Map2k4δ — Identification and functional characterization of a novel Map2k4 splice variant

Mitogen-activated protein kinase kinase 4 (Map2k4) is a dual specificity serin/threonine protein kinase that is unique among all MAP2Ks in activating two different subfamilies of mitogen-activated protein kinases, the c-Jun N-terminal kinases (JNKs) and p38 kinases. Map2k4 is essential during embryogenesis and involved in a variety of physiological and pathological processes. However, studies on its role in cancer development revealed partially conflicting data. In the present study, we report the identification of a novel splice variant of Map2k4, Map2k4δ, with an additional exon in front of the substrate binding D-domain. Map2k4δ is expressed together with Map2k4 in various tissues from rat, mouse and human. In PC12 cells, both splice variants control cell cycle progression and basal apoptosis by using different signaling pathways. If expression and activation of Map2k4 and Map2k4δ are at a certain, cell type-specific equilibrium, an appropriate cell growth is ensured. Overexpression of one kinase disrupts the intricate balance and either results in a highly proliferative or pro-apoptotic phenotype, partially reflecting the discrepancies in the literature on Map2k4 and its role in tumor development. Our findings contribute to the understanding of previous studies and point out that Map2k4 has not always a definite function, but rather triggers a cellular reaction in concert with other modulators.     

Inducible Deletion of Protein Kinase Map4k4 in Obese Mice Improves Insulin Sensitivity in Liver and Adipose Tissues

Studies in vitro suggest that mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) attenuates insulin signaling, but confirmation in vivo is lacking since Map4k4 knockout is lethal during embryogenesis. We thus generated mice with floxed Map4k4 alleles and a tamoxifen-inducible Cre/ERT2 recombinase under the control of the ubiquitin C promoter to induce whole-body Map4k4 deletion after these animals reached maturity. Tamoxifen administration to these mice induced Map4k4 deletion in all tissues examined, causing decreased fasting blood glucose concentrations and enhanced insulin signaling to AKT in adipose tissue and liver but not in skeletal muscle. Surprisingly, however, mice generated with a conditional Map4k4 deletion in adiponectin-positive adipocytes or in albumin-positive hepatocytes displayed no detectable metabolic phenotypes. Instead, mice with Map4k4 deleted in Myf5-positive tissues, including all skeletal muscles tested, were protected from obesity-induced glucose intolerance and insulin resistance. Remarkably, these mice also showed increased insulin sensitivity in adipose tissue but not skeletal muscle, similar to the metabolic phenotypes observed in inducible whole-body knockout mice. Taken together, these results indicate that (i) Map4k4 controls a pathway in Myf5-positive cells that suppresses whole-body insulin sensitivity and (ii) Map4k4 is a potential therapeutic target for improving glucose tolerance and insulin sensitivity in type 2 diabetes.     

Regulated in Development and DNA Damage Responses -1 (REDD1) Protein Contributes to Insulin Signaling Pathway in Adipocytes

REDD1 (Regulated in development and DNA damage response 1) is a hypoxia and stress response gene and is a negative regulator of mTORC1. Since mTORC1 is involved in the negative feedback loop of insulin signaling, we have studied the role of REDD1 on insulin signaling pathway and its regulation by insulin. In human and murine adipocytes, insulin transiently stimulates REDD1 expression through a MEK dependent pathway. In HEK-293 cells, expression of a constitutive active form of MEK stabilizes REDD1 and protects REDD1 from proteasomal degradation mediated by CUL4A-DDB1 ubiquitin ligase complex. In 3T3-L1 adipocytes, silencing of REDD1 with siRNA induces an increase of mTORC1 activity as well as an inhibition of insulin signaling pathway and lipogenesis. Rapamycin, a mTORC1 inhibitor, restores the insulin signaling after downregulation of REDD1 expression. This observation suggests that REDD1 positively regulates insulin signaling through the inhibition of mTORC1 activity. In conclusion, our results demonstrate that insulin increases REDD1 expression, and that REDD1 participates in the biological response to insulin.     

