3α-hydroxysteroid Oxidoreductase Research Articles

Microbiota modulates the steroid response to acute immune stress in male mice.

Microbiota plays a role in shaping the HPA-axis response to psychological stressors. To examine the role of microbiota in response to acute immune stressor, we stimulated the adaptive immune system by anti-CD3 antibody injection and investigated the expression of adrenal steroidogenic enzymes and profiling of plasma corticosteroids and their metabolites in specific pathogen-free (SPF) and germ-free (GF) mice. Using UHPLC-MS/MS, we showed that 4 hours after immune challenge the plasma levels of pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone (CORT), 11-dehydroCORT and their 3α/β-, 5α-, and 20α-reduced metabolites were increased in SPF mice, but in their GF counterparts, only CORT was increased. Neither immune stress nor microbiota changed the mRNA and protein levels of enzymes of adrenal steroidogenesis. In contrast, immune stress resulted in downregulated expression of steroidogenic genes (Star, Cyp11a1, Hsd3b1, Hsd3b6) and upregulated expression of genes of the 3α-hydroxysteroid oxidoreductase pathway (Akr1c21, Dhrs9) in the testes of SPF mice. In the liver, immune stress downregulated the expression of genes encoding enzymes with 3β-hydroxysteroid dehydrogenase (HSD) (Hsd3b2, Hsd3b3, Hsd3b4, Hsd3b5), 3α-HSD (Akr1c14), 20α-HSD (Akr1c6, Hsd17b1, Hsd17b2) and 5α-reductase (Srd5a1) activities, except for Dhrs9, which was upregulated. In the colon, microbiota downregulated Cyp11a1 and modulated the response of Hsd11b1 and Hsd11b2 expression to immune stress. These data underline the role of microbiota in shaping the response to immune stressor. Microbiota modulates the stress-induced increase in C21 steroids, including those that are neuroactive that could play a role in alteration of HPA axis response to stress in GF animals.

Effect of peripheral neural stimulation with allopregnanolone on ovarian physiology.

Neuroactive steroids can rapidly regulate multiple physiological functions in the central and peripheral nervous systems. The aims of the present study were to determine whether allopregnanolone (ALLO), administered in low nanomolar and high micromolar concentrations, can: (i) induce changes in the ovarian progesterone (P4) and estradiol (E2) release; (ii) modify the ovarian mRNA expression of Hsd3b1 (3β-hydroxysteroid dehydrogenase, 3β-HSD)3β-, Akr1c3 (20α-hydroxysteroid dehydrogenase, 20α-HSD), and Akr1c14 (3α-hydroxy steroid oxidoreductase, 3α-HSOR)); and (iii) modulate the ovarian expression of progesterone receptors A and B, α and β estrogenic receptors, luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR). To further characterize ALLO peripheral actions, the effects were evaluated using a superior mesenteric ganglion-ovarian nervous plexus-ovary (SMG-ONP-O) and a denervated ovary (DO) systems. ALLO SMG administration increased P4 concentration in the incubation liquid by decreasing ovarian 20α-HSD mRNA, and it also increased ovarian 3α-HSOR mRNA expression. In addition, ALLO neural peripheral modulation induced an increase in the expression of ovarian LHR, PRA, PRB, and ERα. Direct ALLO administration to the DO decreased E2 and increased P4 concentration in the incubation liquid. The mRNA expression of 3β-HSD decreased and 20α-HSD increased. Further, ALLO in the OD significantly changed ovarian FSHR and PRA expression. This is the first evidence of ALLO's direct effect on ovarian steroidogenesis. Our results provide important insights about how this neuroactive steroid interacts both with the PNS and the ovary, and these findings might help devise some of the pleiotropic effects of neuroactive steroids on female reproduction. Moreover, ALLO modulation of ovarian physiology might help uncover novel treatment approaches for reproductive diseases.

5alpha-dihydroprogesterone promotes proliferation and migration of human glioblastoma cells

Glioblastomas (GBMs) are the most common and deadliest intracranial tumors. Steroid hormones, such as progesterone (P4), at physiological concentrations, promote proliferation, and migration of human GBM cells in vivo and in vitro. Neuronal and glial cells, but also GBMs, metabolize P4 and synthesize different active metabolites such as 5α-dihydroprogesterone (5α-DHP). However, their contribution to GBM malignancy remains unknown. Here, we determined the 5α-DHP effects on the number of cells, proliferation, and migration of the U87 and U251 human GBM-derived cell lines. Of the tested concentrations (1 nM-1 µM), 5α-DHP 10 nM significantly increased the number of U87 and U251 cells from day 2 of treatment, and proliferation (at day 3) in a similar manner as P4 (10 nM). The treatment with the progesterone receptor (PR) antagonist RU486 (mifepristone), blocked the effects of 5α-DHP on the number of cells and proliferation. Besides, in U251 and LN229 GBM cells, 5α-DHP promoted cell migration (from 12 to 24 h). We also determined that GBM cells expressed the 3α-hydroxysteroid oxidoreductases (3α-HSOR), which reversibly reduce 5α-DHP to allopregnanolone (3α-THP). These data indicate that 5α-DHP induces proliferation and migration of human GBM through the activation of PR.

