Primary Transcript Levels Research Articles

Antagonistic Effects of Cellular Poly(C) Binding Proteins on Vesicular Stomatitis Virus Gene Expression

Immunoprecipitation and subsequent mass spectrometry analysis of the cellular proteins from cells expressing the vesicular stomatitis virus (VSV) P protein identified the poly(C) binding protein 2 (PCBP2) as one of the P protein-interacting proteins. To investigate the role of PCBP2 in the viral life cycle, we examined the effects of depletion or overexpression of this protein on VSV growth. Small interfering RNA-mediated silencing of PCBP2 promoted VSV replication. Conversely, overexpression of PCBP2 in transfected cells suppressed VSV growth. Further studies revealed that PCBP2 negatively regulates overall viral mRNA accumulation and subsequent genome replication. Coimmunoprecipitation and immunofluorescence microscopic studies showed that PCBP2 interacts and colocalizes with VSV P protein in virus-infected cells. The P-PCBP2 interaction did not result in reduced levels of protein complex formation with the viral N and L proteins, nor did it induce degradation of the P protein. In addition, PCBP1, another member of the poly(C) binding protein family with homology to PCBP2, was also found to interact with the P protein and inhibit the viral mRNA synthesis at the level of primary transcription without affecting secondary transcription or genome replication. The inhibitory effects of PCBP1 on VSV replication were less pronounced than those of PCBP2. Overall, the results presented here suggest that cellular PCBP2 and PCBP1 antagonize VSV growth by affecting viral gene expression and highlight the importance of these two cellular proteins in restricting virus infections.

The obesity-associated SNPs in intron 1 of the FTO gene affect primary transcript levels

As shown by genome-wide association studies single-nucleotide polymorphisms (SNPs) within intron 1 of the FTO gene are associated with the body mass index and type II diabetes, although the functional significance of these SNPs has remained unclear. Using primer extension assays, we have determined the ratio of allelic FTO transcript levels in unspliced heterogeneous nuclear RNA preparations from blood of individuals heterozygous for SNP rs9939609. Allelic expression ratios of the neighboring RPGRIP1L gene were investigated in individuals who were heterozygous for SNP rs4784319 and heterozygous or homozygous for rs9939609. In each of five individuals, the FTO transcripts containing the A (risk) allele of rs9939609 were more abundant than those with T allele (mean 1.38; 95% confidence interval 1.31-1.44). Similar results were obtained in a fibroblast sample. We also observed skewed allelic expression of the RPGRIP1L gene in blood, but skewing was independent of the FTO genotype. Our data suggest that increased expression of FTO is associated with increased body mass.

Abstract 4085: Bidirectional noncoding RNAs mediate rRNA processing and cell death in human lung adenocarcinoma cells

Abstract Increase in ribosome biogenesis is a characteristic of proliferating cells and may be causative in tumorigenesis. Significant increases in basal levels of several primary precursor transcripts during ribosomal RNA (rRNA) processing and of a cis antisense noncoding rRNA (AS-ncrRNA) near the main transcription start site were detected in human lung adenocarcinoma cells (H441 and A549) compared with non-transformed peripheral lung epithelial cells (HPL). A cis sense noncoding rRNA (S-ncrRNA) was also discovered but its levels showed cell-specific changes. It was hypothesized that a high level of the AS-ncrRNA is required to maintain the tumor phenotype. The functions of the AS-ncrRNA and S-ncrRNA were assessed using complementary oligonucleotides, termed −86S and −84AS (negative numbers correspond to the target locations in reference to the primary transcription start site) and intended for inhibiting the above targets, respectively. The levels of precursor rRNAs and both ncrRNAs in cells transfected with the −86S and −84AS oligonucleotides were measured using reverse transcription and quantitative polymerase chain reaction. Cell phenotypes and apoptotic cell death were also examined. A two-hit treatment with −86S 24 hrs apart induced cell detachment predominantly in H441 and A549 cancer cells. Detached cancer cells showed several hundred- to a thousand-fold upregulation of S-ncrRNA over the vehicle-treated controls while the S-nrcRNA in rare floating HPL cells was only increased by about 20 fold. Precursor rRNAs and AS-ncrRNA were also upregulated but to a lesser extent after −86S treatment in both adherent and detached cells. For the −84AS treatment, detached cells were fewer than after the −86S treatment; however those few floating cancer cells also exhibited several hundred-fold increase of the S-ncrRNA. Although the mechanism of upregulation is unclear, the S-ncrRNA may possibly act as a cell death regulator. Flow cytometric assays to detect fragmented DNA in cells after 24-hr and 48-hr treatments showed an apparent increase in apoptosis (2- to 4-fold) for the −86S treatment compared to −84AS and scrambled control. Further morphological analysis by transmission electron microscopy identified large and abundant autophagosomes and vacuoles preferentially in cancer cells, suggesting another possible cell death mechanism mediated by the oligonucleotide targeting AS-ncrRNA. In sum, rRNA processing and cis bidirectional noncoding rRNAs may form an interconnected network to maintain tumor phenotype. Interruption of this network by oligonucleotides targeting AS-ncrRNA activates massive elevation of the S-ncRNA level and sensitizes cancer cells preferentially for apoptotic and autophagic cell death. Targeting of AS-ncrRNA could have potential for human lung cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4085.

Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells

Most transcripts in growing cells are ribosomal RNA precursors (pre-rRNA). Here, we show that in mammals, aberrant pre-rRNA transcripts generated by RNA polymerase I (Pol I) are polyadenylated and accumulate markedly after treatment with low concentrations of actinomycin D (ActD), which blocks the synthesis of full-length rRNA. The poly(A) polymerase-associated domain-containing protein 5 is required for polyadenylation, whereas the exosome is partly responsible for the degradation of the short aberrant transcripts. Thus, polyadenylation functions in the quality control of Pol I transcription in metazoan cells. The impact of excessive aberrant RNAs on the degradation machinery is an unrecognized mechanism that might contribute to biological properties of ActD.

The Modulation of MicroRNAs by Type I IFN through the Activation of Signal Transducers and Activators of Transcription 3 in Human Glioma

Type I IFNs are involved in double-stranded RNA responses. Here, we investigated the possibility that IFN-beta may induce or downregulate cellular microRNAs (miRNA) in human neoplasms and thereby use the RNA interference system to show antitumor effects. Because of its known connection to glioma biology, we focused on miR-21 among seven miRNAs influenced by IFN-beta. We analyzed the effect of IFN-beta treatment on miR-21 expression in glioma cells and intracranial glioma xenografts. IFN-beta treatment reduced miR-21 expression in glioma cells markedly, and IFN-beta administration suppressed the growth of glioma-initiating cell-derived intracranial tumors. The levels of primary miR-21 gene transcripts, precursor miR-21, and mature miR-21 decreased 6 hours after the addition of IFN-beta, indicating that the reduction in miR-21 levels was due to transcriptional suppression. We did reporter assays to elucidate the IFN-beta-mediated suppression of miR-21; the addition of signal transducers and activators of transcription 3 (STAT3)-expressing vectors induced the IFN-beta-mediated suppression of miR-21, whereas STAT3-inhibiting agents inhibited the miR-21 suppression. Thus, the results of our study show that the downregulation of miR-21 contributes to the antitumor effects of IFN-beta and that miR-21 expression is negatively regulated by STAT3 activation. These results highlight the importance of understanding the transcriptional regulation of the miRNAs involved in oncogenesis.

Rapid muscle atrophy response to unloading: pretranslational processes involving MHC and actin

