Two Distinctive POMC Promoters Modify Gene Expression in Cushing Disease.

CONTEXT: Mechanisms underlying pituitary corticotroph adenoma adrenocorticotropin (ACTH) production are poorly understood, yet circulating ACTH levels closely correlate with adenoma phenotype and clinical outcomes. OBJECTIVE: We characterized the 5' ends of proopiomelanocortin (POMC) gene transcripts, which encode the precursor polypeptide for ACTH, in order to investigate additional regulatory mechanisms of POMC gene transcription and ACTH production. METHODS: We examined 11 normal human pituitary tissues, 32 ACTH-secreting tumors, as well as 6 silent corticotroph adenomas (SCAs) that immunostain for but do not secrete ACTH. RESULTS: We identified a novel regulatory region located near the intron 2/exon 3 junction in the human POMC gene, which functions as a second promoter and an enhancer. In vitro experiments demonstrated that CREB binds the second promoter and regulates its transcriptional activity. The second promoter is highly methylated in SCAs, partially demethylated in normal pituitary tissue, and highly demethylated in pituitary and ectopic ACTH-secreting tumors. In contrast, the first promoter is demethylated in all POMC-expressing cells and is highly demethylated only in pituitary ACTH-secreting tumors harboring the ubiquitin-specific protease 8 (USP8) mutation. Demethylation patterns of the second promoter correlate with clinical phenotypes of Cushing disease. CONCLUSION: We identified a second POMC promoter regulated by methylation status in ACTH-secreting pituitary tumors. Our findings open new avenues for elucidating subcellular regulation of the hypothalamic-pituitary-adrenal axis and suggest the second POMC promoter may be a target for therapeutic intervention to suppress excess ACTH production.

Transcriptome analysis of the effect of diosgenin on autoimmune thyroiditis in a rat model.

In a mouse model of Graves' disease (GD), diosgenin has been shown to have a therapeutic effect on GD by alleviating goitre. However, research on the effect of diosgenin on autoimmune thyroiditis (AIT) is lacking. In this study, transcriptomics was used to comprehensively analyse the protective effect of diosgenin against AIT in rats and the possible mechanism. The results showed that in the diosgenin-intervention group, compared to the model group, the expression of serum triiodothyronine, thyroxine, free triiodothyronine, and free thyroxine was decreased and that of thyroid-stimulating hormone was increased; these changes were accompanied by the downregulation of thyroglobulin, TSH receptor antibody and thyroid peroxidase expression in serum. Furthermore, transcriptome detection, RT-qPCR and immunohistochemistry verification revealed that in thyroid tissue, the relative mRNA and protein expression of cyclic adenosine 3',5'-monophosphate (cAMP), protein kinase A (PKA) and cAMP response element-binding protein (Creb) were increased and the mRNA expression of S100 calcium-binding protein A9 (S100A9) was decreased in the diosgenin groups. In summary, diosgenin alleviates the development of AIT, possibly via the activation of the cAMP/PKA/Creb pathway and downregulation of S100A9 gene expression.

CREB3L1 overexpression as a potential diagnostic marker of Philadelphia chromosome-negative myeloproliferative neoplasms.

Discrimination of Philadelphia-negative myeloproliferative neoplasms (Ph-MPNs) from reactive hypercytosis and myelofibrosis requires a constellation of testing including driver mutation analysis and bone marrow biopsies. We searched for a biomarker that can more easily distinguish Ph-MPNs from reactive hypercytosis and myelofibrosis by using RNA-seq analysis utilizing platelet-rich plasma (PRP)-derived RNAs from patients with essential thrombocythemia (ET) and reactive thrombocytosis, and CREB3L1 was found to have an extremely high impact in discriminating the two disorders. To validate and further explore the result, expression levels of CREB3L1 in PRP were quantified by reverse-transcription quantitative PCR and compared among patients with ET, other Ph-MPNs, chronic myeloid leukemia (CML), and reactive hypercytosis and myelofibrosis. A CREB3L1 expression cutoff value determined based on PRP of 18 healthy volunteers accurately discriminated 150 driver mutation-positive Ph-MPNs from other entities (71 reactive hypercytosis and myelofibrosis, 6 CML, and 18 healthy volunteers) and showed both sensitivity and specificity of 1.0000. Importantly, CREB3L1 expression levels were significantly higher in ET compared with reactive thrombocytosis (P < .0001), and polycythemia vera compared with reactive erythrocytosis (P < .0001). Pathology-affirmed triple-negative ET (TN-ET) patients were divided into a high- and low-CREB3L1-expression group, and some patients in the low-expression group achieved a spontaneous remission during the clinical course. In conclusion, CREB3L1 analysis has the potential to single-handedly discriminate driver mutation-positive Ph-MPNs from reactive hypercytosis and myelofibrosis, and also may identify a subgroup within TN-ET showing distinct clinical features including spontaneous remission.

