antagonists caused apoptosis and cell death. Gene chip analysis revealed that growth-related gene expressions such as epidermal growth factor (EGF) receptor, cyclin E, p27, and caspase 8 were tightly regulated by LTB(4); LXA(4) gave the opposite gene expressions. In addition to proliferation, LTB(4) induced differentiation of NSCs into neurons as monitored by neurite outgrowth and MAP2 expression. These results indicate for the first time that LTB(4) and LXA(4) directly regulate proliferation and differentiation of NSCs, suggesting these new pathways may be useful in restoring stem cells.

Animals↗

PubMed2005

Fusogenic liposome delivers encapsulated nanoparticles for cytosolic controlled gene release.

Therapeutic agents based on DNA or RNA oligonucleotides (e.g., antisense DNA oligonucleotide, small interfering RNA) require a regulation of their kinetics in cytoplasm to maintain an optimal concentration during the treatment period. In this respect, delivery of functional nanoparticles containing these drugs into cytoplasm has been thought to have a potential for the cytosolic controlled gene release. In this study, we establish a protocol for the encapsulation of nanoparticles into liposome, which is further fused with ultra violet-inactivated Sendai virus to compose fusogenic liposomes. When nanoparticles were encapsulated in conventional liposomes, endocytosis-mediated uptake of nanoparticles was observed. In contrast, numerous amounts of nanoparticles were delivered into the cytoplasm without any cytotoxicity when the particles were encapsulated in fusogenic liposomes. Additionally, fusogenic liposome showed a high ability to deliver nanoparticles containing DNA oligonucleotides into cytoplasm. These results indicate that this combinatorial nanotechnology using fusogenic liposome and nanoparticle is a valuable system for regulating the intracellular pharmacokinetics of gene-based drugs.

Animals↗

PubMed2005

RNA interference of PPARgamma using fiber-modified adenovirus vector efficiently suppresses preadipocyte-to-adipocyte differentiation in 3T3-L1 cells.

The peroxisome proliferator-activated receptor (PPAR) gamma is regarded as a "master regulator" of adipocyte differentiation and is abundantly expressed in adipose. To understand the biological role of PPARgamma in adipose, RNA interference (RNAi) of PPARgamma should be a powerful tool. 3T3-L1 cell line serves an excellent model to investigate the mechanism of preadipocyte-to-adipocyte differentiation. However, this cell line is difficult to transfect by plasmid vectors and viral vectors. We optimized the transduction of both 3T3-L1 preadipocytes and adipocytes by means of fiber-modified adenovirus (Ad) vectors. Among the various vectors tested, polylysine modification of the C-terminal of the fiber knob most markedly improved the transduction efficiency in both 3T3-L1 preadipocytes and adipocytes. Then, we examined whether fiber-modified Ad vectors with polylysine peptides expressing the small interfering RNA (siRNA) for PPARgamma inhibit the differentiation of 3T3-L1 preadipocytes into adipocytes. Oil red O staining and measurement of glycerol-3-phosphate dehydrogenase (GPDH) activity indicated that the vectors effectively suppressed the differentiation of 3T3-L1 preadipocytes to adipocytes. These results suggested that the combination of fiber-modified Ad vectors containing polylysine peptides and RNAi is an effective tool for the study of the biological and physiological mechanism of adipogenesis in adiposity and diabetes using 3T3-L1 models. Ad vector-mediated RNAi for PPARgamma should also be useful to clarify the biological role of the PPARgamma pathway in various tissues in addition to adipose and for therapeutic application to a variety of diseases, including adiposity and diabetes.

3T3-L1 Cells↗

PubMed2005

Nuclear receptors as targets for drug development: the role of nuclear receptors during neural stem cell proliferation and differentiation.

The fate of stem cells, such as neural stem cells and hematopoietic stem cells, depends on strictly regulated signaling events including activation of nuclear receptors, resulting in subsequent gene induction. Recently, we demonstrated that PPARgamma, a ligand-activated nuclear receptor, plays an important role in regulating the proliferation and differentiation of murine neural stem cell (NSC). NSC prepared from heterozygous PPARgamma-deficient mouse exhibited a slower growth rate compared with that of wild-type mouse, which was also demonstrated in PPARgamma-knockdown NSC that was generated by the lentiviral-vector-mediated RNA interference approach. These studies have important implications for understanding central nervous system functions and developing a therapy for neurodegenerative disorders. In this review, recent findings on stem cell biology, especially focusing on nuclear receptors in NSCs, including our current study, will be discussed.

