Protein kinase C-mediated phosphorylation of transient receptor potential melastatin type 2 Thr738 counteracts the effect of cytosolic Ca2+ and elevates the temperature threshold.

The transient receptor potential melastatin type 2 (TRPM2) channel is a non-selective cation channel that has high Ca2+ permeability. TRPM2 is sensitive to warm temperatures and is expressed in cells and tissues that are maintained at core body temperature. TRPM2 activity is also regulated by endogenous factors including redox signalling, cytosolic Ca2+ and adenosine diphosphate ribose. As a result of its wide expression and function at core body temperature, these endogenous factors could regulate TRPM2 activity at body temperature under physiological and pathophysiological conditions. We previously reported that cellular redox signalling can lower TRPM2 temperature thresholds, although the mechanism that regulates these thresholds is unclear. Here, we used biochemical and electrophysiological techniques to explore another regulatory mechanism for TRPM2 temperature thresholds that is mediated by TRPM2 phosphorylation. Our results show that: (1) the temperature threshold for TRPM2 activation is lowered by cytosolic Ca2+ ; (2) protein kinase C-mediated phosphorylation of TRPM2 counteracts the effect of cytosolic Ca2+ ; and (3) Thr738 in mouse TRPM2 that lies near the Ca2+ binding site in the cytosolic cleft of the transmembrane domain is a potential phosphorylation site that may be involved in phosphorylation-mediated elevation of TRPM2 thresholds. These findings provide structure-based evidence to understand how temperature thresholds of thermo-sensitive TRP channels (thermo-TRPs) are determined and regulated. KEY POINTS: The transient receptor potential melastatin type 2 (TRPM2) ion channel is temperature-sensitive and Ca2+ -permeable. Endogenous factors and pathways such as redox signalling can regulate TRPM2 activity at body temperature under physiological and pathophysiological conditions. In the present study, we report the novel finding that cytosolic Ca2+ lowers the temperature threshold for TRPM2 activation in a concentration-dependent manner. Protein kinase C-mediated phosphorylation of TRPM2 at amino acid Thr782 elevates the temperature threshold for activation by counteracting the effects of cytosolic Ca2+ . These findings provide structure-based evidence to understand how temperature thresholds of thermo-sensitive TRP channels are determined and regulated.

Suppression of trabecular meshwork phagocytosis by norepinephrine is associated with nocturnal increase in intraocular pressure in mice.

Intraocular pressure (IOP) is an important factor in glaucoma development, which involves aqueous humor (AH) dynamics, with inflow from the ciliary body and outflow through the trabecular meshwork (TM). IOP has a circadian rhythm entrained by sympathetic noradrenaline (NE) or adrenal glucocorticoids (GCs). Herein, we investigated the involvement of GC/NE in AH outflow. Pharmacological prevention of inflow/outflow in mice indicated a diurnal outflow increase, which was related to TM phagocytosis. NE showed a non-self-sustained inhibition in phagocytosis of immortalized human TM cells, but not GC. The pharmacological and reverse genetic approaches identified ß1-adrenergic receptor (AR)-mediated exchange proteins directly activated by cyclic adenosine monophosphate (EPAC)-SHIP1 signal activation by ablation of phosphatidylinositol triphosphate, regulating phagocytic cup formation. Furthermore, we revealed the phagocytosis involvement in the ß1-AR-EPAC-SHIP1-mediated nocturnal IOP rise in mice. These suggest that TM phagocytosis suppression by NE can regulate IOP rhythm through AH outflow. This discovery may aid glaucoma management.

Tamoxifen Activates Dormant Primordial Follicles in Mouse Ovaries.

Our previous study found that 17ß-estradiol (E2) suppresses primordial follicle activation and growth in cultured mouse ovaries. In this study, we administered tamoxifen, an estrogen receptor antagonist, into the abdominal cavity of mice to clarify the relationship between primordial follicle activation and the physiological concentration of E2 in mouse ovaries. The results showed that tamoxifen promoted primordial follicle activation. Administration of tamoxifen promoted degradation of the extracellular matrix surrounding primordial follicles in the ovaries. Furthermore, tamoxifen decreased the expression of stefin A, an inhibitor of cathepsins that digest some proteins and extracellular matrix, in the ovaries. Mechanical stress produced by the extracellular matrix reportedly suppresses the activation of primordial follicles. The collective results show that tamoxifen can promote primordial follicle activation through the degradation of the extracellular matrix surrounding primordial follicles. Our results indicate that E2 suppresses primordial follicle activation in vivo and that tamoxifen may be useful as a therapeutic agent against infertility.

17ß-Estradiol and cathepsins control primordial follicle growth in mouse ovaries.

