Autophagy Induced by HIF1α Overexpression Supports Trophoblast Invasion by Supplying Cellular Energy

Extravillous trophoblasts (EVTs) characterize the invasion of the maternal decidua under low oxygen and poor nutrition at the early feto-maternal interface to establish a successful pregnancy. We previously reported that autophagy in EVTs was activated under 2% O₂in vitro, and autophagy activation was also observed in EVTs at the early feto-maternal interface in vivo. Here, we show that autophagy is an energy source for the invasion of EVTs. Cobalt chloride (CoCl₂), which induces hypoxia inducible factor 1α (HIF1α) overexpression, activated autophagy in HTR8/SVneo cells, an EVT cell line. The number of invading HTR8-ATG4B^C74A cells, an autophagy-deficient EVT cell line, was markedly reduced by 81 percent with the CoCl₂ treatment through the suppression of MMP9 level, although CoCl₂ did not affect the cellular invasion of HTR8-mStrawberry cells, a control cell line. HTR8-ATG4B^C74A cells treated with CoCl₂ showed a decrease in cellular adenosine triphosphate (ATP) levels and a compensatory increase in the expression of purinergic receptor P2X ligand-gated ion channel 7 (P2RX7), which is stimulated with ATP, whereas HTR8-mStrawberry cells maintained cellular ATP levels and did not affect P2RX7 expression. Furthermore, the decreased invasiveness of HTR8-ATG4B^C74A cells treated with CoCl₂ was neutralized by ATP supplementation to the level of HTR8-ATG4B^C74A cells treated without CoCl₂. These results suggest that autophagy plays a role in maintaining homeostasis by countervailing HIF1α-mediated cellular energy consumption in EVTs.     

Curcumin Attenuates the PERK-eIF2α Signaling to Relieve Acrylamide-Induced Neurotoxicity in SH‑SY5Y Neuroblastoma Cells

Neurochemical Research - Curcumin is a natural polyphenolic compound with neuroprotective and antioxidant properties. Acrylamide (ACR) is a by-product of food processing that produces neurotoxicity...     

Regulation of autophagy by p38α MAPK

Autophagy is induced in mammalian cells by nutrient deprivation, which acts through repression of the protein kinase mammalian target of rapamycin (mTOR) and may involve other unknown mechanisms. Mitogen-activated protein kinases (MAPKs), and in particular p38 MAPK, are implicated in amino acid signalling. Furthermore, the extracellular signal-regulated kinase (ERK) and p38 regulate autophagy in response to various stimuli. However, the molecular mechanisms of p38 regulation of autophagy are still widely unknown. Our recent data suggest that p38α MAPK negatively regulates the interaction of mAtg9 and a novel mAtg9 binding partner, p38IP, to control the levels of autophagy induced in response to starvation.     

Regulation of type IIα phosphatidylinositol phosphate kinase localisation by the protein kinase CK2

Inositol lipid synthesis is regulated by several distinct families of enzymes [1]. Members of one of these families, the type II phosphatidylinositol phosphate kinases (PIP kinases), are 4-kinases and are thought to catalyse a minor route of synthesis of the multifunctional phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) from the inositide PI(5)P [2]. Here, we demonstrate the partial purification of a protein kinase that phosphorylates the type IIα PIP kinase at a single site unique to that isoform – Ser304. This kinase was identified as protein kinase CK2 (formerly casein kinase 2). Mutation of Ser304 to aspartate to mimic its phosphorylation had no effect on PIP kinase activity, but promoted both redistribution of the green fluorescent protein (GFP)-tagged enzyme in HeLa cells from the cytosol to the plasma membrane, and membrane ruffling. This effect was mimicked by mutation of Ser304 to alanine, although not to threonine, suggesting a mechanism involving the unmasking of a latent membrane localisation sequence in response to phosphorylation.     

Structural Basis of Protein Kinase Cα Regulation by the C-Terminal Tail

Protein kinase C (PKC) isoenzymes are multi-modular proteins activated at the membrane surface to regulate signal transduction processes. When activated by second messengers, PKC undergoes a drastic conformational and spatial transition from the inactive cytosolic state to the activated membrane-bound state. The complete structure of either state of PKC remains elusive. We demonstrate, using NMR spectroscopy, that the isolated Ca²⁺-sensing membrane-binding C2 domain of the conventional PKCα interacts with a conserved hydrophobic motif of the kinase C-terminal region, and we report a structural model of the complex. Our data suggest that the C-terminal region plays a dual role in regulating the PKC activity: activating, through sensitization of PKC to intracellular Ca²⁺ oscillations; and auto-inhibitory, through its interaction with a conserved positively charged region of the C2 domain.     

