Notch-induced transcription factors are predictive of survival and 5-fluorouracil response in colorectal cancer patients.

BACKGROUND: The purpose of this study was to evaluate the expression of Notch-induced transcription factors (NTFs) HEY1, HES1 and SOX9 in colorectal cancer (CRC) patients to determine their clinicopathologic and prognostic significance. METHODS: Levels of HEY1, HES1 and SOX9 protein were measured by immunohistochemistry in a nonmalignant and malignant tissue microarray of 441 CRC patients, and the findings correlated with pathologic, molecular and clinical variables. RESULTS: The NTFs HEY1, HES1 and SOX9 were overexpressed in tumours relative to colonic mucosa (OR=3.44, P<0.0001; OR=7.40, P<0.0001; OR=4.08 P<0.0001, respectively). HEY1 overexpression was a negative prognostic factor for all CRC patients (HR=1.29, P=0.023) and strongly correlated with perineural and vascular invasion and lymph node (LN) metastasis. In 5-fluorouracil (5-FU)-treated patients, the tumour overexpression of SOX9 correlated with markedly poorer survival (HR=8.72, P=0.034), but had no predictive effect in untreated patients (HR=0.70, P=0.29). When HEY1, HES1 and SOX9 expression were combined to predict survival with chemotherapy, in treated patients there was an additive increase in the risk of death with each NTF overexpressed (HR=2.09, P=0.01), but no prognostic import in the untreated patient group (HR=0.74, P=0.19). CONCLUSION: The present study is the first to discover that HEY1 overexpression correlates with poorer outcome in CRC, and NTF expression is predictive of CRC patient survival with 5-FU chemotherapy. If confirmed in future studies, testing of NTF expression has the potential to enter routine pathological practice for the selection of patients to undergo chemotherapy alone or in combination with Notch inhibitors.

Iron absorption and hepatic iron uptake are increased in a transferrin receptor 2 (Y245X) mutant mouse model of hemochromatosis type 3.

Hereditary hemochromatosis type 3 is an iron (Fe)-overload disorder caused by mutations in transferrin receptor 2 (TfR2). TfR2 is expressed highly in the liver and regulates Fe metabolism. The aim of this study was to investigate duodenal Fe absorption and hepatic Fe uptake in a TfR2 (Y245X) mutant mouse model of hereditary hemochromatosis type 3. Duodenal Fe absorption and hepatic Fe uptake were measured in vivo by 59Fe-labeled ascorbate in TfR2 mutant mice, wild-type mice, and Fe-loaded wild-type mice (2% dietary carbonyl Fe). Gene expression was measured by real-time RT-PCR. Liver nonheme Fe concentration increased progressively with age in TfR2 mutant mice compared with wild-type mice. Fe absorption (both duodenal Fe uptake and transfer) was increased in TfR2 mutant mice compared with wild-type mice. Likewise, expression of genes participating in duodenal Fe uptake (Dcytb, DMT1) and transfer (ferroportin) were increased in TfR2 mutant mice. Nearly all of the absorbed Fe was taken up rapidly by the liver. Despite hepatic Fe loading, hepcidin expression was decreased in TfR2 mutant mice compared with wild-type mice. Even when compared with Fe-loaded wild-type mice, TfR2 mutant mice had increased Fe absorption, increased duodenal Fe transport gene expression, increased liver Fe uptake, and decreased liver hepcidin expression. In conclusion, despite systemic Fe loading, Fe absorption and liver Fe uptake were increased in TfR2 mutant mice in association with decreased expression of hepcidin. These findings support a model in which TfR2 is a sensor of Fe status and regulates duodenal Fe absorption and liver Fe uptake.

Formation of an alphaCP1-KH3 complex with UC-rich RNA.

