N-acetylglucosamine kinase, HXK1 contributes to white–opaque morphological transition in Candida albicans

Morphological transition (yeast-hyphal and white–opaque) is an important biological process in the life cycle of pathogenic yeast, Candida albicans and is a major determinant of virulence. Earlier reports show that the amino sugar, N-acetylglucosamine (GlcNAc) induces white to opaque switching in this pathogen. We report here a new contributor to this switching phenomenon, namely N-acetylglucosamine kinase or HXK1, the first enzyme of the GlcNAc catabolic cascade. Microarray profile analysis of wild type vs. hxk1 mutant cells grown under switching inducing condition showed upregulation of opaque specific and cell wall specific genes along genes involved in the oxidative metabolism. Further, our qRT-PCR and immunoblot analysis revealed that the expression levels of Wor1, a master regulator of the white–opaque switching phenomenon remained unaltered during this HXK1 mediated transition. Thus the derepression of opaque specific gene expression observed in hxk1 mutant could be uncoupled to the expression of WOR1. Moreover, this regulation via HXK1 is independent of Ras1, a major regulator of morphogenetic transition and probably independent of MTL locus too. These results extend our understanding of multifarious roles of metabolic enzymes like Hxk1 and suggest an adaptive mechanism during host–pathogen interactions.     

Regulation of epithelial–mesenchymal IL-1 signaling by PPARβ/δ is essential for skin homeostasis and wound healing

Skin morphogenesis, maintenance, and healing after wounding require complex epithelial–mesenchymal interactions. In this study, we show that for skin homeostasis, interleukin-1 (IL-1) produced by keratinocytes activates peroxisome proliferator–activated receptor β/δ (PPARβ/δ) expression in underlying fibroblasts, which in turn inhibits the mitotic activity of keratinocytes via inhibition of the IL-1 signaling pathway. In fact, PPARβ/δ stimulates production of the secreted IL-1 receptor antagonist, which leads to an autocrine decrease in IL-1 signaling pathways and consequently decreases production of secreted mitogenic factors by the fibroblasts. This fibroblast PPARβ/δ regulation of the IL-1 signaling is required for proper wound healing and can regulate tumor as well as normal human keratinocyte cell proliferation. Together, these findings provide evidence for a novel homeostatic control of keratinocyte proliferation and differentiation mediated via PPARβ/δ regulation in dermal fibroblasts of IL-1 signaling. Given the ubiquitous expression of PPARβ/δ, other epithelial–mesenchymal interactions may also be regulated in a similar manner.     

Suppression of RhoG activity is mediated by a syndecan 4–synectin–RhoGDI1 complex and is reversed by PKCα in a Rac1 activation pathway

Fibroblast growth factor 2 (FGF2) is a major regulator of developmental, pathological, and therapeutic angiogenesis. Its activity is partially mediated by binding to syndecan 4 (S4), a proteoglycan receptor. Angiogenesis requires polarized activation of the small guanosine triphosphatase Rac1, which involves localized dissociation from RhoGDI1 and association with the plasma membrane. Previous work has shown that genetic deletion of S4 or its adapter, synectin, leads to depolarized Rac activation, decreased endothelial migration, and other physiological defects. In this study, we show that Rac1 activation downstream of S4 is mediated by the RhoG activation pathway. RhoG is maintained in an inactive state by RhoGDI1, which is found in a ternary complex with synectin and S4. Binding of S4 to synectin increases the latter''s binding to RhoGDI1, which in turn enhances RhoGDI1''s affinity for RhoG. S4 clustering activates PKCα, which phosphorylates RhoGDI1 at Ser⁹⁶. This phosphorylation triggers release of RhoG, leading to polarized activation of Rac1. Thus, FGF2-induced Rac1 activation depends on the suppression of RhoG by a previously uncharacterized ternary S4–synectin–RhoGDI1 protein complex and activation via PKCα.     