This study aimed to clarify the physiological mechanism regulating the growth of primordial follicles in mouse ovaries. In a previous study, we found that increasing the fetal bovine serum concentration in the culture medium promoted the growth of primordial follicles in cultured postnatal day 0 ovaries but not in cultured postnatal day 4 ovaries. Based on these results, we hypothesized that the regulatory system repressing the growth of primordial follicles is established in postnatal day 4 ovaries. To confirm this hypothesis, microarray analysis of postnatal day 0 and 4 ovaries was performed. The results revealed that the expression of mRNA of stefin A homologs increased in postnatal day 4 ovaries. Stefin A belonging to the type 1 cystatin superfamily is an inhibitor of cysteine cathepsins. Consistently, the inhibitor of cathepsins repressed the growth of primordial follicles in cultured postnatal day 0 ovaries. Furthermore, we found that 17ß-estradiol promoted the expression of mRNA of stefin A homologs in cultured ovaries and repressed the growth of primordial follicles. Our results demonstrate that 17ß-estradiol and cathepsins regulate the growth of primordial follicles in mouse ovaries.

cAMP response element induces Per1 in vivo.

Light is an important cue for resetting the circadian clock. In mammals, light signals are thought to be transmitted to the cAMP response element (CRE) via a binding protein (CREB) to induce the expression of Per1 and Per2 genes in the mammalian circadian pacemaker, the suprachiasmatic nuclei (SCN). Several in vitro studies have suggested candidate CRE sites that contribute to the Per1 and Per2 induction by light, resulting in a phase shift of the circadian rhythm. However, it remains unclear whether the CREs are responsible for the light-induced Per1/2 induction. To address this question, we generated CRE-deleted mice in the Per1 and Per2 promoter regions. Deletion of a cAMP-responsive CRE in the Per1 promoter blunted light-induced Per1 expression in the SCN at night, while deletion of an ATF4 (CREB-2)-associated CRE in the Per2 promoter had no effect on its expression. These results suggested that the CRE in the Per1 promoter works for light induction but not CRE in the Per2 promoter. Behavioral rhythms observed under some light conditions were not affected by the CRE-deletion in Per1 promoter, suggesting that the attenuated Per1 induction did not affect the entrainment in some light conditions.

Circadian Regulation of IOP Rhythm by Dual Pathways of Glucocorticoids and the Sympathetic Nervous System.

Purpose: Elevated IOP can cause the development of glaucoma. The circadian rhythm of IOP depends on the dynamics of the aqueous humor and is synchronized with the circadian rhythm pacemaker, that is, the suprachiasmatic nucleus. The suprachiasmatic nucleus resets peripheral clocks via sympathetic nerves or adrenal glucocorticoids. However, the detailed mechanisms underlying IOP rhythmicity remain unclear. The purpose of this study was to verify this regulatory pathway. Methods: Adrenalectomy and/or superior cervical ganglionectomy were performed in C57BL/6J mice. Their IOP rhythms were measured under light/dark cycle and constant dark conditions. Ocular administration of corticosterone or norepinephrine was also performed. Localization of adrenergic receptors, glucocorticoid receptors, and clock proteins Bmal1 and Per1 were analyzed using immunohistochemistry. Period2::luciferase rhythms in the cultured iris/ciliary bodies of adrenalectomized and/or superior cervical ganglionectomized mice were monitored to evaluate the effect of the procedures on the local clock. The IOP rhythm of retina and ciliary epithelium-specific Bmal1 knockout mice were measured to determine the significance of the local clock. Results: Adrenalectomy and superior cervical ganglionectomy disrupted IOP rhythms and the circadian clock in the iris/ciliary body cultures. Instillation of corticosterone and norepinephrine restored the IOP rhythm. ß2-Adrenergic receptors, glucocorticoid receptors, and clock proteins were strongly expressed within the nonpigmented epithelia of the ciliary body. However, tissue-specific Bmal1 knock-out mice maintained their IOP rhythm. Conclusions: These findings suggest direct driving of the IOP rhythm by the suprachiasmatic nucleus, via the dual corticosterone and norepinephrine pathway, but not the ciliary clock, which may be useful for chronotherapy of glaucoma.

Effect of expression alteration in flanking genes on phenotypes of St8sia2-deficient mice.

ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 (ST8SIA2) synthesizes polysialic acid (PSA), which is essential for brain development. Although previous studies reported that St8sia2-deficient mice that have a mixed 129 and C57BL/6 (B6) genetic background showed mild and variable phenotypes, the reasons for this remain unknown. We hypothesized that this phenotypic difference is caused by diversity in the expression or function of flanking genes of St8sia2. A genomic polymorphism and gene expression analysis in the flanking region revealed reduced expression of insulin-like growth factor 1 receptor (Igf1r) on the B6 background than on that of the 129 strain. This observation, along with the finding that administration of an IGF1R agonist during pregnancy increased litter size, suggests that the decreased expression of Igf1r associated with ST8SIA2 deficiency caused lethality. This study demonstrates the importance of gene expression level in the flanking regions of a targeted null allele having an effect on phenotype.

