Germ cell-specific disruption of the Meig1 gene causes impaired spermiogenesis in mice.

Meiosis expressed gene 1 (Meig1) was originally identified in a search for mammalian genes potentially involved in meiosis. Seven mouse Meig1 transcripts with the same coding region, but different 5'-UTRs, have been identified. These transcripts have different tissue distributions, two are only present in the testis. In the testis, Meig1 is present in germ cells and Sertoli cells. A Meig1 conditional knockout model has been generated. When Meig1 was inactivated globally by crossing with Cmv-Cre transgenic mice, the Meig1-deficient males were sterile due to severe spermiogenic defects, and had no obvious defects in meiosis. To further study its role in individual cell types in the testis, the Meig1(flox) mice were crossed with Hsp2a-Cre, Prm-Cre, and Amh-Cre mice, in which the Cre recombinase is driven by the heat shock protein 2 (Hsp2a) gene promoter (expressed in spermatocytes), the protamine 1 gene promoter (expressed in post-meiotic spermatids) and the anti-Mullerian hormone (Amh) gene promoter (expressed in Sertoli cells) respectively. Both Meig1 mRNA and protein were undetectable in testis of the Hsp2a-Cre; Meig1(flox/flox) mice and all the mutant adult males tested were sterile. This phenotype mirrors that of the Cmv-Cre; Meig1(flox/flox) mice. Even though the total testicular Meig1 mRNA and protein expression levels were dramatically reduced in testis of the Prm-Cre; Meig1(flox/flox) males, all the mice tested were fertile, and there was no significant difference in sperm count and sperm motility compared with age-matched Meig1(flox/flox) male mice. Disruption of Meig1 in the Sertoli cells did not affect the MEIG1 protein expression. Amh-Cre; Meig1(flox/flox) males were fertile, and produced the same amount of spermatozoa as age-matched Meig1(flox/flox) mice. The testicular histology was also normal. Our results indicate that MEIG1 regulates spermiogenesis through effects in germ cells alone, and that the Meig1 gene must be active during a discrete period in spermatogenesis after which it is dispensable.

Impact of FTO genotypes on BMI and weight in polycystic ovary syndrome: a systematic review and meta-analysis.

AIMS/HYPOTHESIS: FTO gene single nucleotide polymorphisms (SNPs) have been shown to be associated with obesity-related traits and type 2 diabetes. Several small studies have suggested a greater than expected effect of the FTO rs9939609 SNP on weight in polycystic ovary syndrome (PCOS). We therefore aimed to examine the impact of FTO genotype on BMI and weight in PCOS. METHODS: A systematic search of medical databases (PubMed, EMBASE and Cochrane CENTRAL) was conducted up to the end of April 2011. Seven studies describing eight distinct PCOS cohorts were retrieved; seven were genotyped for SNP rs9939609 and one for SNP rs1421085. The per allele effect on BMI and body weight increase was calculated and subjected to meta-analysis. RESULTS: A total of 2,548 women with PCOS were included in the study; 762 were TT homozygotes, 1,253 had an AT/CT genotype, and 533 were AA/CC homozygotes. Each additional copy of the effect allele (A/C) increased the BMI by a mean of 0.19 z score units (95% CI 0.13, 0.24; p = 2.26 × 10(-11)) and body weight by a mean of 0.20 z score units (95% CI 0.14, 0.26; p = 1.02 × 10(-10)). This translated into an approximately 3.3 kg/m(2) increase in BMI and an approximately 9.6 kg gain in body weight between TT and AA/CC homozygotes. The association between FTO genotypes and BMI was stronger in the cohorts with PCOS than in the general female populations from large genome-wide association studies. Deviation from an additive genetic model was observed in heavier populations. CONCLUSIONS/INTERPRETATION: The effect of FTO SNPs on obesity-related traits in PCOS seems to be more than two times greater than the effect found in large population-based studies. This suggests an interaction between FTO and the metabolic context or polygenic background of PCOS.

Connective tissue and related disorders and preterm birth: clues to genes contributing to prematurity.

To identify candidate genes contributing to preterm birth, we examined the existing literature on the association between known disorders of connective tissue synthesis and metabolism and related diseases and prematurity. Our hypothesis was that abnormal matrix metabolism contributes to prematurity by increasing risk of preterm premature rupture of membranes (PPROM) and cervical incompetence. Based on this review, we identified gene mutations inherited by the fetus that could predispose to preterm birth as a result of PPROM. The responsible genes include COL5A1, COL5A2, COL3A1, COL1A1, COL1A2, TNXB, PLOD1, ADAMTS2, CRTAP, LEPRE1 and ZMPSTE24. Marfan syndrome, caused by FBN1 mutations, and polymorphisms in the COL1A1 and TGFB1 genes have been associated with cervical incompetence. We speculate that an analysis of sequence variation at the loci noted above will reveal polymorphisms that may contribute to susceptibility to PPROM and cervical incompetence in the general population.

