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PubMed2024

Exploring the transcriptional cooperation between RUNX2 and its associated elncRNA RAIN.

Recent insights into the mechanisms controlling gene expression identified enhancer-associated long non-coding RNAs (elncRNAs) as master players of transcription in cancers. RUNX2, a mammalian RUNT-related transcription factor, is increasingly recognized in cancer biology for its role in supporting survival and progression also in thyroid cancer (TC). We recently identified, within the RUNX2 locus, a novel elncRNA that we named RAIN (RUNX2 associated intergenic lncRNA). We showed that RAIN and RUNX2 expression correlate in TC, both in vitro and in vivo, and that RAIN promotes RUNX2 expression by interacting with and affecting the activity of the RUNX2 P2 promoter through two distinct mechanisms. Here, we took forward these observations to explore the genome-wide transcriptional function of RAIN and its contribution to the RUNX2-dependent gene expression program in TC. By combining multiple omics data, we demonstrated that RAIN functionally cooperates with RUNX2 to the regulation of a subset of functionally related genes involved in promoting matrix remodeling, migration, and loss of differentiation. We showed that RAIN interacts with RUNX2 and its expression is required for the efficient recruitment of this TF to its target regulatory regions. In addition, our data revealed that besides RUNX2, RAIN governs a hierarchically organized complex transcriptional program by controlling a core of cancer-associated TFs that, in turn, orchestrate the expression of downstream genes. This evidence indicates that the functional cooperation observed between RAIN and RUNX2 can be a diffuse work mechanism for this elncRNA.

Core Binding Factor Alpha 1 Subunit↗
PubMed2005

Knockdown of the cytoprotective chaperone, clusterin, chemosensitizes human breast cancer cells both in vitro and in vivo.

Clusterin is a stress-associated cytoprotective chaperone up-regulated by various apoptotic triggers in many cancers and confers treatment resistance when overexpressed. The objectives of this study were to evaluate clusterin expression levels in human breast cancer and to determine whether antisense oligonucleotides or double-stranded small interfering RNAs (siRNA) targeting the clusterin gene enhance apoptosis induced by paclitaxel. Clusterin immunostaining was evaluated in a tissue microarray of 379 spotted breast cancers. The effect of hormone withdrawal, paclitaxel treatment, clusterin antisense oligonucleotide (OGX-011), and siRNA treatments on clusterin expression was examined in MCF-7 and MDA-MB-231 cells. Northern, quantitative real-time PCR, and Western analyses were used to measure change in clusterin mRNA and protein levels. The effect of OGX-011 or siRNA clusterin treatment on chemosensitivity to paclitaxel was done in both cell lines in vitro, whereas the ability of OGX-011 to chemosensitize in vivo was evaluated in athymic mice bearing MCF-7 tumors. Clusterin was expressed in 62.5% of tumors within the tissue microarray. Clusterin expression increased after estrogen withdrawal and paclitaxel treatment in vitro in MCF-7 cells. OGX-011 or siRNA clusterin decreased clusterin levels by >90% in a dose-dependent, sequence-specific manner and significantly enhanced chemosensitivity to paclitaxel in vitro. When combined, OGX-011 or siRNA clusterin reduced the IC50 by 2-log compared with paclitaxel alone. In vivo administration of OGX-011 enhanced the effects of paclitaxel to significantly delay MCF-7 tumor growth. These data identify clusterin as a valid therapeutic target and provides preclinical proof-of-principle to test OGX-011 in multimodality therapies for breast cancer.

Adult↗
PubMed1992

Mallory stain may indicate differential rates of RNA synthesis: I. A seasonal cycle in the harderian gland of the green frog (Rana esculenta).

When Mallory's trichrome stain is used, acinar nuclei of the Harderian gland of Rana esculenta display different affinities for the dye. Some of the orangiophilic nuclei show affinity for aniline blue (blue nuclei). In the Harderian gland of Rana esculenta their number and the intensity of staining with aniline blue may vary during the year. The affinity for aniline blue disappears following digestion of paraffin sections with RNAase, but not with DNAase or trypsin. Furthermore, in vitro incubation with [5, 6-3H]-Uridine shows a selective incorporation by the majority of blue nuclei. Therefore, the affinity for aniline blue is likely due to increased RNA synthesis. The increment of nuclear RNA shown by these methods is supported by the quantitative determination of total RNAs during the resumption (October) and enhancement (May) of secretory activity, when the percentage of blue nuclei of the acinar cells is at its highest levels of the year. The affinity of RNA-rich nuclei for aniline blue, while others are strictly orangiophil, is discussed on the basis of molecular structure of the dyes used in the staining mixture. Mallory's trichrome stain appears to be an useful tool for detecting changes in cell nuclear status.

