Placental protein 13 (PP13/galectin-13) undergoes lipid raft-associated subcellular redistribution in the syncytiotrophoblast in preterm preeclampsia and HELLP syndrome

Abstract

Objective: To investigate placental protein 13 (PP13) localization in relation to cytoskeleton and lipid rafts in preeclampsia and HELLP syndrome.

Study design: Placental cryosections from patients with preeclampsia and HELLP, and controls were stained for PP13, actin, PLAP (lipid raft marker), and CD71 (nonraft marker). BeWo cells exposed to stress conditions were stained for PP13 and actin. Protein localizations were investigated by confocal microscopy, PP13 concentrations by ELISA.

Results: PP13-actin colocalization was increased in syncytiotrophoblast juxtamembrane regions in term/preterm preeclampsia and HELLP. PP13-CD71 colocalization was decreased and PP13-PLAP proximity was increased in preterm but not term preeclampsia and HELLP. PP13-release from BeWo cells was inhibited by cytoskeleton disruption, and augmented by Ca2+-influx and ischemic stress.

Conclusion: The actin cytoskeleton, probably in connection with lipid rafts, controls trophoblastic "nonclassical" PP13 export. PP13 is released from the syncytiotrophoblast in preterm preeclampsia and HELLP, mimicked in BeWo cells by ischemic stress, suggesting PP13 is a placental alarmin.

Figure 1. The syncytiotrophoblastic localization of PP13 is altered in patients with preeclampsia and HELLP syndrome

Representative confocal fluorescence-DIC composite images of cryosections of placentas from women with preterm preeclampsia, preterm HELLP syndrome, term preeclampsia, and gestational age-matched controls. Cryosections were stained with monoclonal anti-PP13 antibody (green) and PI for nuclei (red). There was a weaker cytoplasmic staining of the syncytiotrophoblast in term and preterm preeclampsia and HELLP syndrome when compared with gestational age-matched controls. Stars indicate PP13 staining of the endothelium of fetal vessels, arrows indicate the strong apical membrane staining of terminal villi in disease cases. DIC, differential interface contrast; HELLP, hemolysis elevated enzymes and low platelets; PI, propidium iodide; PP13, placental protein 13. Balogh. Subcellular relocalizaiton of PP13 in preeclampsia and HELLP syndrome. Am J Obstet Gynecol 2011.

Figure 2. Colocalization of PP13 with actin, CD71 or PLAP in preeclampsia and HELLP syndrome compared to controls

Cryosections of placentas taken from women with HELLP syndrome and controls were stained with A–C, anti-PP13 (green) in combination with A, anti-actin, B, anti-PLAP, or C, anti-CD71 (all in red), and analyzed by confocal microscopy. Cell nuclei were counterstained with PI (blue) on all representative confocal images. HELLP, hemolysis elevated liver enzymes and low platelets; PI, propidium iodide; PLAP, placental alkaline phosphatase; PP13, placental protein 13. Balogh, subcellular relocalizaiton of PP13 in preeclampsia and HELLP syndrome. Am J Obstet Gynecol 2011.

Figure 3. PP13 is redistributed in the placenta of patients with preeclampsia and HELLP syndrome

Cryosections of placentas taken from women with preterm preeclampsia of HELLP syndrome, term preeclampsia, and gestational age-matched controls were stained with anti-PP13 in combination with antiactin, anti-PLAP or anti-CD71, and analyzed by confocal microscopy. Colocalization indices for PP13 (derived from approximately 100 ROIs/group) are shown as mean ± SEM with A, actin, B, PLAP, or C, CD71. HELLP, hemolysis elevated liver enzymes and low platelets; PLAP, placental alkaline phosphatase; PP13, placental protein 13; ROI, regions of interest. Balogh. Subcellular relocalizaiton of PP13 in preeclampsia and HELLP syndrome. Am J Obstet Gynecol 2011.

