Biomedical subjects

D W Hum

Publications and source records attributed to D W Hum.

At least 55 records · Page 3Linked to original sources

PubMed1996

Isolation and characterization of a novel cDNA encoding a human UDP-glucuronosyltransferase active on C19 steroids.

To isolate cDNA clones encoding novel UGT2B enzymes, human prostate and LNCaP cell cDNA libraries were screened using a pool of steroid-specific UGT2B cDNA probes. In approximately 10(6) recombinants, we isolated 3 cDNA clones of 2.1 kilobases that encode a novel UGT2B enzyme. UGT2B17 is 95% identical with UGT2B15 and 91% identical with UGT2B8. Primary structure analysis of UGT2B17 based on the nucleotide sequence revealed a putative amino-terminal membrane insertion signal peptide, a carboxyl-terminal membrane-spanning region, and three potential asparagine-linked glycosylation sites. UGT2B17 cloned in the pBK-CMV expression vector was transfected into HK293 cells to obtain a stable clonal cell line expressing a high level of the active 53-kDa UGT2B17 enzyme. Of the over 60 endogenous and exogenous substances tested, 25 compounds revealed reactivity. The major substrates are eugenol > 4-methylumbelliferone > dihydrotestosterone > androstane-3alpha, 17beta-diol (3alpha-diol) > testosterone > androsterone (ADT). The apparent Km values obtained with tritiated steroids in intact cells were 0.4 microM for ADT, 0.7 microM for dihydrotestosterone, 1.0 microM for 3alpha-diol, and 3.4 microM for testosterone. Southern blot analysis of reverse transcription-polymerase chain reaction products revealed expression of UGT2B17 mRNA in various tissues including the liver, kidney, testis, uterus, placenta, mammary gland, adrenal gland, skin, and prostate. UGT2B17 is the first human uridine diphosphoglucuronosyltransferase enzyme expressed in extrahepatic tissues to have a specificity for ADT as well as testosterone, dihydrotestosterone, and 3alpha-diol.

Amino Acid Sequence↗

PubMed1996

Expression and regulation of the lipoprotein lipase gene in human adrenal cortex.

Lipoprotein lipase (LPL), an enzyme which hydrolyzes triglycerides and participates in the catabolism of remnant lipoproteins, plays a crucial role in energy and lipid metabolism. The goal of this study was to analyze the expression and regulation of the LPL gene in human adrenals. Reverse transcriptase-polymerase chain reaction amplification and sequence analysis demonstrated the presence of LPL mRNA in fetal and adult human adrenal cortex. Furthermore, the human adrenocortical carcinoma cell line, NCI-H295, expresses LPL mRNA and protein, which is localized to the outer cellular membrane as demonstrated by immunofluorescence confocal microscopy and can be released in the medium by heparin addition. To asses whether the LPL gene is regulated by agents regulating adrenal steroidogenesis, NCI-H295 cells were treated with activators of second messenger systems. Whereas the calcium-ionophore A23187 did not affect LPL gene expression, treatment with phorbol 12-myristate 13-acetate decreased LPL mRNA levels in a time- and dose-dependent manner. This decrease after phorbol 12-myristate 13-acetate was associated with diminished heparin-releasable LPL mass and activity in the culture medium. Addition of the cAMP analog 8-Br-cAMP to NCI-H295 cells resulted in a rapid, but transient dose-dependent induction of LPL mRNA. Treatment with the protein synthesis inhibitor cycloheximide gradually induced, whereas simultaneous addition of cAMP and cycloheximide superinduced LPL mRNA levels. Nuclear run-on analysis indicated that the effects of cAMP and cycloheximide occurred at the transcriptional and post-transcriptional level, respectively. Transient co-transfection assays demonstrated that the first 230 base pairs of the proximal LPL promoter contain a cAMP-responsive element activated by protein kinase A and transcription factors belonging to the CREB/CREM family. These data indicate that LPL is expressed in human adrenal cortex and regulated in NCI-H295 adrenocortical carcinoma cells by activators of the protein kinase A and protein kinase C second messenger pathways in a manner comparable to P450scc, which catalyzes the first step in adrenal steroidogenesis. These observations suggest a role for LPL in adrenal energy and/or lipid metabolism and possibly in steroidogenesis.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Evidence for a role of glucuronosyltransferase in the regulation of androgen action in the human prostatic cancer cell line LNCaP.

