[Curative Resection after Chemotherapy for Advanced Extensive Cholangiocarcinoma-A Case Report].

A 73-year-old, male patient presented with the chief complaint of epigastric pain and received the diagnosis of extensive cholangiocarcinoma after a close examination. Extensive extension of the malignancy into the right and left hepatic ducts precluded a curative resection, and the patient received GC therapy. After 11 courses of GC over about 1 year, no new lesions or tumor progression was observed, and a bile duct mapping biopsy was performed to investigate the possibility of resection conversion. The results showed a marked decrease in atypia, and reactive atypia was diagnosed. A pancreaticoduodenectomy was performed, and histopathologically negative margins were obtained. The response to treatment was Grade Ⅱa according to the Evans classification. At 23 months after the start of treatment and 12 months after surgery, the patient is recurrence-free without adjuvant chemotherapy. Although the evidence for conversion surgery for biliary tract cancer has not been established, the long-term outcomes may be favorable.

[A Case of a Two-Stage Robotic-Assisted Surgery for a Neuroendocrine Tumor of the Pancreatic Tail with Simultaneous Single Liver Metastasis].

As medical insurance coverage for robotic surgery has been expanded in the field of gastrointestinal surgery in Japan, the number of cases undergoing robotic surgery for hepato-biliary-pancreatic disease has been increasing. Therefore, cases with malignant tumors and metastatic lesions tend to undergo robotic operation for both primary tumors and metastases. Herein, we report a case of neuroendocrine tumor(NET)in the pancreatic tail with simultaneous single liver metastasis, which was treated with two-stage robotic-assisted surgery. A 67-year-old female underwent a computed tomography scan and a hypovascularized tumor in the pancreatic tail region and liver was found. A biopsy of the pancreatic tumor by endoscopic ultrasound-guided fine needle aspiration demonstrated a NET G1-2. The liver lesion was diagnosed as a metastatic tumor, considering the other examinations. The patient underwent a robotic distal pancreatectomy(RDP)and was histopathologically diagnosed as NET G2. Sixty-three days after the RDP, a two-stage partial liver resection for the metastatic tumor was performed under robotic assistance. Curative resection was achieved through two-stage robot-assisted surgery, there were no postoperative complications.

Upregulation of CIP2A in estrogen depletion-resistant breast cancer cells treated with low-dose everolimus.

Everolimus (EVE), an inhibitor of mammalian target of rapamycin, is an emerging second-line therapeutic option for hormone therapy-resistant breast cancers. However, some patients do not respond to EVE, whereas in others it exacerbates the disease. Cellular inhibitor of protein phosphatase 2A (CIP2A) is a human oncoprotein that can promote cancer cell growth and apoptosis resistance. Although CIP2A is upregulated in hormone-related cancers, such as breast cancer, little is known about potential anti-tumor effects of downregulating CIP2A. As a model to study the resistance of breast cancer cells to hormone treatment, we previously established clones of long-term estrogen depletion-resistant MCF-7 (LTED) cells. Here, we selected three clones highly responsive to EVE and three clones poorly responsive to EVE. When cells were treated with EVE, CIP2A mRNA expression was decreased in highly responsive EVE clones (DC-cells) whereas it was increased in poorly responsive EVE clones (IC-cells). Using Kaplan-Meier survival plots, we report that high expression of CIP2A was associated with significantly reduced overall survival in patients with luminal A breast cancer. In IC-cells, cell growth was enhanced upon EVE treatment whereas an EVE range of 0.1-100 nm decreased growth in DC-cells. The mRNA expression of genes involved in epithelial-mesenchymal transition (EMT) such as CDH1, CLDN3, and CK19 was significantly decreased in IC-cells, but remained unchanged in DC-cells. These findings highlight a relationship between CIP2A and EMT in the intrinsic resistance of hormone therapy-resistant breast cancers to EVE.

The ABO Blood Group Impacts the Survival of Patients Undergoing Pancreatoduodenectomy for Biliary Tract Cancer.

