Transactivation activity of human papillomavirus type 16 E6*I on aldo-keto reductase genes enhances chemoresistance in cervical cancer cells.

The oncogenic E6 proteins produced by high-risk human papillomaviruses (HPVs) are invariably expressed in cervical carcinomas and are multifunctional proteins capable of affecting host-cell proliferation by binding and deregulating key host molecules such as p53. High-risk HPVs, including HPV16, have the unique ability to splice the E6 viral transcript, resulting in the production of a truncated E6 protein known as E6*I whose precise biological function is unclear. This study explored the changes in gene expression of the cervical cancer C33A cell line stably expressing HPV16 E6*I (16E6*I) and observed the upregulation of ten genes. Two of these genes were aldo-keto reductases (AKR1Cs), AKR1C1 and AKR1C3, which have been implicated in drug resistance. The results demonstrated that expression of 16E6*I, but not full-length E6, specifically increased AKR1C1 transcript levels although it did not alter AKR1C2 transcript levels. HPV16 E7 alone also had the ability to cause a moderate increase in AKR1C3 at both mRNA and protein levels. Site-directed mutagenesis of 16E6*I revealed that transactivation activity was abolished in R8A, R10A and T17A 16E6*I mutants without altering their intracellular localization patterns. Loss of transactivation activity of the 16E6*I mutants resulted in a significant loss of AKR1C expression and a decrease in drug resistance. Analysis of the AKR1C1 promoter revealed that, unlike the E6 protein, 16E6*I does not mediate transactivation activity solely through Sp1-binding sites. Taken together, it was concluded that 16E6*I has a novel function in upregulating expression of AKR1C and, in concert with E7, has implications for drug treatment in HPV-mediated cervical cancer.

Role of aldo-keto reductases and other doxorubicin pharmacokinetic genes in doxorubicin resistance, DNA binding, and subcellular localization.

BACKGROUND: Since proteins involved in chemotherapy drug pharmacokinetics and pharmacodynamics have a strong impact on the uptake, metabolism, and efflux of such drugs, they likely play critical roles in resistance to chemotherapy drugs in cancer patients. METHODS: To investigate this hypothesis, we conducted a whole genome microarray study to identify difference in the expression of genes between isogenic doxorubicin-sensitive and doxorubicin-resistant MCF-7 breast tumour cells. We then assessed the degree of over-representation of doxorubicin pharmacokinetic and pharmacodynamic genes in the dataset of doxorubicin resistance genes. RESULTS: Of 27,958 Entrez genes on the array, 7.4 per cent or 2,063 genes were differentially expressed by ≥ 2-fold between wildtype and doxorubicin-resistant cells. The false discovery rate was set at 0.01 and the minimum p value for significance for any gene within the "hit list" was 0.01. Seventeen and 43 per cent of doxorubicin pharmacokinetic genes were over-represented in the hit list, depending upon whether the gene name was identical or within the same gene family, respectively. The most over-represented genes were within the 1C and 1B families of aldo-keto reductases (AKRs), which convert doxorubicin to doxorubicinol. Other genes convert doxorubicin to other metabolites or affect the influx, efflux, or cytotoxicity of the drug. In further support of the role of AKRs in doxorubicin resistance, we observed that, in comparison to doxorubicin, doxorubincol exhibited dramatically reduced cytotoxicity, reduced DNA-binding activity, and strong localization to extra nuclear lysosomes. Pharmacologic inhibition of the above AKRs in doxorubicin-resistant cells increased cellular doxorubicin levels, restored doxorubicin cytotoxicity and re-established doxorubicin localization to the nucleus. The properties of doxorubicinol were unaffected. CONCLUSIONS: These findings demonstrate the utility of using curated pharmacokinetic and pharmacodynamic knowledge bases to identify highly relevant genes associated with doxorubicin resistance. The induction of one or more of these genes was found to be correlated with changes in the drug's properties, while inhibiting one specific class of these genes (the AKRs) increased cellular doxorubicin content and restored drug DNA binding, cytotoxicity, and subcellular localization.

Mefenamic acid enhances anticancer drug sensitivity via inhibition of aldo-keto reductase 1C enzyme activity.

