T. J. Murphy

Overexpressed human mitochondrial thioredoxin confers resistance to oxidant-induced apoptosis in human osteosarcoma cells

Oxidative damage to mitochondria is a central mechanism of apoptosis induced by many toxic chemicals. Thioredoxin family proteins share a conserved Cys-X-X-Cys motif at their active center and play important roles in control of cellular redox state and protection against oxidative damage. In addition to the well studied cytosolic and extracellular form (Trx1), rat and avian mitochondrial forms of thioredoxin (mtTrx) have been reported. In this study, we cloned the full-length human mtTrx cDNA and performed localization and functional studies in 143B human osteosarcoma cells. The coding sequence of human mtTrx consists of a region with homology to Trx1 as well as a putative mitochondrial localization signal (MLS) at its N terminus. In stably transfected cell lines, mtTrx had a mitochondrial localization as measured by subcellular fractionation studies and by confocal fluorescence microscopy. Deletion of the MLS rendered mtTrx to be solely expressed in the cytosolic fraction. On SDS-PAGE, transfected mtTrx had the same apparent molecular weight as the MLS truncated form, indicating that the leader sequence is cleaved during or after mitochondrial import. Treatment with the oxidant tert-butylhydroperoxide induced apoptosis in 143B cells. This oxidant-induced apoptosis was inhibited by overexpressing the full-length mtTrx in 143B cells. Thus, human mtTrx is a member of the thioredoxin family of proteins localized to mitochondria and may play important roles in protection against oxidant-induced apoptosis.

Cell-Type-Dependent Up-Regulation of In Vitro Mineralization After Overexpression of the Osteoblast-Specific Transcription Factor Runx2/Cbfa1†

Functional expression of the transcriptional activator Runx2/Cbfa1 is essential for osteoblastic differentiation and bone formation and maintenance. Forced expression of Runx2 in nonosteoblastic cells induces expression of osteoblast‐specific genes, but the effects of Runx2 overexpression on in vitro matrix mineralization have not been determined. To examine whether exogenous Runx2 expression is sufficient to direct in vitro mineralization, we investigated sustained expression of Runx2 in nonosteoblastic and osteoblast‐like cell lines using retroviral gene delivery. As expected, forced expression of Runx2 induced several osteoblast‐specific genes in NIH3T3 and C3H10T1/2 fibroblasts and up‐regulated expression in MC3T3‐E1 immature osteoblast‐like cells. However, Runx2 expression enhanced matrix mineralization in a cell‐type‐dependent manner. NIH3T3 and IMR‐90 fibroblasts overexpressing Runx2 did not produce a mineralized matrix, indicating that forced expression of Runx2 in these nonosteogenic cell lines is not sufficient to direct in vitro mineralization. Consistent with the pluripotent nature of the cell line, a fraction (25%) of Runx2‐expressing C3H10T1/2 fibroblast cultures produced mineralized nodules in a viral supernatant‐dependent manner. Notably, bone sialoprotein (BSP) gene expression was detected at significantly higher levels in mineralizing Runx2‐infected C3H10T1/2 cells compared with Runx2‐expressing cultures which did not mineralize. Treatment of Runx2‐infected C3H10T1/2 cultures with dexamethasone enhanced osteoblastic phenotype expression, inducing low levels of mineralization independent of viral supernatant. Finally, Runx2 overexpression in immature osteoblast‐like MC3T3‐E1 cells resulted in acceleration and robust up‐regulation of matrix mineralization compared with controls. These results suggest that, although functional Runx2 is essential to multiple osteoblast‐specific activities, in vitro matrix mineralization requires additional tissue‐specific cofactors, which supplement Runx2 activity.

Expression of Matrix Metalloproteinase-9 in Endothelial Cells Is Differentially Regulated by Shear Stress ROLE OF c-Myc

Atherosclerotic plaques preferentially localize to areas of the vasculature with complex laminar or oscillatory blood flow. Prior data implicate matrix metalloproteinases (MMPs) in the initiation and progression of atherosclerotic lesions. In cultured endothelial cells, oscillatory but not unidirectional shear significantly increases MMP-9 mRNA as well as secretion of the MMP-9 protein (p < 0.05). In contrast, cell-associated protein levels of Tissue Inhibitor of MMP 1 (TIMP-1), an inhibitor of MMP-9, are insensitive to the shear regimen. To investigate transcriptional regulation of MMP-9 gene expression, we utilized retroviral-based reporter constructs containing different lengths of the human MMP-9 promoter. The activity of the full MMP-9 promoter is 3-fold higher (p < 0.05) in unidirectional shear compared with static conditions, and the activity is further increased ∼10-fold by oscillatory shear (p < 0.01) over unidirectional flow. Our data identify a shear-sensitive binding site at –152 in the MMP-9 promoter. We show that the c-Myc transcription factor binds specifically to this site and that reporter constructs in which the c-Myc binding site was abolished lacked the shear responsiveness of native MMP-9 reporter constructs. Our results suggest that endothelial MMP-9 expression is flow-sensitive and is up-regulated by oscillatory flow via activation of c-Myc. This effect may contribute to the development and progression of atherosclerotic lesions in areas of vasculature that are subject to disturbed flow.