Figure 4. Coexistence of PP13 and actin in the juxtamembrane area of the syncytiotrophoblast is increased in preeclampsia and HELLP syndrome

Cryosections of placenta taken from women with preeclampsia or HELLP syndrome and gestational age-matched controls were stained for PP13 and actin. A, Representative image and line scan intensity distribution are shown. Relative frequency of B, PP13-actin coexistence and of C, actin, or D, PP13 existence alone in membrane/juxtamembrane and in cytoplasmic regions, respectively, was calculated from approximately 200 ROIs in each group. HELLP, hemolysis elevated liver enzymes and low platelets; PP13, placental protein 13; ROI, regions of interest. Balogh. Subcellular relocalizaiton of PP13 in preeclampsia and HELLP syndrome. Am J Obstet Gynecol 2011.

Figure 5. PP13 accumulates in the vicinity of PLAP-containing lipid rafts in preeclampsia and HELLP syndrome

Samples stained for PP13 (green) and PLAP (red) were analyzed with confocal microscopy. Representative A, confocal images, B, 3D surface plots and, C, line scan intensity distributions of samples derived from preterm or term patients and controls. HELLP, hemolysis elevated liver enzymes and low platelets; PLAP, placental alkaline phosphatase; PP13, placental protein 13. Balogh, subcellular relocalizaiton of PP13 in preeclampsia and HELLP syndrome. Am J Obstet Gynecol 2011.

Figure 6. Disruption of the actin cytoskeleton results in intracellular accumulation of Pp13 in BeWo cells

PP13-transfected, untreated or Latrunculin B-treated cells were stained with A, phalloidin or with B, anti-PP13 (green), and antiactin (red), followed by confocal microscopic analysis (40x or 20x magnifications, respectively). Cell nuclei were counterstained with DRAQ5 (Biostatus, Leicenstershire, UK) (blue). Asterisks denote enlarged areas. Yellow color reflects colocalization of PP13 and actin. C, PP13 content of cell culture supernatants of nontransfected controls, as well as PP13-transfected, untreated or Latrunculin B-treated cells were measured by ELISA (data are displayed as mean ± SEM of 3 independent experiments). ELISA, enzyme-linked immunosorbent assay; PP13, placental protein 13. Balogh. Subcellular relocalizaiton of PP13 in preeclampsia and HELLP syndrome. Am J Obstet Gynecol 2011.

Figure 7. Calcium-influx or ischemic stress decreases PP13 immunostaining in BeWo cells

PP13-transfected BeWo cells were treated with either A, calcium ionophore to increase B, intracellular Ca²⁺ - level, or kept under stress, such as C, TNFα treatment, D, hypoxia, or E, ischemia to mimic placental milieu in preterm preeclampsia. B–E, Treated or A, untreated control cells were stained with anti-PP13 (green), nuclei were counterstained with DRAQ5 (Biostatus, Leicestershire, UK) (red). Representative confocal images of 3 independent experiments are displayed (20x magnifications). Asterisks denote enlarged areas. PP13, placental protein 13. Balogh. Subcellular relocalizaiton of PP13 in preeclampsia and HELLP syndrome. Am J Obstet Gynecol 2011.

Figure 8. Calcium-influx or ischemic stress promotes PP13-release from BeWo cells

PP13-transfected cells were treated with calcium ionophore (black bars), kept under ischemic stress (dark gray bars), or left untreated (light gray bars). Nontransfected cells were used as negative controls (white bars). A, PP13 mRNA expression or B, PP13 protein concentration in culture supernatants and C, in cells were measured by qRT-PCR or ELISA, respectively. Data are displayed as mean ± SEM of 3 independent experiments. ELISA, enzyme-linked immunosorbent assay; PP13, placental protein 13; qRT-PCR, quantitative reverse transcribe-polymerase chain reaction. Balogh. Subcellular relocalizaiton of PP13 in preeclampsia and HELLP syndrome. Am J Obstet Gynecol 2011.