Animals↗

PubMed2005

PEGylated adenovirus vectors containing RGD peptides on the tip of PEG show high transduction efficiency and antibody evasion ability.

BACKGROUND: PEGylation of adenovirus vectors (Ads) is an attractive strategy in gene therapy. Although many types of PEGylated Ad (PEG-Ads), which exhibit antibody evasion activity and long plasma half-life, have been developed, their entry into cells has been prevented by steric hindrance by polyethylene glycol (PEG) chains. Likewise, sufficient gene expression for medical treatment could not be achieved. METHODS: A set of PEG-Ads, which have different PEG modification rates, was constructed, and gene expression was evaluated using A549 cells. A novel PEGylated Ad (RGD-PEG-Ad), which contained RGD (Arg-Gly-Asp) peptides on the tip of PEG, was developed. We evaluated gene expression both in Coxsackie-adenovirus receptor (CAR)-positive as well as -negative cells, and in vivo gene expression was also determined. Furthermore, the antibody evasion ability and the specificity of infection exhibited by this RGD-PEG-Ad were also evaluated. RESULTS: Whereas PEG-Ads decreased gene expression in CAR-positive cells, RGD-PEG-Ad enhanced gene expression notably, to a level about 200-fold higher than that of PEG-Ads. Moreover, gene expression of RGD-PEG-Ad was almost equal to that of Ad-RGD, which contains an RGD-motif in the fiber and exhibits among the highest gene expression of CAR-positive and -negative cells. Furthermore, although Ad-RGD gene expression decreased remarkably in the presence of anti-Ad antiserum, RGD-PEG-Ad maintained its activity against antibodies. In vivo experiments also demonstrated that the modification of Ads with RGD-PEG induced efficient gene expression. CONCLUSIONS: In the present study, we demonstrated that a new strategy, which combined integrin-targeting the RGD peptide on the tip of PEG and modified the Ad using this material, could enhance gene expression in both CAR-positive and -negative cells. At the same time, this novel PEGylated Ad maintained strong protective activity against antibodies. This strategy could also be easily modified for developing other vectors using other targeting molecules.

Adenoviridae↗

PubMed2004

A novel therapy for acute hepatitis utilizing dehydroepiandrosterone in the murine model of hepatitis.

Dehydroepiandrosterone (DHEA), one of the major androgens secreted by the adrenal cortex, has been shown to have potential immunoreguratory properties. In this study, we examined the effect of DHEA in a mouse model of hepatitis. Mice were treated with DHEA and injected with concanavalin A (Con A) or lipopolysaccharide (LPS)/D-galactosamine (GalN). Cytokine expression was measured by quantitative RT-PCR and ELISA. Apoptosis was detected by the TUNEL method and by DNA fragmentation analysis. In the DHEA-treated mice, the serum levels of ALT and expression of inflammatory mediators were significantly decreased. The number of apoptotic cells was also much lower than that observed in control, untreated mouse liver tissue. There were fewer tumor necrosis factor-alpha (TNF-alpha)-induced apoptotic cells in H4IIE hepatoma cells treated with DHEA than in non-treated cells. DHEA decreased the expression levels of mRNA transcripts encoding TNF-alpha and iNOS. These results suggest that DHEA can reduce T-cell-mediated injury in the liver as manifest by inhibition of the expression of several inflammatory mediators and hepatocyte apoptosis. DHEA should, thus, be considered as a novel candidate for the therapy of liver injury.

Animals↗

PubMed2004

RNA interfering approach for clarifying the PPARgamma pathway using lentiviral vector expressing short hairpin RNA.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a central role in adipocyte differentiation and insulin sensitivity. Although PPARgamma also appears to regulate diverse cellular processes in other cell types such as lymphocytes, the detailed mechanisms remain unclear. In this study, we established a lentivirus-mediated short hairpin RNA expression system and identified a potent short hairpin RNA which suppresses PPARgamma expression, resulting in marked inhibition of preadipocyte-to-adipocyte differentiation in 3T3-L1 cells. Our PPARgamma-knockdown method will serve to clarify the PPARgamma pathway in various cell types in vivo and in vitro, and will facilitate the development of therapeutic applications for a variety of diseases.

3T3-L1 Cells↗

PubMed2004

Adenovirus vector-mediated doxycycline-inducible RNA interference.

