Nuclear receptor phosphorylation in xenobiotic signal transduction.

Nuclear pregnane X receptor (PXR, NR1I2) and constitutive active/androstane receptor (CAR, NR1I3) are nuclear receptors characterized in 1998 by their capability to respond to xenobiotics and activate cytochrome P450 (CYP) genes. An anti-epileptic drug, phenobarbital (PB), activates CAR and its target CYP2B genes, whereas PXR is activated by drugs such as rifampicin and statins for the CYP3A genes. Inevitably, both nuclear receptors have been investigated as ligand-activated nuclear receptors by identifying and characterizing xenobiotics and therapeutics that directly bind CAR and/or PXR to activate them. However, PB, which does not bind CAR directly, presented an alternative research avenue for an indirect ligand-mediated nuclear receptor activation mechanism: phosphorylation-mediated signal regulation. This review summarizes phosphorylation-based mechanisms utilized by xenobiotics to elicit cell signaling. First, the review presents how PB activates CAR (and other nuclear receptors) through a conserved phosphorylation motif located between two zinc fingers within its DNA-binding domain. PB-regulated phosphorylation at this motif enables nuclear receptors to form communication networks, integrating their functions. Next, the review discusses xenobiotic-induced PXR activation in the absence of the conserved DNA-binding domain phosphorylation motif. In this case, phosphorylation occurs at a motif located within the ligand-binding domain to transduce cell signaling that regulates hepatic energy metabolism. Finally, the review delves into the implications of xenobiotic-induced signaling through phosphorylation in disease development and progression.

Glucocorticoid receptor and serum- and glucocorticoid-induced kinase-1 in esophageal adenocarcinoma and adjacent Barrett's esophagus.

Barrett's esophagus (BE) is a consequence of gastroesophageal reflux disease and is predisposed to esophageal adenocarcinoma (EAC). EAC is an exemplar model of inflammation-associated cancer. Glucocorticoids suppress inflammation through glucocorticoid receptor (GR) and serum- and glucocorticoid-induced kinase-1 (Sgk1) expressions. Therefore, we immunolocalized GR and Sgk1 in EAC and the adjacent BE tissues and studied their association with clinical disease course in 87 patients with EAC who underwent surgical resection (N = 58) or endoscopic submucosal dissection (N = 29). Low GR and Sgk1 expressions in adjacent BE tissues were associated with adverse clinical outcomes (P = 0.0008 and 0.034, respectively). Patients with low Sgk1 expression in EAC cells exhibited worse overall survival (P = 0.0018). In multivariate Cox regression analysis, low GR expression in the adjacent nonmalignant BE tissues was significantly associated with worse overall survival (P = 0.023). The present study indicated that evaluation of GR and Sgk1 expressions in both the EAC cells and adjacent nonmalignant BE tissues could help to predict clinical outcomes following endoscopic and surgical treatments. In particular, the GR status in BE tissues adjacent to EAC was an independent prognostic factor.

A phosphorylation-deficient mutant of retinoid X receptor α at Thr 167 alters fasting response and energy metabolism in mice.

Retinoid X receptor α (RXRα) has a conserved phosphorylation motif at threonine 162 (humans) and threonine 167 (mice) within the DNA-binding domain. Here we have generated RXRα knock-in mice (RxrαT167A) bearing a single mutation of Thr 167 to alanine and examined the roles of Thr 167 in the regulation of energy metabolism within adipose, muscle, and liver tissues. RxrαT167A mice exhibited down-regulation of metabolic pathways converting glucose to fatty acids, such as acetyl-CoA carboxylase in the white adipose tissue (WAT) and ATP citrate lyase in the muscle. They also reduced gene expression for genes related to fatty acid catabolism and triglyceride synthesis in WAT and controlled heat factors such as adrenergic receptor ß1 in muscles. In contrast, hepatic gluconeogenic pathways and synthetic pathways related to fatty acids remained unaffected by this mutation. Expression of multiple genes that were affected by the Thr 167 mutation in adipose tissue exhibited clear response to LG100268, a synthetic RXR agonist. Thus, the altered gene expression in mutant mice adipose appeared to be a direct effect of RXRα Thr 167 mutation and by some secondary effect of the mutation. Blood glucose levels remained normal in RxrαT167A during feeding, as observed with RXRα wild-type mice. However, RxrαT167A mice exhibited an attenuated decrease of blood glucose levels that occurred after fasting. This attenuation correlated with a concomitant down-regulation of lipid metabolism in WAT and was associated with RXRα phosphorylation at Thr 167. Thus, Thr 167 enabled RXRα to coordinate these three organs for regulation of energy metabolism and maintenance of glucose homeostasis.

