Mise au point d'un essai kinase in vitro pour la MAPKKK[indice supérieur DLK] et identification de ses activateurs

Les "mitogen-activated protein kinases" (MAPKs) forment une famille de protéines exerçant un rôle crucial dans la régulation de nombreux processus physiologiques, tels la prolifération, la différentiation, l'apoptose et la survie cellulaire, en permettant la transmission de signaux extracellulaires jusqu'au noyau. Cette cascade de transduction se compose de trois principales voies et chacune de ces voies est constituée d'un module de trois protéines kinases différentes, que l'on désigne les MAPKKKs, MAPKKs et MAPKs selon leur position dans le module. Notre laboratoire s'intéresse particulièrement à un des membres de la famille des MAPKKKs soit la MAPKKK [indice supérieur DLK]. Cette dernière, qui est située en amont de la cascade, est une sérine/thréonine kinase appartenant à la famille des"mixed-lineage kinases" (MLKs). Bien que les MLKs soient des activateurs d'un sous-groupe de MAPKs [indice supérieur JNK] dont la fonction est généralement associée à l'arrêt de la croissance cellulaire et/ou l'apoptose, il n'existe encore aucune donnée expérimentale démontrant l'implication directe de ces protéines dans l'un ou l'autre de ces processus biologiques. Afin de caractériser ces mécanismes d'action, nous avons tenté d'identifier les activateurs susceptibles de stimuler l'activité kinase de la MAPKKK [indice supérieur DLK]. Pour ce faire, nous avons établi un système in vitro qui reconstitue entièrement la cascade de la voie des MAPKs[indice supérieur JNK]. Ce système a permis de mesurer directement les modulations de l'activité enzymatique endogène et transfectée de la MAPKKK[indice supérieur DLK] suite à certaines stimulations. Grâce à l'efficacité de ce système, ces travaux ont permis de démontrer pour la première fois une activitation de la forme endogène et transfectée de la MAPKKK [indice supérieur DLK], en réponse à des stimuli extracellulaires pro-apoptotiques

Down-regulation of the Mixed-lineage Dual Leucine Zipper-bearing Kinase by Heat Shock Protein 70 and Its Co-chaperone CHIP

Dual leucine zipper-bearing kinase (DLK) is a mixed-lineage kinase family member that acts as an upstream activator of the c-Jun N-terminal kinases. As opposed to other components of this pathway, very little is currently known regarding the mechanisms by which DLK is regulated in mammalian cells. Here we identify the stress-inducible heat shock protein 70 (Hsp70) as a negative regulator of DLK expression and activity. Support for this notion derives from data showing that Hsp70 induces the proteasomal degradation of DLK when both proteins are co-expressed in COS-7 cells. Hsp70-mediated degradation occurs with expression of wild-type DLK, which functions as a constitutively activated protein in these cells but not kinase-defective DLK. Interestingly, the Hsp70 co-chaperone CHIP, an E3 ubiquitin ligase, seems to be indispensable for this process since Hsp70 failed to induce DLK degradation in COS-7 cells expressing a CHIP mutant unable to catalyze ubiquitination or in immortalized fibroblasts derived from CHIP knock-out mice. Consistent with these data, we have found that endogenous DLK becomes sensitive to CHIP-dependent proteasomal degradation when it is activated by okadaic acid and that down-regulation of Hsp70 levels with an Hsp70 antisense attenuates this sensitivity. Therefore, our studies suggest that Hsp70 contributes to the regulation of activated DLK by promoting its CHIP-dependent proteasomal degradation

The Mixed-Lineage Kinase DLK Is a Key Regulator of 3T3-L1 Adipocyte Differentiation

The mixed-lineage kinase (MLK) family member DLK has been proposed to serve as a regulator of differentiation in various cell types; however, its role in adipogenesis has not been investigated. In this study, we used the 3T3-L1 preadipocyte cell line as a model to examine the function of DLK in adipocyte differentiation.Immunoblot analyses and kinase assays performed on 3T3-L1 cells showed that the expression and activity of DLK substantially increase as differentiation occurs. Interestingly, DLK appears crucial for differentiation since its depletion by RNA interference impairs lipid accumulation as well as expression of the master regulators of adipogenesis C/EBPalpha and PPARgamma2 at both the mRNA and protein levels. In contrast, neither the expression nor the DNA binding activity of C/EBPbeta, an activator for C/EBPalpha and PPARgamma, is affected by DLK loss.Taken together, these results suggest that DLK is important for expression of mature adipocyte markers and that its action most likely takes place via regulation of C/EBPbeta transcriptional activity and/or initiation of C/EBPalpha and PPARgamma2 gene transcription

Depletion of DLK in 3T3-L1 cells blocks expression of PPARγ and C/EBPα at the mRNA levels.

