SGK3 sustains ERα signaling and drives acquired aromatase inhibitor resistance through maintaining endoplasmic reticulum homeostasis.

Many estrogen receptor alpha (ERα)-positive breast cancers initially respond to aromatase inhibitors (AIs), but eventually acquire resistance. Here, we report that serum- and glucocorticoid-inducible kinase 3 (SGK3), a kinase transcriptionally regulated by ERα in breast cancer, sustains ERα signaling and drives acquired AI resistance. SGK3 is up-regulated and essential for endoplasmic reticulum (EnR) homeostasis through preserving sarcoplasmic/EnR calcium ATPase 2b (SERCA2b) function in AI-resistant cells. We have further found that EnR stress response down-regulates ERα expression through the protein kinase RNA-like EnR kinase (PERK) arm, and SGK3 retains ERα expression and signaling by preventing excessive EnR stress. Our study reveals regulation of ERα expression mediated by the EnR stress response and the feed-forward regulation between SGK3 and ERα in breast cancer. Given SGK3 inhibition reduces AI-resistant cell survival by eliciting excessive EnR stress and also depletes ERα expression/function, we propose SGK3 inhibition as a potential effective treatment of acquired AI-resistant breast cancer.

Aromatase deficiency in a Chinese adult man caused by novel compound heterozygous CYP19A1 mutations: effects of estrogen replacement therapy on the bone, lipid, liver and glucose metabolism.

OBJECTIVES: Aromatase deficiency is a rare disorder resulting in estrogen insufficiency in humans. It has been reported in remarkably few men with loss-of-function mutations in the CYP19A1 gene encoding the aromatase, a cytochrome P450 enzyme that plays a crucial role in the biosynthesis of estrogens from androgens. We investigated a non-consanguineous family including an adult man with clinical features of aromatase deficiency, and studied the effects of estrogen replacement in the man. METHODS: We investigated the clinical and biochemical phenotype, performed CYP19A1 mutational analysis in the family and 50 unrelated persons, studied the effects of CYP19A1 mutations on aromatase protein structure, functionally characterized the mutations by cell-based aromatase activity assays, and studied the effects of estrogen replacement on the bone, lipid, liver and glucose metabolism. RESULTS: The man with clinical features of aromatase deficiency had novel compound heterozygous CYP19A1 mutations (Y81C and L451P) that were not found in 50 unrelated persons. Three-dimensional modeling predicted that Y81C and L451P mutants disrupted protein structure. Functional studies on the basis of in vitro expression showed that Y81C and L45P mutants significantly decreased the aromatase activity and catalytic efficiency. Estrogen replacement in the man increased bone mineral density, accelerated bone maturation, improved lipid profile and liver steatosis, and improved glucose levels but not insulin resistance. CONCLUSIONS: We have identified two novel CYP19A1 missense mutations in an aromatase-deficient man. Estrogen replacement in the man shows great impact on recovering the impairments in the bone, lipid, liver and glucose metabolism, but fails to improve insulin resistance.

SGK3 is an androgen-inducible kinase promoting prostate cancer cell proliferation through activation of p70 S6 kinase and up-regulation of cyclin D1.

Both androgen and phosphatidylinositol 3-kinase (PI3K) signaling are critical for cell proliferation of androgen receptor (AR)-positive prostate cancer cells, but the underlying mechanisms are still not fully understood. Here we report that serum- and glucocorticoid-inducible kinase 3 (SGK3), a Ser/Thr kinase functioning downstream of PI3K, is an AR transcriptional target and promotes prostate cancer cell proliferation. SGK3 expression is up-regulated by androgen DHT via AR. We identified an AR-binding region at the sgk3 locus, which confers androgen responsiveness of sgk3 promoters. Interestingly, we found that androgen/AR-dependent SGK3 expression requires estrogen receptor (ER) (including both isoforms, ERα and ERß). Depletion of ER blocked DHT-induced SGK3 expression. Functionally, knockdown of SGK3 expression significantly decreased LNCaP prostate cancer cell proliferation by inhibiting G1 to S phase cell cycle progression. We further provided evidence that SGK3 promotes p70 S6 kinase (p70S6K) activation and increases cyclin D1 levels. In summary, our study identifies SGK3 as an AR target and provides a novel androgen-induced cell proliferation mechanism mediated by the AR-SGK3-p70S6K-cyclin D1 pathway in prostate cancer cells.