Animals↗
PubMed1987

Regulation of cytochrome P-450 messenger RNA and apoprotein levels by heme.

2-Allylisopropylacetamide, a porphyrinogen which decreases the microsomal and cytosolic heme pools, is a phenobarbitone-like inducer of cytochrome P-450(b + e) messenger RNAs in rat liver. The porphyrinogen, however, does not affect the nuclear heme pool and enhances the transcription of cytochrome P-450(b + e) messenger RNAs strikingly. Inhibitors of heme biosynthesis, such as CoCl2 and 3-amino-1,2,4-triazole, which decrease the total heme levels including that of the nuclear heme pool, block the 2-allylisopropylacetamide- or phenobarbitone-mediated increase in the transcription of cytochrome P-450(b + e) messenger RNAs. Administration of exogenous heme at a very low concentration (25 micrograms/100 g) is able to counteract the inhibitory effects of the heme biosynthetic inhibitors. Addition of heme in vitro to heme-depleted nuclei leads to a significant increase in the transcription rates for cytochrome P-450(b + e) messenger RNAs. 2-Allylisopropylacetamide, unlike phenobarbitone, fails to increase the levels of cytochrome P-450b protein at 12 h after the drug administration, although there is a striking increase in the messenger RNA levels. Under conditions of 2-allylisopropylacetamide treatment, the cytochrome P-450 messenger RNA is translated, but the newly synthesized apoprotein undergoes rapid degradation. It is concluded that heme is a positive modulator of cytochrome P-450 gene transcription and is also required to stabilize the freshly synthesized apoprotein.

Allylisopropylacetamide↗
PubMed1996

An intron enhancer recognized by splicing factors activates polyadenylation.

Alternative processing of the pre-messenger RNA encoding calcitonin/calcitonin gene-related peptide (CT/CGRP) involves alternative inclusion of a 3'-terminal exon (exon 4) embedded within a six exon primary transcript. Expression of CT/CGRP in transgenic mice indicates that inclusion of exon 4 occurs in a wide variety of tissues, suggesting that the factors responsible for exon 4 inclusion are widely distributed. Inclusion of exon 4 requires an enhancer sequence located within the intron downstream of the poly(A) site of exon 4. Here we show that the intron enhancer activated in vitro polyadenylation cleavage of precursor RNAs containing the CT/CGRP exon 4 poly(A) site or heterologous poly(A) sites. To our knowledge this is the first example of an intron-located enhancer that facilitates polyadenylation. Within the enhancer sequence is a 5' splice site sequence immediately preceded by a pyrimidine tract. This 5' splice site sequence was required for enhanced polyadenylation and was recognized by both U1 small nuclear ribonucleoproteins (snRNPs) and alternative splicing factor/splicing factor 2 (ASF/SF2). Enhancement of polyadenylation required U1 RNA, suggesting that the 5' splice site sequence within the enhancer mediates enhancement via interaction with factors normally associated with functional 5' splice sites. Mutation of the polypyrimidine track of the enhancer also inhibited in vitro polyadenylation cleavage. Oligonucleotide competitions and UV cross-linking indicated that the enhancer pyrimidine track binds the polypyrimidine tract binding protein (PTB), but not U2 snRNP auxiliary factor (U2AF), and that binding of PTB was required for maximal enhancer-mediated polyadenylation. These results suggest that the enhancer binds known splicing factors, and that binding of these factors activates polyadenylation cleavage. Furthermore, these results suggest that regulation of alternative processing of CT/CGRP could occur at the level of polyadenylation, rather than splicing.

Alternative Splicing↗
PubMed1993

A single nucleotide substitution in the internal ribosome entry site of foot-and-mouth disease virus leads to enhanced cap-independent translation in vivo.