Resistance to anticancer medications often leads to poor outcomes. The present study explored an effective approach for enhancing chemotherapy targeted against human cancer cells. Real-time quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed overexpression of members of aldo-keto reductase (AKR) 1C family, AKR1C1, AKR1C2, AKR1C3, and AKR1C4, in cisplatin, cis-diamminedichloroplatinum (II) (CDDP)-resistant human cancer cell lines, HeLa (cervical cancer cells) and Sa3 (oral squamous cell carcinoma cells). The genes were downregulated using small-interfering RNA (siRNA) transfection, and the sensitivity to CDDP or 5-fluorouracil (5-FU) was investigated. When the genes were knocked down, sensitivity to CDDP and 5-FU was restored. Furthermore, we found that administration of mefenamic acid, a widely used non-steroidal anti-inflammatory drug (NSAID) and a known inhibitor of AKR1Cs, enhanced sensitivity to CDDP and 5-FU. The present study suggests that AKR1C family is closely associated with drug resistance to CDDP and 5-FU, and mefenamic acid enhances their sensitivity through its inhibitory activity in drug-resistant human cancer cells. Thus, the use of mefenamic acid to control biological function of AKR1C may lead to effective clinical outcomes by overcoming anticancer drug resistance.

Altered gene expression: a mechanism for reproductive toxicity in zebrafish exposed to benzo[a]pyrene.

Exposure of fish to polycyclic aromatic hydrocarbons (PAHs) has consistently been shown to have a negative impact on reproduction (e.g. decreased spawning success, decreased ovarian somatic index (OSI) and lower circulating levels of 17beta-estradiol and vitellogenin). While an understanding of the mechanism behind these changes has yet to be fully elucidated, it has been proposed that PAHs can alter the expression of genes important in regulating reproduction. The objectives of this study were to: (1) determine the effect of exposure to waterborne benzo[a]pyrene (B[a]P) (0, 1.5 and 3.0 microg/L) for 56 days on egg production and OSI in female zebrafish (Danio rerio) and (2) determine the effect of B[a]P on transcription of genes involved in reproduction including gonadotropins (follicle stimulating hormone (FSH) and luteinizing hormone (LH)), steroidogenic enzymes (CYP11A1, CYP17, CYP19A1, CYP19A2 and 20beta-HSD), estrogen receptor beta (ERbeta) and vitellogenin. Cytochrome P4501A1 (CYP1A1) was also measured in the liver and heads as an indicator of exposure to B[a]P. A reduction in total egg output was observed in B[a]P exposed fish as well as a decrease in OSI in fish exposed to 3.0 microg/L B[a]P. A significant increase in CYP1A1 expression in the heads as compared to the control was observed whereas no significant difference in CYP1A1 was detected in livers. A significant increase in 20beta-HSD mRNA occurred in heads and pre-vitellogenic oocytes from fish exposed to 1.5 and 3.0 microg/L as compared to the controls. CYP19A2 and vitellogenin were significantly increased following exposure to 3.0 microg/L in the heads and liver, respectively. No effects on the expression of FSH, LH, CYP19A1 or ERbeta were observed. Results from this study demonstrate that reproduction in zebrafish is affected by waterborne exposure to B[a]P and that altered transcription of genes important in regulating reproduction (20beta-HSD, CYP19A2 and vitellogenin) may be one of the underlying mechanisms of B[a]P-induced reproductive toxicity.

Selective loss of AKR1C1 and AKR1C2 in breast cancer and their potential effect on progesterone signaling.