AroER tri-screen is a biologically relevant assay for endocrine disrupting chemicals modulating the activity of aromatase and/or the estrogen receptor.

Endocrine disrupting chemicals (EDCs) interfere with the biosynthesis, metabolism, and functions of steroid hormones, including estrogens and androgens. Aromatase enzyme converts androgen to estrogen. Thus, EDCs against aromatase significantly impact estrogen- and/or androgen-dependent functions, including the development of breast cancer. The current study aimed to develop a biologically relevant cell-based high-throughput screening assay to identify EDCs that act as aromatase inhibitors (AIs), estrogen receptor (ER) agonists, and/or ER antagonists. The AroER tri-screen assay was developed by stable transfection of ER-positive, aromatase-expressing MCF-7 breast cancer cells with an estrogen responsive element (ERE) driven luciferase reporter plasmid. The AroER tri-screen can identify: estrogenic EDCs, which increase luciferase signal without 17ß-estradiol (E2); anti-estrogenic EDCs, which inhibit the E2-induced luciferase signal; and AI-like EDCs, which suppress a testosterone-induced luciferase signal. The assay was first optimized in a 96-well plate format and then miniaturized into a 1536-well plate format. The AroER tri-screen was demonstrated to be suitable for high-throughput screening in the 1536-well plate format, with a 6.9-fold signal-to-background ratio, a 5.4% coefficient of variation, and a screening window coefficient (Z-factor) of 0.78. The assay suggested that bisphenol A (BPA) functions mainly as an ER agonist. Results from screening the 446 drugs in the National Institutes of Health Clinical Collection revealed 106 compounds that modulated ER and/or aromatase activities. Among these, two AIs (bifonazole and oxiconazole) and one ER agonist (paroxetine) were confirmed through alternative aromatase and ER activity assays. These findings indicate that AroER tri-screen is a useful high-throughput screening system for identifying ER ligands and aromatase-inhibiting chemicals.

Coordinated regulation of serum- and glucocorticoid-inducible kinase 3 by a C-terminal hydrophobic motif and Hsp90-Cdc37 chaperone complex.

Serum- and glucocorticoid-inducible kinase 3 (SGK3) mediates a variety of cellular processes including membrane transport, cell proliferation, and survival, and it has been implicated in Akt-independent signaling downstream of oncogenic PIK3CA mutations (activating mutations in the α catalytic subunit of PI3K) in human cancers. However, the regulation of SGK3 is poorly understood. Here we report that SGK3 stability and kinase activation are regulated by the Hsp90-Cdc37 chaperone complex. Hsp90-Cdc37 associates with the kinase domain of SGK3 and acts in concert with a C-terminal hydrophobic motif of SGK3 to prevent Hsp70 association and ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein)-mediated degradation. Phosphorylation of hydrophobic motif triggers release of Cdc37 and concomitant association of 3-phosphoinositide dependent kinase 1 (PDK1) to activate SGK3. Our study provides new insights into regulation of SGK3 stability and activation and the rationale for application of Hsp90 inhibitors in treating SGK3-dependent cancers.

PNRC accumulates in the nucleolus by interaction with B23/nucleophosmin via its nucleolar localization sequence.

