Messenger Ribonucleic Acid Synthesis Research Articles

CPEB3 low-complexity motif regulates local protein synthesis via protein–protein interactions in neuronal ribonucleoprotein granules

Biomolecular condensates, membraneless organelles found throughout the cell, play critical roles in many aspects of cellular function. Ribonucleoprotein granules (RNPs) are a type of biomolecular condensate necessary for local protein synthesis and are involved in synaptic plasticity and long-term memory. Most of the proteins in RNPs possess low-complexity motifs (LCM), allowing for increased promiscuity of protein-protein interactions. Here, we describe the importance of protein-protein interactions mediated by the LCM of RNA-binding protein cytoplasmic polyadenylation element binding protein 3 (CPEB3). CPEB3 is necessary for long-term synaptic plasticity and memory persistence, but the mechanisms involved are still not completely elucidated. We now present key mechanisms involved in its regulation of synaptic plasticity. We find that CPEB3-LCM plays a role in appropriate local protein synthesis of messenger ribonucleic acid (mRNA) targets, through crucial protein-protein interactions that drive localization to neuronal Decapping protein 1 (DCP1)-bodies. Translation-promoting CPEB3 and translation-inhibiting CPEB1 are packaged into neuronal RNP granules immediately after chemical long-term potentiation is induced, but only translation-promoting CPEB3 is repackaged to these organelles at later time points. This localization to neuronal RNP granules is critical for functional influence on translation as well as overall local protein synthesis (measured as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) insertion into the membrane and localization to the synapse). We therefore conclude that protein-protein interaction between the LCM of CPEB3 plays a critical role in local protein synthesis by utilizing neuronal RNP granules.

Delivering an mRNA vaccine using a lymphatic drug delivery device improves humoral and cellular immunity against SARS-CoV-2.

The exploration and identification of safe and effective vaccines for the SARS-CoV-2 pandemic have captured the world's attention and remains an ongoing issue due to concerns of balancing protection against emerging variants of concern while also generating long-lasting immunity. Here, we report the synthesis of a novel messenger ribonucleic acid encoding the spike protein in a lipid nanoparticle formulation (STI-7264) that generates robust humoral and cellular immunity following immunization of C57Bl6 mice. In an effort to improve immunity, a clinically focused lymphatic drug delivery device (MuVaxx) was engineered to modulate immune cells at the injection site (epidermis and dermis) and draining lymph node (LN) and tested to measure adaptive immunity. Using MuVaxx, immune responses were elicited and maintained at a 10-fold dose reduction compared to traditional intramuscular (IM) administration as measured by anti-spike antibodies, cytokine-producing CD8 T cells, neutralizing antibodies against the Washington (wild type) strain and South African (Beta) variants, and LN-resident spike-specific memory B cells. Remarkably, a 4-fold-elevated T cell response was observed in MuVaxx-administered vaccination compared to that of IM-administered vaccination. Thus, these data support further investigation into STI-7264 and lymphatic-mediated delivery using MuVaxx for SARS-CoV-2 and VoC vaccines.

Classification of DNA microarrays using artificial neural networks and ABC algorithm

DNA microarray is an efficient new technology that allows to analyze, at the same time, the expression level of millions of genes. The gene expression level indicates the synthesis of different messenger ribonucleic acid (mRNA) molecule in a cell. Using this gene expression level, it is possible to diagnose diseases, identify tumors, select the best treatment to resist illness, detect mutations among other processes. In order to achieve that purpose, several computational techniques such as pattern classification approaches can be applied. The classification problem consists in identifying different classes or groups associated with a particular disease (e.g., various types of cancer, in terms of the gene expression level). However, the enormous quantity of genes and the few samples available, make difficult the processes of learning and recognition of any classification technique. Artificial neural networks (ANN) are computational models in artificial intelligence used for classifying, predicting and approximating functions. Among the most popular ones, we could mention the multilayer perceptron (MLP), the radial basis function neural network (RBF) and support vector machine (SVM). The aim of this research is to propose a methodology for classifying DNA microarray. The proposed method performs a feature selection process based on a swarm intelligence algorithm to find a subset of genes that best describe a disease. After that, different ANN are trained using the subset of genes. Finally, four different datasets were used to validate the accuracy of the proposal and test the relevance of genes to correctly classify the samples of the disease.