Androgens play an important role in the regulation of cell growth and specific protein synthesis in hormone-sensitive prostatic cancer. In this study, we have investigated the metabolism of androgens in LNCaP cells from low passage (LP) and high passage (HP) cultures which were previously shown to possess differential androgen responsiveness. When treated with dihydrotestosterone (DHT), cells showed the characteristic biphasic response of cell proliferation with an ED50 of 1 nM for both the LP and HP cells, but the maximal proliferative response was different with values of 2.65- and 4.29-fold over basal for LP and HP cells, respectively. Metabolism studies indicated no difference in 5alpha-reductase activity between LP and HP cells, while 3alpha-, 3beta- and 17beta-hydroxysteroid dehydrogenase activities were significantly higher in LP cultures. The formation of steroid glucuronides (-G), namely DHT-G, was higher in LP than in HP cells with values of 2.16 and 1.31 pmol of glucuronides formed/microgram DNA/3 h, respectively. Northern blot analysis with a UGT21B15 cDNA probe identified two bands corresponding to two or more UGT transcripts in both LNCaP cells and more transcript was observed in LP than in HP cells. Taken together these results indicate that DHT is deactivated more rapidly in the LP cells, which may explain in part the lower proliferative response to androgens of LP cells compared with HP cells.

Adenocarcinoma↗

PubMed1996

Levels of plasma C19 steroids and 5 alpha-reduced C19 steroid glucuronides in primates, rodents, and domestic animals.

In this study, a comparison of circulating levels of androsterone glucuronide and androstane-3 alpha, 17 beta-diol glucuronide in the male and female of several mammalian species was performed. Glucuronidated steroids were not detected in the circulation of the dog, bovine, swine, and rodent. High levels of circulating glucuronidated steroids were measured in the cynomolgus monkey and found to be 10-fold higher than in humans. The determination of tissue levels of unconjugated and conjugated C19 steroids was then performed in intact and castrated rats treated with androgens. Steroid glucuronides were not detected in the plasma, skin, prostate, or liver of either intact or treated rats, although the levels of unconjugated steroids in the plasma and tissues were increased after steroid treatments. Significant levels were detected in the bile, thus suggesting hepatic formation of steroid glucuronides in the rat. It is suggested that the monkey represents the best animal model to date to study the contribution of diphosphoglucuronosyltransferases present in steroid target peripheral tissues to circulating levels of steroid glucuronides.

Aging↗

PubMed1996

Regulation of steroid glucuronosyltransferase activities and transcripts by androgen in the human prostatic cancer LNCaP cell line.

Although much attention has been focused on the synthesis of dihydrotestosterone (DHT), the inactivation and elimination of active androgens can also be key points in regulating androgen levels in tissues such as the prostate. Recent data suggest that 5alpha-reduced C19 steroids can be converted to glucuronide derivatives in the human prostate, leading to complete inactivation of these steroids. These results are supported by the recent finding of at least two steroid uridine diphosphoglucuronosyltransferase (UGT) enzymes in the prostate as well as in the human prostatic cancer LNCaP cell line. To ascertain the role of UGTs in regulating active steroid levels, we investigated the modulation of UGT levels in response to steroid treatments in LNCaP cells. Results demonstrate the down-regulation of UGT activities specific for 3-hydroxysteroids and 17-hydroxy-steroids after treatment with androgens and estrogens. Treating the cells with DHT or R1881 for 7 days inhibited UGT activity by 60%; however, 80% of the total activity was recovered after 5 days in the absence of the androgens. The inhibition of UGT activities by DHT and R1881 increases with the time of incubation and with increasing concentrations of the androgens used. The decrease in UGT enzyme activity occurred in parallel with a diminution in UGT transcript levels, as observed in Northern blot analyses. A correlation between the effect of steroids on the androgen-dependent growth response of LNCaP cells, the secretion of prostate-specific antigen, and the inhibition of UGT activities was clearly demonstrated, which implicates the androgen signaling pathway. Treating cells with Casodex, an androgen antagonist that binds the mutated androgen receptor expressed in LNCaP cells, partially blocked the androgen- and estrogen-induced decrease in UGT activity, suggesting that the regulation of UGT levels involves the androgen receptor. In addition to the formation of DHT, the inactivation of steroids by glucuronidation, which is regulated by steroids themselves, is an important mechanism controlling the level of androgens in the prostate.