Differential regulation of CIDEA and CIDEC expression by insulin via Akt1/2- and JNK2-dependent pathways in human adipocytes

Both insulin and the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) family play important roles in apoptosis and lipid droplet formation. Previously, we reported that CIDEA and CIDEC are differentially regulated by insulin and contribute separately to insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes. However, the upstream signals of CIDE proteins remain unclear. Here, we investigated the signaling molecules involved in insulin regulation of CIDEA and CIDEC expression. The phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and PI-103 blocked both insulin-induced downregulation of CIDEA and upregulation of CIDEC. The Akt inhibitor API-2 and the c-Jun N-terminal kinase (JNK) inhibitor SP600125 selectively inhibited insulin regulation of CIDEA and CIDEC expression, respectively, whereas the MAPK/ERK kinase inhibitor U0126 and the p38 inhibitor SB203580 did not. Small interfering RNA-mediated depletion of Akt1/2 prevented insulin-induced downregulation of CIDEA and inhibition of apoptosis. Depletion of JNK2, but not JNK1, inhibited insulin-induced upregulation of CIDEC and lipid droplet enlargement. Furthermore, insulin increased both Akt and JNK phosphorylation, which was abrogated by the PI3K inhibitors. These results suggest that insulin regulates CIDEA and CIDEC expression via PI3K, and it regulates expression of each protein via Akt1/2- and JNK2-dependent pathways, respectively, in human adipocytes.     

A Rictor-Myo1c complex participates in dynamic cortical actin events in 3T3-L1 adipocytes

Insulin signaling through phosphatidylinositol 3-kinase (PI 3-kinase) activates the protein kinase Akt through phosphorylation of its threonine 308 and serine 473 residues by the PDK1 protein kinase and the Rictor-mammalian target of rapamycin complex (mTORC2), respectively. Remarkably, we show here that the Rictor protein is also present in cultured adipocytes in complexes containing Myo1c, a molecular motor that promotes cortical actin remodeling. Interestingly, the Rictor-Myo1c complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Furthermore, while RNA interference-directed silencing of Rictor results in the expected attenuation of Akt phosphorylation at serine 473, depletion of Myo1c is without effect. In contrast, loss of either Rictor or Myo1c inhibits phosphorylation of the actin filament regulatory protein paxillin at tyrosine 118. Furthermore, Myo1c-induced membrane ruffling of 3T3-L1 adipocytes is also compromised following Rictor knockdown. Interestingly, neither the mTORC2 inhibitor rapamycin nor the PI 3-kinase inhibitor wortmannin affects paxillin tyrosine 118 phosphorylation. Taken together, our findings suggest that the Rictor-Myo1c complex is distinct from mTORC2 and that Myo1c, in conjunction with Rictor, participates in cortical actin remodeling events.     

YTHDF2 mediates LPS-induced osteoclastogenesis and inflammatory response via the NF-κB and MAPK signaling pathways

Aberrant elevation of osteoclast differentiation and function is responsible for disrupting bone homeostasis in various inflammatory bone diseases. YTH domain family 2 (YTHDF2) is a well-known m⁶A-binding protein that plays an essential role in regulating cell differentiation and inflammatory processes by mediating mRNA degradation. However, the regulatory role of YTHDF2 in inflammatory osteoclast differentiation remains unelucidated. Here, we detected the expression of m⁶A-related genes and found that YTHDF2 was upregulated in RANKL-primed osteoclast precursors stimulated with lipopolysaccharide (LPS). Ythdf2 knockdown in RAW264.7 cells and primary bone marrow-derived macrophages (BMMs) enhanced osteoclast formation and bone resorption, which was assessed by TRAP staining assay and pit formation assay. Ythdf2 depletion upregulated osteoclast-related gene expression and proinflammatory cytokine secretion. In contrast, overexpression of Ythdf2 produced the reverse effect. Furthermore, Ythdf2 knockdown enhanced the phosphorylation of IKKα/β, IκBα, ERK, P38 and JNK. NF-κB and MAPK signaling pathway inhibitors effectively abrogated the enhanced expression of Nfact1, c-Fos, IL-1β and TNF-α caused by Ythdf2 knockdown. Mechanistically, the mRNA stability assay revealed that Ythdf2 depletion led to stabilization of Tnfrsf11a, Traf6, Map4k4, Map2k3, Map2k4 and Nfatc1 mRNA. In summary, our findings demonstrated that YTHDF2 has a negative regulatory role in LPS-induced osteoclast differentiation and the inflammatory response via the NF-κB and MAPK signaling pathways.     

