ABCMdb

PMID: 22434821

Kaltenborn E, Kern S, Frixel S, Fragnet L, Conzelmann KK, Zarbock R, Griese M
Respiratory syncytial virus potentiates ABCA3 mutation-induced loss of lung epithelial cell differentiation.
Hum Mol Genet. 2012 Jun 15;21(12):2793-806. Epub 2012 Mar 20., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Here, we investigated the impact of the clinically relevant ABCA3 mutations, p.Q215K and p.E292V, by stable transfection of A549 lung epithelial cells. Login to comment 38 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys For our study, we chose two clinically relevant ABCA3 mutations, namely p.Q215K and p.E292V. Login to comment 39 ABCA3 p.Glu292Val The p.E292V mutation has a prevalence of 1:277 in the USA, and therefore it is the most common ABCA3 mutation observed in children so far (19). Login to comment 40 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Gln215Lys In vitro studies reported that ABCA3 p.E292V is properly localized to the LB but only has residual ATPase activity, therefore belonging to the class of functional mutations (20). Login to comment 41 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys The ABCA3 mutation p.Q215K has been observed in a neonate that died of respiratory distress. Login to comment 42 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys ABCA3 p.Arg288Lys Immunohistochemical analysis of the lung tissue of this patient who was the compound heterozygote for ABCA3 p.Q215K and p.R288K showed no ABCA3 staining in ATII cells, and electron microscopy revealed the presence of many electron-dense bodies in the absence of LBs (9). Login to comment 43 ABCA3 p.Gln215Lys ABCA3 p.Gln215Lys Previous work from our group showed that ABCA3-p.Q215K caused mislocalization of the protein to the ER and its absence from the LB (21). Login to comment 44 ABCA3 p.Gln215Lys ABCA3 p.Arg288Lys Immunohistochemical analysis of the lung tissue of this patient who was the compound heterozygote for ABCA3 p.Q215K and p.R288K showed no ABCA3 staining in ATII cells, and electron microscopy revealed the presence of many electron-dense bodies in the absence of LBs (9). Login to comment 45 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys ABCA3 p.Gln215Lys RESULTS Stable expression of HA-tagged ABCA3 in A549 cells To study the effect of the two clinically relevant ABCA3 mutations p.Q215K and p.E292V on cellular homeostasis, A549 lung epithelial cells were stably transfected with HA-tagged ABCA3. Login to comment 46 ABCA3 p.Gln215Lys In agreement with our previous report (21), the ABCA3 protein with the p.Q215K mutation is localized to the ER and is not, or only to a very small amount, processed and trafficked to the LBs (Fig. 1C). Login to comment 47 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Gln215Lys While ABCA3 harboring the p.E292V mutation is properly localized to LBs (Fig. 1C), it is functionally impaired (20). Login to comment 48 ABCA3 p.Gln215Lys In agreement with our previous report (21), the ABCA3 protein with the p.Q215K mutation is localized to the ER and is not, or only to a very small amount, processed and trafficked to the LBs (Fig. 1C). Login to comment 49 ABCA3 p.Glu292Val While ABCA3 harboring the p.E292V mutation is properly localized to LBs (Fig. 1C), it is functionally impaired (20). Login to comment 51 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys E-cadherin, a cellular adhesion protein found in adherens junctions and desmosomes, was decreased up to 4-fold in p.Q215K and p.E292V cells when compared with untransfected A549 and cells expressing wild-type (WT) ABCA3 (Fig. 1B, right). Login to comment 53 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys ABCA3 p.Gln215Lys A549 cells expressing p.Q215K had approximately 2.5-fold decreased amounts of ZO-1. Login to comment 55 ABCA3 p.Gln215Lys A549 cells expressing p.Q215K had approximately 2.5-fold decreased amounts of ZO-1. Login to comment 57 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys In p.Q215K and p.E292V cells, E-cadherin overall appeared to be less abundant, thus confirming the results obtained by western blotting. Login to comment 59 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys In p.Q215K and p.E292V cells, E-cadherin overall appeared to be less abundant, thus confirming the