Differential expression and functional analysis of the tristetraprolin family during early differentiation of 3T3-L1 preadipocytes

The tristetraprolin (TTP) family comprises zinc finger-containing AU-rich element (ARE)-binding proteins consisting of three major members: TTP, ZFP36L1, and ZFP36L2. The present study generated specific antibodies against each TTP member to evaluate its expression during differentiation of 3T3-L1 preadipocytes. In contrast to the inducible expression of TTP, results indicated constitutive expression of ZFP36L1 and ZFP36L2 in 3T3-L1 preadipocytes and their phosphorylation in response to differentiation signals. Physical RNA pull-down and functional luciferase assays revealed that ZFP36L1 and ZFP36L2 bound to the 3' untranslated region (UTR) of MAPK phosphatase-1 (MKP-1) mRNA and downregulated Mkp-1 3'UTR-mediated luciferase activity. Mkp-1 is an immediate early gene for which the mRNA is transiently expressed in response to differentiation signals. The half-life of Mkp-1 mRNA was longer at 30 min of induction than at 1 h and 2 h of induction. Knockdown of TTP or ZFP36L2 increased the Mkp-1 mRNA half-life at 1 h of induction. Knockdown of ZFP36L1, but not ZFP36L2, increased Mkp-1 mRNA basal levels via mRNA stabilization and downregulated ERK activation. Differentiation induced phosphorylation of ZFP36L1 through ERK and AKT signals. Phosphorylated ZFP36L1 then interacted with 14-3-3, which might decrease its mRNA destabilizing activity. Inhibition of adipogenesis also occurred in ZFP36L1 and TTP knockdown cells. The findings indicate that the differential expression of TTP family members regulates immediate early gene expression and modulates adipogenesis.     

Differential regulation of ARE-mediated TNFalpha and IL-1beta mRNA stability by lipopolysaccharide in RAW264.7 cells

Messenger RNA degradation is a mechanism by which eukaryotic cells regulate gene expression and influence cell growth and differentiation. Many protooncogene, cytokine, and growth factor RNAs contain AU-rich element (AREs) in the 3'untranslated regions which enable them to be targeted for rapid degradation. To investigate the mechanism of ARE-mediated RNA stability, we demonstrate the expression and regulation of TNFalpha and IL-1beta mRNAs in LPS-stimulated macrophages. TNFalpha mRNA was rapidly induced by LPS and showed short half-life at 2-h induction, whereas IL-1beta mRNA was induced slowly and had longer half-life. Electrophoretic mobility shift assays showed that the LPS-induced destabilization factor tristetraprolin (TTP) could bind to TNFalpha ARE with higher affinity than to IL-1beta ARE. HuR was identified to interact with TNFalpha ARE to exert RNA stabilization activity. The expression and phosphorylation of TTP could be activated by p38 MAPK pathway during LPS stimulation. Moreover, ectopic expression with TTP and kinases in p38 pathway followed by biochemical assays showed that the activation of p38 pathway resulted in the phosphorylation of TTP and a decrease in its RNA-binding activity. The ARE-containing reporter assay presented that the p38 signal could reverse the inhibitory activity of TTP on IL-1beta ARE but not on TNFalpha ARE. The present results indicate that the heterogeneity of AREs from TNFalpha and IL-1beta could reflect distinct ARE-binding proteins to modulate their RNA expression.     

Tristetraprolin Inhibits Poly(A)-Tail Synthesis in Nuclear mRNA that Contains AU-Rich Elements by Interacting with Poly(A)-Binding Protein Nuclear 1

Background

Tristetraprolin binds mRNA AU-rich elements and thereby facilitates the destabilization of mature mRNA in the cytosol.