Fine mapping of genetic susceptibility to polycystic ovary syndrome on chromosome 19p13.2 and tests for regulatory activity.

CONTEXT: Little is known about genes that contribute to polycystic ovary syndrome (PCOS). We previously found linkage and association of PCOS with the dinucleotide marker D19S884 in two independent sets of families; allele 8 of D19S884 confers increased risk. OBJECTIVE/DESIGN: The objectives of the study were: 1) use the transmission/disequilibrium test (TDT) to assess linkage and association between PCOS and D19S884 (and nearby markers) in a third set of families; and 2) test D19S884 and surrounding DNA sequence for in vitro regulatory activity in lymphoblastoid cell lines (LCLs) and granulosa cells. SETTING/SUBJECTS: We studied 98 new families with a PCOS proband, father, mother, and other available offspring. We analyzed data from these families separately and in combination with data obtained previously. INTERVENTIONS: Interventions were venipuncture. MAIN OUTCOME MEASURES: Measures were transmission frequencies and in vitro functional studies. RESULTS: The first result we found was that in the 98 new families, the TDT was significant for allele 8 of D19S884 (P = 0.043). In the total collection of 465 families, the TDT evidence is very strong (nominal P < 7 x 10(-5)). Results for all other genetic markers near D19S884 were nonsignificant after correction for multiple testing. The second result was that an approximately 800-bp fragment containing various alleles of D19S884 showed modest but reproducible promoter activity in LCLs. However, no allelic differences were detected. No activity of this fragment was detected in granulosa cells. CONCLUSIONS: This is the second independent confirmation of linkage and association of D19S884 with PCOS. We found in addition that some sequence in the region of D19S884 confers in vitro promoter activity in LCLs.

Candidate gene region for polycystic ovary syndrome on chromosome 19p13.2.

CONTEXT: Polycystic ovary syndrome (PCOS) is a common endocrine disorder that is believed to have a genetic basis. However, no specific susceptibility gene or region has been conclusively identified. OBJECTIVE: The objective of this study was to duplicate a previous study that localized a PCOS susceptibility region to chromosome 19p13.2 and to narrow the susceptibility region. DESIGN: This study was designed to test for genetic linkage and association between PCOS and short tandem repeat polymorphisms in 367 families, by analysis of linkage and family-based association. SETTING: The study was conducted at academic medical centers. PATIENTS OR OTHER PARTICIPANTS: We studied 367 families of predominantly European origin with at least one PCOS patient. Families included 107 affected sibling (sister) pairs (ASPs) in 83 families, and 390 trios with both parents and an affected daughter. The data set comprises two independent groups. Set 1 consists of 44 ASPs and 163 trios. Set 2 consists of 63 ASPs and 227 trios. INTERVENTION(S): The intervention was the drawing of blood for DNA extraction. MAIN OUTCOME MEASURE: We employed measures of evidence for linkage and association between PCOS and 19 STRs. RESULTS: Linkage with PCOS was observed over a broad region of chromosome 19p13.2. The strongest evidence for association was observed with D19S884 (chi2 = 11.85; nominal P < 0.0006; permutation P = 0.034) and duplicated our earlier findings. CONCLUSIONS: The present analysis suggests that a PCOS susceptibility locus maps very close to D19S884. Additional studies that systematically characterize DNA sequence variation in the immediate area of D19S884 are required to identify the PCOS susceptibility variant.

A serum proteomics approach to the diagnosis of ectopic pregnancy.