results obtained by western blotting. Login to comment 60 ABCA3 p.Gln215Lys ZO-1 was less abundantly detected on the cell surface and more diffusely distributed in cells expressing p.Q215K compared with cells with ABCA3-WT (Fig. 1C, left). Login to comment 62 ABCA3 p.Gln215Lys ZO-1 was less abundantly detected on the cell surface and more diffusely distributed in cells expressing p.Q215K compared with cells with ABCA3-WT (Fig. 1C, left). Login to comment 66 ABCA3 p.Gln215Lys Therefore, we investigated MMP-2 expression and secretion in our type II cell model system. MMP-2 mRNA levels were significantly elevated more than 8-fold in p.Q215K cells when compared with ABCA3-WT cells (Fig. 2B, left). Login to comment 67 ABCA3 p.Glu292Val MMP-2 mRNA levels in p.E292V cells were slightly, but not significantly, 2794 increased when compared with WT cells. Login to comment 68 ABCA3 p.Gln215Lys ABCA3 p.Gln215Lys MMP-2 is secreted to the extracellular matrix (ECM) in tissues or into the cell culture supernatants in our model system. MMP-2 protein in supernatants of ABCA3-WT cells could not be detected, whereas p.Q215K-cells secreted large amounts of MMP-2 (Fig. 2C, left). Login to comment 69 ABCA3 p.Glu292Val ABCA3 p.Glu292Val Into supernatants of cells expressing ABCA3-E292V, about one-tenth of this amount was secreted, which was about in the range of non-transfected A549 cells. Login to comment 70 ABCA3 p.Gln215Lys MMP-2 is secreted to the extracellular matrix (ECM) in tissues or into the cell culture supernatants in our model system. MMP-2 protein in supernatants of ABCA3-WT cells could not be detected, whereas p.Q215K-cells secreted large amounts of MMP-2 (Fig. 2C, left). Login to comment 71 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Cells expressing the ABCA3 p.Q215K mutation produce increased amounts of TGF-b1 TGF-b1 is a protein controlling cellular proliferation and differentiation. Login to comment 73 ABCA3 p.Gln215Lys ABCA3 p.Gln215Lys TGF-b1 mRNA was significantly elevated in p.Q215K-expressing cells when compared with ABCA3-WT cells (Fig. 2B, right). Login to comment 74 ABCA3 p.Gln215Lys Also, significantly elevated levels of secreted TGF-b1 were found in supernatants of p.Q215K cells when compared with supernatants from ABCA3-WT cells (Fig. 2C, right). Login to comment 75 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys TGF-b1 secretion was not increased in E292V cells. Login to comment 76 ABCA3 p.Gln215Lys ABCA3 p.Gln215Lys Expression of ABCA3 p.Q215K protein in A549 cells therefore induces synthesis and secretion of TGF-b1. Login to comment 77 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Src kinase activation is increased in cells with ABCA3-Q215K mutation Next, we wanted to investigate whether phosphorylation of the tyrosine kinase Src, which has important signaling functions in cell proliferation, motility and cytoskeleton assembly (26), was upregulated in cells with ABCA3 mutations as well. Login to comment 78 ABCA3 p.Gln215Lys ABCA3 p.Gln215Lys As shown in Fig. 2D, increased amounts of phosphorylated Src kinase could be detected in cells expressing ABCA3 p.Q215K. Login to comment 79 ABCA3 p.Gln215Lys Src kinase activation is increased in cells with ABCA3-Q215K mutation Next, we wanted to investigate whether phosphorylation of the tyrosine kinase Src, which has important signaling functions in cell proliferation, motility and cytoskeleton assembly (26), was upregulated in cells with ABCA3 mutations as well. Login to comment 80 ABCA3 p.Gln215Lys ABCA3 p.Gln215Lys The most pronounced effect was seen in ABCA3 p.Q215K cells. Login to comment 81 ABCA3 p.Gln215Lys While non-infected p.Q215K cells showed a cell shape similar to untransfected A549 control Figure 1. Login to comment 82 ABCA3 p.Gln215Lys The most pronounced effect was seen in ABCA3 p.Q215K cells. Login to comment 83 ABCA3 p.Gln215Lys While non-infected p.Q215K cells showed a cell shape similar to untransfected A549 control Figure 1. Login to comment 103 ABCA3 p.Gln215Lys 2796 cells, p.Q215K cell morphology changed to a more elongated shape after virus infection. Login to comment 105 ABCA3 p.Glu292Val The cell morphology of A549 and ABCA3 WT and p.E292V transfected cells also changed slightly after RSV infection. Login to comment 106 ABCA3 p.Gln215Lys