A cross-sectional study reveals a chronic low-grade inflammation in achalasia.

BACKGROUND AND AIM: Immune-mediated neuroinflammation has been proposed to underlie the loss of lower esophageal sphincter (LES) myenteric neurons in achalasia. However, the immune status and key pathogenic immune subpopulations remain unclear. This study aims to evaluate the inflammatory status of patients with achalasia and their correlation with clinical characteristics, and further explore the key pathogenic subpopulations. METHODS: We investigated the complete blood cell count and inflammatory markers in a large population of patients with achalasia (n = 341) and healthy controls (n = 80). The subpopulations of lymphocytes were analyzed by flow cytometry. Immunofluorescence was used to determine immune cell infiltration in the LES. Transcriptome changes of the key subpopulation were determined by RNA sequencing analysis. RESULTS: NLR, MLR, CRP, globulin, IL-6 and IL-10 were significantly elevated in patients with achalasia. MLR and globulin were positively correlated with disease duration. The absolute count and percentage of CD8+ T cells in peripheral blood and its infiltration around ganglion in the LES were significantly increased in achalasia. Transcriptome analysis indicated that CD8+ T cells were activated and proliferative. In addition to multiple inflammatory pathways, regulation of neuroinflammatory response pathway was also significantly up-regulated in achalasia. GSEA analysis revealed a close association with autoimmune diseases. CONCLUSIONS: Patients with achalasia suffered from chronic low-grade inflammation with dysregulated immune cells and mediators associated with disease duration. CD8+ T cells might be the key pathogenic subpopulation of achalasia. Our results provide an important immune cell signature of the pathogenesis of achalasia.

The destruction box is involved in the degradation of the NTE family proteins by the proteasome.

Neuropathy target esterase (NTE) and NTE-related esterase (NRE) are endoplasmic reticulum (ER) membrane-anchored proteins belonging to the NTE protein family. NTE and NRE are degraded by macroautophagy and by the ubiquitin-proteasome pathway. However, the regulation of NTE and NRE by proteasome has not been well understood. Western blotting showed that the deletion of the regulatory region of NTE and NRE led to protein accumulation compared with that of the corresponding wild-type proteins. Further, deletion and site-directed mutagenesis experiments demonstrated that the destruction (D) box was required for the proteasomal degradation of NTE and NRE. However, unlike the deletion of the regulatory region, the deletion of the D box did not affect the subcellular localisation of NTE or NRE or disrupt the ER. Moreover, the deletion of the D box or the regulatory region of NTE has similar inhibitory effects on cell growth, which are greater than those produced by the full-length NTE. Here, for the first time, we show that the D box is involved in the regulation of NTE family proteins by the proteasome but not in their subcellular localisation. In addition, these results suggest that the NTE overexpression-mediated inhibition of cell growth is related to active protein levels but not to its ER disruption effect.

Identification of human patatin-like phospholipase domain-containing protein 1 and a mutant in human cervical cancer HeLa cells.

Recently members of mammalian patatin-like phospholipase domain containing (PNPLA) protein family have attracted attention for their critical roles in diverse aspects of lipid metabolism and signal pathway. Until now little has been known about the characteristics of PNPLA1. Here, the full length coding cDNA sequence of human PNPLA1 (hPNPLA1) was cloned for the first time, which encoded a polypeptide with 532 amino acids containing the whole patatin domain. Tissue expression profiles analysis showed that low mRNA levels of hPNPLA1 existed in various tissues, except high expression in the digestive system, bone marrow and spleen. Subcellular distribution of hPNPLA1 tagged with green fluorescence protein mainly localized to lipid droplets. Furthermore, a nonsense mutation of PNPLA1 in human cervical cancer HeLa cells was identified. The hPNPLA1 mutant encoded a protein of 412 amino acids without the C-terminal domain and did not colocalize to lipid droplets, which suggested that the C-terminal region of hPNPLA1 affected lipid droplet binding. These results identified hPNPLA1 and a mutant in HeLa cells, and provided insights into the structure and function of PNPLA1.

Degradation of mouse NTE-related esterase by macroautophagy and the proteasome.

NTE-related esterase (NRE) is a novel endoplasmic reticulum-anchored lysophospholipase with high homology to neuropathy target esterase (NTE). However, little is known about the regulation of NRE protein. In the current study, we investigated the degradation pathways of mouse NRE (mNRE) in mammalian cells. Based on experiments with inhibitors and inducer of protein degradation pathways, we provide here the first evidence that mNRE is degraded by macroautophagy as well as by the proteasome. Moreover, the contribution of protein domains to the degradation of mNRE was investigated, which showed that the transmembrane and regulatory domain played a role in the degradation of mNRE by macroautophagy and the proteasome respectively. In contrast the C-terminal catalytic domain was not involved in both degradation pathways of mNRE. These findings showed for the first time that the degradation pathways in controlling mNRE quantity and may provide further insight into structure and regulation of mNRE.

