Tokiko Ogawa

Neuronal Injury-inducible Gene Is Synergistically Regulated by ATF3, c-Jun, and STAT3 through the Interaction with Sp1 in Damaged Neurons

Nerve injury requires the expression of large ensembles of genes. The key molecular mechanism for this gene transcription regulation in injured neurons is poorly understood. Among many nerve injury-inducible genes, the gene encoding damage-induced neuronal endopeptidase (DINE) showed most marked expression response to various kinds of nerve injuries in central and peripheral nervous system neurons. This unique feature led us to examine the promoter region of the DINE gene and clarify both the injury-responsive element within the promoter and its related transcriptional machinery. This study showed that DINE promoter was activated by leukemia inhibitory factor and nerve growth factor withdrawal, which were pivotal for the up-regulation of DINE mRNA after nerve injury. The injury-inducible transcription factors such as activating transcription factor 3 (ATF3), c-Jun, and STAT3, which were located at the downstream of leukemia inhibitory factor and nerve growth factor withdrawal, seemed to be involved in the activation of the DINE promoter. Surprisingly, these transcription factors did not bind to the DINE promoter directly. Instead, the general transcription factor, Sp1, bound to a GC box within the promoter. ATF3, c-Jun, and STAT3 interacted with Sp1 and are associated with the GC box region of the DINE gene in injured neurons. These findings suggested that Sp1 recruit ATF3, c-Jun, and STAT3 to obtain the requisite synergistic effect. Of these transcription factors, ATF3 may be the most critical, because ATF3 is specifically expressed after nerve injury.

Altered expression of neprilysin family members in the pituitary gland of sleep-disturbed rats, an animal model of severe fatigue

Alterations of the expression of some peptidases in the pituitary gland of a fatigued rat model were identified. Rats were kept in a cage filled with water to a height of 1.5 cm to disturb deep sleep. After 24‐h sleep disturbance, expression of neutral endopeptidase 24.11 (neprilysin) mRNA was increased in the intermediate lobe of the pituitary gland, whereas the mRNA expression of another family member, damage‐induced neuronal endopeptidase, which is normally expressed in a subgroup of anterior pituitary cells, was significantly suppressed. These alterations were demonstrated by RT‐PCR, northern blotting and in situ hybridization. Other family members, such as neprilysin 2 and endothelin converting enzyme‐1, did not show any change in mRNA expression. An increase of neprilysin mRNA expression was not seen in any other tissues of the sleep‐disturbed rats. The enzymatic activity of neprilysin was also increased in the pituitary. The augmentation of neprilysin expression and activity was prolonged as long as the sleep disturbance continued (up to 5 days), and returned to the basal level when rats were allowed to sleep freely. These results suggest that peptide processing and degradation in the pituitary may be an influential factor in fatigued states such as sleep disturbance.

Chronic stress elicits prolonged activation of α-MSH secretion and subsequent degeneration of melanotroph

Prolonged stress affects homeostasis in various organs and induces stress‐associated disorders. We examined the cellular changes of pituitary gland under the continuous stress condition using a rat model in which rats were kept in a cage filled with water to a height of 1.5 cm for up to 5 days. Among the pituitary hormone mRNAs, proopiomelanocortin mRNA was up‐regulated specifically in the intermediate lobe (IL) of this rat model. Additionally, the peripheral blood levels of α‐melanocyte stimulating hormone (α‐MSH), a major product of proopiomelanocortin in IL were increased. The α‐MSH secreting cells, melanotrophs, showed a markedly developed endoplasmic reticulum and Golgi apparatus in the early phase of the experiment. Subsequent continuous stress caused remarkable dilation of the endoplasmic reticulum, disruption of the Golgi structure, and the degeneration of some melanotrophs. In addition the dopaminergic nerve fibers from hypothalamus were markedly decreased in IL. A dopamine antagonist elicited the similar morphologic changes of melanotroph in normal rat. These findings suggest that prolonged stress suppressed hypothalamus‐derived dopamine release in IL, which elicited over‐secretion of α‐MSH from the melanotrophs. The present study also suggests that prolonged hyperactivation of endocrine cells could lead to disorder of secretion mechanisms and eventual degeneration.

Id1, Id2 and Id3 are induced in rat melanotrophs of the pituitary gland by dopamine suppression under continuous stress

In rats under continuous stress (CS) there is decreased hypothalamic dopaminergic innervation to the intermediate lobe (IL) of the pituitary gland, which causes hyperactivation and subsequent degeneration of melanotrophs in the IL. In this study, we investigated the molecular basis for the changes that occur in melanotrophs during CS. Using microarray analysis, we identified several genes differentially expressed in the IL under CS conditions. Among the genes up-regulated under CS conditions, we focused on the inhibitor of DNA binding/differentiation (Id) family of dominant negative basic helix-loop-helix (bHLH) transcription factors. RT-PCR, Western blotting and in situ hybridization confirmed the significant inductions of Id1, Id2 and Id3 in the IL of CS rats. Administration of the dopamine D2 receptor agonist bromocriptine prevented the inductions of Id1-3 in the IL of CS rats, whereas application of the dopamine D2 antagonist sulpiride induced significant expressions of Id1-3 in the IL of normal rats. Moreover, an in vitro study using primary cultured melanotrophs demonstrated a direct effect on Id1-3 inductions by dopamine suppression. These results suggest that the decreased dopamine levels in the IL during CS induce Id1-3 expressions in melanotrophs. Because Id family members inhibit various bHLH transcription factors, it is conceivable that the induced Id1-3 would cooperatively modulate gene expressions in melanotrophs under CS conditions to induce hormone secretion.