Progesterone plays an essential role in breast development and cancer formation. The local metabolism of progesterone may limit its interactions with the progesterone receptor (PR) and thereby act as a prereceptor regulator. Selective loss of AKR1C1, which encodes a 20alpha-hydroxysteroid dehydrogenase [20alpha-HSD (EC 1.1.1.149)], and AKR1C2, which encodes a 3alpha-hydroxysteroid dehydrogenase [3alpha-HSD (EC 1.1.1.52)], was found in 24 paired breast cancer samples as compared with paired normal tissues from the same individuals. In contrast, AKR1C3, which shares 84% sequence identity, and 5alpha-reductase type I (SRD5A1) were minimally affected. Breast cancer cell lines T-47D and MCF-7 also expressed reduced AKR1C1, whereas the breast epithelial cell line MCF-10A expressed AKR1C1 at levels comparable with those of normal breast tissues. Immunohistochemical staining confirmed loss of AKR1C1 expression in breast tumors. AKR1C3 and AKR1C1 were localized on the same myoepithelial and luminal epithelial cell layers. Suppression of ARK1C1 and AKR1C2 by selective small interfering RNAs inhibited production of 20alpha-dihydroprogesterone and was associated with increased progesterone in MCF-10A cells. Suppression of AKR1C1 alone or with AKR1C2 in T-47D cells led to decreased growth in the presence of progesterone. Overexpression of AKR1C1 and, to a lesser extent, AKR1C2 (but not AKR1C3) decreased progesterone-dependent PR activation of a mouse mammary tumor virus promoter in both prostate (PC-3) and breast (T-47D) cancer cell lines. We speculate that loss of AKR1C1 and AKR1C2 in breast cancer results in decreased progesterone catabolism, which, in combination with increased PR expression, may augment progesterone signaling by its nuclear receptors.

Dietary indoles and isothiocyanates that are generated from cruciferous vegetables can both stimulate apoptosis and confer protection against DNA damage in human colon cell lines.

The natural indoles 3,3'-diindolylmethane (DIM), ascorbigen (ASG), indole-3-carbinol (I3C), and indolo[3,2-b]carbazole (ICZ), as well as the natural isothiocyanates sulforaphane (SUL), benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), all possess cancer chemopreventive properties. It is now shown that DIM, ICZ, SUL, and BITC can each stimulate apoptosis in human colon adenocarcinoma LS-174 and Caco-2 cells. Treatment of LS-174 cells with nontoxic doses of DIM, ASG, I3C, or ICZ affected an increase of up to 21-fold in cytochrome P450 1A1 (CYP1A1). None of these indoles caused an elevation in either aldo-keto reductase 1C1 (AKR1C1) or the gamma-glutamylcysteine synthetase heavy subunit (GCS(h)), but DIM, I3C, and ICZ produced a very modest increase in NAD(P)H:quinone oxidoreductase 1 (NQO1). By contrast, nontoxic doses of SUL, BITC, or PEITC failed to induce expression of CYP1A1 in LS-174 cells, but caused an increase of between 11- and 17-fold in the protein levels of AKR1C1, NQO1, and GCS(h). Treatment of the colon cell line with ICZ or SUL caused increases in the levels of mRNA for CYP1A1, AKR1C1, and NQO1 that were consistent with the enzyme data. Exposure of Caco-2 cells to media containing indoles or isothiocyanates gave similar results to those obtained using LS-174 cells. Evidence is presented that the ability of indoles and isothiocyanates to stimulate either xenobiotic response element- or antioxidant response element-driven gene expression accounts for the two groups of phytochemicals inducing different gene batteries. Pretreatment of LS-174 cells for 24 h with ICZ and SUL before exposure for 24 h to benzo(a)pyrene (BaP) reduced to <20% the number of single-strand DNA breaks produced by the carcinogen. Neither ICZ alone nor SUL alone were able to confer the same degree of protection against DNA damage produced by BaP as they achieved in combination. Similar results were obtained with H(2)O(2) as the genotoxic agent. Together, these phytochemicals may prevent colon tumorigenesis by both stimulating apoptosis and enhancing intracellular defenses against genotoxic agents.

Molecular cloning and expression of an abundant rabbit ovarian protein with 20 alpha-hydroxysteroid dehydrogenase activity.

