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J F Strauss

Publications and source records attributed to J F Strauss.

At least 55 records · Page 3Linked to original sources
PubMed1999

Pro-inflammatory cytokines induce expression of matrix-metabolizing enzymes in human cervical smooth muscle cells.

The process of cervical ripening has been likened to an inflammatory reaction associated with the catabolism of cervical extracellular matrix by enzymes released from infiltrating leukocytes. We hypothesized that smooth muscle cells in the cervix also participate in this process and that pro-inflammatory cytokines act on cervical smooth muscle cells (CSMC) to provoke the expression of matrix-degrading enzymes. We treated primary cultures of human CSMC with tumor necrosis factor-alpha (TNF-alpha) and examined expression of the elastinolytic enzyme, cathepsin S, the collagen metabolizing matrix metalloproteinases (MMP)-1, -3, -9, and the tissue inhibitor of metalloproteinase (TIMP)-1 and -2. A time course analysis revealed that 10 ng/ml of TNF-alpha induced cathepsin S, MMP-1, -3, and -9 mRNA expression with the maximal response observed after 24-48 hours. TNF-alpha induced cathepsin S, MMP-1, -3, and -9 mRNA expression in a dose-dependent manner: the maximal effect was observed at a concentration of 10 ng/ml, with appreciable increases observed at concentrations of 0.1 to 1.0 ng/ml. In contrast, TIMP-1 and -2 mRNAs were not significantly increased by TNF-alpha treatment. Interleukin-1beta produced a pattern of gene expression in the CSMC similar to that observed following TNF-alpha treatment. Western blot analysis and zymography confirmed the induction of proMMP-1, -3, and -9 in response to TNF-alpha, but MMP-2 immunoreactivity and zymographic activity were unaffected. TNF-alpha increased secretion of procathepsin S, but did not affect TIMP-1 and reduced TIMP-2 production. We conclude that CSMC are targets of pro-inflammatory cytokines, which induce a repertoire of enzymes capable of degrading the cervical extracellular matrix. The induction of these enzymes may facilitate the normal ripening of the cervix at term and participate in the premature cervical changes associated with preterm labor.

Blotting, Western↗
PubMed1999

Insulin and insulin-like growth factor-I and -II modulate human granulosa-lutein cell steroidogenesis: enhancement of steroidogenic acute regulatory protein (StAR) expression.

Insulin and insulin-like growth factors (IGF)-I and -II stimulate granulosa cell steroidogenesis. Since steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroid hormone biosynthesis, the ability of insulin and IGF to modulate StAR protein and mRNA expression was examined in two human granulosa cell culture systems: (i) proliferating granulosa-lutein cells and (ii) luteinized-granulosa cells derived during in-vitro fertilization (IVF). In proliferating granulosa-lutein cells, IGF-I and IGF-II increased StAR protein approximately 4-5-fold, while insulin and 8-bromoadenosine 3',5'-cAMP (8-Br-cAMP) increased amounts of StAR protein 2.5- and 6-fold respectively. A combination of IGFs/insulin and 8-Br-cAMP increased StAR 7-9-fold. Luteinized granulosa cells also had increased StAR expression after treatment with IGF-I (2. 8-fold), IGF-II (3-fold), insulin (2.5-fold) and 8-Br-cAMP (4. 5-fold). Progesterone production generally followed a pattern similar to StAR protein in both cell systems. In proliferating granulosa-lutein cells, both IGF-I and insulin increased StAR mRNA (3-fold) and 8-Br-cAMP increased StAR mRNA 4-fold, whereas a combination of IGF-I and 8-Br-cAMP had an additive effect on StAR mRNA expression (7-fold). Transient transfection of proliferating granulosa-lutein cells with StAR promoter-luciferase reporter constructs demonstrated that IGF-I, IGF-II, and insulin had no effect on the StAR promoter activity, while 8-Br-cAMP stimulated StAR promoter function. The results indicate that: (i) IGFs and insulin stimulate StAR mRNA and protein expression in human granulosa-lutein cells; (ii) IGF-I and 8-Br-cAMP have an additive effect on StAR gene expression; and (iii) IGF-I increases StAR mRNA and protein by a mechanism that does not involve activation of the proximal StAR gene promoter.

