SUMMARY Macrophage-mediated inflammation is critical in the pathogenesis of non-alcoholic steatohepatitis (NASH). Here, we describe that, with high-fat, high-sucrose-diet feeding, mature TIM4 pos Kupffer cells (KCs) decrease in number, while monocyte-derived Tim4 neg macrophages accumulate. In concert, monocyte-derived infiltrating macrophages enter the liver and consist of a transitional subset that expresses Cx3cr1/Ccr2 and a second subset characterized by expression of Trem2, Cd63, Cd9 , and Gpmnb ; markers ascribed to lipid-associated macrophages (LAMs). The Cx3cr1/Ccr2 -expressing macrophages, referred to as C-LAMs, localize to macrophage aggregates and hepatic crown-like structures (hCLSs) in the steatotic liver. In C-motif chemokine receptor 2 ( Ccr2 )-deficient mice, C-LAMs fail to appear in the liver, and this prevents hCLS formation, reduces LAM numbers, and increases liver fibrosis. Taken together, our data reveal dynamic changes in liver macrophage subsets during the pathogenesis of NASH and link these shifts to pathologic tissue remodeling.

In developing inhibitors of the LIM kinases, the initial lead molecules combined potent target inhibition with potent cytotoxic activity. However, as subsequent compounds were evaluated, the cytotoxic activity separated from inhibition of LIM kinases. A rapid determination of the cytotoxic mechanism and its molecular target was enabled by integrating data from two robust core technologies. Highcontent assays and gene expression profiling both indicated an effect on microtubule stability. Although the cytotoxic compounds are still kinase inhibitors, and their structures did not predict tubulin as an obvious target, these results provided the impetus to test their effects on microtubule polymerization directly. Unexpectedly, we confirmed tubulin itself as a molecular target of the cytotoxic kinase inhibitor compounds. This general approach to mechanism of action questions could be extended to larger data sets of quantified phenotypic and gene expression data. [Mol Cancer Ther 2008;7(11):3490-8]

Background: Ceramide is a critical lipid mediator of cellular stress responses relating to inflammation and insulin resistance. Results: Palmitate and LPS synergistically induce macrophage de novo ceramide biosynthesis. Conclusion: TLR4 signaling via TRIF and MyD88 in combination with palmitate up-regulates ceramide synthesis through a non-transcriptional mechanism. Significance: Understanding how lipid excess intersects with inflammatory signaling may provide insights into the pathogenesis of metabolic disease.

Structure-activity studies in the pyrazolo[1,5-a]-1,3,5-triazine series led to the discovery that compound 11i (DMP696) is a potent hCRF(1) receptor antagonist (K(i) = 1.7 nM vs 7.5 nM for alpha-hel-CRF(9-41), hCRF(1) adenylate cyclase IC(50) = 82 nM vs 286 nM for alpha-hel-CRF(9-41)). Compound 11i has excellent oral pharmacokinetic profiles in rats and dogs (37% and 50% oral bioavailabilities, respectively). This compound displays good activity in the rat situational anxiety model (MED = 3 mg/kg (po)), whereas a literature standard 1 (CP154526-1) was inactive (MED > 30 mg/kg (po)). Analogue 11i reduced stereotypical mouth movements in rhesus monkeys by 50% at 21 mg/kg (po) using the human intruder paradigm. Overall, the profile of pyrazolotriazine 11i indicates that hCRF(1) receptor antagonists may be anxiolytic agents, which have reduced motor side effect profiles.

These data suggest that the CRF(1) antagonist DMP696 might retain the therapeutic benefits of classical benzodiazepines but have fewer motoric side effects.

Background: Long noncoding RNAs (lncRNAs) can act as microRNA (miRNA) sponges to regulate protein-coding gene expression; therefore, lncRNAs are considered a major part of the competitive endogenous RNA (ceRNA) network and have attracted growing attention. The present study explored the regulatory mechanisms and functional roles of lncRNAs as ceRNAs in hepatocellular carcinoma (HCC) and their potential impact on HCC patient prognosis. Methods: In this study, we systematically studied the expression profiles and prognostic value of lncRNA, miRNA, and mRNA from a total of 838 HCC patients from five HCC cohorts (TCGA, GSE54236, GSE76427, GSE64041 and GSE14520). The TCGA, GSE54236 and GSE76427 HCC cohorts were utilized to establish a prognosis-related network of dysregulated ceRNAs by bioinformatics methods. The GSE64041 and GSE14520 HCC cohorts were utilized to verify the expression of candidate genes. Results: In total, 721 lncRNAs, 73 miRNAs, and 1563 mRNAs were aberrantly expressed in HCC samples. A ceRNA network including 26 lncRNAs, four miRNAs, and six mRNAs specific to HCC was established. The survival analysis showed that four lncRNAs (MYCNOS, DLX6-AS1, LINC00221, and CRNDE) and two mRNAs (CCNB1 and SHCBP1) were prognostic biomarkers for patients with HCC in both the TCGA and GEO databases. Conclusion: The proposed ceRNA network may help elucidate the regulatory mechanism by which lncRNAs function as ceRNAs and contribute to the pathogenesis of HCC. Importantly, the candidate lncRNAs, miRNAs, and mRNAs involved in the ceRNA network can be further evaluated as potential therapeutic targets and prognostic biomarkers for HCC.

To identify the factors mediating the progression of diabetic nephropathy (DN), we performed RNA sequencing of kidney biopsy samples from patients with early DN, advanced DN, and normal kidney tissue from nephrectomy samples. A set of genes that were upregulated at early but downregulated in late DN were shown to be largely renoprotective, which included genes in the retinoic acid pathway and glucagon-like peptide 1 receptor. Another group of genes that were downregulated at early but highly upregulated in advanced DN consisted mostly of genes associated with kidney disease pathogenesis, such as those related to immune response and fibrosis. Correlation with estimated glomerular filtration rate (eGFR) identified genes in the pathways of iron transport and cell differentiation to be positively associated with eGFR, while those in the immune response and fibrosis pathways were negatively associated. Correlation with various histopathological features also identified the association with the distinct gene ontological pathways. Deconvolution analysis of the RNA sequencing data set indicated a significant increase in monocytes, fibroblasts, and myofibroblasts in advanced DN kidneys. Our study thus provides potential molecular mechanisms for DN progression and association of differential gene expression with the functional and structural changes observed in patients with early and advanced DN.

Triptolide (TPL), a key biologically active component of the Chinese medicinal herb Tripterygium wilfordii Hook. f., has potent anti-inflammation and anti-cancer activities. Its anti-proliferative and pro-apoptotic effects have been reported to be related to the inhibition of Nuclear Factor κB (NF-κB) and Nuclear Factor of Activated T-cells (NFAT) mediated transcription and suppression of HSP70 expression. The direct targets and precise mechanisms that are responsible for the gene expression inhibition, however, remain unknown. Here, we report that TPL inhibits global gene transcription by inducing proteasome-dependent degradation of the largest subunit of RNA polymerase II (Rpb1) in cancer cells. In the presence of proteosome inhibitor MG132, TPL treatment causes hyperphosphorylation of Rpb1 by activation of upstream protein kinases such as Positive Transcription Elongation Factor b (P-TEFb) in a time and dose dependent manner. Also, we observe that short time incubation of TPL with cancer cells induces DNA damage. In conclusion, we propose a new mechanism of how TPL works in killing cancer. TPL inhibits global transcription in cancer cells by induction of phosphorylation and subsequent proteasome-dependent degradation of Rpb1 resulting in global gene transcription, which may explain the high potency of TPL in killing cancer.