An abundant 37-kDa protein, which comprises up to 30% of the soluble proteins of the ovary, has been found to have 20 alpha-hydroxysteroid dehydrogenase (20 alpha HSD) activity. The steroidogenic enzyme 20 alpha HSD regulates the conversion of progesterone to 20 alpha-hydroxyprogesterone in many mammalian species. Complimentary DNA clones encoding a unique and abundant 20 alpha HSD were isolated from a mature rabbit ovary library using guinea pig antisera generated to the purified 37-kDa protein and from a 5' EcoRI fragment from the initial positive clone. A full-length cDNA clone of 1217 basepairs encoding a 323-amino acid protein with an estimated mol wt of 37 kilodaltons was obtained. Amino acid sequence data indicate a similarity to human chlordecone reductase, bovine lung prostaglandin F synthase, human aldose reductase, human aldehyde reductase, and frog lens rho-crystallin, placing rabbit ovarian 20 alpha HSD in the aldo-keto reductase family of proteins. Northern blot analysis demonstrated a 1.2-kilobase mRNA in the interstitial tissue of mature rabbit ovaries and, to a lesser extent, in corpora luteal tissue. 20 alpha HSD was expressed in bacteria as a recombinant protein and was shown to possess enzymatic activity, preferring NADP as a cofactor. These studies demonstrate that an abundant ovarian protein belonging to the superfamily of NADP-dependent aldo-keto reductases has 20 alpha HSD activity. This is the first example of an abundant crystallin-related protein with known enzymatic activity in a tissue other than the lens.

Expression, purification and characterization of the rat luteal 20 alpha-hydroxysteroid dehydrogenase.

The enzyme, rat ovarian 20 alpha-hydroxysteroid dehydrogenase (20 alpha HSD), plays a central role in luteolysis and parturition. It catalyzes the reduction of progesterone, leading to the formation of progestationally inactive steroid, 20 alpha-hydroxypregn-4-ene-3-one (20 alpha-hydroxyprogesterone). Recently, we reported the cloning, sequencing, and deduced amino acid sequence of the rat luteal 20 alpha HSD. To further investigate whether phosphorylation and/or glycosylation affect the activity of 20 alpha HSD and to study its kinetic and biochemical properties, we established both bacterial and insect expression systems for obtaining large quantities of enzyme. The recombinant (rec) 20 alpha HSD expressed as glutathione-S-transferase-20 alpha HSD fusion protein was purified from bacterial lysates by affinity binding to glutathione-Sepharose beads followed by thrombin digestion, whereas the rec enzyme expressed in baculovirus-insect cell system was purified to apparent homogeneity by ion exchange chromatography, followed by dye affinity chromatographies. Both rec preparations of 20 alpha HSD demonstrated a single polypeptide chain of 37 kDa with similar K(m) values for 20 alpha-hydroxyprogesterone and NADP, although the corresponding maximum velocity values were slightly lower for the rec 20 alpha HSD expressed in the insect cells. The rec 20 alpha-HSD showed preference for progesterone/20 alpha-hydroxyprogesterone. 17 alpha-Hydroxyprogesterone was only 30% as effective. The enzyme also used various substrates specific for aldo-keto reductases, although with much less efficiency. The rec enzyme preparations showed an absolute requirement for NADP(H). In vitro phosphorylation of rec bacterial enzyme with either protein kinase A or protein kinase C had no demonstrable effect on its activity. Finally, no differences in enzyme activity were noted between glycosylated (expressed in insect cells) and nonglycosylated (expressed in bacteria) forms of the enzyme. In conclusion, these studies demonstrate that rat luteal 20 alpha HSD can be prepared in large amounts from either bacterial or insect expression systems in a catalytically active form. Indirect evidence also suggests that the catalytic activity of 20 alpha HSD may be independent of phosphorylation and glycosylation states of the enzyme protein, i.e. posttranslational modification of 20 alpha HSD may not be required for the maximal expression of enzyme activity.

20 alpha-hydroxysteroid dehydrogenase expression in a murine virus-induced myeloproliferative syndrome.

The myeloproliferative sarcoma virus (MPSV) infection in DBA/2 mice leads to important quantitative and qualitative changes in their hemopoiesis. These findings suggest a disturbance in the production and action of a certain hemopoietic factor similar to IL3. Here, we show that the level of the 20 alpha-hydroxysteroid dehydrogenase (20 alpha-SDH) expression, which can be induced by IL3, is dramatically increased in spleen and thymus of MPSV-infected mice. Our results suggest that quantification of 20 alpha-SDH activity can be used to indicate abnormal production of a growth factor similar to IL3 in hemopoietic system diseases.

Sex-related expression of 20alpha-hydroxysteroid dehydrogenase mRNA in the adult mouse.

