Calcineurin serves in the circadian output pathway to regulate the daily rhythm of L-type voltage-gated calcium channels in the retina

The L-type voltage-gated calcium channels (L-VGCCs) in avian retinal cone photoreceptors are under circadian control, in which the protein expression of the α1 subunits and the current density are greater at night than during the day. Both Ras-mitogen-activated protein kinase (MAPK) and Ras-phosphatidylionositol 3 kinase-protein kinase B (PI3K-AKT) signaling pathways are part of the circadian output that regulate the L-VGCC rhythm, while cAMP-dependent signaling is further upstream of Ras to regulate the circadian outputs in photoreceptors. However, there are missing links between cAMP-dependent signaling and Ras in the circadian output regulation of L-VGCCs. In this study, we report that calcineurin, a Ca2+/calmodulin-dependent serine (ser)/threonine (thr) phosphatase, participates in the circadian output pathway to regulate L-VGCCs through modulating both Ras-MAPK and Ras-PI3K-AKT signaling. The activity of calcineurin, but not its protein expression, was under circadian regulation. Application of a calcineurin inhibitor, FK-506 or cyclosporine A, reduced the L-VGCC current density at night with a corresponding decrease in L-VGCCα1D protein expression, but the circadian rhythm of L-VGCCα1D mRNA levels were not affected. Inhibition of calcineurin further reduced the phosphorylation of ERK and AKT (at thr 308) and inhibited the activation of Ras, but inhibitors of MAPK or PI3K signaling did not affect the circadian rhythm of calcineurin activity. However, inhibition of adenylate cyclase significantly dampened the circadian rhythm of calcineurin activity. These results suggest that calcineurin is upstream of MAPK and PI3K-AKT but downstream of cAMP in the circadian regulation of L-VGCCs.     

Forskolin induces circadian gene expression of rPer1, rPer2 and dbp in mammalian rat-1 fibroblasts

Mammalian culture cells have the potential for periodicity, since high concentrations of serum can elicit the circadian expression of clock genes in rat-1 fibroblasts. However, the mechanism by which serum affects circadian gene expression remains unclear. In the present study, we incubated rat-1 cells with forskolin and successfully induced the rhythmic expression of Per1, Per2 and dbp. In the initial step of the circadian gene expression, a marked transient induction of Per1 was observed accompanied with CREB phosphorylation. Thus the present study strongly suggests that CREB activation through the cAMP/PKA pathway is involved in the generation of circadian rhythm in rat-1 cells     

Modeling the VPAC2-activated cAMP/PKA signaling pathway: from receptor to circadian clock gene induction

Increasing evidence suggests an important role for VPAC2-activated signal transduction pathways in maintaining a synchronized biological clock in the suprachiasmatic nucleus (SCN). Activation of the VPAC2 signaling pathway induces per1 gene expression in the SCN and phase-shifts the circadian clock. Mice without the VPAC2 receptor lack an overt, coherent circadian rhythm in clock gene expression, SCN neuron firing rate, and locomotor behavior. Using a systems approach, we have developed a kinetic model integrating VPAC2 signaling mediated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway and leading to induced circadian clock gene expression. We fit the model to experimental data from the literature for cAMP accumulation, PKA activation, cAMP-response element binding protein phosphorylation, and per1 induction. By linking the VPAC2 model to a published circadian clock model, we also simulated clock phase shifts induced by vasoactive intestinal polypeptide (VIP) and matched experimental data for the VIP response. The simulated phase response curve resembled the hamster response to a related neuropeptide, GRP1-27, and light. Simulations using pulses of VIP revealed that the system response is extraordinarily robust to input signal duration, a result with physiologically relevant consequences. Lastly, simulations using varied receptor levels matched literature experimental data from animals overexpressing VPAC2 receptors.     

