Intracrine Regulation of Estrogen and Other Sex Steroid Levels in Endometrium and Non-gynecological Tissues; Pathology, Physiology, and Drug Discovery

Our understanding of the intracrine (or local) regulation of estrogen and other steroid synthesis and degradation expanded in the last decades, also thanks to recent technological advances in chromatography mass-spectrometry. Estrogen responsive tissues and organs are not passive receivers of the pool of steroids present in the blood but they can actively modify the intra-tissue steroid concentrations. This allows fine-tuning the exposure of responsive tissues and organs to estrogens and other steroids in order to best respond to the physiological needs of each specific organ. Deviations in such intracrine control can lead to unbalanced steroid hormone exposure and disturbances. Through a systematic bibliographic search on the expression of the intracrine enzymes in various tissues, this review gives an up-to-date view of the intracrine estrogen metabolisms, and to a lesser extent that of progestogens and androgens, in the lower female genital tract, including the physiological control of endometrial functions, receptivity, menopausal status and related pathological conditions. An overview of the intracrine regulation in extra gynecological tissues such as the lungs, gastrointestinal tract, brain, colon and bone is given. Current therapeutic approaches aimed at interfering with these metabolisms and future perspectives are discussed.


INTRODUCTION
The term "intracrinology, " coined in 1988 by prof Labrie, refers to the ability of peripheral tissues to use blood precursors and generate steroids (Labrie, 1991). Several studies have been published but several controversies still exist and relate to the following technical and biological aspects: (a) some intracrine enzymes in peripheral tissues have low expression (300-50,000-times lower than in endocrine glands Stoffel-Wagner, 2001;Murakami et al., 2006, close to the detection limit of standard methods like western blotting and immunohistochemistry -IHC); (b) the technology to robustly quantify steroids (liquid-/gas-chromatography tandem mass-spectrometry -LC-MS or GC-MS), became available during the last 5-10 years only (Rosner et al., 2013); (c) intracrine pathways are highly complex.
This review summarizes our knowledge of intracrinology in peripheral tissues like the endometrium, lungs, gastrointestinal tract (GIT), bone and central nervous system (CNS), with special attention to the metabolism of estrogens. Drug development and potential therapeutic approaches are discussed. In this review, the enzymes involved in steroid deactivation/clearance (Rižner, 2013(Rižner, , 2016 with the exclusion of steroid sulphotransferases) and those involved in the transport of conjugated steroids through the plasma membrane (Rižner et al., 2017) are not described. Studies on serum/tissue steroid levels are reported and discussed only if based on gold standard GC/LC-MS.

FROM OVARIAN ESTROGEN SYNTHESIS TO INTRACRINOLOGY
Local steroid metabolism is possible because those enzymes responsible for steroid synthesis in classical glands (ovaries, adrenals, testes) are expressed in peripheral tissues, where additional and alternative routes for metabolizing steroids are present and make intracrine networks intricate and flexible (Figures 1, 2, Tables 1, 2). In particular, several compounds generated through these pathways, although not being estrogens, can have estrogenic action, because able to bind and activate the estrogen receptors. The biologic activity of the various compounds is given in Table 1, and in Figure 2, by the color codes.

Ovarian Steroidogenesis
Transformation of cholesterol to 17β-estradiol (E2) involves first the production of dehydroepiandrosterone (DHEA) in theca cells through the action of steroidogenic acute regulatory protein (StAR) that facilitates the transport of cholesterol into mitochondria, followed by CYP11A1 (ratelimiting) and CYP17A1 (Figure 2); the ovarian pathway is indicated by the yellow background; reviewed by (Miller and Auchus, 2011;Andersen and Ezcurra, 2014). CYP11A1 is a type I CYP localized in mitochondria that uses nicotineadenine-dinucleotide-phosphate (NADPH) and ferredoxin (Fdx)/ferredoxin reductase (FdR) to cleave the cholesterol side chain and produce pregnenolone (P5). Type II CYP17A1, localized in the endoplasmic reticulum (EndRet), has both 17α-hydroxylase and 17,20-lyase activities. It uses NADPH and P450 oxidoreductase (POR) to first hydroxylate P5 to 17α-hydroxypregnenolone (17OHP5) (17α-hydroxylase action), followed by 17,20-lyase action to release DHEA. Gonad specific type 2 3β-hydroxysteroid dehydrogenase (3βHSD2) has 3β-dehydrogenase and 5 to 4 isomerase activities and converts DHEA to androstenedione (A4). Next, CYP19A1 catalyzes the oxidative demethylation of C 19 androgens to C 18 estrogens, with A-ring aromatisation; hence A4 is converted to estrone (E1). The final conversion of E1 (with low affinity for the estrogen-receptors -ERs) to E2 (high affinity for ERs and high estrogenic potency) is catalyzed by 17βHSD1 that reduces 17-keto to 17β-hydroxyl steroids. In the ovary, the 17-keto group of A4 can be reduced to 17β-hydroxyl by AKR1C3/17βHSD5 yielding testosterone (T) that is converted to E2 by CYP19A1. Upon ovulation, high 3βHSD2 levels in the corpus luteum lead to high progesterone (P) generation from P5.