Nucleosome regulatory dynamics in response to TGF

Nucleosomes play important roles in a cell beyond their basal functionality in chromatin compaction. Their placement affects all steps in transcriptional regulation, from transcription factor (TF) binding to messenger ribonucleic acid (mRNA) synthesis. Careful profiling of their locations and dynamics in response to stimuli is important to further our understanding of transcriptional regulation by the state of chromatin. We measured nucleosome occupancy in human hepatic cells before and after treatment with transforming growth factor beta 1 (TGFβ1), using massively parallel sequencing. With a newly developed method, SuMMIt, for precise positioning of nucleosomes we inferred dynamics of the nucleosomal landscape. Distinct nucleosome positioning has previously been described at transcription start site and flanking TF binding sites. We found that the average pattern is present at very few sites and, in case of TF binding, the double peak surrounding the sites is just an artifact of averaging over many loci. We systematically searched for depleted nucleosomes in stimulated cells compared to unstimulated cells and identified 24 318 loci. Depending on genomic annotation, 44–78% of them were over-represented in binding motifs for TFs. Changes in binding affinity were verified for HNF4α by qPCR. Strikingly many of these loci were associated with expression changes, as measured by RNA sequencing.

Antifibrinolytics attenuate inflammatory gene expression after cardiac surgery

Anti-inflammatory effects of tranexamic acid and aprotinin, used to abate perioperative blood loss, are reported and might be of substantial clinical relevance. The study of messenger ribonucleic acid synthesis provides a valuable asset in evaluating the inflammatory pathways involved. Whole-blood messenger ribonucleic acid expression of 114 inflammatory genes was compared pre- and postoperatively in 35 patients randomized to receive either placebo, tranexamic acid, or aprotinin. These results were further confirmed by reverse transcription-polymerase chain reaction. Of the 23 genes exhibiting independently altered postoperative gene expression levels, 8 were restricted to the aprotinin group only (growth differentiation factor 3, interleukin 19, interleukin 1 family member 7, transforming growth factor α, tumor necrosis factor superfamily 10, tumor necrosis factor superfamily 12, tumor necrosis factor superfamily 13B, vascular endothelial growth factor α), whereas both aprotinin and tranexamic acid altered gene expression of 3 genes as compared with placebo (FMS-related tyrosine kinase 3 ligand, growth differentiation factor 5, interferon-α8). In general, less upregulation of pro-inflammatory, and more upregulation of anti-inflammatory, genes was observed for patients treated with antifibrinolytics. Gene expression affected by aprotinin coded mostly for proteins that function through serine proteases. This study demonstrates that the use of tranexamic acid and aprotinin results in altered inflammatory pathways on the genomic expression level. We further demonstrate that the use of aprotinin leads to significant attenuation of the immune response, with several inhibitory effects restricted to the use of aprotinin only. The results aid in a better understanding of the targets of these drugs, and add to the discussion on which antifibrinolytic can best be used in the cardiac surgical patient.

RVX-208: A Small Molecule That Increases Apolipoprotein A-I and High-Density Lipoprotein Cholesterol In Vitro and In Vivo

The aim of this study was to determine whether a novel small molecule RVX-208 affects apolipoprotein (apo)A-I and high-density lipoprotein cholesterol (HDL-C) levels in vitro and in vivo. Increased apoA-I and HDL-C levels are potential therapeutic targets for reducing atherosclerotic disease. HepG2 cells were treated with 0 to 60 mumol/l RVX-208 followed by assays for apoA-I and HDL-C production. For in vivo studies, African green monkeys (AGMs) received 15 to 60 mg/kg/day RVX-208, and the serum was analyzed for lipoprotein levels, HDL-subparticle distribution, cholesterol efflux, and activity of lipid-modifying enzymes. A phase I clinical trial was conducted in healthy volunteers (given 1 to 20 mg/kg/day of RVX-208) to assess safety, tolerability, and pharmacokinetics. The RVX-208 induced apoA-I messenger ribonucleic acid and protein synthesis in HepG2 cells, leading to increased levels of pre-beta-migrating and alpha-lipoprotein particles containing apoA-I (LpA-I) in spent media. Similarly, in AGMs, RVX-208 treatment for 63 days increased serum apoA-I and HDL-C levels (60% and 97%, respectively). In addition, the levels of pre-beta(1)-LpA-I and alpha1-LpA-I HDL-subparticles were increased as well as adenosine triphosphate binding cassette AI, adenosine triphosphate binding cassette G1, and scavenger receptor class B type I-dependent cholesterol efflux. These changes were not mediated by cholesteryl-ester-transfer protein. Treatment of humans for 1 week with oral RVX-208 increased apoA-I, pre-beta-HDL, and HDL functionality. RVX-208 increases apoA-I and HDL-C in vitro and in vivo. In AGMs, RVX-208 raises serum pre-beta(1)-LpA-I and alpha-LpA-I levels and enhances cholesterol efflux. Data in humans point to beneficial features of RVX-208 that might be useful for treating atherosclerosis.

