Where is aromatase expression

Calcitriol 1,dihydroxyvitamin D 3 , the hormonally active form of vitamin D, suppresses proliferation and promotes apoptosis and differentiation of malignant cells and inhibits tumor angiogenesis and invasion, raising the possibility of its use as an anticancer agent 9 — Calcitriol has been shown to increase aromatase expression in human osteoblasts and fibroblasts 14 , Interestingly, our observations reveal that calcitriol down-regulates aromatase expression in BCa cells.

In BCa cells, calcitriol also decreases the levels of biologically active prostaglandins PGs , which are major stimulators of estrogen production, by suppressing the expression of the PG-synthesizing enzyme cyclooxygenase COX -2 and increasing that of hydroxyprostaglandin dehydrogenase PGDH , which initiates PG catabolism.

The mechanism of calcitriol-mediated aromatase down-regulation in BCa cells therefore appears to be 2-fold: 1 a direct transcriptional repression of the aromatase gene by calcitriol and 2 an indirect effect to reduce aromatase transcription by reducing the levels PGs, which are known stimulators of aromatase transcription in BCa cells 1.

Importantly our data reveal that calcitriol regulation of aromatase expression is tissue specific with a significant suppression in BCa cells and adipocytes surrounding breast tumors, whereas a substantial increase is seen in osteosarcoma cells, used as a model for bone cells.

Due to its ability to suppress both the synthesis and biological activity of estrogens, calcitriol exhibits significant inhibitory effects on BCa cell growth. Furthermore, in this paper, we show that combinations of calcitriol and AIs are cooperative and cause enhanced inhibition of BCa cell growth. We therefore postulate that combining an AI enzyme inhibitor with calcitriol transcriptional regulator would be a useful therapeutic strategy in BCa.

We hypothesize that cumulatively these actions contribute to the beneficial effect of combining calcitriol with an AI to treat BCa patients.

Herndon, VA. Total RNA was isolated from cells treated with vehicle 0. Promoter-specific aromatase transcript levels were determined using primers designed for each of the promoter regions of aromatase exon I and PCR conditions as described Aromatase enzyme activity has been assessed in cell cultures and recombinant enzyme preparations by measuring the conversion of androgenic substrates androstenedione or testosterone T into estrone or estradiol E 2 24 , We determined aromatase activity in cultures of BCa and osteosarcoma cells by measuring the conversion of T to E 2.

After 24 h, the growth media were replaced with serum-free medium OptiMEM; Life Technologies containing the aromatase substrate T n m and various concentrations of calcitriol or letrozol and incubated for 48 h. Aromatase activity was expressed as picograms of E 2 synthesized per hour per milligram protein. COX-2 proteins was visualized as an approximately kDa immunoreactive band.

After 24 h the growth media were replaced with serum-free medium OptiMEM; Life Technologies containing vehicle or 10 or n m calcitriol. A plasmid containing the aromatase promoter I.

After transfections the cells were treated with vehicle 0. Reporter and renilla luciferase activities were measured using the dual-luciferase assay kit Promega. ZR cells treated with vehicle or calcitriol were subjected to ChIP analysis as described Fresh media and the drugs were replenished every other day and cell proliferation was assessed at the end of 6 d by measuring the DNA content When tumors attained a size of about 70 mm 3 or more, the mice were given ip injections of vehicle 0.

This calcitriol dosing regimen has been established to be effective in eliciting changes in tumor gene expression in xenograft models of squamous cell carcinoma and prostate cancer in nude mice 28 , IC 50 values for growth inhibition by individual drugs were calculated from dose-response data.

Dose-response analyses were also carried out for the combinations of calcitriol and the various AIs. Higher calcitriol concentrations up to n m did not further decrease aromatase mRNA levels in these cells.

This calcitriol concentration n m was used to assess its effects in MDA-MB cells in various experiments described below. In all other cells 10 n m calcitriol was used as a single high dose unless otherwise stated. The levels of promoter-specific aromatase transcripts were determined in MCF-7 cells Fig. Tissue-selective regulation of aromatase mRNA by calcitriol. B, Calcitriol decreases various promoter-specific aromatase transcript levels in BCa cells.