Intracrine Steroidogenesis
The expression of StAR, CYP11A1 and CYP17A1 is demonstrated in a limited number of peripheral tissues (see later and Tables 6-8). However, pregnenes, pregnanes, androstenes and androstanes generated from these initial steps (but also abundantly available as circulating precursors) can be further metabolized locally thus generating a plethora of compounds with various biological activities (estrogenic, androgenic, progestogenic and neuroactive; Tables 1, 2 and Figure 2). The 5 to 4 isomerization of androstenes (DHEA, androstenediol -A5-and 17αA5) and pregnenes (P5, 17OHP5) is catalyzed by 3βHSD1, which is the peripheral counterpart of ovarian 3βHSD2. Also 3βHSD2, whose expression was initially considered to be restricted to endocrine tissues, is detected peripherally in recent reports (Stoffel-Wagner, 2001;Tsai et al., 2001;Attar et al., 2009;Huhtinen et al., 2014;Osinski et al., 2018). Due to the high concentration of DHEA (both in blood and tissues), its conversion to A4 by 3βHSDs is relevant to the formation of downstream androgens and of estrogens. Additionally, 3βHSDs convert A5 and the isomer 17αA5 to T and epitestosterone (EpiT). Although minor, in the context of women's health, these pathways are relevant. A5, together with 3α and 3βDIOL (generated by AKR1Cs from DHT and AN, see below) activate both ERs and have estrogenic action (especially 3βDIOL, a potent ERβ binder). A5 possesses immune stimulatory activity whereas its 17α isomer (17αA5) has androgenic, antitumor and neuroactivity. FIGURE 1 | Steroid structure. (A) structure of the C21 steroid progesterone (P, used as an example), with carbon numbering and steroid ring numbering. In the storied graphics in Figures 1B and 2, the H groups and the relative bonds will be omitted (with the exclusion of the H in 5α-reduced steroids -androstanes and pregnanes). Methyl groups will be indicated by the bonds only without the CH 3 group. (B) structures of C21 pregnene ( 4 and 5 , i.e., double bond between C4 and C5 or between C5 and C6, respectively), pregnane (5α-reduced steroid), C19 androstene ( 4 , 5 ) and androstane and C18 (A-ring)-aromatic estrogens. Chemical structures were designed with the aid of Sketcher V2.4 (Ihlenfeldt et al., 2009), available online at PubChem (www.ncbi.nlm.nih.gov; pubchem.ncbi.nlm.nih.gov) (Kim et al., 2016).
Additionally, EpiT is a weak AR binder and a strong endogenous inhibitor of SRD5As (Loria and Graf, 2012). The endogenous occurrence of 17αA5 is demonstrated in humans (Laatikainen et al., 1971) but its route of synthesis is unclear (Shimizu, 1979). A 17αHSD able to convert A4 to EpiT and DHEA to 17αA5 is characterized in mice (Bellemare et al., 2005) but no human homologous is described yet. Similarly to the ovaries, androgen to estrogen conversion is catalyzed by CYP19A1.
A particularly important reaction is controlled by oxidative and reductive 17βHSDs, which interconvert 17-keto and 17βhydroxysteroids. Since 17β-hydroxysteroids (T and E2) have higher affinity for the receptors than the keto-steroids (A4 and E1), this balance determines the final androgenic/estrogenic activity. Fourteen 17βHSDs exist, whose specificity is determined by tissue distribution, intracellular localization and biochemistry ( Table 2); reviewed thoroughly in (Mindnich et al., 2004;Adamski, 2006, 2009;Prehn et al., 2009;Miller and Auchus, 2011). Unpublished data also refer to a 15th 17βHSD (see Table 2; reported in Luu-The et al., 2008) with a putative role in androgen metabolism. With the exclusion of 17βHSD5 (AKR1C3, see below), all other 17βHSDs belong to the shortchain dehydrogenase (SRD) family.
Although all 17βHSDs have been postulated to use steroids as substrates based on cell-free or in vitro assays, recent investigations based on substrate specificity (Laplante et al., 2009) and knock-out (KO) models (Table 4) better clarified their roles. Type 1 17βHSD is the estrogenic enzyme and coverts E1 to E2 both in the ovary and in peripheral tissue. Type 2 17βHSD oxidizes 17-hydroxyl groups (E2 and T) to the 17keto forms (E1 and A4), and possesses also a 20α-hydroxyl oxidative action, through which this enzyme generates P from 20αDHP. Type 6 17βHSD uses 5α-reduced androgens and has 17-hydroxyl oxidative activity (converting androsterone -AN-to androstanedione) and 3-hydroxyl oxidative activity (converting 3αDIOL to the most potent androgen dihydrotestosterone -DHT). Additional catalytic actions for 17βHSD6 (epimerase or 17-hydroxydehydrogenase) are demonstrated in vitro ( Table 2). Type 14 17βHSD is postulated to have 17β-hydroxyl oxidative action on various steroids, type 7 is involved in cholesterol metabolism as indicated by KO mice (Table 4), whereas there is apparently little/no in vivo role of types 8, 9, 10, 11 and 12 17βHSDs on steroid metabolism ( Table 2 and indicated by KO mice, Table 4). Recently, a novel SRD, DHRS11, was shown to possess in vitro 17-keto to 17β-hydroxyl reductive action (able to use E1, 5 or 4 androstenes, androstanes), plus reductive 3βHSD activity toward 4 pregnenes and other compounds (5β-steroids, bile acids; Table 2 and Figure 2; Endo et al., 2016).
SRD5As convert 3-keto 4 androstene and pregnene to 5αreduced steroids (androstanes and pregnanes), hence they are important in progestogen, androgen (DHT production) and neurosteroid metabolism (Di Costanzo et al., 2009). SRD5A1 and 3 are widely expressed, in contrast to SRD5A2. Human 5βreductase activity, catalyzed by AKR1D1, is restricted to the liver, where 5β-steroids are directed to clearance/catabolism. However, some 5β-compounds are neuroactive and recent studies indicate FIGURE 2 | Intracrine networks. Major intracrine networks metabolizing steroids. In this figure, each reaction reports the catalyzing enzymes whose role in that specific reaction is established based on robust evidences (in vitro, ex vivo, in vivo). Additional enzymes whose involvement in the same reactions is less robustly demonstrated or based only on in silico or cell-free assay are reported in Table 2. The role of 17βHSD3 is disregarded in this figure because restricted to tissues that are not assessed in the present review (testes, prostate, Table 2). Color codes: OV ovarian specific referring to 3β-HSD2 (see text); -dotted arrows indicate reactions that are not fully demonstrated to occur or for which the responsible enzyme is not identified yet; (enzyme name) enzymes indicated by brackets are supposed to catalyze the indicated reaction based on the theoretical assumptions, no experimental proof is yet available; & these compounds (THP, 3αDHP and allopregnandiol) exist as various hydroxyl α/β isomers (3,5,17) with no activity, classic action or neuroactivity (see Table 2); * the role of DHRS11 in steroid metabolism is reported only recently by one publication (Endo et al., 2016).
In conclusions, intracrinology presents redundant and complex pathways, which generate compounds with various activities. Genetic variants in intracrine genes are associated with various diseases (classically endocrine and not; Table 5). Even in the absence of the enzymatic machinery to metabolize cholesterol (StAR, steroidogenic factor, CYP17A1 and CYP11A1), DHEA, P5 and especially their sulphated-conjugates have high blood concentrations (Table 1), and are used to generate all other steroids in peripheral tissues.

DRUG DEVELOPMENT
Natural hormones have been historically used as drugs, and depending on definitions, approximately 90 marketed drugs share a steroidal core (see https://www.drugbank. ca). Steroids (T, E2, cortisol, DHEA), simple derivatives (ethinylestrogen, prednisolone) or more complex analogs (abiraterone, fulvestrant) are used in various conditions. This old-and-proven steroidal chemistry based approach is used even in modern era.
By targeting steroid intracrine metabolism, the effects of steroids can be modulated locally. Table 3 overviews the available drugs targeting intracrine enzymes and their developmental status. CYP19A1 (aromatase) inhibitors, currently at their third generation, started to be used for breast cancer during the 80's of last century (Lønning and Eikesdal, 2013), and was followed by drugs able to target other enzymes (CYP11A1, CYP17A1, SRD5As; Table 3).
More recently, there is a re-emerging interest in developing novel intracrine drugs. A number of compounds are in their clinical phases, like STS inhibitors (Maltais and Poirier, 2011;Woo et al., 2011;Purohit and Foster, 2012;Pohl et al., 2014;Pautier et al., 2017) or inhibitors of AKR1C3/17βHSD5, which are of particular interest because this enzyme has crucial role in androgen/estrogen and prostaglandin biosynthesis (Penning, 2017). Bayer's AKR1C3/17βHSD5 inhibitor BAY 1128688 has a modified estrogen core, it interferes with both pathways, and is in phase II clinical trial for endometriosis (Bothe et al., 2017). Astellas Pharma potent and selective AKR1C3/17βHSD5 inhibitor ASP-9521 had only modest effect in a phase II study on prostate cancer as single drug, but combination therapy approaches remain to be studied (Kikuchi et al., 2014;Loriot et al., 2014).
HSD inhibitors are being studied in the area of hormonedependent diseases, with 11βHSD inhibitors being in clinical trials for metabolic disorders (Ye et al., 2017) and 17βHSD inhibitors approaching the clinical phase for a number of gynecological indications (Table 3; Abdelsamie et al., 2017).

INTRACRINOLOGY IN PERIPHERAL TISSUES
In this paragraph, intracrinology of endometrium, GIT, bone, lungs, and CNS is reviewed. To comprehensively understand the ability of these tissues and systems to generate estrogens and other steroids, we have performed a systematic search of all original papers published in English until June 2018 that described the levels of intracrine enzymes (those indicated in Table 2-mRNA, protein or activity) in healthy tissues. In total 177 if the four extra ref are allowed papers were reviewed, and for details of this search, see Supplemental panel: "Systematic Review." The results of this systematic review are summarized in Tables 6-8 and are briefly overviewed in each section dedicated to the distinct tissues or systems. Reports describing the enzymes in cultured cells or cell lines were excluded (may have been discussed elsewhere, though). Each section follows then with a non-systematic overview of the role of intracrinology in pathophysiology. A brief non-systematic description of the intracrinology of the skin, immune system and adipose tissue is also given. We will not describe the intracrinology of breast, prostate and liver (where steroid catabolism is the most relevant aspect), and we redirect the reader to recent reviews (Foster et al., 2008a;Luu-The et al., 2008;Luu-The and Labrie, 2010;Labrie and Labrie, 2013;Labrie, 2015;Mueller et al., 2015;Zhao et al., 2016;Hilborn et al., 2017;Penning, 2017).