PNRC (proline-rich nuclear receptor coregulatory protein) was primarily identified as a coactivator of nuclear receptors (NRs) by our laboratory, which enhances NR-mediated transcription by RNA polymerase II. Recent study has shown that PNRC also stimulates RNA polymerase III-dependent transcription through interaction with the subunit RPC39 of RNA polymerase III. Here, we report that PNRC accumulates in the nucleolus and its depletion by small interfering RNA (siRNA) impairs pre-rRNA transcription by RNA polymerase I. We identified the sequence at position 94-101 ((94)PKKRRKKK(101)) of PNRC as its nucleolar localization sequence (NoLS). Fusion of this sequence to GFP directed GFP to the nucleolus. Characterization of the NoLS revealed that the stretches of six successive basic residues are sufficient to function as a NoLS. Through co-immunoprecipitation assay, we demonstrated that the NoLS is necessary and sufficient to mediate the association of PNRC with B23/nucleophosmin. Moreover, B23 depletion by siRNA disrupted the accumulation of PNRC in the nucleolus. Together, our study indicates that PNRC is a novel nucleolar protein that might be involved in regulation of pre-rRNA synthesis, and it localizes to the nucleolus by interaction with B23 via its NoLS. Our study also suggests that the stretches of six successive basic residues (lysine and/or arginine) could function as NoLS.

SGK3 is an estrogen-inducible kinase promoting estrogen-mediated survival of breast cancer cells.

Serum- and glucocorticoid-inducible kinase 3 (SGK3) is a protein kinase of the AGC family of protein kinase A, protein kinase G, and protein kinase C and functions downstream of phosphatidylinositol 3-kinase (PI3K). Recent study revealed that SGK3 plays a pivotal role in Akt/protein kinase B independent signaling downstream of oncogenic PI3KCA mutations in breast cancer. Here we report that SGK3 is an estrogen receptor (ER) transcriptional target and promotes estrogen-mediated cell survival of ER-positive breast cancer cells. Through a meta-analysis on 22 microarray studies of breast cancer in the Oncomine database, we found that the expression of SGK3 is significantly higher (5.7-fold, P < 0.001) in ER-positive tumors than in ER-negative tumors. In ER-positive breast cancer cells, SGK3 expression was found to be induced by 17ß-estradiol (E(2)) in a dose- and time-dependent manner, and the induction of SGK3 mRNA by E(2) is independent of newly synthesized proteins. We identified two ERα-binding regions at the sgk3 locus through chromatin immunoprecipitation with massively parallel DNA sequencing. Promoter analysis revealed that ERα stimulates the activity of sgk3 promoters by interaction with these two ERα-binding regions on E(2) treatment. Loss-of-function analysis indicated that SGK3 is required for E(2)-mediated cell survival of MCF-7 breast carcinoma cells. Moreover, overexpression of SGK3 could partially protect MCF-7 cells against apoptosis caused by antiestrogen ICI 182,780. Together, our study defines the molecular mechanism of regulation of SGK3 by estrogen/ER and provides a new link between the PI3K pathway and ER signaling as well as a new estrogen-mediated cell survival mechanism mediated by SGK3 in breast cancer cells.

Characterization of the weak estrogen receptor alpha agonistic activity of exemestane.

Third generation aromatase inhibitors (AI) have shown good clinical efficacy in comparison to the anti-estrogen tamoxifen. The steroidal AI, exemestane (EXE) has previously been shown to act as an androgen, but this report demonstrates the estrogen-like activity of EXE. Based on genome-wide microarray analysis, high correlation was seen between EXE-Only (EXE O, hormone-free) and hormone-containing AI-resistant lines. In addition, the top regulated genes in the EXE O lines were mostly estrogen-responsive genes. This estrogen-like activity of EXE was further validated using estrogen receptor (ER) activity assays, where in comparison to 17beta-estradiol (E2), EXE was able to induce ER activity, though at a higher concentration. Also, this EXE-mediated ER activity was blocked by the ER antagonist ICI as well as the ERalpha-specific antagonist methyl-piperidino-pyrazole (MPP). Similarly, EXE was able to induce proliferation of breast cancer cell lines, MCF-7 and MCF-7aro, as well as activate transcription of known estrogen-responsive genes, i.e., PGR, pS2 and AREG. These results suggest that EXE does have weak estrogen-like activity.