The levels of promoter-specific aromatase transcripts were determined in MCF-7 cells treated as in A. C, Calcitriol increases aromatase mRNA in human osteosarcoma and ovarian cancer cells. D, Calcitriol increases various promoter-specific aromatase transcript levels in osteosarcoma cells. MG cells were treated as in A and the levels of various promoter-specific aromatase transcripts determined.

E and F, Calcitriol decreases aromatase expression in 3T3-L1 preadipocytes. The cultures were then exposed to a differentiation-inducing medium with and without calcitriol addition for the final 24 h and aromatase mRNA was measured E, Post-diff. In contrast to BCa cells, in the human osteosarcoma cells MG and SaoS-2, which exhibit osteoblastic features in culture, calcitriol induced an approximately fold and approximately 7-fold increase in total aromatase mRNA levels, respectively Fig.

The levels of transcripts derived from the bone-specific aromatase promoter I. Calcitriol also increased promoter I. To examine calcitriol effects in adipocytes, we used 3T3-L1 mouse fibroblasts preadipocytes , which can be induced to undergo differentiation into mature adipocytes by manipulating culture conditions.

Calcitriol treatment of undifferentiated preadipocytes resulted in a significant decrease in aromatase mRNA Fig. The induction of differentiation Fig. Addition of 10 n m calcitriol to the differentiated cells for 24 h maintained the suppression of aromatase mRNA without causing a further decrease Fig. The effect of calcitriol on aromatase activity in BCa cells and osteosarcoma cells was examined by measuring E 2 production after the addition of the substrate T.

The changes in aromatase activity in the various cells due to calcitriol treatment reflected the changes seen in aromatase mRNA levels Fig. Calcitriol decreased aromatase activity in a dose-dependent manner in MCF-7 cells with an IC 50 of 1. In contrast, in MG osteosarcoma cells, calcitriol at 10 and n m caused an approximately 4-fold and an approximately fold increase in aromatase activity respectively Fig. Tissue-selective regulation of aromatase activity by calcitriol.

A, Calcitriol decreases aromatase activity in MCF-7 cells. MCF-7 cells were treated with vehicle Cal, 0 or various concentrations of calcitriol Cal, 0. Aromatase activity was determined by measuring the concentrations of E 2 formed in the conditioned media by an EIA and expressed as picograms of E 2 formed per hour per milligram protein.

B, Calcitriol decreases aromatase activity in other BCa cells. C, Calcitriol increases aromatase activity in MG cells. MG human osteosarcoma cells were treated with 10 and n m calcitriol and aromatase activity was determined as in A. Calcitriol administration to nude mice carrying MCF-7 xenografts in their mammary fat pads decreased total aromatase mRNA in the xenograft tumors in both intact mice Fig.

No significant change was observed in ovarian aromatase mRNA in the intact mice Fig. COX-2 mRNA expression in the tumors also showed a significant decrease after calcitriol treatment in the intact mice Fig. The same negative trend in COX-2 expression was also seen in OVX mice treated with calcitriol, although the decrease did not attain statistical significance Fig.

Serum calcium levels in the mice receiving calcitriol at a high dose for 3 d assayed approximately 14 h after the final calcitriol injection registered only a slight increase, which, however, was not statistically significant. Vehicle Veh or calcitriol Cal, 0. Total aromatase mRNA was measured in tumors, mammary fat tissue surrounding the tumors, and ovaries. Calcitriol decreases PG synthesis in BCa cells. BCa cells were treated with calcitriol as described in Fig. B, Calcitriol decreases COX-2 protein levels.

BCa cells were treated as described in Fig. PGE 2 levels in conditioned media were measured. In silico analysis of the aromatase promoter I. We transiently transfected a plasmid containing an aromatase promoter I. In MCF-7 cells, addition of Fsk and Bu 2 cAMP caused an approximately 2-fold increase reporter activity, which was significantly attenuated by calcitriol cotreatment Fig.

