Novel Targeting of Cyclooxygenase-2 (COX-2) Pre-mRNA Using Antisense Morpholino Oligonucleotides Directed to the 3Ј Acceptor and 5Ј Donor Splice Sites of Exon 4: Suppression of COX-2 Activity in Human Amnion-Derived WISH and Myometrial Cells

ABSTRACT Increased expression of cyclooxygenase-2 (COX-2) has been implicated in the onset of both term and preterm labor. In this context, both selective and nonselective COX-2 inhibitors have been used in clinical trials to determine their efficacy in delaying preterm labor. However, recent evidence indicates that these tocolytics may have potentially adverse fetal and maternal side effects. Therefore, the development of more specific and nontoxic agents to inhibit COX-2 needs to be considered. We have evaluated whether antisense morpholino oligonucleotides have therapeutic potential in inhibiting COX-2 by specifically targeting both the 3Ј and 5Ј acceptor and donor sites of exon 4 of COX-2's pre-mRNA sequence. Confocal microscopy on "live" cells illustrated high levels of penetrance of antisense morpholino oligonucleotides using the Endo-Porter formula (GeneTools, LLC, Philomath, OR), with delivery efficiencies of 82 and 78%, respectively, in amnion-derived WISH and myometrial cells. Substantial inhibition by the morpholino oligonucleotides of COX-2 expression, induced by lipopolysaccharide administration, was observed at both the mRNA and protein levels. Loss of enzymic activity of COX-2 was confirmed using a sensitive COX enzyme activity assay, which reflects the rate of conversion of arachidonic acid to prostaglandin H 2. Our results indicate that antisense morpholino oligonucleotides significantly inhibit expression and activity of this enzyme in in vitro cultures of amnion-WISH and myometrial cells. The potential thus exists that a similar approach can be mimicked in vivo to produce a highly specific and nontoxic strategy to inhibit COX-2 activity with its subsequent effects on the better management of preterm labor and other inflammatory conditions

Alternative splicing: an important mechanism for myometrial gene regulation that can be manipulated to target specific genes associated with preterm labour

Considerable effort has been expended in attempting to distinguish genes that contribute to initiating the onset of term and preterm labour (PTL) from those that change in expression as a consequence of the progression of labour. The ability to define more clearly the genes involved in triggering labour contractions should lead to the development of new effective and safer strategies to prevent preterm birth. There is ample evidence to suggest that specific genes are co-ordinately regulated within the upper and lower regions of the myometrium prior to and during parturition and many of these genes are regulated by alternative pre-mRNA splicing. This mini-review highlights that expression of a range of different splicing factors, with defined roles in pre-mRNA splicing, is both temporally and spatially regulated within the uterine smooth muscle during pregnancy and labour. Moreover, several of these splicing factors play key roles in controlling the differential expression of specific regulatory proteins involved in uterine signalling and uterine quiescence. In addition, antisense morpholino oligonucleotide manipulation of pre-mRNA splicing may have potential in defining and targeting uterine pro-labour genes and thus contribute to the development of new therapeutic approaches to prevent PTL

Chemotactic and osmotic signals share acGMP transduction pathway in Dictyostelium discoideum

In the ameboid eukaryote Dictyostelium discoideum, chemotactic stimulation by cAMP induces an increase of intracellular cGMP and subsequently the phosphorylation of myosin heavy chain II. Resistance to high osmotic stress also requires transient increases of intracellular cGMP and phosphorylation of myosin heavy chain II, although the kinetics is much slower than for chemotaxis. To examine if chemotaxis and osmotic stress share common signaling components we systematically analyzed the osmotic cGMP response and survival in chemotactic mutants with altered cGMP signaling. Null mutants with deletions of cell surface cAMP receptors or the associated GTP-binding proteins Gα2 and Gβ show no cAMP-induced cGMP response and chemotaxis; in contrast, osmotic stress induces the normal cGMP accumulation and survival. The same result was obtained with the non-chemotactic mutant KI-10, which lacks the activation of guanylyl cyclase by cAMP. This indicates that these components are required for chemotaxis but not osmotic cGMP signaling and survival. The potential guanylyl cyclase null mutant KI-8 shows no chemotaxis, no osmotic cGMP increase and reduced survival in high osmolarity. Two types of cGMP-binding protein mutants, KI-4 and KI-7, also show reduced tolerance during high osmotic stress. Taken together, these observations clarify that chemotactic and osmotic signals are detected by different mechanisms, but share a cGMP signaling pathway.

