BBA - Molecular Cell Research (v.1823, #5)

Regulatory mechanism of transforming growth factor beta receptor type II degradation by interleukin-1 in primary chondrocytes by Catherine Baugé; Nicolas Girard; Sylvain Leclercq; Philippe Galéra; Karim Boumédiene (983-986).
Interleukin-1β (IL-1β), a key-cytokine in osteoarthritis, impairs TGFβ signaling through TβRII down-regulation by increasing its degradation. Here, we investigated the molecular mechanism that controls TßRII fate in IL-1ß treated cells.Chondrocytes were treated with IL-1ß in the presence of different inhibitors. TßRII and Cav-1 expression were assayed by Western blot and RT-PCR.We showed that IL-1ß-induced degradation of TßRII is dependent on proteasome and on its internalization in caveolae. In addition, IL-1ß enhances Cav-1 expression, a major constituent of lipid raft.In conclusion, we enlighten a new mechanism by which IL-1ß antagonizes TGFß pathway and propose a model of TßRII turnover regulation upon IL-1ß treatment.► IL-1ß increases degradation of TßRII. ► IL-1ß-induced degradation of TßRII is dependent on proteasome pathways. ► IL-1ß-induced degradation of TßRII is blocked by nystatin.
Keywords: TGFß receptor; Interleukin-1; Turnover regulation; Caveolin-1; Proteasome; Osteoarthritis;

The aim of the present study is to estimate the role played by cortisol, prolactin (PRL) and epidermal growth factor (EGF) in the synthesis of adipocyte differentiation-related protein (ADRP) as compared to the well-studied regulation of β-casein synthesis by these hormones in the mammary epithelial cell line HC11. This comparison between a cytoplasmic lipid droplet-associated protein, which is strictly specific to both lipid accumulation and secretion by lactating mammary epithelial cells, and an archetypal milk protein is useful for evaluating the extent to which a mechanistic relationship exists between biosynthesis, transport and secretion of these two major milk components. We found that cortisol inhibits PRL-stimulated ADRP synthesis, as opposed to its known stimulating effect on β-casein synthesis. The involvement of PRL and EGF in ADRP synthesis was explored by means of a battery of inhibitors. The Jak2 inhibitor AG490 provoked a stimulation of ADRP synthesis whereas it totally suppressed that of β-casein. The use of AG1478, a specific inhibitor of EGF receptor phosphorylation, or of PD98059, a specific MEK inhibitor, revealed that the Ras/Raf/MEK/ERK1/2 pathway has no significant influence on ADRP levels. Inhibition of JNK was also ineffective. In contrast, incubation of the cells with SB 203580, a specific inhibitor of p38, slightly stimulated ADRP synthesis and induced a proportional dose–response inhibition of PRL-induced β-casein synthesis. Finally, cell treatment with wortmannin or LY294002 revealed that both PRL and EGF positively regulate ADRP and β-casein synthesis through PI3-kinase signaling. Because both the Akt inhibitor MK-2206 and the mTOR inhibitor rapamycin provoked a strong diminution of PRL-induced synthesis of the two proteins, and because oleate induced phosphorylation of Akt, we concluded that, in the mammary epithelial cell line HC11, the PI3-kinase/Akt/mTOR signaling pathway strongly participates in β-casein synthesis and is a main regulator of ADRP expression.► Multiple hormones regulate lipid and protein production by mammary epithelial cells. ► PRL (and EGF) stimulates the expression of ADRP, independently of that of ß-casein. ► The PI3-kinase/Akt/mTOR signaling pathway is a main regulator of ADRP expression. ► The regulation of ADRP expression is crucial to control milk lipid production.
Keywords: PI3K/Akt; Mammary epithelial cell; Lactation; Prolactin; Casein; HC11 cell;

