The 26S proteasome system degrades the ERM transcription factor and regulates its transcription-enhancing activity.

ERM is a member of the ETS transcription factor family. High levels of the corresponding mRNA are detected in a variety of human breast cancer cell lines, as well as in aggressive human breast tumors. As ERM protein is almost undetectable in these cells, high degradation of this transcription factor has been postulated. Here we have investigated whether ERM degradation might depend on the proteasome pathway. We show that endogenous and ectopically expressed ERM protein is short-lived protein and undergoes proteasome-dependent degradation. Deletion mutagenesis studies indicate that the 61 C-terminal amino acids of ERM are critical for its proteolysis and serve as a degradation signal. Although ERM conjugates with ubiquitin, this post-translational modification does not depend on the C-terminal domain. We have used an Ets-responsive ICAM-1 reporter plasmid to show that the ubiquitin-proteasome pathway can affect transcriptional function of ERM. Thus, ERM is subject to degradation via the 26S proteasome pathway, and this pathway probably plays an important role in regulating ERM transcriptional activity.

Viral oncoproteins target the DNA methyltransferases.

Small DNA tumour viruses have evolved a number of mechanisms to drive nondividing cells into S phase. Virally encoded oncoproteins such as adenovirus E1A and human papillomavirus (HPV) E7 can bind an array of cellular proteins to override proliferation arrest. The DNA methyltransferase Dnmt1 is the major mammalian enzyme responsible for maintaining CpG methylation patterns in the cell following replication. One of the hallmarks of tumour cells is disrupted DNA methylation patterns, highlighting the importance of the proper regulation of DNA methyltransferases in normal cell proliferation. Here, we show that adenovirus 5 E1A and HPV-16 E7 associate in vitro and in vivo with the DNA methyltransferase Dnmt1. Consistent with this interaction, we find that E1A and E7 can purify DNA methyltransferase activity from nuclear extracts. These associations are direct and mediated by the extreme N-terminus of E1A and the CR3 zinc-finger domain of E7. Furthermore, we find that a point mutant at leucine 20 of E1A, a residue known to be critical for its transformation functions, is unable to bind Dnmt1 and DNA methyltransferase activity. Finally, both E1A and E7 can stimulate the methyltransferase activity of Dnmt1 in vitro. Our results provide the first indication that viral oncoproteins bind and regulate Dnmt1 enzymatic activity. These observations open up the possibility that this association may be used to control cellular proliferation pathways and suggest a new mechanism by which small DNA tumour viruses can steer cells through the cell cycle.

The methyl-CpG-binding protein MECP2 is required for prostate cancer cell growth.

The incidence of prostate cancer is increasing in western countries because of population aging. Prostate cancer begins as an androgen-dependent disease, but it can become androgen independent at a later stage or in tumors recurring after an antihormonal treatment. Although many genetic events have been described to be involved in androgen-dependent and/or -independent prostate cancer growth, little is known about the contribution of epigenetic events. Here we have examined the possibility that the methyl-CpG-binding protein MECP2 might play a role in controlling the growth of prostate cancer cells. Inhibition of MECP2 expression by stable short hairpin RNA stopped the growth of both normal and cancer human prostate cells. In addition, ectopic expression of the MECP2 conferred a growth advantage to human prostate cancer cells. More importantly, this expression allowed androgen-dependent cells to grow independently of androgen stimulation and to retain tumorigenic properties in androgen-depleted conditions. Analysis of signaling pathways showed that this effect is independent of androgen receptor signaling. Instead, MECP2 appears to act by maintaining a constant c-myc level during antihormonal treatment. We further show that MECP2-expressing cells possess a functional p53 pathway and are still responsive to chemotherapeutic drugs.

[Multiple post-translational modifications implied in the gene expression regulation: example of the sumoylation of Ets factors]. / Multiples modifications post-traductionnelles impliquées dans la régulation de l'expression génique: exemple de la sumoylation des facteurs Ets.

To regulate the spatiotemporal expression of their target genes, the transcription factors undergo post-translational modifications of which the most studied is phosphorylation. Acetylation and ubiquitinylation on lysine residues also exert a role in the transcription, as it is the case for the regulation of the activity of the huge family of Ets transcription factors. Recently, sumoylation, a post-translational modification similar to ubiquitinylation, was described as playing a crucial role in the inhibition of the activity of these factors.

Effect of prolactin and estradiol on cell proliferation in the uterus and the MXT mouse mammary neoplasm.

With the use of an in vivo tritiated thymidine [( 3H]dThd) nuclear labeling followed by autoradiography, the effects at different times before sacrifice of prolactin (PRL) and/or 17 beta-estradiol (E2) were studied in C57BL X DBA/2f)F1 mice given transplants of the MXT hormone-sensitive mammary tumor whose growth was previously shown to be influenced by E2 and/or progesterone. Uteri were chosen as controls for the methodology. Experiments were conducted on ovariectomized mice submitted to endocrine manipulation to achieve plasma PRL modifications. In addition to E2, the proliferation of cancer cells, assessed by the measurement of thymidine labeling indices (TLIs), was demonstrated to be enhanced by ovine prolactin (oPRL) and Sulpiride and strongly slowed down by castration and 2-bromo-alpha-ergokryptin treatment, thus emphasizing the great importance of PRL in mammary cancer development. Moreover, a pulse of 1 mg oPRL/animal produced a marked TLI rise in tumors, lasting from the 6th to the 48th hour after its injection and reaching a maximum at 24 hours. PRL had no proliferative effect on the uterine luminal epithelium. When PRL and E2 were injected concomitantly, the profile of stimulation was quite similar to that obtained with E2 alone; i.e., a maximum stimulation was observed at the 24th and 36th hours after hormonal pulse. From these data it is concluded that, in spayed mice, not only E2 but also PRL is of major importance leading to enhanced proliferation of MXT mammary neoplastic cells. Further investigations are needed to throw light on the cellular events presiding over the action of PRL and E2 at the cancer cell level.

Influence of pituitary grafts or prolactin administrations on the hormone sensitivity of ovarian hormone-independent mouse mammary MXT tumors.

The sensitivity of MXT mouse mammary tumors to ovarian hormones was assessed by the following parameters: growth in vivo; presence or absence of estrogen receptors and progesterone receptors; and histological differentiation. A spontaneous evolution from hormone sensitivity (HS) to hormone independence (HI) was observed when the tumors underwent monthly transplantation onto intact recipient mice, with the tumors fulfilling the criteria of HI tumors after the 12th transplantation. In contrast, the tumors recovered most of the criteria of hormone sensitivity when pituitary isografts were placed under the kidney capsules of HI tumor-bearing animals or when these animals received daily administrations of prolactin over several months. Sensitivity to 17 beta-estradiol, progesterone, or prolactin was further assessed by actinomycin binding on the nucleus and thymidine labeling index, both measured by autoradiography. These technical approaches revealed that 17 beta-estradiol and prolactin stimulated the thymidine labeling index of both HI (despite the lack of detectable estrogen receptors) and HS MXT tumors whereas progesterone influenced only that of HS cancers. The three hormones significantly stimulated [3H]actinomycin D binding within HS tumors but not within HI ones. However, such "HI" tumors were characterized by increased actinomycin binding and thymidine labeling index in comparison with HS neoplasms. Thus, all the data presently reported strongly suggest that prolactin is able to restore the hormone-sensitive phenotype within so-called MXT hormone-independent tumors.

Stimulation of apolipoprotein D secretion by steroids coincides with inhibition of cell proliferation in human LNCaP prostate cancer cells.

Although steroid hormones are known to play a predominant role in the regulation of cell growth in hormone-sensitive cancers, their mechanisms of action, especially their interaction with growth factors and/or growth inhibitors, is poorly understood. We have recently observed that the effects of androgens and estrogens on the expression of the major protein found in human breast gross cystic disease fluid, protein-24, are opposite to their respective action on cell proliferation in human breast cancer cell lines. Somewhat surprisingly, the recent elucidation of the amino acid sequence of this progesterone binding protein reveals that this tumor marker is apolipoprotein D (apo D), a member of a superfamily of lipophilic ligand carrier proteins. The present study was designed to determine whether apo D is secreted by human prostate cancer cells and could thus be a new marker of steroid action in these cancer cells, and whether the sex steroid-induced stimulation of apo D secretion coincides with inhibition of cell proliferation. We took advantage of the biphasic pattern of the effect of steroids on the proliferation of the human prostate cancer LNCaP cell line, which offers the opportunity to discriminate between positive and negative steroid receptor-regulated cell growth processes. A 10-day exposure to low concentrations of dihydrotestosterone and testosterone caused a potent stimulation of LNCaP cell proliferation, whereas incubation with higher concentrations of these androgens led to a progressive decrease in cell proliferation towards basal levels. The biphasic action of androgens was also observed on apo D secretion, the effects on apo D secretion being inversely related to their action on LNCaP cell proliferation. Similar opposite biphasic effects were also observed with 9 other steroids, thus indicating that the stimulation of secretion of this new biochemical marker coincides with inhibition of cell proliferation in LNCaP human prostatic cancer cells.

