Modest induction of endoplasmic reticulum (ER) stress confers resistance to inflammation in glomeruli. Recently, we found that ER stress leads to mesangial insensitivity to cytokine-induced activation of NF-B, but the underlying mechanisms are incompletely understood. ER stress can trigger expression of CCAAT/enhancer-binding proteins (C/EBPs), which interact with transcription factors including NF-B. Here, we investigated a role for C/EBPs in the ER stress-induced resistance to cytokines. Mesangial cells preferentially induced C/EBP␤ after exposure to thapsigargin or tunicamycin; induction of C/EBP␦ was modest and transient, and expression of C/EBP␣ was absent. The induction of C/EBP␤ correlated with accumulation of C/EBP␤ protein and enhanced transcriptional activity of C/EBP. Overexpression of C/EBP␤ markedly suppressed TNF-␣-induced activation of NF-B, independent of its transacting potential. Knockdown of C/EBP␤ by small interfering RNA reversed the suppressive effect of ER stress on NF-B. In vivo, preconditioning of mice with ER stress induced renal C/EBP␤ and suppressed NF-Bdependent gene expression in response to LPS. Using dominant negative mutants and null mutants for individual branches of the unfolded protein response, we identified the RNA-dependent protein kinaselike ER kinase (PERK) and the inositol-requiring ER-to-nucleus signal kinase 1 (IRE1) pathways as the unfolded protein response responsible for ER stress-induced C/EBP␤. These results suggest that ER stress blunts cytokine-triggered activation of NF-B, in part through PERK-and IRE1-mediated preferential induction of C/EBP␤.

BACKGROUND AND PURPOSECordyceps militaris has the potential to suppress differentiation of pre-adipocytes. However, the active entities in the extract and the underlying mechanisms of its action are not known. Hence, we investigated whether and how cordycepin (3′-deoxyadenosine), a constituent of C. militaris, inhibits adipogenesis. EXPERIMENTAL APPROACHDifferentiation of 3T3-L1 pre-adipocytes and pre-adipocytes in primary cultures was induced by Insulin, dexamethasone and IBMX, and these were used as in vitro models of adipogenesis. The effects of cordycepin on adipogenesis were examined with particular focus on the regulation of CCAAT/enhancer-binding protein b (C/EBPb) and PPARg. KEY RESULTSCordycepin suppressed the lipid accumulation and induction of adipogenic markers that occurred on differentiation of pre-adipocytes and also blocked the down-regulation of a pre-adipocyte marker. This anti-adipogenic effect was reversible and mediated by an adenosine transporter, but not A1, A2 or A3 adenosine receptors. This effect of cordycepin was not reproduced by other adenosine-related substances, including ATP, ADP and adenosine. Early induction of the adipogenic C/EBPb-PPARg pathway was suppressed by cordycepin. Blockade of mTORC1 via inhibition of PKB (Akt) and activation of AMP kinase was identified as the crucial upstream event targeted by cordycepin. In addition to its negative effect on adipogenesis, cordycepin suppressed lipid accumulation in mature adipocytes. CONCLUSIONS AND IMPLICATIONSThese results suggest that the anti-adipogenic effects of cordycepin occur through its intervention in the mTORC1-C/EBPb-PPARg pathway. Cordycepin, by blocking both adipogenesis and lipid accumulation, may have potential as a therapeutic agent for effective treatment of obesity and obesity-related disorders.

Therapy resistance in leukemia may be due to cancer cell-intrinsic and/or -extrinsic mechanisms. Mutations within BCR-ABL1 , the oncogene giving rise to chronic myeloid leukemia (CML), lead to resistance to tyrosine kinase inhibitors (TKI), and some are associated with clinically more aggressive disease and worse outcome. Using the retroviral transduction/transplantation model of CML and human cell lines we faithfully recapitulate accelerated disease course in TKI resistance. We show in various models, that murine and human imatinib-resistant leukemia cells positive for the oncogene BCR-ABL1 T315I differ from BCR-ABL1 native ( BCR-ABL1 ) cells with regards to niche location and specific niche interactions. We implicate a pathway via integrin β3, integrin-linked kinase (ILK) and its role in deposition of the extracellular matrix (ECM) protein fibronectin as causative of these differences. We demonstrate a trend towards a reduced BCR-ABL1 T315I+ tumor burden and significantly prolonged survival of mice with BCR-ABL1 T315I+ CML treated with fibronectin or an ILK inhibitor in xenogeneic and syngeneic murine transplantation models, respectively. These data suggest that interactions with ECM proteins via the integrin β3/ILK-mediated signaling pathway in BCR-ABL1 T315I+ cells differentially and specifically influence leukemia progression. Niche targeting via modulation of the ECM may be a feasible therapeutic approach to consider in this setting.

