Expression and Activity of the Transcription Factor CCAAT/Enhancer-Binding Protein ß (C/EBPß) Is Regulated by Specific Pulse-Modulated Radio Frequencies in Oligodendroglial Cells.

The rapid growth of wireless electronic devices has raised concerns about the harmful effects of leaked electromagnetic radiation (EMR) on human health. Even though numerous studies have been carried out to explore the biological effects of EMR, no clear conclusions have been drawn about the effect of radio frequency (RF) EMR on oligodendrocytes. To this end, we exposed oligodendroglia and three other types of brain cells to 2.4 GHz EMR for 6 or 48 h at an average input power of 1 W in either a continuous wave (CW-RF) or a pulse-modulated wave (PW-RF, 50 Hz pulse frequency, 1/3 duty cycle) pattern. RNA sequencing, RT-qPCR, and Western blot were used to examine the expression of C/EBPß and its related genes. Multiple reaction monitoring (MRM) was used to examine the levels of expression of C/EBPß-interacting proteins. Our results showed that PW-RF EMR significantly increased the mRNA level of C/EBPß in oligodendroglia but not in other types of cells. In addition, the expression of three isoforms and several interacting proteins and targeted genes of C/EBPß were markedly changed after 6-h PW-RF but not CW-RF. Our results indicated that RF EMR regulated the expression and functions of C/EBPß in a waveform- and cell-type-dependent manner.

Glucose and lipid metabolisms in human endometrial stromal cells during decidualization.

Decidualization is a process of differentiation of human endometrial stromal cells (hESCs) accompanied by dramatic changes in cellular functions. This process is critical for embryo implantation and the establishment of pregnancy. Impairment of decidualization of hESCs leads to implantation failure, miscarriage, and unexplained infertility. The present review focuses on the metabolic changes in hESCs during decidualization. One of the changes taking place is in the glucose metabolism. Glucose uptake increases during decidualization because glucose is essential for the decidualization of hESCs. In hESCs, GLUT1 is highly expressed and involved in the increase of glucose uptake during decidualization. The up-regulation of GLUT1 is mediated by an epigenetic mechanism, which is regulated by CCAAT enhancer-binding protein ß (C/EBPß) and Wilms tumor 1 (WT1). Another metabolic change is in the lipid metabolism. Lipid accumulation in hESCs increases during decidualization. This increase is mediated by very low-density lipoprotein receptor (VLDLR). The up-regulation of VLDLR is regulated by WT1. In contrast to glucose, lipid is not essential for decidualization of hESCs. Endometrial cells have been implicated as important sources of nutrition for the embryo. hESCs may increase glucose and lipid storage so that they can supply them to the embryo during the implantation process. Taken together, decidualization is the process accompanied by metabolic changes, which may be associated with successful implantation.

The essential glucose transporter GLUT1 is epigenetically upregulated by C/EBPß and WT1 during decidualization of the endometrium.

Human endometrial stromal cells (ESCs) differentiate into decidual cells by the action of progesterone, which is essential for implantation and maintenance of pregnancy. We previously reported that glucose uptake by human ESCs increases during decidualization and that glucose is indispensable for decidualization. Although glucose transporter 1 (GLUT1) is upregulated during decidualization, it remains unclear whether it is involved in glucose uptake. Here, we attempted to determine the role of GLUT1 during decidualization as well as the factors underlying its upregulation. ESCs were incubated with cAMP to induce decidualization. Knockdown of GLUT1 suppressed cAMP-increased glucose uptake and the expressions of specific markers of decidualization, IGF-binding protein-1 (IGFBP-1), and prolactin (PRL). To investigate the regulation of GLUT1 expression, we focused on CCAAT enhancer-binding protein ß (C/EBPß) and Wilms' tumor 1 (WT1) as the upstream transcription factors regulating GLUT1 expression. Knockdown of either C/EBPß or WT1 suppressed cAMP-increased GLUT1 expression and glucose uptake. cAMP treatment also increased the recruitment of C/EBPß and WT1 to the GLUT1 promoter region. Interestingly, cAMP increased the H3K27 acetylation (H3K27ac) and p300 recruitment in the GLUT1 promoter region. Knockdown of C/EBPß or WT1 inhibited these events, indicating that both C/EBPß and WT1 contribute to the increase of H3K27ac by recruiting p300 to the GLUT1 promoter region during decidualization. These findings indicate that GLUT1 is involved in glucose uptake in ESCs during decidualization, thus facilitating the establishment of pregnancy.

Transcription factor C/EBPß induces genome-wide H3K27ac and upregulates gene expression during decidualization of human endometrial stromal cells.