Endometrium
The actions of steroid hormones in the endometrium are mediated by hormone-receptors via the classical mechanisms, although non-genomic and rapid signaling are also present (Groothuis et al., 2007;Zwart et al., 2011;Flach and Zwart, 2016;Hewitt et al., 2016). Estrogens and P control the menstrual cycle (Groothuis et al., 2007;Andersen and Ezcurra, 2014) and the endometrium during the window of implantation (WOI), occurring in the mid-luteal phase (Wang and Dey, 2006).
In rats, the WOI is characterized by high E2 plasma levels, and endometrial ERα and PR expression shows specific and varying cytosolic/nuclear patterns (Singh et al., 1996). ERα and PR expression decreases after ovulation and in preimplantation stages in both mice (Vasquez and DeMayo, 2013) and primates (Macaca mulatta) (Ghosh et al., 1999).
Rodent genetic models unraveled some molecular mechanisms underlying the estrogen-dependency of these processes. ERα-KO mice are infertile, no implantation occurs, endometrium is hypoplastic and estrogen response is absent (Couse and Korach, 1999;Walker and Korach, 2004). Not only its absence, but also sustained estrogen signaling has deleterious effects on endometrial receptivity, as recapitulated by mice with uterine COUP-TFII ablation. These mice exhibit increased estrogen signaling and asynchrony between embryo competency and uterine receptivity with consequent implantation defects. This effect is rescued by treatment with the antiestrogen ICI-182780 . Additionally, the duration of E2 exposure and its dosage affect endometrial receptivity and WOI length in mice (Ma et al., 2003).
Available human data, mostly obtained in the context of assisted reproduction technologies (ART), also indicate that steroid stimulation retards or shortens the luteal phase, the WOI, causes shifts in the appearance of pinopodes (a classical WOI marker) and causes asynchrony between ovarian and menstrual cycles (Devroey et al., 2004).

Intracrinology in Healthy Endometrium-Systematic Search
Initial studies on steroid hormone metabolism in the endometrium date back to 1965 with first demonstration of the STS activity, followed by investigation on the oxidative and reductive 17βHSD activities ( Table 6).
-lack of steroid production.
-decreased corticosterone and aldosterone levels.

Additional
Decreased litter size; small prostate.

Additional
Decreased litter size; small prostate.

Additional
Disturbed platelet physiology. Reproductive system: leyding cell hyperplasia and abnormal morphology; abnormal testis morphology; abnormal placentation and amniotic fluid composition.
Bold text indicates the system affected, italics text indicates the name of the disease.

Intratissue Steroid Levels
Endometrial steroid levels were recently profiled by LC-MS. E2 levels differ between tissue and serum during the menstrual cycle, being up to five-times higher in tissue than in serum during the proliferative phase and 1.5-fold higher in the luteal period (Huhtinen et al., 2012a(Huhtinen et al., , 2014. T levels were lower in tissue than in serum with no cyclic changes. The levels P and P5 (and their 17-hydroxy derivatives) did not vary between serum and tissue, indicating that, contrarily to estrogens, progestogen intratissue levels are determined by passive diffusion from the blood (Huhtinen et al., 2014).

Intracrinology and Reproduction
Animal models show not only that intracrine enzymes are expressed in the endometrium, but also they vary the expression levels during the endometrial phases and during implantation, as shown already during the 80's in rhesus monkeys for the oxidizing 17βHSD activity (Kreitmann et al., 1979). In rodents, STS activity measured with [3H]E1-S in 6-days pregnant rats was lower around the implantation site compared with non-implantation sites (Loza, 1995). In situ hybridisation signal of 17βHSD7 mRNA varied spatio-temporally throughout implantation and early gestation, being initially detected on luminal epithelium around the implantation site and absent in decidua (embryonic day, Ed5.5). At Ed8 and Ed9.5, 17βHSD7 expression increased in the decidua capsularis (the part that interacts with the trophoblast) and later (after E9) in the junctional zone of the developing placenta and in the spongiotrophoblasts (Nokelainen et al., 2000).
A brilliant study in mice showed that decidualization is dependent on local E2 produced through CYP19A1. CYP19A1 expression increased during pregnancy and decidualization was unaffected by ovariectomy. In contrast, treatment with the aromatase inhibitor (AI) letrozole impaired decidualization and decreased decidual marker expression (e.g., PRP, BMP2 and CX43) (Das et al., 2009).
The expression of intracrine enzymes in endometriosis was reviewed in 2012, (Huhtinen et al., 2012b) and among other studies, 20 papers published between 1996 and 2009 specifically described the levels of intracrine enzymes in eutopic and ectopic endometrium from patients and control women. With the exclusion of one study that included over 100 patients (Colette et al., 2009), the rest included small study populations, and in most cases, the various endometriosis types (ovarian, peritoneal and deep infiltrating) were pooled together. Various techniques were used (RT-qPCR, immunohistochemistry, enzyme activity assay). Overall, no clear conclusion could be drawn from these studies. Comparing endometriosis with controls, CYP19A1 was up-regulated (six studies), unchanged (three studies) and one study found no expression of this gene. With respect to oxidative and reductive 17βHSDs, 17βHSD1 was reported upregulated (three studies), 17βHSD2 was reported down-regulated or unchanged and two studies reported an up-regulation of 17βHSD7 and 12 in endometriosis vs. controls (Huhtinen et al., 2012b).
Subsequent investigations also continued to report inconsistent results. No change in mRNA (Delvoux et al., 2014) or increased expression of CYP19A1 in ovarian endometriosis vs. controls (Huhtinen et al., 2012a) were reported. An increased expression of CYP19A1 was also described using in vitro spheroids derived from endometrial stroma cells from patients compared with controls (Mori et al., 2015).
The mRNA expression of 17βHSD1 was higher in endometriosis compared with normal tissue using patient biopsies as well as spheroid cultures derived from endometrial stroma cells of patients and controls (Delvoux et al., 2014;Mori et al., 2015). One study assessing the three endometriosis types separately (60 patients in total) described that the increased 17βHSD1 level was restricted to endometrioma during the secretory phase of the menstrual cycle (Huhtinen et al., 2012a), whereas a second study on 79 patients and 41 controls, found no change in 17βHSD1 level, but described an increased 17βHSD1/2 ratio (Colette et al., 2013).
A few studies reported detectable levels of the enzymes involved in the generation of DHEA from cholesterol (StAR, CYP11A1 and CYP17A1) in endometriosis (Tsai et al., 2001;Rhee et al., 2003;Bukulmez et al., 2008a;Attar et al., 2009;Sinreih et al., 2013Sinreih et al., , 2017bHuhtinen et al., 2014), suggesting that, in contrast to eutopic endometrium, endometriosis is able to produce steroids from cholesterol. However, it has also been argued that the presence of paracrine confounders of ovarian origin in studies using endometriomas could bias the results (Noël et al., 2011).
The contribution of STS, SULT1E1 and other SULTs was investigated by numerous studies and also in this case, conclusions are unclear (recently reviewed, Rižner, 2016). A recent investigation using 78 specimens described increased STS levels in endometriosis vs. control samples and found that the overall balance between STS and SULT1E1 differed between eutopic and ectopic tissue, implying an unbalanced flux of sulpho-conjugated estrogens in this disease (Piccinato et al., 2016b). The same research group also described an aberrant regulation of the enzymes involved in the estrogen oxidative metabolism in endometriosis (Piccinato et al., 2016a).
Although the level of the single enzymes in the intracrine machinery varies with apparently no clear association with the disease condition, the intracrinological nature of endometriosis was recently proven by comparison between serum and tissue levels of steroids in 60 patients (eutopic and ectopic endometrium) and 16 controls. Although E2 changed cyclically in eutopic tissue, E2 levels remained constant in the lesions and inversely correlated with the mRNA level of 17βHSD2 and 17βHSD6 suggesting an impairment in E2 deactivation to E1. P levels were equal in serum and control tissues, but resulted higher in patients and correlated with high 3βHSD2 mRNA. T, low in the tissue of controls, was over 13-times more concentrated at ectopic locations and correlated with low expression of SRD5A3 (Huhtinen et al., 2012a(Huhtinen et al., , 2014.