The concentration-dependent effect of progesterone on follicle growth in the mouse ovary.

Follicle growth in the mammalian ovary is coordinately controlled by multiple factors to sustain periodic ovulation. In this study, we investigated the role of progesterone on follicle growth in the mouse ovary. As the concentration of progesterone changes during the estrus cycle, we cultured the sliced mouse ovary in a medium containing 10 ng/ml, 100 ng/ml, and 1 µg/ml progesterone. Progesterone promoted the growth of primordial to primary follicles at 100 ng/ml, while it suppressed the growth of secondary follicles at 1 µg/ml. Follicles at other developmental stages in the cultured ovary were unaffected with different concentrations of progesterone. The number of ovulated oocytes increased in the medium containing 100 ng/ml progesterone but decreased in the presence of 1 µg/ml progesterone. Follicles expressed two types of progesterone receptors, progesterone receptor (PGR) and PGR membrane component 1 (PGRMC1). While PGR shows transient expression on granulosa cells of Graafian follicles, PGRMC1 expresses in granulosa cells of developing follicles. These results suggest that progesterone controls the growth of developing follicles through PGRMC1. Our study shows that the effect of progesterone on ovulation and follicle growth in mouse ovary is dependent on the concentration of progesterone and the follicle stage.

Licarin A is a candidate compound for the treatment of immediate hypersensitivity via inhibition of rat mast cell line RBL-2H3 cells.

OBJECTIVES: We previously demonstrated that some phenylpropanoids are capable of inhibiting activated mast cells. This study evaluated the anti-allergic effects of licarin A, a neolignan isolated from various plants, on antigen-stimulated rat mast cell line. METHODS: The inhibitory effects of licarin A on histamine release, tumour necrosis factor-α (TNF-α) and prostaglandin D2 (PGD2) production, and cyclooxygenase-2 (COX-2) expression in dinitrophenyl-human serum albumin (DNP-HSA) rat basophilic leukemia cells (DNP-HSA-stimulated RBL-2H3 cells), were investigated by spectrofluorometry, ELISA and immunoblotting. KEY FINDINGS: Licarin A significantly and dose-dependently reduced TNF-α production (IC50 12.6 ± 0.3 µm) in DNP-HSA-stimulated RBL-2H3 cells. Furthermore, the levels of PGD2 secretion in DNP-HSA-stimulated cells pretreated with licarin A were lower than those stimulated with DNP-HSA alone (positive control). Treatment with licarin A at 20 µm produced slight suppression of DNP-HSA-induced increases in COX-2 mRNA and protein levels. We identified several signalling pathways that mediated these pharmacological effects. Licarin A treatment tended to reduce phosphorylated protein kinase C alpha/beta II (PKCα/ßII) and p38 mitogen-activated protein kinase (MAPK) protein levels. CONCLUSIONS: Our results demonstrate that licarin A reduces TNF-α and PGD2 secretion via the inhibition of PKCα/ßII and p38 MAPK pathways; this compound may be useful for attenuating immediate hypersensitivity.

Cubic Rashba spin-orbit interaction of a two-dimensional hole gas in a strained-Ge/SiGe quantum well.

The spin-orbit interaction (SOI) of a two-dimensional hole gas in the inversion symmetric semiconductor Ge is studied in a strained-Ge/SiGe quantum well structure. We observe weak antilocalization (WAL) in the magnetoconductivity measurement, revealing that the WAL feature can be fully described by the k-cubic Rashba SOI theory. Furthermore, we demonstrate electric field control of the Rashba SOI. Our findings reveal that the heavy hole (HH) in strained Ge is a purely cubic Rashba system, which is consistent with the spin angular momentum m(j) = ± 3/2 nature of the HH wave function.

Circadian biology: an unexpected invitee to new time zones.

Adaptation to changing light conditions is a hallmark of the circadian clock. A new study points to the critical role played by a transcriptional repressor previously implicated in cell differentiation, highlighting unappreciated links between the clock and the control of development and tumorigenesis.

Light activates the adrenal gland: timing of gene expression and glucocorticoid release.

Light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. In the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (SCN)-sympathetic nervous system. Moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamo-adenohypophysial axis. The abolishment after SCN lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. The magnitude of corticostereone response is dose dependently correlated with the light intensity. The light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment.