Iron is an important biological catalyst and is critical for DNA synthesis during cell proliferation. Cellular iron uptake is enhanced in tumor cells to support increased DNA synthesis. Circadian variations in DNA synthesis and proliferation have been identified in tumor cells, but their relationship with intracellular iron levels is unclear. In this study, we identified a 24-h rhythm in iron regulatory protein 2 (IRP2) levels in colon-26 tumors implanted in mice. Our findings suggest that IRP2 regulates the 24-h rhythm of transferrin receptor 1 (Tfr1) mRNA expression post-transcriptionally, by binding to RNA stem-loop structures known as iron-response elements. We also found that Irp2 mRNA transcription is promoted by circadian clock genes, including brain and muscle Arnt-like 1 (BMAL1) and the circadian locomotor output cycles kaput (CLOCK) heterodimer. Moreover, growth in colon-26(⌬19) tumors expressing the clock-mutant protein (CLOCK ⌬19 ) was low compared with that in wild-type colon-26 tumor. The time-dependent variation of cellular iron levels, and the proliferation rate in wild-type colon-26 tumor was decreased by CLOCK ⌬19 expression. Our findings suggest that circadian organization contributes to tumor cell proliferation by regulating iron metabolism in the tumor.Circadian rhythms affect blood pressure, locomotor activity, core body temperature, and the sleep-wake cycle. These circadian controls of physiology and behavior are driven by a master pacemaker located in the suprachiasmatic nucleus of the hypothalamus, which relies on the interplay of interconnected transcriptional and translational feedback loops (1). The brain and muscle Arnt-like 1 (BMAL1) 3 and circadian locomotor output cycles kaput (CLOCK) heterodimer drive the transcription of cryptochrome (CRY) and period (PER) genes by binding to the E-box in their promoter regions. CRY and PER homodimers and heterodimers inhibit the function of BMAL1/CLOCK, thus decreasing their own expression (2-5). This core loop creates a 24-h rhythmic oscillation of clock-controlled gene expression in normal and tumor cells.Iron is an important metal for cell proliferation, metabolism, respiration, and DNA synthesis (6 -8). Iron homeostasis is dependent on the expression of transferrin receptor 1 (TfR1) (9, 10). TfR1 is a membrane receptor responsible for iron uptake and ferritin is an intracellular protein that stores iron. When present in excess, cellular iron is toxic (11-13). Therefore, iron concentration must be tightly regulated. In general, the expression of TfR1 is regulated by iron-regulatory proteins (IRPs), which are sensors of intracellular iron levels. IRPs control TfR1 and divalent metal transporter 1 (Dmt1) at the post-transcriptional level by binding to RNA stem-loop structures known as iron-responsive elements (IREs) in the 3Ј-untranslated region (UTR) of mRNA. IRP binding stabilizes transcripts in these genes (14 -16).Iron metabolism is critical for the rapid growth of tumors and iron is also required for DNA synthesis in tumor cells. Therefor...

Cancer cells have acquired several pathways to escape from host immunity in the tumor microenvironment. Programmed death 1 (PD-1) receptor and its ligand PD-L1 are involved in the key pathway of tumor immune escape, and immune checkpoint therapy targeting PD-1 and PD-L1 has been approved for the treatment of patients with certain types of malignancies. Although PD-1 is a well-characterized receptor on T cells, the immune checkpoint receptor is also expressed on tumor-associated macrophages (TAM), a major immune component of the tumor microenvironment. In this study, we found significant diurnal oscillation in the number of PD-1–expressing TAMs collected from B16/BL6 melanoma-bearing mice. The levels of Pdcd1 mRNA, encoding PD-1, in TAMs also fluctuated in a diurnal manner. Luciferase reporter and bioluminescence imaging analyses revealed that a NF-κB response element in the upstream region of the Pdcd1 gene is responsible for its diurnal expression. A circadian regulatory component, DEC2, whose expression in TAMs exhibited diurnal oscillation, periodically suppressed NF-κB–induced transactivation of the Pdcd1 gene, resulting in diurnal expression of PD-1 in TAMs. Furthermore, the antitumor efficacy of BMS-1, a small molecule inhibitor of PD-1/PD-L1, was enhanced by administering it at the time of day when PD-1 expression increased on TAMs. These findings suggest that identification of the diurnal expression of PD-1 on TAMs is useful for selecting the most appropriate time of day to administer PD-1/PD-L1 inhibitors. Implications: Selecting the most appropriate dosing time of PD-1/PD-L1 inhibitors may aid in developing cancer immunotherapy with higher efficacy.

Fluorescence imaging of fatty acid beta oxidation (FAO) with a fluorescent probe metabolically degraded by sequential enzyme reactions of FAO.

Dysfunction of the circadian clock has been implicated in the pathogenesis of cardiovascular disease. The CLOCK protein is a core molecular component of the circadian oscillator, so that mice with a mutated Clock gene (Clk/Clk) exhibit abnormal rhythms in numerous physiological processes. However, here we report that chronic kidney disease (CKD)-induced cardiac inflammation and fibrosis are attenuated in Clk/Clk mice even though they have high blood pressure and increased serum angiotensin II levels. A search for the underlying cause of the attenuation of heart disorder in Clk/Clk mice with 5/6 nephrectomy (5/6Nx) led to identification of the monocytic expression of G protein-coupled receptor 68 (GPR68) as a risk factor of CKD-induced inflammation and fibrosis of heart. 5/6Nx induces the expression of GPR68 in circulating monocytes via altered CLOCK activation by increasing serum levels of retinol and its binding protein (RBP4). The high-GPR68-expressing monocytes have increased potential for producing inflammatory cytokines, and their cardiac infiltration under CKD conditions exacerbates inflammation and fibrosis of heart. Serum retinol and RBP4 levels in CKD patients are also sufficient to induce the expression of GPR68 in human monocytes. Our present study reveals an uncovered role of monocytic clock genes in CKD-induced heart failure.

Chronic kidney disease (CKD) is a global health problem, and novel therapies to treat CKD are urgently needed. Here, we show that inhibition of G0/G1 switch 2 (G0s2) ameliorates renal inflammation in a mouse model of CKD. Renal expression of chemokine (C-C motif) ligand 2 (Ccl2) was increased in response to p65 activation in the kidneys of wild-type 5/6 nephrectomy (5/6Nx) mice. Moreover, 5/6Nx Clk/Clk mice, which carry homozygous mutations in the gene encoding circadian locomotor output cycles kaput (CLOCK), did not exhibit aggravation of apoptosis or induction of F4/80-positive cells. The renal expression of G0s2 in wild-type 5/6Nx mice was important for the transactivation of Ccl2 by p65. These pathologies were ameliorated by G0s2 knockdown. Furthermore, a novel small-molecule inhibitor of G0s2 expression was identified by high-throughput chemical screening, and the inhibitor suppressed renal inflammation in 5/6Nx mice. These findings indicated that G0s2 inhibitors may have applications in the treatment of CKD.