Endometrial Cancer (EC)
EC is the most common gynecological malignancy in western society and 80% of all cases are estrogen-driven (Amant et al., 2005;Morice et al., 2015). Major serum steroids are increased in patients with EC, including several substrates for intracrine E2 synthesis (Lépine et al., 2010;Audet-Walsh et al., 2011). In addition, tissue-steroid levels differ between cancer, normal tissue and serum and correlate with the levels of specific intracrine enzymes (see below) .
Recent studies exploring the association between enzyme levels and tumor characteristics found a correlation between STS with tumor grade and lymphovascular invasion (Sinreih et al., 2017a) and described an association between high CYP19A1 or 17βHSD1 and poor patient prognosis (Segawa et al., 2005;Cornel et al., 2017).
Other investigations emphasized the potential antiestrogenic and protective roles of androgens and P. Formation of DHT (via conversion of A4 to T by AKR1C3/17βHSD5 and of T to DHT by SRD5As) has potential antiestrogenic action because it devoids tissue from T (substrate of CYP19A1 yielding E2) and because it has direct endometrial antiproliferative effects via AR (Ito et al., 2016). Similar to the AKR1C3/17βHSD5 data reported earlier, results on SDR5A expression are inconclusive as SRD5A2 was down-regulated in a study on 47 tumor specimens compared with adjacent normal tissue (Sinreih et al., 2013), but both SRD5A1 and SRD5A2 resulted unchanged in another study on 122 tumors (although only five controls were studied) . This last study found however increased androgen levels (T and DHT) in tissue vs. blood. High DHT levels were restricted to samples with high SRD5A1 immunohistochemical staining. In addition, AR and SRD5A1 positivity was associated with good patient prognosis . The prognostic value of AR is confirmed by independent investigations (Tangen et al., 2016). P is well-known for its antiestrogenic action, PR positivity is a good prognostic marker (Tangen et al., 2014) and P synthesis and metabolism are disturbed in EC (Sinreih et al., 2013). Interestingly, in a study on 47 tumors and adjacent normal tissues, EC had decreased StAR and CYP11A1 mRNA levels, indicative of diminished de novo steroid synthesis (Sinreih et al., 2013(Sinreih et al., , 2017b. At the same time, EC showed decreased SRD5A2 and increased 17βHSD2 indicative of a diminished rate of conversion of P to 5αDHP and of 20αDHP to 5α-pregnan-20-ol-3-one, but increased conversion of 20αDHP to P (see Figure 2).

Intracrine Drug Targets
Endometriosis: blocking the systemic estrogen signaling via P, or GnRH agonist is standard care (Vercellini et al., 2014). Blocking the intracrine E2 generation is the future approach with on-going preclinical/clinical research.
STS inhibition showed promising results. Irosustat (Table 3) inhibited up to 100% the formation of free steroids using ex-vivo material from 27 patients (Purohit et al., 2008) and STS inhibition showed good results in a mouse model of endometriosis, where decreased size and weight of the lesions was observed (Colette et al., 2011). A phase-I clinical trial on 24 volunteers proved the safety of the STS inhibitor E2MATE (PLG2001), which reduced STS activity by over 90% and induced changes in endometrial markers (both alone or co-administered with norethindrone acetate) (Pohl et al., 2014).
Inhibitors of 17βHSD1 are in preclinical phase, and promising results are described using a primate model of endometriosis, where decreased behavior/pain symptoms were reported (Arnold and Einspanier, 2013) and using ex-vivo material from endometriosis patient (over 70% of the patients showed over 80% of enzyme inhibition) (Delvoux et al., 2014).
AKR1C3/17βHSD5 inhibition can interfere with E2, androgen synthesis, and reduce prostaglandin-associated inflammation/proliferation and an inhibitor has recently entered a phase II trial for endometriosis (Table 3). Overall, AIs have limited efficacy for endometriosis (Ferrero et al., 2011;Dunselman et al., 2014), EC: only in case of advanced stage/metastatic disease hormonal care is given (progestogen, tamoxifen or AIs). AIs alone have limited efficacy with low response rates (Rose et al., 2000;Ma et al., 2004;Lindemann et al., 2014). Promising data were obtained using dual regimen (AI and mTOR inhibitor; Slomovitz et al., 2015) and additional trials on combinatory regimen are on-going. STS inhibitors showed promising results in a mouse subcutaneous model of EC, with decreased tumor growth by 48-67% (Foster et al., 2008b). However, a phase II trial on advanced stage EC was stopped because of the absence of added benefit compared with progestogen treatment (Purohit and Foster, 2012;Pautier et al., 2017).
Preclinical studies on 17βHSD1 inhibitors showed promising results in a mouse model of endometrial hyperplasia (Saloniemi et al., 2010;Järvensivu et al., 2015) and in various models of EC (Konings et al., 2018).

Endometrium: Conclusions
The ability to synthesize DHEA from cholesterol (reported by few studies) needs confirmation. However, the endometrium possesses the enzymatic machinery to metabolize sulphatedcompounds and DHEA and form androgens and estrogens, (although this contention is wrangled by other authors: Labrie and Labrie, 2013;Labrie, 2015). Further, the endometrium can metabolize androgens and progestogens via AKR1Cs and SRD5As to produce a wide range of compounds, including estrogens ( Table 6 and Figure 3). The morphological changes during the menstrual cycle are accompanied by cyclic changes in intracrine steroid and enzyme levels, indicating that steroid exposure needs to be cyclically regulated to support endometrial physiology.

Gastrointestinal Tract (GIT) and Digestive System (DS)
ERα and ERβ are expressed throughout the GIT and DS (esophagus, stomach, colon, gallbladder, pancreas) and epidemiological studies show important influence of sex hormones in DS physiology and disturbances, with a clear gender-dependency. In the duodenum, estrogens regulate bicarbonate secretion (Nayeb-Hashemi and Kaunitz, 2009;Tuo et al., 2011). This is an important defense mechanism of the mucosa against acids discharged from the stomach, and men develop duodenal ulcer two/three-times more often than premenopausal women (Wu et al., 2008). Such estrogen protective effect is recapitulated in animal studies exposed to estrogens and anti-estrogens, and is mediated by a rapid action (i.e., non genomic) of ERα on membrane ion channels (Smith et al., 2008).
Men are also more likely than women to develop cancer in the esophagus, stomach and colon. Accordingly, estrogen treatment for prostate cancer decreases the incidence of gastric cancer and menopausal status in women is associated with colorectal cancer CRC risk (Freedman et al., 2007;Kennelly et al., 2008;Hogan et al., 2009;Duell et al., 2010). ERβ results oncoprotective at several GIT sites (Kennelly et al., 2008;Barzi et al., 2013;Caiazza et al., 2015) and low expression correlate with high CRC stage in mice and with poor differentiated gallbladder cancer in humans (Hogan et al., 2009).
The association between estrogens and DS cancer risk is however controversial. The Women's Health Initiative and other large studies showed that combined estrogens plus P hormone replacement therapy (HRT) decreases CRC risk, but increases that of gallbladder. In addition, CRC during HRT has a higher grade (Kennelly et al., 2008;Hogan et al., 2009;Rennert et al., 2009;Foster, 2013;Mueller et al., 2015). However, a recent randomized, placebo-controlled trial enrolling over 10,000 women receiving estrogens alone vs. placebo found no difference in CRC incidence (Lavasani et al., 2015). Such complexity is recapitulated in animal studies where estrogens and androgens can have distinct and opposite effects on colitis and CRC (Amos-Landgraf et al., 2014;Heijmans et al., 2014). Overall, the association between DS disturbances/cancers with estrogens depends on the moment in life, extent and nature (endogenous or exogenous) of exposure and is influenced by the relative balance of the receptors (Foster, 2013). Similarly, androgens influence DS pathophysiology via complex and unclear mechanisms involving classical, membrane signaling, level of free and SHBG bound T (Roshan et al., 2016).
The lack of clear conclusion and the fact that the levels of circulating endogenous estrogens in women do not influence CRC risk indicates that intracrine steroids may have a predominant role irrespective of their circulating levels (Sato et al., 2009;Falk et al., 2015).

Intracrinology in Healthy GIT-Systematic Search
In total, 29 original papers were retrieved that described the levels of the intracrine enzymes in the GIT, published from the late 80's ( Table 7 and Supplemental panel: "Systematic Review").

Stomach intracrinology.
The stomach is an endocrine tissue, and in rodents it produces steroids starting at birth and throughout adulthood (Kobayashi et al., 2013). Human gastric mucosa expresses 17βHSD1, 2, 12 and AKR1C3/17βHSD5 ( Table 7). The mRNA for 17βHSD2 in mucosa surface and glandular epithelium inversely correlates with age in both genders (Oduwole et al., 2003a). Luminal gastric mucosa has strong AKR1C3/17βHSD5 immunoreactivity that decreases toward the gastric pits (Chang et al., 2013). Weak immunoreactivity for 17βHSD12 localizes in the fundic glands and in the squamous epithelium of the esophagus (Sakurai et al., 2006).
Sulphatases in parietal cells of the gastric glands have a protective role in detoxification. Estrogenic SULT1E1 is not expressed whereas data for SULT2A1 are inconsistent. SULT2A1 was detected in the gastric mucosa in a study on seven subjects (Tashiro et al., 2000), but it was low/absent in other studies on 39 (Teubner et al., 2007) and 23 subjects (Chen et al., 2003).