We previously reported that H3K27 acetylation (H3K27ac) increases throughout the genome during decidualization of human endometrial stromal cells (ESCs). However, its mechanisms have not been clarified. We also reported that C/EBPß acts as a pioneer factor initiating chromatin remodeling by increasing H3K27ac of IGFBP-1 and PRL promoters. Therefore, C/EBPß may be involved in the genome-wide increase of H3K27ac during decidualization. In this study, we investigated whether C/EBPß causes genome-wide H3K27ac modifications and regulates gene expressions during decidualization. cAMP was used to induce decidualization. Three types of cells (control cells, cAMP-treated cells, and cAMP-treated + C/EBPß-knockdowned cells by siRNA) were generated. Of 4190 genes that were upregulated by cAMP, C/EBPß knockdown inhibited these upregulation in 2239 genes (53.4%), indicating that they are under the regulation of C/EBPß. cAMP increased H3K27ac in 1272 of the 2239 genes. C/EBPß knockdown abolished the increase of H3K27ac in almost all genes (1263 genes, 99.3%), suggesting that C/EBPß can upregulate gene expression by increasing H3K27ac. To investigate how C/EBPß regulates H3K27ac throughout the genome, we tested the hypothesis that C/EBPß binds to its binding regions and recruits cofactors with histone acetyltransferase activities. To do this, we collated our ChIP-sequence data with public ChIP-sequence database of transcription factors, and found that p300 is the most likely cofactor that binds to the H3K27ac-increased-regions with C/EBPß. ChIP-qPCR of several genes confirmed that C/EBPß binds to the target regions, recruits p300, and increases H3K27ac. Our genome-wide analysis revealed that C/EBPß induces H3K27ac throughout the genome and upregulates gene expressions during decidualization by recruiting p300 to the promoters.

Activating transcription factor 4 (ATF4) promotes skeletal muscle atrophy by forming a heterodimer with the transcriptional regulator C/EBPß.

Skeletal muscle atrophy is a highly-prevalent and debilitating condition that remains poorly understood at the molecular level. Previous work found that aging, fasting, and immobilization promote skeletal muscle atrophy via expression of activating transcription factor 4 (ATF4) in skeletal muscle fibers. However, the direct biochemical mechanism by which ATF4 promotes muscle atrophy is unknown. ATF4 is a member of the basic leucine zipper transcription factor (bZIP) superfamily. Because bZIP transcription factors are obligate dimers, and because ATF4 is unable to form highly-stable homodimers, we hypothesized that ATF4 may promote muscle atrophy by forming a heterodimer with another bZIP family member. To test this hypothesis, we biochemically isolated skeletal muscle proteins that associate with the dimerization- and DNA-binding domain of ATF4 (the bZIP domain) in mouse skeletal muscle fibers in vivo Interestingly, we found that ATF4 forms at least five distinct heterodimeric bZIP transcription factors in skeletal muscle fibers. Furthermore, one of these heterodimers, composed of ATF4 and CCAAT enhancer-binding protein ß (C/EBPß), mediates muscle atrophy. Within skeletal muscle fibers, the ATF4-C/EBPß heterodimer interacts with a previously unrecognized and evolutionarily conserved ATF-C/EBP composite site in exon 4 of the Gadd45a gene. This three-way interaction between ATF4, C/EBPß, and the ATF-C/EBP composite site activates the Gadd45a gene, which encodes a critical mediator of muscle atrophy. Together, these results identify a biochemical mechanism by which ATF4 induces skeletal muscle atrophy, providing molecular-level insights into the etiology of skeletal muscle atrophy.

Casein kinase 2 phosphorylates and stabilizes C/EBPß in pancreatic ß cells.

During the development of type 2 diabetes, endoplasmic reticulum (ER) stress leads to pancreatic ß cell failure. CCAAT/enhancer-binding protein (C/EBP) ß is highly induced by ER stress and AMP-activated protein kinase (AMPK) suppression in pancreatic ß cells, and its accumulation reduces pancreatic ß cell mass. We investigated the phosphorylation state of C/EBPß under these conditions. Casein kinase 2 (CK2) was found to co-localize with C/EBPß in MIN6 cells. It phosphorylated S222 of C/EBPß, a previously unidentified phosphorylation site. We found that C/EBPß is phosphorylated by CK2 under AMPK suppression and ER stress, which are important from the viewpoint of the worsening pathological condition of type 2 diabetes, such as decreased insulin secretion and apoptosis of pancreatic ß cells.

Accelerated apoptosis of peripheral blood monocytes in Cebpb-deficient mice.

The CCAAT/enhancer-binding protein ß (C/EBPß) transcription factor is required for granulopoiesis under stress conditions. However, little is known about its roles in steady state hematopoiesis. Here, we analyzed the peripheral blood and bone marrow of Cebpb(-/-) mice at steady state by flow cytometry and unexpectedly found that the number of peripheral blood monocytes was severely reduced, while the number of bone marrow monocytes was maintained. The ability of Cebpb(-/-) bone marrow cells to give rise to macrophages/monocytes in vitro was comparable to that of wild-type bone marrow cells. Apoptosis of monocytes was enhanced in the peripheral blood, but not in the bone marrow of Cebpb(-/-) mice. These results indicate that C/EBPß is required for the survival of monocytes in peripheral blood.