Methodology/Principal Findings

To understand how tristetraprolin mechanistically functions, we biopanned with a phage-display library for proteins that interact with tristetraprolin and retrieved, among others, a fragment of poly(A)-binding protein nuclear 1, which assists in the 3''-polyadenylation of mRNA by binding to immature poly(A) tails and thereby increases the activity of poly(A) polymerase, which is directly responsible for polyadenylation. The tristetraprolin/poly(A)-binding protein nuclear 1 interaction was characterized using tristetraprolin and poly(A)-binding protein nuclear 1 deletion mutants in pull-down and co-immunoprecipitation assays. Tristetraprolin interacted with the carboxyl-terminal region of poly(A)-binding protein nuclear 1 via its tandem zinc finger domain and another region. Although tristetraprolin and poly(A)-binding protein nuclear 1 are located in both the cytoplasm and the nucleus, they interacted in vivo in only the nucleus. In vitro, tristetraprolin bound both poly(A)-binding protein nuclear 1 and poly(A) polymerase and thereby inhibited polyadenylation of AU-rich element–containing mRNAs encoding tumor necrosis factor α, GM-CSF, and interleukin-10. A tandem zinc finger domain–deleted tristetraprolin mutant was a less effective inhibitor. Expression of a tristetraprolin mutant restricted to the nucleus resulted in downregulation of an AU-rich element–containing tumor necrosis factor α/luciferase mRNA construct.

Conclusion/Significance

In addition to its known cytosolic mRNA–degrading function, tristetraprolin inhibits poly(A) tail synthesis by interacting with poly(A)-binding protein nuclear 1 in the nucleus to regulate expression of AU-rich element–containing mRNA.     

Orphan nuclear receptor Nur77 accelerates the initial phase of adipocyte differentiation in 3T3-L1 cells by promoting mitotic clonal expansion

Nur77 is an orphan member of the nuclear receptor superfamily that is expressed in various types of cells and mediates diverse biological processes. Although Nur77 mRNA is induced in the early stage of adipogenesis of 3T3-L1 cells, its roles are not known. To address this issue, we closely inspected the expression of Nur77 mRNA and protein during differentiation of 3T3-L1 cells. Nur77 was induced rapidly and transiently at both mRNA and protein levels only in the initial phase of differentiation induction, and localized almost exclusively in the nuclei. Isobutylmethylxanthine was essential for the induction of Nur77 protein, acting by at least in part protecting the protein from rapid degradation by proteasome. Nur77 siRNA resulted in delayed adipogenesis in 3T3-L1, accompanied by retarded mitotic clonal expansion. These effects were mediated at least partly by decreased expression of cyclins D and E. Constitutive expression of Nur77 inhibited adipogenesis of 3T3-L1, accompanied by enhanced expression of cyclin D1 and prolonged mitotic clonal expansion. Moreover, constitutive expression of Nur77 inhibited, but transient induction of Nur77 promoted, adipogenesis in NIH-3T3 cells. These results suggest that Nur77 accelerates adipocyte differentiation by regulating cell cycle progression and the rapid and transient induction is crucial for its action.     

Resveratrol Induces Glioma Cell Apoptosis through Activation of Tristetraprolin

Tristetraprolin (TTP) is an AU-rich elements (AREs)-binding protein, which regulates the decay of AREs-containing mRNAs such as proto-oncogenes, anti-apoptotic genes and immune regulatory genes. Despite the low expression of TTP in various human cancers, the mechanism involving suppressed expression of TTP is not fully understood. Here, we demonstrate that Resveratrol (3,5,4′-trihydroxystilbene, Res), a naturally occurring compound, induces glioma cell apoptosis through activation of tristetraprolin (TTP). Res increased TTP expression in U87MG human glioma cells. Res-induced TTP destabilized the urokinase plasminogen activator and urokinase plasminogen activator receptor mRNAs by binding to the ARE regions containing the 3′ untranslated regions of their mRNAs. Furthermore, TTP induced by Res suppressed cell growth and induced apoptosis in the human glioma cells. Because of its regulation of TTP expression, these findings suggest that the bioactive dietary compound Res can be used as a novel anti-cancer agent for the treatment of human malignant gliomas.     

