Chiho Kudo

Peroxisome Proliferator-activated Receptor γ-mediated Regulation of Neural Stem Cell Proliferation and Differentiation

Peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in insulin sensitivity, tissue homeostasis, and regulating cellular functions. We found high-level expression of PPARγ in embryo mouse brain and neural stem cells (NSCs), in contrast to extremely low levels in adult mouse brain. Here, we show that PPARγ 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 PPARγ-deficient mouse brains, PPARγ-RNA-silenced NSCs, and PPARγ dominant-negative NSCs were significantly decreased compared with those of wild-type NSCs. Physiological concentrations of PPARγ 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 PPARγ 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 PPARγ by agonists inhibited the differentiation of NSCs into neurons. The inhibition of differentiation was associated with an activation of STAT3. These data indicate that PPARγ regulates the development of the central nervous system during early embryogenesis via control of NSC proliferation.

PPARγ 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.

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 LTB4 and LXA4 regulated proliferation and differentiation of murine NSCs that were isolated from embryo brains. Proliferation of NSCs was stimulated by LTB4 (3 to 100 nM) and blocked by receptor antagonist (IC50=2.7 µM). In contrast, LXA4, and its aspirin‐triggered‐15‐epi‐LXA4 stable analog attenuated growth of NSCs at as little as 1 nM. Both lipoxygenase (LOX) inhibitors and LTB4 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 LTB4; LXA4 gave the opposite gene expressions. In addition to proliferation, LTB4 induced differentiation of NSCs into neurons as monitored by neurite outgrowth and MAP2 expression. These results indicate for the first time that LTB4 and LXA4 directly regulate proliferation and differentiation of NSCs, suggesting these new pathways may be useful in restoring stem cells.—Wada, K., Arita, M., Nakajima, A., Katayama, K., Kudo, C., Kamisaki, Y., Serhan, C. N. Leukotriene B4 and lipoxin A4 are regulatory signals for neural stem cell proliferation and differentiation. FASEB J. 20, 1785–1792 (2006)

The collagen-binding protein of Streptococcus mutans is involved in haemorrhagic stroke

Although several risk factors for stroke have been identified, one-third remain unexplained. Here we show that infection with Streptococcus mutans expressing collagen-binding protein (CBP) is a potential risk factor for haemorrhagic stroke. Infection with serotype k S. mutans, but not a standard strain, aggravates cerebral haemorrhage in mice. Serotype k S. mutans accumulates in the damaged, but not the contralateral hemisphere, indicating an interaction of bacteria with injured blood vessels. The most important factor for high-virulence is expression of CBP, which is a common property of most serotype k strains. The detection frequency of CBP-expressing S. mutans in haemorrhagic stroke patients is significantly higher than in control subjects. Strains isolated from haemorrhagic stroke patients aggravate haemorrhage in a mouse model, indicating that they are haemorrhagic stroke-associated. Administration of recombinant CBP causes aggravation of haemorrhage. Our data suggest that CBP of S. mutans is directly involved in haemorrhagic stroke.

Nonylphenol induces the death of neural stem cells due to activation of the caspase cascade and regulation of the cell cycle

Endocrine disruptors (EDs) are a great concern throughout the world, because they have adverse effects on human health and wildlife. In the present study, we investigated the effects of EDs on the proliferation and survival of murine neural stem cells (NSCs). In contrast to bisphenol A, phthalic acid benzyl n‐butyl ester, phthalic acid di‐n‐butyl ester and phthalic acid di(2‐ethylhexyl) ester, the treatment of NSCs with 4‐nonylphenol for 24 h inhibited cell growth in a concentration‐dependent manner. In addition, treatment with 4‐nonylphenol resulted in nuclear condensation and DNA fragmentation (morphological changes due to apoptosis) in NSCs after 12 h of exposure, and activated caspase‐3 after 6 h and 9 h of exposure. Furthermore, an exposure to 4‐nonylphenol led to the accumulation of cells at the G2/M phase interface and down‐regulated the protein levels of cyclin A and B1, which are the major regulatory proteins at the G2 to M transition of the cell cycle. Together, these results indicate that, in contrast to other EDs, 4‐nonylphenol may exhibit a potent cytotoxicity through apoptosis via the caspase cascade and cell cycle arrest at the G2/M phase, and suggest that 4‐nonylphenol may affect neurogenesis in the CNS.

Critical Role of Peroxisome Proliferator-Activated Receptor γ on Anoikis and Invasion of Squamous Cell Carcinoma

Purpose: Peroxisome proliferator-activated receptor γ (PPARγ) plays a important role in various physiological functions. We examined whether PPARγ is expressed in primary squamous cell carcinoma and lymph node metastasis and whether PPARγ is a potential target for tumor therapy. Experimental Design and Results: A high-level expression of PPARγ was observed in tumor cells of human primary squamous cell carcinoma, lymph node metastasis, and squamous cell carcinoma cell lines. Treatment with PPARγ-specific antagonists, but not agonists, caused apoptotic cell death on squamous cell carcinoma cell lines in a concentration-dependent manner. Small interfering RNA for PPARγ also inhibited cell adhesion and growth of squamous cell carcinomas. The phosphorylation of focal adhesion kinase (FAK) was decreased by treatment with PPARγ antagonists, and resulted in decreases in phosphorylation of Erk and mitogen-activated protein kinase. Furthermore, PPARγ antagonists decreased the adhesion of squamous cell carcinomas into fibronectin-coated plates, indicating the inhibition of interaction between squamous cell carcinomas and fibronectin. Expression of integrin α5, a counter adhesion molecule for fibronectin, was inhibited by the treatment with PPARγ antagonists. These results indicate that the decrease in integrin α5 and following inhibition of cell adhesion may cause the inhibition of FAK signaling pathways. PPARγ antagonists also strongly inhibited invasion of squamous cell carcinoma via down-regulation of CD151 expression. Conclusions: The cell death caused by the PPARγ antagonists was a result of direct interference with cell adhesion “anoikis” involving intracellular FAK signaling pathways. These results imply a potentially important and novel role for the inhibition of PPARγ function via the use of specific antagonists in the treatment of squamous cell carcinoma and the prevention of tumor invasion and metastasis.