Mutants of foot-and-mouth disease virus (FMDV) with altered biological properties can be selected during the course of persistent infection of BHK-21 cells with FMDV C-S8c1 (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). Two nucleotide substitutions, U to C at position -376 and A to G at position -15, (counting as +1 the A of the first functional AUG), were fixed within the internal ribosome entry site (IRES) of R100, the virus rescued after 100 passages of the carrier BHK-21 cells. IRES-directed cap-independent protein synthesis was quantitated by using bicistronic constructs of the form chloramphenicol acetyltransferase gene-IRES-luciferase gene. The IRES from R100 was 1.5- to 5-fold more active than that of C-S8c1 in directing cap-independent luciferase synthesis. This enhanced translational activity was observed when the RNAs were transcribed either in the nucleus or in the cytoplasm by a weak or a strong promoter, respectively. C-S8c1 and R100 IRES elements were functional in both FMDV-sensitive and FMDV-resistant cells (including persistently infected R cells), indicating that factors mediating cap-independent protein synthesis are not limited in any of the analyzed cell lines. Constructs in which each of the two mutations in the R100 IRES were analyzed separately indicate that the transition at position -376 is responsible for the enhanced activity of the R100 IRES. By estimating the effect that an increase in the initial translation efficiency may have on subsequent RNA replication steps, we suggest that the modifications in the IRES elements can account for the previously described hypervirulence of FMDV R100 for BHK-21 cells. The results show that a single point mutation in an IRES element of a picornavirus can cause an increase in translation efficiency.

Animals↗
PubMed1999

Enhancer-like properties of an RNA element that modulates Tombusvirus RNA accumulation.

Prototypical defective interfering (DI) RNAs of the plus-strand RNA virus tomato bushy stunt virus contain four noncontiguous segments (regions I-IV) derived from the viral genome. Region I corresponds to 5'-noncoding sequence, regions II and III are derived from internal positions, and region IV represents a 3'-terminal segment. We analyzed the internally located region III in a prototypical DI RNA to understand better its role in DI RNA accumulation. Our results indicate that (1) region III is not essential for DI RNA accumulation, but molecules that lack it accumulate at significantly reduced levels ( approximately 10-fold lower), (2) region III is able to function at different positions and in opposite orientations, (3) a single copy of region III is favored over multiple copies, (4) the stimulatory effect observed on DI RNA accumulation is not due to region III-mediated RNA stabilization, (5) DI RNAs lacking region III permit the efficient accumulation of head-to-tail dimers and are less effective at suppressing helper RNA accumulation, and (6) negative-strand accumulation is also significantly depressed for DI RNAs lacking region III. Collectively, these results support a role for region III as an enhancer-like element that facilitates DI RNA replication. A scanning-type mutagenesis strategy was used to define portions of region III important for its stimulatory effect on DI RNA accumulation. Interestingly, the results revealed several differences in the requirements for activity when region III was in the forward versus the reverse orientation. In the context of the viral genome, region III was found to be essential for biological activity. This latter finding defines a critical role for this element in the reproductive cycle of the virus.

Base Sequence↗
PubMed2003

Translation enhancer in the 3'-untranslated region of rotavirus gene 6 mRNA promotes expression of the major capsid protein VP6.

The eleven rotavirus mRNAs contain 5'-cap structures and most end with the 3'-consensus sequence 5'-UGACC-3'. The UGACC functions as a common translation enhancer (3'-TE-con) that upregulates viral protein expression through a process mediated by the nonstructural protein NSP3. To address the possibility that gene-specific enhancers are also contained in the untranslated regions (UTRs) of the rotavirus mRNAs, we used rabbit reticulocyte lysates to investigate the translation efficiencies of analog RNAs containing viral-specific 5'-and 3'-UTRs and the open reading frame for chloramphenicol acetyltransferase. These experiments combined with the analysis of full-length viral RNAs and RNAs containing 3'-truncations showed that a highly active enhancer was present near the 5'-end of the 139-nucleotide 3'-UTR of the gene 6 mRNA (3'-TEg6). The 3'-TEg6 represents a functionally independent enhancer, as no other portion of the gene 6 mRNA was required for its activity. The 3'-TEg6 differs significantly from the 3'-TE-con in that the gene 6-specific enhancer does not require viral protein for activity and is formed by a sequence unique to only one of the eleven viral mRNAs. Together, our findings suggest that the 3'-UTR of the gene 6 mRNA contains two TEs, one is gene-specific (3'-TEg6) and the other is common to nearly all rotavirus genes (3'-TE-con). The activity of the 3'-TEg6 is likely important for directing the efficient translation of the gene 6 mRNA at levels sufficient to provide the 780 copies of VP6 necessary for the assembly of each progeny virion.

