Phosphatidic acid-enabled MKL1 contributes to liver regeneration: Translational implication in liver failure

Liver regeneration following injury aids the restoration of liver mass and the recovery of liver function. In the present study we investigated the contribution of megakaryocytic leukemia 1 (MKL1), a transcriptional modulator, to liver regeneration. We report that both MKL1 expression and its nuclear translocation correlated with hepatocyte proliferation in cell and animal models of liver regeneration and in liver failure patients. Mice with MKL1 deletion exhibited defective regenerative response in the liver. Transcriptomic analysis revealed that MKL1 interacted with E2F1 to program pro-regenerative transcription. MAPKAPK2 mediated phosphorylation primed MKL1 for its interaction with E2F1. Of interest, phospholipase d2 promoted MKL1 nuclear accumulation and liver regeneration by catalyzing production of phosphatidic acid (PA). PA administration stimulated hepatocyte proliferation and enhanced survival in a MKL1-dependent manner in a pre-clinical model of liver failure. Finally, PA levels was detected to be positively correlated with expression of pro-regenerative genes and inversely correlated with liver injury in liver failure patients. In conclusion, our data reveal a novel mechanism whereby MKL1 contributes to liver regeneration. Screening for small-molecule compounds boosting MKL1 activity may be considered as a reasonable approach to treat acute liver failure.

Identification and functional analysis of a novel phospholipase D2 gene mutation associated with familial systemic lupus erythematosus / 中南大学学报(医学版)

OBJECTIVES@#Systemic lupus erythematosus (SLE) is a kind of autoimmune inflammatory connective tissue disease which seriously endangers human health. Genetic factors play a key role in the pathogenesis of SLE. This study aims to investigate a novel phospholipase D2 (PLD2) mutation associated with familial SLE, and further explore the underlying mechanism of the mutation in SLE.@*METHODS@#The blood samples from a SLE patient, the patient's parents, and 147 normal controls were collected and DNA was extracted. Whole genome high-throughput sequencing was performed in the patient and her parents and the results were further analyzed by various bioinformatics methods. The wild type (wt), mutant type (mu), and negative control PLD2 plasmids were further constructed and transfected into 293 cells. The expression level of HRAS protein in 293 cells was detected by Western blotting.@*RESULTS@#In this SLE family, the female SLE patient and her mother, 1 in generation II and 1 in generation III had typical clinical manifestations of SLE, and all of them had lupus nephritis at early stage. The genetic characteristics are consistent with autosomal dominant inheritance. A novel PLD2 heterozygous mutation (c.2722C>T) was found in the patient and her mother, but not in her father and other normal controls. Compared with wtPLD2 plasmid and negative control PLD2 plasmid, the expression of HRAS in 293 cells transfected with muPLD2 plasmid was significantly up-regulated (both @*CONCLUSIONS@#PLD2 c.2722C>T mutation may be one of the pathogeny of SLE in this family.

Expresson of the N-terminus truncated phosphotase D in Escherichia coli and characterization of its anti inflammatory activity / 中国人兽共患病学报

To investigate the immunological activities of the recombinant human phosphatase D2 (rhPLD2) in vitro and in vivo, especially its ability to reduce inflammatory reactions, the cDNA fragment encoding rhPLD2 was cloned into prokaryotic expression vector pET30a by RT-PCR and the recombinant protein rhPLD2 expressed in E.coli was purified from the inclusion bodies, while the anti inflammatory activity of rhPLD2 was determined by the amount of eosinophils in bronchoalveolar fluid(BALF) and blood and the expression of IL-5 and MMP-9 in lung tissues of guinea pig model of chronic asthma. It was found that the rhPLD2 recombinant protein was obtained from human Daudi cells by cloning to E.coli, which contained no membrane-binding site and signal peptide. The cDNA sequence encoded 631 amino acid residues (GenBank Accession Number: AY178289). The purity of the rhPLD2 approached up to 76% with a bioactivity of 50.9745 units/L (0.9212 g/L). In addition, the anti inflammatory effect of rhPLD2 protein could be demonstrated in the guinea pig model of chronic asthma after treatment with rhPLD2 protein, such as down regulation in the expression of the inflammatory cytokine IL-5. It is concluded that the anti-inflammator activity of the recombinant human truncated PLD2 protein produced from the E.coli plasmid can be demonstrated both in vitro and in vivo.