Diclofenac inhibits proliferation and differentiation of neural stem cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in clinical situations as anti-inflammatory, analgesic and antipyretic drugs. However, it is still unknown whether NSAIDs have effects on the development of the central nervous system. In the present study, we investigated the effects of NSAIDs on neural stem cell (NSC) proliferation and differentiation into neurons. In contrast to aspirin, naproxen, indomethacin and ibuprofen, treatment with diclofenac (10 microM) for 2 days induced the death of NSCs in a concentration-dependent manner. Diclofenac also inhibited the proliferation of NSCs and their differentiation into neurons. Treatment with diclofenac resulted in nuclear condensation (a morphological change due to apoptosis of NSCs) 24hr after the treatment and activated caspase-3 after 6 hr, indicating that diclofenac may cause apoptosis of neuronal cells via activation of the caspase cascade. These results suggest that diclofenac may affect the development of the central nervous system.

Involvement of NMDA-nitric oxide pathways in the development of tactile hypersensitivity evoked by the loose-ligation of inferior alveolar nerves in rats.

To investigate whether or not NMDA/nitric oxide (NO) pathways in the trigeminal system are involved in the development and/or maintenance of such pathological pain states as the hyperalgesia and allodynia observed after dental surgery, loose-ligation on the left inferior alveolar nerves of rats were performed. The responses to mechanical stimulation were then measured using von Frey filaments. Hypersensitivity to tactile stimulation developed on the ipsilateral side in ligated animals 5 days after surgery and lasted for at least 30 days. In addition, the effects of drugs on these pain states during the period 2-3 weeks following surgery were investigated. As a result, it was observed that tactile hypersensitivity was inhibited by the intraperitoneal (i.p.) administration of both MK-801 hydrogen maleate (0.05-0.1 mg/kg) and N(G)-monomethyl-L-arginine acetate (L-NMMA: 10-100 mg/kg). Still further, NO production and the number of neuronal NO synthase (nNOS)-positive neurons in the trigeminal nucleus caudalis (SpVc) was evaluated. As a result of these experiments, it was found that the changes in NO levels evoked by the intravenous infusions of N-methyl-D-aspartate (NMDA; 10 mg/kg) and MK-801 (0.5 mg/kg) were significantly larger in the loose-ligated rats compared to the sham-operated rats. Moreover, the number of nNOS-positive neurons was found to have increased on the ipsilateral side in layers I/II of the SpVc. These results would suggest that tactile hypersensitivity develops after inferior alveolar nerve injury and that NMDA receptor/NOS/NO production pathways in the SpVc may be involved in the development of such pathophysiological states.

Anesthetic effects on susceptibility to cortical spreading depression.

Cortical spreading depression (CSD) is a transient neuronal and glial depolarization and disruption of membrane ionic gradients that propagates slowly across the cerebral cortex. Recent clinical and experimental evidence has implicated CSD in the pathophysiology of migraines and neuronal injury states. In the current study, we examined the influence of four different anesthetics (propofol, dexmedetomidine, isoflurane, pentobarbital) on CSD susceptibility in a KCl application animal model. We found that isoflurane and dexmedetomidine suppressed CSD frequency, and tended to reduce the CSD propagation speed. Our data suggest that these anesthetics may be therapeutically beneficial in preventing CSD in diverse neuronal injury states.

Influence of acute hypoxia combined with nitrous oxide on cardiovascular variability in conscious hypertensive rats

Anesthetics have been reported to depress autonomic nervous system (ANS) responses to hypoxia. The mechanisms by which cardiovascular variability responds to acute progressive hypoxia (APH) under nitrous oxide (N(2)O) inhalation, however, remain unclear. Additionally, the effect of hypertension on ANS responses in such cases has not been fully clarified. The present study examined the influence of APH (10% O(2)) under 60% N(2)O inhalation on cardiovascular variability in conscious, spontaneously hypertensive rats (SHR). Twenty-seven male SHR were randomly assigned to 3 treatment groups receiving N(2)O inhalation alone, APH stress alone or APH stress under N(2)O inhalation, using Wistar Kyoto rats (WKY) or non-N(2)O inhalation rats as controls. Systolic blood pressure (SBP) and heart rate (HR) variability were evaluated time-dependently using the wavelet method. While inhalation of N(2)O alone induced more powerful sympathomimetic actions in SHR than in WKY, circulatory and parasympathetic reactions were weaker. APH stress alone evoked significant inhibition of cardiac parasympathetic activity from immediately after exposure to hypoxic stress in SHR in contrast to WKY, facilitating tachycardia. This inhibition of parasympathetic activity in SHR continued without coupled changes in sympathetic activity. In SHR, APH under N(2)O inhalation decreased SBP and sympathetic activity more prominently and earlier than APH alone, and earlier than APH under N(2)O inhalation in WKY. Additionally, APH under N(2)O inhalation inhibited cardiac parasympathetic activity in SHR as compared to APH stress alone. In conclusion, APH under N(2)O inhalation in SHR potentially results in exacerbation of circulatory suppression from the earlier hypoxic phase, compared with non-N(2)O inhalation.