The SufBCD protein complex is the scaffold for iron–sulfur cluster assembly in Thermus thermophiles HB8

Iron–sulfur (Fe–S) clusters are the oldest and most versatile inorganic cofactors that are required to sustain fundamental life processes. Bacteria have three systems of [Fe–S] cluster biogenesis, designated ISC, NIF, and SUF. In contrast, the Thermus thermophiles HB8 has only one system, formed mostly by SUF homologs that contain six proteins: SufA, SufB, SufC, SufD, SufS and SufE. The kinetics of SufC ATPase was studied using a linked enzyme assay method. In the presence of SufB, SufD or SufBD complexes, the activity of SufC was enhanced. The cysteine desulfurase activity of SufS was also stimulated by the presence of the SufBCD complex. The results obtained through enzymology revealed that aconitase activity was activated by [Fe–S] clusters reconstituted on the SufBCD complex. Consolidated results from spectral and enzymatic analysis suggest that the SufBCD complex is a novel type of Fe–S scaffold system that can assemble Fe/S clusters de novo.     

Aging-related hyperinflammation in endotoxemia is mediated by the α2A-adrenoceptor and CD14/TLR4 pathways

AimsSepsis is a major cause of morbidity and mortality in the elderly population. In prior studies, we have shown that in vivo, the inflammatory response in aged animals is exaggerated as compared to young animals and that this response likely accounts for the increased morbidity and mortality. Part of this uncontrolled inflammatory response in sepsis is due to the innate immune response. However, recent studies have shown that the pathogenesis of sepsis is much more complex. The adrenergic autonomic nervous system is now thought to play a key role in modulating the inflammatory response in sepsis. In this study, we hypothesize that not only is the innate immune response enhanced in response to lipopolysaccharide (LPS) in aged animals, but that the adrenergic nervous system also plays a role in the release of excess inflammatory cytokines.Main methodsMale Fischer-344 rats (young: 3 months; aged: 24 months) were used. Endotoxemia was induced by intravenous injection of lipopolysaccharide (LPS, 15 mg/kg BW). Splenic tissues were harvested and mRNA and protein were extracted. The protein expression of CD14 and TLR4, key mediators of LPS in the innate response, as well as alpha-2A adrenergic receptor (α_2A-AR) and phosphodiesterase 4D (PDE4D), as the means by which the autonomic nervous system exerts its effects were analyzed.Key findingsSplenic tissue concentrations of α_2A-AR, PDE4D, CD14, and TLR4 were significantly increased in septic aged rats as compared to aged sham rats and septic young rats. The increased expression of α_2A-AR in septic aged rats was further confirmed by immunohistochemical staining of splenic tissues.SignificanceThese data support the hypothesis that not only is the innate immune response increased in aged animals during sepsis, but that there is also an upregulated response of the adrenergic autonomic nervous system that contributes to excess proinflammatory cytokine release.     

Scribble is required for normal epithelial cell–cell contacts and lumen morphogenesis in the mammalian lung

During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell–cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical–basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, ‘open’ lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in Scrib^Crc/Crc lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell–cell association, we show that Scrib associates with β-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell–cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion.     

Acsl1 is essential for skin barrier function through the activation of linoleic acid and biosynthesis of ω-O-acylceramide in mice

The long-chain acyl-CoA synthase1 (Acsl1) is a major enzyme that converts long-chain fatty acids to acyl-CoAs. The role of Acsl1 in energy metabolism has been elucidated in the adipose tissue, heart, and skeletal muscle. Here, we demonstrate that systemic deficiency of Acsl1 caused severe skin barrier defects, leading to embryonic lethality. Acsl1 mRNA and protein are expressed in the Acsl1^+/+ epidermis, which are absent in Acsl1^?/? mice. In Acsl1^?/? mice, epidermal ceramide [EOS] (Cer[EOS]) containing ω-O-esterified linoleic acid, a lipid essential for the skin barrier, was significantly reduced. Conversely, ω-hydroxy ceramide (Cer[OS]), a precursor of Cer[EOS], was increased. Moreover, the levels of triglyceride (TG) species containing linoleic acids were lower in Acsl1^?/? mice, whereas those not containing linoleic acid were comparable to Acsl1^+/+ mice. As TG is considered to work as a reservoir of linoleic acid for the biosynthesis of Cer[EOS] from Cer[OS], our results suggest that Acsl1 plays an essential role in ω-O-acylceramide synthesis by providing linoleic acid for ω-O-esterification. Therefore, our findings identified a new biological role of Acsl1 as a regulator of the skin barrier.     

