Tuyen Hoang

Switch from stress response to homeobox transcription factors in adipose tissue after profound fat loss.

Background In obesity, impaired adipose tissue function may promote secondary disease through ectopic lipid accumulation and excess release of adipokines, resulting in systemic low-grade inflammation, insulin resistance and organ dysfunction. However, several of the genes regulating adipose tissue function in obesity are yet to be identified. Methodology/Principal Findings In order to identify novel candidate genes that may regulate adipose tissue function, we analyzed global gene expression in abdominal subcutaneous adipose tissue before and one year after bariatric surgery (biliopancreatic diversion with duodenal switch, BPD/DS) (n = 16). Adipose tissue from lean healthy individuals was also analyzed (n = 13). Two different microarray platforms (AB 1700 and Illumina) were used to measure the differential gene expression, and the results were further validated by qPCR. Surgery reduced BMI from 53.3 to 33.1 kg/m2. The majority of differentially expressed genes were down-regulated after profound fat loss, including transcription factors involved in stress response, inflammation, and immune cell function (e.g., FOS, JUN, ETS, C/EBPB, C/EBPD). Interestingly, a distinct set of genes was up-regulated after fat loss, including homeobox transcription factors (IRX3, IRX5, HOXA5, HOXA9, HOXB5, HOXC6, EMX2, PRRX1) and extracellular matrix structural proteins (COL1A1, COL1A2, COL3A1, COL5A1, COL6A3). Conclusions/Significance The data demonstrate a marked switch of transcription factors in adipose tissue after profound fat loss, providing new molecular insight into a dichotomy between stress response and metabolically favorable tissue development. Our findings implicate homeobox transcription factors as important regulators of adipose tissue function.

The nuclear receptors NUR77, NURR1 and NOR1 in obesity and during fat loss

Background:Adipose tissue is critical for systemic metabolic health. Identifying key factors regulating adipose tissue function is a research priority. The NR4A subfamily of nuclear receptors (NRs) (NR4A1/NUR77, NR4A2/NURR1 and NR4A3/NOR1) has emerged as important proteins in different disease states and in the regulation of metabolic tissues, particularly in liver and muscle. However, the expression of the NR4A members in human adipose tissue has not previously been described, and their target genes are largely unknown.Objective:To determine whether the NR4As are differentially expressed in human adipose tissue in obesity, and identify potential NR4A target genes.Design:Prospective analysis of s.c. adipose tissue before and 1 year after fat loss, and during in vitro differentiation of primary human preadipocytes. Case-control comparison of omental (OM) adipose tissue.Subjects:A total of 13 extremely obese patients undergoing biliopancreatic diversion with duodenal switch for fat loss, 12 extremely obese patients undergoing laparoscopic sleeve gastrectomy and 37 lean individuals undergoing hernia repair or laparotomy were included in the study. Measurements were done by quantitative PCR gene expression analysis of the NR4A members and in silico promoter analysis based on microarray data.Results:There was a strong upregulation of the NR4As in extreme obesity and normalization after fat loss. The NR4As were expressed at the highest level in stromal–vascular fraction compared with adipocytes, but were downregulated in both fractions after fat loss. Their expression levels were also significantly higher in OM compared with s.c. adipocytes in obesity. The NR4As were downregulated during differentiation of primary human preadipocytes. Moreover, the NR4As were strongly induced within 30 min of tissue incubation. Finally, promoter analysis revealed potential NR4A target genes involved in stress response, immune response, development and other functions. Our data show altered adipose tissue expression of the NR4As in obesity, suggesting that these stress responsive nuclear receptors may modulate pathogenic potential in humans.

Characterization of receptor-interacting protein RIP140 in the regulation of SF-1 responsive target genes.

Receptor-interacting protein (RIP) 140 interacts with several nuclear receptors, but its function in regulation of nuclear receptor action has been debated. Here we have examined the role of RIP140 in regulation of Steroidogenic factor-1 (SF-1)-dependent transcription. SF-1 interacts with RIP140 through its activation function-2 (AF-2) domain. Several domains of RIP140 interact directly with SF-1, but the carboxyl-terminal region containing 4 of its 9 LXXLL motifs showed the strongest SF-1 interaction. Coexpression of RIP140 and SF-1 in different cell types demonstrated that RIP140 acts as a potent corepressor of transcription from the SF-1 responsive cAMP regulatory sequence 2 (CRS2) element of the CYP17 gene and a variety of SF-1 responsive promoter genes. RIP140 also counteracted the stimulatory action of p160/SRC coactivators. The inhibitory effect of RIP140 was partially reversed by Trichostatin A, suggesting a role of histone deacetylase (HDAC) activity in RIP140-mediated repression of SF-1. Quantitation of endogenous coregulator mRNA levels revealed cell type specific differences that could affect the repressor action by overexpressed RIP140.