3' Untranslated Regions↗
PubMed2000

Current developments and future prospects for HIV gene therapy using interfering RNA-based strategies.

Acquired immunodeficiency syndrome (AIDS) is a slow, progressive, degenerative disease of the human immune system, ultimately leading to premature death of the patient. This disease is primarily caused by human immunodeficiency virus type-1 (HIV-1). The major targets of HIV infection are blood cells, namely lymphocytes and macrophages. While the immune response fails to eliminate the infected cells, the virus continues to spread. The purpose of HIV gene therapy is to provide "anti-HIV" genes to cells that are susceptible to HIV infection. Anti-HIV genes may be designed to express RNAs or proteins that interfere with the function of viral or cellular RNA(s)/protein(s), thereby inhibiting virus replication. Whereas interfering proteins may be cytotoxic and/or immunogenic, interfering RNAs are not. Interfering protein-based strategies requiring inducible gene expression (under the control of HIV regulatory proteins) can only be designed to block steps subsequent to the viral regulatory protein production. In contrast, interfering RNAs can be produced in a constitutive manner, which further enhances their antiviral activity and allows one to design strategies to inhibit virus replication before viral regulatory protein production occurs. Thus, interfering RNAs are of particular interest and are the focus of this review. Genes expressing interfering RNAs were designed to inhibit syncytium formation to prevent the death of the gene-modified cells. Strategies may also be developed to prevent gene-modified cells from becoming infected by HIV or from supporting HIV replication. Genes expressing interfering RNAs have been designed to inhibit HIV-1 entry and to cleave the incoming virion RNA, thus blocking virus replication before provirus DNA synthesis can be completed. A number of genes were also designed to express interfering RNAs that inhibit HIV replication at a post-integration step, by inhibiting the function of HIV RNAs or proteins produced in the infected cell. Also in development are anti-HIV genes that produce RNAs that would not only inhibit HIV replication in the gene-modified cell, but also prevent HIV RNA packaging and/or reverse transcription such that the progeny virus produced would be non-infectious. Further refinements to these strategies may lead to the development of "self-propagating" anti-HIV genes. These genes would express interfering RNAs that not only inhibit virus replication in the cell and prevent HIV RNA packaging and/or reverse transcription in the progeny virus, but also make use of the HIV itself to deliver the anti-HIV gene(s) to other cells. Thus, more and more cells susceptible to HIV infection would become resistant. Such "self-propagation" of anti-HIV-1 genes would only occur in cells that are susceptible to HIV infection, and would continue to take place for as long as HIV exists in the body.

Clinical Trials as Topic↗
PubMed1992

Association of AUUUA-binding protein with A+U-rich mRNA during nucleo-cytoplasmic transport.

Resealed nuclear envelope (NE) vesicles from rat liver containing entrapped exogenous RNA were used to study the effect of adenosine+uridine binding factor (AUBF), present in cytosolic cell extracts, on ATP-dependent transport of A+U-rich RNA (AU+RNA) and A+U-free RNA (AU-RNA) across the NE. This factor specifically binds to A+U-rich sequences present in the 3' untranslated regions of lymphokine and cytokine mRNAs, containing overlapping AUUUA boxes (granulocyte-macrophage colony stimulating factor, interleukin-3). Addition of AUBF to the extravesicular compartment markedly increased the efflux of the in vitro transcribed, capped and polyadenylated AU+ RNAs. Export of entrapped AU- control RNA, such as beta-globin RNA, was not affected by AUBF, in contrast to chimeric AU+ beta-globin RNA containing the A+U-rich sequence of human interferon-alpha mRNA (6 reiterated AUUUA motifs). Competition experiments revealed that AUBF enhances the affinity of poly(A)-containing AU+ RNAs to the NE poly(A)-binding component (poly(A)-recognizing mRNA carrier p106), and thereby accelerates nuclear export of these RNAs. We could demonstrate that AUBF added to the extravesicular space forms stable complexes with polyadenylated AU+ RNA with relative molecular masses of about 45,000, 62,000 and 70,000 inside the vesicles or during ATP-dependent export. In addition we determined that AUBF may affect mRNA stability by protecting A+U-rich RNA against degradation by trans-acting, nuclear matrix-associated and A+U-specific endoribonuclease V.

Adenosine↗
PubMed1996

Suppression of Alzheimer amyloid precursor protein (APP) expression by exogenous APP mRNA.

