Intermittent Hypoxia Increased the Expression of DBH and PNMT in Neuroblastoma Cells via MicroRNA-375-Mediated Mechanism

Abstract

Sleep apnea syndrome (SAS), characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia (IH)), is a risk factor for hypertension and insulin resistance. We report a correlation between IH and insulin resistance/diabetes. However, the reason why hypertension is induced by IH is elusive. Here, we investigated the effect of IH on the expression of catecholamine-metabolizing enzymes using an in vitro IH system. Human and mouse neuroblastoma cells (NB-1 and Neuro-2a) were exposed to IH or normoxia for 24 h. Real-time RT-PCR revealed that IH significantly increased the mRNA levels of dopamine β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in both NB-1 and Neuro-2a. Western blot showed that the expression of DBH and PNMT in the NB-1 cells was significantly increased by IH. Reporter assays revealed that promoter activities of DBH and PNMT were not increased by IH. The miR-375 level of IH-treated cells was significantly decreased relative to that of normoxia-treated cells. The IH-induced up-regulation of DBH and PNMT was abolished by the introduction of the miR-375 mimic, but not by the control RNA. These results indicate that IH stress increases levels of DBH and PNMT via the inhibition of miR-375-mediated mRNA degradation, potentially playing a role in the emergence of hypertension in SAS patients.

Downregulation of the Cd38-Cyclic ADP-Ribose Signaling in Cardiomyocytes by Intermittent Hypoxia via Pten Upregulation

“…The prevalence of SAS in patients with heart failure ranges from 15% to 59%, and the mortality rate among patients with severe SAS is significantly high [ 27 , 28 , 29 , 30 ]. We recently reported that IH increases renin expression in juxtaglomerular cells [ 46 ], as well as the expression of dopamine β-hydroxylase and phenylethanolamine N -methyltransferase in catecholamine-synthesizing neuroblastoma cells [ 64 ], causing SAS patients to become hypertensive. However, how cardiac dysfunction develops in SAS patients remains elusive.…”

“…The cells were kept at 37 °C, 5% CO 2 , and 95% humidity, and the medium was changed every day. The cells were exposed to either normoxia (21% O 2 , 5% CO 2 , and balanced N 2 ) or intermittent hypoxia (IH: 70 cycles of 5 min sustained hypoxia (1% O 2 , 5% CO 2 , and balanced N 2 ) and 10 min normoxia) using a custom-designed, computer-controlled incubation chamber attached to an external O 2 -CO 2 -N 2 computer-driven controller (O 2 programmable control, 9200EX, Wakenbtech Co., Ltd., Kyoto, Japan), as previously described [ 4 , 40 , 46 , 64 , 80 , 81 ]. These conditions are similar to the conditions reported in patients with severe degrees of SAS; in severe SAS cases, patients are repeatedly exposed to severe hypoxemia followed by mild hypoxemia or normoxia (i.e., IH).…”

“…Total RNA was isolated from H9c2 and P19.CL6 cells using an RNeasy Plus Cell Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesized from total RNA as a template using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), as previously described [ 40 , 46 , 64 , 80 , 81 ]. Real-time PCR was performed using an SYBR ® Fast qPCR Kit (KAPA Biosystems, Boston, MA, USA) and a Thermal Cycler Dice Real Time System (Takara Bio, Kusatsu, Japan).…”

“…PCR was performed with an initial step of 3 min at 95 °C, followed by 40 cycles of 3 s at 95 °C and 20 s at 60 °C for Rig/RpS15 , Cd38, Ryr2, Fkbp12.6, and Pten . The mRNA expression levels were normalized to the mRNA level of Rig/RpS15 , as previously described [ 40 , 46 , 64 , 80 , 81 ].…”

“…H9c2 cardiomyocyte extract (5 × 10 5 cells) was subjected to immunoblot analysis as previously described [ 46 , 64 ], using an anti-Cd38 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) raised against a peptide fragment of mouse Cd38 (residues 279–301 in [ 46 ]), anti-Ryr2 polyclonal antibody (PeproTech, Canbury, NJ, USA) [ 67 ], anti-Fkbp12.6 monoclonal antibody (Santa Cruz Biotechnology) raised against the amino acids 38–108 of human/rat/mouse FKBP12.6, anti-Pten monoclonal antibody raised against the full-length human PTEN protein (Abcam, Cambridge, UK), and anti-β-actin monoclonal antibody (Sigma, St. Louis, MO, USA) raised against Ac-Asp-Asp-Asp-Ile-Ala-Ala-Leu-Val-Ile-Asp-Asn-Gly-Ser-Gly-Lys. A SNAP id ® 2.0 Protein Detection System (Merck Millipore, Burlington, MA, USA) was used for the analysis.…”