Recruitment of Coactivator Glucocorticoid Receptor Interacting Protein 1 to an Estrogen Receptor Transcription Complex Is Regulated by the 3′,5′-Cyclic Adenosine 5′-Monophosphate-Dependent Protein Kinase

Steroid receptor coactivators (SRCs), such as glucocorticoid receptor interacting protein 1 (GRIP1) are recruited to the DNA-bound nuclear receptors (NRs) and are also shown to enhance the gene transactivation by other transcription factors. In contrast to the two other members of the SRC family, SRC-1 and SRC-3/amplified in breast cancer 1, SRC-2/GRIP1 is regulated by the cAMP-dependent protein kinase [protein kinase A (PKA)] that stimulates its ubiquitination and degradation. In this report we demonstrate that COS-1 and MCF-7 cells treated with cAMP-elevating agents and 8-para-chlorophenylthio-cAMP for short periods of time showed an increase in GRIP1 coactivator function, whereas prolonged stimulation of the cAMP/PKA pathway led to a decline in GRIP1-mediated activation and protein levels. Furthermore, MCF-7 breast cancer cells were subjected to chromatin immunoprecipitation assays after stimulation of the cAMP/PKA pathway. cAMP/PKA initiated a rapid recruitment of GRIP1 to the endogenous estrogen receptor (ER)-alpha target pS2 gene promoter. In contrast to the estradiol-induced recruitment of GRIP1 to pS2, we observed an additional increase in GRIP1 recruitment on inhibition of the proteasome, suggesting that inhibition of GRIP1 degradation leads to accumulation at the pS2. Real-time PCR experiments confirmed that cAMP/PKA enhanced the expression of pS2. Moreover, confocal imaging of COS-1 cells transfected with yellow fluorescent protein-GRIP1 and cyan fluorescent protein-ERalpha revealed that PKA led to redistribution and colocalization of yellow fluorescent protein-GRIP1 and cyan fluorescent protein-ERalpha in subnuclear foci. In conclusion, these results suggest that activation of the cAMP/PKA pathway stimulates recruitment of GRIP1 to an ER-responsive gene promoter. The initial stimulation of GRIP1 coactivator function is followed by an increased turnover and subsequent degradation of GRIP1 protein.

cAMP-mediated regulation of HNF-4α depends on the level of coactivator PGC-1α

Hepatocyte nuclear factor-4 alpha (HNF-4alpha) is a member of the nuclear receptor superfamily with important roles in hepatic metabolism. Fasting induces the cAMP/protein kinase A (PKA)-signaling pathway. The mechanisms whereby cAMP regulates HNF-4alpha transcriptional activity are incompletely understood. We have therefore investigated the role of cAMP/PKA in regulation of HNF-4alpha in COS-1 cells and the hepatoma HepG2 cell line. cAMP/PKA inhibited the transcriptional activity of HNF-4alpha in COS-1 cells, whereas a stimulatory effect was observed in HepG2 cells. The cAMP-induced inhibition of HNF-4alpha in COS-1 cells was counteracted by overexpression of the nuclear receptor coactivator PGC-1alpha, and cAMP/PKA-dependent induction of the PGC1A gene in HepG2 cells seems to explain the cell specific differences. This was further supported by knock-down of PGC-1alpha in HepG2 cells, which abolished the stimulatory effect of PKA on HNF-4alpha transcriptional activity. Similar to the cAMP/PKA-mediated regulation of HNF-4alpha, overexpression of the cAMP-response element binding protein (CREB) inhibited the transcriptional activity of HNF-4alpha in COS-1 cells, regardless of cAMP/PKA activation and CREB phosphorylation. Moreover, activation of CREB by cAMP/PKA further stimulated HNF-4alpha transactivation in HepG2 cells. cAMP induced the expression of the HNF-4alpha target genes PCK1 and G6Pase in these cells. In conclusion, our results suggest that the level of PGC-1alpha determines whether the cAMP/PKA-pathway overall stimulates or inhibits HNF-4alpha transcriptional activation.

DIFFERENTIAL REGULATION OF SF-1-COFACTOR INTERACTIONS

The orphan nuclear receptor steroidogenic factor-1 (SF-1) plays pivotal roles in the development and function of steroidogenic organs. Here we describe the differential effect of protein kinase A (PKA) on coregulation of SF-1 dependent transcription by two p160 family members, p300/CBP co-integrator-associated protein (p/CIP) and transcription intermediary factor-2 (TIF2). Thus, whereas p/CIP-stimulated SF-1 dependent transcription is further potentiated by PKA, we show that activation of PKA leads to selective downregulation of TIF2 protein and a subsequent repression of TIF2 coactivator function. Using a yeast two-hybrid screen we also identified a novel zinc finger containing protein, which interacts with SF-1 via the AF-2 domain.