Small intestine: duodenum-jejunum-ileum.
Due to its high exposure to food components and harmful xenobiotics, the duodenum expresses several phase I/II enzymes including DHEA/estrogenic SULT1E1, 2A1, 1A1 (Table 7). Protein and enzyme activity of SULT1E1 and 2A1 are present in human jejunum and ileum but absent in duodenum (Teubner et al., 2007), mRNA and protein levels vary with no relation either with age or gender (Her et al., 1996;Nishimura and Naito, 2006). In a study on 23 subjects, SULT1E1 and 2A1 varied inter-individually and between different intestine tracts (Chen et al., 2003). The duodenal mucosa expresses 17βHSD2, but not 17βHSD1 (Casey et al., 1994;Oduwole et al., 2003a) and shows strong luminal AKR1C3/17βHSD5 (Chang et al., 2013) and weak 17βHSD12 immunoreactivity (Sakurai et al., 2006) that decreases toward the Brunner's gland (Chang et al., 2013).
FIGURE 3 | Effect of steroids (mainly estrogens) and intracrine networks in central nervous system, lungs, digestive system, uterus and bone. Italics and by brackets are those metabolism/reactions that need conformation by independent authors (because validated at the mRNA level only or in few studies). * EC, epithelial cells; **SMC, smooth muscle cells; # Chol, cholesterol. The drawing was kindly generated by Dr. Margaretha A. Skowron (Department of Urology, University Düsseldorf, Germany) for this review.

Large intestine: Colon, Cecum, Rectum
The intracrinology of healthy colon mucosa and its relation to CRC was recently reviewed (Foster, 2013). Studies dating from 1987 demonstrated the presence of CYP19A1, 17βHSD reductive and oxidative enzymatic activities, plus the expression of 17βHSD1, 2, 4, CYP19A1, STS and SULT1E1 (Table 7). Most 17βHSDs tend to have higher levels at the surface than in cryptal epithelial cells as indicated for 17βHSD2 mRNA (Oduwole et al., 2002;Foster, 2013), and for the immunoreactivity of AKR1C3/17βHSD5 (very strong; Chang et al., 2013) and 17βHSD12 (weak;Sakurai et al., 2006).

Association With Diseases
SNPs in genes controlling estrogen synthesis, response and deactivation are associated with GIT cancers (Freedman et al., 2009;Cho et al., 2012;Zeng et al., 2016) and AKR1C4 is a candidate gene in hereditary CRC (Gylfe et al., 2013; Table 5). Also variations in the expression of these genes associate with GIT disturbances. Low 17βHSD10 levels are associated with aberrant butyrate β-oxidation and ulcerative colitis (De Preter et al., 2012). The epithelial 17βHSD2 level is low in case of stomach, duodenal cancer and chronic gastritis, though it is high in regenerating epithelium close to active gastritis and ulcers (Oduwole et al., 2003a). In a study on 34 gastric tumors and adjacent healthy tissue, the mRNA and protein levels of 17βHSD2 and AKR1C3/17βHSD5 were down-regulated in cancer (Frycz et al., 2015(Frycz et al., , 2016. Some studies showed lower oxidative 17βHSD activity and mRNA level of 17βHSD2 (and 4) in CRC vs. adjacent normal tissue, suggesting a protective role of estrogen deactivation. However, another study on 35 women and 39 men found that high 17βHSD2 levels were associated with poor prognosis in female patients with distal CRC (reviewed in Foster, 2013). Also 17βHSD1 level measured by RT-qPCR and western blotting in specimens from 52 patients was lower in CRC than adjacent normal mucosa (Rawłuszko et al., 2011). CRC show also higher CYP19A1 mRNA compared with adjacent normal mucosa (n = 31) .
Although no clear target for drugs has been identified in the intracrine network, intracrine enzymes showed some values as biomarkers. In CRC, high STS/SULT1E1 ratio correlates with poor prognosis (Foster, 2013) and AKR1C3/17βHSD5 expression with lymph-node metastasis (Nakarai et al., 2015). In addition, AKR1C1 and AKR1C3/17βHSD5 associate with cisplatin resistance in CRC, hence inhibitors of these AKR1Cs may be used to re-sensitize patients to chemotherapy (Matsunaga et al., 2013). In a study were the levels of E1, E2 and DHEA-S were measured in CRC specimens and adjacent normal mucosa of men and women by LC-MS, intra-tumor estrogens were elevated and (in particular E1) correlated with poor prognosis. In line with an unfavorable role of intra-tissue estrogens, absence on STS was associated with long survival (Sato et al., 2009).

GIT: Conclusions
Human GIT/DS is unable to metabolize cholesterol and there is no clear evidence that it expresses 3βHSDs, hence DHEA cannot be used to generate androgens and estrogens (Table 7 and Figure 3). Several SULTs are expressed throughout GIT and involved in detoxification and STS is regulated by estrogen in vitro via non-classical GPER signaling (Gilligan et al., 2017).
The role of steroids in pathology is complex, with divergent effects that depend on time, length and extent of exposure. In line with this, intracrine networks have unclear roles in pathogenesis. In the GIT these networks are strongly involved in the metabolisms of fatty acids and bile acids (outside the scope of this review).

Bone Tissue and Skeletal System
Bones consist of mineralized connective tissue with structural and supportive functions. The hard exterior part (cortical bone) and the trabecular and spongy cancellous tissue filling the bone interior are identical but differ in the level of mineralization. Osteoblasts, derived from multipotent mesenchymal stem cells, build the bone tissue through deposition of Type-I collagen and through the release of ions that combine chemically forming the bone mineral. Osteoclasts differentiate from hematopoietic stem cells and cause resorption of the mineralized bone mass. The balance between osteoblasts and osteoclasts regulates mineral deposition and resorption. Sex steroid hormones contribute to control bone development during puberty, contribute to bone physiology, bone mass maintenance and regulate the rate of mineral bone deposition and resorption (Svoboda et al., 2010).
The presence of the ERs as well as other hormone-receptors in normal osteoblastic cells, osteoclasts and osteoblasts is documented (Gruber et al., 2002) and estrogens and androgens stimulate bone formation and inhibit bone resorption in both males and females. During human puberty and throughout adulthood, E2 and T induce osteoblast proliferation (Kassem et al., 1998), which is mediated by IGF and GH (Riggs et al., 2002;Svoboda et al., 2010). Such human effects are well recapitulated in animal models. ERα-KO (Vidal et al., 2000) and CYP19A1-KO mice (Oz et al., 2000) exhibit low BMD in both genders and E2 treatment rescues the CYP19A1-KO phenotype (Miyaura et al., 2001). Additionally, ovariectomy stimulates osteoclast differentiation through (indirect) increased levels of IL-1, 6 and TNF in osteoblasts and other bone-derived stromal cells (Gruber et al., 2002;Svoboda et al., 2010).

Intracrinology in Healthy Bone-Systematic Search
Bone expresses CYP19A1 and 17βHSD1, and mRNA in situ hybridisation and immunohistochemistry signals were seen in lining cells, osteoblasts, chondrocytes of articular cartilage, and adipocytes adjacent to bone trabeculae in both male and female tibiae. CYP19A1 mRNA was also widely present in various bones (ribs, femurs) with inter-individual variability, but no relation with gender or age (Sasano et al., 1997). STS and 17βHSD activities were demonstrated by recovery of [3H]E1 and [3H]E2 after incubating femur-head fragments with [3H]E1-S (15 women and 12 men with osteoarthritis indicated for hip replacement). No gender-related differences were observed and E2 formation from androgens was lower than that from E1-S, indicating a predominant role of the sulphatase pathway in bone estrogen supply (Muir et al., 2004). Subsequent studies also demonstrated the presence of CYP11A1, CYP17A1, 17βHSD reductive and oxidative activity in bone tissues (Table 8). Overall, however, only six papers describing the level of intracrine enzymes in bone tissues were retrieved by the systemic search ( Table 8) and most studies on bone intracrinology used in vitro cell cultures. In vitro studies were not included in our systematic review, but those on bone are briefly described in the next paragraph. These studies demonstrate the presence of a complex intracrine networks.
In vitro evidence using osteoblastic cells show that E2 has mitogenic effects, which is blocked by the ERα antagonist fulvestrant. Since both E1-S and DHEA-S elicit effects similar to E2, which are blocked by STS inhibition (Selcer and Difrancesca, 2012;Dias and Selcer, 2014), these studies demonstrate that conjugated steroids are activated and that DHEA is converted to E2. Studies in rat osteoblast with [14C]T demonstrated that T is converted by SRD5As and AKR1Cs to 3α/3βDIOLs, which induce proliferation via activation of ER (and not AR) (Enríquez et al., 2013).
In vitro models of osteoblast differentiation showed that various differentiation stages are accompanied by declines in STS, CYP19A1 and 17βHSD1 (Janssen et al., 1999;Dias and Selcer, 2016).
In rats, during and after sexual maturation, in situ hybridization showed that ERα and ERβ localize in osteoblasts, osteoclasts and osteocytes covering the tibia metaphysis (responsible for elongation of long bones), and co-localize with STS. Starting at sexual maturation (e.g., 7-week-old), ERs also co-localize with CYP19A1, 17βHSD1, 2 and SRD5A1 (van der Eerden et al., 2004). In addition, male transgenic mice overexpressing 17βHSD2 show disturbed IGF-I/steroid actions in bone, with growth retardation, decreased bone formation at prepuberty and decreased serum levels of IGF-I, osteocalcin and T (Shen et al., 2008).