<p>3T3-L1 cells infected with an empty lentivirus (EV), a lentivirus expressing human DLK shRNA (hDLK) or a lentivirus expressing mouse DLK shRNA (mDLK) were induced to differentiate, and at the indicated times, total RNA was extracted. C/EBPβ, C/EBPα, PPARγ, adiponectin and FAS mRNA levels were analyzed by quantitative RT-PCR with specific primers. The expression level of each gene was normalized to the level of the 36B4 housekeeping gene. Results are expressed as fold induction of mRNA levels relative to cells harvested on day 0 and they are representative of at least three independent experiments.</p

Up-regulation of DLK expression and activity during 3T3-L1 adipocyte differentiation.

<p>(A) Homogenates of mouse heart, white adipose tissue from various depots (gonadal, retroperitoneal, omental, mesenteric and inguinal) and brown adipose tissue (BAT) were subjected to Western blot analysis with specific antibodies against DLK or GAPDH. (B) and (D) 3T3-L1 cells induced to differentiate for 2, 4, 6, 8 and 10 days were lysed and subjected to immunoblotting analysis with antibodies to DLK, adiponectin, fatty acid synthase (FAS), PPARγ, ERK, phospho-ERK, p38, phospho-p38, JNK, phospho-JNK and tubulin as the loading control. The antibody raised against PPARγ reveals the presence of both PPARγ isoforms, PPARγ1 and PPARγ2. (C) 3T3-L1 cells induced to differentiate for the indicated times were lysed for immunoprecipitation analysis with anti-DLK antibody and subjected to an immunocomplex kinase assay using myelin basic protein (MBP) as a substrate. <i>Diff: Days of differentiation</i>. <i>WB: Western Blot</i>.</p

Knockdown of DLK does not interfere with the binding of C/EBPβ to the <i>cebpα</i> and <i>pparγ2</i> promoters.

<p>EV-, hDLK- and mDLK-infected 3T3-L1 cells induced to differentiate for two days were subjected to chromatin immunoprecipitation with an antibody specific to C/EBPβ. The precipitated chromatin was analyzed by quantitative PCR using primers spanning the binding site of C/EBPβ within the <i>cebpα</i> and <i>pparγ2</i> promoters. The data represent the mean±SEM of three independent experiments, relative to control EV-infected cells.</p

DLK depletion in 3T3-L1 impairs lipid accumulation in response to differentiation inducers.

<p>3T3-L1 cells infected with an empty lentiviral vector (EV) or with a lentivirus expressing human (hDLK) or mouse (mDLK) DLK shRNA were induced to differentiate for 6 days. After differentiation, cells were subjected to Western blot analysis with an antibody directed against DLK (A) or stained for lipids with ORO and photographed (B). As a control for protein loading, immunoblots were probed in parallel with an antibody specific for tubulin. The inset at the upper right corner of each photograph represents unstained cells at a 20× magnification. (C) Lipids extracted from ORO-stained cells were quantified by spectrophotometry at 520 nm. The data represent the mean±SEM of three independent experiments, relative to control EV-infected cells.</p

PPARγ activation by rosiglitazone rescues adipocyte differentiation in DLK-depleted 3T3-L1 cells.

<p>(A) 3T3-L1 cells infected with an empty lentiviral vector (EV) or with a lentivirus expressing human (hDLK) or mouse (mDLK) DLK shRNA were induced to differentiate for 6 days in the presence of 1 µM rosiglitazone. After differentiation, cells were stained for lipids with ORO and photographed. Lipids extracted from ORO-stained cells were quantified by spectrophotometry at 520 nm. The data represent the mean±SEM of three independent experiments, relative to EV-infected cells. (B) 3T3-L1 cells infected with an empty lentiviral vector (EV) or with a lentivirus expressing human (hDLK) or mouse (mDLK) DLK shRNA were induced to differentiate for 2, 4 or 6 days in the presence of 1 µM rosiglitazone or DMSO (vehicule). After differentiation, cells were subjected to Western blot analysis with antibodies directed against DLK, C/EBPβ, C/EBPα, PPARγ, adiponectin and FAS. As a control for protein loading, immunoblots were probed in parallel with an antibody specific for tubulin.</p

Loss of DLK in 3T3-L1 cells prevents expression of C/EBPα, PPARγ, adiponectin and fatty acid synthase proteins but not that of C/EBPβ.

<p>3T3-L1 cells infected with an empty lentivirus, a lentivirus expressing human DLK shRNA (hDLK) or a lentivirus expressing mouse DLK shRNA (mDLK) were induced to differentiate for the indicated times. After differentiation, cells were subjected to Western blot analysis with specific antibodies against DLK, C/EBPβ, C/EBPδ, C/EBPα, PPARγ, adiponectin, fatty acid synthase (FAS), phospho-JNK, JNK and Akt as the loading control.</p

Proposed model for the role of DLK in adipogenesis.

<p>Early events in adipogenesis lead to the expression of C/EBPβ and C/EBPδ (step 1), which then bind to the <i>cebpα</i> and <i>pparγ2</i> promoters (step 2). DLK (step 3) in turn appears to contribute to the subsequent increase in the expression of PPARγ and C/EBPα (step 4) by a mechanism that remains to be identified. The latter two proteins then direct the expression of various adipocyte-specific genes (step 5).</p