Characteristics of the biphasic action of androgens and of the potent antiproliferative effects of the new pure antiestrogen EM-139 on cell cycle kinetic parameters in LNCaP human prostatic cancer cells.

The most potent steroid in human prostatic carcinoma LNCaP cells, i.e., dihydrotestosterone (DHT), has a biphasic stimulatory effect on cell proliferation. At the maximal stimulatory concentration of 0.1 nM DHT, analysis of cell kinetic parameters shows a decrease of the G0-G1 fraction with a corresponding increase of the S and G2 + M fractions. In contrast, concentrations of 1 nM DHT or higher induce a return of cell proliferation to control levels, reflected by an increase in the G0-G1 fraction at the expense of the S and especially the G2 + M fractions. Continuous labeling for 144 h with the nucleotide analogue 5'-bromodeoxyuridine shows that the percentage of cycling LNCaP cells rises more than 90% after treatment with stimulatory concentrations of DHT, whereas in control cells as well as in cells treated with high concentrations of the androgen, this value remains below 50%. Although LNCaP cells do not contain detectable estrogen receptors, the new pure steroidal antiestrogen EM-139 not only reversed the stimulation of cell proliferation and cell kinetics induced by stimulatory doses of DHT but also inhibited basal cell proliferation.

Inhibition of cell cycle kinetics and proliferation by the androgen 5 alpha-dihydrotestosterone and antiestrogen N,n-butyl-N-methyl-11-[16' alpha-chloro-3',17 beta-dihydroxy-estra-1',3',5'-(10')triene-7' alpha-yl] undecanamide in human breast cancer ZR-75-1 cells.

We have investigated the effects of the pure antiestrogen EM-139 and the nonaromatizable androgen dihydrotestosterone (DHT) alone or in combination with estradiol (E2) on cell proliferation and cell kinetic parameters in human ZR-75-1 breast cancer cells. Following a 24- to 30-h exposure to E2, a decrease in the proportion of G0-G1 cells was observed, this effect being accompanied by the well-known stimulatory effect of the estrogen on cell proliferation at later time intervals. By contrast, DHT or EM-139 alone inhibited basal cell proliferation without a significant influence on cell cycle distribution. Moreover, pretreatment with DHT for 8 days, while decreasing ZR-75-1 cell number, did not cause a loss in E2 sensitivity. In fact, as early as after 24 h of E2 treatment, a decrease in the G0-G1 cell fraction accompanied by a corresponding increase of the S-phase was observed in both control and DHT-pretreated cells. When added concomitantly with E2, DHT or EM-139 inhibited the E2 stimulatory effect on cell proliferation, but only EM-139 significantly reversed the G0-G1 decrease induced by E2. Although DHT and EM-139 did not affect the distribution of ZR-75-1 cells between the different phases of the cell cycle, continuous labeling with 5'-bromodeoxyuridine showed that EM-139 and DHT had a global slowing effect on the duration of the cell cycle, thus explaining the potent inhibitory effect of these compounds on cell proliferation. The present data demonstrate that DHT and EM-139 are both potent inhibitors of the stimulatory effect on E2 on cell proliferation, their main action being related to a general increase in the duration of the cell cycle.

The ETS-related transcription factor ERM is a nuclear target of signaling cascades involving MAPK and PKA.