Relationship between normal weight obesity and mild cognitive impairment is reflected in cognitive-related genes in human peripheral blood mononuclear cells.

AIM: Obesity contributes to the development of mild cognitive impairment, but the potential role of normal weight obesity in this disease has not been explored in humans. The aim of the study was to reveal the relationship between normal weight obesity and mild cognitive impairment in elderly individuals. METHODS: This study consisted of 360 patients with amnestic mild cognitive impairment and 360 cognitively normal controls. Normal weight obesity was defined as having metabolic syndrome but a normal weight. Metabolic health meant having no metabolic syndrome. Reverse transcription quantitative real-time polymerase chain reaction was adopted to measure the messenger RNA expression of four cognitive-related genes (amyloid precursor protein, cyclic adenosine monophosphate-responsive element-binding protein 1, sortilin-related receptor 1, and synapsin I) in peripheral blood mononuclear cells. RESULTS: Normal weight obesity was related to a higher risk of amnestic mild cognitive impairment (odds ratio = 3.14, 95% confidence interval: 2.13-4.60). In the patients, the expression of each gene in the peripheral blood mononuclear cells was linearly related to Mini-Mental State Examination and Montreal Cognitive Assessment scores (P < 0.05). The expression of these genes in the patients with metabolic health deviated from the normal levels found in the controls (P < 0.05), and the deviations were more significant in the patients with normal weight obesity (P < 0.05). CONCLUSION: Normal weight obesity may be a potential risk factor for amnestic mild cognitive impairment in elderly. This relationship was reflected in the abnormal expression of several cognitive-related genes in peripheral blood mononuclear cells.

Clinical significance of serum MicroRNA-203 in patients with acute myeloid leukemia.

This study aimed to detect serum miR-203 expression levels in AML and explore its potential clinical significance. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to measure the serum miR-203 levels in 134 patients with AML and 70 healthy controls. The results demonstrated that serum miR-203 expression was significantly reduced in AML patients compared with healthy controls. Receiver operating characteristic curve (ROC) analysis revealed miR-203 could distinguish AML cases from normal controls. Low serum miR-203 levels were associated with worse clinical features, as well as poorer overall survival and relapse free survival of AML patients. Moreover, multivariate analysis confirmed low serum miR-203 expression to be an independent unfavorable prognostic predictor for AML. The bioinformatics analysis showed that the downstream genes and pathways of miR-203 was closely associated with tumorigenesis. Downregulation of miR-203 in AML cell lines upregulated the expression levels of oncogenic promoters such as CREB1, SRC and HDAC1. Thus, these findings demonstrated that serum miR-203 might be a promising biomarker for the diagnosis and prognosis of AML.

Neuroprotection of reduced thyroid hormone with increased estrogen and progestogen in postpartum depression.

Background: Postpartum depression (PPD) is a common serious mental health problem. Recent studies have demonstrated that hormone therapy serves as a promising therapeutic approach in managing PPD. The present study aims at exploring the role of thyroid hormone (TH), estrogen and progestogen in patients with PPD.Methods: Initially, PPD patients were enrolled and a PPD mouse model was established. The serum levels of estradiol (E2), progesterone (P), triiodothyronine (T3), thyroxine (T4), free triiodothyronine (FT3), free thyroxine (FT4), and thyroid-stimulating hormone (TSH) were subsequently measured. Next, in order to identify the effects of TH, estrogen and progestogen on PPD progression, mice were administrated with E2, P, contraceptives (CA), Euthyrox and methimazole (MMI). Besides, the body weight, activities, basolateral amygdala (BLA) neuron cell structure and the related gene expression of mice were analyzed.Results: The PPD patients and the mice showed elevated serum levels of T3, T4, FT3 and FT4 along with diminished E2, P and TSH levels. In the mice administered with a combination of E2, P, and MMI, decreased TH and increased estrogen and progestogen were detected, which resulted in increased body weight, normal activities, and BLA neuron cell structure. Moreover, brain-derived neurotrophic factor (BDNF) and cAMP-responsive element-binding protein (CREB) were both up-regulated in PPD mice administrated with a combination of E2, P, and MMI, which was accompanied by decreased TH and elevated estrogen and progestogen.Conclusion: Taken together, reduced TH combined with enhanced estrogen and progestogen confers neuroprotection in PPD, highlighting a potential target in prevention and treatment of PPD.

Research on changes in cognitive function, ß-amyloid peptide and neurotrophic factor in stroke patients.

