Yin Wang1, Kui-Yi Xing, Marjorie F Lou. 1. School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
Abstract
PURPOSE: To investigate the molecular mechanism for cytosolic phospholipase A2 (cPLA(2)α) regulation and its association to platelet-derived growth factor (PDGF)-induced cell proliferation. METHODS: cPLA(2)α was examined using human lens epithelial (HLE) B3 cells. Reactive oxygen species (ROS) generation induced by PDGF was analyzed by luminescence assay. Cell proliferation was measured by cell counting and by BrdU assay. Human cPLA(2)α gene was cloned via RT-PCR followed by site-directed mutagenesis to construct HLE B3 cells expressing either inactive cPLA(2)α enzyme with S228A mutation (S228A), or cPLA(2)α truncated at the calcium-binding C2 domain (C2D). Activity of cPLA(2)α was measured by arachidonic acid (AA) release from cell membranes using [(3)H]-arachidonic acid prelabeled cells. The effect of intracellular calcium level on cPLA(2)α function was examined by treating cells with ionomycin (calcium influx), thapsgargin (endoplasmic reticulum [ER] calcium store release) or 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (BAPTA; calcium chelator). Activation of extracellular signal-regulated kinases (ERK), JNK, p38, or Akt was detected by Western blot analysis using specific antibodies. RESULTS: S228A mutant showed suppressed PDGF-induced reactive oxygen species generation, ERK and JNK activation (no effect on p38 or Akt), and cell proliferation in comparison with the vector alone (Vec) control. Calcium-binding C2 domain cells lost the ability of membrane translocation and activation of cPLA(2)α. PDGF cell signaling was calcium-dependent, and the calcium was supplied either from the external flux or endoplasmic reticulum store. However, enrichment of cellular calcium not only augmented PDGF function, but also demonstrated a cPLA(2)α-dependent calcium-signaling cascade that led to cell proliferation. CONCLUSIONS: cPLA(2)α is regulated by calcium mobilization and mitogen-activated protein kinases (MAPK) activation. Both PDGF mitogenic action and calcium signaling are cPLA(2)α-dependent.
PURPOSE: To investigate the molecular mechanism for cytosolic phospholipase A2 (cPLA(2)α) regulation and its association to platelet-derived growth factor (PDGF)-induced cell proliferation. METHODS: cPLA(2)α was examined using human lens epithelial (HLE) B3 cells. Reactive oxygen species (ROS) generation induced by PDGF was analyzed by luminescence assay. Cell proliferation was measured by cell counting and by BrdU assay. Human cPLA(2)α gene was cloned via RT-PCR followed by site-directed mutagenesis to construct HLE B3 cells expressing either inactive cPLA(2)α enzyme with S228A mutation (S228A), or cPLA(2)α truncated at the calcium-binding C2 domain (C2D). Activity of cPLA(2)α was measured by arachidonic acid (AA) release from cell membranes using [(3)H]-arachidonic acid prelabeled cells. The effect of intracellular calcium level on cPLA(2)α function was examined by treating cells with ionomycin (calcium influx), thapsgargin (endoplasmic reticulum [ER] calcium store release) or 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (BAPTA; calcium chelator). Activation of extracellular signal-regulated kinases (ERK), JNK, p38, or Akt was detected by Western blot analysis using specific antibodies. RESULTS:S228A mutant showed suppressed PDGF-induced reactive oxygen species generation, ERK and JNK activation (no effect on p38 or Akt), and cell proliferation in comparison with the vector alone (Vec) control. Calcium-binding C2 domain cells lost the ability of membrane translocation and activation of cPLA(2)α. PDGF cell signaling was calcium-dependent, and the calcium was supplied either from the external flux or endoplasmic reticulum store. However, enrichment of cellular calcium not only augmented PDGF function, but also demonstrated a cPLA(2)α-dependent calcium-signaling cascade that led to cell proliferation. CONCLUSIONS: cPLA(2)α is regulated by calcium mobilization and mitogen-activated protein kinases (MAPK) activation. Both PDGF mitogenic action and calcium signaling are cPLA(2)α-dependent.
Authors: Said Movahedi Naini; Gabriel J Choukroun; James R Ryan; Dirk M Hentschel; Jagesh V Shah; Joseph V Bonventre Journal: FASEB J Date: 2015-12-07 Impact factor: 5.191