Androgen Antagonists↗

PubMed1996

Gene conversion in the CYP11B2 gene encoding P450c11AS is associated with, but does not cause, the syndrome of corticosterone methyloxidase II deficiency.

Cytochrome P450c11AS (aldosterone synthase) has 11 beta-hydroxylase, 18-hydroxylase, and 18-oxidase activities and is expressed solely in the adrenal zona glomerulosa. Corticosterone methyloxidase II (CMOII) deficiency denotes a rare disorder of adrenal steroidogenesis in which only the 18-oxidase activity of P450c11AS is disrupted, while the 11 beta-hydroxylase and 18-hydroxylase activities persist. Such patients have elevated serum concentrations of corticosterone and 18-hydroxycorticosterone and very low or unmeasurable concentrations of aldosterone, often resulting in a clinical salt-losing crisis in infancy. One pair of point mutations, Arg181-->Trp and Val386-->Ala, has been previously characterized to cause this disorder in an inbred Iranian Jewish population. We have sought mutations causing CMOII deficiency in outbred populations. In three of four unrelated P450c11AS alleles from two unrelated patients with CMOII deficiency, we found a gene conversion event in which exons 3 and 4 of the CYP11B2 gene encoding P450c11AS were changed to the sequence of the nearby CYP11B1 gene, which encodes the related enzyme P450c11 beta. This conversion resulted in a mutant P450c11AS protein carrying three changes: Asp141-->Glu, Lys151-->Asn, and Ile246-->Thr. We built seven vectors expressing P450c11AS carrying each mutation singly, each of the three possible pairs of mutations, and the triple mutation as found in the proband. The activities of both the normal P450c11AS and the various mutants in transfected nonsteroidogenic COS-1 cells were very low, but their activities in steroidogenic MA-10 and JEG-3 cells were 10- to 20-fold higher. In these systems all of the mutants retained normal 18-oxidase activity, indicating that the detected gene conversion event is associated with but does not cause CMOII deficiency. None of the four CYP11B2 alleles in these two patients bore other identifiable mutations. These patients might have mutations in the promoters or other noncoding regions, or mutations in genes other than CYP11B2 may cause the syndrome of CMOII deficiency.

Base Sequence↗

PubMed1995

Expression of transcripts encoding steroid UDP-glucuronosyltransferases in human prostate hyperplastic tissue and the LNCaP cell line.

The UDP-glucuronosyltransferase (EC 2.4.1.17) enzymes transform many lipophilic compounds to more water-soluble products via conjugation with glucuronic acid. This conversion is responsible for enhancing the excretion of endogenous aglycones such as steroids. To date, several distinct isoforms of steroid UDP-glucuronosyltransferases (UGTs) have been isolated in the human liver. Among these UGTs, UGT2B7 is specific for estriol and 3,4-catechol estrogens, UGT2B15 glucuronidates 17beta-hydroxy-C19 steroids while UGT2B10 has as yet an undescribed activity. To further demonstrate the presence of UGTs in peripheral tissues we studied the expression of these enzymes in human prostate hyperplastic tissue and the LNCaP cell line. Metabolism studies using intact LNCaP cells in culture indicate the presence of UGT activities involved in the glucuronidation of 3alpha-hydroxysteroids (androsterone) and 17beta-hydroxysteroids (testosterone and dihydrotestosterone). Northern blot analysis of poly(A+) RNA from LNCaP cells and prostate using a UGT2B15 cDNA probe revealed two bands of 2.0 and 2.3 kb. In order to identify more specifically the mRNAs detected in Northern blot analysis we used RNase protection and RT-PCR, although, these approaches did not allow detection of UGT2B7 transcripts. Our studies demonstrate the presence of two UGT activities and at least two types of UGT transcripts in both the human prostate and the LNCaP.