The alphaCP family of proteins [also known as poly(C)-binding or heterogeneous nuclear ribonucleoprotein E proteins] are involved in the regulation of messenger RNA (mRNA) stability and translational efficiency. They bind via their triple heterologous nuclear ribonucleoprotein K homology (KH) domain structures to C-rich mRNA, and are thought to interact with other mRNA-binding proteins as well as provide direct nuclease protection. In particular, alphaCP1 and alphaCP2 have been shown to bind to a specific region of androgen receptor (AR) mRNA, resulting in its increased stability. The roles of each of the KH motifs in the binding affinity and the specificity is not yet understood. We report the beginning of a systematic study of each of the alphaCP KH domains, with the cloning and expression of alphaCP1-KH2 and alphaCP1-KH3. We report the ability of alphaCP1-KH3, but not alphaCP1-KH2, to bind the target AR mRNA sequence using an RNA electrophoretic mobility gel shift assay. We also report the preparation of an alphaCP1-KH3/AR mRNA complex for structural studies. (1)H-(15)N heteronuclear single quantum correlation NMR spectra of (15)N-labelled alphaCP1-KH3 verified the integrity and good solution behaviour of the purified domain. The titration of the 11-nucleotide RNA target sequence from AR mRNA resulted in a rearrangement of the (1)H-(15)N correlations, demonstrating the complete binding of the protein to form a homogeneous protein/RNA complex suitable for future structural studies.

Structure and RNA binding of the third KH domain of poly(C)-binding protein 1.

Poly(C)-binding proteins (CPs) are important regulators of mRNA stability and translational regulation. They recognize C-rich RNA through their triple KH (hn RNP K homology) domain structures and are thought to carry out their function though direct protection of mRNA sites as well as through interactions with other RNA-binding proteins. We report the crystallographically derived structure of the third domain of alphaCP1 to 2.1 A resolution. alphaCP1-KH3 assumes a classical type I KH domain fold with a triple-stranded beta-sheet held against a three-helix cluster in a betaalphaalphabetabetaalpha configuration. Its binding affinity to an RNA sequence from the 3'-untranslated region (3'-UTR) of androgen receptor mRNA was determined using surface plasmon resonance, giving a K(d) of 4.37 microM, which is indicative of intermediate binding. A model of alphaCP1-KH3 with poly(C)-RNA was generated by homology to a recently reported RNA-bound KH domain structure and suggests the molecular basis for oligonucleotide binding and poly(C)-RNA specificity.

cAMP controls human renin mRNA stability via specific RNA-binding proteins.

It is now recognized that post-transcriptional mechanisms are pivotal to renin production. These involve factors that modulate renin mRNA stability. In 2003 new data has emerged from work in Australia and Germany that has identified several of the, as many as, 20 or so proteins involved. These include CP1 (hnRNP E1), HuR, HADHB, dynamin, nucleolin, YP-1, hnRNP K and MINT-homologous protein. Cyclic AMP (cAMP) is a crucial regulator of renin secretion as well as transcriptional and post-transcriptional control of expression. Many of the RNA-binding proteins that were identified responded to forskolin, increasing in amount by two to 10-fold. The cAMP mechanisms that regulate renin mRNA target, at least in large part, other genes that presumably encode some of these proteins. The increase in the expression of these then facilitates, sequentially, renin mRNA stabilization and destabilization. Our data, using a battery of different techniques, confirm that CP1 and HuR stabilize renin mRNA, whereas HADHB causes destabilization. These proteins target cis-acting C-rich sequences (in the case of CP1) and AU-rich sequences (HuR) in the distal region of the 3'-untranslated region of renin mRNA. We found HADHB was enriched in juxtaglomerular cells and that that within Calu-6 cells HADHB, HuR and CP1 all localized in nuclear subregions, as well as cytoplasm (HADHB and CP1) and mitochondria (HADHB) commensurate with the role each plays in control of renin mRNA stability. The specific proteins that bind to human renin mRNA have begun to be revealed. Cyclic AMP upregulates the binding of several of these proteins, which in turn affect renin mRNA stability and thus overall expression of renin.