Skeletal muscles, especially weight-bearing muscles, are very sensitive to changes in loading state. The aim of this paper was to characterize the dynamic changes in the unloaded soleus muscle in vivo following a short bout of hindlimb suspension (HS), testing the hypothesis that transcriptional events respond early to the atrophic stimulus. In fact, we observed that after only 1 day of HS, primary transcript levels of skeletal alpha-actin and type I myosin heavy chain (MHC) genes were significantly reduced by more than 50% compared with ground control levels. The degree of the decline for the mRNA expression of actin and type I MHC lagged behind that of the pre-mRNA levels after 1 day of HS, but by 2 and 7 days of HS, large decreases were observed. Although the faster MHC isoforms, IIx and IIb, began to be expressed in soleus after 1 day of HS, a relatively significant shift in mRNA expression from the slow MHC isoform type I toward these fast MHC isoforms did not emerge until 7 days of HS. One day of HS was sufficient to show significant decreases in mRNA levels of putative signaling factors serum response factor (SRF), suppressor of cytokine signaling-3 (SOCS3), and striated muscle activator of Rho signaling (STARS), although transcription factors yin-yang-1 (YY1) and transcriptional enhancing factor-1 (TEF-1) were not significantly affected by HS. The protein levels of actin and type I MHC were significantly decreased after 2 days of HS, and SRF protein was significantly decreased after 7 days HS. Our results show that after only 1 day of unloading, pre-mRNA and mRNA expression of muscle proteins and muscle-specific signaling factors are significantly reduced, suggesting that the downregulation of the synthesis side of the protein balance equation that occurs in atrophying muscle is initiated rapidly.

Upregulated INHBA Expression May Promote Cell Proliferation and Is Associated with Poor Survival in Lung Adenocarcinoma

The expression, mechanisms of regulation, and functional impact of INHBA (activin A) in lung adenocarcinoma (AD) have not been fully elucidated. INHBA expression was examined in 96 lung samples (86 ADs, 10 normal lung) using oligonucleotide microarrays and 187 lung samples (164 ADs, 6 bronchioalveolar carcinomas, and 17 normal lung) using immunohistochemistry. The proliferation of AD cell lines H460 and SKLU1 was examined with WST-1 assays after treatment with recombinant activin A, follistatin, and INHBA-targeting small-interfering RNA. Cells were also treated with 5-aza-2' deoxycytidine and trichostatin A to investigate the role of epigenetic regulation in INHBA expression. Primary ADs expressed 3.1 times more INHBA mRNA than normal lung. In stage I AD patients, high levels of primary tumor INHBA transcripts were associated with worse prognosis. Immunohistochemistry confirmed higher inhibin betaA protein expression in ADs (78.7%) and bronchioalveolar carcinomas (66.7%) compared with normal lung (11.8%). H460 and SKLU1 demonstrated increased proliferation when treated with exogenous activin A and reduced proliferation when treated with follistatin or INHBA-targeting small-interfering RNA. INHBA mRNA expression in H460 cells was upregulated after treatment with trichostatin A and 5-aza-2' deoxycytidine. INHBA is overexpressed in AD relative to controls. Inhibin betaA may promote cell proliferation, and its overexpression is associated with worse survival in stage I AD patients. In addition, overexpression of INHBA may be affected by promoter methylation and histone acetylation in a subset of lung ADs.

Transcriptional and translational dynamics of light neurofilament subunit RNAs during Xenopus laevis optic nerve regeneration

Neurofilaments (NFs), which comprise one of three cytoskeletal polymers of vertebrate axons, are heteropolymers of multiple NF subunit proteins. During Xenopus laevis optic nerve regeneration, NF subunit composition undergoes progressive changes that correlate with regenerative success. Understanding the relative contributions of transcriptional and post-transcriptional gene regulatory mechanisms to these changes should therefore provide insights into the control of the axonal growth program. Previously, we examined this issue with respect to the medium neurofilament protein (NF-M). Because the integrity of NF heteropolymers depends upon maintaining properly balanced expression among multiple subunits, we have now extended this analysis to include the four light NF subunits — peripherin, the light NF triplet protein (NF-L), and two additional α-internexin-like proteins. Within 3 days after an optic nerve crush injury to one eye, primary transcript levels of NF subunits increased in both eyes. Levels of mRNA, however, increased in only the operated eye and did so later than did increases in primary transcript, indicating that mRNA levels are under significant post-transcriptional regulation. As measured by polysome profiling, the translational efficiencies of individual NF subunit mRNAs also shifted throughout regeneration, with operated eye mRNAs being generally more translationally active than those of unoperated eyes. Also, in operated eyes, the precise mix of efficiently and poorly translated messages throughout regeneration varied independently for each subunit, indicating that their translations are fine-tuned separately. These results suggest a model whereby traumatic disruption of visual circuitry leads to increased expression of NF primary transcripts in both eyes. These increases are subsequently modulated post-transcriptionally to accommodate shifting demands at each phase of regeneration for NF heteropolymers of differing composition in regrowing axons.