Androsterone↗

PubMed1995

Specificity of glucuronosyltransferase activity in the human cancer cell line LNCaP, evidence for the presence of at least two glucuronosyltransferase enzymes.

Recent findings obtained by our group showed that incubation of LNCaP cells with labeled steroids leads to the formation of 3- and 17-hydroxysteroid glucuronides. In this study, the specificity and the kinetic properties of 3-hydroxy-C19steroid uridine diphospho-glucuronosyltransferase (3-OH-UGT) and 17-hydroxy-C19steroid UGT (17-OH-UGT) activities in LNCaP cells were investigated. Results indicate that the UGT has a high affinity for testosterone, dihydrotestosterone (DHT), androsterone (ADT) and androstane-3 alpha, 17 beta-diol (3 alpha-DIOL), with Km values ranging from 0.25 to 0.68 microM. The Km values are approx. 10-fold higher for androst-5-ene-3 beta,17 beta-diol (5-ene-DIOL) and androstane-3 beta,17 beta-diol (3 beta-DIOL). The relative specificities (Vmax/Km) also showed higher turnover rates for testosterone, DHT, ADT and 3 alpha-DIOL with values ranging from 2.93 to 5.71, than for 3 beta-DIOL and 5-ene-DIOL with ratios of 0.41 and 1.10, respectively. Dixon plot and Cornish-Bowden analysis demonstrate that testosterone, DHT, ADT, and 3 alpha-DIOL inhibit the glucuronidation of DHT and ADT in a competitive fashion. In contrast, when the studies are performed with 3 beta-diol and 5-ene-DIOL the inhibition of ADT glucuronidation is uncompetitive while the glucuronidation of DHT is inhibited competitively, suggesting the presence of two UGT enzymes, one for glucuronidation of the 17 beta-OH group and a second for the 3 alpha-OH group. Further evidence for the presence of two UGTs in LNCaP cells was obtained by incubation with a variety of 3 beta-OH-C19 steroids which caused a marked inhibition of DHT-G formation but had no effect on the glucuronidation of ADT. In summary, our data demonstrate the presence of at least two UGTs in the human prostate cancer cell line LNCaP. The relative specificity of the 17-OH-UGT in LNCaP cells is 3 alpha-DIOL > DHT > testosterone, while ADT is glucuronidated by the 3-OH-UGT.

Adenocarcinoma↗

PubMed1995

Characterization of placental transcriptional activation of the human gene for P450scc.

Steroid hormones, which are ubiquitous regulators of physiologic processes, are produced primarily in the adrenals, gonads, and placenta. Each steroidogenic cell type produces different steroids due to cell-specific expression of various steroidogenic enzymes, but all steroidogenesis is initiated by P450scc, the mitochondrial enzyme that converts cholesterol to pregnenolone. We previously showed the unique segments of the P450scc promoter that are responsible for basal and cAMP-induced expression of this gene in the placenta are not employed for expression in the adrenal (C.C.D. Moore, D.W. Hum, and W.L. Miller, Mol. Endocrinol. 6, 2045-2058, 1992). We now show that sequences between -142 and -153 exhibit placental-specific activator activity. Sequences between -131 and -155 can confer activator activity to a 32-bp promoter from the thymidine kinase gene of herpes simplex virus in an orientation-independent fashion. Two protein complexes, termed IV and VII, interact specifically with DNA from -131 to -155. Mutating bases -142 to -151 abolishes formation of complex VII and partially inhibits complex IV, suggesting that the proteins forming these complexes bind neighboring segments of DNA. Mutating only two cytosines at bases 141 and 142 also eliminates the formation of complex VII and reduces the transcriptional activity of the activator by about 75-80%, indicating that complex VII is important for placental expression of P450scc. The sequence from -140 to -149 on the antisense strand resembles an NF-kappa B binding site. Antibodies to NF-kappa B subunit p50, but not to p52, p65, or c-Rel, will supershift some but not all of complex IV, whereas none of these antibodies interact with complex VII. A consensus NF-kappa B oligonucleotide does not form complex IV, suggesting that p50 interacts with the protein component, but not the DNA component of complex IV. Photoaffinity UV cross-linking yielded single bands of cross-linked DNA-protein complexes at approximately 85 kD for complex IV and approximately 70 kD for complex VII, indicating that separate proteins form complexes IV and VII. Southwestern blotting identified a single protein of 55 kD forming complex VII but did not identify the protein forming complex IV. Bandshifts and Southwestern blots with nuclear extracts from steroidogenic human placental JEG-3 cells and human adrenal NCI-H295 cells show that this 55-kD protein is found in placental but not adrenal cells. This 55-kD nuclear protein appears to be a trans-acting factor necessary for placental but not adrenal expression of P450scc.

