Mitochondrial metabolism regulates macrophage biology

Mitochondria are critical for regulation of the activation, differentiation, and survival of macrophages and other immune cells. In response to various extracellular signals, such as microbial or viral infection, changes to mitochondrial metabolism and physiology could underlie the corresponding state of macrophage activation. These changes include alterations of oxidative metabolism, mitochondrial membrane potential, and tricarboxylic acid (TCA) cycling, as well as the release of mitochondrial reactive oxygen species (mtROS) and mitochondrial DNA (mtDNA) and transformation of the mitochondrial ultrastructure. Here, we provide an updated review of how changes in mitochondrial metabolism and various metabolites such as fumarate, succinate, and itaconate coordinate to guide macrophage activation to distinct cellular states, thus clarifying the vital link between mitochondria metabolism and immunity. We also discuss how in disease settings, mitochondrial dysfunction and oxidative stress contribute to dysregulation of the inflammatory response. Therefore, mitochondria are a vital source of dynamic signals that regulate macrophage biology to fine-tune immune responses.     

Spectroscopic and kinetic studies of the reaction of bromopropanesulfonate with methyl-coenzyme M reductase

Methyl-coenzyme M reductase (MCR) catalyzes the final step of methanogenesis in which coenzyme B and methyl-coenzyme M are converted to methane and the heterodisulfide, CoMS-SCoB. MCR also appears to initiate anaerobic methane oxidation (reverse methanogenesis). At the active site of MCR is coenzyme F430, a nickel tetrapyrrole. This paper describes the reaction of the active MCR(red1) state with the potent inhibitor, 3-bromopropanesulfonate (BPS; I50 = 50 nM) by UV-visible and EPR spectroscopy and by steady-state and rapid kinetics. BPS was shown to be an alternative substrate of MCR in an ionic reaction that is coenzyme B-independent and leads to debromination of BPS and formation of a distinct state ("MCR(PS)") with an EPR signal that was assigned to a Ni(III)-propylsulfonate species (Hinderberger, D., Piskorski, R. P., Goenrich, M., Thauer, R. K., Schweiger, A., Harmer, J., and Jaun, B. (2006) Angew. Chem. Int. Ed. Engl. 45, 3602-3607). A similar EPR signal was generated by reacting MCR(red1) with several halogenated sulfonate and carboxylate substrates. In rapid chemical quench experiments, the propylsulfonate ligand was identified by NMR spectroscopy and high performance liquid chromatography as propanesulfonic acid after protonolysis of the MCR(PS) complex. Propanesulfonate formation was also observed in steady-state reactions in the presence of Ti(III) citrate. Reaction of the alkylnickel intermediate with thiols regenerates the active MCR(red1) state and eliminates the propylsulfonate group, presumably as the thioether. MCR(PS) is catalytically competent in both the generation of propanesulfonate and reformation of MCR(red1). These results provide evidence for the intermediacy of an alkylnickel species in the final step in anaerobic methane oxidation and in the initial step of methanogenesis.     

Proton pump-rich cell secretes acid in skin of zebrafish larvae

The mammalian kidney excretes its metabolic acid load through the proton-transporting cells, intercalated cells, in the distal nephron and collecting duct. Fish excrete acid through external organs, gill, or skin; however, the cellular function is still controversial. In this study, molecular and electrophysiological approaches were used to identify a novel cell type secreting acid in skin of zebrafish (Danio rerio) larvae. Among keratinocytes covering the larval surface, novel proton-secreting ionocytes, proton pump (H⁺-ATPase)-rich cells, were identified to generate strong outward H⁺ flux. The present work demonstrates for the first time, with a noninvasive technique, H⁺-secreting cells in an intact animal model, the zebrafish, showing it to be a suitable model in which to study the functions of vertebrate transporting epithelia in vivo. hydrogen-adenosinetriphosphatase; ionocytes; epithelial transport; ion-selective electrode     

The P174L mutation in human Sco1 severely compromises Cox17-dependent metallation but does not impair copper binding