Hypothesis

Hypothesis

Effect of ATP-Sensitive Potassium Channel Agonists on Ventricular Remodeling in Healed Rat Infarcts

The purpose of this study was to determine whether ATP-sensitive potassium (K(ATP)) channel agonists exert a beneficial effect on the structural, functional, and molecular features of the remodeling heart in infarcted rats. Myocardial K(ATP) channels have been implicated in the ventricular remodeling after myocardial infarction by inhibition of 70-kDa S6 (p70S6) kinase. Male Wistar rats after induction of myocardial infarction were randomized to either vehicle, agonists of K(ATP) channels nicorandil and pinacidil, an antagonist of K(ATP) channels glibenclamide, or a combination of nicorandil and glibenclamide or pinacidil and glibenclamide for 4 weeks. To verify the role of p70S6 kinase in ventricular remodeling, rapamycin was also assessed. Significant ventricular hypertrophy was detected by increased myocyte size at the border zone isolated by enzymatic dissociation after infarction. Increased synthesis of p70S6 kinase messenger ribonucleic acid after infarction in vehicle-treated rats was confirmed by reverse transcription-polymerase chain reaction, consistent with the results of immunohistochemistry and Western blot for phosphorylated p70S6 kinase. Rats in the nicorandil- and pinacidil-treated groups significantly attenuated cardiomyocyte hypertrophy and phosphorylated p70S6 kinase expression with similar potency, as compared with vehicle. The beneficial effects of nicorandil and pinacidil were abolished by administering either glibenclamide or 5-hydroxydecanoate. Addition of rapamycin attenuated ventricular remodeling and did not have additional beneficial effects compared with those seen in rats treated with either nicorandil or pinacidil alone. Activation of K(ATP) channels by either nicorandil or pinacidil can attenuate ventricular remodeling, probably through a p70S6 kinase-dependent pathway after infarction.

Long‐term stability of fear memory depends on the synthesis of protein but not mRNA in the amygdala

Synaptic modification supporting memory formation is thought to depend on gene expression and protein synthesis. Disrupting either process around the time of learning prevents the formation of long-term memory. Recent evidence suggests that memory also becomes susceptible to disruption upon retrieval. Whether or not the molecular events involved in the formation of new memory are the same as what is needed for memory to persist after retrieval has yet to be determined. In the present set of experiments, rats were given inhibitors of protein or messenger ribonucleic acid (mRNA) synthesis into the amygdala just after training or retrieval of fear memory. Results showed that blocking mRNA or protein synthesis immediately after learning prevented the formation of long-term memory, while stability of memory after retrieval required protein, but not mRNA, synthesis. These data suggest that the protein needed for memory reconsolidation after retrieval may be transcribed from pre-existing stores of mRNA.

Mechanistic Insights into Sulfur Relay by Multiple Sulfur Mediators Involved in Thiouridine Biosynthesis at tRNA Wobble Positions

The wobble bases of bacterial tRNAs responsible for NNR codons are modified to 5-methylaminomethyl-2-thiouridine (mnm5s2U). 2-thio modification of mnm5s2U is required for accurate decoding and essential for normal cell growth. We identified five genes yhhP, yheL, yheM, yheN, and yccK (named tusA, tusB, tusC, tusD, and tusE, respectively) that are essential for 2-thiouridylation of mnm5s2U by a systematic genome-wide screen ("ribonucleome analysis"). Efficient 2-thiouridine formation in vitro was reconstituted with recombinant TusA, a TusBCD complex, TusE, and previously identified IscS and MnmA. The desulfurase activity of IscS is stimulated by TusA binding. IscS transfers the persulfide sulfur to TusA. TusE binds TusBCD complex and stimulates sulfur transfer from TusA to TusD. TusE also interacts with an MnmA-tRNA complex. This study revealed that 2-thiouridine formation proceeds through a complex sulfur-relay system composed of multiple sulfur mediators that select and facilitate specific sulfur flow to 2-thiouridine from various pathways of sulfur trafficking.