Identification of two novel splicing variants of murine NTE-related esterase.

NTE-related esterase (NRE) is an insulin-regulated lysophospholipase with homology to neuropathy target esterase (NTE), which plays a role in energy metabolism. Here, we reported two alternative splicing variants of the murine NRE (mNRE) gene, termed mNREV1 and mNREV2. Genomic organization analysis indicated that 5' splice site of mNRE intron 33 was changed in both mNREV1 and mNREV2, and mNRE exon 21 was deleted in mNREV2. mNREV1 had the same protein domains with mNRE, while mNREV2 lacked the patatin domain in the C-terminal catalytic region. Green fluorescent protein-mNREV1 or mNREV2 fusion proteins localized to the endoplasmic reticulum. mNREV1 and mNRE exhibited equal hydrolytic activity to the substrate phenyl valerate, whereas mNREV2 did not have any catalytic activity. The expression profiles of mNRE and its splicing isoforms in white adipose tissue, cardiac muscle, skeletal muscle, and testis tissues were further analyzed by real time quantitative-PCR in fed and fasted states, which indicated that the major isoform of mNRE mRNA generated switched from mNREV2 to mNREV1 during fasting. Thus there was a nutritional regulation of mNRE expression at the mRNA levels via alternative splicing.

The role of cell cycle-dependent neuropathy target esterase in cell proliferation.

Neuropathy target esterase (NTE) is a novel phospholipase B and plays a role in phospholipid homeostasis. Although over-expression of NTE inhibits cell division, the role of NTE in cell proliferation is still unknown. In the current study, we firstly used synchronous HeLa cells to study the expression profile of NTE during the cell cycle. NTE protein and activity are regulated during the cell cycle with highest level at G1 and lowest at G2/M phase. However, NTE mRNA levels are constant during the cell cycle. The role of NTE in cell proliferation was investigated by short hairpin RNA (shRNA) to suppress the expression of NTE. Knockdown of NTE significant down-regulated of NTE expression and reduced the glycerophosphocholine level. However, suppression of NTE did not affect phosphatidylcholine content or cell cycle progression. In addition, NTE was demonstrated to be degraded by the ubiquitin-proteasome pathway. These results suggested for the first time that NTE is a cell cycle-dependent protein, but is not essential for cell proliferation, and the ubiquitin-mediated proteolysis may be involved in the regulation of NTE during the cell cycle.

Protein domains, catalytic activity, and subcellular distribution of mouse NTE-related esterase.

A mammalian family of lipid hydrolases, designated "patatin-like phospholipase domain containing (PNPLA)" recently has attracted attention. NTE-related esterase (NRE) as a member of PNPLA is an insulin-regulated lysophospholipase with homology to neuropathy target esterase (NTE). Mouse NRE (mNRE) has a predicted amino-terminal transmembrane region (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. In the current study, we described the expression of green fluorescent protein (GFP)-tagged constructs of mNRE and mutant proteins lacking the specific protein domains. Esterase assays indicated that neither the TM nor R-domain was essential for mNRE esterase activity, but the TM significantly contributed to its activity. Subcellular distribution showed that mNRE was anchored in ER via its TM domain and that its C-domain was associated with ER. Furthermore, experiments involving proteinase treatment revealed that most of mNRE molecule was exposed on the cytoplasmic face of ER membranes. Collectively, our results for the first time revealed the protein domains, catalytic activity, and subcellular location of mNRE and a simplified model for mNRE was proposed.

Identification of side population cells in human hepatocellular carcinoma cell lines with stepwise metastatic potentials.

PURPOSE: To identify the side population (SP) cells from four hepatocellular carcinoma (HCC) cell lines with stepwise metastatic potentials. METHODS: SP cells were sorted from HCCLM3, MHCC97-H, MHCC97-L and Hep3B by flow cytometry, and then analyzed by differentiation study, clonogenic assay, chemoresistance study and tumorigenicity assay in vivo. The expression of ABCG(2) in SP cells was detected by immunocytochemistry, western blotting and real-time quantitative PCR, respectively. RESULTS: There was significant difference in SP proportion among HCCLM3, MHCC97-H, MHCC97-L and Hep3B (28.7 +/- 1.6%, 14.5 +/- 0.6%, 4.2 +/- 0.4%, 0.9 +/- 0.1%, respectively, P < 0.01). All the SP cells showed similar characteristics of self-renewal, high clonogenicity, remarkable chemo-resistance and high expression of ABCG(2). As low as 2,000 SP cells could initiate tumors in non-obese diabetic/severe combined immunodeficiency mice successfully. CONCLUSIONS: SP cells purified from HCC cell lines harbors cancer stem cell-like properties, and may be related to the metastatic potentials and therapeutic-resistance of HCC.