Blotting, Northern↗
PubMed1999

Rat amnion type IV collagen composition and metabolism: implications for membrane breakdown.

We report here that rat amnion type IV collagens are composed primarily of alpha1(IV) and alpha2(IV) chains. Amnion basement membrane collagens were more sensitive to degradation by collagenases than were adult rat kidney basement membrane collagens, which are enriched in alpha3(IV), alpha4(IV), and alpha6(IV) chains. Amnion type IV collagen content per unit of protein was markedly reduced by Day 21 of pregnancy, the day of delivery. Increased amnion levels of matrix metalloproteinase (MMP)-2 and MMP-9, gelatinases that degrade type IV collagen, were found by Day 21, suggesting that collagen breakdown was responsible, in part, for the decline in amnion type IV collagen. Infection of organ cultures of Day 18 rat amnions with a recombinant adenovirus expressing MMP-9 (AdMMP-9) caused release of collagen fragments detected as hydroxyproline in the culture fluid, amnion cell detachment, and apoptosis. The AdMMP-9-induced apoptosis was prevented by the MMP inhibitor batimastat. These findings suggest that MMPs are implicated in anoikis and apoptotic death of amnion cells, and may be part of a complex program of fetal membrane remodeling that occurs before delivery.

Adenoviridae↗
PubMed1999

Induction of matrix metalloproteinases and collagenolysis in chick embryonic membranes before hatching.

The membranes surrounding the chick embryo undergo striking morphological changes before hatching, which include structural degradation of the allantoic membrane. The fibrillar collagen content of the membranes declined by embryonic day (ED) 20 (the day of hatching). By ED 19, a 55-kDa matrix metalloproteinase (MMP) activity appeared in the extraembryonic fluid, and by ED 20 there was substantial 55-kDa MMP activity in embryonic membrane extracts. Reverse transcription-polymerase chain reaction was employed to clone a partial cDNA representing the chicken homologue of MMP-13, a 55- to 57-kDa enzyme. MMP-13 mRNA dramatically increased in abundance in embryonic membranes by ED 19, reaching a peak on ED 20. Introduction of the MMP inhibitor batimastat into the extraembryonic fluid prevented the structural changes in the embryonic membranes before hatching. We conclude that, like mammalian fetal membranes, chick embryonic membranes undergo terminal remodeling before hatching, in part as a result of increased MMP activity. The chicken egg system represents a novel in vivo model for exploring biochemical events leading to embryonic membrane remodeling prior to birth and to test inhibitors of MMPs for their ability to prevent collagenolysis and fetal membrane rupture.

Allantois↗
PubMed1999

Augmented androgen production is a stable steroidogenic phenotype of propagated theca cells from polycystic ovaries.

To test the hypothesis that the hyperandrogenemia associated with polycystic ovary syndrome (PCOS) results from an intrinsic abnormality in ovarian theca cell steroidogenesis, we examined steroid hormone production, steroidogenic enzyme activity, and mRNA expression in normal and PCOS theca cells propagated in long-term culture. Progesterone (P4), 17alpha-hydroxyprogesterone (17OHP4), and testosterone (T) production per cell were markedly increased in PCOS theca cell cultures. Moreover, basal and forskolin-stimulated pregnenolone, P4, and dehydroepiandrosterone metabolism were increased dramatically in PCOS theca cells. PCOS theca cells were capable of substantial metabolism of precursors into T, reflecting expression of an androgenic 17beta-hydroxysteroid dehydrogenase. Forskolin-stimulated cholesterol side chain cleavage enzyme (CYP11A) and 17alpha-hydroxylase/17,20-desmolase (CYP17) expression were augmented in PCOS theca cells compared with normal cells, whereas no differences were found in steroidogenic acute regulatory protein mRNA expression. Collectively, these observations establish that increased CYP11A and CYP17 mRNA expression, as well as increased CYP17, 3beta-hydroxysteroid dehydrogenase, and 17beta-hydroxysteroid dehydrogenase enzyme activity per theca cell, and consequently increased production of P4, 17OHP4, and T, are stable properties of PCOS theca cells. These findings are consistent with the notion that there is an intrinsic alteration in the steroidogenic activity of PCOS thecal cells that encompasses multiple steps in the biosynthetic pathway.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