SERPINA3 is a marker of cartilage differentiation and is essential for the expression of extracellular matrix genes during early chondrogenesis

\ua9 2024Serine proteinase inhibitors (serpins) are a family of structurally similar proteins which regulate many diverse biological processes from blood coagulation to extracellular matrix (ECM) remodelling. Chondrogenesis involves the condensation and differentiation of mesenchymal stem cells (MSCs) into chondrocytes which occurs during early development. Here, and for the first time, we demonstrate that one serpin, SERPINA3 (gene name SERPINA3, protein also known as alpha-1 antichymotrypsin), plays a critical role in chondrogenic differentiation. We observed that SERPINA3 expression was markedly induced at early time points during in vitro chondrogenesis. We examined the expression of SERPINA3 in human cartilage development, identifying significant enrichment of SERPINA3 in developing cartilage compared to total limb, which correlated with well-described markers of cartilage differentiation. When SERPINA3 was silenced using siRNA, cartilage pellets were smaller and contained lower proteoglycan as determined by dimethyl methylene blue assay (DMMB) and safranin-O staining. Consistent with this, RNA sequencing revealed significant downregulation of genes associated with cartilage ECM formation perturbing chondrogenesis. Conversely, SERPINA3 silencing had a negligible effect on the gene expression profile during osteogenesis suggesting the role of SERPINA3 is specific to chondrocyte differentiation. The global effect on cartilage formation led us to investigate the effect of SERPINA3 silencing on the master transcriptional regulator of chondrogenesis, SOX9. Indeed, we observed that SOX9 protein levels were markedly reduced at early time points suggesting a role for SERPINA3 in regulating SOX9 expression and activity. In summary, our data support a non-redundant role for SERPINA3 in enabling chondrogenesis via regulation of SOX9 levels

Regulators of G protein signalling proteins in the human myometrium

The contractile state of the human myometrium is controlled by extracellular signals that promote relaxation or contraction. Many of these signals function through G proteincoupled receptors at the cell surface, stimulating heterotrimeric G proteins and leading to changes in the activity of effector proteins responsible for bringing about the response. G proteins can interact with multiple receptors and many different effectors and are key players in the response. Regulators of G protein signalling (RGS) proteins are GTPase activating proteins for heterotrimeric G proteins and help terminate the signal. Little is known about the function of RGS proteins in human myometrium and we have therefore analysed transcript levels for RGS proteins at various stages of pregnancy (non-pregnant, preterm, term non-labouring, term labouring). RGS2 and RGS5 were the most abundantly expressed isolates in each of the patient groups. The levels of RGS4 and RGS16 (and to a lesser extent RGS2 and RGS14) increased in term labouring samples relative to the other groups. Yeast two-hybrid analysis and co-immunoprecipitation in myometrial cells revealed that both RGS2 and RGS5 interact directly with the cytoplasmic tail of the oxytocin receptor, suggesting they might help regulate signalling through this receptor. Key words: G protein-coupled receptors; labour; myometrium; RGS protein

Regulation of GTP-binding Protein (Galpha s) Expression in Human Myometrial Cells A ROLE FOR TUMOR NECROSIS FACTOR IN MODULATING G s PROMOTER ACETYLATION BY TRANSCRIPTIONAL COMPLEXES

The onset of parturition is associated with a number of proinflammatory mediators that are themselves regulated by the nuclear factor κB (NF-κB) family of transcription factors. In this context, we previously reported that the RelA NF-κB subunit represses transcription and mRNA expression of the proquiescent Gαs gene in human myometrial cells following stimulation with the proinflammatory cytokine TNF. In the present study, we initially defined the functional consequence of this on myometrial contractility. Here we show that, contrary to our initial expectations, TNF did not induce myometrial contractility but did inhibit the relaxation produced by the histone deacetylase inhibitor trichostatin A, an effect that in turn was abolished by the NF-κB inhibitor N4-[2-(4-phenoxyphenyl)ethyl]-4,6-quinazolinediamine. This result suggested a role for TNF in regulating Gαs expression via activating NF-κB and modifying histone acetylation associated with the promoter region of the gene. In this context, we show that the −837 to −618 region of the endogenous Gαs promoter is occupied by cAMP-response element-binding protein (CREB), Egr-1, and Sp1 transcription factors and that CREB-binding protein (CBP) transcriptional complexes form within this region where they induce histone acetylation, resulting in increased Gαs expression. TNF, acting via NF-κB, did not change the levels of CREB, Sp1, or Egr-1 binding to the Gαs promoter, but it induced a significant reduction in the level of CBP. This was associated with increased levels of histone deacetylase-1 and surprisingly an increase in H4K8 acetylation. The latter is discussed herein