The enzyme 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) catalyzes the conversion of progesterone into its inactive form, 20alpha-hydroxyprogesterone. To gain information about the exact sites of 20alpha-HSD mRNA expression, we performed in situ hybridization using a (35)S-labeled cRNA probe in tissues of adult mice of both sexes. 20alpha-HSD mRNA was expressed in both male and female gonads. In the ovary, high expression was found in luteal cells of corpora lutea, while much lower expression could be detected in granulosa cells of growing follicles. In the testis, a specific hybridization signal was detected only in Leydig cells. In the female reproductive tract, 20alpha-HSD mRNA was found in the epithelial cells of the uterine cervix. In the adrenal cortex, only the zona reticularis exhibited specific radiolabeling, the expression being very high in the female and very low in the male. In the skin, specific labeling was restricted to sebaceous glands, the hybridization signal being much higher in the female than in the male. In the liver, 20alpha-HSD mRNA was found in hepatocytes, with a higher degree of expression in the female. In the kidney, specific labeling was observed in the epithelial cells of distal convoluted tubules, the signal being also much more striking in the female than in the male. In non-reproductive tissues, it clearly appears that the expression of 20alpha-HSD mRNA is higher in the female than in the male, suggesting that 20alpha-HSD may play an important role in reducing the intracellular concentration of progesterone originating from the circulation at a much higher level in the female.

Diurnal variation in the inducibility of luteal 20 alpha-hydroxysteroid dehydrogenase in pregnant rats.

Luteolysis was induced in rats during late pregnancy by fetoplacental removal, and was monitored by the increased activity of luteal 20 alpha-hydroxysteroid dehydrogenase (20 alpha-OHSDH). The extent of enzyme induction over a given length of time varied according to the time of day at which the fetuses and placentae were removed, 11.00 and 21.00 h appearing to give optimal and minimal enzyme activities respectively. The 20 alpha-OHSDH was more readily induced on day 19 than on day 18.

Induction of rat granulosa cell steroidogenic enzyme activities and their messenger ribonucleic acids by a splenocyte-derived factor.

We have previously identified and purified a splenocyte-derived factor (PSF) that stimulates the accumulation of progesterone and 20 alpha-dihydroprogesterone (20 alpha-OH-P) in rat ovarian granulosa cells independently of FSH. In the present study, time course experiments comparing the response to PSF with that to FSH revealed that PSF-stimulated progesterone accumulation was slower than that of FSH, but PSF-stimulated 20 alpha-OH-P accumulation had a time course similar to that of FSH. To determine the basis for the slower progesterone response to PSF, the effect of these two agents on each step of the steroidogenic pathway was assessed. First, to examine whether PSF-stimulated cholesterol mobilization was limiting, cultured granulosa cells were treated with 22(R)-hydroxycholesterol. While both FSH- and PSF-stimulated progesterone and 20 alpha-OH-P accumulation approximately doubled, the overall time courses did not change indicating that cholesterol availability was not the factor limiting the response to PSF. Next, PSF and FSH induction of steroidogenic enzyme activities and messenger RNAs were compared. While FSH-stimulated cytochrome P450 side chain cleavage enzyme (SCC) activity rapidly increased (peaking at 2 days) and then slowly declined, PSF-stimulated SCC activity gradually increased over 5 days to approximately 35% of the maximal activity stimulated by FSH. PSF also induced slower increases in P450scc mRNA levels than did FSH. In addition, PSF stimulated 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity more slowly than did FSH, but after 3 days of culture, PSF-stimulated activity was significantly higher than that induced by FSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of aldo-keto reductases as NRF2-target marker genes in human cells.

