Deficiency in the extracellular signal-regulated kinase 1 (ERK1) protects leptin-deficient mice from insulin resistance without affecting obesity.

AIMS/HYPOTHESIS: Extracellular signal-regulated kinase (ERK) activity is increased in adipose tissue in obesity and type 2 diabetes mellitus and strong evidences suggests that it is implicated in the downregulation of insulin signalling and action in the insulin-resistant state. To determine the role of ERK1 in obesity-associated insulin resistance in vivo, we inactivated Erk1 (also known as Mapk3) in obese leptin-deficient mice (ob/ob). METHODS: Mice of genotype ob/ob-Erk1⁻(/)⁻ were obtained by crossing Erk1⁻(/)⁻ mice with ob/ob mice. Glucose tolerance and insulin sensitivity were studied in 12-week-old mice. Tissue-specific insulin sensitivity, insulin signalling, liver steatosis and adipose tissue inflammation were determined. RESULTS: While ob/ob-Erk1⁻(/)⁻ and ob/ob mice exhibited comparable body weight and adiposity, ob/ob-Erk1⁻(/)⁻ mice did not develop hyperglycaemia and their glucose tolerance was improved. Hyperinsulinaemic-euglycaemic clamp studies demonstrated an increase in whole-body insulin sensitivity in the ob/ob-Erk1⁻(/)⁻ mice associated with an increase in both insulin-stimulated glucose disposal in skeletal muscles and adipose tissue insulin sensitivity. This occurred in parallel with improved insulin signalling in both tissues. The ob/ob-Erk1⁻(/)⁻ mice were also partially protected against hepatic steatosis with a strong reduction in acetyl-CoA carboxylase level. These metabolic improvements were associated with reduced expression of mRNA encoding inflammatory cytokine and T lymphocyte markers in the adipose tissue. CONCLUSIONS/INTERPRETATION: Our results demonstrate that the targeting of ERK1 could partially protect obese mice against insulin resistance and liver steatosis by decreasing adipose tissue inflammation and by increasing muscle glucose uptake. Our results indicate that deregulation of the ERK1 pathway could be an important component in obesity-associated metabolic disorders.

p38MAP Kinase activity is required for human primary adipocyte differentiation.

Little is known about the role of p38MAPK in human adipocyte differentiation. Here we showed that p38MAPK activity increases during human preadipocytes differentiation. Pharmacological inhibition of p38MAPK during adipocyte differentiation of primary human preadipocytes markedly reduced triglycerides accumulation and adipocyte markers expression. Cell cycle arrest or proliferation was not affected by p38MAPK inhibition. Although induction of C/EBPbeta was not altered by the p38MAPK inhibitor, its phosphorylation on Threonine(188) was decreased as well as PPARgamma expression. These results indicate that p38MAPK plays a positive role in human adipogenesis through regulation of C/EBPbeta and PPARgamma factors.

Stepwise transformation of rat embryo fibroblasts: c-Jun, JunB, or JunD can cooperate with Ras for focus formation, but a c-Jun-containing heterodimer is required for immortalization.

Among the Jun family of transcription factors, only c-Jun displays full transforming potential in cooperation with activated c-Ha-Ras in primary rat embryo fibroblasts. c-Jun in combination with Ras can both induce foci of transformed cells from rat embryo fibroblast monolayers and promote the establishment of these foci as tumoral cell lines. JunB can also cooperate with Ras to induce foci but is unable to promote immortalization. We report here that JunD, in cooperation with Ras, induces foci with an efficiency similar to that of JunB. Artificial Jun/eb1 derivatives from each of the three Jun proteins were also analyzed. These constructs carry a heterologous homodimerization domain from the viral EB1 transcription factor and are thought to form only homodimers in the cell. We show here that these Jun/eb1 chimeras are potent transactivators of AP1 sites and that they can cooperate with c-Ha-Ras to induce foci. However, among all the Ras-Jun and Ras-Jun/eb1 combinations tested, only foci from Ras-c-Jun can be efficiently expanded and maintained as long-term growing cultures. Therefore, we suggest that a heterodimer containing c-Jun might be required for in vitro establishment of these primary mammalian cells.

The transactivating domain of the c-Jun proto-oncoprotein is required for cotransformation of rat embryo cells.