Dimethylaminopurine inhibits metabolic effects of insulin in primary adipocytes

Dimethylaminopurine (DMAP) has previously been used as an inhibitor of phosphorylation in studies of meiotic events, and more recently to investigate TNFalpha signaling, because of its potential to inhibit activation of c-jun N-terminal kinase (JNK). Here we have addressed the effects of DMAP on metabolic insulin responses in adipocytes and on intracellular insulin signaling molecules. At 100 micromol/L, DMAP completely inhibited the ability of insulin to counteract lipolysis in isolated adipocytes. Insulin-induced lipogenesis and glucose uptake was inhibited to a lesser degree in a concentration-dependent manner starting at 10 micromol/L DMAP. Insulin-induced tyrosine phosphorylation of the insulin receptor was not affected by DMAP. Insulin-induced activation of protein kinase B, a known mediator of insulin action, was not inhibited by 100 micromol/L, but to a low extent by 1 mmol/L DMAP in intact cells. This inhibition was not sufficient to affect activation of the downstream protein kinase B substrate phosphodiesterase 3B. The inhibition of activation of JNK as a possible mechanism whereby DMAP affects insulin-induced antilipolysis, lipogenesis, and glucose uptake, was investigated using the JNK inhibitor SP600125. At 100 micromol/L, SP600125 completely reversed the antilipolytic effect of insulin, as well as partially inhibited insulin-induced lipogenesis and glucose-uptake, indicating that JNK may be involved in mediating these actions of insulin. Inhibition of JNK by DMAP may therefore partly explain the negative impact of DMAP on insulin action in adipocytes.     

ASCT2 silencing regulates mammalian target-of-rapamycin growth and survival signaling in human hepatoma cells

System ASC amino acid transporter-2 (ASCT2) was previously demonstrated to be essential for human hepatoma cell growth and survival, as its silencing via inducible antisense RNA expression results in complete apoptosis within 48 h by a mechanism that transcends its role in amino acid delivery. To gain mechanistic insights into the reliance of cancerous liver cells on ASCT2, the aim of this study was to determine the early consequences of its silencing on the growth and survival signaling that presage apoptosis. Induced antisense ASCT2 RNA in SK-Hep1 cells led to >90% suppression of ASCT2 mRNA by 6 h and inhibition of mammalian target-of-rapamycin (mTOR)/raptor (mTOR complex-1; mTORC1) signaling by 8 h, as manifested by diminished p70 ribosomal protein S6 kinase-1 and eukaryotic initiation factor-4E (eIF4E) binding protein-1 phosphorylation, while protein synthesis rates declined by nearly 50% despite no measurable decreases in the cap binding protein eIF4G or cellular ribosomal protein content. Depressed mTORC1 signaling occurred before detectable reduction in ASCT2 activity but coincided with a 30% decline in total cellular ASCT2 protein. By 12 h after ASCT2 silencing, further decrements were observed in protein synthesis rates and ASCT2 protein and activity, each by 50%, while signaling from mTOR/rictor (mTOR complex-2; mTORC2) was stimulated as indexed by enhanced phosphorylation of the Akt/PKB kinase on serine-473 and of its proapoptotic substrate Bad on serine-136. These results suggest that ASCT2 silencing inhibits mTORC1 signaling to the translational machinery followed by an mTORC2-initiated survival response, establishing a link between amino acid transporter expression and mTOR function. amino acid transport; hepatocellular carcinoma; apoptosis; protein synthesis     