OBJECTIVE: To investigate the changes as well as the related mechanism in cognitive function and levels of serum ß-amyloid peptide (Aß) and brain-derived neurotrophic factor (BDNF) in stroke patients. PATIENTS AND METHODS: A total of 30 patients with acute stroke treated in our hospital from June 2015 to September 2016 were selected as stroke group, while 30 volunteers during the same period were enrolled as control group. Changes in cognitive function of patients were evaluated using the Montreal Cognitive Assessment (MoCA) and mini-mental state examination (MMSE) before and after the treatment. At the same time, the concentrations of serum Aß1-40 and BDNF were detected, and their correlations with the MMSE score were analyzed. Finally, levels of serum cyclic adenosine monophosphate (cAMP) and phosphorylated-cAMP-response element binding protein (p-CREB), and the phosphorylation level of Tau protein were detected by Western blotting. RESULTS: MoCA and MMSE scores of patients in stroke group were significantly lower than those in control group (p < 0.01), and the scores were significantly higher in stroke patients after treatment than those before treatment (p < 0.01). Compared with those in control group, the serum Aß1-40 concentration in patients in stroke group was significantly increased (p < 0.01), but the BDNF level was significantly decreased (p < 0.01). Compared with those before treatment, the serum Aß1-40 concentration in patients was significantly decreased after treatment (p < 0.01), but the BDNF concentration was significantly increased (p < 0.01). Correlation analysis showed that the MMSE score was negatively correlated with the concentration of Aß1-40 (r2 = 0.764, p < 0.01), but positively related to the level of BDNF (r2 = 0.827, p < 0.01). Compared with those in control group, the content of serum cAMP and p-CREB in stroke patients was significantly decreased (p < 0.01), but the expression of p-Tau was statistically increased (p < 0.01). CONCLUSIONS: The cognitive function in stroke patients is impaired, with the rising content of serum Aß1-40 and reduction of BDNF, the mechanism of which is related to the decrease of cAMP and p-CREB and the increase of p-Tau. This provides a theoretical basis for searching the new therapeutic targets and new drugs for stroke.

Molecular hydrogen reduces acute exercise-induced inflammatory and oxidative stress status.

Physical exercise induces inflammatory and oxidative markers production in the skeletal muscle and this process is under the control of both endogenous and exogenous modulators. Recently, molecular hydrogen (H2) has been described as a therapeutic gas able to reduced oxidative stress in a number of conditions. However, nothing is known about its putative role in the inflammatory and oxidative status during a session of acute physical exercise in sedentary rats. Therefore, we tested the hypothesis that H2 attenuates both inflammation and oxidative stress induced by acute physical exercise. Rats ran at 80% of their maximum running velocity on a closed treadmill inhaling either the H2 gas (2% H2, 21% O2, balanced with N2) or the control gas (0% H2, 21% O2, balanced with N2) and were euthanized immediately or 3 h after exercise. We assessed plasma levels of inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6] and oxidative markers [superoxide dismutase (SOD), thiobarbituric acid reactive species (TBARS) and nitrite/nitrate (NOx)]. In addition, we evaluated the phosphorylation status of intracellular signaling proteins [glycogen synthase kinase type 3 (GSK3α/ß) and the cAMP responsive element binding protein (CREB)] that modulate several processes in the skeletal muscle during exercise, including changes in exercise-induced reactive oxygen species (ROS) production. As expected, physical exercise increased virtually all the analyzed parameters. In the running rats, H2 blunted exercise-induced plasma inflammatory cytokines (TNF-α and IL-6) surges. Regarding the oxidative stress markers, H2 caused further increases in exercise-induced SOD activity and attenuated the exercise-induced increases in TBARS 3 h after exercise. Moreover, GSK3α/ß phosphorylation was not affected by exercise or H2 inhalation. Otherwise, exercise caused an increased CREB phosphorylation which was attenuated by H2. These data are consistent with the notion that H2 plays a key role in decreasing exercise-induced inflammation, oxidative stress, and cellular stress.

Cyclic adenosine monophosphate responsive element binding protein in post-traumatic stress disorder.

OBJECTIVES: The cyclic adenosine monophosphate responsive element binding (CREB) protein is a transcription factor involved in different neural processes, such as learning, neuroplasticity and the modulation of stress response. Alterations in the CREB pathway have been observed in the brains and lymphocytes of patients affected by depression and alcohol abuse. Given the lack of information, our study aimed at investigating the levels of total and activated CREB protein in lympho-monocytes of 20 drug-free patients suffering from post-traumatic stress disorders (PTSD), as compared with 20 healthy control subjects. METHODS: Blood samples were collected from patients and healthy control subjects on the same time and lympho-monocytes were isolated according to standardized methods. CREB protein levels and activation were measured by means of immunoenzymatic techniques. RESULTS: The results showed that PTSD patients had statistically lower levels of total CREB protein in lympho-monocytes than healthy control subjects. On the contrary, no difference in the activated CREB protein was detected. CONCLUSIONS: These findings, albeit preliminary, would suggest that the CREB pathway might be involved in the pathophysiology of PTSD. Future studies should clarify if specific PTSD symptom clusters might be related to the CREB pathway.