The nuclear phosphoprotein c-Jun, encoded by the proto-oncogene c-jun, is a major component of the AP-1 complex. A potent transcriptional regulator, c-jun is also able to transform normal rat embryo cells in cooperation with an activated c-Ha-ras gene. By deletion analysis, we identified the regions of c-Jun encoding transformation and transactivation functions. Our studies indicate that there is a direct correlation between the ability of the c-Jun protein to activate transcription and cotransform rat embryo cells. The regions involved in these functions include the conserved leucine zipper/DNA binding domain and an effector domain near its N terminus. This N-terminal region spans amino acids 61 to 146 of the c-Jun protein and is highly conserved among all Jun family members. These results support the hypothesis that c-Jun transforms cells by stimulating the expression of transformation-mediating genes.

Oncoprotein-mediated signalling cascade stimulates c-Jun activity by phosphorylation of serines 63 and 73.

In resting cells, c-Jun is phosphorylated on five sites. Three of these sites reside next to its DNA binding domain and negatively regulate DNA binding. In response to expression of oncogenic Ha-Ras, phosphorylation of these sites decreases, while phosphorylation of two other sites within c-Jun's activation domain is greatly enhanced. Phosphorylation of these residues, serines 63 and 73, stimulates the transactivation function of c-Jun and is required for oncogenic cooperation with Ha-Ras. We now show that the same changes in c-Jun phosphorylation are elicited by a variety of transforming oncoproteins with distinct biochemical activities. These oncoproteins, v-Sis, v-Src, Ha-Ras, and Raf-1, participate in a signal transduction pathway that leads to increased phosphorylation of serines 63 and 73 on c-Jun. While oncogenic Ha-Ras is a constitutive stimulator of c-Jun activity and phosphorylation, the normal c-Ha-Ras protein is a serum-dependent modulator of c-Jun's activity. c-Jun is therefore a downstream target for a phosphorylation cascade involved in cell proliferation and transformation.

The c-Jun-induced transformation process involves complex regulation of tenascin-C expression.

In cooperation with an activated ras oncogene, the site-dependent AP-1 transcription factor c-Jun transforms primary rat embryo fibroblasts (REF). Although signal transduction pathways leading to activation of c-Jun proteins have been extensively studied, little is known about c-Jun cellular targets. We identified c-Jun-upregulated cDNA clones homologous to the tenascin-C gene by differential screening of a cDNA library from REF. This tightly regulated gene encodes a rare extracellular matrix protein involved in cell attachment and migration and in the control of cell growth. Transient overexpression of c-Jun induced tenascin-C expression in primary REF and in FR3T3, an established fibroblast cell line. Surprisingly, tenascin-C synthesis was repressed after stable transformation by c-Jun compared to that in the nontransformed parental cells. As assessed by using the tenascin-C (-220 to +79) promoter fragment cloned in a reporter construct, the c-Jun-induced transient activation is mediated by two binding sites: one GCN4/AP-1-like site, at position -146, and one NF-kappaB site, at position -210. Furthermore, as demonstrated by gel shift experiments and cotransfections of the reporter plasmid and expression vectors encoding the p65 subunit of NF-kappaB and c-Jun, the two transcription factors bind and synergistically transactivate the tenascin-C promoter. We previously described two other extracellular matrix proteins, SPARC and thrombospondin-1, as c-Jun targets. Thus, our results strongly suggest that the regulation of the extracellular matrix composition plays a central role in c-Jun-induced transformation.

Role of MAPKs in development and differentiation: lessons from knockout mice.

The ERK, p38MAPK, JNK mitogen-activated protein kinases (MAPKs) are intracellular signaling pathways that play a pivotal role in many essential cellular processes such as proliferation and differentiation. These cascades are activated by a large variety of stimuli and display a high degree of homology. So far, seven MAPK isoforms have been invalidated in mice leading to the discovery of their important functions in development and differentiation. As we could expect because of their multiple and specific properties in vitro, knockout (KO) of MAPK pathways leads to distinct phenotypes in mice. Surprisingly, into a given cascade, KOs of the various isoforms assign specific non-redundant biological functions to each isoform, without compensation by the others. These results emphasize the notion that, although initiated by the same external stimuli, these intracellular cascades activate kinase isoforms each with its own specific role.

