A Role of CREB in BRCA1 Constitutive Promoter Activity and Aromatase Basal Expression
Aromatase is the rate-limiting enzyme in estrogen biosynthesis and a key target in breast cancer treatment. Its ovary-specific promoter, pII, is induced in response to protein kinase A (PKA) activation. It has been proposed that breast cancer susceptibility gene 1, BRCA1, is involved in negative regulation of aromatase pII activity. Surprisingly, inhibition of PKA pathway by inhibitor H89 elevates basal aromatase expression while abolishes cAMP-mediated aromatase induction in an ovarian granulosa cell line, KGN. In this report, we decipher the mechanism by which the PKA pathway negatively regulates aromatase basal expression. We show that PKA pathway plays a positive role in the expression of BRCA1. H89 effectively reduces endogenous BRCA1 mRNA levels as well as reporter gene expression from a BRCA1 promoter. Mutation of a cAMP-responsive element (CRE) in the BRCA1 promoter reduces BRCA1 expression. Chromatin immunoprecipitation (ChIP) shows that CRE-binding protein, CREB, binds to the BRCA1 promoter. Furthermore, knockdown of CREB in KGN cells leads to decreased BRCA1 level as well as elevated basal aromatase mRNA expression. These data demonstrate that both the CRE site in the BRCA1 promoter and CREB are required for BRCA1 constitutive expression. Our study suggests that PKA pathway exerts its negative impact on basal aromatase expression indirectly by contributing to the constitutive expression of BRCA1.

title: Mechanical phenotype is important for stromal aromatase expression
abstract: Evidence that aromatase expression in tumor-associated breast stroma is elevated, provides a rationale for use of aromatase inhibitors (AIs) in breast cancer treatment. However, regulation of local aromatase expression in cancer-free breast stroma is poorly understood. Recent clinical work indicates that stromal cells in dense breast tissue tend to express higher levels of aromatase than their counterpart from non-dense tissue. Consistent with the clinical observation, our cell culture-based study indicated that cell density, cell shape, and extracellular matrix (ECM) significantly induced stromal aromatase expression via a distinct signal transduction pathway. In addition, we identified a number of cell surface markers that are commonly associated with aromatase-expressing stromal cells. As mammographic density is one of the strongest and most prevalent risk factors for breast cancer, these findings provide a potential mechanistic link between alterations in tissue composition of dense breast tissue and increased stromal aromatase expression. Further exploration of the in vitro model system may advance understanding of an important problem in breast cancer biology.

title: An IL-6 link between obesity and cancer.
abstract: Obesity is a growing epidemic all over the world that by virtue of inducation of a chronic, low-grade, and systemic inflammation leads to an increased risk of a number of diseases, including cancer. IL-6 an important cytokine in the increased risk to cancer in obese patients mainly because of its pro-inflammatory activity. Some data suggest that IL-6 might increase the risk of certain cancers such as those that originate from breast, liver, prostate, colon, and esophagus. A better understanding of the regulation and role of IL-6 in obesity-associated cancer is required to develop effective therapeutic approaches.

title: Cell density-dependent transcriptional activation of endocrine-related genes in human adipose tissue-derived stem cells.
abstract: Adipose tissue is recognized as an endocrine organ that plays an important role in human diseases such as type II diabetes and cancer. Human adipose tissue-derived stem cells (ASCs), a distinct cell population in adipose tissue, are capable of differentiating into multiple lineages including adipogenesis. When cultured in vitro under a confluent condition, ASCs reach a commitment stage for adipogenesis, which can be further induced into terminally differentiated adipocytes by a cocktail of adipogenic factors. Here we report that the confluent state of ASCs triggers transcriptional activation cascades for genes that are responsible for the endocrine function of adipose tissue. These include insulin-like growth factor 1 (IGF-1) and aromatase (Cyp19), a key enzyme in estrogen biosynthesis. Despite similar adipogenic potentials, ASCs from different individuals display huge variations in activation of these endocrine-related genes. Bioinformatics and experimental data suggest that transcription factor Foxo1 controls a large number of "early" confluency-response genes, which subsequently induce "late" response genes. Furthermore, siRNA-mediated knockdown of Foxo1 substantially compromises the ability of committed ASCs to stimulate tumor cell migration in vitro. Thus, our work suggests that cell density is an important determinant of the endocrine potential of ASCs.

