LEF1-mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes

Elayyan, Jinan,Lee, Eun-Jin,Gabay, Odile,Smith, Christopher A.,Qiq, Omar,Reich, Eli,Mobasheri, Ali,Henrotin, Yves,Kimber, Susan J.,Dvir-Ginzberg, Mona The Federation of American Societies for Experimen 2017 The FASEB Journal Vol.31 No.7

<P>Reduced SIRT1 activity and levels during osteoarthritis (OA) promote gradual loss of cartilage. Loss of cartilage matrix is accompanied by an increase in matrix metalloproteinase (MMP) 13, partially because of enhanced LEF1 transcriptional activity. In this study, we assessed the role of SIRT1 in LEF1-mediated MMP13 gene expression in human OA chondrocytes. Results showed that MMP13 protein levels and enzymatic activity decreased significantly during SIRT1 overexpression or activation by resveratrol. Conversely, MMP13 gene expression was reduced in chondrocytes transfected with SIRT1 siRNA or treated with nicotinamide (NAM), a sirtuin inhibitor. Chondrocytes challenged with IL-113, a cytokine involved in OA pathogenesis, enhanced LEF1 protein levels and gene expression, resulting in increased MMP13 gene expression; however, overexpression of SIRT1 during IL-113 challenge impeded LEF1 levels and MMP13 gene expression. Previous reports showed that LEF1 binds to the MMP13 promoter and transactivates its expression, but we observed that SIRT1 repressed LEF1 protein and mRNA expression, ultimately reducing LEF1 transcriptional activity, as judged by luciferase assay. Finally, mouse articular cartilage from Sirt1-/presented increased LEF1 and MMP13 protein levels, similar to human OA cartilage. Thus, demonstrating for the first time that SIRT1 represses MMP13 in human OA chondrocytes, which appears to be mediated, at least in part, through repression of the transcription factor LEF1, a known modulator of MMP13 gene expression. Elayyan, J., Lee, E.-J., Gabay, O., Smith, C. A., Qiq, O., Reich, E., Mobasheri, A., Henrotin, Y., Kimber, S. J., Dvir-Ginzberg, M. LEF1-mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes.</P>

Plasma zinc's alter ego is a low-molecular-weight humoral factor

Ou, Ou,Allen-Redpath, Keith,Urgast, Dagmar,Gordon, Margaret-Jane,Campbell, Gill,Feldmann, Jö,rg,Nixon, Graeme F.,Mayer, Claus-Dieter,Kwun, In-Sook,Beattie, John H. The Federation of American Societies for Experimen 2013 The FASEB Journal Vol.27 No.9

<P>Mild dietary zinc deprivation in humans and rodents has little effect on blood plasma zinc levels, and yet cellular consequences of zinc depletion can be detected in vascular and other tissues. We proposed that a zinc-regulated humoral factor might mediate the effects of zinc deprivation. Using a novel approach, primary rat vascular smooth muscle cells (VSMCs) were treated with plasma from zinc-deficient (<1 mg Zn/kg) or zinc-adequate (35 mg Zn/kg, pair-fed) adult male rats, and zinc levels were manipulated to distinguish direct and indirect effects of plasma zinc. Gene expression changes were analyzed by microarray and qPCR, and incubation of VSMCs with blood plasma from zinc-deficient rats strongly changed the expression of >2500 genes, compared to incubation of cells with zinc-adequate rat plasma. We demonstrated that this effect was caused by a low-molecular-weight (∼2-kDa) zinc-regulated humoral factor but that changes in gene expression were mostly reversed by adding zinc back to zinc-deficient plasma. Strongly regulated genes were overrepresented in pathways associated with immune function and development. We conclude that zinc deficiency induces the production of a low-molecular-weight humoral factor whose influence on VSMC gene expression is blocked by plasma zinc. This factor is therefore under dual control by zinc.—Ou, O., Allen-Redpath, K., Urgast, D., Gordon, M.-J., Campbell, G., Feldmann, J., Nixon, G. F., Mayer, C.-D., Kwun, I.-S., and Beattie, J. H. Plasma zinc's alter ego is a low-molecular-weight humoral factor.</P>