Non-selective analysis of the transformation of FR3T3 rat cells by bovine papillomavirus type 1: regulations of viral transcription associated with phenotypic transformation.

Drug-resistant clones selected from FR3T3 rat cells after transfer of neo-BPV1 (Bovine Papillomavirus Type 1) DNA constructs became phenotypically transformed (focal transformation, growth in suspension and tumor formation) soon after selection (approximately 5 generations in culture). A frameshift mutation in ORF E5 abolished transformation, but did not prevent the autonomous maintenance of the DNA construct. A more complex situation was observed when the E2 transactivating function was abrogated. A minority of the E2(-)-neor clones became phenotypically transformed shortly after drug selection, but the majority maintained normal growth properties for 30 to 50 generations. The rate of viral transcription was uniformly high in cells which exhibited transformed growth properties early after selection (the E2- minority class and all the wild type transformants) and low in phenotypically normal cells (the majority of the E2- lines). The same low transcriptional activity and delayed expression of transformed growth properties had been observed after transfection of a similar construct carrying a wild type viral early region (69-T fragment), but lacking the late region. The elevated rate of viral transcription, which correlates with the immediate expression of transformation, appears therefore to require at least two distinct elements, the E2 transactivator function and sequences in the late region of the viral genome. In their absence, high transcription rates and transformation could be established only in a minority of the transfected clones, by an unknown, E2-independent mechanism. Evidence was obtained for a third transformation route which, in the absence of either E2 or the late region, led to the focal occurrence of transformed derivatives after 30 to 50 generations of normal growth, but was not associated with an overall increase in viral expression.

junB promoter regulation: Ras mediated transactivation by c-Ets-1 and c-Ets-2.

The Jun gene family encode components of the AP-1 transcription factor complex that regulate a variety of TRE-containing target promoters. Expression of family members is induced by a wide variety of extracellular stimuli and thought to be important in mediating cellular proliferation and differentiation. We have localized cis-acting DNA sequences in the murine junB promoter capable of mediating transcriptional activation by the proto-oncogene products c-Ets-1 and c-Ets-2. We show by promoter deletion analysis that multiple elements located between -848 and -574, and between -196 and -91 can mediate transactivation by ETS-family members in different cell types. In vitro DNA binding assays indicate that the elements identified can specifically interact with c-Ets-1 protein. Furthermore, we show that ETS-transactivation of a variety of reporter constructs is dramatically enhanced by introduction of oncogenic Ha-ras. The activation of Ras by extracellular stimuli invokes a phosphorylation cascade that includes the downstream mitogen-activated protein (MAP) kinase p44ERK-1. We further show that addition of activated p44ERK-1 MAP kinase can also enhance ETS-transactivation of junB promoter reporter constructs. Here we propose that ETS-family members play a role in the activation of junB transcription by a Ras-stimulated signal transducing pathway that includes MAP kinase(s).

The defective transforming phenotype of c-Jun Ala(63/73) is rescued by mutation of the C-terminal phosphorylation site.

Cotransfection of primary rat embryo fibroblasts (REF) with c-Jun and activated Ras leads to oncogenic transformation and this process requires the phosphorylation of the N-terminal domain of c-Jun. Ras augments this phosphorylation and, consequently activates the c-Jun transactivation property of TRE (TPA Responsive Element)-dependent promoters. To analyse the role of the c-Jun C-terminal phosphorylation site in oncogenic cooperation we tested the activities of N-terminal c-Jun Ala(63/73) (named Nt), C-terminal c-Jun Ala(234/242/246/252) (named Ct) and (Nt+Ct)-with both mutations-non-phosphorylatable c-Jun mutants. In cooperation with Ras, the Ct mutant and wt c-Jun display similar oncogenic properties whereas the Nt form was defective in transforming REF cells. Unexpectedly, the Nt+Ct mutant exhibited identical oncogenic properties to wt c-Jun, demonstrating that the Ct mutation rescues in cis the Nt mutation. The transcriptional activity and the capacity to bind the c-Jun coactivator CREB Binding Protein (CBP) were enhanced by Ras for the wt and Ct proteins but not for the Nt mutant. Interestingly, the Nt+Ct mutant presents identical transactivation and CBP binding activities to wt c-Jun. Therefore the rescue in cis of the defective Nt mutation by the Ct mutation seems to be due to the recovery of CBP binding. Our results revealed that the process of oncogenic cooperation can occur between Ras and the Nt+Ct non-phosphorylatable c-Jun protein.