Direct repression of aromatase transcription through promoter II by calcitriol. A, A schematic diagram of the aromatase promoter I. An aromatase I. Addition of the aromatase substrate T to cell cultures in media depleted of endogenous steroids caused a 2- to 3-fold increase in cell growth Fig. The growth stimulation by T was not abolished by the addition of the androgen receptor AR antagonist bicalutamide Cdx, Fig. Addition of graded concentrations of calcitriol inhibited T stimulation of cell growth Fig.

Complete inhibition down to the growth level in basal media without T was achieved at 10 n m calcitriol. A further increase in calcitriol concentration n m caused a significant reduction in cell growth below basal levels. Similarly when tested individually, the AIs letrozole, anastrozole, and exemestane inhibited T stimulation of cell growth in a dose-dependent manner with IC 50 values of 10—15 n m data not shown.

Enhanced inhibition of BCa cell growth by calcitriol-AI combinations. B—D, Enhanced inhibition of cell growth by calcitriol-AI combinations. Cells were grown as described in A in the absence or presence of calcitriol Cal , exemestane Exe, 6b , anastrozole Ana, 6c , letrozole Let, 6d , or combinations of calcitriol with each AI at the indicated concentrations.

We tested the growth-inhibitory effects of a series of concentrations of calcitriol 0. Figure 6 , B—D, illustrates the effect of combinations of suboptimal concentrations of calcitriol 0. Calcitriol alone at 0. The steroidal AI exemestane Fig. Calcitriol inhibits BCa cell growth and retards the growth of xenografts of human tumors in immunocompromised hosts by multiple pathways 9 , 10 , 13 , Calcitriol reduced total aromatase mRNA levels and decreased aromatase enzymatic activity in BCa cells.

Interestingly, the regulation of aromatase expression by calcitriol is tissue specific. Our data show significant increases in aromatase mRNA and estrogen synthesis in human osteosarcoma cells, confirming earlier reports of calcitriol-mediated up-regulation of aromatase in osteoblasts 14 , Other studies in keratinocytes 34 and prostate cancer cells 35 found no effect of calcitriol on aromatase expression.

Kinuta et al. Their results indicated that calcitriol plays a role in gonadal estrogen synthesis to a large extent by maintaining calcium homeostasis. However, aromatase deficiency in the VDR null mice was not totally corrected by normalization of serum calcium levels, suggesting that the direct regulation of aromatase by calcitriol might also play a role We observed a modest increase in aromatase mRNA in human ovarian cancer cells after calcitriol treatment, whereas in mice bearing BCa tumors, calcitriol administration did not alter ovarian aromatase mRNA.

Studies on calcitriol effects in human ovarian granulosa cells report either no change 37 or an inhibition of aromatase expression Furthermore, as discussed below, calcitriol decreased aromatase expression in mouse preadipocytes and the mammary adipose tissue surrounding human BCa tumors in mice. Distal pup cues evoke dopamine responses in hormonally primed rats in the absence of pup experience or ongoing maternal behavior. Alarcon, G. Adolescent neural response to reward is related to participant sex and task motivation.

Brain Cogn. Altemus, M. Sex differences in anxiety and depression clinical perspectives. Bakker, J. Sexual differentiation of the neuroendocrine mechanisms regulating mate recognition in mammals.

Effects of aromatase mutation ArKO on the sexual differentiation of kisspeptin neuronal numbers and their activation by same versus opposite sex urinary pheromones. Balthazart, J. Steroid control and sexual differentiation of brain aromatase.

Steroid Biochem. Multiple mechanisms control brain aromatase activity at the genomic and non-genomic level. Bell, M. Comparing postnatal development of gonadal hormones and associated social behaviors in rats, mice, and humans. Beyenburg, S. Expression of cytochrome Pscc mRNA in the hippocampus of patients with temporal lobe epilepsy.

Neuroreport 10, — Biegon, A. Unique distribution of aromatase in the human brain: in vivo studies with PET and [N-methylC]vorozole. Synapse 64, — Blakemore, J. Aromatase: contributions to physiology and disease in women and men. Physiology 31, — Boon, W. The multiple roles of estrogens and the enzyme aromatase. Brain Res. Borbelyova, V. Sex-dependent effects of letrozole on anxiety in middle-aged rats. Bowen, R. Effects of aromatase inhibition on the physical activity levels of male mice.

