The daily rhythm of L‐type voltage‐gated calcium channels (L‐VGCCs) is part of the cellular mechanism underlying the circadian regulation of retina physiology and function. However, it is not completely understood how the circadian clock regulates L‐VGCC current amplitudes without affecting channel gating properties. The phosphatidylinositol 3 kinase–protein kinase B (PI3K–Akt) signaling pathway has been implicated in many vital cellular functions especially in trophic factor‐induced ion channel trafficking and membrane insertion. Here, we report that PI3K–Akt signaling participates in the circadian phase‐dependent modulation of L‐VGCCs. We found that there was a circadian regulation of Akt phosphorylation on Thr308 that peaked at night. Inhibition of PI3K or Akt significantly decreased L‐VGCC current amplitudes and the expression of membrane‐bound L‐VGCCα1D subunit only at night but not during the subjective day. Photoreceptors transfected with a dominant negative Ras had significantly less expression of phosphorylated Akt and L‐VGCCα1D subunit compared with non‐transfected photoreceptors. Interestingly, both PI3K–Akt and extracellular signal‐related kinase were downstream of Ras, and they appeared to be parallel and equally important pathways to regulate L‐VGCC rhythms. Inhibition of either pathway abolished the L‐VGCC rhythm indicating that there were multiple mechanisms involved in the circadian regulation of L‐VGCC rhythms in retina photoreceptors.

Ion channels are the gatekeepers to neuronal excitability. Retinal neurons of vertebrates and invertebrates, neurons of the suprachiasmatic nucleus (SCN) of vertebrates, and pinealocytes of non‐mammalian vertebrates display daily rhythms in their activities. The interlocking transcription–translation feedback loops with specific post‐translational modulations within individual cells form the molecular clock, the basic mechanism that maintains the autonomic ∼24‐h rhythm. The molecular clock regulates downstream output signaling pathways that further modulate activities of various ion channels. Ultimately, it is the circadian regulation of ion channel properties that govern excitability and behavior output of these neurons. In this review, we focus on the recent development of research in circadian neurobiology mainly from 1980 forward. We will emphasize the circadian regulation of various ion channels, including cGMP‐gated cation channels, various voltage‐gated calcium and potassium channels, Na+/K+‐ATPase, and a long‐opening cation channel. The cellular mechanisms underlying the circadian regulation of these ion channels and their functions in various tissues and organisms will also be discussed. Despite the magnitude of chronobiological studies in recent years, the circadian regulation of ion channels still remains largely unexplored. Through more investigation and understanding of the circadian regulation of ion channels, the future development of therapeutic strategies for the treatment of sleep disorders, cardiovascular diseases, and other illnesses linked to circadian misalignment will benefit.

The L-type voltage-gated calcium channels (L-VGCCs) are activated under high depolarization voltages. They are vital for diverse biological events, including cell excitability, differentiation, and synaptic transmission. In retinal photoreceptors, L-VGCCs are responsible for neurotransmitter release and are under circadian influences. However, the mechanism of L-VGCC regulation in photoreceptors is not fully understood. Here, we show that retinoschisin, a highly conserved extracellular protein, interacts with the L-VGCC␣1D subunit and regulates its activities in a circadian manner. Mutations in the gene encoding retinoschisin (RS1) cause retinal disorganization that leads to early onset of macular degeneration. Since ion channel activities can be modulated through interactions with extracellular proteins, disruption of these interactions can alter physiology and be the root cause of disease states. Co-immunoprecipitation and mammalian two-hybrid assays showed that retinoschisin and the N-terminal fragment of the L-VGCC␣1 subunit physically interacted with one another. The expression and secretion of retinoschisin are under circadian regulation with a peak at night and nadir during the day. Inhibition of L-type VGCCs decreased membrane-bound retinoschisin at night. Overexpression of a missense RS1 mutant gene, R141G, into chicken cone photoreceptors caused a decrease of L-type VGCC currents at night. Our findings demonstrate a novel bidirectional relationship between an ion channel and an extracellular protein; L-type VGCCs regulate the circadian rhythm of retinoschisin secretion, whereas secreted retinoschisin feeds back to regulate L-type VGCCs. Therefore, physical interactions between L-VGCC␣1 subunits and retinoschisin play an important role in the membrane retention of L-VGCC␣1 subunits and photoreceptor-bipolar synaptic transmission.

Obesity-induced hyperglycemic and prediabetic/early diabetic conditions caused detrimental impacts on retinal light sensitivities and health. The decrease of the ERG components in early diabetes reflects the decreased neuronal activity of retinal light responses, which may be caused by a decrease in neuronal calcium signaling. Since PI3K-AKT is important in regulating calcium homeostasis and neural survival, maintaining proper PI3K-AKT signaling in early diabetes or at the prediabetic stage might be a new strategy for DR prevention.

Diabetic retinopathy (DR) is the leading cause of blindness among American adults above 40 years old. The vascular complication in DR is a major cause of visual impairment, making finding therapeutic targets to block pathological angiogenesis a primary goal for developing DR treatments. MicroRNAs (miRs) have been proposed as diagnostic biomarkers and potential therapeutic targets for various ocular diseases including DR. In diabetic animals, the expression levels of several miRs, including miR-150, are altered. The expression of miR-150 is significantly suppressed in pathological neovascularization in mice with hyperoxia-induced retinopathy. The purpose of this study was to investigate the functional role of miR-150 in the development of retinal microvasculature complications in high-fat-diet (HFD) induced type 2 diabetic mice. Wild type (WT) and miR-150 null mutant (miR-150-/-) male mice were given a HFD (59% fat calories) or normal chow diet. Chronic HFD caused a decrease of serum miR-150 in WT mice. Mice on HFD for 7 months (both WT and miR-150-/-) had significant decreases in retinal light responses measured by electroretinograms (ERGs). The retinal neovascularization in miR-150-/--HFD mice was significantly higher compared to their age matched WT-HFD mice, which indicates that miR-150 null mutation exacerbates chronic HFD-induced neovascularization in the retina. Overexpression of miR-150 in cultured endothelial cells caused a significant reduction of vascular endothelial growth factor receptor 2 (VEGFR2) protein levels. Hence, deletion of miR-150 significantly increased the retinal pathological angiogenesis in HFD induced type 2 diabetic mice, which was in part through VEGFR2.