BACKGROUND/AIM: Although ABO blood group has been reported to be associated with the outcome of patients with pancreatic cancer, little is known about its impact on patients with biliary tract cancer (BTC). We evaluated the prognostic relevance of ABO blood group in patients who had undergone resection of BTC. PATIENTS AND METHODS: A total of 154 patients with BTC undergoing pancreatoduodenectomy were retrospectively reviewed. Associations between ABO blood group and patient survival were evaluated by univariate and multivariate analysis. RESULTS: The 5-year overall survival rate was higher in group O patients (n=46) than in other blood group patients (n=108) (65.8% vs. 47%, p=0.005). Multivariate analysis revealed that a non-O blood group was an independent risk factor for poor survival (p=0.021). CONCLUSION: ABO blood group is associated with the prognosis of patients with resected BTC; group O patients have a better outcome.

ARP-1 Regulates the Transcriptional Activity of the Aromatase Gene in the Mouse Brain.

An important function of aromatase in the brain is conversion of testosterone secreted from the testis into estradiol. Estradiol produced in the brain is thought to be deeply involved in the formation of sexually dimorphic nuclei and sexual behavior as a neurosteroid. We analyzed the brain-specific promoter to elucidate the control mechanisms of brain aromatase expression that may be highly involved in sexual differentiation of the brain. The 202-bp upstream region of the brain-specific exon 1 has three types of cis-acting elements, aro-AI, AII, and B. We isolated ARP-1 as an aro-AII-binding protein by yeast one-hybrid screening from a cDNA library of mouse fetal brains. ARP-1 is a member of the nuclear receptor superfamily and functions as an orphan-type transcription factor. ARP-1 protein synthesized in vitro showed the same binding property to the aro-AII site as nuclear extract from fetal brains. To determine how the promoter is involved in brain-specific transcription of the aromatase gene, we first detected the in vivo occupancy of the aro-AII site by ARP-1 using chromatin immunoprecipitation assays. Diencephalic regions of fetal brains at embryonic day 16 were analyzed, which revealed ARP-1 recruitment to the aro-AII site. To analyze the effects of ARP-1 on transcriptional regulation of the brain-specific aromatase promoter, a luciferase reporter plasmid driven by the brain-specific promoter was transfected into CV-1 cells together with a plasmid expressing ARP-1 protein. These analyses revealed that ARP-1 induced promoter activity in a dose-dependent manner. Furthermore, to determine whether ARP-1 is required for aromatase expression in neurons, ARP-1 knockdown was conducted in neuronal cell primary culture. Knockdown of ARP-1 significantly suppressed the increase in aromatase mRNA observed in cultured neurons. These results indicate that ARP-1 is involved in the transcriptional regulation of the brain-specific promoter of the aromatase gene.

Clinical Significance of Neoadjuvant Chemotherapy With Gemcitabine Plus S-1 for Resectable Pancreatic Ductal Adenocarcinoma.

BACKGROUND/AIM: Little is known about the efficacy of neoadjuvant chemotherapy (NAC) with gemcitabine plus S-1 (GS) for patients with resectable pancreatic ductal adenocarcinoma (R-PDAC). The aim of this study was to investigate differences in the long-term outcome of patients with R-PDAC undergoing pancreatectomy with and without NAC-GS to clarify the clinical significance of NAC-GS. PATIENTS AND METHODS: A total of 77 patients with R-PDAC who were scheduled for pancreatectomy between January 2012 and December 2017 were enrolled. Of these patients, 39 received NAC-GS (GS group) and 38 had upfront surgery (UFS group). RESULTS: Among the 77 patients, one patient in each group did not undergo pancreatectomy due to intraoperative non-curative factors. Median tumor size and the number of lymph nodes with metastasis were significantly lower in the GS group than in the UFS group (p=0.002 and p=0.017). However, the 5-year overall survival rate was similar in the two groups (26.1% versus 21.5%, p=0.930). CONCLUSION: NAC-GS may not be recommended for patients with R-PDAC since it does not seem to offer any survival benefits.

Aromatase and estrogen receptor immunoreactivity in the coronary arteries of monkeys and human subjects.