Sex differences in the brain expression of steroidogenic molecules under basal conditions and after gonadectomy.

The brain is a steroidogenic tissue. It expresses key molecules involved in the synthesis and metabolism of neuroactive steroids, such as steroidogenic acute regulatory protein (StAR), translocator protein 18kDa (TSPO), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenases (3β-HSD), 5α-reductases (5α-R) and 3α-hydroxysteroid oxidoreductases (3α-HSOR). Previous studies have shown that the levels of brain steroids are different in male and female rats under basal conditions and after gonadectomy. In the present study, we assessed gene expression of key neurosteroidogenic molecules in the cerebral cortex and cerebellum of gonadally intact and gonadectomised adult male and female rats. In the cerebellum, the basal mRNA levels of StAR and 3α-HSOR were significantly higher in females than in males. By contrast, the mRNA levels of TSPO and 5α-R were significantly higher in males. In the cerebral cortex, all neurosteroidogenic molecules analysed showed similar mRNA levels in males and females. Gonadectomy increased the expression of 5α-R in the brain of both sexes, although it affected the brain expression of StAR, TSPO, P450scc and 3α-HSOR in females only and with regional differences. Although protein levels were not investigated in the present study, our findings indicate that mRNA expression of steroidogenic molecules in the adult rat brain is sexually dimorphic and presents regional specificity, both under basal conditions and after gonadectomy. Thus, local steroidogenesis may contribute to the reported sex and regional differences in the levels of brain neuroactive steroids and may be involved in the generation of sex differences in the adult brain function.

Gelsemine and koumine, principal active ingredients of Gelsemium, exhibit mechanical antiallodynia via spinal glycine receptor activation-induced allopregnanolone biosynthesis

Gelsemine, the principal active alkaloid from Gelsemium sempervirens Ait., and koumine, the most dominant alkaloids from Gelsemium elegans Benth., produced antinociception in a variety of rodent models of painful hypersensitivity. The present study explored the molecular mechanisms underlying gelsemine- and koumine-induced mechanical antiallodynia in neuropathic pain. The radioligand binding and displacement assays indicated that gelsemine and koumine, like glycine, were reversible and orthosteric agonists of glycine receptors with full efficacy and probably acted on same binding site as the glycine receptor antagonist strychnine. Treatment with gelsemine, koumine and glycine in primary cultures of spinal neurons (but not microglia or astrocytes) concentration dependently increased 3α-hydroxysteroid oxidoreductase (3α-HSOR) mRNA expression, which was inhibited by pretreatment with strychnine but not the glial inhibitor minocycline. Intrathecal injection of gelsemine, koumine and glycine stimulated 3α-HSOR mRNA expression in the spinal cords of neuropathic rats and produced mechanical antiallodynia. Their spinal mechanical antiallodynia was completely blocked by strychnine, the selective 3α-HSOR inhibitor medroxyprogesterone acetate (MPA), 3α-HSOR gene silencer siRNA/3α-HSOR and specific GABAA receptor antagonist isoallopregnanolone, but not minocycline. All the results taken together uncovered that gelsemine and koumine are orthosteric agonists of glycine receptors, and produce mechanical antiallodynia through neuronal glycine receptor/3α-HSOR/allopregnanolone/GABAA receptor pathway.

Aromatase inhibition by letrozole attenuates kainic acid-induced seizures but not neurotoxicity in mice

Evidence shows neurosteroids play a key role in regulating epileptogenesis. Neurosteroids such as testosterone modulate seizure susceptibility through its transformation to metabolites which show proconvulsant and anticonvulsant effects, respectively. Reduction of testosterone by aromatase generates proconvulsant 17-β estradiol. Alternatively, testosterone is metabolized into 5α-dihydrotestosterone (5α-DHT) by 5α-reductase, which is then reduced by 3α-hydroxysteroid oxidoreductase enzyme (3α-HSOR) to form anticonvulsant metabolite 3α-androstanediol (3α-Diol), a potent GABAA receptor modulating neurosteroid. The present study evaluated whether inhibition of aromatase inhibitor letrozole protects against seizures and neuronal degeneration induced by kainic acid (KA) (10 mg/kg, i.p.) in Swiss albino mice. Letrozole (1 mg/kg, i.p.) administered one hour prior to KA significantly increased the onset time of seizures and reduced the% incidence of seizures. Pretreatment with finasteride, a selective inhibitor of 5α-reductase and indomethacin, a selective inhibitor of 3α-hydroxysteroid oxidoreductase enzyme (3α-HSOR), reversed the protective effects of letrozole in KA-induced seizures in mice. Microscopic examination using cresyl violet staining revealed that letrozole did not modify KA-induced neurotoxicity in the CA1, CA3 and DG region of the hippocampus. Letrozole treatment resulted in the reduced levels of 17-β estradiol and elevated the levels of 5α-dihydrotestosterone (DHT) and 3α-Diol in the hippocampus. Finasteride and indomethacin attenuated letrozole-induced elevations of 5α-DHT and 3α-Diol. Our results indicate the potential anticonvulsant effects of letrozole against KA-induced seizures in mice that might be mediated by inhibiting aromatization of testosterone to 17β-estradiol, a proconvulsant hormone and by redirecting the synthesis to anticonvulsant metabolites, 5α-DHT and 3α-Diol. Acute aromatase inhibition, thus, might be used as an adjuvant in the treatment of status epilepticus and can be pursued further.