The Wilms' tumor gene product represses the transcription of thrombospondin 1 in response to overexpression of c-Jun.

Thrombospondin 1 (TSP1) is known for its significant anti-angiogenic properties. In a previous study, we have shown that transient or stable overexpression of the transcription factor c-Jun, in rat fibroblasts, leads to repression of TSP1. We now demonstrate that the c-Jun-induced repression of TSP1 does not occur directly and does not require binding of c-Jun to the TSP1 promoter. Instead, repression involves a factor secreted by c-Jun-overexpressing cells. This secreted factor triggers a signal transduction pathway from the membrane to the nucleus, and these signals lead to the binding of the product of the Wilms' tumor suppressor gene, WT1, to the -210 region of the TSP1 promoter. This region binds WT1 and SP1, but not EGR1, although its sequence fits the consensus binding site for this transcription factor. WT1 overexpression in transfected cells inhibits endogenous TSP1 gene expression and TSP1 transcription in experiments using TSP1 promoter-reporter constructs. The WT1 - KTS isoform is more active in repressing TSP1 transcription than WT1 + KTS, while EGR1 is inactive. Enhancement of WT1 binding to DNA in response to c-Jun does not require de novo protein synthesis. The above mechanism for TSP1 repression could apply to other genes, thus coordinating their regulation in the vicinity of a c-Jun-overexpressing cell. We conclude that WT1, which was discovered as a result of its tumor suppressor properties, may also possess oncogenic characteristics in the c-Jun transformation process, and thus repress the anti-angiogenic protein, TSP1.

The role of MAPKs in adipocyte differentiation and obesity.

The ERK, p38 and JNK mitogen activated protein kinases (MAPKs) are intracellular signalling pathways that play a pivotal role in many essential cellular processes such as proliferation and differentiation. MAPKs are activated by a large variety of stimuli and one of their major functions is to connect cell surface receptors to transcription factors in the nucleus, which consequently triggers long-term cellular responses. This review focuses on their in vitro and in vivo roles in adipocyte differentiation and obesity. Hyperplasia of adipose tissue is a critical event for the development of obesity. Several studies have analysed the role of MAPKs in vitro in adipocyte differentiation of preadipocyte established cell lines. In the case of ERK, although the first data appeared contradictory, a consensus scenario arises: ERK would be necessary to initiate the preadipocyte into the differentiation process and, thereafter, this signal transduction pathway needs to be shut-off to proceed with adipocyte maturation. The limitation of these cellular models is that only terminal adipocyte differentiation can be analysed, eluding the early proliferative steps of adipogenesis. New insights are now emerging by investigations conducted either in vitro with the use of embryonic stem (ES) cells or in vivo with mice where these genes are invalidated. These studies not only confirm and/or precise the various functions of MAPKs in adipogenesis but, importantly, reveal unsuspected roles, for example JNK in obesity or ERK in adipogenesis of ES cells, and, for a given pathway, assign specific functions to each isoform. It appears now that a fine tuning of the MAPKs regulates both normal and pathological adipogenesis. The precise understanding of the cascade of these molecular events and the way to regulate them will be certainly crucial in order to efficiently fight obesity.

Mechanisms in interleukin-2 protection against glucocorticoid-induced apoptosis: regulation of AP-1 and glucocorticoid receptor transcriptional activities.

