Effect of intravenous lipid infusion on biomarkers of insulin resistance and immune functions of dry and nonpregnant dairy cows.

During the transition period, dairy cows often experience negative energy balance, which can induce metabolic and immunological disturbances. Previous work has shown that there is a relationship between the dysfunction of immune cells and the increase in blood nonesterified fatty acid (NEFA) concentration. Nevertheless, it is difficult to determine the exact effect of NEFA on the immune system, as other metabolic and hormonal perturbations occur simultaneously during the transition period. In the present study, we have determined the effect of NEFA on immune functions using an experimental model designed to assess the effects independently of energy balance, as well as hormonal and metabolic changes due to parturition. Six dry and nonpregnant cows were infused with either sterile water (control treatment) or a lipid emulsion (Intralipid 20%, Frenesius Kabi, lipid treatment) at a rate of 1 mL/kg per hour for 6 h according to a crossover design. Blood concentrations of NEFA, ß-hydroxybutyrate (BHB), and glucose were measured every hour throughout the infusion period, and 1 and 18 h after the end of infusion. Proliferation and interferon-γ secretion of lymphocytes, phagocytosis, and oxidative burst of neutrophils and blood insulin concentration were evaluated before, during, and at the end of the infusion. For NEFA, BHB, and glucose, treatment × time interactions were present. When compared with the control condition, NEFA and BHB levels were greater in the plasma of cows infused with lipids from 1 h after the start of infusion until 1 h after the end of infusion. Glucose level also increased in response to lipid infusion from 2 h of infusion until 1 h after the end of treatment. For sterile water and lipid infusions, respectively, maximal concentrations were 0.06 ± 0.10 mM and 1.39 ± 0.10 mM for NEFA, 0.70 ± 0.05 mM and 1.06 ± 0.05 mM for BHB, and 4.56 ± 0.27 mM and 6.90 ± 0.27 mM for glucose. For all blood metabolites, there were no differences between treatments 18 h postinfusion. Lipid infusion significantly increased blood insulin concentration at 3 and 6 h of infusion. However, it returned to its basal concentration 18 h after the end of the infusion. Lymphoproliferation declined as early as 3 h after the start of the lipid infusion. At 3 and 6 h of infusion, lipid treatment significantly reduced INF-γ concentration in the culture cell supernatant. The lipid infusion did not affect neutrophil phagocytosis. Nevertheless, the efficacy of the response was affected by a reduction of neutrophils' oxidative burst. These results confirm that NEFA inhibits immune functions independently of energy balance and other changes that occur during the transition period. They also indicate that high blood lipid concentration causes insulin resistance.

Effect of different fatty acids on the proliferation and cytokine production of dairy cow peripheral blood mononuclear cells.

During the transition period, dairy cows often experience negative energy balance, which induces metabolic and immunological disturbances. Our previous work has shown a relationship between the inhibition of immune functions and increased blood nonesterified fatty acid (NEFA) levels. In this study, we evaluated the effect of 11 fatty acids (palmitoleic, myristic, palmitic, stearic, oleic, linoleic, docosahexaenoic, conjugated linoleic, lauric, eicosapentaenoic, and linolenic acids) as well as a mix that represented the NEFA profile observed during the transition period at different concentrations (0, 50, 100, and 250 µM) on proliferation and cytokines secretion of lymphocytes. To assess lymphoproliferation, peripheral blood mononuclear cell (PBMC) from 5 healthy cows (166-189 d in milk) were isolated, stimulated with the mitogenic lectin concanavalin A (ConA), incubated for 72 h with or without fatty acids, and subjected to flow cytometry analysis. Our results showed that all fatty acids, except lauric acid, significantly reduced proliferation of PBMC in a dose-dependent manner. The most detrimental effect was observed with conjugated linoleic and stearic acids, where proliferation of PBMC was already inhibited at the lowest dose (50 µM). For cytokine secretion, we found that levels of IL-4 in culture supernatant of ConA-stimulated PBMC were reduced after a 24-h exposure to the lowest dose (50 µM) of oleic and palmitoleic acids. A dose of 100 µM of eicosapentaenoic acid, NEFA mixture, and myristic acid was necessary to observe a reduction in IL-4 levels. The PBMC also showed a decrease in the secretion of IFN-γ in response to lauric, linolenic, palmitoleic, and stearic acids at 50 µM and myristic acid at 100 µM. Overall, polyunsaturated fatty acids were more potent inhibitors of cytokine secretions than saturated fatty acids. In addition, we detected an inverse relationship between the melting points of fatty acids and their ability to inhibit IL-4 and IFN-γ secretions, as evidenced by greater inhibition with low-melting point fatty acids. Overall, our study confirmed that NEFA have a negative effect on some lymphocyte functions, and that their inhibitory effect on cytokine secretions increases with the degree of unsaturation.

