Differentiating agents regulate cathepsin B gene expression in HL‐60 cells

We utilized HL‐60 cells as a model system to examine the regulation of ctsb gene expression by differentiating agents. Inducers of monocytic differentiation [phorbol ester (PMA), calcitriol (D3), and sodium butyrate (NaB)] and inducers of granulocytic differentiation [all‐trans retinoic acid (RA) and 9‐cis retinoic acid (9‐cis RA)] increase ctsb mRNA levels in a dose‐dependent manner as determined by Northern blot hybridization. D3 and retinoids exert additive effects, suggesting that these agents act in part through distinct pathways. Actinomycin D decay experiments indicate that D3, NaB, RA, and 9‐cis RA do not alter mRNA stability. In contrast, PMA markedly increases the half‐life of ctsb mRNA. In transient transfection assays, PMA and NaB both stimulate transcription of the luciferase reporter gene placed under the control of ctsb promoter fragments. Thus, inducers of HL‐60 cell differentiation can regulate the expression of the ctsb gene at both transcriptional and posttranscriptional levels. J. Leukoc. Biol. 66: 609–616; 1999.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142263/1/jlb0609.pd

Genome Characterization of the Oleaginous Fungus Mortierella alpina

Mortierella alpina is an oleaginous fungus which can produce lipids accounting for up to 50% of its dry weight in the form of triacylglycerols. It is used commercially for the production of arachidonic acid. Using a combination of high throughput sequencing and lipid profiling, we have assembled the M. alpina genome, mapped its lipogenesis pathway and determined its major lipid species. The 38.38 Mb M. alpina genome shows a high degree of gene duplications. Approximately 50% of its 12,796 gene models, and 60% of genes in the predicted lipogenesis pathway, belong to multigene families. Notably, M. alpina has 18 lipase genes, of which 11 contain the class 2 lipase domain and may share a similar function. M. alpina's fatty acid synthase is a single polypeptide containing all of the catalytic domains required for fatty acid synthesis from acetyl-CoA and malonyl-CoA, whereas in many fungi this enzyme is comprised of two polypeptides. Major lipids were profiled to confirm the products predicted in the lipogenesis pathway. M. alpina produces a complex mixture of glycerolipids, glycerophospholipids and sphingolipids. In contrast, only two major sterol lipids, desmosterol and 24(28)-methylene-cholesterol, were detected. Phylogenetic analysis based on genes involved in lipid metabolism suggests that oleaginous fungi may have acquired their lipogenic capacity during evolution after the divergence of Ascomycota, Basidiomycota, Chytridiomycota and Mucoromycota. Our study provides the first draft genome and comprehensive lipid profile for M. alpina, and lays the foundation for possible genetic engineering of M. alpina to produce higher levels and diverse contents of dietary lipids

Exon 2 of human cathepsin B derives from an Alu element

AbstractTranscripts for the cysteine protease cathepsin B are alternatively spliced in the untranslated regions (UTRs). We show that a cathepsin B probe containing 5′-UTR sequences hybridized to an RNA of ∼300 nt in addition to the typical 2.2 and 4.0 kbp mRNAs. Within this 5′-UTR, exon 2 was found to be homologous to Alu repetitive elements. Specifically, exon 2 was part of an Alu element interspersed with the cathepsin B gene. The ∼300 nt band that hybridized to our cathepsin B probe likely corresponds to Alu transcripts, which are known to accumulate in human cells. Indeed, a similarly migrating band was detected with an authentic Alu probe. Thus, we suggest that primary transcripts for cathepsin B contain Alu sequences which are preserved as exon 2 in some fully spliced mRNAs

Decorin Suppresses Prostate Tumor Growth through Inhibition of Epidermal Growth Factor and Androgen Receptor Pathways1

Epidermal growth factor receptor (EGFR) and androgen receptor (AR) pathways play pivotal roles in prostate cancer progression. Therefore, agents with dual-targeting ability may have important therapeutic potential. Decorin, a proteoglycan present in the tumor microenvironment, is known to regulate matrix assembly, growth factor binding, and receptor tyrosine kinase activity. Here, we show that in prostate-specific PtenP-/- mice, a genetically defined, immune-competent mouse model of prostate cancer, systemic delivery of decorin inhibits tumor progression by targeting cell proliferation and survival pathways. Moreover, in human prostate cancer cells, we show that decorin specifically inhibits EGFR and AR phosphorylation and cross talk between these pathways. This prevents AR nuclear translocation and inhibits the production of prostate specific antigen. Further, the phosphatidylinositol-3 kinase (PI3K)/Akt cell survival pathway is suppressed leading to tumor cell apoptosis. Those findings highlight the effectiveness of decorin in the presence of a powerful genetic cancer risk and implicate decorin as a potential new agent for prostate cancer therapy by targeting EGFR/AR-PI3K-Akt pathways

Y-box binding protein-1 serine 102 is a downstream target of p90 ribosomal S6 kinase in basal-like breast cancer cells

Introduction: Basal-like breast cancers (BLBC) frequently overexpress the epidermal growth factor receptor (EGFR) and subsequently have high levels of signaling through the MAP kinase pathway, which is thought to contribute to their aggressive behavior. While we have previously reported the expression of Y-box binding protein-1 (YB-1) in 73% of BLBC, it is unclear whether it can be regulated by a component of the MAP kinase signaling pathway. Phosphorylation of YB-1 at the serine 102 residue is required for transcriptional activation of growth-enhancing genes, such as EGFR. Using Motifscan we identified p90 ribosomal S6 kinase (RSK) as a potential candidate for activating YB-1. Methods Inhibition of RSK1 and RSK2 was achieved using siRNA and the small molecule SL0101. RSK1, RSK2, activated RSK and kinase-dead RSK were expressed in HCC1937 cells. Kinase assays were performed to illustrate direct phosphorylation of YB-1 by RSK. The impact of inhibiting RSK on YB-1 function was measured by luciferase assays and chromatin immunoprecipitation. Results Using an in vitro kinase assay, RSK1 and RSK2 were shown to directly phosphorylate YB-1. Interestingly, they were more effective activators of YB-1 than AKT or another novel YB-1 kinase, PKCα. Phosphorylation of YB-1 (serine 102 residue) is blocked by inhibition of the MAP kinase pathway or by perturbing RSK1/RSK2 with siRNA or SL0101. In immortalized breast epithelial cells where RSK is active yet AKT is not, YB-1 is phosphorylated. Supporting this observation, RSK2-/- mouse embryo fibroblasts lose the ability to phosphorylate YB-1 in response to epidermal growth factor. This subsequently interfered with the ability of YB-1 to regulate the expression of EGFR. The RSK inhibitor SL0101 decreased the ability of YB-1 to bind the promoter, transactivate and ultimately reduce EGFR expression. In concordance with these results the expression of constitutively active RSK1 increased YB-1 phosphorylation, yet the kinase-dead RSK did not. Conclusions We therefore conclude that RSK1/RSK2 are novel activators of YB-1, able to phosphorylate the serine 102 residue. This provides a newly described mechanism whereby YB-1 is activated in breast cancer. This implicates the EGFR/RSK/YB-1 pathway as an important component of BLBC, providing an important opportunity for therapeutic intervention.Medicine, Faculty ofPediatrics, Department ofNon UBCReviewedFacult