A Novel Splicing Variant of Mouse Interleukin (IL)-24 Antagonizes IL-24-induced Apoptosis

Alternative splicing of mRNA enables functionally diverse protein isoforms to be expressed from a single gene, allowing transcriptome diversification. Interleukin (IL)-24/MDA-7 is a member of the IL-10 gene family, and FISP (IL-4-induced secreted protein), its murine homologue, is selectively expressed and secreted by T helper 2 lymphocytes. A novel splice variant of mouse IL-24/FISP, designated FISP-sp, lacks 29 nucleotides from the 5'-end of exon 4 of FISP. The level of FISP-sp expression is 10% of the level of total primary FISP transcription. Unlike FISP, FISP-sp does not induce growth inhibition and apoptosis. FISP-sp is exclusively localized in endoplasmic reticulum, and its expression is up-regulated by endoplasmic reticulum stress. Our results suggest that the novel splicing variant FISP-sp dimerizes with FISP and blocks its secretion and inhibits FISP-induced apoptosis in vivo.

Identification and Characterization of microRNAs modulated by Oxidative Stress

Reactive oxygen species (ROS) play important roles in regulating cellular functions. Some growth factors, such as PDGF and EGF, have been discovered to signal through ROS and activate the MAP kinase cascades to regulate the expression of transcription factors. Several laboratories have found that ROS could directly regulate the binding of transcription factors, like AP‐1, NF‐κB, P21 and P53, to DNA and thus influence the downstream gene expression. MicroRNAs (miRNAs) are a class of regulatory RNAs of approximately 21∼25 nucleotides that posttranscriptionally regulate gene expression by directing mRNA cleavage or translational inhibition. The relationship between oxidative stress and miRNAs has not been clearly defined. Toward this end, we used colo205 as a cell model to explore the effect of oxidative stress on miRNA expression. Cells were exposed to 200 μM H2O2 for 1hr and the expression levels of 225 human miRNAs were determined by quantitative RT‐PCR. H2O2 treatment caused a 5‐fold increase in the level of has‐miR‐145. Similar increase was detected in the level of primary transcript of miR‐145 (pri‐miR‐145). These results indicate that the transcription level of miR‐145 is stimulated by ROS in Colo205. The biological significance of miR‐145 up‐regulation is currently under investigation.

Coordinated expression of microRNA-155 and predicted target genes in diffuse large B-cell lymphoma

MicroRNAs (miRNAs) attenuate gene expression by pairing to the 3′UTR of target transcripts inducing RNA cleavage or translational inhibition. Overexpression of microRNA-155 (miR-155), measured either at the primary ( BIC gene) or mature transcript level, was recently described in diffuse large B-cell lymphomas (DLBCL). These studies have been limited in size, however, and have not attempted to link miR-155 expression to that of putative target genes. To start to address these issues, we examined a collection of 22 well-characterized DLBCL cell lines. The expression of miR-155 is heterogeneous in these cell lines and associates with NF-κB activity. We found that the expression of the primary miR-155 transcript reliably reflects that of the functional mature miR-155. Because many gene array platforms include probe sets for the primary miR-155 sequences, these findings allowed us to confidently examine large array-based expression datasets of primary DLBCLs in the context of miR-155 levels. Our investigation revealed that miR-155 expression segregates with specific molecular subgroups of DLBCL and it is highest in activated B-cell (ABC)-type lymphomas. These tumors are characterized by constitutive activation of NF-κB signals, which supports the data derived from our cell lines. More importantly, using supervised learning algorithms, we identified a robust gene signature driven by the differential expression of miR-155. These profiles contained several gene markers, including predicted targets, consistently downregulated in tumors expressing high levels of miR-155. Our data start to unveil the genome-wide effects of miR-155 expression in DLBCL and indicate the utility of this strategy in the identification and validation of miRNA target genes.