Regulation of DNA topoisomerase IIα stability by the ECV ubiquitin ligase complex

In this study, we attempted to elucidate the E3 ubiquitin ligase for topo IIα. When cullins and VHL were ectopically expressed in HT1080 and HEK293T cells, topo IIα was degraded most prominently in cullin 2- and VHL-expressing cells. Cullin 2 and the β domain (aa 114-123) of VHL, a subunit of the ECV (Elongin B/C-cullin 2-VHL protein) complex, specifically interact with the ATPase domain of topo IIα. We identified that topo IIα associated with endogenous Elongin C. In HT1080 cells co-transfected with deletion mutants of topo IIα GRDD (glucose-regulated destruction domain) and VHL, topo IIα was degraded by VHL expression. These results demonstrate that ECV acts as E3 ubiquitin ligase targeting GRDD-independent topo IIα to the ubiquitin-proteasome pathway.     

Regulation of ΔNp63α by NFκB

ΔNp63α, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be downregulated in response to several DNA damaging agents, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying the regulation of ΔNp63α expression in response to extrinsic stimuli, such as chemotherapeutic agents. We show that ΔNp63α interacts with NFκB in presence of cisplatin. We find that NFκB promotes ubiquitin-mediated proteasomal degradation of ΔNp63α. Chemotherapy-induced stimulation of NFκB leads to degradation of ΔNp63α and augments trans-activation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, inhibition of NFκB with siRNA-mediated silencing NFκB expression attenuates chemotherapy induced degradation of ΔNp63α. These data demonstrate that NFκB plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. Our findings suggest that the activation of NFκB may be a mechanism by which levels of ΔNp63α are reduced, thereby rendering the cells susceptible to cell death in the face of cellular stress or DNA damage.Key words: ΔNp63α, NFκB, ubiquitination, cisplatin, head and neck cancer     

Comparative Analysis of Gene Regulation by the Transcription Factor PPARα between Mouse and Human

Background

Studies in mice have shown that PPARα is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARα in human liver. Here we set out to compare the function of PPARα in mouse and human hepatocytes via analysis of target gene regulation.

Methodology/Principal Findings

Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARα agonist Wy14643 and gene expression profiling was performed using Affymetrix GeneChips followed by a systems biology analysis. Baseline PPARα expression was similar in human and mouse hepatocytes. Depending on species and time of exposure, Wy14643 significantly induced the expression of 362–672 genes. Surprisingly minor overlap was observed between the Wy14643-regulated genes from mouse and human, although more substantial overlap was observed at the pathway level. Xenobiotics metabolism and apolipoprotein synthesis were specifically regulated by PPARα in human hepatocytes, whereas glycolysis-gluconeogenesis was regulated specifically in mouse hepatocytes. Most of the genes commonly regulated in mouse and human were involved in lipid metabolism and many represented known PPARα targets, including CPT1A, HMGCS2, FABP1, ACSL1, and ADFP. Several genes were identified that were specifically induced by PPARα in human (MBL2, ALAS1, CYP1A1, TSKU) or mouse (Fbp2, lgals4, Cd36, Ucp2, Pxmp4). Furthermore, several putative novel PPARα targets were identified that were commonly regulated in both species, including CREB3L3, KLF10, KLF11 and MAP3K8.

Conclusions/Significance

Our results suggest that PPARα activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARα as master regulator of hepatic lipid metabolism is generally well-conserved between mouse and human. Overall, however, PPARα regulates a mostly divergent set of genes in mouse and human hepatocytes.     

Triptolide Inhibited Cytotoxicity of Differentiated PC12 Cells Induced by Amyloid-Beta25–35 via the Autophagy Pathway