Graves' disease during immune reconstitution after highly active antiretroviral therapy for HIV infection: evidence of thymic dysfunction.

A patient with HIV infection who experienced immune reconstitution after highly active antiretroviral therapy (HAART) [increase in CD4 T cell count from <1/microl to >600/microl] presented with severe Graves' disease 32 months after commencing HAART. A comprehensive clinical and laboratory study demonstrated pronounced regional lymphadenopathy and thymic enlargement at presentation, and that the onset of thyrotropin receptor antibody production was associated with increased production of soluble CD30 (a marker of type 2 immune responses). Blood naive CD8 T cell counts and TREC levels in both CD4 and CD8 T cells were increased at multiple time points compared with carefully selected controls. We conclude that the Graves' disease in this patient was associated with abnormally high blood counts of thymus-derived T cells, and propose that Graves' disease after HAART in this and other HIV patients may result from failure to delete autoreactive T cell clones in the regenerating thymus.

Thyroid hormone receptor-interacting protein 1 modulates cytokine and nuclear hormone signaling in erythroid cells.

Erythropoietin (Epo) and thyroid hormone (T(3)) are key molecules in the development of red blood cells. We have shown previously that the tyrosine kinase Lyn is involved in differentiation signals emanating from an activated erythropoietin receptor. Here we demonstrate that Lyn interacts with thyroid hormone receptor-interacting protein 1 (Trip-1), a transcriptional regulator associated with the T(3) receptor, providing a link between the Epo and T(3) signaling pathways. Trip-1 co-localized with Lyn and the T(3) receptor alpha in the cytoplasm/plasma membrane of erythroid cells but translocated to discrete nuclear foci shortly after Epo-induced differentiation. Our data reveal that T(3) stimulated the proliferation of immature erythroid cells, and inhibited maturation promoted by erythropoietin. Removal of T(3) reduced cell division and enhanced terminal differentiation. This was accompanied by large increases in the cell cycle inhibitor p27(Kip1) and by increasing expression of erythroid transcription factors GATA-1, EKLF, and NF-E2. Strikingly, a truncated Trip-1 inhibited both erythropoietin-induced maturation and T(3)-initiated cell division. This mutant Trip-1 acted in a dominant negative fashion by eliminating endogenous Lyn, elevating p27(Kip1), and blocking T(3) response elements. These data demonstrate that Trip-1 can simultaneously modulate responses involving both cytokine and nuclear receptors.

Identification of a novel AU-Rich element in the 3' untranslated region of epidermal growth factor receptor mRNA that is the target for regulated RNA-binding proteins.

The epidermal growth factor receptor (EGF-R) plays an important role in the growth and progression of estrogen receptor-negative human breast cancers. EGF binds with high affinity to the EGF-R and activates a variety of second messenger pathways that affect cellular proliferation. However, the underlying mechanisms involved in the regulation of EGF-R expression in breast cancer cells are yet to be described. Here we show that the EGF-induced upregulation of EGF-R mRNA in two human breast cancer cell lines that overexpress EGF-R (MDA-MB-468 and BT-20) is accompanied by stabilization (>2-fold) of EGF-R mRNA. Transient transfections using a luciferase reporter identified a novel EGF-regulated approximately 260-nucleotide (nt) cis-acting element in the 3' untranslated region (3'-UTR) of EGF-R mRNA. This cis element contains two distinct AU-rich sequences (~75 nt), EGF-R1A with two AUUUA pentamers and EGF-R2A with two AUUUUUA extended pentamers. Each independently regulated the mRNA stability of the heterologous reporter. Analysis of mutants of the EGF-R2A AU-rich sequence demonstrated a role for the 3' extended pentamer in regulating basal turnover. RNA gel shift analysis identified cytoplasmic proteins (~55 to 80 kDa) from breast cancer cells that bound specifically to the EGF-R1A and EGF-R2A cis-acting elements and whose binding activity was rapidly downregulated by EGF and phorbol esters. RNA gel shift analysis of EGF-R2A mutants identified a role for the 3' extended AU pentamer, but not the 5' extended pentamer, in binding proteins. These EGF-R mRNA-binding proteins were present in multiple human breast and prostate cancer cell lines. In summary, these data demonstrate a central role for mRNA stabilization in the control of EGF-R gene expression in breast cancer cells. EGF-R mRNA contains a novel complex AU-rich 260-nt cis-acting destabilizing element in the 3'-UTR that is bound by specific and EGF-regulated trans-acting factors. Furthermore, the 3' extended AU pentamer of EGF-R2A plays a central role in regulating EGF-R mRNA stability and the binding of specific RNA-binding proteins. These findings suggest that regulated RNA-protein interactions involving this novel cis-acting element will be a major determinant of EGF-R mRNA stability.