Identification of single nucleotide polymorphisms in carnosine-related genes and effects of genotypes on pork meat quality attributes.

Carnosine has pH-buffering and antioxidant properties that may bring advantages in terms of meat quality attributes. This study aimed at identifying polymorphisms in carnosine-related genes (CARNS1, SLC6A6, SLC15A3, SLC15A4) that might associate with muscle carnosine content and meat quality traits in pigs (Duroc, Landrace, Yorkshire). Twenty seven SNPs were identified and association analyses performed for SLC15A3 c.*35C>T and c.*52C>T (3' UTR region), and SLC15A4 c.658A>G (Ile220Val) and c.818G>A (Ser273Asn) SNPs. Associations were observed for SNP c.658A>G with carnosine content, color b* and L*, drip and cooking losses, pH24h and glycolytic potential values (P≤0.05). The same associations were observed for SNP c.818G>A, but they were not significant after FDR correction. Results suggest that specific SLC15A4 gene variants might increase muscle carnosine content and improve meat quality. With a minor allele frequency of 0.17 for SNP c.658A>G in Yorkshire pigs, selection in favor of the c.658A allele may be considered as a mean to improve pork quality attributes.

Linoleic acid, α-linolenic acid and enterolactone affect lipid oxidation and expression of lipid metabolism and antioxidant-related genes in hepatic tissue of dairy cows.

Although beneficial effects have been attributed to PUFA supplementation in high-yielding dairy cows, diets rich in PUFA may also increase oxidative stress in tissues such as the liver. To fully exploit the health benefits of PUFA, we believe that the addition of natural antioxidants could help in preventing oxidative damage. Using an in vitro precision-cut liver slices (PCLS) tissue culture system, we investigated the effects of different linoleic acid (LA, n-6):α-linolenic acid (ALA, n-3) ratios (LA:ALA ratio of 4, LA:ALA ratio of 15 and LA:ALA ratio of 25) in the presence or absence of the antioxidant enterolactone (ENL) on (1) the mRNA abundance of genes with key roles in hepatic lipid metabolism, oxidative stress response and inflammatory processes, (2) oxidative damages to lipids and proteins and (3) superoxide dismutase activity in early-lactating dairy cows. The addition of LA and ALA to PCLS culture media increased oxidative damage to lipids as suggested by higher concentrations of thiobarbituric acid reactive substances and increased the expression of nuclear factor erythroid 2-related factor 2 target genes. The addition of ENL was effective in preventing lipid peroxidation caused by LA and ALA. Transcript abundance of sterol regulatory element-binding transcription factor 1 and its lipogenic target genes acetyl-CoA carboxylase α, fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) was decreased with LA and ALA, whereas ENL decreased FASN and SCD gene expression. Our results show that addition of LA and ALA to PCLS culture media lowers hepatic lipogenic gene expression and increases oxidative damages to lipids. On the other hand, addition of ENL prevents oxidative damages provoked by these PUFA.

Carnosine content in the porcine longissimus thoracis muscle and its association with meat quality attributes and carnosine-related gene expression.