CBX7 inhibits breast tumorigenicity through DKK-1-mediated suppression of the Wnt/<i>β</i>-catenin pathway

Kim, Hey-Yon,Park, Ji-Hye,Won, Hee-Young,Lee, Jeong-Yeon,Kong, Gu The Federation of American Societies for Experimen 2015 The FASEB Journal Vol.29 No.1

<P>Polycomb protein chromobox homolog 7 (CBX7) is involved in several biologic processes including stem cell regulation and cancer development, but its roles in breast cancer remain unknown. Here, we demonstrate that CBX7 negatively regulates breast tumor initiation. CD44<SUP>+</SUP>/CD24<SUP>−</SUP>/ESA<SUP>+</SUP> breast stem-like cells showed diminished CBX7 expression. Furthermore, small hairpin RNA-mediated CBX7 knockdown in breast epithelial and cancer cells increased the CD44<SUP>+</SUP>/CD24<SUP>−</SUP>/ESA<SUP>+</SUP> cell population and reinforced <I>in vitro</I> self-renewal and <I>in vivo</I> tumor-initiating ability. Similarly, CBX7 overexpression repressed these effects. We also found that CBX7 inhibits the Wnt/<I>β</I>-catenin/T cell factor pathway by enhancing the expression of Dickkopf-1 (DKK-1), a Wnt antagonist. In particular, CBX7 increased <I>DKK-1</I> transcription by cooperating with p300 acetyltransferase and subsequently enhancing the histone acetylation of the <I>DKK-1</I> promoter. Furthermore, pharmacologic inhibition of DKK-1 in CBX7-overexpressing cells showed recovery of Wnt signaling and consequent rescue of the CD44<SUP>+</SUP>/CD24<SUP>−</SUP>/ESA<SUP>+</SUP> cell population. Taken together, these findings indicate that CBX7-mediated epigenetic induction of DKK-1 is crucial for the inhibition of breast tumorigenicity, suggesting that CBX7 could be a potential tumor suppressor in human breast cancer. —Kim, H.–Y., Park, J.–H., Won, H.–Y., Lee, J.–Y., Kong, G. CBX7 inhibits breast tumorigenicity through DKK-1-mediated suppression of the Wnt/<I>β</I>-catenin pathway.</P>

Promiscuous gating modifiers target the voltage sensor of K_v7.2, TRPV1, and H_v1 cation channels

Kornilov, Polina,Peretz, Asher,Lee, Yoonji,Son, Karam,Lee, Jin Hee,Refaeli, Bosmat,Roz, Netta,Rehavi, Moshe,Choi, Sun,Attali, Bernard The Federation of American Societies for Experimen 2014 The FASEB Journal Vol.28 No.6

<P>Some of the fascinating features of voltage-sensing domains (VSDs) in voltage-gated cation channels (VGCCs) are their modular nature and adaptability. Here we examined the VSD sensitivity of different VGCCs to 2 structurally related nontoxin gating modifiers, NH17 and NH29, which stabilize K<SUB>v</SUB>7.2 potassium channels in the closed and open states, respectively. The effects of NH17 and NH29 were examined in Chinese hamster ovary cells transfected with transient receptor potential vanilloid 1 (TRPV1) or K<SUB>v</SUB>7.2 channels, as well as in dorsal root ganglia neurons, using the whole-cell patch-clamp technique. NH17 and NH29 exert opposite effects on TRPV1 channels, operating, respectively, as an activator and a blocker of TRPV1 currents (EC<SUB>50</SUB> and IC<SUB>50</SUB> values ranging from 4 to 40 μM). Combined mutagenesis, electrophysiology, structural homology modeling, molecular docking, and molecular dynamics simulation indicate that both compounds target the VSDs of TRPV1 channels, which, like vanilloids, are involved in π-π stacking, H-bonding, and hydrophobic interactions. Reflecting their promiscuity, the drugs also affect the lone VSD proton channel mVSOP. Thus, the same gating modifier can promiscuously interact with different VGCCs, and subtle differences at the VSD-ligand interface will dictate whether the gating modifier stabilizes channels in either the closed or the open state.—Kornilov, P., Peretz, A., Lee, Y., Son, K., Lee, J. H., Refaeli, B., Roz, N., Rehavi, M., Choi, S., Attali, B. Promiscuous gating modifiers target the voltage sensor of K<SUB>v</SUB>7.2, TRPV1, and H<SUB>v</SUB>1 cation channels.</P>