Dictyostelium discoideum as a Model to Study Inositol Polyphosphates and Inorganic Polyphosphate

The yeast Saccharomyces cerevisiae has given us much information on the metabolism and function of inositol polyphosphates and inorganic polyphosphate. To expand our knowledge of the metabolic as well as functional connections between inositol polyphosphates and inorganic polyphosphate, we have refined and developed techniques to extract and analyze these molecules in a second eukaryotic experimental model, the amoeba Dictyostelium discoideum. This amoeba, possessing a well-defined developmental program, is ideal to study physiological changes in the levels of inositol polyphosphates and inorganic polyphosphate, since levels of both molecules increase at late stages of development. We detail here the methods used to extract inositol polyphosphates using perchloric acid and inorganic polyphosphate using acidic phenol. We also present the postextraction procedures to visualize and quantify these molecules by polyacrylamide gel electrophoresis and by malachite green assay

Agonist-stimulated high-affinity GTPase in Dictyostelium membranes

AbstractGTP hydrolysis in Dictyostelium discoideum membranes is caused by a low (Km> 1 mM) and a high affinity (Km 6.5 μM) GTPase. cAMP enhances GTP hydrolysis apparently by increasing the affinity of the high affinity GTPase (stimulated Km 4.5 μM); the low affinity GTPase was not affected by cAMP. Stimulation of GTP hydrolysis by cAMP was maximal at early time points and declined thereafter. A half-maximal stimulation of GTPase occurred at 3 μM cAMP and the specificity of cAMP derivatives for stimulation of GTPase activity showed a close correlation with the specificity for binding to the cell surface cAMP receptor. Treatment of D. discoideum cells with pertussis toxin decreased the cAMP-induced stimulation of GTPase from 42 ± 6% in control cells to 17 ± 9% in pertussis toxin-treated cells. These results suggest that the interaction of cAMP with its surface receptor leads to stimulation of high affinity GTPase in D. discoideum membranes. At least one of those enzymes may represent a guanine nucleotide-binding protein sensitive to pertussis toxin

Estrogen Alters the Splicing of Type 1 Corticotropin-Releasing Hormone Receptor in Breast Cancer Cells

Hormonal stress response is associated with the pathogenesis of disease, including cancer. The role of the stress hormone CRH (corticotropin-releasing hormone) in breast cancer is complex, and its abundance and biological activity may be modulated by estrogen. In the estrogen receptor–positive (ER+) malignant mammary epithelial cell line MCF7, CRH activated numerous kinases and downstream effectors, at least some of which were mediated by the CRH receptor type 1 (CRH-R1). CRH also increased the transcription of many genes that encode effectors, transcriptional targets, or regulators associated with estrogen signaling. Estrogen increased the abundance of the mRNA encoding CRH-R2 and an alternative splice variant encoding CRH-R1 in which exon 12 was deleted [CRH-R1(Δ12)]. Estrogen inhibited the expression SRSF6, which encodes serine/arginine-rich splicing factor 55 (SRp55). An increase in CRH-R1(Δ12), in response to either estrogen or SRp55 knockdown, dampened the cellular response to CRH and prevented its inhibitory effects on cell invasion. SRp55 knockdown also induced additional splicing events within exons 9 to 12 of CRH-R1, whereas overexpression of SRp55 prevented estrogen-induced generation of CRH-R1(Δ12). ER+ breast tumors had increased CRH-R2 and CRH-R1(Δ12) mRNA abundance, which was associated with decreased abundance of the mRNA encoding SRp55, compared with the amounts in ER– tumors, suggesting that estrogen contributes to the pathophysiology of ER+ breast cancer by altering CRH receptor diversity and disrupting CRH-mediated signaling