Molecular components and mechanism of adrenergic signal transduction in mammalian pineal gland: regulation of melatonin synthesis

Rhythmic neural outputs from the hypothalamic suprachiasmatic nucleus (SCN), which programme the rhythmic release of norepinephrine (NE) from intrapineal nerve fibers, regulate circadian rhythm of melatonin synthesis. Increased secretion of NE with the onset of darkness during the first half of night stimulates melatonin synthesis by several folds. NE binds to both alpha1- and beta-adrenergic receptors present on the pinealocyte membrane and initiates adrenergic signal transduction via cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) generating pathways. The NE-induced adrenergic signal transduction switches 'on' melatonin synthesis during the early hours of night by stimulating expression of the rate-limiting enzyme of melatonin synthesis, N-acetyltransferase (AA-NAT) via cAMP-protein kinase A (PKA)-cAMP response element binding protein (CREB)-cAMP response element (CRE) pathway as well as by increasing AA-NAT activity via cAMP-PKA-14-3-3 protein pathway. Simultaneously, adrenergically-induced expression of inducible cAMP early repressor (ICER) negatively regulates aa-nat gene expression and controls the amplitude of melatonin rhythm. In the second half of night, increased release of acetylcholine from central pinealopetal projections, inhibition of NE secretion by SCN, withdrawal of adrenergic inputs and reversal of events that took place in the first half lead to switching 'off' of melatonin synthesis. Adrenergic signal transduction via cGMP-protein kinase G (PKG)-mitogen activated protein kinase (MAPK)-ribosomal S6 kinase (RSK) pathway also seems to be fully functional, but its role in modulation of melatonin synthesis remains unexplored. This article gives a critical review of information available on various components of the adrenergic signal transduction cascades involved in the regulation of melatonin synthesis.     

Regulation and possible role of serotonin N-acetyltransferase in the retina

The activity of retinal serotonin N-acetyltransferase (NAT) (arylamine acetyltransferase, EC 2.3.1.5), the penultimate enzyme in melatonin biosynthesis, exhibits properties of a circadian rhythm comparable to that seen in the pineal gland. Using an eye cup preparation we have found that circadian properties persist in vitro, which indicates that an endogenous circadian oscillator controlling NAT is present in the eye. Nighttime increases in NAT activity are suppressed by light, protein synthesis inhibitors, and catecholamines. In light, NAT activity is induced by conditions expected to increase intracellular levels of cyclic AMP (cAMP). This suggests that catecholamines and cAMP are normally involved in the regulation of NAT. Circadian indoleamine metabolism may play a role in the control of rhythmic photoreceptor metabolism as evidenced by the observation that melatonin and related compounds are potent activators of disk shedding.     

Cyclic AMP inhibits p38 activation via CREB-induced dynein light chain

The mitogen-activated protein kinase p38 plays a critical role in inflammation, cell cycle progression, differentiation, and apoptosis. The activity of p38 is stimulated by a variety of extracellular stimuli, such as the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha), and subjected to regulation by other intracellular signaling pathways, including the cyclic AMP (cAMP) pathway. Yet the underlying mechanism by which cAMP inhibits p38 activation is unknown. Here we show that the induction of dynein light chain (DLC) by cAMP response element-binding protein (CREB) is required for cAMP-mediated inhibition of p38 activation. cAMP inhibits p38 activation via the protein kinase A-CREB pathway. The inhibition is mediated by the CREB target gene Dlc, whose protein product, DLC, interferes with the formation of the MKK3/6-p38 complex, thereby suppressing p38 phosphorylation activation by MKK3/6. The inhibition of p38 activation by cAMP leads to suppression of NF-kappaB activity and promotion of apoptosis in response to TNF-alpha. Thus, our results identify DLC as a novel inhibitor of the p38 pathway and provide a molecular mechanism by which cAMP suppresses p38 activation and promotes apoptosis.     

A novel pineal-specific product of the oligopeptide transporter PepT1 gene: circadian expression mediated by cAMP activation of an intronic promoter

The oligopeptide transporter 1, PepT1, is a member of the Slc15 family of 12 membrane-spanning domain transporters; PepT1 has proton/peptide cotransport activity and is selectively expressed in intestinal epithelial cells, where it is responsible for the nutritional absorption of di- and tri-peptides. Here, a novel PepT1 gene product has been identified in the rat pineal gland, termed pgPepT1. It encodes a 150-amino acid protein encompassing the C-terminal 3 membrane-spanning domains of intestinal PepT1 protein, with 3 additional N-terminal residues. Expression of pgPepT1 appears to be restricted to the pineal gland and follows a marked circadian pattern with >100-fold higher levels of mRNA occurring at night; this is accompanied by an accumulation of membrane-associated pgPepT1 protein ( approximately 16 kDa). The daily rhythm in pgPepT1 mRNA is regulated by the well described neural pathway that controls pineal melatonin production. This includes the retina, the circadian clock in the suprachiasmatic nucleus, central structures, and projections from the superior cervical ganglia; activation of this pathway results in the release of norepinephrine. Here it was found that pgPepT1 expression is mediated by a norepinephrine-->cyclic AMP mechanism that activates an alternative promoter located in intron 20 of the gene. pgPepT1 protein was found to have transporter-modulator activity; it could contribute to circadian changes in pineal function through this mechanism.     