Gene expression analysis of the Tao kinase family of Ste20p-like map kinase kinase kinases during early embryonic development in Xenopus laevis

Development of the vertebrate embryo requires strict coordination of a highly complex series of signaling cascades, that drive cell proliferation, differentiation, migration, and the general morphogenetic program. Members of the Map kinase signaling pathway are repeatedly required throughout development to activate the downstream effectors, ERK, p38, and JNK. Regulation of these pathways occurs at many levels in the signaling cascade, with the Map3Ks playing an essential role in target selection. The thousand and one amino acid kinases (Taoks) are Map3Ks that have been shown to activate both p38 and JNK and are linked to neurodevelopment in both invertebrate and vertebrate organisms. In vertebrates, there are three Taok paralogs (Taok1, Taok2, and Taok3) which have not yet been ascribed a role in early development. Here we describe the spatiotemporal expression of Taok1, Taok2, and Taok3 in the model organism Xenopus laevis. The X. laevis Tao kinases share roughly 80% identity to each other, with the bulk of the conservation in the kinase domain. Taok1 and Taok3 are highly expressed in pre-gastrula and gastrula stage embryos, with initial expression localized to the animal pole and later expression in the ectoderm and mesoderm. All three Taoks are expressed in the neural and tailbud stages, with overlapping expression in the neural tube, notochord, and many anterior structures (including branchial arches, brain, otic vesicles, and eye). The expression patterns described here provide evidence that the Tao kinases may play a central role in early development, in addition to their function during neural development, and establish a framework to better understand the developmental roles of Tao kinase signaling.     

Activation of mammalian target of rapamycin (mTOR) by insulin is associated with stimulation of 4EBP1 binding to dimeric mTOR complex 1

Insulin stimulates protein synthesis by promoting phosphorylation of the eIF4E-binding protein, 4EBP1. This effect is rapamycin-sensitive and mediated by mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a signaling complex containing mTOR, raptor, and mLST8. Here we demonstrate that insulin produces a stable increase in the kinase activity of mTORC1 in 3T3-L1 adipocytes. The response was associated with a marked increase in 4EBP1 binding to raptor in mTORC1, and it was abolished by disrupting the TOR signaling motif in 4EBP1. The stimulatory effects of insulin on both 4EBP1 kinase activity and binding occurred rapidly and at physiological concentrations of insulin, and both effects required an intact mTORC1. Results of experiments involving size exclusion chromatography and coimmunoprecipitation of epitope-tagged subunits provide evidence that the major insulin-responsive form is dimeric mTORC1, a structure containing two heterotrimers of mTOR, raptor, and mLST8.     

MAP4K4 gene silencing in human skeletal muscle prevents tumor necrosis factor-alpha-induced insulin resistance

Tumor necrosis factor-alpha (TNF-alpha) induces skeletal muscle insulin resistance by impairing insulin signaling events involved in GLUT4 translocation. We tested whether mitogenic-activated protein kinase kinase kinase kinase isoform 4 (MAP4K4) causes the TNF-alpha-induced negative regulation of extracellular signal-regulated kinase-1/2 (ERK-1/2), c-Jun NH2-terminal kinase (JNK), and the insulin receptor substrate-1 (IRS-1) on the insulin signaling pathway governing glucose metabolism. Using small interfering RNA (siRNA) to suppress the expression of MAP4K4 protein 85% in primary human skeletal muscle cells, we provide evidence that TNF-alpha-induced insulin resistance on glucose uptake was completely prevented. MAP4K4 silencing inhibited TNF-alpha-induced negative signaling inputs by preventing excessive JNK and ERK-1/2 phosphorylation, as well as IRS-1 serine phosphorylation. These results highlight the MAPK4K4/JNK/ERK/IRS module in the negative regulation of insulin signaling to glucose transport in response to TNF-alpha. Depletion of MAP4K4 also prevented TNF-alpha-induced insulin resistance on Akt and the Akt substrate 160 (AS160), providing evidence that appropriate insulin signaling inputs for glucose metabolism were rescued. Silencing of MAP2K1 and MAP2K4, signaling proteins downstream of MAP4K4, recapitulated the effect of MAP4K4 siRNA in TNF-alpha-treated cells. Thus, strategies to inhibit MAP4K4 may be efficacious in the prevention of TNF-alpha-induced inhibitory signals that cause skeletal muscle insulin resistance on glucose metabolism in humans. Moreover, in myotubes from insulin-resistant type II diabetic patients, siRNA against MAP4K4, MAP2K4, or MAP2K1 restored insulin action on glucose uptake to levels observed in healthy subjects. Collectively, our results demonstrate that MAP4K4 silencing prevents insulin resistance in human skeletal muscle and restores appropriate signaling inputs to enhance glucose uptake.     

