Caroline J. Speed

Liver receptor homologue-1 (LRH-1) regulates expression of aromatase in preadipocytes

Estrogen biosynthesis from C19steroids is catalyzed by aromatase cytochrome P450. Aromatase is expressed in breast adipose tissue through the use of a distal, cytokine-responsive promoter (promoter I.4). Breast tumors, however, secrete soluble factors that stimulate aromatase expression through an alternative proximal promoter, promoter II. In other estrogenic tissues such as ovaries, transcription from promoter II requires the presence of the Ftz-F1 homologue steroidogenic factor-1 (SF-1); adipose tissue, however, does not express SF-1. We have explored the hypothesis that in adipose tissue, an alternative Ftz-F1 family member, liver receptor homologue-1 (LRH-1), substitutes for SF-1 in driving transcription from promoter II. In transient transfection assays using 3T3-L1 preadipocytes, promoter II reporter constructs were modestly (2–3-fold) stimulated by either treatment with activators of protein kinases A or C (PKA/C) or by cotransfection with LRH-1. In combination, these treatments synergistically activated promoter II (>30-fold). Induction by LRH-1 (but not by PKA/C) required an AGGTCA motif at −130 base pairs, to which LRH-1 bound in gel shift assays. Activity of GAL4-LRH-1 fusion proteins was not altered by activators of PKA or PKC. Quantitative real-time PCR revealed that LRH-1 (but not SF-1) is expressed in the preadipocyte fraction of human adipose tissue at levels comparable with that of liver. Differentiation of cultured human preadipocytes into mature adipocytes was associated with a time-dependent induction of peroxisome proliferator-activated receptor-γ (PPARγ), and rapid loss of LRH-1 and aromatase expression. We conclude that LRH-1 is a preadipocyte-specific nuclear receptor that regulates expression of aromatase in adipose tissue. Alterations in LRH-1 expression and/or activity in adipose tissue could therefore have considerable effects on local estrogen production and breast cancer development.

Regulation of aromatase expression by the nuclear receptor LRH-1 in adipose tissue

Aromatase is the enzyme responsible for estrogen production, and is the product of the CYP19 gene. This gene is under the control of many tissue-specific promoters, each of which is regulated by different cohorts of factors. In normal breast adipose tissue, relatively low levels of aromatase are expressed via the action of the adipose specific promoter I.4. Breast tumor-derived factors such as prostaglandin E(2) (PGE(2)) strongly stimulate aromatase expression via an alternative promoter, promoter II, leading to increased estrogenic drive and tumor growth. Understanding the mechanisms that regulate promoter II activity in tumorous breast may therefore identify new targets for breast cancer drug discovery. The current study describes the role of the orphan nuclear receptor LRH-1 and its co-regulators in modulating aromatase expression in breast adipose tissue.

Cloning and Characterization of a 72-kDa Inositol-polyphosphate 5-Phosphatase Localized to the Golgi Network

The inositol-polyphosphate 5-phosphatase enzyme family removes the 5-position phosphate from both inositol phosphate and phosphoinositide signaling molecules. We have cloned and characterized a novel 5-phosphatase, which demonstrates a restricted substrate specificity and tissue expression. The 3.9-kb cDNA predicts for a 72-kDa protein with an N-terminal proline rich domain, a central 5-phosphatase domain, and a C-terminal CAAX motif. The 3.9-kilobase mRNA showed a restricted expression but was abundant in testis and brain. Antibodies against the sequence detected a 72-kDa protein in the testis in the detergent-insoluble fraction. Indirect immunofluorescence of the Tera-1 cell line using anti-peptide antibodies to the 72-kDa 5-phosphatase demonstrated that the enzyme is predominantly located to the Golgi. Expression of green fluorescent protein-tagged 72-kDa 5-phosphatase in COS-7 cells revealed that the enzyme localized predominantly to the Golgi, mediated by the N-terminal proline-rich domain, but not the C-terminal CAAX motif. In vitro, the protein inserted into microsomal membranes on the cytoplasmic face of the membrane. Immunoprecipitated recombinant 72-kDa 5-phosphatase hydrolyzed phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,5-bisphosphate, forming phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3-phosphate, respectively. We propose that the novel 5-phosphatase hydrolyzes phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,5-bisphosphate on the cytoplasmic Golgi membrane and thereby may regulate Golgi-vesicular trafficking.