Activation of Stat3 in endothelial cells following hypoxia–reoxygenation is mediated by Rac1 and protein kinase C by Subhendra N. Mattagajasingh; Xiao Ping Yang; Kaikobad Irani; Ilwola Mattagajasingh; Lewis C. Becker (997-1006).
Stat3 is an important transcription factor that regulates both proinflammatory and anti-apoptotic pathways in the heart. This study examined the mechanisms of activation of Stat3 in human endothelial cells following hypoxia/reoxygenation (H/R). By expression of constitutively active Rac1 mutant protein, and by RNA silencing of Rac1, we found that Stat3 Y705 and S727 phosphorylation following H/R is dependent on Rac1. Reactive oxygen species produced during H/R, and direct physical association with Rac1 both contribute to Stat3 activation. Stat3 forms a multiprotein complex with Rac1 and PKC in an H/R-dependent manner, which at least in part, appears to regulate Stat3 S727 phosphorylation. Selective inhibition of PKC with calphostin C produces a marked suppression of Stat3 S727 phosphorylation. The association of Stat3 with Rac1 occurs predominantly at the cell membrane, but also inside the nucleus, and occurs through the binding of the coiled-coil domain of Stat3 to the 54 NH2-terminal residues of Rac1. Transfection with a peptide comprising the NH2-terminal 17 amino acid residues of Rac1 inhibits Stat3 S727 phosphorylation after H/R. Thus, Stat3 is activated in endothelial cells by H/R through Rac1-dependent signaling pathways resulting in physical association between Rac1 and Stat3 and the formation of a novel multiprotein complex with PKC.► Stat3 activation by hypoxia/reoxygenation is mediated by both Rac1 and PKC. ► Activation of Stat3 occurs through a multiprotein complex with Rac1 and PKC. ► Stat3 binds through its coiled-coil domain to the 54 NH2-terminal residues of Rac1. ► A peptide of the 17 NH2-terminal amino acids of Rac1 can inhibit Stat3 activation.
Keywords: Endothelial cell; Stat3; Protein kinase C; Inflammation; Protein–protein interaction; Reactive oxygen species;

Complement modulates the function of the ubiquitin–proteasome system and endoplasmic reticulum-associated degradation in glomerular epithelial cells by Thomas M. Kitzler; Joan Papillon; Julie Guillemette; Simon S. Wing; Andrey V. Cybulsky (1007-1016).
In experimental membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 also activates mechanisms that restrict injury or facilitate recovery. The ubiquitin–proteasome system (UPS) selectively degrades damaged or abnormal proteins, while misfolded proteins in the endoplasmic reticulum (ER) undergo ER-associated degradation (ERAD). In this study, we investigated the effect of complement on the UPS and ERAD. We monitored UPS function by transfection of rat GECs with a UPS reporter, GFPu (CL1 degron fused with green fluorescent protein). By analogy, CD3δ-yellow fluorescent protein (YFP) was employed as a reporter of ERAD. We demonstrated decreased GFPu levels in GECs after incubation with antibody and complement, compared with control. Using C8-deficient serum with or without purified C8, cycloheximide (an inhibitor of protein synthesis), and the proteasome inhibitor, MG132, we confirmed that the decrease of GFPu was mediated by C5b-9, and subsequent proteasomal degradation of the reporter. Inhibition of the c-Jun N-terminal kinase attenuated the effect of complement on GFPu degradation. Complement, however, increased the level of CD3δ-YFP in GECs, implying an impairment of ERAD, likely due to an overabundance of misfolded proteins in the ER. The overall ubiquitination of proteins was enhanced in complement-treated GECs and in glomeruli of rats with experimental membranous nephropathy, although ubiquitin mRNA was unchanged in GECs. Proteasome inhibition with MG132 increased the cytotoxic effect of complement in GECs. Complement-stimulated UPS function, by accelerating removal of damaged proteins, may be a novel mechanism to limit complement-induced injury.► The effect of complement on the ubiquitin–proteasome system (UPS) was investigated. ► Complement stimulated UPS function in glomerular epithelial cells. ► Ubiquitination increased in complement-treated cells and rat membranous nephropathy. ► Complement impaired endoplasmic reticulum-associated degradation. ► Proteasome inhibition exacerbated the cytotoxic effect of complement.
Keywords: Cell injury; Glomerulonephritis; Kidney; Protein kinase; Signal transduction;

