Carolyn R. Fisher

Bone Has a Sexually Dimorphic Response to Aromatase Deficiency

Aromatase synthesizes estrogen from androgen precursors. To better understand the role of estrogen in skeletal metabolism and growth, we have assessed long bone growth and histomorphometry in aromatase‐deficient (ArKO) mice. The age range for the animals was 5–7 months. At this age mice have already achieved peak bone density but continue slow bone growth. Femur length, an index of long bone growth, showed decreased growth in ArKO males compared with wild‐type (wt) littermates but no significant difference in females. Radiographically, compared with age‐ and sex‐ matched littermates both ArKO males and females showed osteopenia in the lumbar spine. Histologically, both ArKO males and females showed an osteoporotic‐type picture, characterized by significant decreases in trabecular bone volume and trabecular thickness. However, compared with wt littermates female ArKO animals showed a bone remodeling picture consistent with increased bone turnover, much like early postmenopausal osteoporosis in humans. On the other hand, male ArKO animals showed decreases in both osteoblastic and osteoclastic surfaces compared with wt littermates, similar to age‐related osteopenia. These findings suggest that osteoporosis seen in aromatase‐deficient mice may arise from different bone remodeling activities between males and females. These results also show that the ArKO model exhibits the expected results of estrogen deficiency and may be a good model for investigating sex‐specific responses to estrogen deficiency. Furthermore, they imply that estrogen is important for attaining peak bone mass in male as well as in female mice.

Endocrine disorders associated with inappropriately high aromatase expression

Aromatase P450 (P450arom) is responsible for conversion of C19 steroids to estrogens in a number of human tissues, such as the placenta, gonads, adipose tissue, skin and the brain. Aromatase expression in human tissues is regulated by use of alternative promoters in the placenta (promoter I.1), adipose tissue (promoters I.4, I.3 and II) and gonads (promoter II). Aromatase expression is absent in the disease-free adult liver, adrenal and uterine tissues. Excessive or inappropriate aromatase expression in adipose fibroblasts and endometriosis-derived stromal cells, as well as in testicular, hepatic, adrenal and uterine tumors, is associated with abnormally high circulating estrogen levels and/or with increased local estrogen concentrations in these tissues. Whether systemically delivered or locally produced, elevated estrogen levels will in turn promote the growth of hormone-responsive tissues. We recently studied aromatase expression in testicular tumor and adipose tissue samples from prepubertal boys with gynecomastia, in hepatocellular cancer and adrenocortical tumor samples from adult men with gynecomastia, in breast adipose tissue samples proximal to breast tumors, and in endometrial cancer, leiomyoma and endometriosis tissues. Excessive aromatase activity and P450arom transcript levels were found in these tissue samples or in cultured cells derived from these tissues. In these neoplastic or non-neoplastic tissues or cells, the regulation of aromatase expression was studied in terms of alternative promoter use, both in vivo and in response to various hormonal stimuli. Our results were suggestive of a common metabolic abnormality associated with activation of a cyclic AMP-dependent signalling pathway that gives rise to transcriptional transactivation of aromatase expression via promoters I.3 and II in all of the above tissues. This article describes the common pathophysiological and molecular features of excessive aromatase expression in these disease states.

Upregulation of estrogen receptors in the forebrain of aromatase knockout (ArKO) mice