OBJECTIVE: The objective of this study was to determine whether estrogen could be formed locally in the coronary arteries. DESIGN: Coronary arteries were examined from monkeys (Macaca fascicularis, one male and one female) and human subjects (one premenopausal woman, one postmenopausal woman, and one man) by immunocytochemistry, using purified antisera against human placental estrogen synthetase (aromatase) and ER α. The arteries were graded for the amount of atherosclerosis. RESULTS: There was clear immunopositivity for both aromatase and estrogen receptors in all arteries studied. Although all endothelial cells (CD31 positive) stained for both antigens, the staining in macrophages, fibroblasts, and smooth muscle cells was irregular. CONCLUSION: The present results provide the first evidence for the local formation of estrogen in the coronary arteries. In addition to complementing the evidence of a cardioprotective effect of estrogen on the coronary circulation, our results highlight the potential importance of local regulation of estrogen formation and the role of available precursor androgens in maintaining the cardiovascular system.

HMGA1a Induces Alternative Splicing of the Estrogen Receptor-αlpha Gene by Trapping U1 snRNP to an Upstream Pseudo-5' Splice Site.

Objectives: The high-mobility group A protein 1a (HMGA1a) protein is known as a transcription factor that binds to DNA, but recent studies have shown it exerts novel functions through RNA-binding. We were prompted to decipher the mechanism of HMGA1a-induced alternative splicing of the estrogen receptor alpha (ERα) that we recently reported would alter tamoxifen sensitivity in MCF-7 TAMR1 cells. Methods: Endogenous expression of full length ERα66 and its isoform ERα46 were evaluated in MCF-7 breast cancer cells by transient expression of HMGA1a and an RNA decoy (2'-O-methylated RNA of the HMGA1a RNA-binding site) that binds to HMGA1a. RNA-binding of HMGA1a was checked by RNA-EMSA. In vitro splicing assay was performed to check the direct involvement of HMGA1a in splicing regulation. RNA-EMSA assay in the presence of purified U1 snRNP was performed with psoralen UV crosslinking to check complex formation of HMGA1a-U1 snRNP at the upstream pseudo-5' splice site of exon 1. Results: HMGA1a induced exon skipping of a shortened exon 1 of ERα in in vitro splicing assays that was blocked by the HMGA1a RNA decoy and sequence-specific RNA-binding was confirmed by RNA-EMSA. RNA-EMSA combined with psoralen UV crosslinking showed that HMGA1a trapped purified U1 snRNP at the upstream pseudo-5' splice site. Conclusions: Regulation of ERα alternative splicing by an HMGA1a-trapped U1 snRNP complex at the upstream 5' splice site of exon 1 offers novel insight on 5' splice site regulation by U1 snRNP as well as a promising target in breast cancer therapy where alternative splicing of ERα is involved.

Forskolin increases the effect of everolimus on aromatase inhibitor-resistant breast cancer cells.

Aromatase inhibitor (AI) resistance is a major obstacle in the treatment of estrogen receptor-positive breast cancer. Everolimus (EVE) ameliorates AI-resistant breast cancer and is therefore used in cancer treatment. However, some patients show resistance to EVE. Here, we used 30 clones of long-term estrogen-deprived (LTED) MCF-7 cells as a model of AI-resistant breast cancer. We examined changes in protein phosphatase type 2A (PP2A) and cancerous inhibitor of PP2A (CIP2A), a negative regulator of PP2A, in LTED cells treated with EVE. In LTED cells with high sensitivity to EVE, CIP2A expression decreased at low EVE concentrations; however, in LTED cells poorly sensitive to EVE, CIP2A and PP2A did not change upon exposure to EVE. Therefore, we hypothesized that there is a relation between expression of CIP2A and sensitivity to EVE. Knockdown of CIP2A increased the sensitivity to EVE in three clones poorly sensitive to EVE. Additionally, we found that treatment with FSK, which activates PP2A, increased the sensitivity of the cells to EVE. Our data point to CIP2A and PP2A as novel therapeutic targets for AI-resistant breast cancer.

HMGA1a induces alternative splicing of estrogen receptor alpha in MCF-7 human breast cancer cells.