Muscle carnosine has pH-buffering, antioxidant and carbonyl scavenging properties, which may affect pork quality attributes. Study objectives were to: (1) compare muscle carnosine content and carnosine-related gene mRNA abundance in purebred pigs (n=282), (2) study the effect of muscle carnosine content on pork quality attributes and gene expression across breeds, and (3) study transcript abundance of carnosine-related genes in various tissues. Pigs were raised under similar conditions and slaughtered at 120±4.5kg. Longissimus thoracis muscles were sampled on the dressing line for gene expression and at 24h for meat quality measurements. Muscle carnosine content and carnosine-related gene mRNA abundance were modulated according to pig breeds. Greater pH24h, better water holding capacity and improved meat color values were found in pigs with high muscle carnosine content. Data suggest that high muscle carnosine is associated with improved pork meat quality attributes. The pig genetic background may be a key determinant for muscle carnosine content regulation.

A Role for DLK in Microtubule Reorganization to the Cell Periphery and in the Maintenance of Desmosomal and Tight Junction Integrity.

Dual leucine zipper-bearing kinase (DLK) is an inducer of keratinocyte differentiation, a complex process also involving microtubule reorganization to the cell periphery. However, signaling mechanisms involved in this process remain to be elucidated. Here, we demonstrate that DLK enhances and is required for microtubule reorganization to the cell periphery in human cell culture models and in Dlk knockout mouse embryos. In tissue-engineered skins with reduced DLK expression, cortical distribution of two microtubule regulators, LIS1 and HSP27, is impaired as well as desmosomal and tight junction integrity. Altered cortical distribution of desmosomal and tight junction proteins was also confirmed in Dlk knockout mouse embryos. Finally, desmosomal and tight junction defects were also observed after microtubule disruption in nocodazole-treated tissue-engineered skins, thus confirming a role for microtubules in the maintenance of these types of cell junctions. Globally, this study demonstrates that DLK is a key regulator of microtubule reorganization to the cell periphery during keratinocyte differentiation and that this process is required for the maintenance of desmosomal and tight junction integrity.

Dual leucine zipper kinase regulates expression of axon guidance genes in mouse neuronal cells.

BACKGROUND: Recent genetic studies in model organisms, such as Drosophila, C. elegans and mice, have highlighted a critical role for dual leucine zipper kinase (DLK) in neural development and axonal responses to injury. However, exactly how DLK fulfills these functions remains to be determined. Using RNA-seq profiling, we evaluated the global changes in gene expression that are caused by shRNA-mediated knockdown of endogenous DLK in differentiated Neuro-2a neuroblastoma cells. RESULTS: Our analysis led to the identification of numerous up- and down-regulated genes, among which several were found to be associated with system development and axon guidance according to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, respectively. Because of their importance in axonal growth, pruning and regeneration during development and adult life, we then examined by quantitative RT-PCR the mRNA expression levels of the identified axon guidance genes in DLK-depleted cells. Consistent with the RNA-seq data, our results confirmed that loss of DLK altered expression of the genes encoding neuropilin 1 (Nrp1), plexin A4 (Plxna4), Eph receptor A7 (Epha7), Rho family GTPase 1 (Rnd1) and semaphorin 6B (Sema6b). Interestingly, this regulation of Nrp1 and Plxna4 mRNA expression by DLK in Neuro-2a cells was also reflected at the protein level, implicating DLK in the modulation of the function of these axon guidance molecules. CONCLUSIONS: Collectively, these results provide the first evidence that axon guidance genes are downstream targets of the DLK signaling pathway, which through their regulation probably modulates neuronal cell development, structure and function.

Effects of different n-6:n-3 fatty acid ratios and of enterolactone on gene expression and PG secretion in bovine endometrial cells.