Echium and Pontechium specific floral cues for host–plant recognition by the oligolectic bee Hoplitis adunca

Flowering plants often have specific floral cues, which allow bees and other pollinators to differentiate between them. Many bee species exhibit specialised associations with flowers (oligolecty) and it is important for them to find and recognise their specific host plants. In this study we compared the visual and olfactory floral cues of different Echium and Pontechium (Boraginaceae) species with the closely related Anchusa officinalis (Boraginaceae). We tested whether plant-specific cues occur in Echium and Pontechium which may allow oligolectic Hoplitis adunca (Megachilidae) to recognise its host plants and to distinguish them from Anchusa non-hosts. Our investigations showed that Echium/Pontechium provides a specific scent bouquet. Furthermore, we identified compounds which were not described as floral scent before ((Z)-3-nonenal and 1,4-benzoquinone). These unique volatiles and the specific bouquet could act as a recognition cue for H. adunca. The corolla colours differed between all species, but were grouped together in the bee colour categories blue and UV-blue and can indicate potential host flowers for H. adunca.     

TSC1/2 signaling complex is essential for peripheral naïve CD8+ T cell survival and homeostasis in mice

The PI3K-Akt-mTOR pathway plays crucial roles in regulating both innate and adaptive immunity. However, the role of TSC1, a critical negative regulator of mTOR, in peripheral T cell homeostasis remains elusive. With T cell-specific Tsc1 conditional knockout (Tsc1 KO) mice, we found that peripheral na?ve CD8(+) T cells but not CD4(+) T cells were severely reduced. Tsc1 KO na?ve CD8(+) T cells showed profound survival defect in an adoptive transfer model and in culture with either stimulation of IL-7 or IL-15, despite comparable CD122 and CD127 expression between control and KO CD8(+) T cells. IL-7 stimulated phosphorylation of Akt(S473) was diminished in Tsc1 KO na?ve CD8(+)T cells due to hyperactive mTOR-mediated feedback suppression on PI3K-AKT signaling. Furthermore, impaired Foxo1/Foxo3a phosphorylation and increased pro-apoptotic Bim expression in Tsc1 KO na?ve CD8(+)T cells were observed upon stimulation of IL-7. Collectively, our study suggests that TSC1 plays an essential role in regulating peripheral na?ve CD8(+) T cell homeostasis, possible via an mTOR-Akt-FoxO-Bim signaling pathway.     

Chemoattractant stimulation of TORC2 is regulated by receptor/G protein–targeted inhibitory mechanisms that function upstream and independently of an essential GEF/Ras activation pathway in Dictyostelium

Global stimulation of Dictyostelium with different chemoattractants elicits multiple transient signaling responses, including synthesis of cAMP and cGMP, actin polymerization, activation of kinases ERK2, TORC2, and phosphatidylinositide 3-kinase, and Ras-GTP accumulation. Mechanisms that down-regulate these responses are poorly understood. Here we examine transient activation of TORC2 in response to chemically distinct chemoattractants, cAMP and folate, and suggest that TORC2 is regulated by adaptive, desensitizing responses to stimulatory ligands that are independent of downstream, feedback, or feedforward circuits. Cells with acquired insensitivity to either folate or cAMP remain fully responsive to TORC2 activation if stimulated with the other ligand. Thus TORC2 responses to cAMP or folate are not cross-inhibitory. Using a series of signaling mutants, we show that folate and cAMP activate TORC2 through an identical GEF/Ras pathway but separate receptors and G protein couplings. Because the common GEF/Ras pathway also remains fully responsive to one chemoattractant after desensitization to the other, GEF/Ras must act downstream and independent of adaptation to persistent ligand stimulation. When initial chemoattractant concentrations are immediately diluted, cells rapidly regain full responsiveness. We suggest that ligand adaptation functions in upstream inhibitory pathways that involve chemoattractant-specific receptor/G protein complexes and regulate multiple response pathways.     

