Miyahiko Murata

Distinct roles of thioredoxin in the cytoplasm and in the nucleus. A two-step mechanism of redox regulation of transcription factor NF-kappaB

Oxidative stresses such as UV irradiation to mammalian cells triggers a variety of oxistress responses including activation of transcription factors. Recently, activation of nuclear factor-κB (NF-κB) has been shown to be under oxidoreduction (redox) regulation controlled by thioredoxin (TRX), which is one of major endogenous redox-regulating molecules with thiol reducing activity. In order to elucidate where in the cellular compartment TRX participates in NF-κB regulation, we investigated the intracellular localization of TRX. UVB irradiation induced translocation of TRX from the cytoplasm into the nucleus. In our in vitro diamide-induced cross-linking study, we showed that TRX can associate directly with NF-κB p50. Overexpression of wild-type TRX suppressed induction of luciferase activity under NF-κB-binding sites in response to UV irradiation compared with the mock transfectant. In contrast, overexpression of nuclear-targeted TRX enhanced the luciferase activity. Thus, TRX seems to play dual and opposing roles in the regulation of NF-κB. In the cytoplasm, it interferes with the signals to IκB kinases and blocks the degradation of IκB. In the nucleus, however, TRX enhances NF-κB transcriptional activities by enhancing its ability to bind DNA. This two-step TRX-dependent regulation of the NF-κB complex may be a novel activation mechanism of redox-sensitive transcription factors.

Alpha-Klotho as a regulator of calcium homeostasis

α-klotho was identified as a gene associated with premature aging–like phenotypes characterized by short lifespan. In mice, we found the molecular association of α-Klotho (α-Kl) and Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) and provide evidence for an increase of abundance of Na+,K+-ATPase at the plasma membrane. Low concentrations of extracellular free calcium ([Ca2+]e) rapidly induce regulated parathyroid hormone (PTH) secretion in an α-Kl- and Na+,K+-ATPase–dependent manner. The increased Na+ gradient created by Na+,K+-ATPase activity might drive the transepithelial transport of Ca2+ in cooperation with ion channels and transporters in the choroid plexus and the kidney. Our findings reveal fundamental roles of α-Kl in the regulation of calcium metabolism.

Survival of fetal rat otocyst cells grafted into the damaged inner ear

Hair cell loss induced by aging, ototoxic drugs and noise leads to irreversible hearing loss and balance disorders in mammals due to the failure of hair cells to regenerate. To investigate the possibility of transplantation therapy to repair damaged inner ear, we have examined whether grafted fetal otocyst cells could survive and migrate into injured sensory organs. We obtained otocyst cells from green fluorescein protein (GFP)-transgenic rats on embryonic day 12.5, then transplanted these cells into the inner ears of young rats previously exposed to intense sound. One month after transplantation, the grafted inner ear sensory organs were examined immunohistochemically. Grafted otocyst cells had survived and demonstrated special morphological features in the host organs; cells that migrated into the organ of Corti were similar to supporting cells. These results indicate that injured sensory organs express some kind of scaffolding that plays important roles in the survival and differentiation of the grafted otocyst cells

n-Propyl gallate activates hypoxia-inducible factor 1 by modulating intracellular oxygen-sensing systems

HIF-1 (hypoxia-inducible factor 1) is a master regulator of cellular adaptive responses to hypoxia. The expression and transcriptional activity of the HIF-1alpha subunit is stringently controlled by intracellular oxygen tension through the action of prolyl and asparaginyl hydroxylases. In the present study we demonstrate that PG (n-propyl gallate) activates HIF-1 and expression of its downstream target genes under normoxic conditions in cultured cells and in mice. The stability and transcriptional activity of HIF-1alpha are increased by PG. PG treatment inhibits the interaction between HIF-1alpha and VHL (von Hippel-Lindau protein) and promotes the interaction between HIF-1alpha and p300, indicating that PG inhibits the activity of both prolyl and asparaginyl HIF-1alpha hydroxylases. We conclude that PG activates HIF-1 and enhances the resultant gene expression by directly affecting the intracellular oxygen sensing system in vitro and in vivo and that PG represents a lead compound for the development of a non-toxic activator of HIF-1.

Modification of the N-terminus of membrane fusion-active peptides blocks the fusion activity.