Effects of cyclophosphamide on the prognosis of Japanese patients with renal vasculitis associated with anti-neutrophil cytoplasmic antibody-positive microscopic polyangiitis.

BACKGROUND: The aim of this study was to determine the efficacy of cyclophosphamide (CY) on anti-neutrophil cytoplasmic antibody (ANCA)-positive microscopic polyangiitis (MPA) with renal involvement in Japanese patients. METHODS: Eighty-two patients with newly diagnosed ANCA-positive MPA were enrolled in this retrospective study. Patients were divided into two groups based on whether they received combination therapy with a corticosteroid (CS) plus CY (CY group) or CS alone or with other therapies (non-CY group). The primary outcome was defined as the combination of death and end-stage renal disease (ESRD). RESULTS: The CY and non-CY groups included 29 and 53 patients, respectively. In the non-CY group, 31 patients were treated with CS alone, and 22 with a combination of CS and other therapeutics. The percentage of males and mean Birmingham vasculitis activity scores were higher in the CY group than those in the non-CY group, but other factors such as age, serum creatinine, serum albumin, or CRP at baseline were equivalent in the two groups. No differences were observed in remission rates using induction therapy for the two groups. However, the survival rate 5 years after induction therapy was lower in the CY group than in the non-CY group (0.50 vs. 0.73; P = 0.041), although the hazard ratio of CY for the primary outcome adjusted for all confounding factors was 1.321 [95 % confidence interval (CI), 0.662-2.637; P = 0.171]. CONCLUSIONS: CY may not have an additive effect on induction therapy with CS for Japanese patients with renal vasculitis associated with ANCA-positive MPA.

[Clinical features and pathophysiology of acute esophageal mucosal lesion].

Acute esophageal mucosal lesions (AEMLs) are categorized into black esophagitis (type B) and non-black esophagitis (type NB) on endoscopy. To clarify the distinct pathophysiology, we compared the clinical features and hematological findings at onset among 17 patients with type B esophagitis and 6 patients with type NB esophagitis. In type B esophagitis, time to endoscopy after onset was significantly shorter, and blood levels of lactate, urea nitrogen, creatinine, and glucose were higher than in type NB esophagitis. However, there were no significant intergroup differences in the incidences of other predisposing factors, such as diabetic ketoacidosis or esophageal hernias. These findings suggest that AEMLs are caused by acid reflux and peripheral vascular insufficiency, the latter being more associated with type B esophagitis by its etiology. In addition, blood lactate may indicate the severity of AEML, leading to black esophagitis.

Postoperative drainage with one chest tube is appropriate for pulmonary lobectomy: a randomized trial.

To expand postoperative residual lungs after pulmonary lobectomy, thoracic drainage with two chest tubes has been recommended. Several studies recently demonstrated that postoperative drainage with one chest tube (PD1) was as safe as that with two chest tubes (PD2). However, most of the patients in those studies underwent lobectomy by standard thoracotomy. Although the number of pulmonary lobectomies by video-assisted thoracic surgery (VATS) has been increasing in recent years, there have been no reports that compared PD1 with PD2 after pulmonary lobectomy, including that by VATS. To elucidate whether postoperative management with PD1 is as safe as that with PD2, we conducted a randomized controlled trial. Lung cancer patients who underwent lobectomies with mediastinal nodal dissection in our hospital were assigned to one of two groups: one chest tube placed in PD1 group and two chest tubes placed in PD2 group. A total of 108 patients were registered in the study. There were no significant differences in the age, gender, pathological stage or histological type between two groups. Since the residual lung expansion was good in both groups, there were no patients who needed thoracentesis. There were no significant differences in the number of cases with pleurodesis, the amount/duration of drainage or the pain of the patients between two groups. In conclusion, since PD1 has advantages in saving cost and time and in low risk of transcutaneous infection, PD1 is appropriate after pulmonary lobectomy by VATS and by open thoracotomy.

Cardiac Malignant Lymphoma with Diffuse Extension to the Left Ventricle: A Case Report.