To establish a cell line expressing enhanced levels of beta-amyloid precursor protein (beta-APP), we constructed plasmid DNAs expressing beta-APP-751 mRNA and transfected them into COS-1 cells. Using a modified version of the reverse transcriptase polymerase chain reaction which is RNA sensitive to study the beta-APP iso-RNAs, we have made the unexpected observation that enhanced expression of beta-APP-751 mRNA resulted in a significant reduction of beta-APP-770 and -695 mRNA levels. Suppression of beta-APP-770 and -695 was also observed in cells expressing truncated and chimeric beta-App-751 mRNAs. Similar observations were made in P19 cells expressing a chimeric beta-APP-751 mRNA where endogenous beta-APP-751 mRNA levels also were decreased. Also, suppression of beta-APP-770 and -751 mRNAs was observed in human kidney cells expressing exogenous beta-APP-695 mRNA.

Amyloid beta-Protein Precursor↗
PubMed1996

Regulation of herpes simplex virus poly (A) site usage and the action of immediate-early protein IE63 in the early-late switch.

The essential herpes simplex virus type 1 (HSV-1) immediate-early IE63 (ICP27) is pleiotropic in function, promoting the switch from the early to late phase of virus gene expression, and has effects on the posttranscriptional processes of mRNA splicing and 3' processing. We have investigated the role of IE63 in the regulation of viral mRNA 3' processing and of late gene expression. Our in vitro 3' processing studies demonstrated that HSV-1 infection induces an activity, which requires IE63 gene expression, responsible for an observed increase in 3' processing of selected HSV-1 poly(A) sites. Processing efficiencies at the poly(A) sites of two late genes, UL38 and UL44, shown to be inherently weak processing sites, were increased by the IE63-induced activity. In contrast, 3' processing at the poly(A) sites of selected immediate-early and early genes, stronger processing sites, was unaffected by IE63 expression. UV cross-linking experiments demonstrated that HSV infection caused enhanced binding of protein factors, including the 64-kDa component of cleavage stimulation factor (CstF), to poly(A) site RNAs from virus genes of all temporal classes and that this enhanced binding required expression of IE63. By immunofluorescence, the homogeneous pattern of the 64-kDa CstF protein distribution became slightly clumped with infection, whereas the splicing small nuclear ribonucleoprotein particles were recognized into a highly punctate distribution away from the sites of virus transcription. This effect could create an increase in the relative concentration of 3' processing factors available to pre-mRNAs. Western blot (immunoblot) analysis showed that IE63 was required for expression of several true late genes and for the efficient and timely expression of the UL29 and UL42 early genes, integral components on the viral DNA synthesis machinery. Our data are consistent with two effects of IE63 on late gene regulation: firstly, a stimulation of pre-mRNA 3' processing and, secondly, as a requirement for expression of functions necessary for viral DNA synthesis.

Animals↗
PubMed1993

Methylphosphate cap structure increases the stability of 7SK, B2 and U6 small RNAs in Xenopus oocytes.

We studied the role of the methylphosphate cap structure in the stability and nucleocytoplasmic transport by microinjecting U6, 7SK and B2 RNAs into the Xenopus oocytes. In every case, the methylphosphate capped RNAs were 3 to 9 times more stable than the uncapped RNAs. When a methylphosphate cap structure was placed on human H1 RNA which is normally not capped, its stability was improved 2-7 fold. These data show that the methylphosphate cap enhances the stability of 7SK, B2, H1 and U6 RNAs. The methylphosphate-capped 7SK RNA was transported into the nucleus from cytoplasm, but remained in the nucleus when injected into the nucleus; in this respect, 7SK RNA exhibited properties previously shown for U6 RNA. Both U6 and 7SK RNAs with ppp on their 5' ends were transported from cytoplasm to the nucleus suggesting that the methylphosphate cap structure is not required for transport of these RNAs across the nuclear membrane.

Animals↗
PubMed1989

Multiple inducers of the Drosophila heat shock locus 93D (hsr omega): inducer-specific patterns of the three transcripts.