A single dose of allopregnanolone affects the ovarian morphology and steroidogenesis.

Allopregnanolone, a progesterone metabolite, is one of the best characterized neurosteroids. In a dose that mimics serum levels during stress, allopregnanolone inhibits sexual receptivity and ovulation and induces a decrease in luteinizing hormone levels. The aim of this work was to examine the effect of an intracerebroventricular administration of allopregnanolone on ovarian morphophysiology, serum and tissue levels of progesterone and estrogen, and enzymatic activity of 3β-hydroxysteroid dehydrogenase, 20α-hydroxysteroid dehydrogenase and 3α-hydroxysteroid oxido-reductase in the ovary and in the medial basal hypothalamus on the morning of estrus. Ovarian morphology was analyzed under light microscopy. The hormone assays were performed by radioimmunoassay. The enzymatic activities were measured by spectrophotometric analysis. The morphometric analysis revealed that, in allopregnanolone-treated animals, the number of secondary and Graafian follicles was decreased while that of atretic follicles and cysts was significantly increased. Some cysts showed luteinized unruptured follicles. There were no differences in the number of tertiary follicles or corpora lutea in comparison with the corresponding control groups. In allopregnanolone-treated animals, progesterone serum levels were increased, while ovarian progesterone levels were decreased. Moreover, 3β-HSD and 3α-HSOR enzymatic activities were increased in the medial basal hypothalamus while ovarian levels were decreased. The enzyme 20α-hydroxysteroid dehydrogenase showed the opposite profile. The results of this study showed that allopregnanolone interferes on ovarian steroidogenesis and ovarian morphophysiology in rats, providing a clear evidence for the role of this neurosteroid in the control of reproductive function under stress situations.

Koumine Enhances Spinal Cord 3α-Hydroxysteroid Oxidoreductase Expression and Activity in a Rat Model of Neuropathic Pain

BackgroundKoumine is an alkaloid monomer found abundantly in Gelsemium plants. It has been shown to reverse thermal hyperalgesia and mechanical allodynia induced by sciatic nerve chronic constriction injury (CCI) in rats in a dose-dependent manner. Interestingly, this effect is mediated by elevated allopregnanolone levels in the spinal cord (SC). Since 3α-hydroxysteroid oxidoreductase (3α-HSOR), the key synthetase of allopregnanolone, is responsible for allopregnanolone upregulation in the SC, the objective of the present study was to investigate the role of its expression in the SC in koumine-induced analgesia using a rat model of neuropathic pain following peripheral nerve injury.ResultsTime-course investigations of immunohistochemistry and real-time polymerase chain reaction revealed that the immunoreactivity and mRNA expression of 3α-HSOR markedly increased in a time-dependent manner in the SC of koumine-treated CCI rats. Furthermore, 3α-HSOR activity in the SC of koumine-treated CCI rats increased by 15.8% compared to the activity in untreated CCI rats. Intrathecal injection of medroxyprogesterone acetate, a selective 3α-HSOR inhibitor, reversed the analgesic effect of koumine on CCI-induced mechanical pain perception. Our results confirm that koumine alleviates neuropathic pain in rats with CCI by enhancing 3α-HSOR mRNA expression and bioactivity in the SC.ConclusionThis study demonstrates that 3α-HSOR is an important molecular target of koumine for alleviating neuropathic pain. Koumine may prove a promising compound for the development of novel analgesic agents effective against intractable neuropathic pain.

Progesterone Exerts a Neuromodulatory Effect on Turning Behavior of Hemiparkinsonian Male Rats: Expression of 3 α -Hydroxysteroid Oxidoreductase and Allopregnanolone as Suggestive of GABAA Receptors Involvement.