An ectopic pregnancy (EP) occurs when implantation of the embryo occurs outside of the uterus. If left untreated, the developing fetus will continue to grow, leading to life-threatening consequences for the mother. A major difficulty with the diagnosis of ectopic pregnancy is that methods of detection are limited, and some, such as ultrasound, are not very reliable in the earliest days of gestation. Currently, no effective serum test exists to distinguish an ectopic pregnancy from a normal intrauterine pregnancy. The incidence of ectopic pregnancy is increasing and has doubled in the last 20 years. It is now the second most common cause of maternal death in the first trimester of pregnancy. To address this issue, we initiated a project to identify serum markers of ectopic pregnancy. The subjects for these studies presented at the Hospital of the University of Pennsylvania. We obtained over 140 serum samples from women with suspected ectopic pregnancy: women presenting with pain and/or bleeding in the first trimester of pregnancy. The approximate racial breakdown of the subjects is as follows: African American, 36%; Caucasian, 3%; Asian, 2%; Hispanic, 1%; unknown, 58%. Serum samples from 139 women (62 with ectopic pregnancy and 77 with a normal intrauterine pregnancy) were applied to WCX2 (weak ion exchange) protein chip surfaces and analyzed for serum markers using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Several proteins in the 7500-18,000 Da mass range were identified that may discriminate an ectopic pregnancy from an intrauterine pregnancy. The most promising markers were analyzed using classification and regression tree analysis (CART) with and without clinical variables (serum hCG value, length of amenorrhea). Two different algorithms were developed that classify the patients on the basis of sensitivity (number of EPs who screen positive/# of EPs) or specificity (# of healthy patients who screen negative/# of healthy). Our current approach is to refine these two "rule sets" to segregate patients into three groups: those who need immediate intervention for a probable ectopic pregnancy, those who appear to have a normal pregnancy, and those who need further monitoring for diagnosis.

In-vitro secretion of proinflammatory cytokines by human amniochorion carrying hyper-responsive gene polymorphisms of tumour necrosis factor-alpha and interleukin-1beta.

The identification of polymorphisms in genes encoding proinflammatory cytokines that affect transcription or the secretion rate has opened new ways to understand the variation in responses to infection during pregnancy. In this study, human amniochorion carrying hyper-responsive alleles of tumour necrosis factor-alpha (TNF-alpha: TNF*2 at -308) and interleukin-1beta (IL-1beta: IL-1*2 at +3953) were stimulated in vitro with bacterial lipopolysaccharide (LPS) and compared with tissues carrying the common alleles (TNF*1 and IL-1*1). Fetal membranes carrying the TNF*1 allele displayed an identical dose-response pattern to tissues carrying a TNF*2 allele, except at the highest dose of LPS tested (50 ng/ml) there was a significantly greater production of TNF-alpha in the presence of a TNF*2 allele. Membranes carrying the IL-1*2 polymorphism secreted IL-1beta in a dose-response curve that was different from IL-1* tissues when challenged with 5, 10 and 50 ng/ml LPS. These observations support the hypothesis that reproductive tissues carrying hyper-responsive proinflammatory cytokine genes may over-respond to intrauterine infection secreting higher amounts of cytokines, which in turn, may lead to adverse pregnancy outcomes.

The biochemical basis for increased testosterone production in theca cells propagated from patients with polycystic ovary syndrome.

Ovarian theca cells propagated from patients with polycystic ovary syndrome (PCOS) convert steroid precursors into T more efficiently than normal theca cells. To identify the basis for increased T production by PCOS theca cells, we examined type I-V 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) isoform expression in long-term cultures of theca and granulosa cells isolated from normal and PCOS ovaries. RT-PCR analysis demonstrated that theca cells express type V 17 beta HSD a member of the aldo-keto reductase (AKR) superfamily (17 beta HSDV, AKR1C3), whereas expression of type I, II, and IV 17 beta HSD, which are members of the short-chain dehydrogenase/reductase superfamily, was limited to granulosa cells. Type III 17 beta HSD, the testicular isoform, was not detected in either granulosa or theca cells. Northern and real-time PCR analyses demonstrated that 17 beta HSDV transcripts were not significantly increased in PCOS theca cells compared with normal theca cells. RT-PCR analysis revealed that theca cells also express another AKR, 20 alpha HSD (AKR1C1). Both basal and forskolin-stimulated 20 alpha HSD mRNA levels were increased in PCOS theca cells compared with normal theca cells. However, 17 beta HSD enzyme activity per theca cell was not significantly increased in PCOS, suggesting that neither AKR1C3 nor AKR1C1 contributes to the formation of T in this condition. In contrast, 17 alpha-hydroxylase/C17,20 lyase and 3 beta HSD enzyme activities were elevated in PCOS theca cells, driving increased production of T precursors. These findings indicate that 1) increased T production in PCOS theca cells does not result from dysregulation of "androgenic" 17 beta HSD activity or altered expression of AKRs that may express 17 beta HSD activity; and 2) increased synthesis of T precursors is the primary factor driving enhanced T secretion in PCOS.