Methylglyoxal attenuates isoproterenol-induced increase in uncoupling protein 1 expression through activation of JNK signaling pathway in beige adipocytes

Methylglyoxal (MG) is a metabolite derived from glycolysis whose levels in the blood and tissues of patients with diabetes are higher than those of healthy individuals, suggesting that MG is associated with the development of diabetic complications. However, it remains unknown whether high levels of MG are a cause or consequence of diabetes. Here, we show that MG negatively affects the expression of uncoupling protein 1 (UCP1), which is involved in thermogenesis and the regulation of systemic metabolism. Decreased Ucp1 expression is associated with obesity and type 2 diabetes. We found that MG attenuated the increase in Ucp1 expression following treatment with isoproterenol in beige adipocytes. However, MG did not affect protein kinase A signaling, the core coordinator of isoproterenol-induced Ucp1 expression. Instead, MG activated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases. We found that JNK inhibition, but not p38, recovered isoproterenol-stimulated Ucp1 expression under MG treatment. Altogether, these results suggest an inhibitory role of MG on the thermogenic function of beige adipocytes through the JNK signaling pathway.     

TGF-β-activated kinase 1 mediates mechanical stress-induced IL-6 expression in osteoblasts

Mechanical stress plays a key role in bone remodeling. Previous studies showed that loading of mechanical stretch induces a rapid Ca²⁺ influx and subsequent activation of stress-activated protein kinase pathways in osteoblasts. However, the activation mechanism and its significance in bone remodeling have not been fully elucidated. Here we show that TAK1 MAPKKK was activated by cyclic stretch loading of MC3T3-E1 cells. Knockdown of TAK1 attenuated the stretch-induced activation of JNK, p38, and NF-κB. Extracellular (EGTA) or intracellular (BAPTA/AM) Ca²⁺ chelator prevented the stretch-induced activation of TAK1. Activation of TAK1 and its associated downstream signaling pathways were also suppressed by CaMKII inhibitors (KN-93 and KN-62). Furthermore, TAK1-mediated downstream pathways cooperatively induced the expression of IL-6 mRNA in the stretched MC3T3-E1 cells. We also confirmed that TAK1 mediates cyclic stretch-induced IL-6 protein synthesis in the cells using immunoblotting and ELISA. Finally, stretch loading of murine primary osteoblasts induced the expression of IL-6 mRNA via TAK1. Collectively, these data suggest that stretch-dependent Ca²⁺ influx activates TAK1 via CaMKII, leading to the enhanced expression of IL-6 through JNK, p38, and NF-κB pathways in osteoblasts.     

Pharmacological inhibition of Polo-like kinase 1 (PLK1) by BI-2536 decreases the viability and survival of hamartin and tuberin deficient cells via induction of apoptosis and attenuation of autophagy