There is a growing amount of evidence for a neuroprotective role of progesterone and its neuroactive metabolite, allopregnanolone, in animal models of neurodegenerative diseases. By using a model of hemiparkinsonism in male rats, injection of the neurotoxic 6-OHDA in left striatum, we studied progesterone's effects on rotational behavior induced by amphetamine or apomorphine. Also, in order to find potential explanatory mechanisms, we studied expression and activity of nigrostriatal 3α-hydroxysteroid oxidoreductase, the enzyme that catalyzes progesterone to its active metabolite allopregnanolone. Coherently, we tested allopregnanolone for a possible neuromodulatory effect on rotational behavior. Also, since allopregnanolone is known as a GABAA modulator, we finally examined the action of GABAA antagonist bicuculline. We found that progesterone, in addition to an apparent neuroprotective effect, also increased ipsilateral expression and activity of 3α-hydroxysteroid oxidoreductase. It was interesting to note that ipsilateral administration of allopregnanolone reversed a clear sign of motor neurodegeneration, that is, contralateral rotational behavior. A possible GABAA involvement modulated by allopregnanolone was shown by the blocking effect of bicuculline. Our results suggest that early administration of progesterone possibly activates genomic mechanisms that promote neuroprotection subchronically. This, in turn, could be partially mediated by fast, nongenomic, actions of allopregnanolone acting as an acute modulator of GABAergic transmission.

Allopregnanolone and puberty: Modulatory effect on glutamate and GABA release and expression of 3α-hydroxysteroid oxidoreductase in the hypothalamus of female rats

The hypothalamic release of glutamate and GABA regulates neurosecretory functions that may control the onset of puberty. This release may be influenced by neurosteroids such as allopregnanolone. Using superfusion experiments we examined the role of allopregnanolone on the K+-evoked and basal [3H]-glutamate and [3H]-GABA release from mediobasal hypothalamus and anterior preoptic area in prepubertal, vaginal opening and pubertal (P) rats and evaluated its modulatory effect on GABAA and NMDA (N-methyl-d-aspartic acid) receptors. Also, we examined the hypothalamic activity and mRNA expression of 3α-hydroxysteroid oxidoreductase (3α-HSOR) – enzyme that synthesizes allopregnanolone – using a spectrophotometric method and RT-PCR, respectively. Allopregnanolone increased both the K+-evoked [3H]-glutamate and [3H]-GABA release in P rats, being the former effect mediated by the modulation of NMDA receptors – as was reverted by Mg2+ and by the NMDA receptor antagonist AP-7 and the latter by the modulation of NMDA and GABAA receptors – as was reverted by Mg2+ and the GABAA receptor antagonist bicuculline. The neurosteroid also increased the basal release of [3H]-glutamate in VO rats in an effect that was dependent on the modulation of NMDA receptors as was reverted by Mg2+. On the other hand we show that allopregnanolone reduced the basal release of [3H]-GABA in P rats although we cannot elucidate the precise mechanism by which the neurosteroid exerted this latter effect. The enzymatic activity and the mRNA expression of 3α-HSOR were both increased in P rats regarding the other two studied stages of sexual development. These results suggest an important physiological function of allopregnanolone in the hypothalamus of the P rat where it might be involved in the ‘fine tuning’ of neurosecretory functions related to the biology of reproduction of the female rats.

P.4.004 Schizophrenia-like phenotypes induced by genetic over-expression of brain andenosine kinase - a potential novel antipsychotic target

The neurosteroid 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) induced catalepsy in mice is modified by dopaminergic, adenosinergic and GABAergic agents. In light of serotonergic agents being implicated in antipsychotic-induced catalepsy and their ability to increase brain neurosteroid content, the present study was undertaken to investigate the effect of various 5-HT agents on catalepsy induced by 3α,5α-THP in mice. Pretreatment with selective serotonin reuptake inhibitor, fluoxetine (5 mg/kg, i.p.), 5-HT releaser, fenfluramine (10 mg/kg, i.p.), 5-HT1A receptor agonist, 8-OH-DPAT (0.3 mg/kg, s.c.), 5-HT1B/1C receptor agonist, TFMPP (3 mg/kg, i.p.), 5-HT2A/1C receptor agonist, DOI (1.5 mg/kg, s.c.) and 5-HT3 agonist, 2-methylserotonin (5 mg/kg, i.p.) potentiated the catalepsy induced by exogenous administration of 3α,5α-THP. Furthermore, FGIN 1–27, an MDR agonist that increases endogenous content of 3α,5α-THP although per se failed to exhibit any cataleptic effect but enhanced the cataleptic response in combination with these serotonergic agents. The potentiating action of 5-HT1A, 5-HT2A/1C or 5-HT3 receptor agonist on 3α,5α-THP induced catalepsy was not blocked by prior administration of sub-effective dose (1 mg/kg, s.c.) of their respective receptor antagonists pindolol, ritanserin or ondansetron or by pretreatment with serotonergic neurotoxin 5,7-DHT (100 μg/mouse, i.c.v.). However this effect of different serotonergic agents was antagonized by the GABAA receptor antagonist, bicuculline (1 mg/kg, i.p.) or the 3α-hydroxysteroid oxidoreductase enzyme inhibitor, indomethacin (5 mg/kg, i.p.). The 5-HT agents enhance neurosteroid-induced catalepsy by increasing GABAergic tone, likely as a consequence of increased brain content of 3α,5α-THP.