Steroids Horm. Bowers, J. A developmental sex difference in hippocampal neurogenesis is mediated by endogenous oestradiol. Sex Differ. Bulun, S. Aromatase, breast cancer and obesity: a complex interaction. Trends Endocrinol. Butler, H. Cao, M. Sexually dimorphic and ontogenetic expression of dmrt1, cyp19a1a and cyp19a1b in Gobiocypris rarus. A Mol. Chavez, C. Psychotropic drug-induced locomotor hyperactivity and prepulse inhibition regulation in male and female aromatase knockout ArKO mice: role of dopamine D1 and D2 receptors and dopamine transporters.

Psychopharmacology , — Cherrier, M. Testosterone supplementation improves spatial and verbal memory in healthy older men. Neurology 57, 80— The role of aromatization in testosterone supplementation: effects on cognition in older men.

Neurology 64, — Choate, J. Prenatal inhibition of aromatase activity affects luteinizing hormone feedback mechanisms and reproductive behaviors of adult guinea pigs. Connolly, P. Aromatase activity in developing guinea pig brain: ontogeny and effects of exogenous androgens. Corbo, R. Dalla, C. Oestrogen-deficient female aromatase knockout ArKO mice exhibit depressive-like symptomatology. El Hajj Chehadeh, S.

Gestational methyl donor deficiency alters key proteins involved in neurosteroidogenesis in the olfactory bulbs of newborn female rats and is associated with impaired olfactory performance. Fester, L. Synaptopodin is regulated by aromatase activity. Garcia-Segura, L. Aromatase in the brain: not just for reproduction anymore. Aromatase expression by astrocytes after brain injury: implications for local estrogen formation in brain repair.

Neuroscience 89, — Gray, T. Functional and anatomical relationships among the amygdala, basal forebrain, ventral striatum, and cortex. An integrative discussion. Hong, J. Differential susceptibility to obesity between male, female and ovariectomized female mice.

Howdeshell, K. A model of the development of the brain as a construct of the thyroid system. Health Perspect. Jones, M. Aromatase-deficient ArKO mice accumulate excess adipose tissue.

Aromatase-deficient ArKO mice have a phenotype of increased adiposity. Kadioglu, P. Aromatase cytochrome P enzyme expression in human pituitary. Pituitary 11, 29— Kanaya, M. Temporal expression patterns of genes related to sex steroid action in sexually dimorphic nuclei during puberty.

Kokras, N. Sex differences in behavioral and neurochemical effects of gonadectomy and aromatase inhibition in rats. Psychoneuroendocrinology 87, 93— Konkle, A. Developmental time course of estradiol, testosterone, and dihydrotestosterone levels in discrete regions of male and female rat brain. Kramer, K. Sex steroids are necessary in the second postnatal week for the expression of male alloparental behavior in prairie voles Microtus ochragaster.

Lavaque, E. Sex differences, developmental changes, response to injury and cAMP regulation of the mRNA levels of steroidogenic acute regulatory protein, cytochrome pscc, and aromatase in the olivocerebellar system.

Lephart, E. A review of brain aromatase cytochrome P Li, R. Lonstein, J. Influence of gonadal hormones on the development of parental behavior in adult virgin prairie voles Microtus ochrogaster. Lorsch, Z. Estrogen receptor alpha drives pro-resilient transcription in mouse models of depression. Lu, J. Sex differences in the stress response in SD rats. Martin, S. Impaired spatial reference memory in aromatase-deficient ArKO mice. Neuroreport 14, — Martinez-Cerdeno, V.

Estradiol stimulates progenitor cell division in the ventricular and subventricular zones of the embryonic neocortex. Matsuda, K. Histone deacetylation during brain development is essential for permanent masculinization of sexual behavior. McAllister, C. Genetic targeting aromatase in male amyloid precursor protein transgenic mice down-regulates beta-secretase BACE1 and prevents Alzheimer-like pathology and cognitive impairment. McCarthy, M. Neuroimmunology and neuroepigenetics in the establishment of sex differences in the brain.

