View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1843 (2014) 797–805 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr Dissociated presenilin-1 and TACE processing of ErbB4 in lung alveolar type II cell differentiation Najla Fiaturi a,⁎, Anika Ritzkat b,c, Christiane E.L. Dammann b,c,d, John J. Castellot a,d,1, Heber C. Nielsen b,d,1 a Program in Pharmacology and Experimental Therapeutics, Department of Integrative Physiology and Pathobiology, Sackler School of Graduate Biomedical Studies, Tufts University, Boston, MA 02111, USA b Department of Pediatrics, Tufts Medical Center, Boston, MA 02111 USA c Hannover Medical School, Hannover, Germany d Graduate Program in Cell, Molecular and Developmental Biology, Department of Integrative Physiology and Pathobiology, Sackler School of Graduate Biomedical Studies, Tufts University, Boston, MA 02111, USA article info abstract Article history: Neuregulin (NRG) stimulation of ErbB4 signaling is important for type II cell surfactant synthesis. ErbB4 may me- Received 25 October 2013 diate gene expression via a non-canonical pathway involving enzymatic cleavage releasing its intracellular do- Received in revised form 18 December 2013 main (4ICD) for nuclear trafficking and gene regulation. The accepted model for release of 4ICD is consecutive Accepted 13 January 2014 cleavage by Tumor necrosis factor alpha Converting Enzyme (TACE) and γ-secretase enzymes. Here, we show Available online 24 January 2014 that 4ICD mediates surfactant synthesis and its release by γ-secretase is not dependent on previous TACE cleav- age. We used siRNA to silence Presenilin-1 (PSEN-1) expression in a mouse lung type II epithelial cell line (MLE12 Keywords: fi ErbB receptor cells), and both siRNA knockdown and chemical inhibition of TACE. Knockdown of PSEN-1 signi cantly de- Gamma secretase creased baseline and NRG-stimulated surfactant phospholipid synthesis, expression of the surfactant proteins Presenilin-1 SP-B and SP-C, as well as 4ICD levels, with no change in ErbB4 ectodomain shedding. Neither siRNA knockdown Neonatal lung nor chemical inhibition of TACE inhibited 4ICD release or surfactant synthesis. PSEN-1 cleavage of ErbB4 for non- Type II cell canonical signaling through 4ICD release does not require prior cleavage by TACE. Surfactant protein © 2014 Elsevier B.V. All rights reserved. 1. Introduction expressed by type II cells, play a prominent role in stimulation of type II cell maturation and surfactant synthesis [4,5]. NRG is expressed in Respiratory distress syndrome (RDS), formerly known as hyaline the midtrimester human fetal lung [6] and increases in fetal lung at membrane disease, is a common problem in preterm infants born be- the onset of surfactant synthesis [7]. fore 28 weeks. This disease is caused primarily by deficiency of pulmo- ErbB4 is a member of the ErbB receptor tyrosine kinase family, nary surfactant in immature lungs and is more common the earlier the which also includes the epidermal growth factor receptor, also called infant is born [1]. Despite the beneficial effects of prenatal glucocorti- ErbB1, ErbB2, and ErbB3 [8]. The ErbB receptors are transmembrane ty- coids and postnatal surfactant replacement therapies, RDS remains rosine kinase proteins and act as important regulators of cell prolifera- one of the significant causes of morbidity and mortality in premature in- tion and differentiation during fetal organ development including lung fants [2,1]. Pulmonary surfactant is a mixture of surface active phospho- development [9]. ErbB4 signal transduction is a complex process that in- lipids and proteins (termed surfactant protein B (SP-B) and SP-C) which volves both canonical and non-canonical signaling pathways. Binding of is produced in alveolar type II epithelial cells [3]. The development of NRG to its extracellular ligand-binding site causes ErbB4 to form homo surfactant synthesis is under multifactorial control, in which paracrine or heterodimers with other ErbB receptors linked by disulfide bonds mesenchyme-type II cell communication mechanisms play a central in the extracellular domain [10]. These receptor dimers then undergo role. We have shown that the growth factor Neuregulin (NRG-1), auto phosphorylation on tyrosine residues within the intracellular do- which is secreted by fibroblasts, and its target receptor ErbB4, which is main. In the canonical signal pathway tyrosine phosphorylation acti- vates signal cascades through specific intracellular signaling pathways such as the phosphatidylinositol-3 (PI3) Kinase/Akt pathway to ulti- Abbreviations: SP-B, Surfactant protein B; SP-C, Surfactant protein C; NRG-1, fl Neuregulin-1; TACE, Tumor necrosis factor alpha converting enzyme; DSPC, Disaturated mately in uence gene expression [11]. However, within the ErbB fami- phosphatidyl choline ly, ErbB4 is unique in that it may undergo proteolytic processing to ⁎ Corresponding author at: 136 Harrison Ave., Tufts University, Boston, MA 02148, USA. initiate non-canonical