Steroidogenic acute regulatory protein: an update on its regulation and mechanism of action.

Steroidogenic acute regulatory (StAR) protein controls the rate-limiting step in steroidogenesis: the transport of cholesterol from the outer to the inner mitochondrial membrane. Early studies indicated that rate of transcription of the StAR gene is a primary determinant of steroidogenesis. The transcription factors that govern basal and cAMP-dependent StAR expression are reviewed, as are new findings regarding chromatin modifications associated with activation of the StAR promoter. Molecular genetic studies of congenital lipoid adrenal hyperplasia, a rare disease caused by mutations in the StAR gene, and structure-function studies defined two major domains within the StAR protein, the N-terminal mitochondrial targeting sequence and the C-terminal StAR-related lipid transfer (START) domain, which promotes the translocation of cholesterol between the two mitochondrial membranes. Several models of StAR's mechanism of action have been proposed based on a combination of structure/function studies or on the crystal structure of a related START domain. The models-intermembrane shuttle hypothesis, and cholesterol desorption hypothesis-are discussed with respect to the known biochemical and biophysical events associated with steroidogenesis and the structure of StAR.

Expression of steroidogenic acute regulatory protein in the human corpus luteum throughout the luteal phase.

The expression of the steroidogenic acute regulatory protein (StAR) in the human corpus luteum (CL) was examined throughout the luteal phase. The primary 1.6-kb StAR transcript was in greater abundance in early (3.1-fold) and mid (2.2-fold) luteal phase CL compared with late luteal phase CL. The larger StAR transcript (4.4 kb) was found in early and midluteal phase CL, but was not detected in late luteal phase specimens. Mature StAR protein (30 kDa) was present in lower amounts within late CL compared with early and midluteal phase CL. The StAR preprotein (37 kDa) was also detected in greater abundance in early and midluteal CL. Immunohistochemistry revealed that StAR staining was most prominent in thecal-lutein cells throughout the luteal phase. The intensity of the signal for StAR exhibited significant changes throughout the luteal phase, being most intense during the midluteal phase and least during the late luteal phase. Plasma progesterone concentrations were highly correlated (r = 0.73 and r = 0.79) with luteal expression of the preprotein and mature StAR isoforms, respectively, throughout the luteal phase. To examine the LH dependency of StAR expression, the GnRH antagonist, Cetrorelix, was administered during the midluteal phase. Cetrorelix caused a decline in serum LH levels within 2 h, which, in turn, caused a pronounced decline in plasma progesterone within 6 h. The StAR 4.4-kb transcript was not detectable, and the 1.6-kb transcript was reduced by approximately 50% within 24 h of Cetrorelix treatment. The mature 30-kDa StAR protein level declined approximately 30% after Cetrorelix treatment. We conclude that 1) StAR mRNA and protein are highly expressed in early and midluteal phase CL; 2) StAR protein is present in both thecal-lutein and granulosa-lutein cells throughout the luteal phase; 3) StAR protein levels in the CL are highly correlated with plasma progesterone levels; 4) declining StAR mRNA and protein levels are characteristic of late luteal phase CL; and 5) suppression of LH levels during the midluteal phase results in a marked decline in plasma progesterone and a diminished abundance of StAR transcripts in the CL without a corresponding significant decline in StAR protein. Collectively, these data are consistent with the idea that StAR gene expression is a key determinant of luteal progesterone during the normal menstrual cycle. However, the pharmacologically induced withdrawal in the midluteal phase of LH support diminishes luteal progesterone output by mechanisms others than reduced StAR protein levels.

Amniotic fluid matrix metalloproteinase-8 indicates intra-amniotic infection.

OBJECTIVE: This study was undertaken to determine whether matrix metalloproteinase-8, which is produced by neutrophils, is a useful marker for the detection of intra-amniotic infection. STUDY DESIGN: We performed a case-control study using enzyme-linked immunosorbent assays to detect matrix metalloproteinase-8 in 77 amniotic fluid specimens that were obtained by amniocentesis from women with preterm contractions or preterm labor and intact fetal membranes (n = 66) and from women with preterm premature rupture of membranes (n = 11). RESULTS: Thirty women had culture-proven intra-amniotic infection (cases), 21 of whom had intact membranes. After constructing receiver operating characteristic curves to establish the optimal threshold concentration of matrix metalloproteinase-8 for a positive test result, we detected matrix metalloproteinase-8 in 27 of 30 women with intra-amniotic infection; only 10 of 47 control specimens contained matrix metalloproteinase-8 (P <.001; odds ratio, 33.3; 95% CI, 8.4, 132.7). Matrix metalloproteinase-8 was present in 20 of 21 women with intact membranes and intra-amniotic infection and in only 10 of 45 control subjects (P <.001; odds ratio, 70.0; 95% CI, 8.3, 587.6). Among women with intact membranes, the sensitivity of the assay was 0.95 and the specificity was 0.78. CONCLUSION: Our results indicate that matrix metalloproteinase-8 is highly correlated with intra-amniotic infection and that enzyme-linked immunosorbent assay for matrix metalloproteinase-8 may be a clinically useful test for the diagnosis of intra-amniotic infection in women with preterm contractions and preterm labor.