Malignant cardiac lymphoma is rare and commonly involves nodules on the right side of the heart. We herein report a case of malignant cardiac lymphoma with diffuse extension into the left ventricle. The patient was a woman in her 60s who complained of dyspnea and malaise. Echocardiography revealed left ventricular hypertrophy (LVH), and magnetic resonance imaging revealed diffuse contrast enhancement on delayed contrast. Cardiac catheterization and a myocardial biopsy suggested heart failure due to cardiac malignant lymphoma, and diastolic dysfunction was mild despite LVH. The patient underwent chemotherapy, and her cardiac function improved and was maintained.

Advantages of diffusion-weighted imaging over positron emission tomography-computed tomography in assessment of hilar and mediastinal lymph node in lung cancer.

BACKGROUND: The significance of diffusion-weighted imaging (DWI) is uncertain for the diagnosis of nodal involvement. The purpose of this study was to examine diagnostic capability of DWI compared with PET-CT for nodal involvement of lung cancer. METHODS: A total of 160 lung cancers (114 adenocarcinomas, 36 squamous cell carcinomas, and 10 other cell types) were analyzed in this study. DWI and PET-CT were performed preoperatively. RESULTS: The optimal cutoff values to diagnose metastatic lymph nodes were 1.70 × 10(-3) mm(2)/s for ADC value and 4.45 for SUVmax. DWI correctly diagnosed N staging in 144 carcinomas (90 %) but incorrectly diagnosed N staging in 16 (10 %) [3 (1.9 %) had overstaging, 13 (8.1 %) had understaging]. PET-CT correctly diagnosed N staging in 133 carcinomas (83.1 %) but incorrectly diagnosed N staging in 27 (16.8 %) [4 (2.5 %) had overstaging, 23 (14.4 %) had understaging]. Sensitivity, accuracy, and negative predictive value for N staging by DWI were significantly higher than those by PET-CT. Of the 705 lymph node stations examined, 61 had metastases, and 644 did not. The maximum diameter of metastatic lesions in lymph nodes were 3.0 ± 0.9 mm in 21 lymph node stations not detected by either DWI or PET-CT: 7.2 ± 4.1 mm in 39 detected by DWI, and 11.9 ± 4.1 mm in 24 detected by PET-CT. There were significant differences among them. The sensitivity (63.9 %) for metastatic lymph node stations by DWI was significantly higher than that (39.3 %) by PET-CT. The accuracy (96.2 %) for all lymph node stations by DWI was significantly higher than that (94.3 %) by PET-CT. CONCLUSIONS: DWI has advantages over PET-CT in diagnosing malignant from benign lymph nodes of lung cancers.

[Retrospective analysis concerning AFP-producing gastric cancer].

The α-fetoprotein(AFP)-producing gastric cancer is a group of gastric cancers with poor prognosis because of its rapid growth and aggressive metastatic character. We examined AFP-producing gastric cancer in our department from 2008 to 2010. Of 12 patients studied, the median of the AFP level was 16, 038(96. 1-167, 360)ng/mL. All patients had liver metastasis. Four patients were ECOG performance status(PS)3, and were unable to receive chemotherapy. Eight patients received chemotherapy. Two cases who received cisplatin+paclitaxel(CDDP+PTX)therapy showed partial response(PR). Median survival time was 5. 6 months. Compared to AFP non-producing gastric cancer, this disease is definitely considered to have a poorer prognosis. The clinical effect and survival time seemed to have a relative correlation. PR and SD groups tend to have longer survival. Those with a decline of serum LDH levels at the first three weeks after chemotherapy have a strong tendency to be PR and SD(p=0. 11). Changing the serum LDH level may enable us to estimate the clinical effect while still in the early stages of chemotherapy.

Constitutive androstane receptor-responsive elements for mouse Cyp1a2 transcriptional activation induced by constitutive androstane receptor ligands.

The mouse cytochrome P450 1A2 (Cyp1a2) gene is one of the constitutive androstane receptor (CAR, NR1I3) activator-inducible genes, and the regions involved in induction were examined herein. A reporter gene assay indicated the involvement of the -0.2-kb region in the induction of transcriptional activation by the mouse CAR agonist ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). Some putative nuclear receptor-binding elements were identified in this region, and mutations in the elements located at -160/-155 or -153/-148 abolished this induction. An electrophoretic mobility shift assay demonstrated that a fragment comprised of three elements was capable of binding to the CAR/retinoid X receptor alpha (RXRα) heterodimer. The three elements comprise the two elements indicated above and one located at -146/-141. A chromatin immunoprecipitation assay confirmed CAR binding to the region including these elements in chromatin after treatment with TCPOBOP. These results indicate that mouse Cyp1a2 is the direct target of CAR, and binding of the CAR/RXRα heterodimer to the newly identified region in the promoter may be involved in transcriptional activation. Binding motifs were estimated as ER1 (everted repeat with a spacing of 1 nucleotide, -160/-155 and -153/-148) and ER8 (everted repeat with a spacing of 8 nucleotides, formed with -160/-155 and -146/-141).