Reciprocal regulation of brain and muscle Arnt-like protein 1 and peroxisome proliferator-activated receptor alpha defines a novel positive feedback loop in the rodent liver circadian clock

Recent evidence has emerged that peroxisome proliferator-activated receptor alpha (PPARalpha), which is largely involved in lipid metabolism, can play an important role in connecting circadian biology and metabolism. In the present study, we investigated the mechanisms by which PPARalpha influences the pacemakers acting in the central clock located in the suprachiasmatic nucleus and in the peripheral oscillator of the liver. We demonstrate that PPARalpha plays a specific role in the peripheral circadian control because it is required to maintain the circadian rhythm of the master clock gene brain and muscle Arnt-like protein 1 (bmal1) in vivo. This regulation occurs via a direct binding of PPARalpha on a potential PPARalpha response element located in the bmal1 promoter. Reversely, BMAL1 is an upstream regulator of PPARalpha gene expression. We further demonstrate that fenofibrate induces circadian rhythm of clock gene expression in cell culture and up-regulates hepatic bmal1 in vivo. Together, these results provide evidence for an additional regulatory feedback loop involving BMAL1 and PPARalpha in peripheral clocks.     

cAMP mediated upregulation of CYP2A5 in mouse hepatocytes

CYP2A5 is induced by a large number of chemicals including some cAMP modifiers. In a primary hepatocyte model, stimulation of the cAMP signal transduction pathway by glucagon and isoproterenol, acting via specific G-protein coupled plasma membrane receptors, produced up to 17-fold increases in the marker activity of CYP2A5, coumarin 7-hydroxylase. In contrast, glucagon and isoproterenol caused no significant effects on two other major CYP forms, CYP2B10 and CYP1A1/2. Phenobarbital (PB) elicited a 3-fold increase in CYP2A5 expression (catalytic activity and mRNA), while the cAMP and protein kinase A (PKA) stimulators dibutyryl-cAMP, forskolin and Sp-cAMPs caused up to 18-fold increases in the amount of CYP2A5 mRNA. Coadministration of PB and cAMP/PKA stimulating agents produced an additive inducing effect. The expression of CYP2A5, but not CYP2B10 or CYP1A1/2, in DBA/2 mice displayed a marked circadian rhythm, the level of expression being highest in the evening. These results suggest that among xenobiotic metabolizing CYP enzymes, CYP2A5 is uniquely upregulated by cAMP, possibly having the physiological function of priming the olfactory and digestive systems for nocturnal feeding.     

The pseudo-receiver domain of CikA regulates the cyanobacterial circadian input pathway

CikA (circadian input kinase) is a component of the cyanobacterial circadian clock that aids in synchronizing the endogenous oscillator with the external environment. cikA mutants of the prokaryotic circadian model organism Synechococcus elongatus PCC 7942 fail to reset the phase of the circadian rhythm of gene expression after an environmental time cue, and also exhibit reduced amplitude and shortened period of circadian oscillation. CikA has histidine protein kinase (HPK) activity that is modulated in vitro by GAF and pseudo-receiver (PsR) domains. Here we show that the PsR domain negatively regulates HPK activity in vivo and also serves as an interaction module to dock CikA at a specific subcellular location. Phenotypes conferred by alleles that encode CikA variants showed that all domains except the featureless N-terminus are required for CikA function. Overexpression of all alleles that encode the PsR domain, whether or not the HPK is functional, caused a dominant arrhythmic phenotype, whereas overexpressed variants that lack PsR did not. Subcellular localization of intact CikA identified a polar focus whereas a variant without PsR showed uniform distribution in the cell, consistent with a model in which PsR mediates interaction with other input pathway components.