cells, p.Q215K cell morphology changed to a more elongated shape after virus infection. Login to comment 108 ABCA3 p.Glu292Val The cell morphology of A549 and ABCA3 WT and p.E292V transfected cells also changed slightly after RSV infection. Login to comment 110 ABCA3 p.Gln215Lys When these cells were infected with RSV, the arrangement of microtubules inside the cells was more organized and the tubulin fibers in p.Q215K-transfected cells almost had a parallel arrangement in order to support their elongated cell shape (Fig. 3B, right). Login to comment 113 ABCA3 p.Gln215Lys When these cells were infected with RSV, the arrangement of microtubules inside the cells was more organized and the tubulin fibers in p.Q215K-transfected cells almost had a parallel arrangement in order to support their elongated cell shape (Fig. 3B, right). Login to comment 120 ABCA3 p.Gln215Lys Collagen type I mRNA increased significantly in p.Q215K cells after RSV infection when compared with non-infected cells. Login to comment 123 ABCA3 p.Gln215Lys ABCA3 p.Gln215Lys In RSV-infected p.Q215K cells, we detected significantly increased levels of SNAI1 mRNA when compared with the other cell lines (Fig. 5B). Login to comment 124 ABCA3 p.Gln215Lys In addition to SNAI1 mRNA, we also found elevated amounts of SNAI2 mRNA in p.Q215K cells (Fig 5C). Login to comment 126 ABCA3 p.Gln215Lys In RSV-infected p.Q215K cells, we detected significantly increased levels of SNAI1 mRNA when compared with the other cell lines (Fig. 5B). Login to comment 127 ABCA3 p.Gln215Lys In addition to SNAI1 mRNA, we also found elevated amounts of SNAI2 mRNA in p.Q215K cells (Fig 5C). Login to comment 134 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Gln215Lys ABCA3 p.Gln215Lys After infection with RSV, MMP-2 mRNA levels in p.Q215K and p.E292V cells increased even further when compared with ABCA3-WT, the change being more than 15-fold between p.Q215K and WT and more than 7-fold between p.E292V and WT (Fig. 6A, left and B). Login to comment 137 ABCA3 p.Glu292Val ABCA3 p.Glu292Val ABCA3 p.Gln215Lys ABCA3 p.Gln215Lys After infection with RSV, MMP-2 mRNA levels in p.Q215K and p.E292V cells increased even further when compared with ABCA3-WT, the change being more than 15-fold between p.Q215K and WT and more than 7-fold between p.E292V and WT (Fig. 6A, left and B). Login to comment 150 ABCA3 p.Glu292Val When MMP-2 protein levels in UV-inactivated samples from non-infected cells were analyzed, the MMP-2 protein amount in untransfected A549 cells and A549 cells transfected with ABCA3 WT and p.E292V decreased below the detection limit. Login to comment 151 ABCA3 p.Gln215Lys Therefore, MMP-2 protein in RSV-infected UV-inactivated samples was detectable only in p.Q215K supernatants (Fig. 6A, right). Login to comment 153 ABCA3 p.Glu292Val When MMP-2 protein levels in UV-inactivated samples from non-infected cells were analyzed, the MMP-2 protein amount in untransfected A549 cells and A549 cells transfected with ABCA3 WT and p.E292V decreased below the detection limit. Login to comment 154 ABCA3 p.Gln215Lys Therefore, MMP-2 protein in RSV-infected UV-inactivated samples was detectable only in p.Q215K supernatants (Fig. 6A, right). Login to comment 155 ABCA3 p.Gln215Lys When both types of samples, non-infected and RSV-infected, were UV-inactivated and analyzed for TGF-b1 secretion, we found that RSV infection significantly induced TGF-b1 secretion in all cells, with p.Q215K samples having the significantly highest TGF-b1 amounts secreted (Fig. 6C and D, right). Login to comment 157 ABCA3 p.Gln215Lys Even though Src phosphorylation was increased significantly in all cells after RSV infection, it was higher in p.Q215K cells when compared with all other cells (Fig. 7A and B). Login to comment 158 ABCA3 p.Gln215Lys ABCA3 p.Gln215Lys Additionally, increased phosphorylation of the mitogen-activated protein kinases (MAPKs) JNK1 and ERK1/2 was observed in p.Q215K cells when compared with ABCA3-WT cells after RSV infection (Fig. 7A, C, D). Login to comment 160 ABCA3 p.Gln215Lys Even though Src phosphorylation was increased significantly in all cells after RSV infection, it was higher in p.Q215K cells when compared with all other cells (Fig. 7A and B). Login to comment 161 ABCA3 p.Gln215Lys ABCA3 p.Gln215Lys However, TGF-b1 