Hormonal regulation of mRNA stability and RNA-protein interactions in the pituitary.

Regulating gene expression from DNA to protein is a complex multistage process with multiple control mechanisms. Transcriptional regulation has been considered the major control point of protein production in eukaryotic cells; however, there is growing evidence of pivotal posttranscriptional regulation for many genes. This has prompted extensive investigations to elucidate the mechanisms controlling RNA processing, mRNA nuclear export and localization, mRNA stability and turnover, in addition to translational rates and posttranslational events. The regulation of mRNA stability has emerged as a critical control step in determining the cellular mRNA level, with individual mRNAs displaying a wide range of stability that has been linked to discrete sequence elements and specific RNA-protein interactions. This review will focus on current knowledge of the determinants of mRNA stability and RNA-protein interactions in the pituitary. This field is rapidly expanding with the identification of regulated cis-acting stability-modifying elements within many mRNAs, and the cloning and characterization of trans-acting proteins that specifically bind to their cognate cis elements. We will present evidence for regulation of multiple pituitary genes at the level of mRNA stability and some examples of the emerging data characterizing RNA-protein interactions.

Thyrotropin-releasing hormone and epidermal growth factor regulate iron-regulatory protein binding in pituitary cells via protein kinase C-dependent and -independent signaling pathways.

Intracellular iron homeostasis is regulated, in part, by interactions between iron-regulatory proteins (IRP1 and IRP2) and iron-responsive elements (IREs) in ferritin and transferrin receptor mRNAs. In addition to iron, cellular oxidative stress induced by H(2)O(2), nitric oxide, and hypoxia, and hormonal activation by thyroid hormone and erythropoeitin have each been shown to regulate IRP binding to IREs. Hormonal signals, in particular mediated through protein kinase C (PKC), play a central role in the modulation of IRP/IRE interactions since phorbol esters were shown to activate IRP binding (Eisenstein, R. S., Tuazon, P. T., Schalinske, K. L., Anderson, S. A., and Traugh, J. A. (1993) J. Biol. Chem. 268, 27363-27370). In pituitary thyrotrophs (TtT97), we found that thyrotropin releasing hormone (TRH) and epidermal growth factor (EGF) increased IRP binding to a ferritin IRE, dependent on PKC and mitogen-activated protein kinase (MAPK) activity. In contrast, TRH and EGF decreased IRP binding in pituitary lactotrophs (GH3), despite activation of PKC and MAPK. IRP1 and IRP2 levels remained constant and IRP2 binding was predominant throughout. TRH and EGF markedly decreased IRP binding in MAPK kinase inhibitor-treated GH3 cells, whereas, they increased IRP binding in phosphatase inhibitor-treated GH3 cells. IRE-dependent CAT reporter translational expression closely reflected IRP binding to the ferritin IRE in both GH3 and TtT97 cells. Interestingly, ferritin protein levels were regulated similarly by TRH in both cell lines. These data link two different cell receptor systems to common signaling pathways that regulate IRP binding and ferritin expression. Remarkably, for TRH and EGF, these effects may be PKC-dependent or -independent determined by the cell type.

A comparison of three methods for measuring thoracic kyphosis: implications for clinical studies.