A pilot study of an oral phosphodiesterase inhibitor (apremilast) for atopic dermatitis in adults.

OBJECTIVE: To investigate the preliminary safety and efficacy of apremilast, an oral phosphodiesterase 4 inhibitor, for atopic dermatitis. DESIGN: This investigator-initiated, open-label pilot study evaluated 2 doses of apremilast in patients with atopic dermatitis. Differential gene analysis was performed from peripheral whole blood using data before and after treatment. SETTING: University-based dermatology clinical research unit. PATIENTS: Sixteen adult patients with atopic dermatitis. INTERVENTION: A specific phosphodiesterase 4 inhibitor, apremilast. MAIN OUTCOME MEASURES: The primary outcome was incidence of adverse events. Secondary outcomes included the differences in pruritus, Dermatology Life Quality Index (DLQI), and Eczema Area and Severity Index (EASI) scores between the baseline visit and end-ofstudy visit for each cohort. RESULTS: The group receiving apremilast, 20 mg twice daily, displayed a significant reduction from baseline of pruritus (P=.02) and the DLQI (P=.003) at 3 months. The group receiving apremilast, 30 mg twice daily, displayed a significant reduction of the EASI (P=.008) and the DLQI (P=.01) at 3 months. At 6 months, there was a significant reduction of the EASI (P=.002), the visual analog scale (P=.03), and the DLQI (P=.03). Gene ontologic analyses comparing baseline with samples during treatment revealed alterations in immune response pathways, especially those related to cyclic adenosine monophosphate­mediated signaling. CONCLUSIONS: These results support further development of apremilast for treatment of atopic dermatitis. Larger randomized controlled studies are needed to more adequately evaluate both safety and efficacy. Limitations include the small sample size and absence of a control. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01393158.

Calcium-activated pathways and oxidative burst mediate zymosan-induced signaling and IL-10 production in human macrophages.

Outside of the TLR paradigm, there is little understanding of how pathogen recognition at the cell surface is linked to functional responses in cells of the innate immune system. Recent work in this area demonstrates that the yeast particle zymosan, by binding to the beta-glucan receptor Dectin-1, activates an ITAM-Syk-dependent pathway in dendritic cells, which is required for optimal cytokine production and generation of an oxidative burst. It remains unclear how activation of Syk is coupled to effector mechanisms. In human macrophages, zymosan rapidly activated a calcium-dependent pathway downstream of Dectin-1 and Syk that led to activation of calmodulin-dependent kinase II and Pyk2. Calmodulin-dependent kinase and Pyk2 transduced calcium signals into activation of the ERK-MAPK pathway, CREB, and generation of an oxidative burst, leading to downstream production of IL-10. These observations identify a new calcium-mediated signaling pathway activated by zymosan and link this pathway to both inflammatory and anti-inflammatory responses in macrophages.

Smoking behaviour is associated with expression and phosphorylation of CREB in human buffy coat.

Nicotine induces various acute und chronic pharmacological effects which can be long lasting and might lead to nicotine dependence. Nicotinic acetylcholine receptors (nAChRs) are involved in nicotine-induced phosphorylation of CREB (cyclic AMP response element-binding protein) in PC12h cells. Several studies, mainly done in animal models, report that CREB plays a role in anxiety, memory and substance abuse as well as in affective disorders. Information regarding nicotine effects on gene expression in humans in vivo is rare. The aim of our study was to determine whether or not there are differences between smokers and non-smoking controls in terms of CREB expression and phosphorylation in human buffy coat. Comparing 32 smokers with 76 non-smoking controls we found significantly elevated relative (p=0.043) and absolute (p=0.040) CREB phosphorylation in the blood of smokers who had smoked two cigarettes in the past 6 h. In contrast, the score of the State and Trait Anxiety Inventory, total-CREB and mRNA-CREB were not significantly different. Multiple regression analysis revealed a significant relation between the number of cigarettes smoked daily (R2=0.143, p=0.023), the Fagerström Test for Nicotine Dependence score (R2=0.145, p=0.022) and the expression of CREB. Moreover, in accord with previously published data our analysis suggests gender and age as factors that significantly influence expression and phosphorylation of CREB. It appears that human buffy coat is suitable for studying pharmacological effects of substances such as nicotine on selected signal transduction pathways in humans in vivo. This kind of study may be helpful for translating findings from animal models and cell cultures.