Development of a Dual-Luciferase Reporter System for In Vivo Visualization of MicroRNA Biogenesis and Posttranscriptional Regulation

MicroRNAs (miRNAs) function in mammalian cells via translational repression or messenger RNA (mRNA) cleavage of target genes by base-pairing with 3' untranslated regions (UTRs) of target mRNAs. Although miRNAs are involved in cell differentiation or organ development, posttranscritptional regulation of miRNA is not well understood. Here, we developed a dual-luciferase reporter system for monitoring in vivo endogenous transcription of primary miRNA (pri-miRNA) and also the mature miRNA activity simultaneously. miR23P639/Fluc plasmid carrying firefly luciferase (Fluc) under the control of miR-23a promoter was used to monitor the transcriptional level of miR-23a, and a cytomegalovirus (CMV)/Gluc/3xPT_mir23 recombinant containing 3 copies of the target sequence of miR-23a in the 3' UTR of Gaussia luciferase (Gluc) before the poly(A) tail was used to monitor the targeting activity of mature miR-23a. This dual-luciferase reporter system transfected to the same population of cells was used to monitor the increased transcriptional level of the pri-miR-23a reflected in the Fluc activity and the decreased Gluc activity affected by mature miR-23a action. Fluc and Gluc activities were also imaged in vivo using the respective substrates in grafted cells in the same nude mice using an in vivo bioluminescence imager. In HeLa cells and undifferentiated P19 cells, the increased Fluc activity representing the primary miR-23a transcript level reflected the resultant increase in repression of Gluc activity representing mature miR-23a activity. However, 293 cells showed Gluc activity was not repressed as much as Fluc activity was increased, suggesting a block in the posttranscriptional processing of miR-23a transcript in 293 cells. The miR-23a expression in P19 cells before and after neuronal differentiation with retinoic acid treatment showed an increase in Fluc activity and a concomitant decrease in Gluc activity in vitro. HeLa, 293 cells and undifferentiated P19 cells grafted to the nude mice showed exactly the same pattern of luciferase activities in vivo and in vitro. We developed a dual-luciferase reporter system to monitor expression and posttranscriptional regulation of a miR-23a in cells in vitro and in vivo. This dual-luciferase reporter system is intended to be used to monitor the expression and regulation of miRNAs noninvasively, especially to understand the differentiation of grafted cells in vivo.

Pulsatile Gonadotropin-Releasing Hormone Stimulation of Gonadotropin Subunit Transcription in Rat Pituitaries: Evidence for the Involvement of Jun N-Terminal Kinase But Not p38

We investigated whether Jun N-terminal kinase (JNK) and p38 mediate gonadotropin subunit transcriptional responses to pulsatile GnRH in normal rat pituitaries. A single pulse of GnRH or vehicle was given to female rats in vivo, pituitaries collected, and phosphorylated JNK and p38 measured. GnRH stimulated an increase in JNK phosphorylation within 5 min, which peaked 15 min after GnRH (3-fold). GnRH also increased p38 phosphorylation 2.3-fold 15 min after stimulus. Rat pituitary cells were given 60-min pulses of GnRH or media plus the JNK inhibitor SP600125 (SP, 20 microM), p38 inhibitor SB203580 (20 microM), or vehicle. In vehicle-treated groups, GnRH pulses increased LHbeta and FSHbeta primary transcript (PT) levels 3-fold. SP suppressed both basal and GnRH-induced increases in FSHbeta PT by half, but the magnitude of responses to GnRH was unchanged. In contrast, SP had no effect on basal LHbeta PT but suppressed the stimulatory response to GnRH. SB203580 had no effect on the actions of GnRH on either LH or FSHbeta PTs. Lbeta-T2 cells were transfected with dominant/negative expression vectors for MAPK kinase (MKK)-4 and/or MKK-7 plus a rat LHbeta promoter-luciferase construct. GnRH stimulated a 50-fold increase in LHbeta promoter activity, and the combination of MKK-4 and -7 dominant/negatives suppressed the response by 80%. Thus, JNK (but not p38) regulates both LHbeta and FSHbeta transcription in a differential manner. For LHbeta, JNK is essential in mediating responses to pulsatile GnRH. JNK also regulates FSHbeta transcription (i.e. maintaining basal expression) but does not play a role in responses to GnRH.

Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription

We have previously demonstrated that the expression of human ribosomal RNA genes (rDNA) in normal and cancer cells is differentially regulated by methylation of the promoter CpG islands. Furthermore, we showed that the methyl CpG-binding protein MBD2 plays a selective role in the methylation-mediated block in rDNA expression. Here, we analyzed the role of three functional mammalian DNA methyltransferases (DNMTs) in regulating the rDNA promoters activity. Immunofluorescence analysis and biochemical fractionation showed that all three DNMTs (DNMT1, DNMT3A, and DNMT3B) are associated with the inactive rDNA in the nucleolus. Although DNMTs associate with both methylated and unmethylated rDNA promoters, DNMT1 preferentially associates with the methylated genes. The rDNA primary transcript level was significantly elevated in DNMT1-/- or DNMT3B-/- human colon carcinoma (HCT116) cells. Southern blot analysis demonstrated a moderate level of rDNA promoter hypomethylation in DNMT1-/- cells and a dramatic loss of rDNA promoter methylation in double knockout cells. Transient overexpression of DNMT1 or DNMT3B suppressed the luciferase expression from both methylated and unmethylated pHrD-IRES-Luc, a reporter plasmid where the rDNA promoter drives luciferase expression. DNMT1-mediated suppression of the unmethylated promoter involves de novo methylation of the promoter, whereas histone deacetylase 2 cooperates with DNMT1 to inhibit the methylated rDNA promoter. Unlike DNMT1, both the wild type and catalytically inactive DNMT3B mutant can suppress rDNA promoter irrespective of its methylation status. DNMT3B-mediated suppression of the rDNA promoter also involves histone deacetylation. Treatment of HCT116 cells with Decitabine (a DNMT inhibitor) or trichostatin A (a histone deacetylase inhibitor) up-regulated endogenous rDNA expression. These inhibitors synergistically activated methylated pHrD-IRES-Luc, whereas they exhibited additive effects on the unmethylated promoter. These results demonstrate localization of DNMTs with the inactive rDNA in the nucleolus, the specific role of DNMT1 and DNMT3B in rDNA expression and the differential regulation of rDNA expression from the methylated and unmethylated rDNA promoters.

Paracrine regulation of growth hormone gene expression by gonadotrophin release in grass carp pituitary cells: functional implications, molecular mechanisms and signal transduction

Growth hormone (GH) is known to stimulate luteinizing hormone (LH) release via paracrine interactions between somatotrophs and gonadotrophs. However, it is unclear if LH can exert a reciprocal effect to modulate somatotroph functions. Here we examined the paracrine effects of LH on GH gene expression using grass carp pituitary cells as a cell model. LH receptors were identified in grass carp somatotrophs and their activation by human chorionic gonadotropin (hCG) increased 'steady-state' GH mRNA levels. Removal of endogenous LH by immunoneutralization using LH antiserum inhibited GH release and GH mRNA expression. GH secretagogues, including gonadotrophin releasing hormone (GnRH), pituitary adenylate cyclase-activating polypeptide (PACAP) and apomorphine, were effective in elevating GH mRNA levels but these stimulatory actions were blocked by LH antiserum. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was not affected by hCG but was enhanced by LH immunoneutralization. Treatment with LH antiserum also suppressed basal levels of mature GH mRNA and primary transcripts. hCG increased cAMP synthesis in carp pituitary cells and hCG-induced GH mRNA expression was mimicked by forskolin but suppressed by inhibiting adenylate cyclase and protein kinase A. Similarly, the stimulatory actions of hCG and forskolin on GH mRNA expression were blocked by inhibiting Janus kinase 2 (JAK2) and MAP kinase (MAPK), including P42/44(MAPK) and P38 (MAPK). These results suggest that LH is essential for the maintenance of GH release, GH gene expression, and somatotroph responsiveness to GH-releasing factors. The paracrine actions of LH on GH mRNA expression are mediated by a concurrent increase in GH gene transcription and GH mRNA turnover, probably through JAK2/MAPK coupled to the cAMP-dependent pathway.

Chronic nicotine treatment leads to induction of tyrosine hydroxylase in locus ceruleus neurons: the role of transcriptional activation.