Base Sequence↗

PubMed1995

Artificial mutations in P450c11AS (aldosterone synthase) can increase enzymatic activity: a model for low-renin hypertension?

The conversion of 11-deoxycorticosterone (DOC) to aldosterone is catalyzed by a single enzyme, termed P450c11AS, which has 11 beta-hydroxylase, 18-hydroxylase and 18-oxidase activities. The normotensive Dahl salt-resistant (R) rat has two mutation in P450c11AS that increase its aldosterone synthase activity. If such a mutation were to occur in human patients the predicted phenotype would be low-renin hypertension with elevated ratios of plasma aldosterone to plasma renin activity. Before searching for P450c11AS mutations in such patients we sought to determine if mutations in human P450c11AS could increase enzymatic activity in a fashion analogous to the Dahl R rat. We used site-directed mutagenesis of the human P450c11AS cDNA to create the mutants Glu 136-->Asp, Lys 251-->Arg and the combination of the two; these mutations correspond to those seen in the Dahl R rat. Cells transfected with these mutant human P450c11AS sequences could convert [14C]DOC to corticosterone, 18OH-corticosterone, and aldosterone. In particular the Lys 251-->Arg mutant produced 4 times as much 18OH-corticosterone and 50-80% more aldosterone than the wild type. These data show that mutations of human P450c11AS can increase enzymatic activity, suggesting that such mutations could, in theory, be the basis of some forms of human low-renin hypertension.

Aldosterone↗

PubMed1994

Point mutation of Arg440 to His in cytochrome P450c17 causes severe 17 alpha-hydroxylase deficiency.

Genetic disorders in the gene encoding P450c17 cause 17 alpha-hydroxylase deficiency. The consequent defects in the synthesis of cortisol and sex steroids cause sexual infantilism and a female phenotype in both genetic sexes as well as mineralocorticoid excess and hypertension. A 15-yr-old patient from Germany was seen for absent pubertal development and mild hypertension with hypokalemia, high concentrations of 17-deoxysteroids, and hypergonadotropic hypogonadism. Analysis of her P450c17 gene by polymerase chain reaction amplification and direct sequencing showed mutation of codon 440 from CGC (Arg) to CAC (His). Expression of a vector encoding this mutated form of P450c17 in transfected nonsteroidogenic COS-1 cells showed that the mutant P450c17 protein was produced, but it lacked both 17 alpha-hydroxylase and 17,20-lyase activities. To date, 15 different P450c17 mutations have been described in 23 patients with 17 alpha-hydroxylase deficiency, indicating that mutations in this gene are due to random events.

Adolescent↗

PubMed1993

Basal transcriptional activity and cyclic adenosine 3',5'-monophosphate responsiveness of the human cytochrome P450scc promoter transfected into MA-10 Leydig cells.

Mouse Leydig MA-10 tumor cells are a good model of testicular steroidogenesis. The endogenous murine P450scc mRNA in these cells accumulated in response to 8-bromo-cAMP, forskolin, cholera toxin, and 1-methyl-3-isobutylxanthine, but not in response to 1,9-dideoxyforskolin, indicating that this accumulation was stimulated by the protein kinase-A pathway. Inhibiting transcription with actinomycin-D showed that the half-life of cytochrome P450scc mRNA in these cells was not altered by cAMP, consistent with earlier nuclear run-on data showing that the effect of cAMP on P450scc is at the transcriptional level. A series of 17 fragments of 5'-flanking DNA from the human P450scc gene were fused to the gene for firefly luciferase and transiently transfected into MA-10 cells. The longest construct, containing 2327 basepairs of 5'-flanking DNA, responded 4-fold to forskolin and, hence, was used to optimize the forskolin dose response, showing that 30 microM forskolin elicited a 90% maximal effect. Examination of the activity of the deletion constructs located basal and cAMP-responsive sequences. Constructions containing 79 basepairs of 5'-flanking DNA had basal activity; adding sequences between -79 and -110 had minimal effect, but adding sequences between -110 and -127 increased basal activity 3-fold. Adding sequences beyond -127 did not increase basal transcription further, indicating the presence of a basal transcription element between -110 and -127. These serial deletion mutants were used similarly to locate cAMP responsiveness between -1620 and -1676, indicating the presence of a cAMP response element in this region. The locations of these basal and cAMP-responsive sequences correspond well with those previously identified when human P450scc promoter/reporter constructions were transfected into mouse adrenocortical Y-1 cells, but differ from those identified when such constructions were transfected into human JEG-3 choriocarcinoma cells.