Evidence shows that an abnormal deposition of amyloid beta-peptide_25–35 (Aβ_25–35) was the primary cause of the pathogenesis of Alzheimer’s disease (AD). And the elimination of Aβ_25–35 is considered an important target for the treatment of AD. Triptolide (TP), isolated from Tripterygium wilfordii Hook.f. (TWHF), has been shown to possess a broad spectrum of biological profiles, including neurotrophic and neuroprotective effects. In our study investigating the effect and potential mechanism of triptolide on cytotoxicity of differentiated rat pheochromocytoma cell line (the PC12 cell line is often used as a neuronal developmental model) induced by Amyloid-Beta_25–35 (Aβ_25–35), we used 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay, flow cytometry, Western blot, and acridine orange staining to detect whether triptolide could inhibit Aβ_25–35–induced cell apoptosis. We focused on the potential role of the autophagy pathway in Aβ_25–35-treated differentiated PC12 cells. Our experiments show that cell viability is significantly decreased, and the apoptosis increased in Aβ_25–35-treated differentiated PC12 cells. Meanwhile, Aβ_25–35 treatment increased the expression of microtubule-associated protein light chain 3 II (LC3 II), which indicates an activation of autophagy. However, triptolide could protect differentiated PC12 cells against Aβ_25–35-induced cytotoxicity and attenuate Aβ_25–35-induced differentiated PC12 cell apoptosis. Triptolide could also suppress the level of autophagy. In order to assess the effect of autophagy on the protective effects of triptolide in differentiated PC12 cells treated with Aβ_25–35, we used 3-Methyladenine (3-MA, an autophagy inhibitor) and rapamycin (an autophagy activator). MTT assay showed that 3-MA elevated cell viability compared with the Aβ_25–35-treated group and rapamycin inhibits the protection of triptolide. These results suggest that triptolide will repair the neurological damage in AD caused by deposition of Aβ_25–35 via the autophagy pathway, all of which may provide an exciting view of the potential application of triptolide or TWHF as a future research for AD.     

Thioredoxin-independent Regulation of Metabolism by the ��-Arrestin Proteins

Thioredoxin-interacting protein (Txnip), originally characterized as an inhibitor of thioredoxin, is now known to be a critical regulator of glucose metabolism in vivo. Txnip is a member of the α-arrestin protein family; the α-arrestins are related to the classical β-arrestins and visual arrestins. Txnip is the only α-arrestin known to bind thioredoxin, and it is not known whether the metabolic effects of Txnip are related to its ability to bind thioredoxin or related to conserved α-arrestin function. Here we show that wild type Txnip and Txnip C247S, a Txnip mutant that does not bind thioredoxin in vitro, both inhibit glucose uptake in mature adipocytes and in primary skin fibroblasts. Furthermore, we show that Txnip C247S does not bind thioredoxin in cells, using thiol alkylation to trap the Txnip-thioredoxin complex. Because Txnip function was independent of thioredoxin binding, we tested whether inhibition of glucose uptake was conserved in the related α-arrestins Arrdc4 and Arrdc3. Both Txnip and Arrdc4 inhibited glucose uptake and lactate output, while Arrdc3 had no effect. Structure-function analysis indicated that Txnip and Arrdc4 inhibit glucose uptake independent of the C-terminal WW-domain binding motifs, recently identified as important in yeast α-arrestins. Instead, regulation of glucose uptake was intrinsic to the arrestin domains themselves. These data demonstrate that Txnip regulates cellular metabolism independent of its binding to thioredoxin and reveal the arrestin domains as crucial structural elements in metabolic functions of α-arrestin proteins.Thioredoxin-interacting protein (Txnip),³ an inhibitor of thioredoxin disulfide reductase activity in vitro (1–3), is robustly induced by glucose (4–6) and a critical regulator of metabolism in vivo (7–10). In humans, Txnip expression is suppressed by insulin and strongly up-regulated in diabetes (7). Txnip-deficient mice have fasting hypoglycemia and ketosis (8, 9, 11, 12) with a striking enhancement of glucose uptake by peripheral tissues (8, 9). We have proposed that Txnip inhibits thioredoxin by forming a mixed disulfide with thioredoxin at its catalytic active site cysteines in a disulfide exchange reaction (13). However, it is not known how Txnip metabolic functions relate to its ability to bind thioredoxin.Structurally, Txnip belongs to the arrestin superfamily of proteins (14). The prototypical arrestins (the visual arrestins and the β-arrestins) are key regulators of receptor signaling. The β-arrestins, named for their interaction with the β-adrenergic receptor, are now known to control signaling through the multiple families of receptors (15). These arrestin proteins have two wing-like arrestin domains arranged around a central core that detects and binds selectively to the charged phosphates of activated receptors (16). The arrestin domains then act as multifunctional scaffolds that cannot only quench receptor signals by recruiting endocytotic machinery and ubiquitin ligases, but also start new signal cascades (15). Recently, arrestin-β2 has also been shown to play a key role in metabolism as a controller of insulin receptor signaling that is deficient in diabetes (17).In addition to the classical visual/β-arrestins, a large number of arrestins more closely related to Txnip are present throughout multicellular evolution. These proteins have been termed the “α-arrestins,” as they are of more ancient origin than the visual/β family (14). Although no structures are known of the α-arrestins to date, they appear highly likely to share the overall fold: two β-sheet sandwich arrestin domains connected by a short linker sequence (14, 18). Confidence in this prediction has been enhanced by the surprising finding that the vps26 family of proteins, even more distantly related to the classical arrestins than Txnip, also share the arrestin fold (19). The vps26 proteins are a component of the retromer complex that controls retrograde transport of recycling endosomes to the trans-Golgi network. This functional overlap with visual/β-arrestin regulation of endocytosis suggests that control of endosome formation and transport may be a conserved function of the arrestin superfamily fold.The functions of the mammalian α-arrestins remain unclear. Humans have six α-arrestins: Txnip and five other proteins, which have been assigned the names Arrdc1–5 (arrestin domain-containing 1–5) (13). Very little is known about these other α-arrestins; thioredoxin binding is not conserved beyond Txnip (13, 20). More is known in yeast: recent reports suggest that α-arrestins function in regulation of endocytosis and protein ubiquitination through PXXY motifs in their C-terminal tails (21–25). However, as all the vertebrate α-arrestins have diverged from the ancestral α-arrestins (14), their structure-function relationships may differ from yeast α-arrestins.Given that other α-arrestins are not thioredoxin-binding proteins, we hypothesized that Txnip metabolic functions may be conserved in mammalian α-arrestins and independent of its interaction with thioredoxin. Overexpression of Txnip in vitro can decrease levels of available thioredoxin and increase levels of reactive oxygen species (1, 3, 26). However, in vivo studies of two different Txnip-deficient mouse models found no change in available thioredoxin levels (8, 27). Txnip reportedly binds to other proteins including Jab1 (28) and Dnajb5 (29), but it is not clear to what extent these interactions are themselves independent of a Txnip-thioredoxin complex (30).Using overexpression of a mutant Txnip that does not bind thioredoxin, we show here that a major metabolic function of Txnip, its inhibition of glucose uptake, does not require interaction with thioredoxin. Instead, we show that inhibition of glucose uptake is a conserved function of another human α-arrestin, Arrdc4. Studies of Txnip mutants and chimeric α-arrestins suggest that the metabolic functions of Txnip and Arrdc4 are intrinsic to the arrestin domains.     