Selective regulation of 3α-hydroxysteroid oxido-reductase expression in dorsal root ganglion neurons: A possible mechanism to cope with peripheral nerve injury-induced chronic pain

The enzyme 3α-hydroxysteroid oxido-reductase (3α-HSOR) catalyzes the synthesis and bioavailability of 3α,5α-neurosteroids as allopregnanolone (3α,5α-THP) which activates GABA A receptors and blocks T-type calcium channels involved in pain mechanisms. Here, we used a multidisciplinary approach to demonstrate that 3α-HSOR is a cellular target the modulation of which in dorsal root ganglia (DRG) may contribute to suppress pain resulting from peripheral nerve injury. Immunohistochemistry and confocal microscope analyses showed 3α-HSOR-immunostaining in naive rat DRG sensory neurons and glial cells. Pulse-chase, high performance liquid chromatography and Flo/One characterization of neurosteroids demonstrated 3α,5α-THP production in DRG. Behavioral methods allowed identification of pain symptoms (thermal and mechanical hyperalgesia and/or allodynia) in rats subjected to sciatic nerve chronic constriction injury (CCI). Reverse transcription and real-time polymerase chain reaction revealed that 3α-HSOR mRNA concentration in CCI-rat ipsilateral DRG, 5-fold higher than in contralateral DRG, was also 4- to 6-fold elevated than that in sham-operated or naive rat DRG. Consistently, Western blotting confirmed increased 3α-HSOR protein levels in CCI-rat ipsilateral DRG and double immunolabeling showed that 3α-HSOR overexpression occurred in DRG neurons but not in glia. Functional plasticity of 3α-HSOR leading to increased 3α,5α-THP production was evidenced in CCI-rat DRG. Interestingly, behavioral and molecular time-course investigations revealed that 3α-HSOR gene upregulation was correlated to pain symptom development. Most importantly, in vivo knockdown of 3α-HSOR expression in healthy rat DRG using 6-carboxyfluorescein-3α-HSOR-siRNA exacerbated thermal and mechanical pain perceptions. This paper is the first to show that siRNA-induced knockdown of a key neurosteroid-synthesizing enzyme directly affects an important function as nociception. Hopefully, these results may be useful for the development of novel analgesics.

Regulation of neurosteroid allopregnanolone biosynthesis in the rat spinal cord by glycine and the alkaloidal analogs strychnine and gelsemine

The neurosteroid allopregnanolone (3α,5α-THP) is well characterized as a potentially therapeutic molecule which exerts important neurobiological actions including neuroprotective, antidepressant, anxiolytic, anesthetic and analgesic effects. We have recently observed that neurons and glial cells of the rat spinal cord (SC) contain various key steroidogenic enzymes such as 5α-reductase and 3α-hydroxysteroid oxido-reductase which are crucial for 3α,5α-THP biosynthesis. Furthermore, we demonstrated that the rat SC actively produces 3α,5α-THP. As the key factors regulating neurosteroid production by nerve cells are unknown and because glycine is one of the pivotal inhibitory neurotransmitters in the SC, we investigated glycine effects on 3α,5α-THP biosynthesis in the rat SC. Glycine markedly stimulated [ 3H]-progesterone conversion into [ 3H]3α,5α-THP by SC slices. The alkaloid strychnine, well-known as a glycine receptor (Gly-R) antagonist, blocked glycine stimulatory effect on 3α,5α-THP formation. Gelsemine, another alkaloid containing the same functional groups as strychnine, increased 3α,5α-THP synthesis. The stimulatory effects of glycine and gelsemine on 3α,5α-THP production were additive when the two drugs were combined. These results demonstrate that glycine and gelsemine, acting via Gly-R, upregulate 3α,5α-THP biosynthesis in the SC. The data also revealed a structure-activity relationship of the analogs strychnine and gelsemine on neurosteroidogenesis. Possibilities are opened for glycinergic agents and gelsemine utilization to stimulate selectively 3α,5α-THP biosynthetic pathways in diseases evoked by a decreased neurosteroidogenic activity of nerve cells.

The biological activity of 3α-hydroxysteroid oxido-reductase in the spinal cord regulates thermal and mechanical pain thresholds after sciatic nerve injury

Identification of cellular targets pertinent for the development of effective therapies against pathological pain constitutes a difficult challenge. We combined several approaches to show that 3α-hydroxysteroid oxido-reductase (3α-HSOR), abundantly expressed in the spinal cord (SC), is a key target, the modulation of which markedly affects nociception. 3α-HSOR catalyzes the biosynthesis and oxidation of 3α,5α-reduced neurosteroids as allopregnanolone (3α,5α-THP), which stimulates GABA A receptors. Intrathecal injection of Provera (pharmacological inhibitor of 3α-HSOR activity) in naive rat SC decreased thermal and mechanical nociceptive thresholds assessed with behavioral methods. In contrast, pain thresholds were dose-dependently increased by 3α,5α-THP. In animals subjected to sciatic nerve injury-evoked neuropathic pain, molecular and biochemical experiments revealed an up-regulation of 3α-HSOR reductive activity in the SC. Enhancement of 3α,5α-THP concentration in the SC induced analgesia in neuropathic rats while Provera exacerbated their pathological state. Possibilities are opened for chronic pain control with drugs modulating 3α-HSOR activity in nerve cells.