Recent studies support a model for signal transduction from activated receptor tyrosine kinases to Ras which, in turn, activates the pathway of the mitogen-activated protein kinase (MAPK). Although some members of the Ets transcription factor family have been shown to be activated by this signaling pathway, no data are available on the activation of the PEA3 group of Ets proteins. This group is composed of three members -- PEA3, ER81 and ERM -- which are very similar in the DNA-binding domain, the ETS domain, in the 32 residue amino-terminal acidic domain and in the 61 residue carboxy-terminal domain. First of all we demonstrated that ERM-transfected cells contain a positive labeling in the nucleus, and we concluded that a nuclear localization signal might be situated in the ETS domain. We then showed that of four putative reporter plasmids, ERM activated the artificial 3 x TORU plasmid which contains an Ets binding site contiguous to an AP1 one. This transactivation enhancement requires the presence of the ERM amino-terminal domain. In contrast, although the lack of the carboxy-terminal domain induced a decrease in transactivation, this latter domain is not crucial. By using the E74-reporter plasmid system which is not basically activated by ERM, we showed that the activation of the Ras/Raf-1/MAPK pathway significantly enhanced ERM-mediated transactivation. The deletion of the amino-terminal transactivation domain abolished the capacity of stimulated MAPK to activate ERM. We also demonstrated that ERM can also be activated through the protein kinase A (PKA), another signaling pathway. Nevertheless, the MAPK and PKA activation of ERM are not synergistic. Finally, we showed that this Ets transcription factor is in vitro phosphorylated by both activated ERK-2 and activated PKA. ERM has thus been identified as a transcription factor which is a target for two different signaling pathways and might therefore be involved in the mitogenic response of cells.

The transcription of the intercellular adhesion molecule-1 is regulated by Ets transcription factors.

The Ets family of transcription factors comprises several members which are involved to regulate gene transcription. Although several consensus binding sites for Ets proteins can be found in a wide series of promoter, only a limited number of them are indeed activated by these transcription factors. The human intercellular adhesion molecule-1 (ICAM-1) plays a crucial role in immune responses by enabling the binding of effector cells to various target cell types. ICAM-1 is constitutively expressed at different levels in the absence of stimuli in different cell types, and its expression is upregulated by several proinflammatory cytokines. We have here examined the transcriptional regulation of human ICAM-1 expression by Ets proteins, and more particularly by ERM, a member of this family of transcription factors. Transient transfection assays revealed that Ets-2 and ERM significantly activate the transcription of ICAM-1 promoter, whereas the less-related Ets family member, Spi-1/Pu.1, failed to do so. Transfection of a series of ICAM-1 promoter deletion mutants together with ERM expression plasmids have shown that an Ets responsive element is located within the first 176 bp upstream from the translational start site. Electrophoretic mobility shift assays and DNase I footprinting analysis have enabled us to identify two Ets binding sites at positions -158 and -138 from the ATG, respectively. Site directed mutagenesis of these elements has shown that the distal site is the major element required for the ERM-mediated activation of the ICAM-1 promoter. We can thus conclude that expression of ICAM-1 can be regulated by Ets transcription factors.

Differential expression patterns of the PEA3 group transcription factors through murine embryonic development.

ERM, ER81 and PEA3 are three highly related transcription factors belonging to the ETS family. Together they form the PEA3 group within this family. Little data is yet available regarding the roles of these three genes during embryonic development. A prerequisite to investigations in this field is to obtain an accurate spatio-temporal expression map for the erm, er81 and pea3 genes. To this end, we have used in situ hybridization to compare their expression patterns during critical stages of murine embryogenesis. We report that all three genes are expressed in numerous developing organs coming from different embryonic tissues. The three genes appeared co-expressed in different organs but presented specific sites of expression, so that the resultant expression pattern could in fact reveal several distinct functions depending upon isolated and/or various combinations of the PEA3 member expression. These results suggest that erm, er81 and pea3 genes are differentially regulated, probably to serve important functions as cell proliferation control, tissue interaction mediator or cell differentiation, all over successive steps of the mouse organogenesis.

Molecular cloning and characterization of human ERM, a new member of the Ets family closely related to mouse PEA3 and ER81 transcription factors.