title: Cell density is a critical determinant of aromatase expression in adipose stromal cells
abstract: Obesity is associated with an increased risk of breast cancer among postmenopausal women. This is at least partly due to excessive estrogen production in adipose tissue of obese women. Aromatase, the key enzyme in estrogen biosynthesis, is an important target in endocrine therapy for estrogen receptor (ER)-positive postmenopausal breast cancer. In this study we show that high confluency of human adipose stromal cells (ASCs) cultured in vitro can significantly stimulate aromatase gene expression and reduce the expression of breast tumor suppressor BRCA1 and members of the NR4A orphan nuclear family. Furthermore, small interfering RNA (siRNA)-mediated knockdown of Nurr1, a member of the NR4A family, substantially increased aromatase expression. Lastly, we found that the cell density-triggered inducibility of aromatase expression varies in ASCs isolated from different disease-free individuals. Our finding highlights the impact of increased cell number on estrogen biosynthesis as in the case of excessive adiposity.

title: A two-stage variable-stringency semiparametric method for mapping quantitative-trait loci with the use of genomewide-scan data on sib pairs.
abstract: Genomewide scans for mapping loci have proved to be extremely powerful and popular. We present a semiparametric method of mapping a quantitative-trait locus (QTL) or QTLs with the use of sib-pair data generated from a two-stage genomic scan. In a two-stage genomic scan, either the entire genome or a large portion of the genome is saturated with low-density markers at the first stage. At the second stage, the intervals that are identified as probable locations of the trait loci, by means of analysis of data from the first stage, are then saturated with higher-density markers. These data are then analyzed for fine mapping of the loci. Our statistical strategy for analysis of data from the first stage is a low-stringency method based on the rank correlation of squared trait-difference values of the sib pairs and the estimated identity-by-descent scores at the marker loci. We suggest the use of a low-stringency method at the first stage, to save on computational time and to avoid missing any marker interval that may contain the trait loci. For analysis of data from the second stage, we have developed a high-stringency nonparametric-regression approach, using the kernel-smoothing technique. Through extensive simulations, we show that this approach is more powerful than is a currently used method for mapping QTLs by use of sib pairs, particularly in the presence of dominance and epistatic effects at the trait loci.

title: Resolution of the spectroscopy versus crystallography issue for NO intermediates of nitrite reductase from Rhodobacter sphaeroides.
abstract: Copper nitrite reductase (NiR) is a homotrimeric enzyme, containing a T1 copper site, which transfers electrons to the T2 catalytic site, where nitrite is reduced by one electron to nitric oxide (NO2− + 2H+ + e− → NO + H2O).1–3 Recently a side-on bound copper nitrosyl complex of NiR has been crystallized by reacting the reduced enzyme with excess NO.4 Based on the EPR spectrum of the species generated by the reaction of reduced NiR with saturated NO in solution, it has been assigned as an η 2-NO−Cu2+species. However, spectroscopic data on several Cu-nitrosyl model complexes5 and an NO adduct formed by reacting reduced NiR with nitrite6 and DFT calculations on an η2-NOCu species7 describe these complexes as Cu+NO•. In this study, we define the species generated in solution by reacting reduced NiR with NO, using EPR, MCD and ENDOR spectroscopy and correlate these with that observed by crystallography. 
 
Reduced NiR reacted with saturated NO solution generates the EPR spectrum in Figure 1A red, which is equivalent to the spectrum reported by Tocheva et al.4 This species has an EPR signal characteristic of an oxidized T2 Cu (g|| = 2.30 and A|| = 117 G). The MCD spectrum  has low energy d-d bands, also indicative of a T2 cupric complex. Both EPR and MCD spectra show a negligible T1 contribution indicating that this site is reduced . 
 
 
 
Figure 1 
 
Overlay of reduced NiR with excess NO (red) and nitrite bound NiR (blue) A) EPR B) MCD, C) N14 ENDOR, D) H1 ENDOR, with exchangeable features with 8.5 MHz coupling. Parts A and B have nitrite bound to T2 site with the T1 Cu reduced to remove intense overlaying ... 
 
 
 
Nitrite binds to the T2 site of the resting WT enzyme,8,9 but the EPR and, in particular, the MCD features of the nitrite bound T2 site are obscured by the dominant T1 Cu signals. However under these conditions (nitrite bound), the T1 Cu could be selectively reduced with acsorbate. The EPR spectrum of the nitrite bound T2 species  is identical to that produced above by reacting reduced NiR with excess NO. The MCD spectrum  of the T1 reduced T2-nitrite bound form is also identical to that generated by the reduced enzyme with NO. The T2 EPR spectrum and the MCD d-d bands of the resting WT enzyme are very sensitive to nitrite binding . Thus the EPR and MCD data strongly indicate that the species generated by the reaction of excess NO with reduced NiR has T1 reduced and T2 oxidized with nitrite bound. 
 