Feeding flaxseed to dairy cows can modulate gene expression and PG synthesis in the uterus at the time of peri-implantation. The objectives of the present study were to determine which flaxseed components are responsible for these effects and how different endometrial cell types are affected. We evaluated the effects of six different linoleic acid (n-6):α-linolenic acid (n-3) ratios and three concentrations of the lignan enterolactone (ENL) on endometrial stromal cells (SC) and epithelial cells (EC). The mRNA abundance of genes with known or suspected roles in embryo survival or PG synthesis was evaluated, along with PGE2 and PGF2α concentrations in culture media. The mRNA abundance of several genes was modulated by different fatty acid (FA) ratios and/or ENL, and this modulation differed between cell types. The FA4 (FA at an n-6:n-3 ratio of 4) treatment (rich in n-3 FA) increased the mRNA abundance of genes that have positive effects on uterine receptivity and implantation when compared with the FA25 (FA at an n-6:n-3 ratio of 25) treatment (rich in n-6 FA). ENL decreased PGE2 and PGF2α concentrations in both cell types, and this reduction was associated with lower mRNA abundance of the PG synthase genes AKR1B1 and PTGES in SC. The combination of ENL with FA (FA4 treatment) resulted in the greatest reduction in PGF2α concentrations when compared with the addition of FA (FA4) or ENL alone. Because of the known luteolytic properties of PGF2α, a reduction in endometrial PGF2α secretion would favour the establishment and maintenance of pregnancy.

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death.

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.

Transglutaminase 2 facilitates or ameliorates HIF signaling and ischemic cell death depending on its conformation and localization.

Transglutaminase 2 (TG2) is a widely expressed and multifunctional protein that modulates cell death/survival processes. We have previously shown that TG2 binds to hypoxia inducible factor 1ß (HIF1ß) and decreases the upregulation of HIF responsive genes; however, the relationship between these observations was not investigated. In this study, we investigated whether endogenous TG2 is sufficient to suppress HIF activity and whether the interaction between TG2 and HIF1ß is required for this suppression. shRNA-mediated silencing of TG2 significantly enhanced HIF activation in response to hypoxia. In addition, nuclear localization of TG2 is required for its suppressive effect on HIF activity, with TG2 being recruited to HIF responsive promoters in hypoxic conditions. These observations suggest that TG2 directly regulates hypoxic transcriptional machinery; however, its interaction with HIF1ß was not required for this regulation. We also examined whether TG2's effect on cell death/survival processes in ischemia is due to its effects on HIF signaling. Our results indicate that TG2 mediated HIF suppression can be separated from TG2's effect on cell survival in hypoxic/hypoglycemic conditions. Lastly, here we show that nuclear TG2 in the closed conformation and non-nuclear TG2 in the open conformation have opposing effects on hypoxic/hypoglycemic cell death, which could explain previous controversial results. Overall, our results further clarify the role of TG2 in mediating the cellular response to ischemia and suggest that manipulating the conformation of TG2 might be of pharmacological interest as a therapeutic strategy for the treatment of ischemia-related pathologies.

The p400/Brd8 chromatin remodeling complex promotes adipogenesis by incorporating histone variant H2A.Z at PPARγ target genes.

Adipogenesis, the biological process by which preadipocytes differentiate into mature fat cells, is coordinated by a tightly regulated gene expression program. Indeed, it has been reported that a large number of genetic events, from fat cell-specific transcription factors expression, such as the master regulator of fat cell differentiation peroxisome proliferator-activated receptor (PPAR)γ2 to epigenetic modifications, govern the acquisition of a mature adipocyte phenotype. Here, we provide evidence that the E1A-binding protein p400 (p400) complex subunit bromo-containing protein 8 (Brd8) plays an important role in the regulation of PPARγ target genes during adipogenesis by targeting and incorporating the histone variant H2A.Z in transcriptional regulatory regions. The results reported here indicate that expression of both Brd8 and p400 increases during fat cell differentiation. In addition, small hairpin RNA-mediated knockdown of Brd8 or H2A.Z completely abrogated the ability of 3T3-L1 preadipocyte to differentiate into mature adipocyte, as evidenced by a lack of lipid accumulation. Chromatin immunoprecipitation experiments also revealed that the knockdown of Brd8 blocked the accumulation of PPARγ, p400, and RNA polymerase II and prevented the incorporation of H2A.Z at two PPARγ target genes. Taken together, these results indicate that the incorporation of the histone variant H2A.Z at the promoter regions of PPARγ target genes by p400/Brd8 is essential to allow fat cell differentiation.

Loss of DLK expression in WI-38 human diploid fibroblasts induces a senescent-like proliferation arrest.