Diseases and Treatments
Genetic variants of estrogen and intracrine pathways are associated with bone disturbances ( Table 5). Defects in the CYP19A1 and ERα are associated with low BMD and other skeletal disturbances (e.g., high stature, delayed bone age) and estrogen therapy ameliorates some bone abnormalities caused by CYP19A1 deficiencies in men (Smith et al., 1994;Morishima et al., 1995;Carani et al., 1997;Mullis et al., 1997;Bilezikian et al., 1998). In lumbar vertebrae, CYP19A1 levels correlate with changes in osteoporotic degree (Sasano et al., 1997).
Inhibitors of 17βHSD2 attracted attention as potential drugs to oppose the effects of low E2 on BMD, fracture and osteoporosis. Ovariectomised female macaques receiving a 17βHSD2 inhibitor display desirable bone balance, bone strength and lower bone resorption compared with untreated controls (Bagi et al., 2008). Several compounds targeting this enzyme have been developed and their use and challenges in osteoporosis were recently reviewed (Soubhye et al., 2015).
In a study on 35 chondrosarcoma biopsies (a malignant bone cancer occurring in middle aged patients), ERα (mRNA and IHC) and CYP19A1 (mRNA and activity) were demonstrated in the majority of the samples, and the AI exemestane impaired the E2and androgen-induced proliferation of primary chondrosarcoma cells . Although AIs were proposed as novel drugs to treat this condition (Bovée et al., 2005), a pilot study on six patients with progressive disease showed no benefit of exemestane in progression-free survival compared with untreated patients (Meijer et al., 2011).
In a study of 28 osteosarcoma specimens (one of the most common bone cancers developing at young age) strong ERβ and PR immunoreactivity was seen in over 80% of the samples (and also correlated with Ki67). ERα and AR staining was seen in 30% of the samples, whereas CYP19A1 was undetected (Dohi et al., 2008). In another study, 20 osteosarcoma specimens, including 11 good responders to chemotherapy and nine poor responders, were subjected to cDNA microarray and 17βHSD10 resulted unregulated in the poor responder group. Results were further confirmed by IHC on 69 archival biopsies, hence targeting 17βHSD10 may be a valuable approach for drug (re)sensitisation (Salas et al., 2009). Additional intracrine imbalances are described in bone diseases, such as higher androgen reducing 17βHSD activity in benign vs. malignant tumors, declines of CYP19A1 from normal bone to osteosarcoma and expression of SULT1E1 in the majority of the skeletal benign and malignant lesions, originated in bones or from primary tumors elsewhere (Svoboda et al., 2007).

Bone Tissue: Conclusions
In vitro, animal and human studies show that intracrinology controls bone development, benign and malignant conditions, and offer novel potential drug targets (Table 8 and Figure 3). Steroids can be synthesized in situ from cholesterol (Rodríguez-Sanz et al., 2015) and can be recruited from the serum via the sulphatase pathway. DHEA is substrate for androgen and estrogen production. The action of androgens is partly mediated by their conversion to estrogens via CYP19A1 or to estrogenic 3α/βDIOLs (Vanderschueren et al., 2008).

Lungs
Sex steroids play an important role in lung development and homeostasis. Androgens, progestogens and estrogens are present and exert genomic and non-genomic actions via their hormonereceptors. Classical ERs (with ERβ as predominant form) and membrane GPER are expressed (Couse et al., 1997;Prossnitz and Barton, 2011;Konings et al., 2017). Sex steroids remain active in the lungs throughout lifetime and modulate lung function in both a beneficial or detrimental way, extensively reviewed (González-Arenas and Agramonte-Hevia, 2012; Townsend et al., 2012;Sathish et al., 2015).
E2 and P regulate epithelial sodium channel expression in alveolar epithelial cells (Luo et al., 2015). In alveolar smooth muscle cells, E2 induces bronchodilation via the reduction of intracellular Ca 2+ (Townsend et al., 2010).
Both human and animal studies support a promoting role for estrogens and inhibitory role for androgens in lung development and maturation. During gestation and neonatal period, AR is expressed in mesenchymal and epithelial cells. Androgens inhibit the production of surfactants, which starts later in male than in female neonatal lungs (Carey et al., 2007), but also support the developing lung during branching morphogenesis (Kimura et al., 2003).
Intracrinology controls lung development and maturation as shown in various animal models (Boucher et al., 2009) and intracrine enzymes are expressed already during fetal stages. Human fetal lungs possess StAR, CYP11A1, 3βHSD1 mRNA (Pezzi et al., 2003), SULT1E1 activity (Jones et al., 1992) and show 17βHSD1 and 2 mRNAs expression at 13 and 20 weeks of gestational age (Takeyama et al., 2000). High mRNA levels of AR, 17βHSD2 and 17βHSD5/AKR1C3 in mid-late gestation period and adult lungs indicate the present of androgen metabolism (Simard et al., 2010). Immunoreactivity for 17βHSD11 is detected in bronchioles of 14 and 31 weeks old fetuses, whereas other structures are negative (e.g., alveoli, ciliated epithelium, acini of the trachea). The expression of 17βHSD11 increases during the second half of pregnancy and maintains similar patterns in neonatal (14 days) and adult lugs (Brereton et al., 2001).

Intracrinology and Lung Diseases
Altered intracrinology is involved in lung disorders already from neonatal stages toward adulthood, and SNPs in intracrine genes are associated with the onset of diseases (Zhang et al., 2013). Higher concentration of estrogens were measured by LC-MS in women with multiple-synchronous-lung adenocarcinoma compared with single adenocarcinoma (Ikeda et al., 2016) and in neoplastic tissue compared with adjacent normal lungs (Niikawa et al., 2008;Verma et al., 2013). Type 1 17βHSD mRNA, protein and activity are present in various non-smallcell-lung-cancer (NSCLC) cell lines where the mitogenic effect of E1 is abrogated by 17βHSD1 knockdown (Drzewiecka and Jagodzinski, 2012;Verma et al., 2013). In specimens from 48 NSCLC patients, 17βHSD1 expression was associated with squamous cell carcinoma and stage 3A disease (Drzewiecka et al., 2015). In another study on 103 NSCLC specimens, high 17βHSD1 immunoreactivity was associated with low intratumoural E1 and high E2:E1 ratio, whereas higher 17βHSD2 immunoreactivity was associated with high intratumoural E1. Multivariate regression analysis also demonstrated that increased 17βHSD1 immunoreactivity in tumors was an independent negative prognostic factor (Verma et al., 2013).
CYP19A1 is expressed in lung cancer and has potential therapeutic value (Niikawa et al., 2008;Verma et al., 2011;Siegfried and Stabile, 2014). A recent IHC study on 335 NSCLC specimens found an inverse association between CYP19A1 expression with disease specific survival (Skjefstad et al., 2016). Similar data, although restricted to women only, were confirmed in an independent study on 150 primary lung adenocarcinoma specimens, where CYP19A1 was found as the main driver of local estrogen supply (Tanaka et al., 2016). Another study on 110 lung adenocarcinoma specimens found an association between CYP19A1 mRNA (RT-qPCR) and poor prognosis in females, never-smokers and harboring EGFR mutations (Kohno et al., 2014). However, a recent mRNA study on 96 NSCLC patients showed that CYP19A1 in combination with ER is a good prognostic marker (Aresti et al., 2014).
STS and SULT1E1 immunoreactivity is detected in the majority of NSCLC cases, and STS is a good prognostic marker (Iida et al., 2013).
Lymphangioleiomyomatosis (LAM) is a rare, potentially fatal disease affecting predominantly young women. It is strongly hormone sensitive and it is hypothesized to originate from the uterus as lung metastasis (Prizant and Hammes, 2016). The levels of ERs, PR, AR, CYP19A1, STS, 17βHSD1 and SRD5A2 were recently assessed among 30 LAM biopsies. CYP19A1 expression resulted a useful classification marker with implication for potential therapy (Adachi et al., 2015). A recent study on specimens from 73 patients with chronic obstructive pulmonary disease (COPD) and 48 controls described an association between both CYP19A1 and 17βHSD1 with COPD (Konings et al., 2017). CYP19A1 is also implicated in interstitial pneumonia interstitial pneumonia, where local E2 concentration and CYP19A1 activity and immunoreactivity were elevated in diseased compared with normal tissue (Taniuchi et al., 2014).