Sco1 is a metallochaperone that is required for copper delivery to the Cu(A) site in the CoxII subunit of cytochrome c oxidase. The only known missense mutation in human Sco1, a P174L substitution in the copper-binding domain, is associated with a fatal neonatal hepatopathy; however, the molecular basis for dysfunction of the protein is unknown. Immortalized fibroblasts from a SCO1 patient show a severe deficiency in cytochrome c oxidase activity that was partially rescued by overexpression of P174L Sco1. The mutant protein retained the ability to bind Cu(I) and Cu(II) normally when expressed in bacteria, but Cox17-mediated copper transfer was severely compromised both in vitro and in a yeast cytoplasmic assay. The corresponding P153L substitution in yeast Sco1 was impaired in suppressing the phenotype of cells harboring the weakly functional C57Y allele of Cox17; however, it was functional in sco1delta yeast when the wild-type COX17 gene was present. Pulse-chase labeling of mitochondrial translation products in SCO1 patient fibroblasts showed no change in the rate of CoxII translation, but there was a specific and rapid turnover of CoxII protein in the chase. These data indicate that the P174L mutation attenuates a transient interaction with Cox17 that is necessary for copper transfer. They further suggest that defective Cox17-mediated copper metallation of Sco1, as well as the subsequent failure of Cu(A) site maturation, is the basis for the inefficient assembly of the cytochrome c oxidase complex in SCO1 patients.     

Stereoelectronic effects on polyproline conformation

The polyproline type II (PPII) helix is a prevalent conformation in both folded and unfolded proteins, and is known to play important roles in a wide variety of biological processes. Polyproline itself can also form a type I (PPI) helix, which has a disparate conformation. Here, we use derivatives of polyproline, (Pro)10, (Hyp)10, (Flp)10, and (flp)10, where Hyp is (2S,4R)-4-hydroxyproline, Flp is (2S,4R)-4-fluoroproline, and flp is (2S,4S)-4-fluoroproline, to probe for a stereoelectronic effect on the conformation of polyproline. Circular dichroism spectral analyses show that 4R electron-with-drawing substituents stabilize a PPII helix relative to a PPI helix, even in a solvent that favors the PPI conformation, such as n-propanol. The stereochemistry at C4 ordains the relative stability of PPI and PPII helices, as (flp)10 forms a mixture of PPI and PPII helices in water and a PPI helix in n-propanol. The conformational preferences of (Pro)10 are intermediate between those of (Hyp)10/(Flp)10 and (flp)10. Interestingly, PPI helices of (flp)10 exhibit cold denaturation in n-propanol with a value of T(s) near 70 degrees C. Together, these data show that stereoelectronic effects can have a substantial impact on polyproline conformation and provide a rational means to stabilize a PPI or PPII helix.     

Crassocolides G–M,Cembranoids from the Formosan Soft Coral Sarcophyton crassocaule

Seven new polyoxygenated cembranoids possessing an α‐methylene‐γ‐lactone group, crassocolides G–M ( 1 – 7 , resp.), have been isolated from the AcOEt extract of the Formosan soft coral Sarcophyton crassocaule. The structures of compounds 1 – 7 were established by detailed spectroscopic analyses, including 2D‐NMR spectroscopy (¹H,¹H‐COSY, HMQC, HMBC, and NOESY), while the absolute configuration of 1 was determined using a modified reaction of Mosher's method. The cytotoxicity of compounds 1 – 7 against a limited panel of cancer cell lines was also determined.     

Osteopontin increases migration and MMP‐9 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and proliferation via interaction with its receptor, that is, αvβ3 integrin. However, the effect of OPN on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that OPN increased the migration and expression of matrix metalloproteinase (MMP)‐9 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the OPN‐induced increase of the migration and MMP‐9 up‐regulation of chondrosarcoma cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited OPN‐induced cell migration and MMP‐9 up‐regulation. Stimulation of JJ012 cells with OPN also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The OPN‐mediated increases in MMP‐9 and κB‐luciferase activities were inhibited by RGD peptide, PD98059 or FAK and ERK2 mutant. Taken together, our results indicated that OPN enhances the migration of chondrosarcoma cells by increasing MMP‐9 expression through the αvβ3 integrin, FAK, MEK, ERK and NF‐κB signal transduction pathway. J. Cell. Physiol. 221: 98–108, 2009. © 2009 Wiley‐Liss, Inc     

Stromal cell‐derived factor‐1/CXCR4 enhanced motility of human osteosarcoma cells involves MEK1/2, ERK and NF‐κB‐dependent pathways