Chronic nicotine treatment (two daily subcutaneous injections administered approximately 12 h apart for 14 days) is associated with long-term inductions of tyrosine hydroxylase (TH) protein and TH mRNA in locus ceruleus (LC) neurons. These increases persist for at least 3 days after the final nicotine injection in LC cell bodies and for at least 7 to 10 days in LC nerve terminal regions. We tested whether this long-term response is due to sustained stimulation of TH gene transcription rate. A semiquantitative reverse transcription-polymerase chain reaction assay was developed to assess changes in the levels of TH RNA primary transcripts; these changes are an indirect measurement of changes in TH gene transcription rate. TH RNA primary transcript levels increase rapidly in the LC after a single nicotine administration and return to basal levels by 24 h. A similar rapid and transient induction of LC TH RNA primary transcripts occurs after chronic nicotine administration. In contrast, TH RNA primary transcript levels remain elevated for a sustained period of time (at least 1 day) in the adrenal medulla after chronic nicotine administration. Similar rapid, but transient changes in LC TH RNA primary transcript levels are observed after repeated immobilization stress. These results suggest that TH gene transcription rate in the LC is stimulated rapidly after each nicotine injection; however, in contrast to the adrenal medulla, there is no sustained transcriptional response elicited by chronic nicotine treatment or repeated immobilization stress in the LC, suggesting that post-transcriptional mechanisms may also play a role in these long-term responses.

Site of action of acute alcohol administration in stimulating the rat hypothalamic-pituitary-adrenal axis: comparison between the effect of systemic and intracerebroventricular injection of this drug on pituitary and hypothalamic responses.

The peripheral injection of alcohol stimulates the activity of the hypothalamic-pituitary-adrenal (HPA) axis, but the ready penetration of this drug in most bodily compartments has made it difficult to identify its specific sites of action. Here we determined whether alcohol can directly influence the corticotropes. We first determined whether alcohol acted within the brain to stimulate neurons in the paraventricular nucleus (PVN) of the hypothalamus, which synthesizes corticotropin-releasing factor (CRF) and vasopressin (VP). To test this hypothesis, we injected alcohol intracerebroventricularly (icv; 5 microl of 200-proof; 86 micromol) and compared these results with those obtained after its ip administration (3.0 g/kg). Although not causing neuronal damage and not leading to detectable levels of the drug in the general circulation, icv alcohol significantly up-regulated PVN CRF heteronuclear RNA levels and increased plasma ACTH levels, a change comparable to the one observed in the ip model. To determine whether alcohol stimulated the corticotropes independently of CRF and/or VP, we injected the drug ip or icv and measured changes in anterior pituitary proopiomelanocortin (POMC) transcripts and ACTH release in the presence or absence of endogenous CRF and/or VP. Intracerebroventricular and ip alcohol significantly increased POMC primary transcript levels, measured by ribonuclease protection assay, over a time-course that corresponded to ACTH release. Both the POMC and the ACTH responses were completely abolished by removal of CRF and VP. Collectively, these results indicate that alcohol-induced activation of the corticotropes does not represent a direct influence of the drug on the pituitary but requires CRF and VP.

Differential effects of forced swim-stress on the corticotropin-releasing hormone and vasopressin gene transcription in the parvocellular division of the paraventricular nucleus of rat hypothalamus

Corticotropin-releasing hormone (CRH) and vasopressin (AVP) colocalize in the parvocellular division of the paraventricular nucleus of the hypothalamus (PVN). We examined the effect of forced swim-stress on the CRH and AVP primary transcript (hnRNA) levels in the rat PVN by semi-quantitative in situ hybridization. CRH hnRNA increased markedly following 10-min swim-stress and returned to the basal level by 2 h. AVP hnRNA in the parvocellular division of the PVN, where AVP colocalizes with CRH, did not change significantly immediately after the swim-stress, but it did increase significantly 2 h after the stress. Pretreatment with dexamethasone abolished the increases in CRH and AVP hnRNA levels after the swim-stress. The present results demonstrate the differential effects of forced swim-stress on the CRH and AVP gene transcription in the parvocellular PVN, confirming the diverse response of the dual peptide-containing system in the face of acute stressful events.

Both SMAD2 and SMAD3 mediate activin-stimulated expression of the follicle-stimulating hormone beta subunit in mouse gonadotrope cells.