Steroidogenic acute regulatory protein binds cholesterol and modulates mitochondrial membrane sterol domain dynamics.

The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step of steroidogenesis, delivery of cholesterol to the inner mitochondrial membrane. However, the mechanism whereby cholesterol translocation is accomplished has not been resolved. Recombinant StAR proteins lacking the first N-terminal 62 amino acids comprising the mitochondrial-targeting sequence were used to determine if StAR binds cholesterol and alters mitochondrial membrane cholesterol domains to enhance sterol transfer. First, a fluorescent NBD-cholesterol binding assay revealed 2 sterol binding sites (K(d) values near 32 nm), whereas the inactive A218V N-62 StAR mutant had only a single binding site with 8-fold lower affinity. Second, NBD-cholesterol spectral shifts and fluorescence resonance energy transfer from StAR Trp residues to NBD-cholesterol showed (i) close molecular interaction between these molecules (R(2/3) = 33 A) and (ii) sensitized NBD-cholesterol emission from only one of the two sterol binding sites. Third, circular dichroism showed that cholesterol binding induced a change in StAR secondary structure. Fourth, a fluorescent sterol transfer assay that did not require separation of donor and acceptor mitochondrial membranes demonstrated that StAR enhanced mitochondrial sterol transfer as much as 100-fold and induced/increased the formation of rapidly transferable cholesterol domains in isolated mitochondrial membranes. StAR was 67-fold more effective in transferring cholesterol from mitochondria of steroidogenic MA-10 cells than from human fibroblast mitochondria. In contrast, sterol carrier protein-2 (SCP-2) was only 2.2-fold more effective in mediating sterol transfer from steroidogenic cell mitochondria. Taken together these data showed that StAR is a cholesterol-binding protein, preferentially enhances sterol transfer from steroidogenic cell mitochondria, and interacts with mitochondrial membranes to alter their sterol domain structure and dynamics.

Quantitative analysis of the hormone-induced hyperacetylation of histone H3 associated with the steroidogenic acute regulatory protein gene promoter.

Transcriptional regulation of steroidogenic acute regulatory protein (StAR) determines adrenal and gonadal cell steroidogenesis. Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the StAR promoter. MA-10 cells treated with 8-bromo-cAMP had increased acetylated histone H3 associated with the proximal (but not distal) StAR promoter, nascent StAR transcripts, and progesterone production within 15 min, whereas StAR mRNA increased at 30 min. At 360 min, steroidogenesis remained elevated, but mRNA, nascent RNA, and StAR promoter-associated H3 acetylation all declined. StAR promoter-associated H4 acetylation was unchanged by 8-bromo-cAMP treatment of MA-10 cells. In vivo analysis of macaque and human granulosa cells showed that luteinization was associated with increased StAR promoter-associated H3 acetylation. We conclude that acetylation of H3 (but not H4) associated with the proximal promoter is associated with StAR gene transcription, that chromatin modification occurs in discrete regions of the promoter, that the initial steroidogenic response to 8-bromo-cAMP occurs prior to increased StAR mRNA accumulation, and that MA-10 cell StAR gene transcription and promoter-associated H3 acetylation are biphasic during a 6-h treatment period. The union of the chromatin immunoprecipitation assay with quantitative real-time polymerase chain reaction described and validated here should enhance the analysis of gene expression.

The ERK signaling cascade inhibits gonadotropin-stimulated steroidogenesis.

The response of granulosa cells to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH- and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the steroidogenic acute regulatory protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.

The proteasome inhibitor MG132 promotes accumulation of the steroidogenic acute regulatory protein (StAR) and steroidogenesis.