secretion and phosphorylation of Src as well as MAPKs were elevated in cells harboring the ABCA3-p-Q215K mutation when compared with A549 cells transfected with ABCA3-WT. Login to comment 163 ABCA3 p.Gln215Lys Phosphorylation of Src and MAPKs is induced by RSV infection and is increased in Q215K cells. Login to comment 164 ABCA3 p.Gln215Lys However, TGF-b1 secretion and phosphorylation of Src as well as MAPKs were elevated in cells harboring the ABCA3-p-Q215K mutation when compared with A549 cells transfected with ABCA3-WT. Login to comment 166 ABCA3 p.Gln215Lys Phosphorylation of Src and MAPKs is induced by RSV infection and is increased in Q215K cells. Login to comment 171 ABCA3 p.Glu292Val Significant changes compared with the corresponding non-infected cells were observed in p.E292V cells for both markers. Login to comment 174 ABCA3 p.Glu292Val Significant changes compared with the corresponding non-infected cells were observed in p.E292V cells for both markers. Login to comment 179 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Thus, ER stress does not seem to account for the changes in p.Q215K cell morphology and protein expression as observed after RSV infection when compared with A549 cells expressing ABCA3 WT or p.E292V. Login to comment 182 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Thus, ER stress does not seem to account for the changes in p.Q215K cell morphology and protein expression as observed after RSV infection when compared with A549 cells expressing ABCA3 WT or p.E292V. Login to comment 190 ABCA3 p.Gln215Lys In addition to upregulation of SNAI1, we found increased phosphorylation of Src in cells with the p.Q215K mutation. Login to comment 193 ABCA3 p.Gln215Lys In addition to upregulation of SNAI1, we found increased phosphorylation of Src in cells with the p.Q215K mutation. Login to comment 194 ABCA3 p.Gln215Lys ABCA3 mutations (especially p.Q215K) lead to downregulation of epithelial markers accompanied by acquisition of mesenchymal characteristics and activation of signaling molecules. Login to comment 197 ABCA3 p.Gln215Lys ABCA3 mutations (especially p.Q215K) lead to downregulation of epithelial markers accompanied by acquisition of mesenchymal characteristics and activation of signaling molecules. Login to comment 206 ABCA3 p.Gln215Lys Both TGF-b1 production and secretion were increased in cells expressing the ABCA3 p.Q215K mutation. Login to comment 209 ABCA3 p.Gln215Lys Both TGF-b1 production and secretion were increased in cells expressing the ABCA3 p.Q215K mutation. Login to comment 219 ABCA3 p.Gln215Lys Moreover, RSV infection also induced SNAI2 and collagen type I mRNA in cells harboring the ABCA3-p.Q215K mutation. Login to comment 222 ABCA3 p.Gln215Lys Moreover, RSV infection also induced SNAI2 and collagen type I mRNA in cells harboring the ABCA3-p.Q215K mutation. Login to comment 229 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Interestingly, we and others found decreased levels of mature SP-C protein in immunohistochemistry staining of lung tissue and in lavage samples from patients (compound) heterozygote for the ABCA3 mutations Q215K and E292V (data not shown), respectively (9). Login to comment 232 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys Interestingly, we and others found decreased levels of mature SP-C protein in immunohistochemistry staining of lung tissue and in lavage samples from patients (compound) heterozygote for the ABCA3 mutations Q215K and E292V (data not shown), respectively (9). Login to comment 250 ABCA3 p.Gln215Lys We found elevated MAPK phosphorylation in p.Q215K cells after RSV infection, supporting a role for MAPK signaling in ABCA3-mediated EMT. Login to comment 253 ABCA3 p.Gln215Lys We found elevated MAPK phosphorylation in p.Q215K cells after RSV infection, supporting a role for MAPK signaling in ABCA3-mediated EMT. Login to comment 275 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys A549 cells were transfected with the pUB6-ABCA3-WT/Q215K/E292V vectors using ExGene 500 (Fermentas) according to the manufacturer`s protocol. Login to comment 278 ABCA3 p.Glu292Val ABCA3 p.Gln215Lys A549 cells were transfected with the pUB6-ABCA3-WT/Q215K/E292V vectors using ExGene 500 (Fermentas) according to the manufacturer`s protocol. Login to comment