A new therapeutic strategy against hormone-dependent breast cancer: the preclinical development of a dual aromatase and sulfatase inhibitor.

PURPOSE: The production of E2 is paramount for the growth of estrogen receptor-positive breast cancer. Various strategies have been used, including the use of enzyme inhibitors against either aromatase (AROM) or steroid sulfatase (STS), in an attempt to ablate E2 levels. Both these enzymes play a critical role in the formation of estrogenic steroids and their inhibitors are now showing success in the clinic. EXPERIMENTAL DESIGN: We show here, in a xenograft nude mouse model, that the inhibition of both enzymes using STX681, a dual AROM and STS inhibitor (DASI), is a potential new therapeutic strategy against HDBC. MCF-7 cells stably expressing either AROM cDNA (MCF-7(AROM)) or STS cDNA (MCF-7(STS)) were generated. Ovariectomized MF-1 female nude mice receiving s.c. injections of either androstenedione (A(4)) or E2 sulfate and bearing either MCF-7(AROM) or MCF-7(STS) tumors were orally treated with STX64, letrozole, or STX681. Treatment was administered for 28 days. Mice were weighed and tumor measurements were taken weekly. RESULTS: STX64, a potent STS inhibitor, completely blocked MCF-7(STS) tumor growth but failed to attenuate MCF-7(AROM) tumor growth. In contrast, letrozole inhibited MCF-7(AROM) tumors but had no effect on MCF-7(STS) tumors. STX681 completely inhibited the growth of both tumors. AROM and STS activity was also completely inhibited by STX681, which was accompanied by a significant reduction in plasma E2 levels. CONCLUSIONS: This study indicates that targeting both the AROM and the STS enzyme with a DASI inhibits HDBC growth and is therefore a potentially novel treatment for this malignancy.

Identification and characterization of PNRC splicing variants.

Nuclear receptor (NR) dependent transcriptional action requires recruitment of diverse factors characterized as coregulators. PNRC (proline-rich nuclear receptor coregulatory protein) is a member of coregulators that are capable of potentiating the transcriptional activity of NRs. Here we identified three human PNRC splicing variants designated PNRC1c, PNRC1d and PNRC1f. PNRC1c and PNRC1f are generated through alternative recognition of the 3'-splice site in exon 1, leading to in-frame deletion of 79 amino acids (aa) and an altered reading frame, respectively. PNRC1d is generated through the alternate promoter usage and forms a truncated protein containing C-terminus 142 aa of full-length PNRC. These isoforms differ in their abilities to bind NRs and potentiate NR mediated transcriptions. Moreover, PNRC1d can modulate the activity of full-length PNRC in enhancing ER mediated transcription. Our results suggest that PNRC exists as functionally distinct isoforms and alternative splicing serves as a regulatory mechanism of PNRC coactivator activity.

MCF-7aro/ERE, a novel cell line for rapid screening of aromatase inhibitors, ERalpha ligands and ERRalpha ligands.

We have previously generated a breast cancer cell line, MCF-7aro, which over-expresses aromatase and is also ER positive. Recently, this MCF-7aro cell line was stably transfected with a promoter reporter plasmid, pGL3-Luc, containing three repeats of estrogen responsive element (ERE). Experiments using MCF-7aro/ERE have demonstrated that it is a novel, non-radioactive screening system for aromatase inhibitors (AIs), ERalpha ligands and ERRalpha ligands. The screening is carried out in a 96-well plate format. To evaluate this system, the cells were cultured overnight in charcoal-dextran stripped FBS medium supplemented with 0.1 nM testosterone or 17beta-estradiol, and various concentrations of antiestrogens or AIs. We found that the luciferase activity was induced when the cells were cultured either in the presence of testosterone or 17beta-estradiol. Furthermore, a 50% decrease in luciferase activity could be achieved when the cells were cultured in the presence of testosterone together with letrozole, anastrozole, tamoxifen or fulvestrant (concentrations being 75 nM, 290 nM, 100 nM, and 5 nM, respectively), compared to the testosterone-only cultured cells. Using this assay system, we confirmed that 3(2'-chlorophenyl)-7-methoxy-4-phenylcoumarin is an agonist of ER. Furthermore, genestein has been shown to be a ligand of ERRalpha because its binding could be blocked by an ERRalpha inverse agonist, XCT790. These results indicate that MCF-7aro/ERE is a novel cell line for rapid screening of AIs, ERalpha ligands and ERRalpha ligands.