The steroidogenic acute regulatory protein (StAR): a window into the complexities of intracellular cholesterol trafficking.

Stimulation of steroid-producing cells of the gonads and adrenals with tropic hormone results in a marked increase in steroid hormone synthesis within minutes. The rate-limiting step in this acute steroidogenic response is the transport of cholesterol from the outer to the inner mitochondrial membrane, where the first committed step in steroid synthesis is performed by the side-chain cleavage enzyme system. This process of cholesterol translocation is blocked by inhibitors of protein synthesis, suggesting that the effect of trophic hormones, acting through the intermediacy of cAMP, most likely involves the de novo synthesis of a protein that is rapidly inactivated. The recently identified steroidogenic acute regulatory (StAR) protein appears to be the most likely candidate for the "labile" protein, based on the following observations: 1) Expression of StAR in COS-1 cells engineered to contain the cholesterol side-chain cleavage system substantially augments pregnenolone formation; 2) StAR protein is expressed almost exclusively in steroid-producing cells, except the trophoblast of the human placenta, and its presence is correlated with steroid hormone production; 3) StAR mRNA increases in response to cAMP; 4) StAR is a target for serine phosphorylation mediated by protein kinase A, a process that is essential for maximizing StAR activity; and 5) lack of functional StAR causes the autosomal recessive disease, congenital lipoid adrenal hyperplasia, characterized by markedly impaired gonadal and adrenal steroid hormone synthesis. Studies on the mechanism of action of StAR revealed that import into mitochondria is not essential to its steroidogenesis-enhancing activity and more likely represents a means of rapidly inactivating StAR. Truncation mutations and site-directed mutations established that the C-terminus of the StAR protein contains the functionally important domains. The demonstration of steroidogenic activity of recombinant StAR protein on isolated mitochondria from bovine corpus luteum using protein that lacks the mitochondrial targeting sequence confirmed that StAR import is not essential for its steroidogenic activity and suggested that StAR acts directly on the outer mitochondrial membrane in the absence of intermediary cytosolic factors. Evidence that StAR functions as a cholesterol transfer protein raises the possibility that StAR acts directly on lipids of the outer mitochondrial membrane, probably stimulating cholesterol desorption from the sterol-rich outer membrane and its movement to the relatively sterol-poor inner membrane.

Animals↗
PubMed1998

Evidence for a genetic basis for hyperandrogenemia in polycystic ovary syndrome.

Our preliminary family studies have suggested that some female first-degree relatives of women with polycystic ovary syndrome (PCOS) have hyperandrogenemia per se. It was our hypothesis that this may be a genetic trait and thus could represent a phenotype suitable for linkage analysis. To investigate this hypothesis, we examined 115 sisters of 80 probands with PCOS from unrelated families. PCOS was diagnosed by the combination of elevated serum androgen levels and </=6 menses per year with the exclusion of secondary causes. The sisters were compared with 70 healthy age- and weight-comparable control women with regular menses, no clinical evidence of hyperandrogenemia, and normal glucose tolerance. Twenty-two percent of the sisters fulfilled diagnostic criteria for PCOS. In addition, 24% of the sisters had hyperandrogenemia and regular menstrual cycles. Circulating testosterone (T) and nonsex hormone-binding globulin-bound testosterone (uT) levels in both of these groups of sisters were significantly increased compared with unaffected sisters and control women (P < 0.0001 for both T and uT). Probands, sisters with PCOS, and hyperandrogenemic sisters had elevated serum luteinizing hormone levels compared with control women. We conclude that there is familial aggregation of hyperandrogenemia (with or without oligomenorrhea) in PCOS kindreds. In affected sisters, only one-half have oligomenorrhea and hyperandrogenemia characteristic of PCOS, whereas the remaining one-half have hyperandrogenemia per se. This familial aggregation of hyperandrogenemia in PCOS kindreds suggests that it is a genetic trait. We propose that hyperandrogenemia be used to assign affected status in linkage studies designed to identify PCOS genes.