Medway, C. Melcangi, R. Levels and actions of neuroactive steroids in the nervous system under physiological and pathological conditions: sex-specific features. Moraga-Amaro, R. Sex steroid hormones and brain function: PET imaging as a tool for research. Naftolin, F. Brain aromatization of androgens. Google Scholar. Aromatization of androstenedione by limbic system tissue from human foetuses.

Aromatization of androstenedione by the anterior hypothalamus of adult male and female rats. Endocrinology 90, — Nelson, L. Estrogen production and action. Northcutt, K. Sex differences and effects of neonatal aromatase inhibition on masculine and feminine copulatory potentials in prairie voles.

Nugent, B. Brain feminization requires active repression of masculinization via DNA methylation. Olvera-Hernandez, S. Prenatal letrozole produces a subpopulation of male rats with same-sex preference and arousal as well as female sexual behavior. Op de Macks, Z. Risky decision-making in adolescent girls: the role of pubertal hormones and reward circuitry. Psychoneuroendocrinology 74, 77— Ordyan, N.

Effects of impaired testosterone metabolism during prenatal ontogenesis on the level of anxiety and behavior of rats in a novel environment. Overk, C. Article Google Scholar. Weihua, Z. Mouriec, K. Androgens upregulate cyp19a1b aromatase B gene expression in the brain of zebrafish Danio rerio through estrogen receptors.

Honda, S. Novel exon 1 of the aromatase gene specific for aromatase transcripts in human brain. Lephart, E. A review of brain aromatase cytochrome P Brain Research Reviews 22 , 1—26 Yilmaz, M. Aromatase promoter I. Burgoyne, P. Wijchers, P. Epigenetic regulation of autosomal gene expression by sex chromosomes. Trends Genet. Nugent, B. Brain feminization requires active repression of masculinization via DNA methylation.

Schwarz, J. Romano, E. Genes and sex hormones interaction in neurodevelopmental disorders. Xu, X. Prenatal hyperandrogenic environment induced autistic-like behavior in rat offspring. Pfaffl, M. Nucleic Acids Res. Lowry, O. Protein measurement with the Folin phenol reagent. J Biol Chem.

Aromatase-immunoreactivity is localised specifically in neurones in the developing mouse hypothalamus and cortex. Ghorbanpoor, S. Aromatase inhibition exacerbates pain and reactive gliosis in the dorsal horn of the spinal cord of female rats caused by spinothalamic tract injury. Endocrinology , —55 Download references. MJC would like to thank Dr. We are also indebted to Dr. Nancy Forger and Dr.

Hugo F. Carrer for critical reading of this manuscript. You can also search for this author in PubMed Google Scholar. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Reprints and Permissions. Sci Rep 7, Download citation. Received : 28 February Accepted : 01 June Published : 13 July Anyone you share the following link with will be able to read this content:.

Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Cellular and Molecular Life Sciences Scientific Reports By submitting a comment you agree to abide by our Terms and Community Guidelines.

If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Advanced search. Skip to main content Thank you for visiting nature.

Download PDF. Subjects Cellular neuroscience Endocrinology. Abstract During development sex differences in aromatase expression in limbic regions of mouse brain depend on sex chromosome factors. Introduction The field of sexual differentiation has been receiving increasing contributions since the pioneering work of Phoenix et al.

Figure 1. Full size image. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Discussion Previously we have demonstrated a sex difference in aromatase expression in the anterior amygdaloid area of the MF1 FCG mouse brain and culture 7.

Figure 9. Table 1 Primer sequences and conditions of qPCR assay. Full size table. References 1. Google Scholar Article PubMed Google Scholar Article Google Scholar View author publications. Ethics declarations Competing Interests The authors declare that they have no competing interests. Additional information Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

About this article. Cite this article Cisternas, C. Copy to clipboard. Further reading X-linked histone H3K27 demethylase Kdm6a regulates sexually dimorphic differentiation of hypothalamic neurons Lucas E. Cabrera Zapata , Carla D.

Comments By submitting a comment you agree to abide by our Terms and Community Guidelines. Publish with us For authors Submit manuscript. Search Search articles by subject, keyword or author.