Further N14 and H1 ENDOR data for this species generated by the reaction of reduced NiR with an NO solution  and those for a form with T1+T2 oxidized and nitrite bound (collected at g=2.266 where there is no contribution from T1, Fig. 1C, D, blue) have the same features. The nitrogen features  from Cu2+NO2− bound T2 are from its histidines, and these features all show lower ENDOR frequencies and smaller hyperfine couplings due to the change of the T2 axial ligand from aquo to nitrite.10 
 
From the above results the reaction of reduced NiR with excess NO gives T1 reduced T2 Cu2+-NO2−. However, the reaction of the reduced enzyme with limited NO has an EPR signal , characteristic of a Cu+NO•  This species lacks d-d bands in the MCD spectrum , further confirming that the Cu is reduced. Therefore, reduced NiR forms a Cu+NO• species at low concentrations of NO. 
 
 
 
Figure 2 
 
EPR spectra A) following the reaction of reduced NiR with sub-stoichiometric NO solution (10:1 enzyme: NO) trapped at 1 second (green) 4 second (red) and 18 seconds (purple). B) following the reaction of the resting oxidized enzyme (black) with NO gas ... 
 
 
 
The reaction of reduced NiR with excess NO involves two one-electron oxidations; reduced T2 → oxidized T2, and NO → NO2−. A similar reaction has been observed for the coupled binuclear Cu active sites in hemocyanins11 (Scheme S1). Deoxyhemocyanin (2Cu+) reacts with NO generating N2O, and a met (2Cu2+) form, which further reacts with NO to generate the half-met (Cu2+Cu+) nitrite bound form. N2O evolution is in fact detected during the reaction of reduced NiR with excess NO, using nitrous oxide reductase (35 μmols N2O reduced min−1mg−1of enzyme), which requires N2O as its substrate.12 Reaction of the resting WT NiR (analogous to the met form of hemocyanin) with NO generates the T1 reduced, T2 oxidized form of the enzyme (g||=2.30, A||=150G), which converts to the nitrite bound T2 form , indicating NO is oxidized to nitrite and nitrite binding is slow. 
 
 
 
Scheme 1 
 
Proposed mechanism for the formation of nitrite bound T2 NiR from reduced enzyme with excess NO. Red boxes = species observed. 
 
 
 
To evaluate the entire reaction mechanism, reduced NiR was reacted with limited NO solutions and intermediates were trapped at different times. We were able to trap the Cu+NO• form at shorter times, leading to the formation of the T1 reduced, T2 oxidized form, and ultimately generating the T1 reduced, T2 oxidized nitrite bound form  at longer times. A reaction mechanism consistent with these results is presented in Scheme 1. Note that in the scheme, intermediate E is not observed because of rapid ET from T2 → T1.13 The experimental details and kinetic rates are provided in the supporting information. 
 
There is still one issue to resolve on correlating the spectroscopy with crystallography. The Cu-NO species trapped in the crystal structure has a side-on  DFT calculations indicate that this species is best described as a Cu+-NO• species.7 However an end-on Cu-NO species is also trapped after limited exposure to NO gas (B, in scheme 1) which is equivalent to the species generated by reacting reduced NiR with nitrite (η1-CuNO).6 Geometry optimized DFT calculations (B3LYP/6–311g* on Cu, N, O and 6–31g* on C, H)14 of both side-on  and end-on  Cu-NO adducts indicate that both forms have a Cu+NO• electronic configuration and that the end-on structure is more stable by 7 kcal/mol. However the orientation of the 2nd sphere aspartic acid residue (Asp129)9,15 appears to play a key role in determining the geometry of the Cu-NO species. In the side-on adduct this residue is perpendicular to the Cu-N-O plane and H-bonds to the O of N- O  while in the end-on structure the Asp129 residue H-bonds to the O of NO and is parallel to the Cu-N-O plane . Both orientations of the Asp129 residue have been observed in crystal structures.15 The Asp129 residue exists in the perpendicular orientation in the crystal structures of the oxidized and reduced NiR and H-bonds to the axial ligand. However, to form the more stable end-on Cu+NO• adduct this aspartate residue has to rotate 90° which can occur in solution but may not be feasible in a crystal. 
 