Increased androgen receptor (AR) levels are associated with prostate cancer progression to androgen independence and therapy resistance. Evidence has suggested that chronic inflammation is closely linked to various cancers including prostate cancer. Herein we show that the proinflammatory cytokine TNFalpha negatively regulates AR mRNA and protein expression and reduces androgen sensitivity in androgen-dependent LNCaP human prostate cancer cells. Decreased AR expression results from transcription repression involving essential in cis interaction of nuclear factor-kappaB (NF-kappaB) with the B-myb transcription factor at a composite genomic element in the 5'-untranslated region of AR. The negative regulation was abrogated when NF-kappaB activity was inhibited by a superrepressor of the inhibitory kappaB protein. In contrast, androgen-independent C4-2 (LNCaP-derived) cells fail to show AR down-regulation by TNFalpha, despite expression of B-myb and TNFalpha-induced NF-kappaB activity similar to that in LNCaP cells. The negatively regulated AR gene chromatin region showed TNFalpha-dependent enrichment of B-myb and the NF-kappaB proteins p65 and p50. In parallel, the histone deacetylase 1, corepressor silencing mediator of retinoid and thyroid hormone receptor and the corepressor-associated scaffold protein mSin3A were recruited to the inhibitory site. In C4-2 cells, neither NF-kappaB and B-myb, nor any of the corepressor components, were detected at the negative site in response to TNFalpha. Apoptosis was induced in TNFalpha-treated LNCaP cells, likely in part due to the down-regulation of AR. The androgen-independent, AR-expressing C4-2 and C4-2B (derived from C4-2) cells were resistant to TNFalpha-induced apoptosis. The results linking androgen dependence to the NF-kappaB and AR pathways may be insightful in identifying novel treatment targets for prostate cancer.

PurposeThe purpose of this study was to determine the effects of metformin on dysfunctional retinas in obesity-induced type 2 diabetic mice.MethodsA high-fat diet (HFD)-induced diabetic mouse model (C57BL/6J) was used in this study. After 2 months of the HFD regimen, HFD mice were given daily metformin through oral gavage. Body weights, glucose tolerance, and retinal light responses were monitored regularly. Fluorescein angiography (FA) was used to assess changes in retinal vasculature. Ocular tissues (retina, vitreous, and lens) were harvested and analyzed for molecular changes as determined by immunofluorescent staining, Western blot analysis, and cytokine profiling.ResultsStarting 1 month after the diet regimen, mice fed the HFD had mildly compromised retinal light responses as measured by electroretinography (ERG), which worsened over time compared to that in the control. In HFD mice treated with metformin, systemic glucose levels reverted back to normal, and their weight gain slowed. Metformin reversed HFD-induced changes in phosphorylated protein kinase B (pAKT), extracellular signal-regulated kinase (pERK), and 5′AMP-activated protein kinase (pAMPK) in the retina. However, metformin treatments for 3 months did not restore the retinal light responses nor lessen the HFD-induced retinal neovascularization, even though it did reduce intraocular inflammation.ConclusionsAlthough metformin was able to reverse systemic changes induced by HFD, it was not able to restore HFD-caused retinal light responses or deter neovascularization.

The vitamin D receptor (VDR) regulates steroid and drug metabolism by inducing the genes encoding phase I and phase II enzymes. SULT2A1 is a liver- and intestine-expressed sulfo-conjugating enzyme that converts the alcohol-OH of neutral steroids, bile acids, and drugs to water-soluble sulfated metabolites. 1alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces SULT2A1 gene transcription after the recruitment of VDR to the vitamin D-responsive chromatin region of SULT2A1. A composite element in human SULT2A1 directs the 1,25-(OH)2D3-mediated induction of natural and heterologous promoters. This element combines a VDR/retinoid X receptor-alpha-binding site [vitamin D response element (VDRE)], which is an imperfect inverted repeat 2 of AGCTCA, and a CAAT/enhancer binding protein (C/EBP)-binding site located 9 bp downstream to VDRE. The binding sites were identified by EMSA, antibody supershift, and deoxyribonuclease I footprinting. C/EBP-alpha at the composite element plays an essential role in the VDR regulation of SULT2A1, because 1) induction was lost for promoters with inactivating mutations at the VDRE or C/EBP element; 2) SULT2A1 induction by 1,25-(OH)2D3 in C/EBP-alpha-deficient cells required the expression of cotransfected C/EBP-alpha; and 3) C/EBP-beta did not substitute for C/EBP-alpha in this regulation. VDR and C/EBP-alpha were recruited concurrently to the composite element along with the coactivators p300, steroid receptor coactivator 1 (SRC-1), and SRC-2, but not SRC-3. VDR and C/EBP-alpha associated endogenously as a DNA-dependent, coimmunoprecipitable complex, which was detected at a markedly higher level in 1,25-(OH)2D3-treated cells. These results provide the first example of the essential role of the interaction in cis between C/EBP-alpha and VDR in directing 1,25-(OH)2D3-induced expression of a VDR target gene.