The Drosophila hsr omega locus produces one of the largest and most active heat shock puffs, yet it does not encode a heat shock protein. Instead, this locus produces a distinctive set of three transcripts, all from the same start site. The largest transcript, omega 1, is limited to the nucleus and appears to have a role there. A second nuclear transcript, omega 2, is produced by alternative termination and contains the sequence found in the 5' 20-25% of omega 1 (depending on the Drosophila species). The cytoplasmic transcript, omega 3, is produced by removal of a 700-bp intron from omega 2. All three hsr omega RNAs are produced constitutively and production is enhanced by heat shock. In addition to being a member of the set of heat shock puffs, the hsr omega puff is induced by agents that do not affect other heat shock loci, suggesting that hsr omega is more sensitive to environmental changes than other loci. We report here that agents that induce puffing of hsr omega loci in polytene nuclei also lead to an increase in hsr omega transcripts in diploid cells. We also show that the relative levels of omega 1 and omega 3 can be modulated independently by several agents. All drugs that inhibit translation, either initiation or elongation, stabilize the omega 3 transcript, which normally turns over within minutes in control cells. Drugs (such as benzamide and colchicine) that induce puffing of hsr omega, but not other heat shock loci, lead to large increases in omega 1. Although the constitutive level of omega 1 is relatively stable, the drug-induced excess is lost rapidly when the drug is withdrawn. The relative levels of hsr omega transcripts may reflect different states in cellular metabolism.

Animals↗
PubMed2004

Changes of the secondary structure of the 5' end of the Sindbis virus genome inhibit virus growth in mosquito cells and lead to accumulation of adaptive mutations.

Both the 5' end of the Sindbis virus (SIN) genome and its complement in the 3' end of the minus-strand RNA synthesized during virus replication serve as parts of the promoters recognized by the enzymes that comprise the replication complex (RdRp). In addition to the 5' untranslated region (UTR), which was shown to be critical for the initiation of replication, another 5' sequence element, the 51-nucleotide (nt) conserved sequence element (CSE), was postulated to be important for virus replication. It is located in the nsP1-encoding sequence and is highly conserved among all members of the Alphavirus genus. Studies with viruses containing clustered mutations in this sequence demonstrated that this RNA element is dispensable for SIN replication in cells of vertebrate origin, but its integrity can enhance the replication of SIN-specific RNAs. However, we showed that the same mutations had a deleterious effect on virus replication in mosquito cells. SIN with a mutated 51-nt CSE rapidly accumulated adaptive mutations in the nonstructural proteins nsP2 and nsP3 and the 5' UTR. These mutations functioned synergistically in a cell-specific manner and had a stimulatory effect only on the replication of viruses with a mutated 51-nt CSE. Taken together, the results suggest the complex nature of interactions between nsP2, nsP3, the 5' UTR, and host-specific protein factors binding to the 51-nt CSE and involved in RdRp formation. The data also demonstrate an outstanding potential of alphaviruses for adaptation. Within one passage, SIN can adapt to replication in cells of a vertebrate or invertebrate origin.

5' Untranslated Regions↗
PubMed1998

Tumor necrosis factor-alpha causes insulin receptor substrate-2-mediated insulin resistance and inhibits insulin-induced adipogenesis in fetal brown adipocytes.

Treatment of fetal brown adipocytes with 0.6 nM tumor necrosis factor (TNF)-alpha for 24 h resulted in a partial impairment in the expression of fatty acid synthase, glycerol-3-phosphate dehydrogenase, and glucose transporter (GLUT)-4 messenger RNAs (mRNAs), as well as in the enhancement in the cytoplasmic lipid content in response to insulin. However, the expression of the tissue-specific gene, uncoupling protein 1, is increased by the presence of TNF-alpha. The antiadipogenic effect of TNF-alpha was accompanied by a down-regulation of CCAAT/enhancer-binding protein-alpha and beta mRNAs and up-regulation of CCAAT/enhancer-binding protein-delta, with the expression of peroxisome proliferator-activated receptor-gamma remaining essentially unmodified. Moreover, TNF-alpha caused an insulin resistance on the insulin-induced glucose uptake in brown adipocytes. Pretreatment with TNF-alpha resulted in hypophosphorylation of the insulin receptor in response to insulin, without affecting the number of insulin receptors per cell or its molecular mass. However, insulin receptor substrate (IRS)-1 and IRS-2 signaling in response to insulin showed functional differences. Thus, TNF-alpha pretreatment induced a hypophosphorylation of IRS-2 but not of IRS-1. This effect leads to an impairment in the IRS-2-associated phosphatidylinositol (PI) 3-kinase activation due to a decreased association of alpha-p85 regulatory subunit of PI 3-kinase with IRS-2 but not in the IRS-1-associated PI 3-kinase activation in response to insulin. Our results indicate that TNF-alpha induced an IRS-2- but not IRS-1-mediated insulin resistance on glucose transport and lipid synthesis in fetal brown adipocytes.