The high-mobility group A protein 1a (HMGA1a) protein is known as an oncogene whose expression level in cancer tissue correlates with the malignant potential, and known as a component of senescence-related structures connecting it to tumor suppressor networks in fibroblasts. HMGA1 protein binds to DNA, but recent studies have shown it exerts novel functions through RNA-binding. Our previous studies have shown that sequence-specific RNA-binding of HMGA1a induces exon-skipping of Presenilin-2 exon 5 in sporadic Alzheimer disease. Here we show that HMGA1a induced exon-skipping of the estrogen receptor alpha (ERα) gene and increased ERα46 mRNA expression in MCF-7 breast cancer cells. An RNA-decoy of HMGA1a efficiently blocked this event and reduced ERα46 protein expression. Blockage of HMGA1a RNA-binding property consequently induced cell growth through reduced ERα46 expression in MCF-7 cells and increased sensitivity to tamoxifen in the tamoxifen-resistant cell line, MCF-7/TAMR1. Stable expression of an HMGA1a RNA-decoy in MCF-7 cells exhibited decreased ERα46 protein expression and increased estrogen-dependent tumor growth when these cells were implanted in nude mice. These results show HMGA1a is involved in alternative splicing of the ERα gene and related to estrogen-related growth as well as tamoxifen sensitivity in MCF-7 breast cancer cells.

Estrogen-Related Factors in the Frontal Lobe of Alzheimer's Disease Patients and Importance of Body Mass Index.

Estrogens play a physiologically important role in the brain, but controversies exist regarding the association between Alzheimer's disease (AD) and estrogens. Estrogen-related factors were comprehensively examined in frontal lobe tissues from autopsied AD patients, and compared with controls. Concentrations of estrogens, expression of estrogen receptors (ERs), and estrogen-metabolizing enzymes (EMEs) which are important for determining the peripheral estrogen concentrations, were examined using liquid chromatography tandem mass spectrometry, immunohistochemistry, and quantitative real-time PCR, respectively. Body mass index (BMI), known to correlate with the serum estrogen concentrations, was also taken into consideration. There were no significant differences in estrogen concentrations or each EME level between the two groups in both the cortex and white matter, whereas glial nuclear ER-ß expression was significantly lower in white matter from the AD group than the control group (Allred score, 3.2 ± 0.3 and 6.5 ± 0.3, respectively. P < 0.0001). Estrogen concentrations were found to closely correlate with BMI, particularly in controls. ER-ß loss in the white matter from the AD group suggests the necessity of studying the effects of estrogens on glias as well as neurons in the etiology of AD. The correlation between BMI and estrogen concentrations in the frontal lobe suggests the importance of non-brain sources of estrogens.

Estradiol suppresses phosphorylation of ERα serine 167 through upregulation of PP2A in breast cancer cells.

Aromatase inhibitors (AIs) are effective endocrine therapeutics for postmenopausal women with estrogen receptor (ER)α-positive breast cancer. However, the efficacy of the treatment is often limited by the onset of AI resistance, owing to the phosphorylation of ERα serine 167 (Ser167). Previous studies have indicated that hyperactivation of the phosphoinositide-3 kinase/RAC serine/threonine-protein kinase signaling pathway occurs in AI-resistant breast cancer models, which coincides with elevated levels of ERα phosphorylation at Ser167. The tumor suppressor serine/threonine-protein phosphatase 2A (PP2A) regulates the phosphatidylinositol 3-kinase/RAC serine/threonine-protein kinase signaling pathway. A previous study indicated that PP2A inhibition decreased ERα Ser167 phosphorylation and estradiol (E2)-independent cell growth. The present study investigated the potential relevance of PP2A in E2 deprivation-resistant MCF-7 cells. E2 depletion reduced the susceptibility of MCF-7 cells to inhibitors of mechanistic target of rapamycin (mTOR) and significantly increased ERα Ser167 phosphorylation and decreased expression of PP2A. Conversely, long-term E2-deprived (LTED) MCF-7 cells, a model of AI-resistant breast cancer, exhibited decreased ERα Ser167 phosphorylation and further upregulation of PP2A in E2-containing medium. The PP2A activator forskolin (FSK) significantly inhibited LTED cell proliferation by increasing the effect of everolimus (Eve), an mTOR inhibitor. In summary, the present study provides further evidence that PP2A represents a therapeutic target for AI-resistant breast cancer.