Phosphorylation of nuclear receptors: Novelty and therapeutic implications.

Nuclear receptors (NR) collectively regulate several biological functions in various organs. While NRs can be characterized by activation of the transcription of their signature genes, they also have other diverse roles. Although most NRs are directly activated by ligand binding, which induces cascades of events leading to gene transcription, some NRs are also phosphorylated. Despite extensive investigations, primarily focusing on unique phosphorylation of amino acid residues in different NRs, the role of phosphorylation in the biological activity of NRs in vivo has not been firmly established. Recent studies on the phosphorylation of conserved phosphorylation motifs within the DNA- and ligand-binding domains confirmed has indicated the physiologically relevance of NR phosphorylation. This review focuses on estrogen and androgen receptors, and highlights the concept of phosphorylation as a drug target.

A randomized controlled trial on the efficacy of thoracic CT screening for lung cancer in non-smokers and smokers of <30 pack-years aged 50-64 years (JECS study): research design.

In order to assess the efficacy of lung cancer screening using low-dose thoracic computed tomography, compared with chest roentgenography, in people aged 50-64 years with a smoking history of <30 pack-years, a randomized controlled trial is being conducted in Japan. The screening methods are randomly assigned individually. The duration of this trial is 10 years. In the intervention arm, low-dose thoracic computed tomography is performed for each participant in the first and the sixth years. In the control arm, chest roentgenography is performed for each participant in the first year. The participants in both arms are also encouraged to receive routine lung cancer screening using chest roentgenography annually. The interpretation of radiological findings and the follow-up of undiagnosed nodules are to be carried out according to the guidelines published in Japan. The required sample size is calculated to be 17 500 subjects for each arm.

Proliferative glomerulonephritis with monoclonal IgM deposits without Waldenström's macroglobulinemia: case report and review of the literature.

A 38-year-old man was presented with nephrotic syndrome associated with hematuria and mild hypocomplementemia. Renal biopsy revealed lobular mesangial proliferation, thickened capillary walls, and intraluminal protein thrombi. Immunofluorescence showed marked and mild depositions of immunoglobulin (Ig) M heavy chains and complement C3, respectively, in a peripheral lobular pattern. On light chain staining, only kappa (κ) light and IgM heavy chains showed a similar pattern. Electron microscopy showed fine granular electrondense deposits in subendothelial areas and numerous globular deposits (varying size and density) in glomerular capillary lumens. Serum levels of Ig κ, but not of free κ, light chains were significantly increased. Bone marrow aspiration revealed a normocellular marrow. Waldenström's macroglobulinemia and cryoglobulinemia were ruled out. Clinically, steroid therapy was ineffective and proteinuria persisted. This report demonstrates that glomerular deposition of monoclonal IgM-κ can produce membranoproliferative- like changes in the glomeruli. This condition may be recognized as proliferative glomerulonephritis with monoclonal IgM deposits similar to the recently recognized proliferative glomerulonephritis with monoclonal IgG deposits.

κ I light chain AL amyloidosis presenting with rapidly progressive renal and hepatic failure with unusual renal amyloid distribution.

A 65-year-old man suffering from generalized edema and jaundice was admitted to our hospital. Laboratory findings revealed marked renal dysfunction with heavy proteinuria as well as liver dysfunction with severe obstructive jaundice. On renal biopsy, the diagnosis of AL amyloidosis associated with κ I light chain was made. Interestingly, amyloid deposits were restricted to the glomeruli. Although hemodialysis was initiated, the patient died due to further deterioration of hepatic function. On autopsy, severe intrahepatic cholestasis was observed, and there was marked deposition of AL amyloid in the liver. Literature reviews showed that rapidly progressive renal failure is common in AL amyloidosis patients who presented with acute hepatic failure due to severe intrahepatic cholestasis. However, the detailed renal pathology in this condition has not been documented. The present case is very interesting because rapidly progressive renal and hepatic failure was simultaneously observed, and renal amyloid deposition was restricted to the glomeruli.

[Screening for lung cancer: present and future].