Is preconditioning by oxytocin administration mediated by iNOS and/or mitochondrial KATP channel activation in the in vivo anesthetized rabbit heart?

AimsOxytocin (OXT) pretreatment protects the heart during ischemia–reperfusion injury by activating ATP-dependent potassium (K_ATP) channels. The aim of the current study was to elucidate the roles of nitric oxide synthaseNOS and myocardial biochemistry in the cardioprotective effects of OXT and ischemic preconditioning (IPC).Main methodsMale New Zealand White anesthetized rabbits (13 groups) were subjected to 30 min of occlusion of the left coronary artery and 120 min of reperfusion with or without IPC.Key findingsIPC (1 cycle), OXT (0.03 μg/kg, i.p.) or IPC + OXT yield significant infarct size reductions (21.8 ± 1.5%, 20.5 ± 1.2% and 19.4 ± 1.4%, respectively, versus 38.9 ± 3.5% in the S-CONT group; P < 0.01) and antiarrhythmic effects, including VF (0%, 0% and 0%, versus 50% in S-CONT group; P < 0.05) sustained VT (13%, 13% and 13%, versus 100% in S-CONT group; P < 0.005) and other arrhythmias (25%, 13% and 25%, versus 100% in S-CONT group; P < 0.005, P < 0.01 and P < 0.005, respectively). Atosiban (ATO, a selective OXT receptor antagonist), 5-HD and l-NAME (a nonspecific NOS inhibitor) abolished the beneficial effects of IPC and OXT, suggesting that the benefits are achieved via selective activation of OXT receptors, mitochondrial K_ATP channels and NO. An iNOS inhibitor (1400 W) blocked the beneficial effects of IPC but not OXT. The IPC, OXT, IPC + OXT and 1400 W + OXT interventions significantly preserved ATP levels in the heart.SignificanceThis study demonstrates similarities between acute OXT pretreatment and IPC in terms of infarct size reduction, antiarrhythmic activity, and metabolic status.     

FRS2α is essential for the fibroblast growth factor to regulate the mTOR pathway and autophagy in mouse embryonic fibroblasts

Although the fibroblast growth factor (FGF) signaling axis plays important roles in cell survival, proliferation, and differentiation, the molecular mechanism underlying how the FGF elicits these diverse regulatory signals is not well understood. By using the Frs2α null mouse embryonic fibroblast (MEF) in conjunction with inhibitors to multiple signaling pathways, here we report that the FGF signaling axis activates mTOR via the FGF receptor substrate 2α (FRS2α)-mediated PI3K/Akt pathway, and suppresses autophagy activity in MEFs. In addition, the PI3K/Akt pathway regulated mTOR is crucial for the FGF signaling axis to suppress autophagy in MEFs. Since autophagy has been proposed to play important roles in cell survival, proliferation, and differentiation, the findings suggest a novel mechanism for the FGF signaling axis to transmit regulatory signals to downstream effectors.     