The amphiphilic anionic peptides E5 and E5L can mimic the fusogenic activity of influenza hemagglutinin(HA). These peptides induced fusion of egg yolk phosphatidylcholine small or large unilamellar vesicles only at acidic pH in a similar manner to viral HA. Acetylation or acetimidylation of the N-terminus of the peptides drastically reduced the fusion activity of the intact peptides, while C-terminal amidation left the activity unchanged. The binding assay suggested that the interaction of the modified peptides with lipid membranes was almost unchanged in comparison with those of the parent peptides, and the CD spectra showed that these peptides were alpha-helical. The results showed the importance of the N-terminus of the peptides on the membrane fusion activity, although why the N-terminal modifications affect the activity is still unclear.

Cerebellotectal projection in the rat; anterograde and retrograde WGA-HRP study of individual cerebellar nuclei

Cerebellotectal projections were studied in the rat by the anterograde and retrograde tracing methods using wheat-germ-agglutinin-conjugated horseradish peroxidase. The pathway arises from all four cerebellar nuclei on the contralateral side; mainly from the posterior interpositus nucleus and lateral nucleus and to a lesser extent from the medial nucleus and anterior interpositus nucleus. The fibers arising from the medial nucleus and the posterior interpositus nucleus terminate mainly in the deeper zone of layer IV and in layer VI throughout the entire rostrocaudal extent of the contralateral superior colliculus. Those arising from the anterior interpositus nucleus and the lateral nucleus terminate mainly in the superficial zone of layer IV in the rostral three-fourths of the contralateral superior colliculus. In addition, the fibers from the lateral nucleus terminate densely in a zone extending from the deep part of layer III through layer VII in the lateral portion of the rostral half of the superior colliculus. In comparison with data on other species the present findings are discussed with respect to the evolutional changes from monocular to binocular vision.

Regeneration and recovery of the hearing function of the central auditory pathway by transplants of embryonic brain tissue in adult rats

The present study is the first report of successful regeneration and recovery of hearing function of the central auditory pathway after transection in the adult rat. The ventral cochlear tract in the brain stem to pons was transected on one side in adult rats. Tissue from embryos (E14 to E16) was used to cover the lesion site. In 30% of the rats examined, the axons regrew beyond the transected site and regenerated into the denervated side and terminated at the normal targets. The hearing function of rats was elucidated by recording the auditory brain stem response (ABR). Rats with successful regeneration showed nearly normal ABR. In rats receiving simple transection without covering embryonic tissue, there was no regeneration and hearing function did not recover. Thus, the present findings contradict the widely held view that the adult mammalian central auditory system cannot be restored following damage.

An endogenous redox molecule, thioredoxin, regulates transactivation of epidermal growth factor receptor and activation of NF-κB by lysophosphatidic acid

Lysophosphatidic acid (LPA) is the smallest and simplest of all the glycerophospholipids that activates a specific GTP‐binding protein coupled receptor to evoke multiple cellular responses. In this paper, we have demonstrated that LPA stimulates nuclear factor (NF)‐κB‐dependent gene induction in a neuronal cell line, NG108‐15 and that this is under redox regulation by an endogenous molecule, thioredoxin. We also have shown that redox‐sensitive transactivation of epidermal growth factor receptor by LPA confers NF‐κB activation and small GTPase proteins are involved in this pathway.

Spontaneous regeneration and recovery of hearing function of the central auditory pathway in young rats.

Spontaneous regeneration of the auditory pathway after transection of the ventral cochlear tract of the brain stem to pons was examined in young rats by the anterograde tracing method with wheat germ agglutinin-conjugated horseradish peroxidase. Care was taken to cut the tract as sharply as possible to minimize traumatic injuries. About 59% of the young rats examined showed successful regeneration. Functional recovery of hearing by the regenerated fibers was confirmed by the auditory brain stem response. Thus, the present findings contradict the widely held view that the mammalian central auditory system cannot be restored following damage.

Regeneration of the auditory pathway in adult rats by transplants of fetal brain tissue.

THE ventral cochlear tract in the brain stem to pons was transected on one side in two groups of adult rats. In one group simple transection was performed, while in the other group tissue from embryos (E14–E16) was used to cover the lesion site. While rats receiving simple tran-section without transplantation showed no evidence of regeneration, in 30% of the rats receiving transplants of embryonic tissue the axons regrew beyond the transected site and regenerated into the denervated side and terminated at the normal targets. The present findings contradict the widely held view that the adult mammalian central auditory system cannot be restored following damage.