BACE1 and presenilin/γ-secretase regulate proteolytic processing of KCNE1 and 2, auxiliary subunits of voltage-gated potassium channels

Sachse, Carolyn C.,Kim, Young Hye,Agsten, Marianne,Huth, Tobias,Alzheimer, Christian,Kovacs, Dora M.,Kim, Doo Yeon The Federation of American Societies for Experimen 2013 The FASEB Journal Vol.27 No.6

<P>BACE1 and presenilin (PS)/γ-secretase play a major role in Alzheimer's disease pathogenesis by regulating amyloid-β peptide generation. We recently showed that these secretases also regulate the processing of voltage-gated sodium channel auxiliary β-subunits and thereby modulate membrane excitability. Here, we report that KCNE1 and KCNE2, auxiliary subunits of voltage-gated potassium channels, undergo sequential cleavage mediated by either α-secretase and PS/γ-secretase or BACE1 and PS/γ-secretase in cells. Elevated α-secretase or BACE1 activities increased C-terminal fragment (CTF) levels of KCNE1 and 2 in human embryonic kidney (HEK293T) and rat neuroblastoma (B104) cells. KCNE-CTFs were then further processed by PS/γ-secretase to KCNE intracellular domains. These KCNE cleavages were specifically blocked by chemical inhibitors of the secretases in the same cell models. We also verified our results in mouse cardiomyocytes and cultured primary neurons. Endogenous KCNE1- and KCNE2-CTF levels increased by 2- to 4-fold on PS/γ-secretase inhibition or BACE1 overexpression in these cells. Furthermore, the elevated BACE1 activity increased KCNE1 processing and shifted KCNE1/KCNQ1 channel activation curve to more positive potentials in HEK cells. KCNE1/KCNQ1 channel is a cardiac potassium channel complex, and the positive shift would lead to a decrease in membrane repolarization during cardiac action potential. Together, these results clearly showed that KCNE1 and KCNE2 cleavages are regulated by BACE1 and PS/γ-secretase activities under physiological conditions. Our results also suggest a functional role of KCNE cleavage in regulating voltage-gated potassium channels.—Sachse, C. C., Kim, Y. H., Agsten, M., Huth, T., Alzheimer, C., Kovacs, D. M., and Kim, D. Y. BACE1 and presenilin/γ-secretase regulate proteolytic processing of KCNE1 and 2, auxiliary subunits of voltage-gated potassium channels.</P>

Transglutaminase 2 inhibition found to induce p53 mediated apoptosis in renal cell carcinoma

Ku, Bo Mi,Kim, Dae-Seok,Kim, Kyung-Hee,Yoo, Byong Chul,Kim, Seok-Hyun,Gong, Young-Dae,Kim, Soo-Youl The Federation of American Societies for Experimen 2013 The FASEB Journal Vol.27 No.9