1-Methyl-3-isobutylxanthine↗

PubMed1993

Regulation of steroidogenesis in NCI-H295 cells: a cellular model of the human fetal adrenal.

NCI-H295 is a recently described human adrenocortical carcinoma cell line that makes a variety of steroid hormones. We sought to determine if steroidogenesis in these cells employs the same enzymes as those used in normal adrenal steroidogenesis, and if the genes encoding those enzymes exhibit characteristic responsiveness to activators of the protein kinase-A and -C pathways of intracellular second messengers. Northern blots show that NCI-H295 cells contain abundant mRNAs for three key steroidogenic enzymes, cytochrome P450scc, cytochrome P450c17, and cytochrome P450c21. These mRNAs accumulated in a time- and dose-dependent fashion in response to 8-bromo-cAMP (8Br-cAMP), forskolin, cholera toxin, and 3-isobutyl-1-methylxanthine, all activators of the protein kinase-A pathway. Nuclear run-on assays and actinomycin-D transcriptional inhibition experiments show that cAMP regulates the expression of all three genes primarily at the transcriptional level. Inhibition of protein synthesis with cycloheximide did not prevent the cAMP-induced accumulation of P450scc or P450c17 mRNAs, but did inhibit accumulation of P450c21 mRNA, suggesting that cAMP is acting through a mechanism dependent on protein synthesis to promote accumulation of P450c21 mRNA. Stimulation of the protein kinase-C pathway with phorbol ester decreased P450scc and P450c17 mRNAs, but stimulated the accumulation of P450c21 mRNA. RNase protection experiments, Northern blot hybridizations, and reverse transcription-polymerase chain reaction show that NCI-H295 cells express both the 11 beta-hydroxylase (P450c11 beta) encoded by the P450c11B1 gene and the aldosterone synthetase (P450c11AS) encoded by the P450c11B2 gene. 8Br-cAMP increased the abundance of both of these mRNAs with similar kinetics, with maximal accumulation of both after about 24 h. NCI-H295 cells also contain the mRNAs for aromatase and insulin-like growth factor-II. 8Br-cAMP increased the abundance of aromatase mRNA and decreased the abundance of IGF-II mRNA. These studies show that NCI-H295 cells express most of the enzymes needed for human adrenal steroidogenesis, and that the genes encoding these enzymes respond to stimulation of second messenger pathways in a manner similar to that of human adrenals. NCI-H295 cells appear to be a good model for studying the molecular regulation of human adrenal steroidogenesis.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1993

Transcriptional regulation of human genes for steroidogenic enzymes.

Steroid hormones are synthesized from cholesterol in the adrenals, gonads, and placenta by a complex series of reactions. The human genes encoding each of these biosynthetic enzymes have been cloned, permitting study of their regulation. Tropic hormones, such as corticotropin and the gonadotropins, exert their chronic effects on steroidogenesis by increasing the amounts of steroidogenic enzymes; this in turn occurs primarily through increased gene transcription. Our studies have emphasized the cholesterol side-chain cleavage enzyme P450scc, which catalyzes the first and rate-limiting step in steroidogenesis, and P450c17, which determines what class of steroids is synthesized. By fusing the promoters of the genes for these enzymes to readily assayed reporter genes and transiently transfecting cultured cells with these constructions, we have identified the regions of each promoter that confer basal expression, induction by cAMP, and repression by activators of protein kinase C. Different segments of the P450scc promoter are used for each of these purposes in different cell types, indicating that the regulation of this gene is very complex. Transcription is not the only level at which steroidogenesis is regulated. The abundance of mRNA for adrenodoxin reductase, a flavoprotein needed for P450scc activity, is post-transcriptionally regulated by cAMP.