The Cyclosporin A-sensitive Nuclear Factor of Activated T Cells (NFAT) Proteins Are Expressed in Vascular Smooth Muscle Cells DIFFERENTIAL LOCALIZATION OF NFAT ISOFORMS AND INDUCTION OF NFAT-MEDIATED TRANSCRIPTION BY PHOSPHOLIPASE C-COUPLED CELL SURFACE RECEPTORS

Expression of the antigen-regulated, cyclosporin A-sensitive nuclear factor of activated T cells (NFAT) is not restricted to lymphoid cells, as thought initially, but the physiological inducers of NFAT-mediated transcription in non-lymphoid cells are unknown. Here, cultured vascular smooth muscle cells (VSMC) are shown to express two isoforms of the NFAT family endogenously, which are localized differentially in cells under resting conditions. Using a retroviral NFAT-specific luciferase reporter, we show that VSMC support previously unrecognized complexities in NFAT-mediated transcription, including evidence for negative regulation by Ca2+ signaling and positive regulation through co-activation of adenylyl cyclase and Ca2+ mobilization. The VSMC mitogen platelet derived growth factor-BB (PDGF-BB) induces NFAT-mediated transcription in VSMC. Thrombin and angiotensin II, which activate Gαq-coupled receptors, are significantly weaker inducers of NFAT-mediated luciferase expression than is PDGF-BB. However, co-stimulation studies show that Gαq receptor agonists augment the NFAT-mediated transcriptional response to PDGF-BB. This synergy can be explained in part by augmented intracellular Ca2+ transients elicited by multiple agonist challenges. These data indicate that agonists for phospholipase C-coupled receptors stimulate NFAT-mediated transcription in VSMC differentially, and that NFAT can function to integrate co-activating signals in the extracellular environment.

Recognition of polyadenosine RNA by zinc finger proteins

Messenger RNA transcripts are coated from cap to tail with a dynamic combination of RNA binding proteins that process, package, and ultimately regulate the fate of mature transcripts. One class of RNA binding proteins essential for multiple aspects of mRNA metabolism consists of the poly(A) binding proteins. Previous studies have concentrated on the canonical RNA recognition motif-containing poly(A) binding proteins as the sole family of poly(A)-specific RNA binding proteins. In this study, we present evidence for a previously uncharacterized poly(A) recognition motif consisting of tandem CCCH zinc fingers. We have probed the nucleic acid binding properties of a yeast protein, Nab2, that contains this zinc finger motif. Results of this study reveal that the seven tandem CCCH zinc fingers of Nab2 specifically bind to polyadenosine RNA with high affinity. Furthermore, we demonstrate that a human protein, ZC3H14, which contains CCCH zinc fingers homologous to those found in Nab2, also specifically binds polyadenosine RNA. Thus, we propose that these proteins are members of an evolutionarily conserved family of poly(A) RNA binding proteins that recognize poly(A) RNA through a fundamentally different mechanism than previously characterized RNA recognition motif-containing poly(A) binding proteins.

Immediate-early MEK-1-dependent Stabilization of Rat Smooth Muscle Cell Cyclooxygenase-2 mRNA by Gαq-coupled Receptor Signaling

Activation of Gαq-coupled P2Y nucleotide receptors strongly (>100-fold) induces the rat vascular smooth muscle cell cyclooxygenase-2 (COX-2) mRNA, yet transcription is induced only ∼3-fold over 1 h. Intact cell decay analysis of tetracycline-suppressible luciferase chimera mRNAs shows that regulated stabilization of the intrinsically unstable mRNA contributes to this response. Deletion mapping of the 2468-base COX-2 mRNA 3′-untranslated region (UTR) shows that a distal, 130-base AU-rich region functions as a cis-acting regulated stabilization response element, which under basal conditions serves as the dominant instability determinant for the 3′-UTR. Regulation of this response is through the p42/44 MAP kinases, whereas the p38 MAP kinases are not involved. The stabilization response element binds avidly and specifically to a prominent nuclear-enriched ∼90-kDa factor and several less abundantly labeled mRNA binding proteins that are unaffected by P2Y receptor signaling. Although other instability determinants are located throughout the rat COX-2 mRNA 3′-UTR, mitogen signaling only interferes with rapid decay mediated by its most distal 130 bases. A complex of nuclear factors that bind this mRNA region specifically may include candidate targets for regulatory modulation. These observations support the general notion that the rapid induction of immediate-early gene expression through mitogenic receptors involves simultaneous activation of transcriptional and post-transcriptional mechanisms.