Induction of Vascular Endothelial Growth Factor Messenger Ribonucleic Acid Expression in Stored Micrografts by Aminoguanidine

Aminoguanidine (AMG) has been found to inhibit apoptotic cell death in hair follicle micrografts and improves the viability of isolated micrografts during the storage period in hair restoration surgery. In this study, we investigated the effect of AMG on messenger ribonucleic acid (mRNA) synthesis of growth factors in stored micrografts and primary cultures of follicle-derived cell populations. Hair follicles were obtained from 10 different patients undergoing routine micrograft transplant and were stored for 5 hours at room temperature in phosphate-buffered saline containing different concentrations of AMG. After a culture period of 72 hours, quantitative changes of mRNA for basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) were determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Primary cell cultures of dermal papilla and outer root sheath cells were stimulated for 72 hours with AMG followed by RT-PCR measurement of growth factor mRNA. A dose-dependent induction of VEGF mRNA could be demonstrated in stored micrografts after stimulation with AMG (unstimulated: 1.0 [0.7-2.2]; AMG 10 pg/mL: 5.6 [3.1-9.7], p < .05; AMG 50 pg/mL: 6.9 [5.7-10.0], p < .05; AMG 100 microg/mL: 17.1 [14.1-22.3], p < .001). Expression of bFGF mRNA and IGF-1 mRNA levels was not influenced by AMG stimulation. Stimulation of cultured dermal papilla and outer root sheath cells demonstrated 14-fold induction of VEGF mRNA by AMG in outer root sheath cells (unstimulated: 1.0 [0.8-1.4]; AMG 100 pg/mL: 14.0 [12.5-16.1], p < .01), and no changes in VEGF mRNA levels were detected in dermal papilla cells. Our study demonstrated induction of VEGF mRNA in stored micrografts by AMG. Although the clinical relevance in post-transplant hair growth and wound healing needs further evaluation, the possibility of actively influencing growth factor production in isolated micrografts during the storage period is the basis for the development of hair follicle growth-promoting storage solutions in the future.

Effects of progestins and relaxin on glycodelin gene expression in human endometrial cells

Objective: Glycodelin is an endometrial protein proposed to play an important role in embryonic implantation. We examined the effects of progestins and relaxin on glycodelin transcription, synthesis, and secretion. Study Design: Northern blotting, metabolic labeling, and fluorography were used to assess glycodelin messenger ribonucleic acid and protein synthesis in endometrial tissue and cells. Luciferase reporter constructs transfected into endometrial adenocarcinoma cells (Ishikawa cells) were used to determine whether progestins or relaxin could activate the glycodelin gene promoter. Results: Progestins but not relaxin stimulated glycodelin secretion in primary epithelial cell cultures. A 452–base pair fragment of the glycodelin gene promoter was activated 4.3 ± 0.7 times normal by 10-nmol/L promegestone; however, addition of relaxin to the same construct repressed progestin-stimulate promoter activation by >30%. Conclusion: Glycodelin transcription, synthesis, and secretion by endometrial epithelial cells were stimulated by progestins. However, relaxin failed to stimulate production of this immunomodulatory protein and, in fact, repressed progestin-stimulated activation of the glycodelin gene promoter. (Am J Obstet Gynecol 2000;182:841-9.)

Estradiol amplifies interleukin-1-induced monocyte chemotactic protein-1 expression by ectopic endometrial cells of women with endometriosis.