Progesterone metabolites in breast cancer

In the 70 years since progesterone (P) was identified in corpus luteum extracts, its metabolism has been examined extensively in many tissues and cell lines from numerous species. In addition to the reproductive tissues and adrenals, every other tissue that has been investigated appears to have one or more P-metabolizing enzyme, each of which is specific for a particular site on the P molecule. In the past, the actions of the P metabolizing enzymes generally have been equated to a means of reducing the P concentration in the tissue microenvironment, and the products have been dismissed as inactive waste metabolites. In human breast tissues and cell lines, the following P-metabolizing enzymes have been identified: 5alpha-reductase, 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO), 3beta-HSO, 20alpha-HSO, and 6alpha-hydroxylase. Rather than providing diverse pathways for inactivating and controlling the concentration of P in breast tissue microenvironments, it is proposed that the enzymes act directly on P to produce two types of autocrines/paracrines with opposing regulatory roles in breast cancer. Evidence is reviewed which shows that P is directly converted to the 4-pregnenes, 3alpha-hydroxy-4-pregnen-20-one (3alpha-dihydroprogesterone; 3alphaHP) and 20alpha-dihydroprogesterone (20alphaHP), by the actions of 3alpha-HSO and 20alpha-HSO respectively and to the 5alpha-pregnane, 5alpha-pregnane-3,20-dione(5alpha-dihydroprogesterone; 5alphaP), by the irreversible action of 5alpha-reductase. In vitro studies on a number of breast cell lines indicate that 3alphaHP promotes normalcy by downregulating cell proliferation and detachment, whereas 5alphaP promotes mitogenesis and metastasis by stimulating cell proliferation and detachment. The hormones bind to novel, separate, and specific plasma membrane-based receptors and influence opposing actions on mitosis, apoptosis, and cytoskeletal and adhesion plaque molecules via cell signaling pathways. In normal tissue, the ratio of 4-pregnenes:5alpha-pregnanes is high because of high P 3alpha- and 20alpha-HSO activities/expression and low P 5alpha-reductase activity/expression. In breast tumor tissue and tumorigenic cell lines, the ratio is reversed in favor of the 5alpha-pregnanes because of altered P-metabolizing enzyme activities/expression. The evidence suggests that the promotion of breast cancer is related to changes in in situ concentrations of cancer-inhibiting and -promoting P metabolites. Current estrogen-based theories and therapies apply to only a fraction of all breast cancers; the majority (about two-thirds) of breast cancer cases are estrogen-insensitive and have lacked endocrine explanations. As the P metabolites, 5alphaP and 3alphaHP, have been shown to act with equal efficacy on all breast cell lines tested, regardless of their tumorigenicity, estrogen sensitivity, and estrogen receptor/progesterone receptor status, it is proposed that they offer a new hormonal basis for all forms of breast cancer. New diagnostic and therapeutic possibilities for breast cancer progression, control, regression, and prevention are suggested.

Dutasteride affects progesterone metabolizing enzyme activity/expression in human breast cell lines resulting in suppression of cell proliferation and detachment

Recent evidence indicates that progesterone metabolites play important roles in regulating breast cancer. Previous studies have shown that breast carcinoma and tumorigenic breast cell lines have higher 5α-reductase and lower 3α-hydroxysteroid oxidoreductase (3α-HSO) and 20α-HSO activities and mRNA expression levels than normal tissue and non-tumorigenic cell lines. The 5α-reduced progesterone metabolites such as 5α-dihydroprogesterone (5αP) promote both mitogenic and metastatic activity in breast cell lines in culture, whereas the 4-pregnene metabolites, 4-pregnen-3α-ol-20-one (3αHP) and 4-pregnen-20α-ol-3-one (20αHP) have the opposite (anti-cancer-like) effects. The 5α-reductase inhibitor dutasteride has been shown to inhibit 5α-reduction of testosterone to 5α-dihydrotestosterone in prostate tissue, resulting in decreased prostate volume. The aim of this study was to determine if dutasteride is an effective inhibitor of progesterone 5α-reduction in human breast cell lines and if such inhibition reduces mammary cell proliferation and detachment. The effect of dutasteride on progesterone metabolizing enzyme activities and mRNA expression were examined in tumorigenic MCF-7 and non-tumorigenic MCF-10A human breast cell lines. Dutasteride (10 −6 M) inhibited progesterone conversion to 5α-pregnanes by >95% and increased 4-pregnene production. The results indicated that effects of dutasteride on the progesterone metabolizing enzymes are due to direct inhibition of 5α-reductase activity and to altered levels of expression of 5α-reductase and HSO mRNAs. Treatment of cells with progesterone without medium change for 72 h resulted in significant conversion to 5α-pregnanes and increases in cell proliferation and detachment. The increases in proliferation and detachment were blocked by dutasteride and were reinstated by concomitant treatment with 5αP, providing proof-of-principle that the effects were due not to progesterone but to the 5α-reduced metabolites. This study provides the first evidence that dutasteride is a potent progesterone 5α-reductase inhibitor and that such inhibition may be beneficial in breast cancer.