Tissue-specific estrogen biosynthesis and metabolism

Abstract: While the ovaries are the principal source of systemic estrogen in the premenopausal nonpregnant woman, other sites of estrogen biosynthesis are present throughout the body and these become the major sources of estrogen beyond menopause. These extragonadal sources of estrogen are small, but may play an important, though hitherto largely unrecognized, physiological and pathophysiological role. Aromatase activity in extragonadal sites contributes to this source of estrogen and may contribute to breast tumor development and/or growth. Selective aromatase modulators (SAMs) may have a role to play in the treatment of estrogen‐dependent diseases, such as breast cancer.

Underexpression of the 43 kDa inositol polyphosphate 5-phosphatase is associated with cellular transformation.

The 43 kDa inositol polyphosphate 5‐phosphatase (5‐phosphatase) hydrolyses the second messenger molecules inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4]. We have underexpressed the 43 kDa 5‐phosphatase by stably transfecting normal rat kidney cells with the cDNA encoding the enzyme, cloned in the antisense orientation into the tetracycline‐inducible expression vector pUHD10–3. Antisense‐transfected cells demonstrated a 45% reduction in Ins(1,4,5)P3 5‐phosphatase activity in the total cell homogenate upon withdrawal of tetracycline, and an approximately 80% reduction in the detergent‐soluble membrane fraction of the cell, as compared with antisense‐transfected cells in the presence of tetracycline. Unstimulated antisense‐transfected cells showed a concomitant 2‐fold increase in Ins(1,4,5)P3 and 4‐fold increase in Ins(1,3,4,5)P4 levels. The basal intracellular calcium concentration of antisense‐transfected cells (170 +/− 25 nM) was increased 1.9‐fold, compared with cells transfected with vector alone (90 +/− 25 nM). Cells underexpressing the 43 kDa 5‐phosphatase demonstrated a transformed phenotype. Antisense‐transfected cells grew at a 1.7‐fold faster rate, reached confluence at higher density and demonstrated increased [3H]thymidine incorporation compared with cells transfected with vector alone. Furthermore, antisense‐transfected cells formed colonies in soft agar and tumours in nude mice. These studies support the contention that a decrease in Ins(1,4,5)P3 5‐phosphatase activity is associated with cellular transformation.

Aromatase, Nuclear Receptors, Adipose Tissue, And Breast Cancer

Models of estrogen insufficiency have revealed new and often unexpected roles for estradiol in both females and males [1]. These models include natural mutations in humans of the aromatase gene, of which there are some ten cases known, of whom two are men, as well as one man with a mutation in the estrogen receptor (ER) α. They also include mice with targeted disruptions of the ERα and ERβ, the double ERα and β knockout [2 – 4], as well as the aromatase knockout (ArKO) mouse [5]. Some of these roles challenge the definition of the terms estrogen and androgen. For example the lipid and carbohydrate phenotype of estrogen insufficiency is nonsexually dimorphic and appears to apply equally to males and females [6,7], as does the bone phenotype of undermineralization and failure of epiphyseal closure. Even more dramatically, the role of estradiol in male germ cell development would indicate that at least in this local context estradiol would be more appropriately defined as an androgen [8]. The second important point is that in men and in postmenopausal women when the ovaries cease to produce estrogens, estradiol does not function as a circulating hormone. It is no longer an endocrine factor, instead estradiol is produced in a number of extragonadal sites and acts locally at these sites as a paracrine or even intracrine factor [9,10]. These sites include the mesenchymal cells of adipose tissue, osteoblasts and chondrocytes of bone, numerous sites in the brain, and also the Leydig cells and germ cells of the testes. Thus circulating levels of estrogens in postmenopausal women and in men do not drive estrogen action.