Adolescent↗
PubMed1998

Oxysterol regulation of steroidogenic acute regulatory protein gene expression. Structural specificity and transcriptional and posttranscriptional actions.

Oxysterols exert a major influence over cellular cholesterol homeostasis. We examined the effects of oxysterols on the expression of steroidogenic acute regulatory protein (StAR), which increases the delivery of cholesterol to sterol-metabolizing P450s in the mitochondria. 22(R)-hydroxycholesterol (22(R)-OHC), 25-OHC, and 27-OHC each increased steroidogenic factor-1 (SF-1)-mediated StAR gene transactivation by approximately 2-fold in CV-1 cells. In contrast, cholesterol, progesterone, and the 27-OHC metabolites, 27-OHC-5beta-3-one and 7alpha,27-OHC, had no effect. Unlike our findings in CV-1 cells, SF-1-dependent StAR promoter activity was not augmented by 27-OHC in COS-1 cells, Y-1 cells, BeWo choriocarcinoma cells, Chinese hamster ovary (CHO) cells, and human granulosa cells. Studies examining the metabolism of 27-OHC indicated that CV-1 cells formed a single polar metabolite, 3beta-OH-5-cholestenoic acid from radiolabeled 27-OHC. However, this metabolite inhibited StAR promoter activity in CV-1, COS-1 and CHO cells. Because 7alpha,27-OHC was unable to increase SF-1-dependent StAR promoter activity, we examined 27-OHC 7alpha-hydroxylase in COS-1 and CHO cells. COS-1 cells contained high 7alpha-hydroxylase activity, whereas the enzyme was undetectable in CHO cells. The hypothesis that oxysterols act in CV-1 cells to increase StAR promoter activity by reducing nuclear levels of sterol regulatory element binding protein was tested. This notion was refuted when it was discovered that sterol regulatory element binding protein-1a is a potent activator of the StAR promoter in CV-1, COS-1, and human granulosa cells. Human granulosa and theca cells, which express endogenous SF-1, contained more than 5-fold more StAR protein following addition of 27-OHC, whereas StAR mRNA levels remained unchanged. We conclude that 1) there are cell-specific effects of oxysterols on SF-1-dependent transactivation; 2) the ability to increase transactivation is limited to certain oxysterols; 3) there are cell-specific pathways of oxysterol metabolism; and 4) oxysterols elevate StAR protein levels through posttranscriptional actions.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Unveiling the mechanism of action and regulation of the steroidogenic acute regulatory protein.