Transcriptional responses to growth factor and G protein-coupled receptors in PC12 cells : Comparison of α1-Adrenergic receptor subtypes

Abstract: Transcriptional responses to growth factor and G protein‐coupled receptors were compared in PC12 cells using retroviral luciferase reporters. In cells stably expressing α1A‐adrenergic receptors, norepinephrine activated all five reporters [AP1 (activator protein‐1), SRE (serum response element), CRE (cyclic AMP response element), NFκB) (nuclear factor‐κB), and NFAT (nuclear factor of activated T cells)], whereas nerve growth factor (NGF) and epidermal growth factor activated only AP1 and SRE. Activation of P2Y2 receptors by UTP did not activate any reporters. Protein kinase C inhibition blocked NFκB activation by norepinephrine, but potentiated CRE. Mitogen‐activated protein kinase kinase inhibition blocked AP1 activation by norepinephrine, but also potentiated CRE. p38 mitogen‐activated protein kinase inhibition reduced most norepinephrine responses, but not NGF responses. inhibition of Src eliminated SRE responses to norepinephrine and NGF, and reduced all responses except CRE. Phosphatidylinositol 3‐kinase inhibitors markedly potentiated CRE activation by norepinephrine, with only small effects on the other responses. Comparison of the three human subtypes showed that the α1A activated all five reporters, the α1B showed smaller effects, and the α1D was ineffective. Cell differentiation caused by norepinephrine, but not NGF, was reduced by all inhibitors studied. These experiments suggest that α1A‐adrenergic receptors activate a wider array of transcriptional responses than do growth factors in PC12 cells. These responses are not linearly related to second messenger production, and different subtypes show different patterns of activation.

Platelet-derived Growth Factor-induced Stabilization of Cyclooxygenase 2 mRNA in Rat Smooth Muscle Cells Requires the c-Src Family of Protein-tyrosine Kinases

Cyclooxygenases (COXs) are crucial rate-limiting enzymes required for the biosynthesis of prostaglandins. COX-2 is an inducible isoform of this enzyme, which is believed to play important roles in the development of atherosclerotic vascular disease. We found that COX-2 expression rapidly increases in response to various signaling events, including activation of the platelet-derived growth factor (PDGF) pathway. Activation of PDGF receptor (PDGFR) in rat aortic vascular smooth muscle cells leads to c-Src-dependent stabilization of COX-2 mRNA requiring an AU-rich region within the 3′-untranslated region of this transcript. This regulation correlates with tyrosine phosphorylation of the RNA-associated protein, CUG-binding protein 2 (CUGBP2), which appears to enhance its interaction with COX-2 mRNA. Site-directed mutagenesis of putative tyrosine phosphorylation sites in CUGBP2 identified tyrosine 39 as a c-Src target, and a CUGBP2 with a mutated tyrosine 39 displayed an attenuated ability to bind COX-2 mRNA. We further show that silencing of CUGBP2 with specific small interference RNAs significantly reduces PDGF-dependent induction of COX-2 at both mRNA and protein levels. Furthermore, forced expression of CUGBP2 or constitutively active c-Src leads to stabilization of co-expressed COX-2 mRNA. Finally, in vitro RNA decay assay demonstrates that CUGBP2 is functionally required for the stabilization of COX-2 mRNA. Therefore, our data suggest that tyrosine phosphorylation of CUGBP2 is an important underlying mechanism for the ability of PDGFR/c-Src signaling to control the stability of COX-2 mRNA.

Retroviral vectors applied to gene regulation studies.

There are instances in G-protein-coupled receptor studies when it is desirable to express a transgene in some specialized cell phenotype to understand a process better. Most such cells are more resistant to plasmid transfection than are the transformed libroblasts often used in G protein signaling studies (for example, COS, 293, and Chinese hamster ovary (CHO)). This often presents a substantial barrier against understanding context-specific mechanisms of Gα protein-coupled receptor signaling and cellular regulation. Highly efficient gene transfer methods applicable to broader ranges of cell phenotypes can surmount this problem. Retroviruses use naturally evolved mechanisms to gain entry into cells, release their transgene cargoes, and permanently tag the new host. Their most significant advantages over plasmid transfection include high gene transfer efficiencies in many cell types, with the genes integrating into host cell chromatin permanently. One limitation of the Moloney murine leukemia-based retroviruses is that cell division is necessary to take advantage of these properties. For cells that transfect poorly, retroviral methods are more reproducible, easier, and less costly over the long haul.

The mating response cascade does not modulate changes in the steady-state level of target mRNAs through control of mRNA stability.

Many extracellular signals trigger changes in gene expression by altering the steady‐state level of target transcripts. This modulation of transcript levels is typically ascribed to changes in transcription of target genes; however, there are numerous examples of changes in mRNA processing and stability that contribute to the overall change in transcript levels following signalling pathway activation. The α‐factor‐stimulated mating pathway in Saccharomyces cerevisiae is a receptor‐operated MAP kinase cascade that results in increased levels of a large number of target mRNA transcripts when stimulated acutely. A previous study identified many of the transcripts modulated in response to α‐factor and argued, based on genetic studies, that the response occurred solely at the level of gene transcription (Roberts et al., 2000). We directly examined whether enhanced mRNA stability contributes to the increase in the steady‐state level of α‐factor target transcripts by exploiting a temperature‐sensitive RNA Polymerase II mutant, a Ste12 transcription factor import mutant, and tet‐regulated synthetic mating factor minigene reporters. Examination of a panel of α‐factor‐responsive transcripts reveals no change in mRNA stability in response to α‐factor stimulation, providing direct evidence that this signal transduction pathway in S. cerevisiae does not function through modulating transcript stability. Copyright