Assays for monitoring viral manipulation of host ARE-mRNA turnover

Early host responses to viral infection rapidly induce an antiviral gene expression program that limits viral replication and recruits sentinel cells of the innate immune system. These responses are mediated by cytokines. The mRNAs that encode cytokines typically harbor destabilizing adenine- and uridine-rich elements (AREs) that direct their constitutive degradation in the cytoplasm. In response to a variety of signals, including viral infection, small pools of cytoplasmic ARE-mRNAs are rapidly stabilized and translated. Thus, mRNA stability plays a key role in antiviral gene expression. Intriguingly, recent studies have identified viral proteins that specifically target ARE-mRNAs for stabilization, suggesting that certain proteins encoded by ARE-mRNAs may be advantageous for infection. Here, we discuss the development of a suite of sensitive and complementary assays to monitor ARE-mRNA turnover. These include luciferase- and destabilized-GFP-based assays that can be adapted for high-throughput screening applications.     

Expression of estrogen synthetase (P-450 aromatase) during adipose differentiation of 3T3-L1 cells.

The expression of estrogen synthetase (aromatase), catalyzing a rate limiting reaction in estrogen formation, was examined in 3T3-L1 cells during adipose differentiation. The expression of another P-450 enzyme, cholesterol side-chain cleavage enzyme (P-450scc) by the cells was also studied for comparison. The level of specific mRNA for aromatase increased 17-fold during adipogenic conversion and the elevated level was maintained in fully differentiated adipocytes. The level of specific mRNA for P-450scc increased about 5-fold, mainly due to net increase of cellular RNA. Various reagents, such as dexamethasone, testosterone and 1-methyl-3-isobutylxanthine, affected the expression of specific mRNA for aromatase markedly in adipocytes but had scarcely any effect on its level in fibroblasts. In contrast, these reagents caused similar increases in the level of mRNA for P-450scc in the two types of cells. Thus the 3T3-L1 cell line during adipogenic differentiation may be a useful system for studies on the mechanism regulating aromatase gene expression.     

A novel pathway to enhance adipocyte differentiation of 3T3-L1 cells by up-regulation of lipocalin-type prostaglandin D synthase mediated by liver X receptor-activated sterol regulatory element-binding protein-1c

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is expressed in adipocytes and is proposed to be involved in the regulation of glucose tolerance and atherosclerosis in type 2 diabetes, because L-PGDS gene knock-out mice show abnormalities in these functions. However, the role of L-PGDS and the regulation mechanism governing its gene expression in adipocytes remain unclear. Here, we applied small interference RNA of L-PGDS to mouse 3T3-L1 cells and found that it suppressed differentiation of these cells into adipocytes. Reporter analysis of the mouse L-PGDS promoter demonstrated that a responsive element for liver receptor homolog-1 (LRH-1) at -233 plays a critical role in preadipocytic 3T3-L1 cells. Moreover, we identified two sterol regulatory elements (SREs) at -194 to be cis-elements for activation of L-PGDS gene expression in adipocytic 3T3-L1 cells. L-PGDS mRNA was induced in response to synthetic liver X receptor agonist, T0901317, through activation of the expression of SRE-binding protein-1c (SREBP-1c) in the adipocytic 3T3-L1 cells. The results of electrophoretic mobility shift assay and chromatin immunoprecipitation assay revealed that LRH-1 and SREBP-1c bound to their respective binding elements in the promoter of L-PGDS gene. Small interference RNA-mediated suppression of LRH-1 or SREBP-1c decreased L-PGDS gene expression in preadipocytic or adipocytic 3T3-L1 cells, respectively. These results indicate that L-PGDS gene expression is activated by LRH-1 in preadipocytes and by SREBP-1c in adipocytes. Liver X receptor-mediated up-regulation of L-PGDS through activation of SREBP-1c is a novel path-way to enhance adipocyte differentiation.     