Early activation of the β-catenin pathway in osteocytes is mediated by nitric oxide, phosphatidyl inositol-3 kinase/Akt, and focal adhesion kinase

Bone mechanotransduction is vital for skeletal integrity. Osteocytes are thought to be the cellular structures that sense physical forces and transform these signals into a biological response. The Wnt/β-catenin signaling pathway has been identified as one of the signaling pathways that is activated in response to mechanical loading, but the molecular events that lead to an activation of this pathway in osteocytes are not well understood. We assessed whether nitric oxide, focal adhesion kinase, and/or the phosphatidyl inositol-3 kinase/Akt signaling pathway mediate loading-induced β-catenin pathway activation in MLO-Y4 osteocytes. We found that mechanical stimulation by pulsating fluid flow (PFF, 0.7 ± 0.3 Pa, 5 Hz) for 30 min induced β-catenin stabilization and activation of the Wnt/β-catenin signaling pathway. The PFF-induced stabilization of β-catenin and activation of the β-catenin signaling pathway was abolished by adding focal kinase inhibitor FAK inhibitor-14 (50 μM), or phosphatidyl inositol-3 kinase inhibitor LY-294002 (50 μM). Addition of nitric oxide synthase inhibitor l-NAME (1.0 mM) also abolished PFF-induced stabilization of β-catenin. This suggests that mechanical loading activates the β-catenin signaling pathway by a mechanism involving nitric oxide, focal adhesion kinase, and the Akt signaling pathway. These data provide a framework for understanding the role of β-catenin in mechanical adaptation of bone.     

Rapamycin-enhanced mitomycin C-induced apoptotic death is mediated through the S6K1–Bad–Bak pathway in peritoneal carcinomatosis

Peritoneal carcinomatosis (PC) is the most common secondary cancerous disease, and more effective novel regimens are needed. In this study, we identified a novel combination treatment for PC, chemotherapeutic agent mitomycin C in combination with mTOR (mammalian target of rapamycin) inhibitor rapamycin. We observed that the combination of mitomycin C and rapamycin induced synergistic cytotoxicity and apoptosis, which was mediated through an increase in caspase activation. The combination of mitomycin C and rapamycin inactivated p70 S6 ribosomal kinase (S6K1) and dephosphorylated Bad, leading to dissociation of Bcl-xL from Bak, which resulted in Bak oligomerization, mitochondria dysfunction and cytochrome c release. PF-4708671, a S6K1-specific inhibitor, enhanced the combination treatment-induced apoptosis, whereas S6K1 E389 DeltaCT-HA (S6K1 active form) dramatically decreased the induction of apoptosis. In addition, the combination treatment significantly inhibited LS174T intraperitoneal tumor growth in vivo. This study provides a preclinical rationale for apoptosis induction linked with the mTOR pathway through a combination of chemotherapeutic agents and mTOR inhibitor, and will support this combinatorial strategy to PC patients.Peritoneal carcinomatosis (PC) is the most common secondary cancerous disease to affect the peritoneal cavity following colorectal cancer, appendiceal cancer, ovarian cancer, gastric cancer, or diffuse malignant peritoneal mesothelioma, and is regarded as a lethal condition having poor prognosis with a median survival of ∼6 months.^{1, 2} Systemic chemotherapy prolongs survival in PC patients, but is less effective than for patients with hepatic metastasis; thus, an increasing number of patients are treated with cytoreductive surgery followed by hyperthermic intraperitoneal chemoperfusion (HIPEC).³ Nevertheless, more effective novel regimens are still needed to improve the survival of PC patients.Mitomycin C, a potent DNA crosslinker, was used as an anticancer drug and led to pronounced induction of apoptosis, especially through the mitochondrial-dependent pathway.^{4, 5} It has received a great number of preclinical and clinical studies and is mostly widely used for PC.⁶ Combination of cytotoxic drugs, such as mitomycin C with novel biological agents, has achieved improvement in animal models and provides additional survival benefits for patients.^{7, 8, 9}mTOR (mammalian target of rapamycin), a serine/threonine kinase, is ubiquitously involved in cell growth and proliferation. The fact that hyperactivation of mTOR is a common feature of human cancers makes it an attractive target in cancer therapy.¹⁰ It has been reported that some agents inactivate mTOR signaling as an early response prior to the execution of apoptosis.^{11, 12} Recently, it has been found that mTOR can directly control mitochondrial function,¹³ which raises the possibility that targeting mTOR may regulate the apoptotic pathway. Sirolimus, known as rapamycin, an mTOR inhibitor, potently inhibits S6K1 (S6 ribosomal kinase) activity¹⁴ and has been generally well tolerated in clinical trials, often inducing prolonged stable disease or tumor regression, and can be considered as an option for combination with other anticancer agents.^{15, 16}In this study we observed that the combination of chemotherapy agent mitomycin C and mTOR inhibitor rapamycin induced synergistic cytotoxicity and apoptosis in vitro and in vivo. The combination of mitomycin C and rapamycin inactivated S6K1, not Akt, and resulted in dephosphorylation of Bad, which inactivated its pro-apoptotic function, inducing Bak oligomerization and mitochondrial-dependent apoptotic cell death.     