DLK, a serine/threonine kinase that functions as an upstream activator of the mitogen-activated protein kinase (MAPK) pathways, has been shown to play a role in development, cell differentiation, apoptosis and neuronal response to injury. Interestingly, recent studies have shown that DLK may also be required for cell proliferation, although little is known about its specific functions. To start addressing this issue, we studied how DLK expression is modulated during cell cycle progression and what effect DLK depletion has on cell proliferation in WI-38 fibroblasts. Our results indicate that DLK protein levels are low in serum-starved cells, but that serum addition markedly stimulated it. Moreover, RNA interference experiments demonstrate that DLK is required for ERK activity, expression of the cell cycle regulator cyclin D1 and proliferation of WI-38 cells. DLK-depleted cells also show a senescent phenotype as revealed by senescence-associated galactosidase activity and up-regulation of the senescence pathway proteins p53 and p21. Consistent with a role for p53 in this response, inhibition of p53 expression by RNA interference significantly alleviated senescence induced by DLK knockdown. Together, these findings indicate that DLK participates in cell proliferation and/or survival, at least in part, by modulating the expression of cell cycle regulatory proteins.

The DLK gene is a transcriptional target of PPARγ.

DLK (dual leucine zipper-bearing kinase) is a key regulator of development, cell differentiation and apoptosis. Interestingly, recent studies have shown that DLK expression is up-regulated in 3T3-L1 cells induced to differentiate into adipocytes and that DLK knockdown impairs the expression of PPARγ (peroxisome-proliferator-activated receptor γ), a master regulator of adipogenesis. Because the PPARγ agonist rosiglitazone was found to increase DLK expression in 3T3-L1 cells, we hypothesized that PPARγ is required for the transcriptional activation of the DLK gene. To test this hypothesis, we first examined the effects of pharmacological inhibition or shRNA (small-hairpin RNA)-mediated depletion of PPARγ on DLK accumulation in 3T3-L1 cells undergoing differentiation. In addition to blocking adipocyte conversion of 3T3-L1 cells, inhibition of PPARγ suppressed DLK expression at both the mRNA and protein levels. Moreover, supporting a role for PPARγ in DLK regulation, two potential PPARγ-binding sites identified by bioinformatic tools at positions -611 and -767 upstream of the DLK gene transcriptional start site were shown by electrophoretic mobility-shift assay and chromatin immunoprecipitation to bind PPARγ and its essential heterodimer partner retinoid X receptor as differentiation proceeds. Collectively, these results show that DLK is a novel transcriptional target of PPARγ with functional PPARγ-binding sites in its promoter.

Adiponectin action from head to toe.

Adiponectin, the most abundant protein secreted by white adipose tissue, is known for its involvement in obesity-related disorders such as insulin resistance, type 2 diabetes mellitus and atherosclerosis. Moreover, modulation of the circulating adiponectin concentration is observed in pathologies that are more or less obesity-related, such as cancer and rheumatoid arthritis. The wide distribution of adiponectin receptors in various organs and tissues suggests that adiponectin has pleiotropic effects on numerous physiological processes. Besides its well-known insulin-sensitizing, anti-inflammatory and antiatherosclerotic properties, accumulating evidence suggests that adiponectin may also have anticancer properties and be cardioprotective. A beneficial effect of adiponectin on female reproductive function was also suggested. Since adiponectin has numerous beneficial biological functions, its use as a therapeutic agent has been suggested. However, the use of adiponectin or its receptors as therapeutic targets is complicated by the presence of different adiponectin oligomeric isoforms and production sites, by multiple receptors with differing affinities for adiponectin isoforms, and by cell-type-specific effects in different tissues. In this review, we discuss the known and potential roles of adiponectin in various tissues and pathologies. The therapeutic promise of administration of adiponectin and the use of its circulating levels as a diagnostic biomarker are further discussed based on the latest experimental studies.

Sanguinarine induces apoptosis of human osteosarcoma cells through the extrinsic and intrinsic pathways.