Phosphorylation of mRNA Decapping Protein Dcp1a by the ERK Signaling Pathway during Early Differentiation of 3T3-L1 Preadipocytes

Background

Turnover of mRNA is a critical step in the regulation of gene expression, and an important step in mRNA decay is removal of the 5′ cap. We previously demonstrated that the expression of some immediate early gene mRNAs is controlled by RNA stability during early differentiation of 3T3-L1 preadipocytes.

Methodology/Principal Findings

Here we show that the mouse decapping protein Dcp1a is phosphorylated via the ERK signaling pathway during early differentiation of preadipocytes. Mass spectrometry analysis and site-directed mutagenesis combined with a kinase assay identified ERK pathway–mediated dual phosphorylation at Ser 315 and Ser 319 of Dcp1a. To understand the functional effects of Dcp1a phosphorylation, we examined protein-protein interactions between Dcp1a and other decapping components with co-immunoprecipitation. Dcp1a interacted with Ddx6 and Edc3 through its proline-rich C-terminal extension, whereas the conserved EVH1 (enabled vasodilator-stimulated protein homology 1) domain in the N terminus of Dcp1a showed a stronger interaction with Dcp2. Once ERK signaling was activated, the interaction between Dcp1a and Ddx6, Edc3, or Edc4 was not affected by Dcp1a phosphorylation. Phosphorylated Dcp1a did, however, enhanced interaction with Dcp2. Protein complexes immunoprecipitated with the recombinant phosphomimetic Dcp1a(S315D/S319D) mutant contained more Dcp2 than did those immunoprecipitated with the nonphosphorylated Dcp1a(S315A/S319A) mutant. In addition, Dcp1a associated with AU-rich element (ARE)-containing mRNAs such as MAPK phosphatase-1 (MKP-1), whose mRNA stability was analyzed under the overexpression of Dcp1a constructs in the Dcp1a knockdown 3T3-L1 cells.

Conclusions/Significance

Our findings suggest that ERK-phosphorylated Dcp1a enhances its interaction with the decapping enzyme Dcp2 during early differentiation of 3T3-L1 cells.     

G-protein mRNA levels during adipocyte differentiation

G-protein-mediated transmembrane signaling in 3T3-L1 cells is modulated by differentiation. The regulation of G-protein expression in differentiating 3T3-L1 cells was probed at the level of mRNA by DNA-excess solution hybridization. Pertussis toxin-catalyzed ADP-ribosylation of G-protein alpha-subunits increased as fibroblasts differentiate to adipocytes. Steady-state levels of mRNA for Gi alpha 2 and Go alpha, in contrast, declined sharply. Immunoblotting with antipeptide antibodies specific for Gi alpha 2, too, revealed a decline in the steady-state expression of this pertussis toxin substrate. ADP-ribosylation of Gs alpha by cholera toxin was less in the adipocyte than fibroblast. Analysis by immunoblotting revealed only a modest decline in Gs alpha. Analysis of mRNA levels also demonstrated a decline for Gs alpha. mRNA levels for the G beta-subunits rose initially (25%) on day 1, declined from day 1 to day 3, and remained 25% lower in adipocytes than in fibroblasts. In 3T3-L1 adipocytes the molar amounts of subunit mRNAs were: 60.6 (Gs alpha); 2.1 (Gi alpha 2); and 1.5 (Go alpha) amol/microgram total cellular RNA. In rat fat cells these mRNA levels were 19.4 (Gs alpha); 7.0 (Gi alpha 2); and 2.3 (Go alpha). These data demonstrate that for Gi alpha 2 and Go alpha alike mRNA and protein expression decrease, not increase, in differentiation. A substrate for pertussis toxin other than Gi alpha 2 and Go alpha appears to be responsible for the increase in toxin-catalyzed labeling that accompanies differentiation of 3T3-L1 cells.