Probes for LONG

Long noncoding RNAs (lncRNAs) are critical regulators of inflammation with great potential as new therapeutic targets. However, the role of lncRNAs in early atherosclerosis remains poorly characterized. This study aimed to identify the key lncRNA players in activated endothelial cells (ECs). The lncRNAs in response to pro-inflammatory factors in ECs were screened through RNA sequencing. ICAM-1-related non-coding RNA (ICR) was identified as the most potential candidate for early atherosclerosis. ICR is essential for intercellular adhesion molecule-1 (ICAM1) expression, EC adhesion and migration. In a high fat diet-induced atherosclerosis model in mice, ICR is upregulated in the development of atherosclerosis. After intravenous injection of adenovirus carrying shRNA for mouse ICR, the atherosclerotic plaque area was markedly reduced with the declined expression of ICR and ICAM1. Mechanistically, ICR stabilized the mRNA of ICAM1 in quiescent ECs; while under inflammatory stress, ICR upregulated ICAM1 in a nuclear factor kappa B (NF-κB) dependent manner. RNA-seq analysis showed pro-inflammatory targets of NF-κB were regulated by ICR. Furthermore, the chromatin immunoprecipitation assays showed that p65 binds to ICR promoter and facilitates its transcription. Interestingly, ICR, in turn, promotes p65 accumulation and activity, forming a positive feedback loop to amplify NF-κB signaling. Preventing the degradation of p65 using proteasome inhibitors rescued the expression of NF-κB targets suppressed by ICR. Taken together, ICR acts as an accelerator to amplify NF-κB signaling in activated ECs and suppressing ICR is a promising early intervention for atherosclerosis through ICR/p65 loop blockade.

Many extracellular matrix (ECM) associated proteins that influence ECM properties have Thrombospondin type 1 repeats (TSRs) which are modified with O-linked fucose. The O-fucose is added in the endoplasmic reticulum to folded TSRs by the enzyme Protein O-fucosyltransferase-2 (POFUT2) and is proposed to promote efficient trafficking of substrates. The importance of this modification for function of TSR-proteins is underscored by the early embryonic lethality of mouse embryos lacking Pofut2. To overcome early lethality and investigate the impact of the Pofut2 knockout on the secretion of POFUT2 substrates and on extracellular matrix properties in vivo, we deleted Pofut2 in the developing limb mesenchyme using Prrx1-Cre recombinase. Loss of Pofut2 in the limb mesenchyme caused significant shortening of the limbs, long bones and tendons and stiff joint resembling the musculoskeletal dysplasias in human and in mice with mutations in ADAMTS or ADAMTSL proteins. Limb shortening was evident at embryonic day 14.5 where loss of O-fucosylation led to an accumulation of fibrillin 2 (FBN2), decreased BMP and IHH signaling, and increased TGF-β signaling. Consistent with these changes we saw a decrease in the size of the hypertrophic zone with lower levels of Collagen-X. Unexpectedly, we observed minimal effects of the Pofut2 knockout on secretion of two POFUT2 substrates, CCN2 or ADAMTS17, in the developing bone. In contrast, CCN2 and two other POFUT2 substrates important for bone development, ADAMTS6 and 10, showed a decrease in secretion from POFUT2-null HEK293T cells in vitro. These combined results suggest that the impact of the Pofut2 mutation is cell-type specific. In addition, these observations raise the possibility that the O-fucose modification on TSRs extends beyond promoting efficient trafficking of POFUT2 substrates and has the potential to influence their function in the extracellular environment.

BACKGROUND: Elucidation of the molecular mechanisms by which cancer cells overcome hypoxia is potentially important for targeted therapy. Complexation of hypoxia-inducible factors (HIFs) with aryl hydrocarbon receptor nuclear translocators can enhance gene expression and initiate cellular responses to hypoxia. However, multiple molecular mechanisms may be required for cancer cells to adapt to diverse microenvironments. We previously demonstrated that a physical interaction between the ubiquitously expressed transcription factor Sp1 and HIF2 is a major cause of FVII gene activation in poor prognostic ovarian clear cell carcinoma (CCC) cells under hypoxia. Furthermore, it was found that FVII activation is synergistically enhanced when serum-starved cells are cultured under hypoxic conditions. In this study, we investigated whether HIFs and transcription factor Sp1 cooperate to activate multiple genes in CCC cells under conditions of serum starvation and hypoxia (SSH) and then contribute to malignant phenotypes. METHODS: To identify genes activated under hypoxic conditions in an Sp1-dependent manner, we first performed cDNA microarray analyses. We further investigated the molecular mechanisms of synergistic gene activations including the associated serum factors by various experiments such as real-time RT-PCR, western blotting and chromatin immunoprecipitation. The study was further extended to animal experiments to investigate how it contributes to CCC progression in vivo. RESULTS: ICAM1 is one such gene dramatically induced by SSH and is highly induced by SSH and its synergistic activation involves both the mTOR and autonomously activated TNFα-NFκB axes. We identified long chain fatty acids (LCFA) as a major class of lipids that is associated with albumin, a serum factor responsible for synergistic gene activation under SSH. Furthermore, we found that ICAM1 can be induced in vivo to promote tumor growth. CONCLUSION: Sp1 and HIFs collaborate to activate genes required for the adaptation of CCC cells to severe microenvironments, such as LCFA starvation and hypoxia. This study highlights the importance of transcriptional regulation under lipid starvation and hypoxia in the promotion of CCC tumor growth.