Adipocytes↗
PubMed2000

Interactive stimulation by luteinizing hormone and insulin of the steroidogenic acute regulatory (StAR) protein and 17alpha-hydroxylase/17,20-lyase (CYP17) genes in porcine theca cells.

LH and insulin are postulated to jointly stimulate theca-cell androgen biosynthesis in patients with hyperthecosis or polycystic ovarian syndrome. To explore the mechanisms of putative LH and insulin steroidogenic synergy in primary culture of normal theca cells, we have implemented an in vitro serum-free monolayer culture system of Percoll-purified, porcine theca cells harvested from immature ovaries. Initial dose and time course analyses revealed that a maximally effective concentration of LH (100 ng/ml) or insulin (100 ng/ml) individually will drive androstenedione production (at 6 to 48 h) by 1.5- to 2.6- and 1.1- to 1.7-fold, respectively, while combined agonists act synergistically over the interval 12 to 48 h yielding a 3- to 4-fold joint effect. Coadministration of LH and insulin can augment theca-cell concentrations of CYP17 and StAR messenger RNA (mRNA) resulting in 3.4- to 3.9- and 3.8- to 4.1-fold increases at 24 to 48 h, respectively (P < 0.01). Combined LH and insulin stimulation also amplified the nuclear content of intron-specific heterogeneous nuclear (hn)RNAs encoding CYP17 and StAR. Insulin significantly enhanced LH-driven but not basal cAMP accumulation (14-18 vs. 3-5.5 pmol/microg DNA/12-48 h) (P < 0.01). A stable exogenous analog of cAMP, 8 Br-cAMP, mimicked LH's effect on steroidogenesis and StAR and CYP17 gene expression and with insulin stimulated StAR mRNA and hnRNA accumulation synergistically. However, unlike LH, 8 Br-cAMP did not synergize with insulin on theca-cell androstenedione biosynthesis or CYP17 mRNA and hnRNA expression. In summary, the present in vitro data identify molecular interactions of LH and insulin on StAR and CYP17 gene expression, thus establishing potent signaling interfaces between these distinct hormonal agonists in regulating theca-cell steroidogenesis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2002

Involvement of phosphodiesterase isozymes in osteoblastic differentiation.

The cyclic monophosphate nucleotides (cyclic adenosine monophosphate [cAMP] and cyclic guanosine monophosphate [cGMP]) are found ubiquitously in mammalian cells and act as second messenger transducers to effect the intracellular actions of a variety of hormones, cytokines, and neurotransmitters. In turn, these nucleotides also modulate the signal transduction processes regulated by a range of cytokines and growth factors. Previously, we have reported that pentoxifylline, a nonselective phosphodiesterase (PDE) inhibitor, can promote osteoblastic differentiation by elevating intracellular cAMP levels and, consequently, enhance bone formation in vivo and in vitro. In this study, reverse-transcription polymerase chain reaction (RT-PCR) analysis of the osteoblastic cell lines, MC3T3-E1 and ST2 revealed the presence of PDE1, PDE2, PDE3, PDE4, PDE7, PDE8, and PDE9. We examined the effect of selective inhibitors for a respective PDE isozyme on the capacity of bone morphogenetic protein 4 (BMP-4)-induced alkaline phosphatase (ALP) activity, a cellular differentiation marker, in cells with osteogenetic potential. The results indicate that selective inhibitors for PDE2, PDE3, and PDE4 enhanced the BMP-4-induced ALP activity in a dose-dependent manner in ST2 cells but not in MC3T3-E1 cells. Northern blot analysis also revealed that the selective inhibitors for PDE2, PDE3, and PDE4 enhanced the levels of expression of messenger RNAs (mRNAs) of ALP, osteopontin (OP), and collagen type I in ST2 cells but not in MC3T3-E1 cells except for the treatment with PDE4 inhibitor. Given these data, we conclude that PDE isozymes are involved in the modulation of osteoblastic differentiation mainly at an early stage. Additionally, selective inhibitors for PDE2, PDE3, and PDE4 appear to promote the differentiation of osteogenic precursor cells toward an osteoblastic phenotype.

Alkaline Phosphatase↗