StAR, a protein synthesized in the cytoplasm and subsequently imported into mitochondria, regulates the rate-determining step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The active form of StAR is the 37 kDa pre-protein, which has a short half-life. To determine whether proteasomes participate in the turnover of StAR, we incubated primary cultures of preovulatory rat granulosa cells and immortalized human granulosa cells in the presence of MG132, a specific inhibitor to proteasome catalysis. This treatment caused accumulation of StAR in unstimulated cells. Moreover, incubation of the cells with MG132 in the presence of forskolin (FK), luteinizing hormone/chorionic gonadotropin or follicular stimulating hormone augmented the accumulation of both the 37 kDa cytoplasmic protein and the 30 kDa mature mitochondrial protein, compared to cells incubated with FK or the gonadotropic hormones alone. Concomitantly, progesterone production was enhanced. In contrast no elevation in the 37 kDa StAR intracellular levels or progesterone production was observed following incubation of the cells with the cysteine protease inhibitor E-64. The increase of the 37 kDa StAR protein was evident after 15 min and 30 min of incubation with MG132 (143% and 187% of control values, respectively) with no significant elevation of the 30 kDa protein. Accumulation of the intermediate mitochondrial 32 kDa protein was evident after 1-2 h and the accumulation of the 30 kDa protein was evident only after 4 h of incubation with MG132. In contrast, no elevation in adrenodoxin, a component of the cytochrome P450scc enzyme system, was found. These data suggest that StAR protein is either directly or indirectly degraded by the proteasome which may explain, in part, its short half-life. Moreover, it seems that the cytosolic 37 kDa protein, which is responsible for the steroidogenic activity of StAR, is the primary proteasomal substrate and that the inhibition of its degradation by MG132 causes the up-regulation of progesterone production.

The extra domain A of fibronectin activates Toll-like receptor 4.

Cellular fibronectin, which contains an alternatively spliced exon encoding type III repeat extra domain A (EDA), is produced in response to tissue injury. Fragments of fibronectin have been implicated in physiological and pathological processes, especially tissue remodeling associated with inflammation. Because EDA-containing fibronectin fragments produce cellular responses similar to those provoked by bacterial lipopolysaccharide (LPS), we examined the ability of recombinant EDA to activate Toll-like receptor 4 (TLR4), the signaling receptor stimulated by LPS. We found that recombinant EDA, but not other recombinant fibronectin domains, activates human TLR4 expressed in a cell type (HEK 293 cells) that normally lacks this Toll-like receptor. EDA stimulation of TLR4 was dependent upon co-expression of MD-2, a TLR4 accessory protein. Unlike LPS, the activity of EDA was heat-sensitive and persisted in the presence of the LPS-binding antibiotic polymyxin B and a potent LPS antagonist, E5564, which completely suppressed LPS activation of TLR4. These observations provided a mechanism by which EDA-containing fibronectin fragments promote expression of genes involved in the inflammatory response.

Conditional response of the human steroidogenic acute regulatory protein gene promoter to sterol regulatory element binding protein-1a.

The steroidogenic acute regulatory protein (StAR) gene controls the rate-limiting step in the biogenesis of steroid hormones, delivery of cholesterol to the cholesterol side-chain cleavage enzyme on the inner mitochondrial membrane. We determined whether the human StAR promoter is responsive to sterol regulatory element-binding proteins (SREBPs). Expression of SREBP-1a stimulated StAR promoter activity in the context of COS-1 cells and human granulosa-lutein cells. In contrast, expression of SREBP-2 produced only a modest stimulation of StAR promoter activity. One of the SREBP-1a response elements in the StAR promoter was mapped in deletion constructs and by site-directed mutagenesis between nucleotides -81 to -70 from the transcription start site. This motif bound recombinant SREBPs in electrophoretic mobility shift assays, but with lesser affinity than a low density lipoprotein receptor SREBP-binding site. An additional binding site for the transcriptional modulator, yin yang 1 (YY1), was observed within the SREBP-binding site (nucleotides -73 to -70). Mutation of the YY1-binding site increased the responsiveness of the StAR promoter to exogenous SREBP-1a, but did not alter the affinity for SREBP-1a binding in electrophoretic mobility gel shift assays. Manipulations that altered endogenous mature SREBP-1a levels (e.g. culture in lipoprotein-deficient medium and addition of 27-hydroxycholesterol) did not affect StAR promoter function, but influenced low density lipoprotein receptor promoter activity. We conclude that 1) the human StAR promoter is conditionally responsive to SREBP-1a such that promoter activity is up-regulated in the presence of high levels of SREBP-1a, but is unaffected when mature SREBP levels are suppressed; and 2) the human StAR promoter is selectively responsive to SREBP-1a.