Endometriosis, one of the most frequently occurring gynecological disorders, is estrogen dependent and is often associated with immunological changes. These include increased macrophage activation and infiltration into the endometriotic implants themselves as well as the peritoneal cavity where the implants often develop. Despite the critical role estrogens play in the development of endometriosis, the biochemical mechanisms of their action remain unclear. In the present study we report that estradiol (E2) enhances endometriotic cell responsiveness to the proinflammatory cytokine interleukin-1beta by up-regulating interleukin-1-induced monocyte chemotactic protein-1 (MCP-1) expression at the level of both protein secretion and messenger ribonucleic acid (mRNA) synthesis, whereas progesterone had no significant effects. According to mRNA half-life experiments, E2 action does not seem to be due to increased MCP-1 mRNA stability but, rather, to a higher level of transcription, as shown by run-on analysis. Interestingly, immunohistochemical analysis of MCP-1 expression in endometriotic tissue showed intense immunostaining in both epithelial glands and stroma regardless of the menstrual cycle phase, which is consistent with the cell culture data and indicates that MCP-1 expression is not subject to cyclic variation. The findings of the present study for the first time provide evidence that E2 up-regulates, although in an indirect way, the expression of a potent chemotactic and activating factor by ectopic endometrial cells, which may occur locally in the inflammatory site and contribute to peritoneal macrophage recruitment and activation, and reveal a new means of E2 action in the pathophysiology of endometriosis.

Effect of human seminal fluid on production of messenger ribonucleic acid for metalloproteinase 2 and metalloproteinase 9 in cervical epithelial carcinoma cells

Objectives: The risk for cervical cancer is related to sexual intercourse. One factor associated with tumor invasion and metastases is the production of matrix metalloproteinases, which degrade the extracellular matrix. The ability of human semen to influence production of messenger ribonucleic acid for 2 matrix metalloproteinases associated with cervical cancer, MMP-2 and MMP-9, was examined. Study Design: Seminal fluids from 16 men were diluted 1:50 and incubated with the cervical carcinoma cell line HeLa for 18 hours. Cells were harvested, ribonucleic acid was isolated and reverse transcribed into complementary deoxyribonucleic acid, and deoxyribonucleic acid sequences coding for regions of the genes for MMP-2 and MMP-9 were generated by polymerase chain reaction and detected by enzyme-linked immunosorbent assay. Results: All 16 semen samples inhibited production of MMP-2 messenger ribonucleic acid. The MMP-2 messenger ribonucleic acid synthesis in the presence of semen was a mean of 39.5% of the control value. In contrast, MMP-9 messenger ribonucleic acid synthesis was stimulated (mean stimulation, 68.3%) by semen in 10 of the 15 samples tested. Synthesis of β-actin was comparable in each culture, and cell viability was unaffected by the diluted semen. Conclusion: The capacity of semen to influence transcription of the genes for matrix metalloproteinases may be one mechanism whereby sexual activity influences cervical cancer progression. (Am J Obstet Gynecol 1999;181:591-5.)

The nuclear transcription factor NF-κB mediates interleukin-1β–induced expression of cyclooxygenase-2 in human myometrial cells

Objective: Up-regulation of prostaglandin production by gestational tissues in the setting of intrauterine infection has been implicated as an important contributor to preterm labor and parturition. In this study we investigated the possible role of the nuclear transcription factor NF-κB in interleukin-1 signaling, leading to the expression of cyclooxygenase 2 and prostaglandin production in human myometrial cell cultures. Study Design: Human myometrial smooth muscle cells from an immortalized line were used as a model system between passages 20 and 35. Growth-arrested cell cultures were stimulated with human recombinant interleukin 1, and the activation of NF-κB was assessed by the degradation of the inhibitory protein IκB-α (Western analysis), as well as by nuclear binding of NF-κB by using an electrophoretic mobility shift assay. The abundance of cyclooxygenase-2 messenger ribonucleic acid and protein was measured by Northern and Western analyses, whereas prostaglandin (prostaglandin I 2 and prostaglandin E 2 ) production was determined by specific radioimmunoassays. Results: Within 15 minutes of stimulation with interleukin 1, 90% of IκB-α was degraded. This was temporally associated with nuclear translocation and binding of NF-κB. Within 30 minutes, cyclooxygenase 2 messenger ribonucleic acid appeared, with steady-state levels increasing up to 4 hours. This was followed by an up to 80-fold increase in cyclooxygenase 2 protein and a corresponding time-dependent increase in prostaglandin production. When IκB-α degradation was blocked with calpain I inhibitor, NF-κB translocation, cyclooxygenase 2 messenger ribonucleic acid and protein expression, and prostaglandin synthesis were also inhibited. Conclusion: Stimulation of human myometrial cells with interleukin 1 leads to rapid activation of the transcription factor NF-κB, which is functionally linked to the expression of cyclooxygenase 2 messenger ribonucleic acid, protein, and prostaglandin synthesis. (Am J Obstet Gynecol 1999;181:359-66.)