Emerging therapeutic targets in human acute myeloid leukemia (part 2) - bromodomain inhibition should be considered as a possible strategy for various patient subsets.

The recent advances in our understanding of leukemogenesis have clearly demonstrated that human acute myeloid leukemia is a heterogeneous malignancy, and several disease mechanisms should probably be regarded as possible therapeutic targets only for specific subsets of patients and not for acute myeloid leukemia in general. One promising strategy for epigenetic targeting is inhibition of the binding between bromodomain-containing transcription regulators and acetylated lysine residues on histones. This possible approach has been investigated especially for patients with 11q23 and chromosome 8 abnormalities. An alternative target is the histone methyltransferase COT1L. Major challenges for both approaches will be to clarify how these strategies should be combined with each other or with conventional chemotherapy, and whether their use should be limited to certain subsets of patients.

cAMP Response Element-Binding Protein Interacts With and Stimulates the Proteasomal Degradation of the Nuclear Receptor Coactivator GRIP1

The glucocorticoid receptor interacting protein (GRIP1) belongs to the p160 steroid receptor coactivator family that plays essential roles in nuclear receptor-dependent transcriptional regulation. Previously, we reported that the cAMP-dependent protein kinase (PKA) induces ubiquitination leading to degradation of GRIP1. Here we show that the cAMP response element-binding protein (CREB) downregulates GRIP1 and is necessary for the PKA-stimulated degradation of GRIP1, which leads to changes in the expression of a subset of genes regulated by estrogen receptor-α in MCF-7 breast cancer cells. Our data of domain-mapping and ubiquitination analyses suggest that CREB promotes the proteasomal breakdown of ubiquitinated GRIP1 through 2 functionally independent protein domains containing amino acids 347 to 758 and 1121 to 1462. We provide evidence that CREB interacts directly with GRIP1 and that CREB Ser-133 phosphorylation or transcriptional activity is not required for GRIP1 interaction and degradation. The basic leucine zipper domain (bZIP) of CREB is important for the interaction with GRIP1, and deletion of this domain led to an inability to downregulate GRIP1. We propose that CREB mediates the PKA-stimulated degradation of GRIP1 through protein-protein interaction and stimulation of proteasomal degradation of ubiquitinated GRIP1.

Protected sampling is preferable in bronchoscopic studies of the airway microbiome

The aim was to evaluate susceptibility of oropharyngeal contamination with various bronchoscopic sampling techniques. 67 patients with obstructive lung disease and 58 control subjects underwent bronchoscopy with small-volume lavage (SVL) through the working channel, protected bronchoalveolar lavage (PBAL) and bilateral protected specimen brush (PSB) sampling. Subjects also provided an oral wash (OW) sample, and negative control samples were gathered for each bronchoscopy procedure. DNA encoding bacterial 16S ribosomal RNA was sequenced and bioinformatically processed to cluster into operational taxonomic units (OTU), assign taxonomy and obtain measures of diversity. The proportion of Proteobacteria increased, whereas Firmicutes diminished in the order OW, SVL, PBAL, PSB (p<0.01). The alpha-diversity decreased in the same order (p<0.01). Also, beta-diversity varied by sampling method (p<0.01), and visualisation of principal coordinates analyses indicated that differences in diversity were smaller between OW and SVL and OW and PBAL samples than for OW and the PSB samples. The order of sampling (left versus right first) did not influence alpha- or beta-diversity for PSB samples. Studies of the airway microbiota need to address the potential for oropharyngeal contamination, and protected sampling might represent an acceptable measure to minimise this problem. Protected bronchoscopic sampling is most suitable for identification of a distinct airway microbiome http://ow.ly/qIIy30eqB9M

Cyclin C interacts with steroid receptor coactivator 2 and upregulates cell cycle genes in MCF-7 cells.

Steroid receptor coactivator 2 (SRC-2) is a coactivator that regulates nuclear receptor activity. We previously reported that SRC-2 protein is degraded through the action of cAMP-dependent protein kinase A (PKA) and cAMP response element binding protein (CREB). In the study presented here, we aimed to identify proteins that interact with and thereby regulate SRC-2. We isolated cyclin C (CCNC) as an interacting partner with the SRC-2 degradation domain aa 347-758 in a yeast two-hybrid assay and confirmed direct interaction in an in vitro assay. The protein level of SRC-2 was increased with CCNC overexpression in COS-1 cells and decreased with CCNC silencing in COS-1 and MCF-7 cells. In a pulse-chase assay, we further show that silencing of CCNC resulted in a different SRC-2 degradation pattern during the first 6 h after the pulse. Finally, we provide evidence that CCNC regulates expression of cell cycle genes upregulated by SRC-2. In conclusion, our results suggest that CCNC temporarily protects SRC-2 against degradation and this event is involved in the transcriptional regulation of SRC-2 cell cycle target genes.