The ets-related transcription factors PEA3 and ER81 have recently been isolated and characterized in the mouse. They share 95% identity in a 85 amino acid (AA) domain termed the ETS domain which is responsible for DNA binding, and therefore they form an Ets family group. By screening a human testis cDNA library with a probe containing the mouse PEA3 ETS domain, we isolated a 2.2 kb clone containing a 510 AA open reading frame. Since the ETS domain, which is localized in the carboxy terminal region of the encoded protein, is 95% and 96% identical to that of PEA3 and ER81, respectively, we named this new member 'Ets Related Molecule PEA3-like' (ERM). Although the first 120 AA in the amino-terminal region of ERM share 47% identity with PEA3 and 66% with ER81, ERM contains a central region of approximately 35 AA not found in the two mouse proteins. Gel shift analysis indicates that the full-length ERM protein is able to bind specifically to an oligonucleotide containing the consensus nucleotide core sequence GGAA recognized by the Ets proteins. Moreover, in vitro translation of 83 AA of the ERM ETS domain led to the production of a truncated protein which also binds to DNA. Though differential expression is observed in primary tumors and normal lymphocytes do not express ERM, this gene is almost ubiquitously expressed in human normal tissues. ERM mRNA is highly expressed in brain as well as in placenta and, to a lesser degree, in lung, pancreas, and heart. Moreover, almost all human cell lines tested express it at varying levels. In mouse tissues, we showed that PEA3 and ER81 mRNAs display restricted expression, whereas ERM is almost ubiquitously expressed as observed for human tissues. Altogether these results indicate that ERM is clearly a new ets family member and not the human equivalent of PEA3 or ER81.

Molecular characterization of the ets-related human transcription factor ER81.

The PEA3 group is a homogeneous group of the ets transcription factor family and is composed of three known members, PEA3, ERM and ER81, which, on the amino acid (AA) level, are more than 95% identical within the DNA-binding domain (the Ets domain), more than 85% within a 32 AA domain (the acidic domain) localized in the amino-terminus and almost 50% identical overall. By screening a human kidney cDNA library with a specific probe obtained from mouse ER81, we isolated two clones of 1.6 and 1.5 kb in length encoding a 458 AA open reading frame. When compared to mouse ER81, the present human ER81 lacks the last 13 AA of the acidic domain and the 5 AA contiguous to the carboxy-terminal part of the acidic domain. Of the 458 AA of the human ER81 protein, 97% are identical to mouse ER81. Gel shift analysis indicates that the full-length human ER81 protein is able to bind specifically to an oligonucleotide containing the binding sites recognized by most of the Ets proteins. By transient expression in RK13 mammalian cells, human ER81 protein transactivated a reporter plasmid containing Ets binding sites, indicating that this molecule is a bonafide transcriptional activator, while by expression in Cos-1 transfected cells, we detected the presence of human ER81 protein in the nucleus using immunocytochemistry. In human tissues, ER81 mRNA is very highly expressed in brain, highly expressed in testis, lung and heart, moderately in spleen, small intestine, pancreas and colon, weakly in liver, prostate and thymus, very weakly in skeletal muscle, kidney and ovary and not in placenta and peripheral blood leukocytes. Analysis of human solid or haematopoietic tumour cell lines showed that most of them did not express ER81 detectably. Database searches revealed that ETV1 mRNA is highly similar to human ER81 described here, although it contains the full-length acidic domain present in mouse ER81. By screening a genomic DNA library, we characterized the intron-exon junction within the acidic domain of human ER81 and we showed that this junction corresponds to the site where the remaining AA of the acidic domain of ETV1 or mouse ER81 are inserted.

Two functionally distinct domains responsible for transactivation by the Ets family member ERM.

The recently cloned human Ets transcription factor ERM is closely related to the ER81 and PEA3 genes. Here, we report the functional analysis of the DNA-binding and transactivation properties of ERM. Specific DNA-binding by ERM requires the ETS domain, conserved in all members of the Ets family and is inhibited by an 84 residue long central region and the carboxy-terminal tail. Two fragments of ERM are transferrable activation domains: alpha, which sits in the 72 first residues and encompasses the acidic domain conserved between ERM, ER81 and PEA3, and the carboxy-terminal tail which also bears a DNA-binding inhibition function. Deletion of alpha strongly reduces transactivation by ERM. Moreover, alpha and the carboxy-terminal tail exhibit functional synergism, suggesting that they activate transcription through different mechanisms. In support of this idea, we demonstrate that VP16 squelches transactivation by alpha but not by the carboxy-terminal tail. This result also indicates that alpha and VP16 may share common limiting cofactors. alpha and the carboxy-terminal tail do not seem to be conserved within the whole Ets family, indicating that the specificity of ERM may rely on interactions with distinct cofactors.

Characterization of the human and mouse ETV1/ER81 transcription factor genes: role of the two alternatively spliced isoforms in the human.