Progesterone's 5α-reduced metabolite, 3α,5α-THP, mediates lateral displacement of hamsters

5α-pregnan-3α-ol-20-one (3α,5α-THP), progesterone (P 4)'s 5α-reduced, 3α-hydroxysteroid oxidoreduced product, facilitates lordosis of rodents in part via agonist-like actions at GABA A/benzodiazepine receptor complexes in the ventral tegmental area (VTA). Whether 3α,5α-THP influences another reproductively-relevant behavior, lateral displacement, of hamsters was investigated. Lateral displacement is the movement that female hamsters make with their perineum towards male-like tactile stimulation. This behavior facilitates, and is essential for, successful mating. Hamsters in behavioral estrus had greater lateral displacement responses when endogenous progestin levels were elevated compared to when progestin levels were lower. Administration of P 4, a prohormone for 3α,5α-THP, dose-dependently (500 > 200 > 100, 50, or 0 μg) enhanced lateral displacement of ovariectomized hamsters that had been primed with SC estradiol benzoate (5 or 10 μg). Inhibiting P 4's metabolism to 3α,5α-THP by co-administering finasteride, a 5α-reductase inhibitor, or indomethacin, a 3α-hydroxysteroid oxidoreductase inhibitor, either systemically or to the VTA, significantly decreased lateral displacement and midbrain progestin levels of naturally receptive or hormone-primed hamsters compared to controls. These data suggest that lateral displacement is progestin-sensitive and requires the formation of 3α,5α-THP in the midbrain VTA.

The role of progesterone metabolites in breast cancer: Potential for new diagnostics and therapeutics

Proliferative changes in the normal breast are known to be controlled by female sex steroids. However, only a portion of all breast cancer patients respond to current estrogen based endocrine therapy, and with continued treatment nearly all will become unresponsive and experience relapse. Therefore, ultimately for the majority of breast carcinomas, explanations and treatments based on estrogen are inadequate. Recent observations indicate that 5α-pregnane and 4-pregnene progesterone metabolites may serve as regulators of estrogen-responsive as well as unresponsive human breast cancers. The conversion of progesterone to the 5α-pregnanes is increased while conversion to the 4-pregnenes is decreased in breast carcinoma tissue, as a result of changes in progesterone metabolizing 5α-reductase, 3α-hydroxysteroid oxidoreductase (3α-HSO) and 20α-HSO activities and gene expression. The 5α-pregnane, 5α-pregnane-3,20-dione (5αP) stimulates, whereas the 4-pregnene, 3α-hydroxy-4-pregnen-20-one (3αHP), inhibits cell proliferation and detachment, by modulation of cytoskeletal and adhesion plaque molecules via the MAP kinase pathway and involving separate and specific plasma membrane-based receptors. The promotion of breast cancer appears to be related to changes in in situ concentrations of cancer-inhibiting and cancer-promoting progesterone metabolites. New diagnostic and therapeutic possibilities for breast cancer are suggested.

Long-term social isolation enhances picrotoxin seizure susceptibility in mice: up-regulatory role of endogenous brain allopregnanolone in GABAergic systems

Allopregnanolone (ALLO, 3α,5α-tetrahydroprogesterone), a positive allosteric modulator of actions of γ-aminobutyric acid GABA) at GABA A receptors, is synthesized in the brain from progesterone by the sequential action of two enzymes: a type I 5α-reductase and a 3α-hydroxysteroid oxidoreductase. We previously demonstrated that long-term social isolation of mice caused a significant decrease in brain ALLO content via suppression of type I 5α-reductase and its mRNA expression. In this study, to clarify a physiological role of endogenous brain ALLO, we investigated changes in seizure susceptibility of mice following protracted social isolation and compared with those of mice treated with SKF105111 (SKF), an inhibitor of types I and II 5α-reductase. Social isolation of mice for 7 weeks prior to the experiments caused a significant increase of seizure susceptibility to the GABA A receptor antagonist picrotoxin but not to the glycine receptor antagonist strychnine or the glutamate receptor agonist kainic acid. The change in the seizure susceptibility was completely reversed by 2.5 mg/kg ip ALLO, a dose that per se had no effect on picrotoxin-induced seizure. Treatment of mice with SKF (20 mg/kg ip) also reduced a threshold dose of picrotoxin, but not that of strychnine or kainic acid, which was required to elicit seizure in group-housed mice. The effect of SKF was attenuated by ALLO (2.5 mg/kg ip). In contrast, SKF treatment had no effect on picrotoxin-induced seizure in socially isolated mice. These findings suggest that endogenous brain ALLO plays a suppressive role in seizure susceptibility via a positive modulation of GABA A receptor function and that social isolation enhances seizure susceptibility in mice via reduction of GABA A receptor function caused by a decrease of endogenous ALLO.