MiR-217 is involved in Tat-induced HIV-1 long terminal repeat (LTR) transactivation by down-regulation of SIRT1 by Hong-Sheng Zhang; Tong-Chao Wu; Wei-Wei Sang; Zheng Ruan (1017-1023).
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and may contribute to the development and progression of many infective diseases including human immunodeficiency virus 1 (HIV-1) infection. The Tat protein is fundamental to viral gene expression. In this study, our goal was to investigate the regulation of a specific miRNA (known as miR-217) in multinuclear activation of galactosidase indicator (MAGI) cells and explore the mechanisms by which miR-217 influenced Tat-induced HIV-1 transactivation through down-regulation of SIRT1 expression. We showed that miR-217 was up-regulated when Tat was expressed in multinuclear activation of galactosidase indicator cells. Forced expression of “miR-217 mimics” increased Tat-induced LTR transactivation. In addition, miR-217 significantly inhibited SIRT1 protein expression by acting on the 3′-UTR of the SIRT1 mRNA. In turn, the decrease in SIRT1 protein abundance provoked by miR-217 affected two important types of downstream signaling molecules that were regulated by Tat. Lower expression of SIRT1 caused by miR-217 enhanced Tat-induced phosphorylation of IKK and p65-NFkB and also exacerbated the loss of AMPK phosphorylation triggered by Tat. Our results uncover previously unknown links between Tat and a specific host cell miRNA that targets SIRT1. We also demonstrate that this regulatory mechanism impinges on p65-NFkB and AMPK signaling: two important host cell pathways that influence HIV-1 pathogenesis. Our results also suggest that strategies to augment SIRT1 protein expression by down-regulation of miR-217 may have therapeutic benefits to prevent HIV-1 replication.► miR-217 was up-regulated in MAGI cells induced by transfection with Tat. ► Forced expression of miR-217 decreased SIRT1 protein expression. ► MiR-217 diminished Tat induced phosphorylation of p65 and IKK. ► MiR-217 reversed Tat inhibited phosphorylation of AMPK.
Keywords: miR-217; SIRT1; AMPK; NF-κB;

It has been suggested that mouse lbx1 is essential for directing hypaxial myogenic precursor cell migration. In zebrafish, the expression of lbx1a, lbx1b, and lbx2 has been observed in pectoral fin buds. It has also been shown that knocking down endogenous lbx2 in zebrafish embryos diminishes myoD expression in the pectoral fin bud. However, downstream lbxs signals remain largely unexplored. Here, we describe a previously unknown function of zebrafish lbx2 (lbx2) during convergent extension (CE) movements. The abrogation of the lbx2 function by two non-overlapping morpholino oligonucleotides (MOs) resulted in the defective convergence and extension movements in morphants during gastrulation. Our transplantation studies further demonstrated that the overexpression of lbx2 autonomously promotes CE movements. Expression of wnt5b is significantly reduced in lbx2 morphants. We have demonstrated that application of the wnt5b MO, a dominant-negative form of disheveled (Dvl) and a chemical inhibitor of Rho-associated kinase Y27632 in zebrafish embryos have effects reminiscent that are of the CE and hypaxial myogenesis defects observed in lbx2 morphants. Moreover, the CE and hypaxial mesoderm defects seen in lbx2 morphants can be rescued by co-injection with wnt5b or RhoA mRNA. However, this reduced level of active RhoA and hypaxial myogenesis defects in the embryos injected with the dominant-negative form of Dvl mRNA cannot be effectively restored by co-injection with lbx2 mRNA. Our results suggest that the key noncanonical Wnt signaling components Wnt5, Dvl, and RhoA are downstream effectors involved in the regulative roles of lbx2 in CE movement and hypaxial myogenesis during zebrafish embryogenesis.Display Omitted ► Zlbx2 participates in the regulation of CE movements during gastrulation. ► The nc-Wnt signaling can be regulated by zlbx2 during gastrulation. ► Aberrant CE movements during gastrulation results in zebrafish hypaxial myogenesis.
Keywords: Zebrafish; lbx2; Noncanonical Wnt; Gastrulation;