Mycelial extracts have a wide range of biological activities that modulate functions of mammalian cells. In this report, we sought to identify antiadipogenic mycelia with the use of 3T3-L1 cells and found that the extract of Cordyceps militaris exclusively suppressed differentiation of 3T3-L1 preadipocytes into mature adipocytes without affecting cell viability. This inhibitory effect was dose dependent, reversible, and associated with 1) a decrease in lipid accumulation, 2) blunted induction of adipocyte markers including adiponectin, peroxisome proliferator-activated receptor-γ, and CCAAT/enhancer binding protein-α, and 3) sustained expression of a preadipocyte marker, monocyte chemoattractant protein-1. C. militaris also significantly decreased accumulation of lipid and hypertrophy in mature adipocytes and preserved their response to insulin (phosphorylation of Akt) during prolonged culture. Subsequent experiments revealed that C. militaris has the potential to activate the aryl hydrocarbon receptor (AhR). In 3T3-L1 cells, treatment with AhR agonists including benzo[ a]pyrene and 3-methylcholanthrene reproduced the antiadipogenic effect of C. militaris. Furthermore, dominant-negative inhibition of AhR abrogated the suppressive effect of C. militaris on adipocyte differentiation. These results suggest that C. militaris has the potential to interfere with adipocyte differentiation through activation of AhR.

In many cancers, somatic mutations confer tumorigenesis and drug-resistance. The recently established clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a potentially elegant approach to functionally evaluate mutations in cancers. To reproduce mutations by homologous recombination (HR), the HR pathway must be functional, but DNA damage repair is frequently impaired in cancers. Imatinib is a tyrosine kinase inhibitor for BCR-ABL1 in Philadelphia chromosome-positive (Ph+) leukemia, and development of resistance due to kinase domain mutation is an important issue. We attempted to introduce the T315I gatekeeper mutation into three Ph+ myeloid leukemia cell lines with a seemingly functional HR pathway due to resistance to the inhibitor for poly (ADP) ribose polymerase1. Imatinib-resistant sublines were efficiently developed by the CRISPR/Cas9 system after short-term selection with imatinib; resulting sublines acquired the T315I mutation after HR. Thus, the usefulness of CRISPR/Cas9 system for functional analysis of somatic mutations in cancers was demonstrated.

Tumor necrosis factor-a (TNF-a) is a key regulator of adipose tissue mass, but mechanisms underlying this effect have not been fully elucidated. We found that exposure to TNF-a caused a significant decrease in the number of adipocytes, but not preadipocytes. Subsequent experiments revealed that TNF-a selectively deleted adipocytes through induction of apoptosis. Following exposure to TNF-a, rapid activation of nuclear factor-kB (NF-kB) was observed only in preadipocytes, but not in adipocytes. Inhibition of NF-kB rendered preadipocytes susceptible to TNF-a-induced apoptosis, suggesting that different activity of NF-kB is the determinant for the distinct apoptotic responses. During adipocyte differentiation, expression and activity of peroxisome proliferator-activated receptor-g (PPARg) were upregulated. Treatment of preadipocytes with a PPARg agonist attenuated NF-kB activation and rendered the cells vulnerable to TNF-a-induced apoptosis. Conversely, treatment of adipocytes with a PPARg antagonist enhanced NF-kB activation and conferred resistance to TNF-a-induced apoptosis, suggesting involvement of PPARg in the suppression of NF-kB in adipocytes. We also found that, following differentiation, expression and activity of CCAAT/ enhancer binding protein (C/EBP), especially C/EBPa and C/EBPb, were upregulated in adipocytes. Overexpression of individual C/EBPs significantly inhibited activation of NF-kB in preadipocytes. Furthermore, transfection with siRNA for C/EBPa or C/EBPb enhanced activity of NF-kB in adipocytes, suggesting that C/EBP is also involved in the repression of NF-kB in adipocytes. These results suggested novel mechanisms by which TNF-a selectively deletes adipocytes in the adipose tissue. The C/EBP-and PPARg-mediated suppression of NF-kB may contribute to TNF-a-related loss of adipose tissue mass under certain pathological situations, such as cachexia.

Human herpesvirus 6B (HHV-6B) causes exanthema subitum in infants and is known to be mildly pathogenic. However, HHV-6B infection can induce febrile seizures in a high percentage of patients, and in rare cases, result in encephalitis. We detected higher levels of interleukin (IL)-1β and basic fibroblast growth factor (bFGF) in the cerebrospinal fluid (CFS) of patients with HHV-6B encephalitis when compared to those in patients with non-HHV-6B-induced febrile seizures. In vitro, IL-1β and bFGF enhanced HHV-6B gene expression in infected U373 astrocytes during the initial and maintenance phases of infection, respectively. These findings indicated that IL-1β and bFGF contribute to HHV-6B growth and the onset of encephalitis.

No abstract