Estrogens have numerous reproductive and nonreproductive functions in brain. The actions of estrogens are mediated by estrogen receptors (ERs), and estrogens are believed to down-regulate their own receptors in many tissues. Assuming this to be true, if estrogens are removed there should be an upregulation of ERs. We have developed a mouse model in which estrogen synthesis is completely eliminated by homologous recombination to delete the gene encoding aromatase cytochrome P450 (P450(arom)). The P450(arom) enzyme catalyzes the synthesis of estrogens from androgens in the brain. The localization and density of ERs was studied in the brains of aromatase knockout (ArKO) and wild type male mice by using immunohistochemistry with a peptide antibody to ERalpha (ER-21) and computer imaging. In the wild-type animals a high density of ERalpha was found in a small number of hypothalamic cells; in the medial preoptic area, periventricular, arcuate, and ventromedial nuclei. A low and medium density of ERalpha was observed in cells of the lateral preoptic area, supraoptic, bed nucleus of the stria terminalis, and in central, medial and anterior cortical amygdaloid nuclei. The number of cells containing ERalpha-immunoreactivity was significantly increased (244%) in the medial preoptic area of the ArKO mice. In neither wild type nor ArKO animals was immunoreactivity observed in the cerebral cortex or striatum. There was intense ER-immunostaining in the nucleus of neurons in both wild type and ArKO mice. These data indicate that in the absence of estrogens there is as much as a 2-fold increase in the number of cells with ERalpha-immunoreactivity in certain hypothalamic and limbic regions. Thus, estrogens can down-regulate ERalpha in brain.

Maple syrup urine disease in Mennonites. Evidence that the Y393N mutation in E1 alpha impedes assembly of the E1 component of branched-chain alpha-keto acid dehydrogenase complex.

Maple Syrup Urine Disease (MSUD) in Mennonites is associated with homozygosity for a T to A transversion in the E1 alpha gene of the branched-chain alpha-keto acid dehydrogenase complex. This causes a tyrosine to asparagine substitution at position 393 (Y393N). To assess the functional significance of this missense mutation, we have carried out transfection studies using E1 alpha-deficient MSUD lymphoblasts (Lo) as a host. The level of E1 beta subunit is also greatly reduced in Lo cells. Efficient episomal expression in lymphoblasts was achieved using the EBO vector. The inserts employed were chimeric bovine-human cDNAs which encode mitochondrial import competent E1 alpha subunit precursors. Transfection with normal E1 alpha cDNA into Lo cells restored decarboxylation activity of intact cells. Western blotting showed that both E1 alpha and E1 beta subunits were markedly increased. Introduction of Y393N mutant E1 alpha cDNA failed to produce any measurable decarboxylation activity. Mutant E1 alpha subunit was expressed at a normal level, however, the E1 beta subunit was undetectable. These results provide the first evidence that Y393N mutation is the cause of MSUD. Moreover, this mutation impedes the assembly of E1 alpha with E1 beta into a stable alpha 2 beta 2 structure, resulting in the degradation of the free E1 beta subunit.

A 17-bp insertion and a PHE215→ CYS missense mutation in the dihydrolipoyl transacylase (E2) mRNA from a thiamine-responsive maple syrup urine disease patient WG-34

We have amplified the cDNA for the transacylase (E2) subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKAD) complex from a thiamine-responsive MSUD cell line (WG-34) by the polymerase chain reaction. Sequencing of the amplified WG-34 cDNA showed a 17-bp insertion (AAATACCTTGTTACCAG) apparently resulting from an aberrant splicing of the E2 gene, and a missense (T----G) mutation that changes Phe215 to Cys in the E2 subunit. The existence of these two mutations was confirmed by probing the amplified E2 cDNA or genomic DNA with allele-specific oligonucleotides. The above results support the thesis that the thiamine-responsive MSUD patient (WG-34) is a compound heterozygote at the E2 locus. The implication of the E2 mutations for the thiamine-responsiveness observed in this patient is discussed.

The Role of Plasma Protein Binding in Drug Delivery to Brain

Many factors influence drug activity in brain. One of the most important is the ability of a drug to gain access to brain following systemic administration by passage across the blood-brain barrier (BBB). The BBB is formed at the cerebral capillaries by a continuous layer of endothelial cells that are joined together by high resistance tight junctions (Pardridge, 1998). These tight junctions effectively seal off the aqueous paracellular channels between brain endothelial cells, so that if a drug is to gain access to brain it must either be of the appropriate lipid solubility, hydrogen bonding capacity, and size to readily dissolve and diffuse across the lipophilic endothelial cell membranes (Habgood et al.,2000) or be transported across the endothelium by any of 20 or more active or facilitated carrier systems which are expressed in brain capillaries at high levels (Smith et al.,1995).