Base Sequence↗

PubMed1992

Identification of positive and negative placenta-specific basal elements and a cyclic adenosine 3',5'-monophosphate response element in the human gene for P450scc.

The chronic regulation of steroiodgenesis is mediated principally by transcriptional regulation of the genes encoding the various steroidogenic enzymes. The cholesterol side-chain cleavage enzyme, P450scc, is rate limiting and hormonally regulated in a tissue-specific fashion. Human placental steroidogenesis is regulated by LH and hCG through increased intracellular cAMP, and forskolin and 8-bromo-cAMP increase the abundance of human P450scc mRNA in human JEG-3 choriocarcinoma cells. We transfected JEG-3 cells with 24 promoter/reporter constructions to examine the tissue-specific and hormonally induced transcription of the human P450scc gene in these cells. A reporter construction containing only bases -79 to +49 of the human P450scc gene was expressed in JEG-3 cells. This basal expression was increased by four elements, especially by a powerful element between -152 to -142. Adding DNA sequences to -177 suppressed the basal expression seen with the -152 construction, indicating that a repressor element lies between -177 and -152. Thus, basal expression of the human P450scc gene in JEG-3 cells is mediated by the interplay of several separate cis-acting DNA elements. Forskolin induction was conferred by sequences between -108 and -89. The mechanism for cAMP induction appears to be direct, as this induction is rapid and is not blocked by inhibiting protein synthesis with cycloheximide. Gel mobility shift experiments identified six specific DNA-protein complexes. Five of these complexes correlate closely with the basal transcription activities identified by the reporter assays. The powerful basal element, the repressor element, and the cAMP element differ from those identified by similar experiments in mouse adrenal Y1 cells, suggesting that the human P450scc gene is regulated by the tissue-specific use of different regulatory elements.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

A pseudogene on the X chromosome for the human trifunctional enzyme MTHFD (methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase).

The human trifunctional folate-dependent protein MTHFD has been mapped to chromosome 14q24 and to the X chromosome was identified by screening an X-chromosome-specific library. Amplification by the polymerase chain reaction (PCR) and sequencing of PCR products indicate that the sequence is an intronless pseudogene.

Aminohydrolases↗

PubMed1991

Expression of active domains of a human folate-dependent trifunctional enzyme in Escherichia coli.

The cDNA encoding the human trifunctional enzyme methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase was engineered to contain a prokaryotic ribosome binding site and was expressed under the bacteriophage T7 RNA polymerase promoter in Escherichia coli. Site-directed mutagenesis was used to prepare constructs that encode separately the dehydrogenase/cyclohydrolase (D/C) domain as amino acid residues 1-301, and the synthetase (Syn) domain as residues 304-935. Both domains formed active enzymes thereby demonstrating their ability to fold independently. The full-length enzyme, D/C and Syn domains were expressed at levels 4-, 55- and 3-fold higher than the specific activities found in liver. Additional mutagenesis and independent expression of domains further defined the interdomain region to include amino acids 292-310. The D/C domain was purified to homogeneity by a single affinity chromatographic step, and the full-length protein in a two-step procedure. The kinetic properties of the D/C domain appear unaltered from those of the trifunctional enzyme.

Amino Acid Sequence↗

PubMed1989

Chromosomal localization of the gene for the human trifunctional enzyme, methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase.

A trifunctional protein in man, 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyltetrahydrofolate cyclohydrolase-10-formyltetrahydrofolate synthetase, catalyzes three consecutive steps in the interconversion of tetrahydrofolate derivatives; these derivatives supply one-carbon units for intermediary metabolism. Somatic cell hybridization and in situ hybridization were used to localize the functional gene coding for this protein--to human chromosome 14q24, near the c-fos and TGF-beta 3 loci. A second hybridizing sequence, possibly a pseudogene, was identified near the centromere of the X chromosome, at Xp11.

Aminohydrolases↗