Transcription factor NF-E2-related factor 2 (NRF2) plays a crucial role in the cellular defense against oxidative/electrophilic stress by up-regulating multiple antioxidant genes. Numerous studies with genetically modified animals have demonstrated that Nrf2 is a sensitivity determining factor upon the exposure to environmental chemicals including carcinogens. Moreover, recent studies have demonstrated that polymorphism in the human NRF2 promoter is associated with higher risks for developing acute lung injury, gastric mucosal inflammation, and nephritis. Therefore, the identification of reliable and effective human target genes of NRF2 may allow the monitoring of NRF2 activity and to predict individual sensitivity to environmental stress-induced damage. For this purpose, we investigated genes that are tightly controlled by NRF2 to establish markers for NRF2 activity in human cells. Firstly, in the normal human renal epithelial HK-2 cells, the measurement of the expression of 30 previously reported NRF2 target genes in response to NRF2 inducers (sulforaphane, tert-butylhydroquinone, cinnamic aldehyde, and hydrogen peroxide) showed that the aldo-keto reductase (AKR) 1C1 is highly inducible by all treatments. Accordantly, the basal and inducible expressions of AKRs were significantly attenuated in NRF2-silenced HK-2 cells. Whereas, cells with stable KEAP1 knockdown, which causes a modest NRF2 activation, demonstrated substantially increased levels of AKR1A1, 1B1, 1B10, 1C1, 1C2, and 1C3. Secondly, the linkage between NRF2 and the AKRs was confirmed in human monocytic leukemia cell line U937, which can be a model of peripherally available blood cells. The treatment of U937 cells with NRF2 inducers including sulforaphane effectively elevated the expression of AKR1B1, 1B10, 1C1, 1C2, and 1C3. Whereas, the levels of both the basal and sulforaphane-inducible expression of AKR1C1 were significantly reduced in NRF2-silenced stable U937 cells compared to the control cells. Similarly, the inducible expression of AKR1C1 was observed in another human monocytic leukemia cell line THP-1 as well as in human primary blood CD14(+) monocytes. In conclusion, together with the high inducibility and NRF2 dependency shown in renal epithelial cells as well as in peripherally available blood cells, current findings suggest that AKRs can be utilized as a marker of NRF2 activity in human cells.

Proteasome inhibitors MG-132 and bortezomib induce AKR1C1, AKR1C3, AKR1B1, and AKR1B10 in human colon cancer cell lines SW-480 and HT-29.

Aldo-keto reductases (AKRs) play central roles in the reductive metabolism of endogenous signaling molecules and in the detoxification of xenobiotics. AKRC1-1C3, AKR1B1 and AKR1B10 have been shown to be regulated via nuclear factor-erythroid 2 related factor 2 (Nrf2), a transcription factor that is activated upon oxidative stress. Proteasome inhibitors bortezomib and MG-132 produce mild oxidative stress that activates Nrf2-mediated gene expression that in turn may have cytoprotective effects. Bortezomib is clinically approved to treat haematological malignancies and it has also proven activity in solid tumors such as colon cancer. The present study investigated the effect of bortezomib and MG-132 on the expression of AKR1C1-1C4, AKR1B1, and AKR1B10 in colon cancer cell lines HT-29 and SW-480. Human cancer cell lines derived from different organs (lung, colon, pancreas, skin, liver, ovary) were initially assayed for the expression of the AKRs, showing a very unequal distribution. Even among the colon cell lines HT-29, Caco-2, HCT116 and SW-480, the AKRs were expressed quite non-uniformly. HT-29 cells expressed all AKRs on the mRNA level including liver-specific AKR1C4, but AKR1B1 was almost undetectable. In SW-480 cells, treatment with bortezomib (50 nM, 48 h) dramatically increased mRNA levels of AKR1B10 (32-fold), AKR1B1 (5.5-fold), and, to a lesser extent, AKR1C1 and AKR1C3. Drug-efflux transporter MRP2 (ABCC2) and Cox-2 were induced as well. AKR1C2 mRNA was down-regulated in SW-480 but induced in HT-29 cells. MG-132 increased mRNA amounts of AKR1C1, 1C3, 1B1, and 1B10 in a concentration-dependent manner. AKR1B10 and AKR1B1 protein expression was inducible by bortezomib in HT-29 cells, but not detectable in SW-480 cells. In conclusion, treatment with proteasome inhibitors increased the expression of several AKRs as well as of MRP2. It remains to be investigated whether this enzyme induction may contribute to enhanced cell survival and thereby supporting the phenomenon of multidrug resistance upon cancer chemotherapy.

Rat NAD+-dependent 3alpha-hydroxysteroid dehydrogenase (AKR1C17): a member of the aldo-keto reductase family highly expressed in kidney cytosol.