The activins are pleiotropic members of the TGFbeta superfamily. Within the anterior pituitary gland, activins stimulate FSH synthesis in an autocrine/paracrine fashion by stimulating transcription of the FSHbeta subunit gene. Here, the mechanisms mediating this effect were investigated in the murine gonadotrope cell line, LbetaT2. Recombinant activin A and activin B dose- and time-dependently stimulated endogenous FSHbeta mRNA expression. FSHbeta primary transcript and mRNA levels were increased within 30-60 min, but these effects were blocked by preincubation with the transcription inhibitor actinomycin-D, suggesting that the FSHbeta gene is a direct target of the activin signal transduction cascade. In other systems, activin signals are transduced through a heteromeric serine/threonine receptor complex, which includes the signaling activin type IB receptor [activin receptor-like kinase 4 (ALK4)]. Transfection of a constitutively active form of the receptor, ALK4T206D, stimulated FSHbeta mRNA levels. Overexpression of the inhibitory SMAD7 blocked this effect, as well as activin-stimulated FSHbeta transcription. Because SMAD7 functions by preventing access of SMAD2 and SMAD3 to ALK4, these data suggested that both activins and ALK4 require SMAD2 and/or SMAD3 to affect FSHbeta transcription. Consistent with this idea, activin A stimulated SMAD2 and SMAD3 phosphorylation and nuclear translocation within 5-10 min in LbetaT2 cells. Transient transfection of SMAD3, but not SMADs 1, 2, 4, 5, or 8, stimulated endogenous FSHbeta mRNA levels. The results of SMAD2 transfection studies were inconclusive, however, because of a persistent failure to overexpress the full-length SMAD2 protein specifically in LbetaT2 cells. To assess more directly roles for both SMAD2 and SMAD3 in activin-stimulated FSHbeta expression, RNA interference was used to decrease endogenous SMAD protein levels in LbetaT2 cells. Activin A- and ALK4T206D-stimulated transcription of the FSHbeta gene were significantly attenuated by the depletion of either SMAD2 or SMAD3. Collectively, these data suggest that activins use both SMAD2- and SMAD3-dependent mechanisms to stimulate FSHbeta transcription in mouse gonadotrope cells.

Chlamydia trachomatis genes whose products are related to energy metabolism are expressed differentially in active vs. persistent infection

The Chlamydia trachomatis genome encodes glycolysis and pentose phosphate pathway enzymes, two ATP/ADP exchange proteins, and other energy transduction-related components. We asked if and when chlamydial genes specifying products related to energy transduction are expressed during active vs. persistent infection in in vitro models and in synovia from Chlamydia-associated arthritis patients. Hep-2 cells infected with K serovar were harvested from 0–48 h post-infection (active infection). Human monocytes identically infected were harvested at 1, 2, 3, 5 days post-infection (persistent). RNA from each preparation and from synovial samples PCR-positive/-negative for Chlamydia DNA was subjected to RT-PCR targeting (a) chlamydial primary rRNA transcripts and adt1 mRNA, (b) chlamydial mRNA encoding enzymes of the glycolysis ( pyk, gap, pgk) and pentose phosphate ( gnd, tal) pathways, the TCA cycle ( mdhC, fumC), electron transport system ( cydA, cydB), and σ factors ( rpoD, rpsD, rpoN). Primary rRNA transcripts and adt1 mRNA were present in each infected preparation and patient sample; controls were negative for chlamydial RNA. In infected Hep-2 cells, all energy transduction-related genes were expressed by ≈11 h post-infection. In monocytes, pyk, gap, pgk, gnd, tal, cydA mRNA were present in 1–2-day-infected cells but absent at 3 days and after; cydB, mdhC, fumC were expressed through 5 days post-infection. RT-PCR targeting mRNA from σ factor genes indicated that lack of these gene products cannot explain selective transcriptional down-regulation during persistence. Analyses of RNA from synovial tissues mirrored those from the monocyte system. These data suggest that in the first phase of active chlamydial infection, ADP/ATP exchange provides energy required for metabolism; in active growth, glycolysis supplements host ATP. In persistence host, rather than bacterially produced, ATP is the primary energy source. Metabolic rate in persistent C. trachomatis is lower than in actively growing cells, as judged from assays for relative chlamydial primary rRNA transcript levels in persistent vs. actively growing cells.