Stimulation of steroid-producing cells of the gonads and adrenals with trophic hormone (LH, and ACTH, respectively) produces a marked increase in steroid hormone synthesis within minutes. The rate-limiting step in this acute steroidogenic response is the transport of cholesterol from the outer to the inner mitochondrial membrane, where the first committed step in steroid synthesis is performed by the side-chain cleavage enzyme system (P450scc), resulting in the production of pregnenolone. This process of cholesterol translocation is blocked by inhibitors of protein synthesis (i.e. cycloheximide) indicating that the effect of trophic hormones, acting through the intermediacy of cAMP, most likely involves the de novo synthesis of a protein that is rapidly inactivated. The recently identified steroidogenic acute regulatory protein (StAR) appears to be the most likely candidate for the labile protein: (1) StAR is synthesized in response to cAMP and the StAR preprotein disappears rapidly in the presence of inhibitors of protein synthesis; (2) StAR has an N-terminal targeting sequence that directs the protein to the mitochondria; and (3) StAR protein is expressed almost exclusively in steroid-producing cells, its presence is correlated with steroid hormone production, and lack of functional StAR causes the autosomal recessive disease congenital lipoid adrenal hyperplasia (lipoid CAH), characterized by markedly impaired gonadal and adrenal steroid hormone synthesis. We have demonstrated that StAR is a target for serine phosphorylation mediated by protein kinase A (PKA), a process that is essential to maximizing StAR activity. StAR import by mitochondria is not essential to its steroidogenesis enhancing activity, and more likely, represents a means of rapidly inactivating StAR. Truncation mutations and site-directed mutations in StAR demonstrated that the C-terminus of the protein contains the functionally important domains. Further, we have demonstrated potent steroidogenic activity of recombinant StAR protein on isolated mitochondria from bovine corpus luteum using protein that lacks the mitochondrial targeting sequence. These observations confirm that StAR import is not essential for its steroidogenic activity and suggest that StAR acts directly on the outer mitochondrial membrane in the absence of intermediary cytosolic factors. More recently, we have found that StAR functions as a cholesterol transfer protein that does not require a protein receptor or co-factor, suggesting that StAR acts directly on lipids of the outer mitochondrial membrane to promote cholesterol translocation.

Animals↗
PubMed1998

Steroidogenic acute regulatory protein (StAR) is a sterol transfer protein.

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis by enhancing the delivery of substrate cholesterol from the outer mitochondrial membrane to the cholesterol side chain cleavage enzyme system on the inner membrane. A recombinant StAR protein lacking the first N-terminal 62 amino acid residues that includes the mitochondrial targeting sequence was shown to stimulate the transfer of cholesterol and beta-sitosterol from liposomes to heat-treated mitochondria in a dose-, time-, and temperature-dependent manner. A recombinant mutant StAR protein that cannot stimulate steroidogenesis by isolated mitochondria did not promote sterol transfer. Unlike the more promiscuous lipid transfer protein, sterol carrier protein 2 (SCP2), StAR did not stimulate phosphatidylcholine transfer in our assay system. The recombinant StAR protein increased cholesterol transfer to heat-treated microsomes as well as to heat- and trypsin-treated mitochondria. These observations demonstrate that StAR has sterol transfer activity, which may reflect an ability to enhance desorption of cholesterol from sterol-rich donor membranes. We suggest that the ability of StAR to promote sterol transfer explains its steroidogenic activity.

Animals↗
PubMed1998

The mechanism of action of steroidogenic acute regulatory protein (StAR). StAR acts on the outside of mitochondria to stimulate steroidogenesis.

Steroidogenic acute regulatory protein (StAR) plays an essential role in steroidogenesis, facilitating delivery of cholesterol to cytochrome P450scc on the inner mitochondrial membrane. StAR is synthesized in the cytoplasm and is subsequently imported by mitochondria and processed to a mature form by cleavage of the NH2-terminal mitochondrial targeting sequence. To explore the mechanism of StAR action, we produced 6-histidine-tagged N-62 StAR (His-tag StAR) constructs lacking the NH2-terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria. His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system. His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells whereas wild-type StAR was localized to mitochondria. There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes. In vitro import assays demonstrated that wild-type StAR preprotein was imported and processed to mature protein that was protected from subsequent trypsin treatment. In contrast, His-tag StAR was not imported and protein associated with mitochondria was sensitive to trypsin. Using metabolically labeled COS-1 cells transfected with wild-type or His-tag StAR constructs, we confirmed that wild-type StAR preprotein was imported and processed by mitochondria, whereas His-tag StAR remained largely cytosolic and unprocessed. To determine whether cytosolic factors are required for StAR action, we developed an assay system using washed mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in Escherichia coli. Recombinant His-tag StAR stimulated pregnenolone production in a dose- and time-dependent manner, functioning at nanomolar concentrations. A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein. This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria. Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import, and in the absence of cytosol. The ability to produce bioactive recombinant StAR protein paves the way for refined structural studies of StAR and StAR mutants.