Fast skeletal muscle troponin I is a co-activator of estrogen receptor-related receptor alpha.

ERRalpha (estrogen receptor-related receptor alpha) is a member of the nuclear receptor superfamily. To further our understanding of the detailed molecular mechanism of transcriptional regulation by ERRalpha, we searched for ERRalpha-interacting proteins using a yeast two-hybrid system by screening a human mammary gland cDNA expression library with the ligand-binding domain (LBD) of ERRalpha as the "bait". Fast skeletal muscle troponin I (TNNI2), along with several known nuclear receptor co-activators, were isolated. We demonstrated that TNNI2 localizes to the cell nucleus and interacts with ERRalpha in co-immunoprecipitation experiments. GST pull-down assays also revealed that TNNI2 interacts directly with ERRalpha. Through luciferase reporter gene assays, TNNI2 was found to enhance the transactivity of ERRalpha. Combining mutagenesis and yeast two-hybrid assays, we mapped the ERRalpha-interacting domain on TNNI2 to a region encompassing amino acids 1-128. These findings reveal a new function for TNNI2 as a co-activator of ERRalpha.

Transcriptional regulation of the human PNRC promoter by NFY in HepG2 cells.

PNRC (proline-rich nuclear receptor co-activator) was previously identified using bovine SF-1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC has been demonstrated to be a novel co-activator for multiple nuclear receptors. To understand the molecular mechanisms that regulate the expression of human PNRC gene, in this study, potential transcriptional start site was determined by 5' RACE analysis. Functional analysis of the 5' flanking region of the human PNRC gene by deletion mutagenesis, transient transfection and luciferase assays revealed that the -123/+27 region is the minimal promoter of the human PNRC gene. Within this promoter region, there is one putative binding site for the transcription factor NFY (nuclear factor Y). EMSA and ChIP analyses demonstrated the specific binding of NFY protein to the human PNRC promoter. Transient transfection and luciferase assays further revealed that over-expression of NFY represses promoter activity of PNRC gene in a dose-dependent manner. These results indicate that the transcription factor NFY specifically binds to promoter region of PNRC and negatively regulates the transcription of the human PNRC gene.

MicroRNAs play a role in the development of human hematopoietic stem cells.

MicroRNAs (miRNAs) are a class of recently discovered noncoding RNA genes that post-transcriptionally regulate gene expression. It is becoming clear that miRNAs play an important role in the regulation of gene translation during development. However, in mammals, expression data are principally based on whole tissue analysis and are still very incomplete. We isolated CD34(+)CD38(-) hematopoietic stem cells (HSCs) from human umbilical cord blood, on the basis of cell-surface markers using fluorescence-activated cell sorting (FACS). Also, CD34(+) subpopulation was FACS isolated as the control. Next, using microarray containing oligonucleotides corresponding to 517 miRNAs from human, mouse, and rat genomes, we obtained miRNA gene expression profiles of both subpopulations. We focused on the HSCs correlative miRNAs with comparison to the control. The miRNAs of particular interest were confirmed by real-time RT-PCR. HSCs-overexpressed hsa-miR-520h and underexpressed hsa-miR-129 were selected for target prediction and analysis. The result showed that EIF2C3 and CAMTA1, genes related to miRNAs processing or transcription regulation, were proved to be real targets for hsa-miR-129. And ABCG2, involved in stemness maintaining, a real target for hsa-miR-520h. Finally, we chose hsa-miR-520h, enriched in HSCs but low in CD34(+) cells, for functional characterization, because of its possible role in differentiation of HSCs by regulating ABCG2. As a result, hsa-miR-520h transduction into CD34(+) cells greatly increased number of different progenitor colonies in Colony-Forming-Cell assays, suggesting that hsa-miR-520h may promote differentiation of HSCs into progenitor cells by inhibiting ABCG2 expression. This study paves the way for identifying HSC-specific miRNAs and their roles in HSC development.