OBJECTIVES: To compare the Cobb technique for measuring kyphosis with an alternative Cobb method and a computer-assisted curve assessment technique, and to examine the influence of vertebral body and disc shape on kyphosis. METHODS: Kyphosis measurements were derived from 93 lateral spinal radiographs or sagittal computed tomography images of cadaveric spines, using: (i) a computer-assisted method for estimating radius of curvature; (ii) the traditional Cobb method; and (iii) an alternative Cobb method. Regression models were applied for agreement analyses, and to examine the relative contribution of vertebral body and disc shape on the extent of curvature. Results and conclusions. Strong associations existed between curvature and angle data derived from the three methods, confirming the clinical utility of these techniques for the quantification of thoracic kyphosis. However, the traditional Cobb method tended to overestimate kyphosis in the presence of vertebral body end-plate deformation. The degree of kyphosis was strongly reflective of the extent of deformity of the vertebral bodies, and to a lesser extent the shape of the thoracic discs.

Iron-regulatory proteins, iron-responsive elements and ferritin mRNA translation.

Iron plays a central role in the metabolism of all cells. This is evident by its major contribution to many diverse functions, such as DNA replication, bacterial pathogenicity, photosynthesis, oxidative stress control and cell proliferation. In mammalian systems, control of intracellular iron homeostasis is largely due to posttranscriptional regulation of binding by iron-regulatory RNA-binding proteins (IRPs) to iron-responsive elements (IREs) within ferritin and transferrin receptor (TfR) mRNAs. the TfR transports iron into cells and the iron is subsequently stored within ferritin. IRP binding is under tight control so that it responds to changes in intracellular iron requirements in a coordinate manner by differentially regulating ferritin mRNA translational efficiency and TfR mRNA stability. Several different stimuli, as well as intracellular iron levels and oxidative stress, are capable of regulating these RNA-protein interactions. In this mini-review, we shall concentrate on the mechanisms underlying modulation of the interaction of IRPs and the ferritin IRE and its role in regulating ferritin gene expression.

Optimized RNA gel-shift and UV cross-linking assays for characterization of cytoplasmic RNA-protein interactions.

Considerable interest has recently focused on defining the mechanisms involved in the regulation of gene expression at the level of mRNA stability and translational efficiency. However, the assays used to directly investigate interactions between RNA and cytoplasmic proteins have been difficult to establish, and methods are not widely available. Here, we describe a robust method for RNA electrophoretic mobility shift and UV cross-linking assays that allows rapid detection of cytoplasmic RNA-protein interactions. For added convenience to new investigators, these assays use mini-gels with an electrophoresis time of 15-20 min, enabling a high throughput of samples. The method works successfully with many different probes and cytoplasmic extracts from a variety of cell lines. Furthermore, we provide a system to optimize characterization of the RNA-protein complex and troubleshoot most assay difficulties.

The relative influence of vertebral body and intervertebral disc shape on thoracic kyphosis.

OBJECTIVE: The aim of this study was to quantify the morphology or shape of thoracic vertebral bodies and intervertebral discs, and to examine the ex vivo association of thoracic kyphosis with these shape parameters. DESIGN: A quantitative, retrospective study design was applied to define vertebral body and disc influences on thoracic kyphosis. BACKGROUND: Age-related progression of thoracic kyphosis is a well-defined process that is influenced by the morphology of vertebral bodies. However, little is known about the contribution of intervertebral disc shape to the thoracic curvature. METHODS: Vertebral and disc morphology, as represented by antero-posterior height ratios, were quantified in 93 lateral spine radiographs and midsagittal computed tomography films of ex vivo spines. Kyphosis was indicated by the Cobb angle. Linear and stepwise regression were applied to examine relationships for cumulative (T1-T12) and regional (T4-T9) analyses. RESULTS: Vertebral morphology was highly predictive of thoracic curvature, while a poorer association was noted for disc morphology. The combined influence of both accounted for >85% of the variability in kyphosis. There was a trend for a more pronounced anterior wedge configuration of the midthoracic vertebral bodies and discs. Higher associations between variables were also noted in this region. CONCLUSIONS: The normal kyphosis of the thoracic spine reflects the morphological adaptation of both the vertebral bodies and intervertebral discs. RELEVANCE: This study contributes new data on the thoracic spine, particularly the characteristics of thoracic discs and their contribution to kyphosis genesis. Future directions for morphology studies should encompass more detailed examination of the thoracic discs and greater emphasis on the midthoracic segments, considering the prevalence of osteoporosis related fractures and subsequent deformity at these levels.