Animals↗
PubMed1998

Transduction of human trophoblastic cells by replication-deficient recombinant viral vectors. Promoting cellular differentiation affects virus entry.

We investigated the transfer of the lacZ reporter gene into human trophoblastic cells using herpes simplex virus and adeno-associated virus vectors. We used an established choriocarcinoma cell line (BeWo cells) that can be induced to terminally differentiate after treatment with cyclic-AMP. Our results demonstrate that transduction of trophoblastic cells by the herpes simplex virus vector, HSV.CMVlac, and the adeno-associated virus vector, AAV.CMVlac, is affected by cellular differentiation. Treatment of BeWo cells with cyclic-AMP reduced transduction by HSV.CMVlac but increased transduction by the AAV vector. In contrast, when BeWo cells were transfected with herpes simplex virus and adeno-associated virus plasmids, lacZ expression was not affected by treatment with cyclic-AMP. Southern blot analysis demonstrated 2.75 times less herpes simplex virus DNA in cyclic-AMP treated BeWo cells, but 2.0 to 7.4 times more adeno-associated virus DNA in treated cells. We conclude that inefficient transduction of differentiated trophoblastic cells with HSV.CMVlac is because of diminished viral entry, whereas cellular differentiation is associated with increased entry of AAV.CMVlac. These observations suggest that adeno-associated virus vectors may be used to modify trophoblast function and study placental physiology. Additionally, trophoblast differentiation leads to alterations in the mechanisms of virus uptake that may affect maternal-to-fetus transmission.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Phenotype and genotype in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common disorder in premenopausal women and is characterized by hyperandrogenic chronic anovulation. The cause is unknown. PCOS is associated with significant insulin resistance as well as with defects in insulin secretion. These abnormalities place these women at substantial risk for developing type 2 diabetes mellitus. A defect in insulin-mediated receptor autophosphorylation has been found in a substantial proportion of PCOS women. Both PCOS and the insulin resistance that accompanies it appear to have major genetic components. Family studies of PCOS have supported this, although they suffer from incomplete phenotyping of probands and first-degree relatives. The phenotype in males and nonreproductive age females is uncertain. Despite the shortcomings of the family studies of PCOS, they have consistently indicated familial clustering and suggested that the mode of inheritance is dominant. Our initial studies of 50 families of PCOS probands indicate that 24% of sisters are affected with PCOS. There also appears to be an intermediate phenotype of sisters with regular menstrual cycles who are hyperandrogenic per se (22% of sisters). Additionally, there appears to be a major familial defect, with 50% of first-degree relatives having glucose intolerance (impaired glucose tolerance by oral glucose tolerance test or type 2 diabetes mellitus). These findings suggest that hyperandrogenism in females and glucose intolerance may be genetic traits in PCOS kindreds. Systematic phenotyping will allow assignment of affected status for eventual linkage analysis.

Female↗
PubMed1997

Phosphorylation of steroidogenic acute regulatory protein (StAR) modulates its steroidogenic activity.