Osteosarcoma is characterized by a high malignant and metastatic potential. The chemokine stromal‐derived factor‐1α (SDF‐1α) and its receptor, CXCR4, play a crucial role in adhesion and migration of human cancer cells. Integrins are the major adhesive molecules in mammalian cells, and has been associated with metastasis of cancer cells. Here, we found that human osteosarcoma cell lines had significant expression of SDF‐1 and CXCR4 (SDF‐1 receptor). Treatment of osteosarcoma cells with SDF‐1α increased the migration and cell surface expression of αvβ3 integrin. CXCR4‐neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or small interfering RNA against CXCR4 inhibited the SDF‐1α‐induced increase the migration and integrin expression of osteosarcoma cells. Pretreated of osteosarcoma cells with MAPK kinase (MEK) inhibitor PD98059 inhibited the SDF‐1α‐mediated migration and integrin expression. Stimulation of cells with SDF‐1α increased the phosphorylation of MEK and extracellular signal‐regulating kinase (ERK). In addition, NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also inhibited SDF‐1α‐mediated cell migration and integrin up‐regulation. Stimulation of cells with SDF‐1α induced IκB kinase (IKKα/β) phosphorylation, IκB phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. Furthermore, the SDF‐1α‐mediated increasing κB‐luciferase activity was inhibited by AMD3100, PD98059, PDTC and TPCK or MEK1, ERK2, IKKα and IKKβ mutants. Taken together, these results suggest that the SDF‐1α acts through CXCR4 to activate MEK and ERK, which in turn activates IKKα/β and NF‐κB, resulting in the activations of αvβ3 integrins and contributing the migration of human osteosarcoma cells. J. Cell. Physiol. 221: 204–212, 2009. © 2009 Wiley‐Liss, Inc     

Effects of sediment-bound Cd, Pb, and Ni on the growth, feeding, and survival of Capitella sp. I

Anthropogenic metal pollutants bioaccumulated in benthic animals by means of feeding and osmotic diffusion. These metals may affect the physiology of the benthos. In this study, we exposed Capitella sp. I to three metals (Cd, Pb, and Ni), each in eight different concentrations, to determine the effects of metals on the animals. Growth rate, ingestion rate, and percent survival were estimated in three separated experiments. The growth and feeding of the worms were sensitive to even the lowest concentrations of each metal added to the sediments. The lowest observable adverse effect levels for Cd, Ni, and Pb were 0.03, 1.59, and 0.41 μmol g^− 1 sediment, respectively. Growth rates in the elevated metal contaminant treatments decreased drastically at slightly contaminated levels, lessened detrimental effects at moderately contaminated levels, and showed incompensable intoxication at heavily contaminated levels. The trends in ingestion rates were similar to those of growth rates. No significant difference in survivorship was found among the different contaminant levels for any of the three heavy metals. Capitella sp. I was most sensitive to Cd, followed by Ni and Pb, which had similar effects. The rapid physiological responses of Capitella sp. I allowed the animals to survive metal exposure. Sediment productivity remained unchanged at different contamination levels of Ni and Pb, but was drastically reduced at 4.75 μmol g^− 1 Cd in the sediment. This further demonstrated Capitella sp. I can adjust their ingestion rates to maintain constant sediment productivities in moderate pollution conditions; however, when threshold concentration was exceeded, homeostasis collapsed.     

Adiponectin increases BMP‐2 expression in osteoblasts via AdipoR receptor signaling pathway

Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes and involved in energy homeostasis. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. However, the effects of adiponectin on BMPs expression in cultured osteoblasts are largely unknown. Here we found that adiponectin increased mRNA expression of BMP‐2 but not other BMPs in cultured osteoblastic cells. Stimulation of osteoblasts with adiponectin also increased protein levels of BMP‐2 by Western blot and ELISA assay. Adiponectin‐mediated BMP‐2 expression was attenuated by 5′‐AMP‐activated protein kinase (AMPK) small interference RNA and AMPK inhibitor (araA and compound C). Activations of p38 and NF‐κB pathways after adiponectin treatment were demonstrated, and adiponectin‐induced expression of BMP‐2 was inhibited by the specific inhibitor and mutant of p38 and NF‐κB cascades. Taken together, our results provide evidence that adiponectin enhances BMP‐2 expression in osteoblastic cells, and AdipoR1 receptor, AMPK, p38 and NF‐κB signaling pathways may be involved in increasing BMP‐2 expression by adiponectin. J. Cell. Physiol. 224: 475–483, 2010. © 2010 Wiley‐Liss, Inc.