Regulation of amyloid-β production by the prion protein

Alzheimer disease (AD) is characterized by the amyloidogenic processing of the amyloid precursor protein (APP), culminating in the accumulation of amyloid-β peptides in the brain. The enzymatic action of the β-secretase, BACE1 is the rate-limiting step in this amyloidogenic processing of APP. BACE1 cleavage of wild-type APP (APP_WT) is inhibited by the cellular prion protein (PrP^C). Our recent study has revealed the molecular and cellular mechanisms behind this observation by showing that PrP^C directly interacts with the pro-domain of BACE1 in the trans-Golgi network (TGN), decreasing the amount of BACE1 at the cell surface and in endosomes where it cleaves APP_WT, while increasing BACE1 in the TGN where it preferentially cleaves APP with the Swedish mutation (APP_Swe). PrP^C deletion in transgenic mice expressing the Swedish and Indiana familial mutations (APP_Swe,Ind) failed to affect amyloid-β accumulation, which is explained by the differential subcellular sites of action of BACE1 toward APP_WT and APP_Swe. This, together with our observation that PrP^C is reduced in sporadic but not familial AD brain, suggests that PrP^C plays a key protective role against sporadic AD. It also highlights the need for an APP_WT transgenic mouse model to understand the molecular and cellular mechanisms underlying sporadic AD.     

Regulation of ΔNp63α by NFκΒ

ΔNp63α, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be down regulated in response to several DNA damaging agents, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying the regulation of ΔNp63α expression in response to extrinsic stimuli, such as chemotherapeutic agents. We show that ΔNp63α interacts with NF-κΒ in presence of cisplatin. We find that NF-κΒ promotes ubiquitin-mediated proteasomal degradation of ΔNp63α. Chemotherapy-induced stimulation of NF-κΒ leads to degradation of ΔNp63α and augments trans-activation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, inhibition of NF-κΒ with siRNA-mediated silencing NF-κΒ expression attenuates chemotherapy induced degradation of ΔNp63α . These data demonstrate that NF-κΒ plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. Our findings suggest that the activation of NF-κΒ may be a mechanism by which levels of ΔNp63α are reduced, thereby rendering the cells susceptible to cell death in the face of cellular stress or DNA damage.     