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone synthesis. StAR is thought to increase the delivery of cholesterol to the inner mitochondrial membrane where P450scc resides. Tropic hormones acting through the intermediacy of cAMP rapidly increase pregnenolone synthesis, and this rapid steroidogenic response is believed to be due to StAR's action. The StAR protein contains two consensus sequences for phosphorylation catalyzed by protein kinase A that are conserved across all species in which the amino acid sequence of the StAR protein has been determined. We demonstrated that human StAR expressed in COS-1 cells exists in at least four species detectable by two-dimensional gel electrophoresis followed by Western blotting. The two more acidic species disappeared after treatment of the cell extracts with alkaline phosphatase. 32P was incorporated into StAR protein immunoprecipitated from COS-1 cell extracts, and a 10-min treatment with 8-bromo-cAMP increased 32P incorporation into the StAR preprotein. StAR protein generated by in vitro transcription/translation was phosphorylated by the protein kinase A catalytic subunit in the presence of [gamma-32P]ATP. Mutation of potential sites for protein kinase A-mediated phosphorylation at serine 57 and serine 195 to alanines, individually, reduced 32P incorporation from labeled ATP into StAR preprotein produced by in vitro transcription/translation when incubated with protein kinase A catalytic subunit. 32P labeling of StAR protein expressed in COS-1 cells was also reduced when serine 57 or serine 195 were mutated to alanines. A double mutant in which both serine 57 and serine 195 were changed to alanines displayed markedly reduced 32P incorporation. To determine the functional significance of StAR phosphorylation, we tested the steroidogenic activity of the wild-type StAR and mutated StAR proteins in COS-1 cells expressing the human cholesterol side chain cleavage enzyme system. Mutation of the conserved protein kinase A phosphorylation site at serine 57 had no effect on pregnenolone synthesis. However, mutation of the serine residue at 195 resulted in an approximately 50% reduction in pregnenolone production. The S195A mutant construct did not yield the more acidic species of StAR detected in two-dimensional Western blots, indicating that the mutation affected the ability of the protein to be post-translationally modified. Mutation of the corresponding serine residues in murine StAR (Ser56 and Ser194) to alanines yielded results that were similar to those obtained with human StAR; the S56A mutant displayed a modest reduction in steroidogenic activity, whereas the S194A mutant had approximately 40% of the activity of murine wild-type StAR. In contrast to the human S195A mutation, conversion of serine 195 to an aspartic acid residue had no effect on steroidogenic activity, consistent with the idea that a negative charge at this site modulates StAR function. Our observations suggest that phosphorylation of serine 194/195 increases the biological activity of StAR and that this post- or co-translational event accounts, in part, for the immediate effects of cAMP on steroid production.

Amino Acid Sequence↗
PubMed1997

Mechanisms of trophoblast-virus interaction.

The human placenta serves as a barrier to the transmission of some viruses, but allows others to reach the fetal circulation. The resistance and permissiveness of the placenta to viral transmission appears to be determined in large part by the placental trophoblast. In order to define the mechanisms by which human trophoblast cells influence vertical transmission of viruses, we have studied the interaction of replication-deficient recombinant viral vectors with transformed human choriocarcinoma (BeWo) cells and primary trophoblast cell cultures. Recombinant adenovirus vectors efficiently transduce BeWo cells and primary trophoblast cells. However, as BeWo cells differentiate after treatment with cAMP analogs, they become relatively resistant to adenovirus-mediated transduction due to diminished uptake of the virus particles. This differentiation-dependent loss of adenovirus transduction may be related to the down-regulation of the coxsackie adenovirus receptor, which we have detected in undifferentiated trophoblast cells. Recombinant herpes simplex virus vectors also transduce undifferentiated BeWo cells and isolated cytotrophoblast cells, but transduction by herpes simplex virus vectors declines with cAMP-induced trophoblast differentiation, apparently due to reduced viral uptake. In contrast, cAMP treatment augments trophoblast transduction by recombinant adeno-associated virus, a member of the parvovirus family. This augmentation appears to be due to increased virus uptake by a yet to be determined mechanism. The understanding of the molecular basis of these interactions between recombinant viral vectors and trophoblast cells may yield strategies to prevent vertical transmission of viruses as well as to create opportunities to modify trophoblast function through gene transfer using viral vectors.

Adenoviridae↗