RNA interference (RNAi) is a powerful tool for the knockdown of gene expression. Here, we report on the development of an adenovirus (Ad) vector-mediated doxycycline (Dox)-inducible small interfering RNA (siRNA) expression system. We used this siRNA system to control the expression of p53 and c-Myc in human cancer cells. Coinfection of Ad vectors containing the siRNA expression system under the control of the Dox-inducible H1 promoter and Ad vectors expressing a tetracycline repressor inhibited the expression levels of p53 and c-Myc in a dose-dependent manner with both Dox and viral dose. Regulated silencing of p53 and c-Myc expression was obtained. Because an Ad vector-mediated inducible RNAi system can efficiently transduce a variety of cell types in vitro and in vivo, and the degree of loss of gene expression can be modulated according to the dose of Dox, this expression system should be a useful tool for both basic research on the analysis of gene function and therapeutic applications of RNAi.

Adenoviridae↗

PubMed2004

Neutralizing antibody evasion ability of adenovirus vector induced by the bioconjugation of methoxypolyethylene glycol succinimidyl propionate (MPEG-SPA).

Although adenovirus vectors (Ad) which possesses high transduction efficiency are widely used for gene therapy in animal models, clinical use is very limited. One of the main reason is that nearly 80% of human beings possess anti-Ad antibodies. In this study, we tried to modify Ad with methoxypolyethylene glycol (MPEG) activated by succinimidyl propionate, and, the neutralizing antibody evasion ability of PEGylated Ad was evaluated. The results demonstrated that PEG-Ad showed stronger protection ability against anti-Ad neutralizing antibody compared to that with unmodified-Ad. Considering there are many people carrying neutralizing antibody against Ad and readministration of Ad was necessary for treating chronic diseases, this strategy, which was also applicable to other vectors, can be used for developing improved vectors.

Adenoviruses, Human↗

PubMed2003

Antitumor effect by interleukin-11 receptor alpha-locus chemokine/CCL27, introduced into tumor cells through a recombinant adenovirus vector.

In this study, we examined antitumor activity of a mouse CC chemokine ILC/CCL27 and a mouse CX(3)C chemokine fractalkine/CX(3)CL1 in vivo. We generated recombinant adenovirus vectors with a fiber mutation, encoding mILC (Ad-RGD-mILC) and mFKN (Ad-RGD-mFKN). We confirmed tumor cells infected with Ad-RGD-mILC and Ad-RGD-mFKN to express and release these chemokines. Tumor rejection experiments in vivo were carried out by inoculating OV-HM cells infected with Ad-RGD-mILC or Ad-RGD-mFKN into immunocompetent mice. mILC significantly suppressed the tumor growth, whereas no such significant effect was observed by mFKN. The antitumor activity induced by mILC was T cell dependent, involving both CD4(+) and CD8(+) T cells. Immunohistochemical analysis revealed accumulation of both CD3(+) lymphocytes and NK cells in the tumor tissue transduced with mILC and mFKN. However, there was a significant difference in the distribution of infiltrating cells. Furthermore, mFKN appeared to have an angiogenic activity, which might have masked its tumor suppressive activity. Collectively, ILC/CCL27 may be a good candidate molecule for cancer gene therapy.

Adenoviridae↗

PubMed2003

A novel T7 system utilizing mRNA coding for T7 RNA polymerase.

The T7 system dose not require the relocation of a reporter gene to the nucleus for its gene expression in the cytoplasm, but relies on the co-localization of T7 RNA polymerase (T7 RNAP) enzyme and reporter gene DNA that is controlled by the T7 promoter. In the present study, we developed a new T7 system in that gene expression can occur at a higher level than those using conventional systems. Insertion of 5'- and 3'-untranslated regions (UTR) of beta-globin gene into a reporter gene enhanced the reporter gene expression, presumably due to the stability and efficient translation of the mRNA. Instead of the T7 RNAP protein used in conventional methods, moreover, transfection of cells with T7 RNAP mRNA, which has been modified by inserting beta-globin 5'- and 3'-UTR sequences as well as the cap and poly(A) tail structures, further enhanced the reporter gene expression. Thus, this novel T7 system using T7 RNAP mRNA may be powerful for the efficient gene expression of DNA exogenously provided in the cytoplasm.

3' Untranslated Regions↗

PubMed2003

A novel PPAR gamma gene therapy to control inflammation associated with inflammatory bowel disease in a murine model.