<P>Renal cell carcinoma (RCC), the predominant form of kidney cancer, is characterized by high resistance to radiation and chemotherapy. This study shows that expression of protein cross-linking enzyme transglutaminase 2 (TGase 2) is markedly increased in 7 renal cell carcinoma (RCC) cell lines in comparison to HEK293 and other cancer cell lines, such as NCI 60. However, the key role of TGase 2 in RCC was not clear. The down-regulation of TGase 2 was found to stabilize p53 expression, thereby inducing a 3- to 10-fold increase in apoptosis for 786-O, A498, CAKI-1, and ACHN cell lines by DAPI staining. MEF cells from TGase 2<SUP>−/−</SUP> mice showed stabilized p53 under apoptotic stress to compare to MEFs from wild-type mice. TGase 2 directly cross links the DNA binding domain of p53, leading to p53 depletion <I>via</I> autophagy in RCC. TGase 2 and p53 expression showed an inverse relationship in RCC cells. This finding implies that induced expression of TGase 2 promotes tumor cell survival through p53 depletion in RCC.—Ku, B.M., Kim, D.-S. Kim, K.-H., Yoo, B.C., Kim, S.-H., Gong, Y.-D., Kim, S.-Y. Transglutaminase 2 inhibition found to induce p53 mediated apoptosis in renal cell carcinoma.</P>

Secreted protein lipocalin-2 promotes microglial M1 polarization

Jang, Eunha,Lee, Shinrye,Kim, Jong-Heon,Kim, Jae-Hong,Seo, Jung-Wan,Lee, Won-Ha,Mori, Kiyoshi,Nakao, Kazuwa,Suk, Kyoungho The Federation of American Societies for Experimen 2013 The FASEB Journal Vol.27 No.3

<P>Activated macrophages are classified into two different forms: classically activated (M1) or alternatively activated (M2) macrophages. The presence of M1/M2 phenotypic polarization has also been suggested for microglia. Here, we report that the secreted protein lipocalin 2 (LCN2) amplifies M1 polarization of activated microglia. LCN2 protein (EC<SUB>50</SUB> 1 μg/ml), but not glutathione <I>S</I>-transferase used as a control, increased the M1-related gene expression in cultured mouse microglial cells after 8–24 h. LCN2 was secreted from M1-polarized, but not M2-polarized, microglia. LCN2 inhibited phosphorylation of STAT6 in IL-4-stimulated microglia, suggesting LCN2 suppression of the canonical M2 signaling. In the lipopolysaccharide (LPS)-induced mouse neuroinflammation model, the expression of LCN2 was notably increased in microglia. Primary microglial cultures derived from LCN2-deficient mice showed a suppressed M1 response and enhanced M2 response. Mice lacking LCN2 showed a markedly reduced M1-related gene expression in microglia after LPS injection, which was consistent with the results of histological analysis. Neuroinflammation-associated impairment in motor behavior and cognitive function was also attenuated in the LCN2-deficient mice, as determined by the rotarod performance test, fatigue test, open-field test, and object recognition task. These findings suggest that LCN2 is an M1-amplifier in brain microglial cells.—Jang, E., Lee, S., Kim, J.-H., Kim, J.-H., Seo, J.-W., Lee, W.-H., Mori, K., Nakao, K., Suk, K. Secreted protein lipocalin-2 promotes microglial M1 polarization.</P>

An intracellular antifreeze protein from an Antarctic microalga that responds to various environmental stresses

Gwak, Yunho,Jung, Woongsic,Lee, Yew,Kim, Ji Sook,Kim, Chul Geun,Ju, Ji-Hyun,Song, Chihong,Hyun, Jae-Kyung,Jin, EonSeon The Federation of American Societies for Experimen 2014 The FASEB Journal Vol.28 No.11

<P>The structure and function of the Antarctic marine diatom <I>Chaetoceros neogracile</I> antifreeze protein (Cn-AFP), as well as its expression levels and characteristics of the ice-binding site, were analyzed in the present study. <I>In silico</I> analysis revealed that the <I>Cn-AFP</I> promoter contains both light- and temperature-responsive elements. Northern and Western blot analyses demonstrated that both Cn-AFP transcript and protein expression were strongly and rapidly stimulated by freezing, as well as temperature and high light stress. Immunogold labeling revealed that Cn-AFP is preferentially localized to the intracellular space near the chloroplast membrane. Recombinant Cn-AFP had clear antifreeze activity. Protein-folding simulation was used to predict the putative ice-binding sites in Cn-AFP, and site-directed mutagenesis of the Cn-AFP b-face confirmed their identification.—Gwak, Y., Jung, W., Lee, Y., Kim, J. S., Kim, C. G., Ju, J.-H., Song, C., Hyun, J.-K., Jin, E. An intracellular antifreeze protein from an Antarctic microalga that responds to various environmental stresses.</P>