The Ets transcription factors of the PEA3 group--E1AF/PEA3, ETV1/ER81 and ERM--are almost identical in the ETS DNA-binding and the transcriptional acidic domains. To accelerate our understanding of the molecular basis of putative diseases linked to ETV1 such as Ewing's sarcoma we characterized the human ETV1 and the mouse ER81 genes. We showed that these genes are both encoded by 13 exons in more than 90 kbp genomic DNA, and that the classical acceptor and donor splicing sites are present in each junction except for the 5' donor site of intron 9 where GT is replaced by TT. The genomic organization of the ETS and acidic domains in the human ETV1 and mouse ER81 (localized to chromosome 12) genes is similar to that observed in human ERM and human E1AF/PEA3 genes. Moreover, as in human ERM and human E1AF/PEA3 genes, a first untranslated exon is upstream from the first methionine, and the mouse ER81 gene transcription is regulated by a 1.8 kbp of genomic DNA upstream from this exon. In human, the alternative splicing of the ETV1 gene leads to the presence (ETV1 alpha) or the absence (ETV1 beta) of exon 5 encoding the C-terminal part of the transcriptional acidic domain, but without affecting the alpha helix previously described as crucial for transactivation. We demonstrated here that the truncated isoform (human ETV1 beta) and the full-length isoform (human ETV1 alpha) bind similarly specific DNA Ets binding sites. Moreover, they both activate transcription similarly through the PKA-transduction pathway, so suggesting that this alternative splicing is not crucial for the function of this protein as a transcription factor. The comparison of human ETV1 alpha and human ETV1 beta expression in the same tissues, such as the adrenal gland or the bladder, showed no clear-cut differences. Altogether, these data open a new avenue of investigation leading to a better understanding of the functional role of this transcription factor.

Molecular characterization of the zebrafish PEA3 ETS-domain transcription factor.

The PEA3 subfamily of ETS-domain proteins play important roles in regulating transcriptional activation and have been implicated in several tumorigenic processes. Here we describe the identification of a further member of this family from zebrafish which most likely represents a homologue of PEA3. A high degree of sequence conservation is observed in the ETS DNA-binding domain and acidic transcriptional activation domain. The DNA binding specificity of zebrafish PEA3 is virtually identical to that exhibited by mammalian family members and is autoregulated by cisacting inhibitory domains. Transcriptional activation by zebrafish PEA3 is potentiated by the ERK MAP kinase and protein kinase A pathways. During embryogenesis, PEA3 is expressed in complex spatial and temporal patterns in both mesodermal somites and ectodermal tissues including the brain, dorsal spinal chord and neural crest. Our characterisation of zebrafish PEA3 furthers our understanding of its molecular function and its expression profile suggests a novel role in cell patterning in the early vertebrate embryo.

Expression patterns of the Ets transcription factors from the PEA3 group during early stages of mouse development.

erm, er81 and pea3 are three related genes that define a novel Ets-related subfamily of transcription factors. The expression patterns of these genes has been previously characterized in the mouse from embryonic day (E) 9.5 to birth (Oncogene 15 (1997) 937). In this study, we report differential expression patterns of the PEA3 group genes during early mouse post-implantation development. erm and pea3 expression patterns were partly overlapping. erm was activated prior to pea3 in the distal tip of the embryonic epiblast but, at primitive streak-stages, both genes were coexpressed in the posterior region of the embryo in epiblast, primitive streak and adjacent mesoderm. Similar erm and pea3 expression patterns were seen later in posterior neural plate, presomitic and lateral mesoderm, mesonephros, and tail bud. Only erm, however, was expressed in specific brain regions corresponding to prospective midbrain and ventral forebrain. erm was also strongly expressed as early as E8 in the developing branchial region, especially at the level of branchial pouches, whereas pea3 transcripts appeared later in frontonasal and first arch mesenchyme. er81 transcripts were not detected prior to E9.0-9.5, suggesting that this gene may not be involved in early developmental events.

Structure and expression of a new complementary DNA encoding the almost exclusive 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase in human adrenals and gonads.