Modulation of in situ estrogen synthesis by proline-, glutamic acid-, and leucine-rich protein-1: potential estrogen receptor autocrine signaling loop in breast cancer cells.

In situ estrogen synthesis is implicated in tumor cell proliferation through autocrine or paracrine mechanisms especially in postmenopausal women. Several recent studies demonstrated activity of aromatase, an enzyme that plays a critical role in estrogen synthesis in breast tumors. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1/MNAR) is an estrogen receptor (ER) coregulator, and its expression is deregulated in breast tumors. In this study, we examined whether PELP1 promotes tumor growth by promoting local estrogen synthesis using breast cancer cells (MCF7) that stably overexpress PELP1. Immunohistochemistry revealed increased aromatase expression in MCF7-PELP1-induced xenograft tumors. Real-time PCR analysis showed enhanced activation of the aromatase promoter in MCF7-PELP1 clones compared with MCF7 cells. Using a tritiated-water release assay, we demonstrated that MCF7-PELP1 clones exhibit increased aromatase activity compared with control MCF-7 cells. PELP1 deregulation uniquely up-regulated aromatase expression via activation of aromatase promoter I.3/II, and growth factor signaling enhanced PELP1 activation of aromatase. PELP1-mediated induction of aromatase requires functional Src and phosphatidylinositol-3-kinase pathways. Mechanistic studies revealed that PELP1 interactions with ER-related receptor-alpha and proline-rich nuclear receptor coregulatory protein 2 lead to activation of aromatase. Immunohistochemistry analysis of breast tumor array showed increased expression of aromatase in ductal carcinoma in situ and node-positive tumors compared with no or weak expression in normal breast tissue. Fifty-four percent (n = 79) of PELP1-overexpressing tumors also overexpressed aromatase compared with 36% (n = 47) in PELP1 low-expressing tumors. Our results suggest that PELP1 regulation of aromatase represents a novel mechanism for in situ estrogen synthesis leading to tumor proliferation by autocrine loop and open a new avenue for ablating local aromatase activity in breast tumors.

Nuclear receptor coactivator PNRC2 regulates energy expenditure and adiposity.

PNRC2 was identified in our laboratory as a general cofactor for nuclear receptors. To better characterize the physiological function of PNRC2, we used gene-targeting technology to generate PNRC2-null mice (PNRC2(-/-) mice). These PNRC2(-/-) mice are viable and fertile. PNRC2-null mice, especially male mice, are lean and are resistant to high fat diet-induced obesity but without the induction of insulin resistance. Male mice devoid of PNRC2 protein have a higher metabolic rate than wild-type mice. They consume more oxygen and produce more heat. Consistent with reduced adipose mass, the levels of leptin are lower in PNRC2(-/-) mice. This study provides evidence that PNRC2 plays one or more important roles in controlling the energy balance between energy storage and energy expenditure. PNRC2 may be a new target in the treatment of obesity and related metabolic diseases.

PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription.

BACKGROUND: PNRC transcriptionally regulates a wide range of RNA polymerase (pol) II-transcribed genes by functioning as a nuclear receptor coactivator. To search for additional PNRC-interacting proteins other than nuclear receptors, a PNRC fragment was used as bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. RESULTS: RNA pol III/RPC39 fragments were repeatedly identified as PNRC-interacting partners in two independent screenings. The interaction between these RPC39 fragments and PNRC was further confirmed in the independent yeast two-hybrid assays. The association of endogenous PNRC and RPC39 in MCF7 cells was demonstrated by co-immunoprecipitation. Furthermore, ChIP analysis detected co-recruitment of PNRC and RPC39 to tRNA and U6 RNA promoters. The biological consequence of the interaction between PNRC and RPC39 was further studied. Overexpression of PNRC, either by transient or stable transfection, increased RNA pol III-dependent transcription in MCF7 cells, while a decrease in transcription in MCF7 cells treated with PNRC/siRNA was observed. CONCLUSION: Here, we demonstrate that human PNRC stimulates RNA pol III transcription through its interaction with the subunit RPC39 of RNA pol III.PNRC is a unique coactivator that has profound effects on many aspects of cellular function by directly influencing both RNA pol II- and RNA pol III-dependent transcription.