Complex post-transcriptional regulation of EGF-receptor expression by EGF and TGF-alpha in human prostate cancer cells.

The epidermal growth factor receptor (EGFR) plays an important role in the development and progression of prostate cancer and its overexpression is associated with decreased survival. With progression, prostate cancer cells switch from epidermal growth factor (EGF) to transforming growth factor alpha (TGF-alpha) synthesis, which contributes to autocrine growth and unrestrained proliferation. To define the molecular mechanisms involved in the regulation of EGFR expression by EGF and TGF-alpha we studied three human prostate cancer cell lines, androgen-responsive (LNCaP) and -unresponsive (DU145 and PC3). Here we show that TGF-alpha stabilized EGFR mRNA two- to threefold in all three cell lines, whilst EGF stabilized EGFR mRNA approximately twofold in LNCaP and DU145 cells, but not in PC3 cells. Both ligands increased EGFR transcription in LNCaP and DU145 cells, with less effect in PC3 cells. In all three cell lines EGF reduced total EGFR protein levels more than TGF-alpha, but this was associated with a greater increase in de novo protein synthesis with EGF compared to TGF-alpha. Only EGF, however, shortened EGFR protein stability (half-life decreased from 5 h to 120 min), resulting in rapid disappearance of newly synthesized EGFR protein. Both ligands increased total LNCaP and DU145 cell numbers. These studies demonstrate that the EGF- and TGF-alpha-induced upregulation of EGFR mRNA and protein in human prostate cancer cell lines is complex and occurs at multiple, transcriptional and post-transcriptional levels. Taken together, these data provide novel insight into the molecular mechanisms by which TGF-alpha would preferentially maintain an autocrine loop in human prostate cancer cells. Furthermore, this work suggests that in human prostate cancer cells ligand-specific differential intracellular trafficking of the EGFR plays a major role in regulating its expression.

Differential posttranscriptional regulation of androgen receptor gene expression by androgen in prostate and breast cancer cells.

Androgens, via the androgen receptor (AR), modulate the growth and proliferation of prostate and breast cancer cells. However, the molecular mechanisms underlying the regulation of AR gene expression by androgen in these cells remain to be fully elucidated. To explore differences in AR gene expression between these hormone-responsive tumor cell types, we studied androgen-responsive LNCaP prostate cancer and AR positive MDA453 breast cancer cells. Dihydrotestosterone (DHT) 10 nM increased LNCaP cell proliferation and the proportion of LNCaP cells in S-phase of the cell cycle but inhibited MDA453 cell proliferation and reduced the proportion of MDA453 cells in S-phase of cell cycle. In both these cell lines, DHT decreased total AR messenger RNA (mRNA) but increased AR protein. In LNCaP cells, DHT down-regulated AR mRNA transcription but stabilized AR mRNA. In contrast, in MDA453 cells, DHT had no effect on AR mRNA transcription but destabilized AR mRNA. In summary, transcriptional down-regulation induced by androgens in LNCaP cells results in down-regulation of steady-state AR mRNA despite an androgen-induced increase in AR mRNA stability. However, in MDA453 cells, posttranscriptional destabilization of AR mRNA appears to be the predominant mechanism resulting in down-regulation of AR mRNA by androgen. These results demonstrate cell-specific and divergent regulation of AR mRNA turnover by androgen and identify a novel pathway of androgen-induced posttranscriptional destabilization and down-regulation of AR mRNA in human breast cancer cells. Furthermore, these data establish an important role for posttranscriptional pathways in the regulation of AR gene expression by androgen in human prostate and breast cancer cells.