The 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta HSD) enzyme catalyzes the oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into delta 4-ketosteroids, thus leading to the formation of all classes of steroid hormones. In addition, 3 beta HSD catalyzes the interconversion of 3 beta-hydroxy- and 3-keto-5 alpha-androstane steroids. Clinical observations in patients with 3 beta HSD deficiency as well as our recent data obtained by Southern blot analysis using a human placental 3 beta HSD cDNA (type I) as probe suggested the existence of multiple related 3 beta HSD isoenzymes. We now report the isolation and characterization of a second type of cDNA clone (arbitrarily designated type II) encoding 3 beta HSD after screening of a human adrenal lambda gt22A library. The nucleotide sequence of 1676 basepairs of human 3 beta HSD type II cDNA predicts a protein of 371 amino acids with a calculated molecular mass of 41,921 daltons, which displays 93.5% and 96.2% homology with human placental type I and rhesus macaque ovary 3 beta HSD deduced proteins, respectively. To characterize and compare the kinetic properties of the two isoenzymes, plasmids derived from pCMV and containing type I or type II 3 beta HSD full-length cDNA inserts were transiently expressed in HeLa human cervical carcinoma cells. In vitro incubation with NAD+ and 3H-labeled pregnenolone or dehydroepiandrosterone shows that the type I protein possesses a 3 beta HSD/delta 5-delta 4 isomerase activity higher than type II, with respective Km values of 0.24 vs. 1.2 microM for pregnenolone and 0.18 vs. 1.6 microM for dihydroepiandrosterone, while the specific activity of both types is equivalent. Moreover, incubation in the presence of NADH of homogenates from cells transfected with type I or type II 3 beta HSD indicates that dihydrotestosterone is converted into 5 alpha-androstane-3 beta, 17 beta-diol, with Km values of 0.26 and 2.7 microM, respectively. Ribonuclease protection assay using type I- and type II-specific cRNA probes revealed that type II transcripts are the almost exclusive 3 beta HSD mRNA species in the human adrenal gland, ovary, and testis, while type I transcripts correspond to the almost exclusive 3 beta HSD mRNA species in the placenta and skin and represent the predominantly expressed species in mammary gland tissue. The present data show for the first time that adrenals and gonads express a type of 3 beta HSD isoenzyme that is distinct from the type expressed in the placenta.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular basis of congenital adrenal hyperplasia due to 3 beta-hydroxysteroid dehydrogenase deficiency.

Congenital adrenal hyperplasia is the most frequent cause of adrenal insufficiency and ambiguous genitalia in newborn children. In contrast to congenital adrenal hyperplasia due to 21-hydroxylase and 11 beta-hydroxylase deficiencies, which impair steroid formation in the adrenal cortex, exclusively, classical 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency affects steroid biosynthesis in the gonads as well as in the adrenals. The structures of the highly homologous type I and II 3 beta-HSD genes have been analyzed in three male pseudohermaphrodite 3 beta-HSD deficient patients from unrelated families in order to elucidate the molecular basis of classical 3 beta-HSD deficiency from patients exhibiting various degrees of severity of salt losing. The nucleotide sequence of DNA fragments generated by selective polymerase chain reaction amplification that span the four exons, the exon-intron boundaries, as well as the 5'-flanking region of each of the two 3 beta-HSD genes have been determined in the three male patients. The five point mutations characterized were all detected in the type II 3 beta-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3 beta-HSD gene, predominantly expressed in the placenta and peripheral tissues. The two male patients suffering from severe salt-losing 3 beta-HSD deficiency are compound heterozygotes, one bearing the frame-shift mutation 186/insC/187 and the missense mutation Y253N, while the other bears the nonsense mutation W171X and the missense mutation E142K. The influence of the detected missense mutations on enzymatic activity was assessed by in vitro expression analysis of mutant recombinant enzymes generated by site-directed mutagenesis in heterologous mammalian cells. Recombinant mutant type II 3 beta-HSD enzymes carrying Y253N or E142K substitutions exhibit no detectable activity. On the other hand, the nonsalt-losing patient is homozygous for the missense mutation A245P. This mutation decreases 3 beta-HSD activity by approximately 90%. The present findings, describing the first missense mutations in the human type II 3 beta-HSD gene, provide unique information on the structure-activity relationships of the 3 beta-HSD superfamily. Moreover, the present findings provide a molecular explanation for the enzymatic heterogeneity responsible for the severe salt-losing form to the clinically inapparent salt-wasting form of classical 3 beta-HSD deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)