Local ventilation system successfully reduced formaldehyde exposure during gross anatomy dissection classes.

Dear Editor, In the recent Acta Anatomica Nipponica (Kaibogaku Zasshi Vol. 85, No. 1, 2010)—a special issue concerning the improvement of the formaldehyde (FA) environment in gross-anatomy laboratories—four papers were published, two of which described originally developed dissection tables equipped with local ventilation systems (Shinoda and Oba 2010; Kikuta et al. 2010) and two of which outlined the environmental health hazards caused by FA (Uchiyama 2010; Sakamoto and Miyake 2010). Due to its effectiveness and low cost, the FA solution is widely used in Japanese medical and dental schools to embalm human bodies donated for use in gross anatomy dissection classes. However, the gaseous FA that evaporates from embalmed bodies causes not only acute irritation to the eyes and respiratory tract but also chronic non-threshold carcinogenicity. According to a risk-based evaluation of FA, the Ministry of Health, Labor, and Welfare in Japan has set the administrative level of FA to 0.1 ppm in working environments in which FA is handled. In our medical school, the dissection laboratory for students (L23 9 W12 9 H3.3 m, in which 20–23 bodies prepared with 10 l of 5.5% FA/30% ethanol solution are used) had a high-performance general (whole-room) ventilation system with a competence of 25000 m/h; however, the mean FA concentration of the room (‘‘A’’ measurement) and the estimated maximum exposure to FA (‘‘B’’ measurement) during dissection classes were 0.520 and 0.480 ppm, respectively (HPLC analysis performed by Panasonic Health Organization Science Center of Industrial Hygiene, Osaka, Japan). To reduce FA exposure during dissection classes, we introduced 23 dissection tables with local ventilation apparatus (Meiko Medical, Fukuoka, Japan). The details of this system have already been described elsewhere (Shinoda and Oba 2010). Briefly, the system consists of a simple plenum-chambered dissection table and a transparent vinyl duct which connects the table to the pre-existing general ventilation duct via a flow control valve in the ceiling. The 40 pre-existing, randomly oriented air-supply openings in the ceiling were not replaced (no downward flow of air for each dissecting table). The total ventilation flow rate was 18 m/min/table. The local ventilation system we introduced successfully reduced the FA concentration of the room during dissection classes. The A and B FA measurements were 0.035 and 0.054 ppm, respectively. Using a photoelectronic method (FP-30, Rikenkeiki, Tokyo, Japan), we also measured the 30-min mean FA concentration at the center of the dissection room (1.2 m above the floor) and at the corner of a dissection table (0.5 m above the table) in every class 30–60 min after the start of the dissection (Fig. 1). The mean FA concentrations at the center of the room and the corner of the table were 0.056 (n = 41) and 0.057 ppm (n = 41), respectively. FA concentrations higher than 0.1 ppm were recorded for dissection schedules #8 and 9, during which the upper extremities were placed in the abducent position; thus, the source of the FA was out of the effective range of the local ventilation system. Thirty-nine of the 41 measurements of FA concentration at the center of the room and 40 of the 41 measurements at the corner of M. Takahashi M. Abe T. Yamagishi K. Nakatani Y. Nakajima (&) Anatomy and Cell Biology, Graduate School of Medicine, Osaka City University, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan e-mail: yuji@med.osaka-cu.ac.jp

Continuous stress promotes expression of VGF in melanotroph via suppression of dopamine

Prolonged exposure to stress elicits profound effects on homeostasis that may lead to cryptogenic disorders such as chronic fatigue syndrome. To investigate the pathophysiology associated with the syndrome, we used a rat continuous stress (CS) model where the pituitary represents one of the most affected organs. Here we found that mRNA for VGF (non-acronymic), a member of the granin family, was induced specifically in the intermediate lobe (IL). This was matched by a concomitant increase at the peptide/protein level assessed by C-terminal antibody. Furthermore, the up-regulation of VGF was confirmed by immunohistochemistry in a subset of melanotrophs. VGF expression was altered in the IL of rats receivingthe dopamine D2 receptor agonist bromocriptine or the antagonist sulpiride. In vitro, dopamine dose-dependently decreased the mRNA levels in cultured melanotrophs. These findings suggest that VGF expression under CS is negatively regulated by dopaminergic neurons projecting from the hypothalamus.