The mechanistic target of rapamycin complex 1 (mTORC1) increases translation, cell size and angiogenesis, and inhibits autophagy. mTORC1 is negatively regulated by hamartin and tuberin, the protein products of the tumor suppressors TSC1 and TSC2 that are mutated in Tuberous Sclerosis Complex (TSC) and sporadic Lymphangioleiomyomatosis (LAM). Hamartin interacts with the centrosomal and mitotic kinase polo-like kinase 1 (PLK1). Hamartin and tuberin deficient cells have abnormalities in centrosome duplication, mitotic progression, and cytokinesis, suggesting that the hamartin/tuberin heterodimer and mTORC1 signaling are involved in centrosome biology and mitosis. Here we report that PLK1 protein levels are increased in hamartin and tuberin deficient cells and LAM patient-derived specimens, and that this increase is rapamycin-sensitive. Pharmacological inhibition of PLK1 by the small-molecule inhibitor BI-2536 significantly decreased the viability and clonogenic survival of hamartin and tuberin deficient cells, which was associated with increased apoptosis. BI-2536 increased p62, LC3B-I and GFP-LC3 punctae, and inhibited HBSS-induced degradation of p62, suggesting that PLK1 inhibition attenuates autophagy. Finally, PLK1 inhibition repressed the expression and protein levels of key autophagy genes and proteins and the protein levels of Bcl^-2 family members, suggesting that PLK1 regulates both autophagic and apoptotic responses. Taken together, our data point toward a previously unrecognized role of PLK1 on the survival of cells with mTORC1 hyperactivation, and the potential use of PLK1 inhibitors as novel therapeutics for tumors with dysregulated mTORC1 signaling, including TSC and LAM.     

Liver Clock Protein BMAL1 Promotes de Novo Lipogenesis through Insulin-mTORC2-AKT Signaling

The clock protein BMAL1 (brain and muscle Arnt-like protein 1) participates in circadian regulation of lipid metabolism, but its contribution to insulin AKT-regulated hepatic lipid synthesis is unclear. Here we used both Bmal1^−/− and acute liver-specific Bmal1-depleted mice to study the role of BMAL1 in refeeding-induced de novo lipogenesis in the liver. Both global deficiency and acute hepatic depletion of Bmal1 reduced lipogenic gene expression in the liver upon refeeding. Conversely, Bmal1 overexpression in mouse liver by adenovirus was sufficient to elevate the levels of mRNA of lipogenic enzymes. Bmal1^−/− primary mouse hepatocytes displayed decreased levels of de novo lipogenesis and lipogenic enzymes, supporting the notion that BMAL1 regulates lipid synthesis in hepatocytes in a cell-autonomous manner. Both refed mouse liver and insulin-treated primary mouse hepatocytes showed impaired AKT activation in the case of either Bmal1 deficiency or Bmal1 depletion by adenoviral shRNA. Restoring AKT activity by a constitutively active mutant of AKT nearly normalized de novo lipogenesis in Bmal1^−/− hepatocytes. Finally, Bmal1 deficiency or knockdown decreased the protein abundance of RICTOR, the key component of the mTORC2 complex, without affecting the gene expression of key factors of insulin signaling. Thus, our study uncovered a novel metabolic function of hepatic BMAL1 that promotes de novo lipogenesis via the insulin-mTORC2-AKT signaling during refeeding.     

Delayed Cell Cycle Progression in Selenoprotein W-depleted Cells Is Regulated by a Mitogen-activated Protein Kinase Kinase 4-p38/c-Jun NH2-terminal Kinase-p53 Pathway

Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a transient p53- and p21(Cip1)-dependent G(1)-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser-33 in p53, which is associated with decreased p53 ubiquitination and stabilization of p53. We report here that delayed cell cycle progression, Ser-33 phosphorylation, and p53 nuclear accumulation from SEPW1 depletion require mitogen-activated protein kinase kinase 4 (MKK4). Silencing MKK4 rescued G(1) arrest, Ser-33 phosphorylation, and nuclear accumulation of p53 induced by SEPW1 depletion, but silencing MKK3, MKK6, or MKK7 did not. SEPW1 silencing did not change the phosphorylation state of MKK4 but increased total MKK4 protein. Silencing p38γ, p38δ, or JNK2 partially rescued G(1) arrest from SEPW1 silencing, suggesting they signal downstream from MKK4. These results imply that SEPW1 silencing increases MKK4, which activates p38γ, p38δ, and JNK2 to phosphorylate p53 on Ser-33 and cause a transient G(1) arrest.