Techniques of Fluorescence Cholangiography During Laparoscopic Cholecystectomy for Better Delineation of the Bile Duct Anatomy.

To evaluate the clinical and technical factors affecting the ability of fluorescence cholangiography (FC) using indocyanine green (ICG) to delineate the bile duct anatomy during laparoscopic cholecystectomy (LC).Application of FC during LC began after laparoscopic fluorescence imaging systems became commercially available.In 108 patients undergoing LC, FC was performed by preoperative intravenous injection of ICG (2.5  mg) during dissection of Calot's triangle, and clinical factors affecting the ability of FC to delineate the extrahepatic bile ducts were evaluated. Equipment-related factors associated with bile duct detectability were also assessed among 5 laparoscopic systems and 1 open fluorescence imaging system in ex vivo studies.FC delineated the confluence between the cystic duct and common hepatic duct (CyD-CHD) before and after dissection of Calot's triangle in 80 patients (74%) and 99 patients (92%), respectively. The interval between ICG injection and FC before dissection of Calot's triangle was significantly longer in the 80 patients in whom the CyD-CHD confluence was detected by fluorescence imaging before dissection (median, 90  min; range, 15-165  min) than in the remaining 28 patients in whom the confluence was undetectable (median, 47  min; range, 21-205  min; P < 0.01). The signal contrast on the fluorescence images of the bile duct samples was significantly different among the laparoscopic imaging systems and tended to decrease more steeply than those of the open imaging system as the target-laparoscope distance increased and porcine tissues covering the samples became thicker.FC is a simple navigation tool for obtaining a biliary roadmap to reach the "critical view of safety" during LC. Key factors for better bile duct identification by FC are administration of ICG as far in advance as possible before surgery, sufficient extension of connective tissues around the bile ducts, and placement of the tip of laparoscope close and vertically to Calot's triangle.

Aromatase knockout mice reveal an impact of estrogen on drug-induced alternation of murine electrocardiography parameters.

Our in vitro characterization showed that physiological concentrations of estrogen partially suppressed the I(Kr) channel current in guinea pig ventricular myocytes and the human ether-a-go-go-related gene (hERG) channel currents in CHO-K1 cells regardless of estrogen receptor signaling and revealed that the partially suppressed hERG currents enhanced the sensitivity to the hERG blocker E-4031. To obtain in vivo proof-of-concept data to support the effects of estrogen on cardiac electrophysiology, we here employed an aromatase knockout mouse as an in vivo estrogen-null model and compared the acute effects of E-4031 on cardiac electrophysiological parameters with those in wild-type mice (C57/BL6J) by recording surface electrocardiogram (ECG). The ablation of circulating estrogens blunted the effects of E-4031 on heart rate and QT interval in mice under a denervation condition. Our result provides in vivo proof of principle and demonstrates that endogenous estrogens increase the sensitivity of E-4031 to cardiac electrophysiology.

The Proportion of Chromatin Graded between Closed and Open States Determines the Level of Transcripts Derived from Distinct Promoters in the CYP19 Gene.

The human CYP19 gene encodes aromatase, which converts androgens to estrogens. CYP19 mRNA variants are transcribed mainly from three promoters. Quantitative RT-PCR was used to measure the relative amounts of each of the three transcripts and determine the on/off state of the promoters. While some of the promoters were silent, CYP19 mRNA production differed among the other promoters, whose estimated transcription levels were 0.001% to 0.1% of that of the TUBB control gene. To investigate the structural aspects of chromatin that were responsible for this wide range of activity of the CYP19 promoters, we used a fractionation protocol, designated SEVENS, which sequentially separates densely packed nucleosomes from dispersed nucleosomes. The fractional distribution of each inactive promoter showed a similar pattern to that of the repressed reference loci; the inactive regions were distributed toward lower fractions, in which closed chromatin comprising packed nucleosomes was enriched. In contrast, active CYP19 promoters were raised toward upper fractions, including dispersed nucleosomes in open chromatin. Importantly, these active promoters were moderately enriched in the upper fractions as compared to active reference loci, such as the TUBB promoter; the proportion of open chromatin appeared to be positively correlated to the promoter strength. These results, together with ectopic transcription accompanied by an increase in the proportion of open chromatin in cells treated with an H3K27me inhibitor, indicate that CYP19 mRNA could be transcribed from a promoter in which chromatin is shifted toward an open state in the equilibrium between closed and open chromatin.