A certified method for lung cancer screening in Japan is the combination of chest X-ray and sputum cytology. The chest Xray examination is intended primarily for the detection of peripheral-type lung cancer. Interpretation of the films should be performed by two different physicians, and the films of screenees suspected to have abnormal shadow should be compared to the same screenee's films from previous screening visits. Sputum cytology is conducted for heavy smokers, and is useful for early detection of central lung cancer. The efficacy of this lung cancer screening method has been shown in several case control studies. There are some problems to solve i. e., a low rate of attendance and inadequate quality control. Low-dose thoracic CT screening is performed with an exposure within a single breath hold, and its interpretation can be conducted with films, CRT, or a LCD monitor. Even when taken at low doses, the radiation exposure dose is large compared to a chest X-ray, being about 3-10 times greater than the absorbed dose and 20-40 times greater than the effective dose. Since the radiation dose in a usual clinical condition is much higher, the clinical condition is not recommended for screening. Concerning the efficacy of low-dose CT screening for heavy-smokers, a positive result was reported in June 2011, and further detailed analyses are required. There are still some problems to solve i. e., the management of undiagnosed shadows, harm caused by the screening, quality control, and efficacy in non-smokers.

Hepatocyte nuclear factor 4α regulates expression of the mouse female-specific Cyp3a41 gene in the liver.

CYP3A41 is a female-specific cytochrome P450 in mouse liver. A putative hepatocyte nuclear factor 4α (HNF4α)-binding site was found at -99/-87 in the promoter of Cyp3a41 by reporter assays performed in the hepatocytes of female mice. Cotransfection of an HNF4α expression plasmid significantly increased transcription of the reporter gene. Although electrophoretic mobility shift assays with liver nuclear extracts did not show a sex-related difference, chromatin immunoprecipitation (ChIP) assays showed that larger amounts of HNF4α bound to Cyp3a41 in female than in male mice. A relation between the amount of HNF4α on the Cyp3a41 gene and mRNA expression was observed in hepatic tissue sets, which differ in mRNA expression depending on the sex, age, or endocrine status of mice. The degree of histone-3-lysine-4 dimethylation and histone-3-lysine-27 trimethylation around the HNF4α-binding site was higher in females and males, respectively. Moreover, the ChIP assay indicated greater acetylation of histone-4-lysine-8 of the Cyp3a41 chromatin in females than in males. HNF4α plays an important role in the transcriptional activation of the Cyp3a41 gene, and a sex difference in chromatin structure may contribute to the female-specific expression of Cyp3a41 in the livers of mice.

The effect of influenza virus A on th1/th2 balance and alveolar fluid clearance in pregnant rats.

ABSTRACT Pregnant women are more prone to H1N1 infection and often with severe complications. The authors studied the influence of H1N1 infection on T-helper cell type 1/type 2 (Th1/Th2) balance and alveolar fluid clearance (AFC) in pregnant rats. The pregnant rats were infected intranasally with influenza virus. Peripheral blood interferon-γ (IFN-γ) and interleukin-4 (IL-4) were measured by enzyme-linked immunosorbent assay (ELISA) and AFC was estimated by albumin concentration in alveolar lavage. The ratio of IFN-γ/IL-4 in nonpregnant rats was 21 ± 7. There was significant increase in both cytokines in infected pregnant rats compared with noninfected counterparts, with dramatic reduction in IFN-γ/IL-4 ratio (8 ± 3) compared to that (15 ± 8) in normal pregnant group. AFC of normal nonpregnant rats was 17% ± 3% and H1N1 infection reduced it to 11% ± 2%. AFC of normal pregnant rats was 22% ± 2% and H1N1 infection reduced it to 10% ± 2%. Dexamethasone reversed AFC in both nonpregnant and pregnant groups (14% ± 4% and 13% ± 2%, respectively). These results show that influenza virus A infection leads to Th2-biased immunity and reduces AFC in normal rats, and further worsens these in pregnant rats. Dexamethasone reverses these effects in both pregnant and nonpregnant rats.

Pulmonary hypertension confirmed histologically five months prior to scleroderma renal crisis onset.

A 69-year-old man presented shortness of breath and acute renal failure. He had undergone pulmonary partial resection for lung cancer 5 months prior. On examination, severe hypertension, skin sclerosis of his forearms, and anticentromere antibody were observed. A renal biopsy specimen showed characteristic findings for scleroderma renal crisis, and a right heart catheterization revealed severe pulmonary arterial hypertension. Re-examination of the resected lung specimen revealed sclerodermatous vascular involvement was present.