There is growing awareness that androgens and estrogens have general metabolic roles that are not directly involved in reproductive processes. These include actions on vascular function, lipid and carbohydrate metabolism, as well as bone mineralization and epiphyseal closure in both sexes. In postmenopausal women, as in men, estrogen is no longer solely an endocrine factor but instead is produced in a number of extragonadal sites and acts locally at these sites in a paracrine and intracrine fashion. These sites include breast, bone, vasculature, and brain. Within these sites, aromatase action can generate high levels of estradiol locally without significantly affecting circulating levels. Circulating C19 steroid precursors are essential substrates for extragonadal estrogen synthesis. The levels of these androgenic precursors decline markedly with advancing age in women, possible from the mid-to-late reproductive years. This may be a fundamental reason why women are at increased risk for bone mineral loss and fracture, and possibly decline of cognitive function, compared with men. Aromatase expression in these various sites is under the control of tissue-specific promotors regulated by different cohorts of transcription factors. Thus in principle, it should be possible to develop selective aromatase modulators (SAMs) that block aromatase expression, for example, in breast, but allow unimpaired estrogen synthesis in other tissues such as bone.
Estrogen biosynthesis from C 19 steroids 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-␥ (PPAR␥), 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.
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 Cterminal 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.The inositol-polyphosphate 5-phosphatases (5-phosphatases) 1 are a large family of enzymes that remove the 5-position phosphate from the inositol ring of phosphatidylinositols including phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), and the inositol phosphates, inositol 1,4,5-trisphosphate (Ins(1,4,5)-P 3 ) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 ). Nine distinct mammalian 5-phosphatases have been identified and characterized, while four 5-phosphatases have been described in Saccharomyces cerevisiae. All 5-phosphatases are defined by the presence of a conserved 300-amino acid domain, which contains two signature motifs, proposed to mediate substrate binding and catalysis (1, 2). Although all 5-phosphatase enzymes contain these signature motifs, there is considerable diversity in the substrate specificity of 5-phosphatase isoforms. The enzyme family has been subclassified on this basis into four types, I-IV. The type I enzymes, characterized by the 43-kDa 5-phosphatase (also called 5-phosphatase I), hydrolyze Ins(1,4,5)P 3 and Ins(1,3,4,5)P 4 but not any of the 5-position phosphoinositide substrates; the type II 5-phosphatases, including synaptojanin, 5-phosphatase II (originally designated the 75-kDa 5-phosphatase), and the protein product of the oculocerebrorenal syndrome (OCRL) gene, hydrolyze PtdIns-(4,5)P 2 , PtdIns(3,4,5)P 3 , Ins(1,3,4,5)P 4 ...
Corresponding authorThe 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the second messenger molecules inositol 1,4,4,5)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 -80 % reduction in the detergentsoluble 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 5phosphatase 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.
The inositol polyphosphate 5-phosphatases are an enlarging family of enzymes that terminate the signals generated by the phosphoinositide kinases and phospholipase C. Given the diverse signalling functions of both the polyphosphoinositides and Ins(1,4,5)P3, it is predicted that the 5-phosphatases will play a critical role in regulating many cellular events, in particular membrane trafficking and cell growth.
The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyzes and thereby inactivates the second messenger molecules inositol 1,4,5-trisphosphate -Ins(1,4,5)P3- and inositol 1,3,4,5-tetrakisphosphate in a signal terminating reaction. Recent studies have shown that the platelet protein pleckstrin forms a complex with the 43 kDa 5-phosphatase and activates Ins(1,4,5)P3 hydrolysis 2-fold [Auethavekiat, V., Abrams, C. S., & Majerus, P. W. (1997) J. Biol. Chem. 272, 1786-1790]. We now show that another platelet protein, 14-3-3zeta, forms a complex with the 43 kDa 5-phosphatase and thereby activates the hydrolysis of Ins(1,4,5)P3. Both pleckstrin and 14-3-3zeta contain one or more pleckstrin-homology domains, both are present in platelet cytosol, and both dimerize and form complexes with other signalling proteins. Purified platelet pleckstrin and 14-3-3zeta enhanced the rate of the hydrolysis of Ins(1,4,5)P3 by the 43 kDa 5-phosphatase 1.9- and 3.8-fold, respectively, but did not activate the 75 kDa 5-phosphatase. We have demonstrated that the mechanism of 5-phosphatase activation by 14-3-3zeta results from specific complex formation between the 43 kDa 5-phosphatase and 14-3-3zeta. Recombinant 43 kDa 5-phosphatase bound to recombinant glutathione S-transferase (GST)/14-3-3zeta fusion protein, but not GST alone, immobilized on glutathione-Sepharose. A potential 14-3-3 binding motif was located in the 43 kDa, but not the 75 kDa, 5-phosphatase. The motif "363RSESEE" is present in close proximity to the proposed catalytic domain of the 43 kDa 5-phosphatase. A synthetic peptide corresponding to the putative 14-3-3 binding motif demonstrated specific, saturable binding to purified 125I-14-3-3, with a Kd of 92 nM. In addition, platelet cytosolic 5-phosphatase bound to recombinant 14-3-3zeta immobilized on glutathione-Sepharose. Thus, 14-3-3zeta serves in human platelets to activate the 43 kDa 5-phosphatase and may thereby function to prevent generation of Ins(1,4,5)P3 -mediated calcium release in unstimulated platelets.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.