Neurosteroids as Endogenous Regulators of Seizure Suscepibility.

Stress-induced Deoxycorticosterone-derived Neurosteroids Modulate GABAA Receptor Function and Seizure Susceptibility Reddy DS, Rogawski MA J Neurosci 2002;22:3795–3805 Stress affects seizure susceptibility in animals and humans, but the underlying mechanisms are obscure. Here we provide evidence that γ-aminobutyric acid subunit A (GABAA) receptor-modulating neurosteroids derived from deoxycorticosterone (DOC) play a role in stress-related changes in seizure control. DOC, an adrenal steroid, the synthesis of which is enhanced during stress, undergoes sequential metabolic reduction by 5α-reductase and 3α-hydroxysteroid oxidoreductase to form 5α-dihydrode-oxycorticosterone (DHDOC) and allotetrahydrodeoxycorti-costerone (THDOC), a GABAA receptor-modulating neurosteroid with anticonvulsant properties. Acute swim stress in rats significantly elevated plasma THDOC concentrations and raised the pentylenetetrazol (PTZ) seizure threshold. Small systemic doses of DOC produced comparable increases in THDOC and PTZ seizure thresholds. Pretreatment with finasteride, a 5α-reductase inhibitor that blocks the conversion of DOC to DHDOC, reversed the antiseizure effects of stress. DOC also elevated plasma THDOC levels and protected mice against PTZ, methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate, picrotoxin, and amygdala-kindled seizures in mice (ED50 values, 84–97 mg/kg). Finasteride reversed the antiseizure activity of DOC (ED50, 7.2 mg/kg); partial antagonism also was obtained with indomethacin (100 mg/kg), an inhibitor of 3α-hydroxysteroid oxidoreductase. Finasteride had no effect on seizure protection by DHDOC and THDOC, whereas indomethacin partially reversed DHDOC but not THDOC. DHDOC, like THDOC, potentiated GABA-activated Cl– currents in cultured hippocampal neurons (≤1 μ) and directly activated GABAA receptor currents (≤1 μ M), compatible with a role for DHDOC in the antiseizure activity of DOC. DOC is a mediator of the physiological effects of acute stress that could contribute to stress-induced changes in seizure susceptibility through its conversion to neurosteroids with modulatory actions on GABAA receptors including THDOC and possibly also DHDOC. Enhanced Neurosteroid Potentiation of Ternary GABAA Receptors Containing the δ Subunit Wohlfarth KM, Bianchi MT, Macdonald RL J Neurosci 2002;22:1541–1549 Attenuated behavioral sensitivity to neurosteroids has been reported for mice deficient in the γ-aminobutyric acid subunit A [GABA(A)]-receptor δ subunit. We therefore investigated potential subunit-specific neurosteroid pharmacology of the following GABAA-receptor isoforms in a transient expression system: α1β372L, α1β38, α6β372L, and α6β38. Potentiation of submaximal GABAA-receptor currents by the neurosteroid tetrahydrodeoxycorticosterone (THDOC) was greatest for the α1β38 isoform. Whole-cell GABA concentration-response curves performed with and without low concentrations (30 n M) of THDOC revealed enhanced peak GABAA-receptor currents for isoforms tested without affecting the GABA EC50. The α1β38 currents were enhanced the most (>150%), whereas the other isoform currents were enhanced 15–50%. At a higher concentration (1 μ M), THDOC decreased peak α1β382L-receptor current amplitude evoked by GABA (1 m M) concentration jumps and prolonged deactivation but had little effect on the rate or extent of apparent desensitization. Thus the polarity of THDOC modulation depended on GABA concentration for α1β382L GABAA receptors. However, the same protocol applied to α1β38 receptors resulted in peak current enhancement by THDOC of >800% and prolonged deactivation. Interestingly, THDOC induced pronounced desensitization in the minimally desensitizing α1β38 receptors. Single-channel recordings obtained from α1β38 receptors indicated that THDOC increased the channel-opening duration, including the introduction of an additional longer duration open state. Our results suggest that the GABAA-receptor delta subunit confers increased sensitivity to neurosteroid modulation and that the intrinsic gating and desensitization kinetics of α1β38 GABAA receptors are altered by THDOC.