Biology subjects

Pothoulakis, C.

Publications and source records attributed to Pothoulakis, C..

3 recordsLinked to original sources

bioRxiv2022

In vivo selection reveals long non-coding RNAs implicated in colon to liver metastasis

Colorectal cancer (CRC) is the third most common malignancy in both American men and women. Most of the deaths attributed to CRC are a result of metastatic spread to the liver. In this study, colon cancer cells that highly metastasized to liver in vivo were compared to less metastatic parental cells to investigate the role for long non-coding RNAs (lncRNAs) in CRC metastasis. The highly metastatic daughter cells (LS-3B) were found to be 63-fold more metastatic than the parental cell line (LS-PAR) in vivo. A lncRNA microarray comparing LS-PAR and LS-3B cells revealed that 104 lncRNAs had fold changes > 2.0 and an FDR < 0.05. Real time PCR mediated validation revealed many lncRNAs exhibited high fold changes such as a 60-fold increase in LOC101448202, a 20-fold increase in MRPL23-AS1 and 50-fold decreases in GNAS-AS1 and LOC101928131. In vivo metastasis differences could be recapitulated in vitro as LS-3B cells closed wounds faster than their parental LS-PAR cells. However, intestinal epithelial cancer cells with robust downregulation of MRPL23-AS1, C1QTNF1-AS1, GNAS-AS1, LINCR-0002 and LOC101448202 failed to display differences in comparison to controls in in vitro migration assays. Three of the five lncRNAs with microarray probes for currently available GEO-datasets were significantly altered in liver CRC-associated tumor biopsies as compared to the primary tumor of non-metastatic CRC. Further studies on the lncRNAs identified will better define their roles in metastasis and how they might be useful if targeted therapeutically.

cancer biology↗

bioRxiv2022

Substance P and adenosine signaling pathways regulate exosomal sorting of miR-21 in colonic epithelial cells

Background & AimsMicroRNAs (miRNAs) are transported in body fluids within exosomes, and exosomal-miRNA sorting is highly selective. In colonic epithelial cells (CECs), Substance P-neurokinin-1-receptor (SP/NK1R) signaling regulates miR-21 sorting into secreted exosomes. Here, we studied the molecular mechanisms driving miR-21 sorting into colonic epithelial exosomes (CEEs) under SP-stimulation. MethodsWe performed studies with human colonic epithelial NCM460 cells overexpressing neurokinin-1-receptor (NCM460-NK1R) and in intestinal-epithelial-specific NK1R knockout mice. SP-regulated gene targets were validated by real-time polymerase chain reaction (RT-PCR) and immunoblotting. Small non-coding RNAs (sncRNAs) were isolated from NCM460-NK1R cells and secreted exosomes, and 3'-end adenylated and 3'-end uridylated fractions were separated. Cellular and exosomal sncRNA fractions were processed for miR-21 and miR-1307-3p expression and 3'-end adenylation to uridylation (A/U) ratios using RT-PCR. Pharmacological inhibition studies in NCM460-NK1R cells were performed in the presence of the Adenosine A2B receptor (ADORA2B) antagonist, PSB-1115, followed by RT-PCR and immunoblotting. Mass spectrometry validated in silico interacting partners of miR-21 in CECs. ResultsIn CECs, miR-21 is predominantly polyuridylated under SP-stimulation and preferentially sorted to CEEs. SP/NK1R signaling activation upregulates extracellular adenosine (ADO) signaling via ADORA2B concomitant with reduced ADO uptake via epithelial-specific equilibrative nucleoside transporter-2 (ENT-2). Mass spectrometry and immunoblotting revealed upregulation of TUT7 in SP-stimulated CEEs. Knockdown of TUT7 decreased both TUT7 and miR-21 content in these exosomes. Pharmacological inhibition of ADORA2B in CECs resulted in decreased TUT7 and miR-21 in CEEs, regardless of SP stimulation. ConclusionsSP/NK1R coupling in CECs activates ADORA2B and its downstream signaling cascade which mediates TUT7/miR-21 interaction and subsequent miR-21 polyuridylation and exosomal export. SynopsisSubstance P-neurokinin-1 receptor signaling regulates terminal uridyl transferase7-mediated predominant 3-end polyuridylation and exosomal recruitment of miR-21 in human colonic epithelial NCM460 cells overexpressing NK-1R via activation of Adenosine A2B receptor and its downstream signaling cascade. Because Adenosine A2B receptor signaling is involved in IBD pathogenesis, it could therefore be exploited as a pharmacological target for the therapeutic benefits in IBD.

cell biology↗

bioRxiv2022

Aftiphilin regulation of myosin light chain kinase activity promotes actin dynamics and intestinal epithelial barrier function

The expression levels of aftiphilin (AFTPH) are significantly lower in inflamed colonic tissues from patients with ulcerative colitis (UC) and mice with experimental colitis. During colonic inflammation, the selective permeability of the colonic epithelium is compromised largely due to dysregulation of proteins associated with either the tight junction (TJ) complex and actin-myosin contraction rings. Here, we hypothesized that inflammation-associated reduction in AFTPH levels might cause an increase in the selective permeability of the colonic epithelium. In this study, we measured the transepithelial electric resistance (TEER), sodium (Na+) ion flux and dextran permeability in polarized colonic epithelial cells after manipulation of AFTPH. Silencing of AFTPH reduced TEER, increased Na+ ion flow and dextran permeability. Examination of mRNA and protein levels of multiple TJ proteins and Na+ ion transporters suggested that AFTPH deficiency did not significantly change expression of most of these transmembrane proteins. While the gross structure of the TJs in AFTPH gene-silenced cells appeared normal, elevated levels of junctional Occludin were observed. Most notably we observed that AFTPH co-localized with myosin light chain kinase (MLCK) and attenuated cellular MLCK activity as observed by phospho-myosin light chain 2 (pMLC2) western blots. Importantly, inhibition of MLCK activity reversed the reduction of TEER in AFTPH-deficient monolayers. Lastly, examination on transmission electron microcopy on microvilli and immunofluorescent microscopy on actin filament arrangement showed that AFTPH deficiency also affected filament arrangement in colonic epithelial cells. Taken together, these results suggest that AFTPH regulates intestinal epithelial permeability and actin polymerization in colonic epithelium through interfering MLCK/MLC interactions.

cell biology↗