Potential Novel Treatments
Blocking the estrogen signaling showed promising preclinical results in animal models of lung cancer (Verma et al., 2011). In humans, antiestrogen treatments (ER antagonists, GnRH, oophorectomy, P) have been used in LAM (Taveira-DaSilva and Moss, 2014) and lung cancer patients (Verma et al., 2011;Kohno et al., 2014). A phase II study on advanced NSCLC patients non-responsive to platinum-based drugs tested the dual-regimen mTOR/CYP19A1 inhibitors. Unfortunately, this study was prematurely terminated due to high toxicity (Singhal et al., 2015) and one additional trial using ER antagonist plus AI (fulvestrant and anastrozole) as consolidation therapy in postmenopausal women with advanced NSCLC (NCT00932152) was terminated due to poor recruitment.
Better results were obtained using the AI letrozole as single agent or in combination with rapamycin in a phase II trial on 17 postmenopausal women with LAM (NCT01353209). AI treatment was safe and well tolerated also in the dual drug regimen (Lu et al., 2017).

Lungs: Conclusions
Steroids are involved in lung maturation, development and in susceptibility to diseases. Most 17βHSDs, STS/SULT1E1, CYP19A1 are expressed indicating the lung's ability to metabolize androgens, estrogens and progestogens. Evidence of 3βHSDs is limited to fetal tissues (Table 8 and Figure 3). Approaches aimed at decreasing local estrogens may offer future novel treatments for various lung diseases.

Brain and Central Nervous System (CNS)
One of the first CNS actions of sex steroids to be described is the hypothalamus-pituitary-gonadal axes control (Andersen and Ezcurra, 2014). The identification of steroid-receptors outside the hypothalamus, like hippocampus (controlling memory), prefrontal cortex, cerebellum and dopaminergic system regulation indicated that sex steroids have complex and widespread effects in the CNS. They control aggressive behavior, cognitive functions, mood, food intake, appetite, addiction, blood pressure, fine motor skills, motor coordination, pain circuit and both estrogens and androgens are neuroprotective (López and Tena-Sempere, 2015;Soma et al., 2015;McEwen and Milner, 2017). Estrogen deprivation in animals and humans is associated with development of metabolic disorders and estrogen administration has a general catabolic effect (López and Tena-Sempere, 2015). Animal experiments and KO models show that ERα mediates the major actions of estrogens in the CNS, like the metabolic control functions (Musatov et al., 2007) and the negative-feedback on the hypothalamus-pituitary-gonadal axes (Couse et al., 2003). However, both nuclear and non-nuclear ERs are relevant in distinct CNS regions (Almey et al., 2015;López and Tena-Sempere, 2015;McEwen and Milner, 2017).

Intracrinology in CNS-Systematic Search
Intracrine enzymes are widely expressed in human CNS (Table 8) and intratissue concentrations of steroids in distinct regions differ between regions and from the levels in the blood Murakami et al., 2006;Jäntti et al., 2010). In contrast to rodents, however, the presence of the complete steroid biosynthetic pathway is not clearly demonstrated in the human CNS and contrasting data were reported (Table 8). CYP11A1 mRNA was detected in the temporal, frontal neocortex and subcortical white matter of men, women and children (Stoffel-Wagner, 2001). Low mRNA levels of CYP17A1 were detected in the hippocampus, amygdala, caudate nucleus, cerebellum, corpus callosum, spinal cord and thalamus (Stoffel-Wagner, 2001;Yu et al., 2002), but other authors found no expression of this enzyme (Steckelbroeck et al., 2004b(Steckelbroeck et al., , 2010MacKenzie et al., 2008). No 3βHSD1 or 2 was detected in temporal lobes, hippocampus, thalamus and amygdala (Stoffel-Wagner, 2001;Steckelbroeck et al., 2010), although other authors detected low levels in amygdala, caudate nucleus, cerebellum, corpus callosum, hippocampus, spinal cord and thalamus (Yu et al., 2002).
Production of 5α-androstane and pregnane neurosteroids is mediated by the action of SRD5As and AKR1Cs (Figure 2). SRD5A1 (not type 2) mRNA and enzyme activity were demonstrated in temporal neocortex and subcortical white matter, hippocampus, cerebellum, hypothalamus (Steckelbroeck et al., 2001;Stoffel-Wagner, 2001), and AKR1C4 mRNA was detected in both hippocampus and temporal lobe (Stoffel-Wagner, 2001). AKR1C1 and AKR1C2 are widely expressed in CNS and since no specific inhibitors directed against AKR1C1 to 4 could completely inhibit AKR1C brain activity, the involvement of an unidentified enzyme is suggested (Steckelbroeck et al., 2010). Isomeric 5β-neurosteroids require the action of AKR1D1, and it is unknown whether AKR1D1 is expressed in CNS, or liver 5β-steroids reach peripheral regions via the circulation (Jin et al., 2011).
The sulphatase pathway in the CNS is relevant because (although recent studies are revisiting this paradigm; Qaiser et al., 2017), sulphated-steroids do not cross the blood-brain barrier. Therefore, sulphated neurosteroids like DHEA-S and P5-S need to be generated locally, and in line with this, their level in the CNS is independent from the level in the blood (Rajkowski et al., 1997) and varies throughout distinct brain regions (especially hippocampus and hypothalamus) (Jäntti et al., 2010).

Diseases and Treatments
Steroid metabolism is deviated in schizophrenia (Bicikova et al., 2013) and aberrations and unbalances of intracrine enzymes are associated with neurological disorders (Luchetti et al., 2011 and see Table 5). In a study of 49 patients with AD, prefrontal cortex mRNA levels of 17βHSD1, CYP19A1 and AKR1C2 increased at late stages (Luchetti et al., 2011). STS and SULT activities, measured by radioimmunoassay and GC-MS in 55 human brain tumor specimens, varied between tumor types (Kríz et al., 2008b). Immunoreactivity for AKR1C3/17βHSD5 was low in medulloblastomas (n = 10 analyzed), high in 37 glial neoplasms and 18 meningiomas and was absent in intracranial schwannoma (n = 7) (Park et al., 2010). A recent screening of a chemical library of steroid inhibitors using three low grade pediatric glioma cell lines found that inhibition of 17βHSD3 blocked cell growth and induced apoptosis in vitro (Ajeawung et al., 2013) Type 10 17βHSD is associated with AD and is a potential target in diseases like AD, Parkinson, and an X-linked mental retardation, that may arise from the impaired degradation of branched chain fatty acid, isoleucine or aberrant neurosteroid (THP) metabolism (Lim et al., 2011;Yang et al., 2016).
STS has been implicated in ADHD and a recent mouse study indicates that genetic and pharmacological manipulations of the STS axis influence the inhibitory processes and give rise to improvements in response control (Davies et al., 2014). A recent animal experiment using a model of autoimmune encephalomyelitis showed high SULT1A1 mRNA expression in laser-captured-micro-dissected white matter astrocytes, suggesting that deactivation of estrogens (and other phenolic substrates) may be responsible for the resistance to anti-neuroinflammatory treatments in these cells and could be possibly used as new treatments to protect CNS from inflammatory injuries (Guillot et al., 2015).

CNS: Conclusions
CNS can synthesize steroids from cholesterol, although this is restricted to few brain regions. Steroid metabolism in the CNS is particular complex due to the formation of both 5α-/β-reduced and sulpho-conjugated neurosteroids (Table 8 and Figure 3).