Aromatase controls Sjögren syndrome-like lesions through monocyte chemotactic protein-1 in target organ and adipose tissue-associated macrophages.

Several autoimmune diseases are known to develop in postmenopausal women. However, the mechanism by which estrogen deficiency influences autoimmunity is unknown. Aromatase is an enzyme that converts androgens to estrogens. Herein, we used female aromatase gene knockout (ArKO) mice as a model of estrogen deficiency to investigate the molecular mechanism that underlies the onset and development of autoimmunity. Histological analyses showed that inflammatory lesions in the lacrimal and salivary glands of ArKO mice increased with age. Adoptive transfer of spleen cells or bone marrow cells from ArKO mice into recombination activating gene 2 knockout mice failed to induce the autoimmune lesions. Expression of mRNA encoding proinflammatory cytokines and monocyte chemotactic protein-1 increased in white adipose tissue of ArKO mice and was significantly higher than that in wild-type mice. Moreover, an increased number of inflammatory M1 macrophages was observed in white adipose tissue of ArKO mice. A significantly increased monocyte chemotactic protein-1 mRNA expression of the salivary gland tissue in ArKO was found together with adiposity. Furthermore, the autoimmune lesions in a murine model of Sjögren syndrome were exacerbated by administration of an aromatase inhibitor. These results suggest that aromatase may play a key role in the pathogenesis of Sjögren syndrome-like lesions by controlling the target organ and adipose tissue-associated macrophage.

Acoustic radiation force impulse imaging of the pancreas for estimation of pathologic fibrosis and risk of postoperative pancreatic fistula.

BACKGROUND: We sought to evaluate whether pancreatic elasticity, measured using acoustic radiation force impulse (ARFI) imaging, can determine the degree of pancreatic fibrosis and risk of pancreatic fistula (PF) in patients undergoing pancreatic resection. Although soft pancreatic texture is a reliable predictor of postoperative PF, noninvasive, quantitative methods of assessing pancreatic hardness have not been established. STUDY DESIGN: Shear wave velocity (SWV) of the pancreas was preoperatively measured by ARFI imaging in 62 patients undergoing pancreatic resection. Correlations of SWV with pathologic degree of fibrosis in the resected pancreas, exocrine function of the remnant pancreas, and the incidence of postoperative PF were determined. RESULTS: The SWV was positively correlated with the degree of pancreatic fibrosis (Spearman's rank correlation coefficient [ρ] = 0.660, p < 0.001) and inversely correlated with postoperative amylase concentrations and daily output of pancreatic juice. The incidence of postoperative PF was significantly higher in the 32 patients with soft (SWV < 1.54 m/s) than in the 30 with hard (SWV ≥ 1.54 m/s) pancreata (63% vs 17%, p < 0.001). Multivariate analysis showed that a soft pancreas (SWV < 1.54 m/s) was an independent predictor of postoperative PF (odds ratio 38.3; 95% CI 5.82 to 445; p = 0.001). CONCLUSIONS: Pancreatic elasticity on preoperative ARFI imaging accurately reflected the pathologic degree of fibrosis and exocrine function of the pancreas, enabling surgeons to adopt appropriate surgical procedures according to the risk of postoperative PF in each patient undergoing pancreatic resection.

Post-translational dual regulation of cytochrome P450 aromatase at the catalytic and protein levels by phosphorylation/dephosphorylation.