Regulation of cellular Gsα levels and basal adenylyl cyclase activity by expression of the β2-adrenoceptor in neuroblastoma cell lines

Mouse neuroblastoma x rat glioma hybrid NG108-15 and mouse neuroblastoma x embryonic hamster brain NCB20 cells were transfected with a construct containing a human 2 adrenoceptor cDNA under the control of the actin promoter. Clones were selected on the basis of resistance to geneticin sulphate and those expressing a range of levels of the receptor expanded for further study. Membranes from a clone of NG108-15 cells expressing high levels of the receptor (N22) but not one expressing only low levels of the receptor (N17) exhibited a markedly elevated adenylyl cyclase activity when measured in the presence of Mg²⁺ compared to wild type cells. This was not due to elevated levels of the adenylyl cyclase catalytic moiety however as there was no difference in these membranes when the adenylyl cyclase activity was measured in the presence of Mn²⁺. The elevated basal activity was partially reversed by addition of the -adrenoceptor antagonist propranolol. Agonist activation of N22 but not N17 cells led to a large selective down-regulation of cellular G_s levels which was independent of the generation of cyclic AMP. Isoprenaline stimulation of adenylyl cyclase activity and of the specific high affinity binding of [³H] forskolin was achieved with substantially greater potency (some 30 fold) in N22 cell membranes than in N17. By contrast agonist activation of the endogenously expressed IP prostanoid receptor caused stimulation of adenylyl cyclase and stimulation of high affinity [³H] forskolin binding which was equipotent in each of N22, N17 and wild type NG108-15 cells. Agonist activation of the IP prostanoid receptor caused an equivalent degree of G_s down-regulation in each cell type. Expression of an epitope tagged variant of G_s in NG108-15 cells resulted in prostanoid agonist-induced down-regulation of this polypeptide in a manner indistinguishable from that of wild type G_s. Isolation of clones of NCB20 cells expressing high levels of the 2 adrenoceptor also resulted in a specific agonist-induced down-regulation of G_s.     

Regulation of amyloid-β production by the prion protein

Alzheimer disease (AD) is characterized by the amyloidogenic processing of the amyloid precursor protein (APP), culminating in the accumulation of amyloid-β peptides in the brain. The enzymatic action of the β-secretase, BACE1 is the rate-limiting step in this amyloidogenic processing of APP. BACE1 cleavage of wild-type APP (APP_WT) is inhibited by the cellular prion protein (PrP^C). Our recent study has revealed the molecular and cellular mechanisms behind this observation by showing that PrP^C directly interacts with the pro-domain of BACE1 in the trans-Golgi network (TGN), decreasing the amount of BACE1 at the cell surface and in endosomes where it cleaves APP_WT, while increasing BACE1 in the TGN where it preferentially cleaves APP with the Swedish mutation (APP_Swe). PrP^C deletion in transgenic mice expressing the Swedish and Indiana familial mutations (APP_Swe,Ind) failed to affect amyloid-β accumulation, which is explained by the differential subcellular sites of action of BACE1 toward APP_WT and APP_Swe. This, together with our observation that PrP^C is reduced in sporadic but not familial AD brain, suggests that PrP^C plays a key protective role against sporadic AD. It also highlights the need for an APP_WT transgenic mouse model to understand the molecular and cellular mechanisms underlying sporadic AD.     

Repression of Osteoblast Maturation by ERRα Accounts for Bone Loss Induced by Estrogen Deficiency

ERRα is an orphan member of the nuclear receptor family, the complete inactivation of which confers resistance to bone loss induced by ageing and estrogen withdrawal to female mice in correlation with increased bone formation in vivo. Furthermore ERRα negatively regulates the commitment of mesenchymal cells to the osteoblast lineage ex vivo as well as later steps of osteoblast maturation. We searched to determine whether the activities of ERRα on osteoblast maturation are responsible for one or both types of in vivo induced bone loss. To this end we have generated conditional knock out mice in which the receptor is normally present during early osteoblast differentiation but inactivated upon osteoblast maturation. Bone ageing in these animals was similar to that observed for control animals. In contrast conditional ERRαKO mice were completely resistant to bone loss induced by ovariectomy. We conclude that the late (maturation), but not early (commitment), negative effects of ERRα on the osteoblast lineage contribute to the reduced bone mineral density observed upon estrogen deficiency.