Human mast cells activate fibroblasts: tryptase is a fibrogenic factor stimulating collagen messenger ribonucleic acid synthesis and fibroblast chemotaxis.

The effect of human mast cells on fibroblast activity was studied using an organotypic skin-equivalent culture system. Human mast cell-1 (HMC-1) cells were embedded in a collagen gel with neonatal dermal fibroblasts at a ratio of 1:4; keratinocytes then were allowed to stratify above this composite culture. Analysis of type a1(I) procollagen mRNA synthesis by in situ hybridization revealed a substantial increase in mRNA levels in the presence of mast cells and especially following degranulation, induced by calcium ionophore A23187. Tryptase, a major product of human mast cells, could substitute for mast cells in this culture system, up-regulating procollagen mRNA synthesis. Tryptase pretreated with the specific protease inhibitor bis(5-amidino-2-benzimidazo-lyl)methane (BABIM) markedly attenuated the collagen mRNA up-regulation. Further studies revealed HMC-1 cell sonicates stimulated fibroblast chemotaxis and procollagen mRNA synthesis. Inhibition of HMC-1 sonicates with either BABIM or a neutralizing mAb against tryptase resulted in significant reduction of fibroblast chemotaxis and procollagen mRNA, implying that tryptase accounted for the majority of HMC-1 sonicate activity. Tryptase directly stimulated fibroblast chemotaxis with optimal concentrations between 10 pM and 1 nM. The maximal response of optimal concentrations of tryptase was comparable with the known fibrogenic factor, TGF-beta. Inhibition of tryptase with BABIM resulted in approximately 50% reduction in chemotactic activity. Additional studies revealed that tryptase (0.3-3 nM) stimulated procollagen mRNA synthesis in confluent monolayers of dermal fibroblasts.

Expression of gastrin-releasing peptide (GRP) and GRP receptors in the pregnant human uterus at term.

Synthesis of both gastrin-releasing peptide (GRP) and messenger ribonucleic acid (mRNA) has been demonstrated in pregnant sheep, but studies in women have not been reported. Therefore, we examined the uterus and placenta of pregnant women at term for synthesis of GRP and expression of GRP receptor genes BRS-3 and GRP-R. A transcript of 0.95 kilobases, corresponding to GRP mRNA, was detected in endometrium and myometrium, but not in amnion, chorion, placenta, or nonpregnant endometrium. GRP immunoreactivity (GRP-ir) was detected in half (three of six) of the endometrial (1.23 +/- 0.04 pmol/g) and myometrial (0.73 +/- 0.04 pmol/g) samples and in some, but not all, samples of amnion (one of four subjects; 0.6 pmol/g), chorion (four of five subjects; 0.8 +/- 0.2 pmol/g), placenta (two of six subjects; 0.5 +/- 0.2 pmol/g), and amniotic fluid (four of six subjects; 59 +/- 19 fmol/mL). GRP-ir was present in the maternal circulation (44 +/- 12 fmol/mL) and was higher in plasma obtained from the umbilical artery (152 +/- 14 fmol/mL) and vein (143 +/- 24 fmol/mL). The major peak of GRP-ir in pregnant endometrial tissue was larger than GRP-(1-27), as determined by gel filtration chromatography. Minor peaks were also observed: two larger than the main form and one corresponding to GRP-(18-27). mRNA for GRP receptors GRP-R and BRS-3 was detected by semiquantitative reverse transcription-PCR. For both receptors, mRNA was higher in the pregnant endometrium than in the nonpregnant endometrium but was detected in all of the uteroplacental tissues examined. GRP-R mRNA predominated in the pregnant endometrium, whereas BRS-3 mRNA predominated in the membranes and placenta. In these tissues, PCR for BRS-3 mRNA gave rise to an additional product (approximately 50 bp larger). These studies demonstrated that a peptide larger than, but related to, GRP is synthesized in the pregnant human uterus and is secreted into the maternal and fetal circulations. The detection of mRNA for GRP-R, BRS-3, and possibly a transcript variant of BRS-3 as well as the detection of a peptide larger than, but related to, GRP suggest a novel regulatory unit in the human reproductive tract.