Continuous stress-induced dopamine dysregulation augments PAP-I and PAP-II expression in melanotrophs of the pituitary gland

Under continuous stress (CS) in rats, melanotrophs, the predominant cell-type in the intermediate lobe (IL) of the pituitary, are hyperactivated to secrete α-melanocyte-stimulating hormone and thereafter degenerate. Although these phenomena are drastic, the molecular mechanisms underlying the cellular changes are mostly unknown. In this study, we focused on the pancreatitis-associated protein (PAP) family members of the secretory lectins and characterized their expression in the IL of CS model rats because we had identified two members of this family as up-regulated genes in our previous microarray analysis. RT-PCR and histological studies demonstrated that prominent PAP-I and PAP-II expression was induced in melanotrophs in the early stages of CS, while another family member, PAP-III, was not expressed. We further examined the regulatory mechanisms of PAP-I and PAP-II expression and revealed that both were induced by the decreased dopamine levels in the IL under CS. Because the PAP family members are implicated in cell survival and proliferation, PAP-I and PAP-II secreted from melanotrophs may function to sustain homeostasis of the IL under CS conditions in an autocrine or a paracrine manner.

The Absence of Somatotroph Proliferation During Continuous Stress is a Result of the Lack of Extracellular Signal-Regulated Kinase 1/2 Activation

The integrity of homeostasis can be affected by chronic stress, and hyposomatotropism is evident in chronic stress‐associated illnesses. In the present study, we demonstrated that a continuous stress (CS) severely affected somatotrophs among hormone‐secreting cells in the anterior lobe (AL) of the pituitary by using a rat CS model. Among AL cells, the proliferation of somatotrophs was almost entirely suppressed in rats that had 3–5 days of CS (5dCS), although other hormone‐secreting cells continued to proliferate. The cell size of somatotrophs was reduced at 5dCS (P < 0.01), the number of secretory granules was increased at 3dCS (P < 0.01) and serum growth hormone (GH) was on declining trend during 1 to 5dCS, suggesting that GH release was inhibited. GH‐releasing hormone (GHRH) mRNA level in the arcuate nucleus was transiently decreased, whereas its receptor expression in the AL was significantly increased in CS rats. When 5dCS rats were injected with GHRH, transient GH secretion was observed, whereas proliferation of somatotrophs did not occur. The GHRH administration failed to stimulate extracellular signal‐regulated kinase (ERK) 1/2 phosphorylation and the nuclear translocation of ERK in somatotrophs. These results suggest that somatotrophs of 5dCS rats expressed sufficient GHRH receptor, which could transfer a signal for GH release. However, the GHRH‐induced proliferation signal was blocked somewhere between the receptor and ERK1/2. Because significant increase of corticosterone in the initial stage (the 1–3dCS) was observed in this model, the corticosterone may affect the signalling. Although the mechanism underlying the blockage of the proliferation signal in somatotrophs under CS remains unclear, these somatotrophic disorder, suggesting that the present animal model may be useful for understanding the molecular mechanisms of chronic stress‐associated illnesses.

Accumulated fatigue induces over-activation and subsequent degeneration of melanotrophs in rat pituitary gland

Novel peptidergic neurons, metastin neurons, are thought to facilitate sexual maturation and ovulation by stimulating gonadotropin releasing hormone (GnRH) neurons, but their physiological functions are still largely unknown. Here we focused on the relationship between the metastin and hypothalamic/extrahypothalamic GnRH neurons. Taking advantage of well-developed teleost GnRH systems, we used medaka, which is a useful teleost for the application of various molecular genetic tools. First, we cloned and sequenced the metastin gene (Kiss1) in medaka for the first time in non-mammalian species. Then, Kiss1 expression in the brain was analyzed by in situ hybridization. We found two hypothalamic nuclei with Kiss1 expression and discovered prominent sexual difference in cell number (male female) in one of the two nuclei. On the other hand, we found no circadian variation in expression of Kiss1 by real-time PCR, although medaka shows daily ovulatory cyclicity.

1128 Axon-glia interactions during the initial stage of myelination in the rat fimbria

TOKIKO OGAWA’v2, MITSURU SUZUKI’, AKIHIRO KITABATAKE’, TOSHIO MITA’ Ultrastructural interactions between developing axons and astrocytes in the rat fimbria during the early postnatal period (P7, 10, 14) were investigated. Confocal microscopic images of the axons and the astrocytes, which were labeled with DiI and anti-GFAP antibody respectively, were also analyzed three-dimensionally. Myelinated fimbrial axons were first observed at PlO, while processes of astrocytes contacted to unmyelinated axon segments. The astrocytic processes were occasionally observed adjacent to the initial sheaths of oligodendrocytes. The findings suggest that astrocytes correlate with oligodendrocytes for myelination in the central white matter.