Skeletal muscle increases FGF21 expression in mitochondrial disorders to compensate for energy metabolic insufficiency by activating the mTOR–YY1–PGC1α pathway

Fibroblast growth factor 21 (FGF21) is a growth factor with pleiotropic effects on regulating lipid and glucose metabolism. Its expression is increased in skeletal muscle of mice and humans with mitochondrial disorders. However, the effects of FGF21 on skeletal muscle in response to mitochondrial respiratory chain deficiency are largely unknown. Here we demonstrate that the increased expression of FGF21 is a compensatory response to respiratory chain deficiency. The mRNA and protein levels of FGF21 were robustly raised in skeletal muscle from patients with mitochondrial myopathy or MELAS. The mammalian target of rapamycin (mTOR) phosphorylation levels and its downstream targets, Yin Yang 1 (YY1) and peroxisome proliferator-activated receptor γ, coactivator 1α (PGC-1α), were increased by FGF21 treatment in C2C12 myoblasts. Activation of the mTOR–YY1–PGC1α pathway by FGF21 in myoblasts regulated energy homeostasis as demonstrated by significant increases in intracellular ATP synthesis, oxygen consumption rate, activity of citrate synthase, glycolysis, mitochondrial DNA copy number, and induction of the expression of key energy metabolic genes. The effects of FGF21 on mitochondrial function required phosphoinositide 3-kinase (PI3K), which activates mTOR. Inhibition of PI3K, mTOR, YY1, and PGC-1α activities attenuated the stimulating effects of FGF21 on intracellular ATP levels and mitochondrial gene expression. Our findings revealed that mitochondrial respiratory chain deficiency elicited a compensatory response in skeletal muscle by increasing the FGF21 expression levels in muscle, which resulted in enhanced mitochondrial function through an mTOR–YY1–PGC1α-dependent pathway in skeletal muscle.     

Rap1 and Canoe/afadin are essential for establishment of apical–basal polarity in the Drosophila embryo

The establishment and maintenance of apical–basal cell polarity is critical for assembling epithelia and maintaining organ architecture. Drosophila embryos provide a superb model. In the current view, apically positioned Bazooka/Par3 is the initial polarity cue as cells form during cellularization. Bazooka then helps to position both adherens junctions and atypical protein kinase C (aPKC). Although a polarized cytoskeleton is critical for Bazooka positioning, proteins mediating this remained unknown. We found that the small GTPase Rap1 and the actin-junctional linker Canoe/afadin are essential for polarity establishment, as both adherens junctions and Bazooka are mispositioned in their absence. Rap1 and Canoe do not simply organize the cytoskeleton, as actin and microtubules become properly polarized in their absence. Canoe can recruit Bazooka when ectopically expressed, but they do not obligatorily colocalize. Rap1 and Canoe play continuing roles in Bazooka localization during gastrulation, but other polarity cues partially restore apical Bazooka in the absence of Rap1 or Canoe. We next tested the current linear model for polarity establishment. Both Bazooka and aPKC regulate Canoe localization despite being “downstream” of Canoe. Further, Rap1, Bazooka, and aPKC, but not Canoe, regulate columnar cell shape. These data reshape our view, suggesting that polarity establishment is regulated by a protein network rather than a linear pathway.