Tonicity‐responsive enhancer binding protein (TonEBP) regulates TNF‐α‐induced hypothalamic inflammation

Abstract

Tonicity‐responsive enhancer binding protein (TonEBP) is a widely expressed transcription factor and is important in the regulation of inflammatory cytokines. Here, we have identified TonEBP expression in the hypothalamus, which is particularly high in proopiomelanocortin (POMC) neurons. TonEBP overexpression stimulates POMC transcription, and TonEBP haploinsufficiency in TonEBP (+/−) mice results in a decrease in hypothalamic POMC expression. TonEBP (+/−) mice show reduced sickness responses, which include anorexia and hyperthermia, that are initially induced by tumor necrosis factor (TNF)‐α. TonEBP (+/−) mice also show lower levels of TNF‐α‐induced hypothalamic expression of POMC and pro‐inflammatory cytokines. These results suggest that TonEBP is an important molecular regulator in the development of inflammatory sickness responses through the control of POMC and pro‐inflammatory cytokine expression in the hypothalamus.

Transcription Factor TonEBP Stimulates Hyperosmolality-Dependent Arginine Vasopressin Gene Expression in the Mouse Hypothalamus

“…As a next step, we used a TonEBP haploinsufficiency mouse model to investigate the physiological function of the hypothalamic TonEBP–AVP system in dehydration-dependent metabolic adaptation. TonEBP knockout animals die during development, so TonEBP haploinsufficiency mice have been used extensively to study the physiological and behavioral functions of TonEBP in vivo ( 39 , 41 , 44 ). While mRNA levels of TonEBP in the PVN/SON of control mice are increased in response to dehydration, this phenomenon is attenuated in TonEBP haploinsufficiency animals, indicating that TonEBP gene expression in response to dehydration is impaired in the animals with TonEBP haploinsufficiency.…”

“…We have utilized mHypoA 2/28 cell line in this study, as this cell line was originally generated from the hypothalamus, and therefore, includes a broad library of hypothalamic neuronal phenotypes ( 44 , 45 ). mHypoA 2/28 cells (CELLutions Biosystems Inc. Ontario, Canada) were maintained in high-glucose Dulbecco’s Modified Eagle Medium (Hyclone, South Logan, UT, USA) supplemented with 10% fetal bovine serum and 100 U/ml penicillin-streptomycin (Hyclone) in a humidified atmosphere with 5% CO 2 at 37°C.…”

“…The 2,065-nucleotide-long PCR product was inserted into a luciferase reporter plasmid (pGL2-promoter vector, Promega, Madison, WI, USA), and its sequence was confirmed by DNA sequencing. This 5’ flanking sequence of the rat AVP gene includes 4 conserved core domains (TGGAAANNYNY, N = any nucleotide, Y = pyrimidine nucleotide) for the binding of TonEBP ( 44 ). Transfection efficiency was normalized by co-transfecting a β-galactosidase reporter plasmid (pCMV-β-gal; Clontech, Palo Alto, CA) at 100 ng/well.…”

“…In addition, we investigated a dehydration-induced anorectic effect in TonEBP (+/-) mice (Figures 6C, D). Previous investigations, including by our group, have demonstrated that metabolic parameters between TonEBP (+/+) and TonEBP (+/-) mice, such as food intake, body weight gain, glucose and insulin metabolism, and physical activity, are comparable to each other under normal diet conditions(41,44). However, in this study we found that acute dehydration reduced the daily body-weight gain in the TonEBP (+/+) mice, but not in the TonEBP (+/-) mice [on day 2 of dehydration, -3.4 vs. 0.1 g/day in TonEBP (+/+) mice; -1.6 vs. -1.0 g/day in TonEBP (+/-) mice, dehydrated vs. control, respectively].…”

“…To identify the in vivo effects of TonEBP on AVP mRNA expression in the hypothalamus, heterozygous TonEBP haploinsufficiency [TonEBP (+/-)] mice were used (a kind gift from Dr. H.M. Kwon, UNIST, Ulsan, South Korea). TonEBP knockout animals die during development, so TonEBP haploinsufficiency mice have been used extensively from us and other groups to study the physiological functions of TonEBP in vivo (38)(39)(40)(41)(42)(43)(44). To determine whether TonEBP affects dehydration-induced energy homeostasis, drinking water was withdrawn for 48 h, starting at 10:00 a.m., with food available ad libitum, and the food intake and body weight of wild and TonEBP haploinsufficiency mice were measured every day for 2 days.…”