We have used the gibbon ape leukemia cell line MLA-144 and its corticoid-sensitive subclone MLA-E7T to analyze the mechanisms whereby interleukin-2 (IL-2) can protect T cells against dexamethasone-induced apoptosis. MLA cells are characterized by the constitutive expression of intermediate affinity receptors for IL-2, together with IL-4 receptors. MLA-144 cells secrete IL-2 and are insensitive to dexamethasone, whereas MLA-E7T cells do not constitutively produce significant amounts of IL-2 and undergo apoptotic cell death in the presence of dexamethasone. Exogenous IL-2 was shown to protect MLA-E7T cells against the apoptotic effect of dexamethasone and to increase both the DNA binding and transactivating functions of activator protein-1 (AP-1). The functional relationship between AP-1 and glucocorticoid receptors transcriptional activities was further investigated using transient expression of reporter gene constructs whose transcriptions are regulated by promoters containing TPA-responsive elements or glucocorticoid-responsive elements. The data reported here demonstrate that in MLA-144 cells, IL-2 or PMA stimulation antagonizes the glucocorticoid receptor, whereas in MLA-E7T, synergistic effects are observed between dexamethasone and IL-2 or PMA for transactivation of MMTV-CAT. Taken together with the finding that IL-2 but not PMA protects MLA-E7T from dexamethasone-induced apoptosis, our results indicate that IL-2 does not induce such a protection by repressing the transcriptional activity of the glucocorticoid receptor.

A p38MAPK-p53 cascade regulates mesodermal differentiation and neurogenesis of embryonic stem cells.

Embryonic stem cells (ESCs) differentiate in vivo and in vitro into all cell lineages, and they have been proposed as cellular therapy for human diseases. However, the molecular mechanisms controlling ESC commitment toward specific lineages need to be specified. We previously found that the p38 mitogen-activated protein kinase (p38MAPK) pathway inhibits neurogenesis and is necessary to mesodermal formation during the critical first 5 days of mouse ESC commitment. This period corresponds to the expression of specific master genes that direct ESC into each of the three embryonic layers. By both chemical and genetic approaches, we found now that, during this phase, the p38MAPK pathway stabilizes the p53 protein level and that interfering directly with p53 mimics the effects of p38MAPK inhibition on ESC differentiation. Anti-p53 siRNA transient transfections stimulate Bcl2 and Pax6 gene expressions, leading to increased ESC neurogenesis compared with control transfections. Conversely, p53 downregulation leads to a strong inhibition of the mesodermal master genes Brachyury and Mesp1 affecting cardiomyogenesis and skeletal myogenesis of ESCs. Similar results were found with p53(-/-) ESCs compared with their wild-type counterparts. In addition, knockout p53 ESCs show impaired smooth muscle cell and adipocyte formation. Use of anti-Nanog siRNAs demonstrates that certain of these regulations result partially to p53-dependent repression of Nanog gene expression. In addition to its well-known role in DNA-damage response, apoptosis, cell cycle control and tumor suppression, p53 has also been involved in vivo in embryonic development; our results show now that p53 mediates, at least for a large part, the p38MAPK control of the early commitment of ESCs toward mesodermal and neural lineages.

The Lac repressor provides a reversible gene expression system in undifferentiated and differentiated embryonic stem cell.

Control of mammalian gene promoters by the bacterial LacI repressor provides reversible regulation and dose-response levels of derepressed expression by the lactose analog isopropyl thiogalactose (IPTG). Here, we show that insertion of LacI-binding sites in the ubiquitous beta-actin promoter confers a strong and dose-dependent IPTG-regulatable expression of transiently transfected reporter genes in mouse embryonic stem (ES) cells expressing LacI. We established ES cell lines stably expressing reporter genes under inducible control and found a five- to tenfold IPTG induction of transgene expression. The kinetics of induction is rapid and stable, and can be rapidly reversed after IPTG removal. Importantly, this regulatable expression was maintained throughout the differentiation process of ES cells, and observed in individual differentiated cardiomyocyte-like cells and neuronal-like cells. This reversible system is the first to function from undifferentiated to individual well-differentiated ES cells, providing a very useful tool to understand molecular mechanisms underlying ES cell self-renewal, commitment and differentiation.

Ha-Ras augments c-Jun activity and stimulates phosphorylation of its activation domain.

Ha-Ras augments c-Jun-mediated transactivation by potentiating the activity of the c-Jun activation domain. Ha-Ras also causes a corresponding increase in phosphorylation of specific sites in that part of the c-Jun protein. A Ha-Ras-induced protein kinase cascade resulting in hyperphosphorylation of the c-Jun activation domain could explain how these oncoproteins cooperate to transform rat embryo fibroblasts.

Conditional requirement for sequences distinct from the replication origin during episomal establishment of the BPV1 genome.