Regulation of EGF-receptor expression by EGF and TGF alpha in epidermoid cancer cells is cell type-specific.

The epidermal growth factor receptor (EGF-R) and its major ligands EGF and transforming growth factor alpha (TGF alpha) play an important role in the development of multiple human tumors. However, little is known of the comparative effects of each ligand on the regulation of EGF-R expression. To investigate this issue we used two similar human epidermoid cancer cell lines that overexpress EGF-Rs (KB and A431). In KB cells, EGF and TGF alpha increased EGF-R mRNA and protein levels by 2-3 fold over 8 h, associated with a greater than 4-fold stabilization of EGF-R mRNA half-life. EGF and TGF alpha also increased transcription of EGF-R mRNA 2-3-fold in KB cells. In contrast, EGF and TGF alpha only minimally increased EGF-R mRNA and protein in A431 cells, without changing EGF-R mRNA half-life. Basal EGF-R mRNA half-life was 2 fold greater in A431 cells than in KB cells (6-7 h versus 2-3 h), whilst the half-life of a mutant 2.6 kb EGF-R mRNA present in A431 cells, which lacks the 3-untranslated region (3'-UTR), was 2 fold greater than the full-length EGF-R mRNA. RNA gel-shift studies demonstrated that KB and A431 cells contain cytoplasmic proteins that bind specifically to an AU-rich sequence from the 3'-UTR of EGF-R mRNA. Taken together, these results demonstrate that in KB cells EGF and TGF alpha upregulate EGF-R expression at both transcriptional and post-transcriptional levels. The identification of AU-rich EGF-R mRNA-specific RNA-binding proteins from epidermoid cancer cells that overexpress EGF-Rs suggests that regulated RNA-protein interactions involving this region may play a central role in modulating EGF-R mRNA stability.

Posttranscriptional regulation of thyrotropin beta-subunit messenger ribonucleic acid by thyroid hormone in murine thyrotrope tumor cells: a conserved mechanism across species.

Thyroid hormone (T3) negatively regulates TSH beta-subunit (TSHbeta) messenger RNA (mRNA) gene expression in whole rat pituitary, in part at the level of mRNA stability. However, the regulation of TSHbeta mRNA turnover by T3 in pure populations of thyrotropes and in other species is unknown. To further investigate this, we used murine thyrotropic TtT97 tumor cells. Using primary cultures of TtT97 cells, T3 down-regulated TSHbeta mRNA to approximately 35% of the control level by 8 h. Actinomycin D chase revealed that T3 destabilized TSHbeta mRNA, reducing the half-life from approximately 24 to 7 h, and was accompanied by a decrease in TSHbeta mRNA size. Ribonuclease H analysis revealed that this T3-induced decrease in size was due to a shortening of poly(A) tail from approximately 160 to approximately 30 nucleotides and was specific for TSHbeta mRNA. Cycloheximide mimicked the poly(A) tail effect observed with T3. In the absence of T3, actinomycin D deadenylated TSHbeta mRNA without inducing rapid decay. We conclude that T3 reduces the steady state half-life of TSHbeta mRNA in murine TtT97 thyrotropic tumor cells accompanied by a reduction in poly(A) tail length. However, in the absence of T3, deadenylation alone is not sufficient to induce TSHbeta mRNA decay. Together with the high degree of sequence conservation in the 3'-untranslated region of murine and rat TSHbeta mRNA sequences and the similarities of the T3 effect, these data provide the first evidence for a highly conserved posttranscriptional mechanism operative across species. We propose a model in which T3 coordinately regulates shortening of the poly(A) tail and the activity of a transacting RNA-binding protein and/or an exonuclease to accelerate TSHbeta mRNA turnover.