 
 
Figure 3 
 
Overlay of the DFT optimized side-on bound NO (red) and end-on bound NO (blue) structures indicating the different orientations of the aspartate residue. 
 
 
 
In summary, the species generated in solution during the reaction of reduced NiR with excess NO is the T1 reduced, T2 oxidized nitrite bound form and not the side-on Cu-NO species observed in the crystal structure. The latter is best described as Cu+NO• rather than Cu2+NO−. The mechanism for the reaction of reduced NiR with excess NO involves its two electron oxidation, generating N2O. This oxidized form of the enzyme oxidizes NO generating nitrite bound to the oxidized T2 site generated by ET from T2 → T1. DFT calculations provide insight into factors which favor the η2-NO•Cu+ complex in the crystal structure.

title: Study of properties of cholinephosphotransferase from fetal guinea pig lung mitochondria and microsomes
abstract: SummaryWe have reported earlier that cholinephosphotransferase (EC 2.7.8.2) is present in both mitochondria and microsomes of fetal guinea pig lung. This study was designed to compare the properties of mitochondrial and microsomal cholinephosphotransferase in fetal guinea pig lung. Various parameters, such as substrate specificity, Km values, sensitivity to N-ethylmaleimide, dithiothreitol and trypsin were measured. Both showed significant preference for unsaturated diacylglycerols over saturated diacylglycerols. Data on Km and Vmax indicate that the affinity of this enzyme for different diacylglycerols varies between the two forms. The ID50 values for N-ethylmaleimide were 20 mM and 12.5 mM for the mitochondrial and microsomal form of the enzyme, respectively. Dithiothreitol showed an inhibitory effect on both; however, the mitochondrial form was inhibited less than the microsomal form. The effects of N-ethylmaleimide and dithiothreitol on both forms of enzyme indicated that the microsomal cholinephosphotransferase requires a higher concentration of -SH for its activity than the mitochondrial enzyme does. The enzyme was inhibited by trypsin in both mitochondria and microsome under isotonic condition suggesting that this enzyme is on the outside of the membrane in both endoplasmic reticulum and mitochondria.

title: Essential role of tuberous sclerosis genes TSC1 and TSC2 in NF-kappaB activation and cell survival.
abstract: The TSC1-TSC2 complex has recently been implicated in cell survival responses. We observed that NF-kappaB signaling is attenuated in TSC1- and TSC2-deficient MEFs concomitant with reduced survival following DNA damage or TNFalpha stimulation. Reconstitution of TSC2 expression in TSC2(-/-) MEFs rescued survival in an NF-kappaB activity-dependent manner. Furthermore, in TSC2(-/-) MEFs, the rapamycin-mediated inhibition of deregulated mTOR activity restored NF-kappaB activation and survival. This rapamycin-mediated effect was reversed by inhibition of NF-kappaB transcriptional activation or by inhibition of ERK1/2 MAP kinase or PI-3K pathways, which lie on signaling cascades that lead to NF-kappaB activation. These results provide evidence for a crosstalk between the TSC/Rheb/mTOR pathway and the NF-kappaB induction pathways and indicate that NF-kappaB functions as an important survival factor that regulates TSC2-dependent cell survival.

title: Molecular cloning and expression of rat hepatic neutral cholesteryl ester hydrolase.
abstract: The 1923 bp cDNA for rat hepatic cholesteryl ester hydrolase (CEH) was cloned by screening a lambda gt11 expression library with an oligonucleotide containing the consensus active site sequence for cholesteryl esterases. Expression of a fusion protein, cross-reacting with antibody to the purified liver CEH, was demonstrated by Western blot analysis. The cDNA was sequenced and found to have only 44% homology with pancreatic CEH. Although unique, the cDNA sequence exhibited much greater overall homology with liver carboxylesterases, in both coding and 5'/3' non-coding regions. In Northern blot analysis, the cDNA hybridized with a single band from liver mRNA but not with pancreatic mRNA. The 1.7 kb coding sequence, predicting a 62 kDa protein, was cloned into an Escherichia coli expression system with an inducible promoter and into COS-7 cells. Both expression systems produced a protein which comigrated with liver CEH (66 kDa) on SDS-PAGE and immunoreacted with antibodies to liver CEH on Western blots. Whereas the prokaryotic system produced an inactive protein, expression in COS-7 cells was accompanied by a 5-fold increase in CEH activity and a corresponding increase in immunoreactive protein.