BACKGROUND & AIMS: Peroxisome proliferator-activated receptor gamma (PPAR gamma) is one of the nuclear receptors that plays a central role in adipocyte differentiation and insulin sensitivity. PPAR gamma has also recently been recognized as an endogenous regulator of intestinal inflammation. However, its levels are decreased during chronic inflammation in human and mice, thus limiting PPAR gamma ligand therapy during established disease. We sought to determine whether this decrease in PPAR gamma could be counteracted by a gene therapy approach. METHODS: We characterized PPAR gamma levels in experimental colitis associated with dextran sodium sulfate administration to mice. In this model, the therapeutic benefits of PPAR gamma gene therapy using a replication-deficient adenovirus vector expressing PPAR gamma (Ad-PPAR gamma) was assessed. RESULTS: PPAR gamma protein levels were decreased in whole colonic tissue, lamina propria lymphocytes, and peritoneal exudate cells during the course of colitis. PPAR gamma gene delivery using Ad-PPAR gamma restored responsiveness to a PPAR gamma ligand, resulting in marked amelioration of tissue inflammation associated with the colitis, which included attenuation of intercellular adhesion molecule-1, cyclooxygenase-2 and tumor necrosis factor-alpha expression. CONCLUSIONS: Our results suggest that gene delivery of PPAR gamma can be used to restore and/or enhance endogenous anti-inflammatory processes that are normally operative in mammalian tissues such as in the colon.

Adenoviridae↗

PubMed2003

[PPARgamma-gene therapy using an adenovirus vector for inflammatory bowel disease].

Peroxisome proliferator-activated receptor gamma (PPARgamma) is one of the nuclear receptors that plays a central role in adipocyte differentiation and insulin sensitivity. Recently, PPARgamma has also been recognized as a suppressive regulator of inflammation in the gastrointestinal tract. We summarize here the therapeutic benefits of PPARgamma-gene therapy using a replication-deficient adenovirus vector expressing PPARgamma (AdRGD-PPARgamma). We demonstrate that PPARgamma- protein levels are decreased in dextran sodium sulfate-induced colitis and restored in this model by intraperitoneal administration of the AdRGD-PPARgamma. Treatment with AdRGD-PPARgamma and PPARgamma-specific ligands resulted in a marked amelioration of tissue inflammation associated with the colitis, including reduction in intercellular adhesion molecule-1, cyclooxygenase-2, and tumor necrosis factor-alpha expression. Our results suggest that gene delivery of PPARgamma may open up a novel therapeutic approach for inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.

Adenoviridae↗

PubMed2002

Nitration of PPARgamma inhibits ligand-dependent translocation into the nucleus in a macrophage-like cell line, RAW 264.

Nitration of tyrosine residues in proteins has been observed in many inflammatory tissues of arthritis, ulcerative colitis, septic shock and ischemia-reperfusion injury. Although several studies have been carried out, it is still unclear what type of protein is nitrated and whether tyrosine nitration interferes with protein function. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor whose activation is linked to several physiological pathways including regulation of insulin sensitivity and control of inflammation. PPARgamma possesses several tyrosine residues, which might be potential targets for nitration by peroxynitrite during inflammatory responses. Here we have investigated whether PPARgamma is nitrated in macrophage-like RAW 264 cells and the effect of nitration on the translocation of PPARgamma into the nucleus. Western blot analysis showed that tumor necrosis factor-alpha, lipopolysaccharide or peroxynitrite treatment significantly increases the nitration of PPARgamma. Cell fractionation analysis and immunofluorescence coupled with confocal laser microscopy revealed that nitration of PPARgamma inhibits its ligand-dependent translocation from the cytosol into the nucleus. Together, these results indicate that nitration of PPARgamma during inflammation may be involved in a reduction in the control of inflammatory responses and also in the development of resistance to PPARgamma ligand-based therapies against inflammation.