Removal from BPV1 DNA of a short segment (nt. 4786-5045) that contains several protein binding sites and is required for efficient replication in short term assays prevents its autonomous maintenance in cell lines established by selection in G418 medium after cotransfer of neo(r). In contrast, transformed cell lines established from foci, which express the viral genes at higher levels, maintain extrachromosomal copies of the deleted DNA. Two modes of maintenance of the viral genome are thus distinguished by their requirement for sequences in this region.

Recombinant DNA molecules comprising bovine papilloma virus type 1 DNA linked to plasmid DNA are maintained in a plasmidial state both in rodent fibroblasts and in bacterial cells.

Transformed cells obtained after transfecting FR3T3 rat fibroblasts with DNA of bovine papilloma virus type 1 ( BPV1 ) maintained only free copies of the viral genome. Transfection with BPV1 DNA inserted in a bacterial plasmid (pBR322 or pML2 ) did not produce transformants at a detectable rate, unless the viral sequences had been first excised from the plasmid. In contrast, transfer of the same plasmids by polyethylene glycol-induced fusion of bacterial protoplasts with FR3T3 rat or C127 mouse cells led to significant transformation frequencies. A total of eight cell lines were studied, three rat and five mouse transformants, obtained with various BPV1 - pML2 recombinants. In all cell lines, both BPV1 and plasmid sequences were maintained as non-integrated molecules, predominantly as oligomeric forms of the transforming DNA. In the three rat transformants and in two of the mouse lines, parts of the non-transforming viral region and some bacterial sequences were deleted. In the remaining three mouse lines, the monomeric repeat was a non-rearranged plasmid molecule which could be re-established as a plasmid in Escherichia coli after cleavage with "one-cut" restriction endonucleases and circularization of the molecule.

Plasmidial maintenance in rodent fibroblasts of a BPV1-pBR322 shuttle vector without immediately apparent oncogenic transformation of the recipient cells.

A recombinant plasmid was constructed (pV69) which comprises a subgenomic fragment of bovine papilloma virus type 1 (BPV1) DNA, part of plasmid pBR322 DNA and a drug resistance gene expressed in both mammalian fibroblasts and Escherichia coli. This gene (vv2) is a modified form of the bacterial neomycin resistance gene (neo) linked to the herpes simplex virus thymidine kinase (tk) promoter (plasmid pAG60), to which the original bacterial neo promoter from transposon Tn5 was added back, upstream of the eukaryotic promoter. It induced kanamycin resistance in E. coli, as well as resistance to the drug G418 in rat and mouse fibroblasts. Its expression in FR3T3 rat cells was enhanced as compared with the original tk-neo construction. After transfer of plasmid pV69 into C127 mouse cells or FR3T3 rat cells, the number of resistant colonies selected in medium containing G418 was one to two orders of magnitude higher than that of transformed foci in normal medium. In eight independent cell lines selected by drug resistance, pV69 DNA was found to be maintained in a plasmidial state, without any detectable rearrangement or deletion and could be transferred back in E. coli. In contrast, cell lines selected by focus formation in normal medium maintained deleted forms of the original plasmid DNA, and only part of them were resistant to G418. Most of the drug-resistant clones had kept the morphology and growth control of the normal fibroblasts. However, with further passages in culture, these cells spontaneously produced transformed foci with increasing frequencies.

Successive steps in the process of immortalization identified by transfer of separate bovine papillomavirus genes into rat fibroblasts.

Transfer of neor and bovine papillomavirus type 1 (BPV1) DNA into rat embryo fibroblasts led to colony formation in G418-containing medium, with no detectable background in controls with neor DNA alone. More than 50% of the drug-resistant clones could be further propagated in culture. The genetic functions of BPV1 involved in colony formation and in long-term immortalization were investigated by both translation termination mutations in the full-length genome, which inactivate individual open reading frames, and constructs in which these open reading frames were separately expressed under control of long terminal repeat promoter enhancers. Expression of either open reading frame E2 or E5 was sufficient for formation of a drug-resistant colony, but long-term growth in culture required that of E6. No significant cooperative effect was observed upon cotransfection of BPV1 and ras oncogene DNAs. Expression of the early region of the human papillomavirus type 16 also led to immortalization of rat embryo fibroblast cells in the same assay, and, unlike what was previously reported in baby rat kidney cells, it required neither activation by a heterologous promoter, nor a cooperating ras oncogene.