The post-translational regulation of aromatase has not been well characterized as compared with transcriptional regulation. Several studies of post-translational regulation have focused on decreases in catalytic activity following phosphorylation. We report here dual post-translational regulation of aromatase, at the catalytic activity and protein levels. Microsomal aromatase prepared from JEG-3 cells was rapidly inactivated and subsequently degraded in the presence of a cytosolic fraction with calcium, magnesium, and ATP. In a reconstituted system consisting of microsomal and cytosolic fractions, aromatase was protected from protein degradation by treatment with alkaline phosphatase, whereas degradation was enhanced by treatment with calcineurin inhibitors (FK506 and cyclosporin A). Furthermore, aromatase was protected from degradation by treatment with kinase inhibitors, especially the calcium/calmodulin kinase inhibitors KN62 and KN93. Similarly to the reconstituted system, aromatase in cultured JEG-3 cells was protected from degradation by KN93, whereas FK503 increased degradation in the presence of cycloheximide, although cellular aromatase mRNA levels were unchanged by these reagents. Knockdown of calcineurin and calcium/calmodulin kinase II (CaMKII) with small interfering RNAs resulted in a dose-dependent increase in aromatase degradation and protection from degradation, respectively. The cytosol fraction-dependent phosphorylation of microsomal aromatase was inhibited by calcineurin, KN62, and KN93, and promoted by CaMKII and FK506. These results indicate that aromatase is regulated acutely at the catalytic activity level and subsequently at the enzyme content level by CaMKII/calcineurin-dependent phosphorylation/dephosphorylation.

Calcineurin and CRTC2 mediate FSH and TGFß1 upregulation of Cyp19a1 and Nr5a in ovary granulosa cells.

Estrogens are essential for female reproduction and overall well-being, and estrogens in the circulation are largely synthesized in ovarian granulosa cells. Using primary cultures of ovarian granulosa cells from gonadotropin-primed immature rats, we have recently discovered that pituitary FSH and ovarian cytokine transforming growth factor beta 1 (TGFß1) induce calcineurin-mediated dephosphorylation-activation of cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC2) to modulate the expression of Star, Cyp11a1, and Hsd3b leading to increased production of progesterone. This study explored the role of calcineurin and CRTC2 in FSH and TGFß1 regulation of Cyp19a1 expression in granulosa cells. Ovarian granulosa cells treated with FSH displayed increased aromatase protein at 24  h post-treatment, which subsided by 48  h, while TGFß1 acting through its type 1 receptor augmented the action of FSH with a greater and longer effects. It is known that the ovary-specific Cyp19a1 PII-promoter contains crucial response elements for CREB and nuclear receptor NR5A subfamily liver receptor homolog 1 (LRH1/NR5A2) and steroidogenic factor 1 (SF1/NR5A1), and that the Nr5a2 promoter also has a potential CREB-binding site. Herein, we demonstrate that FSH+TGFß1 increased LRH1 and SF1 protein levels, and their binding to the Cyp19a1 PII-promoter evidenced, determined by chromatin immunoprecipitation analysis. Moreover, pretreatment with calcineurin auto-inhibitory peptide (CNI) abolished the FSH+TGFß1-upregulated but not FSH-upregulated aromatase activity at 48  h, and the corresponding mRNA changes in Cyp19a1, and Nr5a2 and Nr5a1 at 24  h. In addition, FSH and TGFß1 increased CRTC2 binding to the Cyp19a1 PII-promoter and Nr5a2 promoter at 24  h, with CREB bound constitutively. In summary, the results of this study indicate that calcineurin and CRTC2 have important roles in mediating FSH and TGFß1 collateral upregulation of Cyp19a1 expression together with its transcription regulators Nr5a2 and Nr5a1 in ovarian granulosa cells.

Application of indocyanine green-fluorescence imaging to full-thickness cholecystectomy.

Fluorescence imaging using indocyanine green (ICG) has recently been applied to laparoscopic surgery to identify cancerous tissues, lymph nodes, and vascular anatomy. Here we report the application of ICG-fluorescence imaging to visualize the boundary between the liver and subserosal tissues of the gallbladder during laparoscopic full-thickness cholecystectomy. A patient with a potentially malignant gallbladder lesion was administered 2.5-mg intravenous ICG just before laparoscopic full-thickness cholecystectomy. Intraoperative fluorescence imaging enabled the real-time delineation of both extrahepatic bile duct anatomy and hepatic parenchyma throughout the procedure, which resulted in complete removal of subserosal tissues between liver and gallbladder. Safe and feasible ICG-fluorescence imaging can be widely applied to laparoscopic hepatobiliary surgery by utilizing a biliary excretion property of ICG.