The quaternary benzo[c]phenanthridine alkaloid sanguinarine inhibits the proliferation of cancerous cells from different origins, including lung, breast, pancreatic and colon, but nothing is known of its effects on osteosarcoma, a primary malignant bone tumour. We have found that sanguinarine alters the morphology and reduces the viability of MG-63 and SaOS-2 human osteosarcoma cell lines in concentration- and time-dependent manner. Incubation with 1 micromol/L sanguinarine for 4 and 24h killed more efficiently MG-63 cells than SaOS-2 cells, while incubation with 5 micromol/L sanguinarine killed almost 100% of both cell populations within 24h. This treatment also changed the mitochondrial membrane potential in both MG-63 and SaOS-2 cells within 1h, caused chromatin condensation and the formation of apoptotic bodies. It activated multicaspases, and increased the activities of caspase-8 and caspase-9 in both MG-63 and SaOS-2 cells. These data highlight sanguinarine as a novel potential agent for bone cancer therapy.

The small heat-shock protein Hsp27 undergoes ERK-dependent phosphorylation and redistribution to the cytoskeleton in response to dual leucine zipper-bearing kinase expression.

Hsp27, a small heat-shock protein, has important roles in many cellular processes, including cytoskeleton dynamics, cell differentiation, and apoptosis. Its expression in normal epidermis correlates with differentiation; however, little is known about the regulatory mechanisms involved. In this study, we report that Hsp27 undergoes upregulation, phosphorylation, and redistribution to the cytoskeleton during the late phase of epidermal keratinocyte differentiation. Our results also show that the expression of the dual leucine zipper-bearing kinase (DLK), an upstream activator of the MAP kinase pathways, is sufficient by itself to induce Hsp27 phosphorylation, cell periphery localization, and redistribution to the insoluble protein fraction (cytoskeleton) in poorly differentiated keratinocytes. This redistribution correlates with the insolubilization of cornified envelope-associated proteins such as involucrin. Interestingly, the effects of DLK on Hsp27 were blocked by PD98059, a selective inhibitor of the extracellular signal-regulated protein kinase (ERK) pathway. Moreover, downregulation of Hsp27 by small interfering RNA in epithelial cells expressing DLK was accompanied by attenuated expression of involucrin in the cytoskeleton. Thus, these observations suggest that the DLK-ERK signaling pathway may act as a regulator of the interaction that occurs between Hsp27 and the cytoskeleton during the formation of the cornified cell envelope, a process conferring to the skin its crucial barrier function.

The mixed-lineage kinase DLK is a key regulator of 3T3-L1 adipocyte differentiation.

BACKGROUND: 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. METHODS AND FINDINGS: 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. CONCLUSIONS: 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.

The mixed-lineage kinase DLK undergoes Src-dependent tyrosine phosphorylation and activation in cells exposed to vanadate or platelet-derived growth factor (PDGF).

Some data in the literature suggest that serine/threonine phosphorylation is required for activation of the mixed-lineage kinases (MLKs), a subgroup of mitogen-activated protein kinase kinase kinases (MAPKKKs). In this report, we demonstrate that the MLK family member DLK is activated and concurrently tyrosine-phosphorylated in cells exposed to the protein tyrosine phosphatase inhibitor vanadate. Tyrosine phosphorylation appears crucial for activation as incubation of vanadate-activated DLK molecules with a tyrosine phosphatase substantially reduced DLK enzymatic activity. Interestingly, the effects of vanadate on DLK are completely blocked by treatment with a Src family kinase inhibitor, PP2, or the expression of short hairpin RNA (shRNA) directed against Src. DLK also fails to undergo vanadate-stimulated tyrosine phosphorylation and activation in fibroblasts which lack expression of Src, Yes and Fyn, but reintroduction of wild-type Src or Fyn followed by vanadate treatment restores this response. In addition to vanadate, stimulation of cells with platelet-derived growth factor (PDGF) also induces tyrosine phosphorylation and activation of DLK by a Src-dependent mechanism. DLK seems important for PDGF signaling because its depletion by RNA interference substantially reduces PDGF-stimulated ERK and Akt kinase activation. Thus, our findings suggest that Src-dependent tyrosine phosphorylation of DLK may be important for regulation of its activity, and they support a role for DLK in PDGF signaling.

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.