signaling [12]. The accepted model for the non- Tel.: +1 781 228 9652. canonical pathway involves two sequential cleavage processes. The fi E-mail addresses: naj [email protected] (N. Fiaturi), [email protected] (A. Ritzkat), fi [email protected] (C.E.L. Dammann), [email protected] rst step in this pathway is performed by a transmembrane (J.J. Castellot), [email protected] (H.C. Nielsen). metalloprotease Tumor necrosis factor alpha Converting Enzyme 1 Co-senior authors. (TACE) which releases the ErbB4 ectodomain by a cleavage that 0167-4889/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamcr.2014.01.015 798 N. Fiaturi et al. / Biochimica et Biophysica Acta 1843 (2014) 797–805 produces two fragments: a 120 kDa ectodomain fragment which is Millipore (Billerica, MA). Anti-prosurfactant protein C (rabbit polyclonal released into the extracellular space and an 80 kDa membrane- antibody, ab90716), anti-prosurfactant protein B (rabbit polyclonal anti- associated fragment that contains the ErbB4 transmembrane domain body ab15011), monoclonal antibody to beta actin (HRP conjugated, and the entire cytoplasmic region, including the tyrosine kinase domain. cat# 20272), Anti-TACE antibody (rabbit polyclonal antibody, cat# Ectodomain cleavage of ErbB4 in cells occurs at a low constitutive or ab2051) were all from Abcam (Cambridge, MA). Peroxidase-conjugated basal level [13] that can be increased by neuregulin or other ErbB4 Affinipure Goat Anti-Rabbit IgG (H + L), (111-035-144) was from Jackson ligands [14]. The ectodomain cleavage of ErbB4 is sensitive to ImmunoResearch. Anti-ERBb4 antibody (cat# sc-283) was obtained from metalloprotease inhibitors [13] and does not occur in cells genetically Santa Cruz (Santa Cruz, CA). Chloroform (spectrophotometric grade) was deficient in TACE. The ErbB4 m80 fragment (membrane associated frag- obtained from Sigma Aldrich (St Louis, MO). Methanol was from Fisher ment) that remains following ectodomain cleavage is further processed Scientific; Silica gel H thin layer chromatography sheets were from by γ-secretase that cleaves at the transmembrane domain to release a Analtech (Newark, DE). Osmium tetroxide 05500-1G, carbon tetrachlo- soluble intracellular s80 fragment (4ICD) into the cytosol from which ride and dipalmitoylphosphatidyl choline (P-5911) were from Sigma it translocates to the nucleus in association with chaperone proteins Aldrich (St Louis, MO). Ultima-Gold scintillation fluid was from Perkin [14,15]. Gamma secretase is an enzyme complex which consists of 4 Elmer (Waltham, MA). For centrifugation we used a Beckman J-6 M. components: presenilin 1 (PSEN-1) or Presenilin 2 (PSEN-2), Nycastrin, anterior pharynx defective-1 (APH-1) and the Presenilin-Enhancer 2. PSEN-1 or PSEN-2 are the active enzymatic component; other compo- 2.2. Cell culture nents behave as scaffolding molecules and essential cofactors [16–19]. Studies with transgenic mice show that PSEN-1 and PSEN-2 have func- MLE12 cells were used as a model for type II alveolar epithelial tionally distinct phenotypes in several organs including the lung cells. MLE12 cells exhibit characteristics of alveolar type II cells, in- [20,21]. cluding the expression of SP-B and SP-C and formation of microvilli Nuclear localization of ErbB4 is the preferred mechanism of ErbB4 and multivesicular bodies. They have a strong response to fetal signaling in several regulatory processes during development [22].The fibroblast-conditioned media (FCM) and NRG with increased DPSC accepted model for γ-secretase activity in ErbB4 processing is that synthesis [7,24]. MLE-12 cells were grown in DMEM containing ectodomain cleavage by TACE is a prerequisite step [12]. In this study 10% FBS, 2% pen/strep and 2% L-glutamine. Media were changed we sought to more specifically define the sequential interactive relation- every second day. ship of PSEN-1 and TACE for ErbB4 processing controlling lung alveolar type II cell surfactant production. Our focus on PSEN-1 was motivated by the more severe developmental phenotype of alveolar maturation in 2.3. Transfection with siRNA the PSEN-1 knockout mouse compared to the PSEN-2 knock out and our previous work showing the importance of PSEN-1 signaling for MLE12 cells were transfected with siRNA using the transfection re- fetal type II cell maturation [22,23]. We studied the effect of PSEN-1 agent Dharmafect 2. To knock down presenilin 1, three pre-designed knockdown and TACE knockdown in MLE12 cells and evaluated the ef- Presenilin-1 siRNA sequences that target three different regions of fects on ErbB4 cleavage in association with the expression of the SP-B Presenilin mRNA were used. To knock down TACE we used a cocktail and SP-C mRNA and protein and synthesis of the major surfactant phos- of 3 siRNAs targeting the TACE gene. The protocol for transfection of pholipid disaturated phosphatidylcholine (DSPC). MLE12 cells was adapted from the manufacturer's guidelines; all steps were done using RNAse-free pipette tips and RNase spray for 2.