Mammalian 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) have been divided into two types: Cytosolic NADP(H)-dependent 3alpha-HSDs belonging to the aldo-keto reductase family, and mitochondrial and microsomal NAD(+)-dependent 3alpha-HSDs belonging to the short-chain dehydrogenase/reductase family. In this study, we characterized a rat aldo-keto reductase (AKR1C17), whose functions are unknown. The recombinant AKR1C17 efficiently oxidized 3alpha-hydroxysteroids and bile acids using NAD(+) as the preferred coenzyme at an optimal pH of 7.4-9.5, and was inhibited by ketamine and organic anions. The mRNA for AKR1C17 was detected specifically in rat kidney, where the enzyme was more highly expressed as a cytosolic protein than NADP(H)-dependent 3alpha-HSD (AKR1C9). Thus, AKR1C17 represents a novel NAD(+)-dependent type of cytosolic 3alpha-HSD with unique inhibitor sensitivity and tissue distribution. In addition, the replacement of Gln270 and Glu276 of AKR1C17 with the corresponding residues of NADP(H)-dependent 3alpha-HSD resulted in a switch in favor of NADP(+) specificity, suggesting their key roles in coenzyme specificity.

Regulation of T cell differentiation: in vitro induction of 20 alpha-hydroxysteroid dehydrogenase in splenic lymphocytes from athymic mice by a unique lymphokine.

The enzyme 20 alpha-hydroxysteroid dehydrogenase (20 alpha SDH) has previously been shown to be a specific enzyme marker of mature T cells. In vivo, the expression of 20 alpha SDH is thymus dependent, in that splenic lymphocytes from athymic mice have only low levels of activity, although the levels of enzyme activity increase gradually with age. In vitro, 20 alpha SDH can be induced in splenic lymphocytes from nu/nu mice by conditioned media from mitogen- or alloantigen-stimulated normal lymphocytes. Induction is rapid in vitro. Beginning after a lag period of approximately 6 hr, enzyme activity increases linearly for approximately 20 to 30 hr resulting in a 5- to 10-fold increase in activity. Induction is blocked by mitomycin C, suggesting a requirement for cell proliferation. The phenotype of both the precursor and the induced lymphocyte populations is Thy 1.2-, Lyt 1-, 2-, suggesting that induction of 20 alpha SDH expression is an early step in T cell differentiation. The factor responsible for 20 alpha SDH induction has been partially purified and is distinct from other known lymphokines in both its biochemical and functional properties. The term interleukin 3 is proposed for this factor.

Steroid desmolase synthesis by Eubacterium desmolans and Clostridium cadavaris.

The synthesis of a steroid desmolase was demonstrated in two obligate anaerobes: a new bacterial species, Eubacterium desmolans, isolated from cat fecal flora, and Clostridium cadavaris, recovered from sewage of New York City. The enzyme cleaves the C-17-C-20 bond of corticoids possessing hydroxyl functions at C-17 and C-21. The conversion is quantitative, provided the substrate concentration is less than 100 micrograms/ml and the organisms are in the log phase. The velocity of transformation parallels the bacterial growth curve and in the log phase is higher for E. desmolans than for C. cadavaris. In addition, both organisms synthesize a 20 beta-hydroxysteroid dehydrogenase.

Expression of ovarian 20alpha-hydroxysteroid dehydrogenase in rat thymus.

Ovarian 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which converts progesterone to a derivative devoid of biological activity, plays a crucial role in achieving the short estrous cycle in rats. Although 20alpha-HSD activity has also been demonstrated in the thymus, its molecular nature, function, and regulation of expression have yet to be determined. In the present study we investigated if 20alpha-HSD activity in the thymus originates in a transcript identical to that expressed in the ovary. RT-PCR analysis indicated the expression of 20alpha-HSD mRNA in rat thymus, and sequencing of the PCR product showed 100% identity to ovarian 20alpha-HSD cDNA. Immunohistochemical study using anti-rat ovarian 20alpha-HSD antibody demonstrated the expression of 20alpha-HSD protein in the thymus. The 20alpha-HSD-expressing cells in the thymus seemed to be some type of lymphocyte by their morphology. These results suggest that the same molecular species as ovarian 20a-HSD is expressed in thymic lymphocytes. Therefore, 20alpha-HSD may play a role in T-lymphocyte proliferation and differentiation processes.