Specific lipids influence the import capacity of the chloroplast outer envelope precursor protein translocon by Rajae Elkehal; Thomas Becker; Maik S. Sommer; Martina Königer; Enrico Schleiff (1033-1040).
Recent studies demonstrated that lipids influence the assembly and efficiency of membrane-embedded macromolecular complexes. Similarly, lipids have been found to influence chloroplast precursor protein binding to the membrane surface and to be associated with the Translocon of the Outer membrane of Chloroplasts (TOC). We used a system based on chloroplast outer envelope vesicles from Pisum sativum to obtain an initial understanding of the influence of lipids on precursor protein translocation across the outer envelope. The ability of the model precursor proteins p(OE33)titin and pSSU to be recognized and translocated in this simplified system was investigated. We demonstrate that transport across the outer membrane can be observed in the absence of the inner envelope translocon. The translocation, however, was significantly slower than that observed for chloroplasts. Enrichment of outer envelope vesicles with different lipids natively found in chloroplast membranes altered the binding and transport behavior. Further, the results obtained using outer envelope vesicles were consistent with the results observed for the reconstituted isolated TOC complex. Based on both approaches we concluded that the lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylinositol (PI) increased TOC-mediated binding and import for both precursor proteins. In contrast, enrichment in digalactosyldiacylglycerol (DGDG) improved TOC-mediated binding for pSSU, but decreased import for both precursor proteins. Optimal import occurred only in a narrow concentration range of DGDG.►The lipid-dependence of the chloroplast outer envelope translocon (TOC) was analyzed. ►A new in vitro method for analysis of sole TOC-function in membranes is developed. ►The lipid-dependence of TOC function is precursor specific. ►The native DGDG concentration in the outer envelope is optimal for TOC function. ►The lipids SQDG and PI stimulate TOC-dependent precursor recognition and import.
Keywords: Precursor protein translocation; Outer envelope vesicle; Lipid composition; TOC;

Manganese superoxide dismutase: A regulator of T cell activation-induced oxidative signaling and cell death by Marcin Mikołaj Kamiński; Daniel Röth; Sabine Sass; Sven Wolfgang Sauer; Peter Heinrich Krammer; Karsten Gülow (1041-1052).
Mitochondrial reactive oxygen species (ROS) are indispensible for T cell activation-induced expression of interleukin 2 (IL-2) and CD95 ligand (CD95L, FasL/Apo-1L) genes, and in turn, for CD95L-mediated activation-induced cell death (AICD). Here, we show that manganese superoxide dismutase (MnSOD/SOD2), a major mitochondrial antioxidative enzyme, constitutes an important control switch in the process of activation-induced oxidative signal generation in T cells. Analysis of the kinetics of T cell receptor (TCR)-triggered ROS production revealed a temporal association between higher MnSOD abundance/activity and a shut-down phase of oxidative signal generation. Transient or inducible MnSOD overexpression abrogated T cell activation-triggered mitochondrial ROS production as well as NF-κB- and AP-1-mediated transcription. Consequently, lowered expression of IL-2 and CD95L genes resulted in decreased IL-2 secretion and CD95L-dependent AICD. Moreover, upregulation of the mitochondrial MnSOD level is dependent on oxidation-sensitive transcription and not on the increase of mitochondrial mass. Thus, MnSOD-mediated negative feedback regulation of activation-induced mitochondrial ROS generation exemplifies a process of retrograde mitochondria-to-nucleus communication. Our finding underlines the critical role for MnSOD and mitochondria in the regulation of human T cell activation.Display Omitted► MnSOD plays a critical role in the regulation of T cell activation. ► MnSOD content and activity are up-regulated in the late phase of TCR-induced oxidative signaling. ► Up-regulated MnSOD blocks TCR-induced mitochondrial ROS release via a feedback loop. ► Decreased oxidative signal shuts down NF-κB-mediated transcription of IL-2 and CD95L. ► Lowering of CD95L expression results in decreased T cell activation-induced apoptosis.
Keywords: T cell activation; Manganese superoxide dismutase (MnSOD/SOD2); Activation-induced cell death (AICD); Mitochondria; Reactive oxygen species (ROS); IL-2 and CD95L/FasL;