Functional analysis on the 5'-flanking region of human FXR gene in HepG2 cells.

The farnesoid X receptor (FXR) is a bile acid (BA)-activated nuclear receptor that plays a major role in the regulation of BA and lipid metabolism. Although modulation of FXR expression has been reported, the mechanisms underlying the regulation of human FXR are yet unclear. Functional assays showed that the -150/+29 nucleotides region from the first nucleotide at the Exon I is the minimal promoter of the human FXR gene by the technique of serial deletion and point mutants of the 5'-flanking region. Chromatin immunoprecipitation analysis and electrophoretic mobility shift assay revealed that hepatic nuclear factor 1alpha (HNF1alpha) interacted with the region. Co-transfection of the promoter with HNF1alpha expression vectors enhanced promoter activity of FXR gene. Over-expression of HNF1alpha up-regulated FXR expression in HepG2 cells. These data indicate that (a) the identified HNF1alpha binding site serves as a positive regulatory sequence, (b) the binding site is functionally active both in vivo and in vitro, and (c) the transcription factor HNF1alpha that binds to this site plays an important role in the regulation of human FXR promoter activity.

Molecular basis for the aromatization reaction and exemestane-mediated irreversible inhibition of human aromatase.

Aromatase converts androgens to aromatic estrogens. Aromatase inhibitors have been used as first-line drugs in the treatment of hormone-dependent breast cancer. Structural basis of the aromatization reaction and drug recognition by aromatase has remained elusive because of its unknown three-dimensional structure. In this study, recombinant human aromatase was expressed and purified from Escherichia coli. Using this purified and active preparation, the three-dimensional folding of aromatase was revealed by proteomic analysis. Combined with site-directed mutagenesis, several critical residues involved in enzyme catalysis and suicide inhibition by exemestane were evaluated. Based on our results, a new clamping mechanism of substrate/exemestane binding to the active site is proposed. These structure-function studies of aromatase would provide useful information to design more effective aromatase inhibitors for the prevention and the treatment of hormone-dependent breast cancer.

The molecular basis of the interaction between the proline-rich SH3-binding motif of PNRC and estrogen receptor alpha.

PNRC and PNRC2 are members of a new family of nuclear receptor coactivators. We systematically determined the molecular basis and the structure/function relationship for the PNRC-ERalpha interaction. PNRC was found to interact with ERalpha mainly through its C-terminus region, amino acids 270-327, and an SH3-binding motif within this region was shown to be essential for PNRC to interact with and function as coactivator of ERalpha. The importance of the flanking sequences of SH3-binding motif in the interaction between PNRC and ERalpha was also investigated. The PNRC-interacting domain(s) on ERalpha was also mapped. PNRC was found to interact with both AF1 and LBD of ERalpha, and to function as a coactivator for both AF1 and AF2 transactivation functions. The interaction of ERalpha mutants, I358R, K362A, V376R, L539R and E542K, with PNRC/PNRC2 was further investigated. ERalpha/HBD/V376R could bind to PNRC or PNRC2, with similar affinity as wild-type ERalpha/HBD, and the transactivation activity of ERalpha/V376R was enhanced 5-fold by PNRC. Since GRIP1, a well-characterized coactivator, was found not to be able to enhance the transactivation function of this mutant, our results indicate that the PNRC-ERalpha interaction interface is not exactly identical to that of GRIP1-ERalpha interaction.