Modulation of the levels of interleukin-8 messenger ribonucleic acid and interleukin-8 protein synthesis in human endometrial stromal cells by transforming growth factor-beta 1

Modulation of the levels of interleukin-8 messenger ribonucleic acid and interleukin-8 protein synthesis in human endometrial stromal cells by transforming growth factor-beta 1

Modulation of the levels of interleukin-8 messenger ribonucleic acid and interleukin-8 protein synthesis in human endometrial stromal cells by transforming growth factor-beta 1.

Selected functions of uterine endometrium of ovulatory women before and during pregnancy appear to be modulated by cytokines and other paracrine-acting factors. Some of these functions are regulated, in turn, by cyclic changes in ovarian steroid secretion or by pregnancy-induced endocrine and paracrine factors. The recruitment of specific types and numbers of bone marrow-derived cells into the endometrium occurs in a predictable manner with hormonal changes of the ovarian cycle, during the process of endometrial decidualization, at the time of blastocyst implantation, and during pregnancy, parturition, and the puerperium. As part of an investigation of the regulation of the leukocyte population of endometrium/decidua, this study was conducted to evaluate further the regulation of interleukin-8 (IL-8) gene expression by transforming growth factor-beta (TGF beta). IL-8 is a neutrophil chemoattractant/activating and T cell chemotactic factor as well as a chemotactic factor for fibroblasts. IL-8 is produced by mesenchymal cells of many tissues, including human endometrial stromal cells in culture. The level of IL-8 messenger ribonucleic acid (mRNA) in endometrial stromal cells and the accumulation of immunoreactive IL-8 in medium are increased by TGF beta 1 treatment of these cells. This response to TGF beta 1 is attributable primarily to an increase in the stability of IL-8 mRNA through a process that is dependent on protein synthesis. Transcription of the IL-8 gene in endometrial stromal cells is not increased, but, rather, is slightly decreased, by treatment with TGF beta 1. The findings of this study indicate that TGF beta may act in endometrial stroma to modulate the stability of IL-8 mRNA.

Opposing actions of transforming growth factor-beta and glucocorticoids in the regulation of fibronectin expression in the human placenta.

Alterations in the expression of extracellular matrix (ECM) proteins in the placenta and fetal membranes have been linked to parturition whether occurring before or at term. In the present study, we examined the individual and combined effects of transforming growth factor (TGF)-beta and dexamethasone (DEX) on the expression of oncofetal fibronectin (onfFN), i.e. a major ECM protein synthesized by placenta, in cytotrophoblasts isolated from human term placentas to establish a model system from which to evaluate the actions of positive and negative regulators of ECM protein expression in the human placenta. Cytotrophoblasts were maintained for 21=62 h in medium supplemented with 4% charcoal-stripped calf serum in the presence or absence of TGF-beta (2 ng/mL) and DEX (10(-7) mol/L). Levels of onfFN in culture media were determined by immunoassay. TGF-beta treatment alone induced approximately a 150% increase in media levels of onfFN after 21 and 45 h of culture when compared with control, whereas DEX treatment alone reduced levels of onfFN to 15% of control levels. Media levels of onfFN in cells treated with both TGF-beta and DEX were 40-90% of control levels. Similarly, treatment of cells with TGF-beta alone promoted a 100-250% increase in rates of FN synthesis and levels of FN messenger ribonucleic acid, whereas DEX treatment alone reduced these indices of FN expression to approximately 10% of control levels. In cells treated with TGF-beta and DEX, levels of ECM protein synthesis and FN messenger ribonucleic acid were between 30 and 100% of control values. Similar patterns of regulation of FN expression by TGF-beta and DEX were observed when experiments were carried out in serum-free medium. Our results suggest that during pregnancy, TGF-beta and glucocorticoids may be important opposing physiological regulators of placental ECM protein expression.