Intracrinology in Other Tissues and Systems
Steroid metabolism is also important in the immune system, skin and adipose tissue. A thorough review of these systems is outside the scope of this study, however, a brief mention is given below.

Immune System and Inflammation
Beside corticosteroids, several other steroids affect the immune system and inflammation. A5 induces white blood cells and platelets production in bone marrow (Chen et al., 2004); estrogens and androgens control B-lymphocyte development in a sex-dependent way and modulate autoimmune diseases (McCarthy, 2000;Calippe et al., 2010;Sakiani et al., 2013).
Lipopolysaccharide-mediated proinflammatory pathway in macrophages and NF-κB activation are blocked by estrogens, which induce T-helper (Th) type 2 responses, whereas androgens stimulate type 1 responses (Iwasa et al., 2015). DHEA and DHEA-S also regulate the maturation of Th1 or Th2 cells. It was shown that plasma Th2 lymphocytes and its major secreted cytokine IL6 increase with age, and this is reversed in mice upon administration of DHEA or DHEA-S (Reed et al., 2005). Such effect was recapitulated in vitro by DHEA but not DHEA-S implicating the involvement of macrophage STS in lymphoid tissues where Th cell maturation occurs. In line with this, the effect of DHEA-S, but not DHEA, was impaired in vivo by an STS inhibitor (Reed et al., 2005). These data prompted to propose STS inhibition as a therapeutic approach for diseases associated with inappropriate immune responses and excess Th1 cytokines such as rheumatoid arthritis (Reed et al., 2005). Whether the action of DHEA is secondary to its conversion to androgens or estrogens is currently unclear. STS activity of peripheral blood leukocytes is higher in women during the follicular phase of the menstrual cycle than in women in the luteal phase or in men and it becomes highest during pregnancy, suggesting a role for P in regulating STS activity (Reed et al., 2005;Mueller et al., 2015). In vitro studies also demonstrated that STS activity is induced by cytokines such as IL6 and TNF (Mueller et al., 2015).
Opposite deregulation of the sulphatase pathway is seen in other chronic inflammatory diseases/cell types. Vascular smooth muscle cells show higher STS activity in women with mild atherosclerosis compared with women with severe disease (and male), whereas SULT1E1 activity is lower in women with severe disease (Mueller et al., 2015).
CYP19A1 is also expressed in macrophages (Konings et al., 2017) and KO mice have increased numbers of peripheral blood and bone marrow cells and inflammatory renal lesions (Shim et al., 2004). CYP19A1 inhibitors exacerbate the autoimmune lesions in a murine model of Sjögren syndrome and estrogen administration reverses such phenotype (Iwasa et al., 2015;Park et al., 2015). Opposite effects are observed in prostate, where elevated intracrine estrogens due to CYP19A1 overexpression induce inflammation and pre-malignant pathology (Ellem et al., 2009) as well as in adipose tissue (Reed et al., 2005).

Skin
The skin is the largest human organ and first barrier against pathogens where important immune functions interconnected with intracrine steroid metabolism take place (Slominski et al., 2013). Keratinocytes and sebocytes express ERs, intracrine enzymes, and the activity of sebaceous glands is influenced by steroids as indicated by the sebum production at andrenarche (Slominski et al., 2013). CYP17A1, CYP19A1, 17βHSD1, 2, 3, 4 (and enzymes metabolizing corticosteroid -outside the scope of this review) are detected in human skin. Some genes are under the influence of vitamin D and sebocytes can synthesize T from adrenal precursors (Hughes et al., 1997;Thiboutot et al., 1998;Slominski et al., 2013). Low 17βHSD oxidative metabolism characterizes sebaceous glands from skin areas prone to develop acne compared with other locations, suggesting a protective role of the oxidative metabolism against androgen excess (Fritsch et al., 2001). Sulphatase pathway is present in the skin (Reed et al., 2005;Simard et al., 2005), and genetic variants in STS and SULTs are associated with skin disturbances, most likely because of unbalanced steroid accumulation ( Table 5).

Adipose Tissue
The adipose tissue is one of the most complex endocrine organs that besides secreting leptin and adiponectin, is a site of steroid metabolism, it establishes interaction with the CNS for glucose and lipid metabolism control, energy homeostasis and inflammation. The implication of sex steroids in adipose tissue is demonstrated by the different fat distribution that characterizes men and women (Mauvais-Jarvis et al., 2013;Varlamov et al., 2014;Tena-Sempere, 2015, 2016;Palmer and Clegg, 2015). ER-KO and CYP19A1-KO mice develop obesity with human-like phenotypes (López and Tena-Sempere, 2015). Estrogens protect against metabolic syndrome and men lacking endogenous estrogens (CYP19A1 or ER-α deficiency) develop hypertriglyceridemia, glucose intolerance and insulin resistance (Kim et al., 2014). In adipose tissue of men, 17βHSD2 levels and androgen inactivation correlate with BMI (Fouad Mansour et al., 2015). A mouse study also showed that increased unsulphated-estrogen availability due to loss of SULT1E1 improved metabolic function in a model of type 2 diabetes, which leads to speculations about a potential role of SULT1E1 inhibition for this disease -at least in women (Gao et al., 2012).
Fat consists of different tissue types (white and brown) and different regional depots with distinct physiological, intracrinological characteristics and distinct relations with pathologies and metabolic disorders (Blouin et al., 2009;Mauvais-Jarvis et al., 2013). White adipose tissue is mainly subcutaneous (abdomen) or visceral (surrounding the inner organs), this last being associated with metabolic risks. A plethora of investigations demonstrated the ability of adipose tissue to aromatise androgens into estrogens and that the intra-tissue steroid levels are higher than the levels in blood (Bélanger et al., 2002). Androgenic and estrogenic 17βHSD activity and the mRNA for 17βHSD1, 2, 3, 7, 12, AKR1C3/17βHSD5 were detected in both intra-abdominal and subcutaneous fat (Bélanger et al., 2002;Quinkler et al., 2004;Bellemare et al., 2009;Wang et al., 2013).
Both subcutaneous and visceral fat tissue of women expresses the androgenic 17βHSD3 (generally considered testis specific) indicating that adipose tissue in women is substantially androgenic. Such characteristic in the visceral depot increased with increasing BMI, suggesting a link between central obesity and metabolic diseases (Corbould et al., 2002).

CONCLUSIONS AND RECOMMENDATIONS
Intracrinology consists of a complex and intricate network of alternative and redundant pathways that generate, deactivate steroids in peripheral tissues and ultimately control steroid exposure in a tissue specific manner. A number of compounds have that ability to bind and activate more than one nuclear receptors thus exerting multiple biological actions. Blood steroids represent a reservoir of substrates that support these intracrine networks. Studies retrieved by the systematic search demonstrated that most investigations rely on RT-PCR or IHC to detect enzyme and protein, and frequently without multiple-technique confirmation of the data. Since both techniques present limitations, and antibodies for IHC often perform sub-optimally (detection limit is not sufficient to detect some intracrine enzymes, crossreactivity between isoforms) these techniques are not always suitable to infer the real biological role of a reaction/enzyme. However, the recent technological advances in steroid profiling together with an improved knowledge of intracrine enzymes and the possibility to validate data using multiple approaches (RNA, protein, activity, steroid profiling) create today unprecedented opportunities to expand our understanding of intracrinology, its relation with endocrinology and to exploit this knowledge in patient care. Improved multiplex platforms allowing to profile in peripheral tissues all steroids depicted in Figure 2 are awaited and will elucidate the relevant tissue-specific networks. It is envisaged that novel prognostic markers and drug targets will become of clinical relevance soon.
We should however be aware that the redundant actions of intracrine enzymes, their substrate promiscuity, the existence of alternative pathways and the patient-to-patient variability might result in drug insensitivity. Dual/triple inhibitors will help solving this problem. In addition, in order to optimize research on novel drugs, the classical preclinical drug discovery pipelines (safety, pharmacokinetics and dynamics), should encompass parallel research lines to learn how to pre-select potentially responsive patients.
Finally, since we know that steroidal and intracrine drugs might have profound effects on the CNS, it is desirable to have in depth research on the neurological effects of potential novel drugs during the nonclinical phase of drug development. This will facilitate to select suitable compounds to the clinical development.

FUNDING
The present study was sponsored by the Dutch Cancer Society (KWF Kankerbestrijding: www.kwf.nl), contract number UM-13-5782 granted to AR.