Lysophosphatidylcholine suppresses apoptosis and induces neurite outgrowth in PC12 cells through activation of phospholipase D2

Lysophosphatidylcholine (LPC) is a bioactive lipid generated by phospholipase A2-mediated hydrolysis of phosphatidylcholine. In the present study, we demonstrate that LPC stimulates phospholipase D2 (PLD2) activity in rat pheochromocytoma PC12 cells. Serum deprivation induced cell death of PC12 cells, as demonstrated by decreased viability, DNA fragmentation, and increased sub-G1 fraction of cell cycle. LPC treatment protected PC12 cells partially from the cell death and induced neurite outgrowth of the cells. Overexpression of PLD2 drastically enhanced the LPC-induced inhibition of apoptosis and neuritogenesis. Pretreatment of the cells with 1-butanol, a PLD inhibitor, completely abrogated the LPC-induced inhibition of apoptosis and neurite outgrowth in PC12 cells overexpressing PLD2. These results indicate that LPC possesses the neurotrophic effects, such as anti-apoptosis and neurite outgrowth, through activation of PLD2.

Development of ligand-dependent regulatory system and its application to gene therapy of insulin-dependent diabetes mellitus

To develop an inducible expression system, the enhanced artificial nuclear receptors and target reporters were constructed. Artificial nuclear receptors were generated by fusing three domains, consisting of DNA-binding domain (DBD) of GAL4, ligand binding domain (LBD) of progesterone or estrogen receptor, and activation domain (AD) of VP16, sterol regulatory element binding protein (SREBP)-1a, or SREBP-2. The activation domain of SREBP-1a showed most potent transcriptional activity. The maximal level of target reporter gene expression was extremely elevated by the usage of ATP citrate-lyase (ACL) minimal promoter -60/+67 in place of artificial TATA promoter, while the SV40 enhancer severely increased the basal transcription in the absence of ligand. The induction system, developed in the present study, was applied to cell therapy, resulting in successful induction of single-chain insulin analogue (SIA) gene expression to correct the hyperglycemia in diabetic animals. By means of subcutaneous cell therapy, the SIA gene expression rapidly occurred after the local topical application of ligand. These results suggest that our system represents a powerful tool for transcriptional regulation of target gene that can be used for diverse applications, ranging from basic research to gene therapy.

Comparative Analysis of Phospholipase D2 Localization in the Pancreatic Islet of Rat and Guinea Pig

To examine the localization pattern of phospholipase D2 (PLD2) in the pancreatic islet (the islet of Langerhans) depending on species, we conducted a morphological experiment in the rat and guinea pig. Since individual islets display a typical topography with a central core of B cell mass and a peripheral boundary of A, D, and PP cells, double immunofluorescent staining with a panel of antibodies was performed to identify PLD2-immunoreactive cells in the islets PLD2 immunoreactivity was mainly present in A and PP cells of the rat pancreatic islets. And yet, in the guinea pig, PLD2 immunoreactivity was exclusively localized in A cells, and not in PP cells. These findings suggest a possibility that PLD2 is mainly located in A cells of rodent pancreatic islets, and that the existence of PLD2 in PP cells is not universal in all species. Based on these results, it is suggested that PLD2 may play a significant role in the function of A and/or PP cells via a PLD-mediated signaling pathway.

Reductive effect of rhPLD2 on PAF content in serum of guinea pig model with chronic asthma / 中国免疫学杂志

Objective:To study the biologic function of rhPLD2 mutation form.Methods:To adapt the guinea pig chronic asthma model was induced by OVA, the functions of rhPLD2 on PAF was observed through the assay of platelet congregating.Results:rhPLD2 remarkably reduced the lever of PAF in the serum of guinea pig chronic asthma model; compares with the NS group, the P