Active Transport, Cell Nucleus↗

PubMed2002

PPAR gamma ligands inhibit nitrotyrosine formation and inflammatory mediator expressions in adjuvant-induced rheumatoid arthritis mice.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor, whose activation has been linked to several physiologic pathways including those related to the regulation of insulin sensitivity. Here, we investigate effects of PPARgamma specific ligands, rosiglitazone and pioglitazone, on formation of nitrotyrosine and increased expression of inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 and intercellular adhesion molecule-1 (ICAM-1) in adjuvant-induced murine arthritis. Administration of rosiglitazone or pioglitazone (30 mg/kg, p.o.) significantly inhibited the adjuvant-induced increase in formation of nitrotyrosine and expression of iNOS on both ankle and temporomandibular joints. Rosiglitazone also inhibited the adjuvant-induced expression of M30 positive cells, as a marker of apoptosis, in the joint tissues. In addition, treatment with rosiglitazone or pioglitazone (30 microM) inhibited lipopolysaccharide plus tumor necrosis factor (TNF)-alpha-induced protein expression of iNOS, cyclooxygenase-2, ICAM-1 and nitrotyrosine formation in RAW 264 cells, a murine macrophage-like cell line. Rosiglitazone or pioglitazone inhibited increase in phosphorylated I-kappaB (pI-kappaB) expression, as an index of activation of nuclear factor (NF)-kappaB, in both joint tissues and RAW264 cells. Furthermore, in PPARgamma-transfected HEK293 cells, rosiglitazone inhibited the TNF-alpha-stimulated response using NF-kappaB-mediated transcription reporter assay. These results indicate that PPARgamma ligands may possess anti-inflammatory activity against adjuvant-induced arthritis via the inhibition of NF-kappaB pathway.

Animals↗

PubMed2002

Lack of antigen-specific immune responses in anti-IL-7 receptor alpha chain antibody-treated Peyer's patch-null mice following intestinal immunization with microencapsulated antigen.

Peyer's patches (PP) represent a well-characterized inductive site in gut-associated lymphoid tissue that actively acquires antigens from the intestinal lumen. It was reported that organized PP are not required for antigen-specific IgA responses induced by oral immunization with soluble antigen mixed with the mucosal adjuvant, cholera toxin. However, the role of PP in the induction of mucosal and systemic immune responses remains to be clarified in the case of particulate antigen. Here, we created PP-null mice by treating them with monoclonal anti-IL-7 receptor alpha chain (IL-7 R alpha) antibody during gestation and then immunized with antigen-encapsulated poly-lactic acid (PLA) microspheres. Brisk OVA-specific antibody responses were noted in serum and fecal extracts of normal mice following direct intestinal immunization with OVA in PBS (OVA-PBS) as well as in PLA-microspheres (OVA-MS). Antibody production was similarly elevated in PP-null mice immunized with OVA-PBS via direct injection into the intestinal tract. In contrast, OVA-specific antibody responses were dramatically decreased in both serum and fecal extracts collected from PP-null mice immunized intestinally with OVA-MS. These results were further supported by the number of OVA-specific antibody-forming cells detected in the spleen and intestinal lamina propria. PP deficiency also resulted in the reduction in OVA-specific Th1/Th2 cell responses in the spleen and mesenteric lymph nodes of mice intestinally immunized with OVA-MS. These results suggested that organized PP do, in fact, play a crucial role in the induction of antigen-specific immune responses against ingested particulate antigen.

Animals↗

PubMed2002

Gene expression profile after peroxisome proliferator activator receptor-gamma ligand administration in dextran sodium sulfate mice.

BACKGROUND: Peroxisome proliferator activator receptor-gamma (PPARgamma) is a member of the nuclear receptor superfamily. Ligands of PPARgamma, thiazolidione derivatives, have been reported to be the one of the candidates for the treatment of inflammatory bowel disease (IBD). Given the fact that PPARgamma is a transcription regulator, expression pharmacogenomics, including differential gene expression profiling of drug responses in a colitis model, is thought to be a useful approach for finding relevant genes that can serve as the target for new drug treatment of IBD. METHODS: We performed a global analysis for differential gene expression of the intestine in a dextran sodium sulfate (DSS) colitis mouse model following PPARgamma ligand administration. By applying a high-density oligonucleotide array method, the expression patterns of approximately 12000 genes were analyzed, and selected genes were confirmed by a real-time quantitative PCR method. RESULTS: The analysis of downregulated genes in the DSS mice following PPARgamma administration revealed several functional gene clusters with altered expression: (1) oncogene families such as GRO1 oncogenes, (2) inflammatory mediator-related genes such as the interferon-gamma gene, (3) water electrolyte-associated genes, and (4) others. CONCLUSIONS: This is the first demonstration of global gene expression analysis using the DSS colitis mouse model with a PPARgamma ligand, and these results provide new insight for finding novel target genes for treating IBD.

Animals↗