Genetic ablation of lymphocytes and cytokine signaling in nonobese diabetic mice prevents diet-induced obesity and insulin resistance

Friedline, Randall H.,Ko, Hwi Jin,Jung, Dae Young,Lee, Yongjin,Bortell, Rita,Dagdeviren, Sezin,Patel, Payal R.,Hu, Xiaodi,Inashima, Kunikazu,Kearns, Caitlyn,Tsitsilianos, Nicholas,Shafiq, Umber,Shultz The Federation of American Societies for Experimen 2016 The FASEB Journal Vol.30 No.3

<P>Obesity is characterized by a dysregulated immune system, which may causally associate with insulin resistance and type 2 diabetes. Despite widespread use of nonobese diabetic (NOD) mice, NOD with severe combined immunodeficiency (scid) mutation (SCID) mice, and SCID bearing a null mutation in the IL-2 common chain receptor (NSG) mice as animal models of human diseases including type 1 diabetes, the underlying metabolic effects of a genetically altered immune system are poorly understood. For this, we performed a comprehensive metabolic characterization of these mice fed chow or after 6 wk of a high-fat diet. We found that NOD mice had similar to 50% less fat mass and were 2-fold more insulin sensitive, as measured by hyperinsulinemic-euglycemic clamp, than C57BL/6 wild-type mice. SCID mice were also more insulin sensitive with increased muscle glucose metabolism and resistant to diet-induced obesity due to increased energy expenditure (similar to 10%) and physical activity (similar to 40%) as measured by metabolic cages. NSG mice were completely protected from diet-induced obesity and insulin resistance with significant increases in glucose metabolism in peripheral organs. Our findings demonstrate an important role of genetic background, lymphocytes, and cytokine signaling in diet-induced obesity and insulin resistance.</P>

Human leucine zipper protein promotes hepatic steatosis <i>via</i> induction of apolipoprotein A-IV

Kang, Minsoo,Kim, Jeonghan,An, Hyoung-Tae,Ko, Jesang The Federation of American Societies for Experimen 2017 The FASEB Journal Vol.31 No.6

<P>The molecular mechanism of stress-induced hepatic steatosis is not well known. Human leucine zipper protein (LZIP) regulates the expression of genes involved in inflammation, cell migration, and stress response. The aim of this study was to determine the regulatory role of LZIP in stress-induced hepatic steatosis. We used a microarray analysis to identify LZIP-induced genes involved in hepatic lipid metabolism. LZIP increased the expression of apolipoprotein A-IV (APOA4) mRNA. In the presence of stress inducer, APOA4 promoter analysis was performed, and LZIP-induced lipid accumulation was monitored in mouse primary cells and human tissues. Under Golgi stress conditions, LZIP underwent proteolytic cleavage and was phosphorylated by AKT to protect against proteasome degradation. The stabilized N-terminal LZIP was translocated to the nucleus, where it directly bound to the APOA4 promoter, leading to APOA4 induction. LZIP-induced APOA4 expression resulted in increased absorption of surrounding free fatty acids. LZIP also promoted hepatic steatosis inmouse liver. Both LZIP and APOA4 were highly expressed in human steatosis samples. Our findings indicate that LZIP is a novel modulator of APOA4 expression and hepatic lipid metabolism. LZIP might be a therapeutic target for developing treatment strategies for hepatic steatosis and related metabolic diseases.-Kang, M., Kim, J., An, H.-T., Ko, J. Human leucine zipper protein promotes hepatic steatosis via induction of apolipoprotein A-IV.</P>