Cell Type‐ and Protein‐Specific Induction of LAMP3

Abstract

RATIONALEThe family of lysosome‐associated membrane proteins (LAMP) includes LAMPs 1, 2, and 3. While LAMPs 1 and 2 are ubiquitously expressed and account for half of the proteins in the lysosomal membrane, LAMP3 (DC‐LAMP) is only expressed in distinct cell types. LAMP3 expression is associated with poor prognosis of certain cancers, Parkinson's disease, and is believed to contribute to the unfolded protein response by promoting protein degradation and cell survival during proteasome dysfunction. LAMP3 is constitutively expressed in and has been used as a subcellular marker for alveolar type 2 (AT2) cells primarily localizing in the lamellar bodies (the principal storage site of pulmonary surfactant). In this study, the expression of LAMP3 in various cell types and the mechanisms regulating its induction were evaluated.METHODSIn vitro cell line models (including lung epithelial A549 and H441 cells, kidney epithelial HEK292 cells, and cervical cancer cell line HeLa cells) transiently expressing fluorescent‐tagged proteins were used for co‐expression studies with immunostained LAMP3 by confocal microscopy. Additionally, LAMP3 mRNA levels in cells prior to and following transient expression were assessed by real‐time PCR.RESULTSWe discovered that none of the cell lines studied constitutively expresses LAMP3 (mRNA or protein). However, upon stimulation of cells by transient expression of certain proteins, A549 and HeLa cells strongly and weakly induced LAMP3 expression, respectively, while no LAMP3 induction was observed in either H441 or HEK293 cells. In A549 cells, transient expression of the AT2 cell specific proteins (ABCA3 and SP‐C) and the huntingtin poly‐Q repeat peptides (Mtt‐25 and Mtt‐72) induced LAMP3 expression. Media‐transfer assays also showed that LAMP3 induction takes place via paracrine signaling. In contrast, LAMP3 protein expression was absent in cells expressing empty fluorescence vector constructs including GFPC1, DsRedC1, and pcDNA3, as well as fluorescent‐tagged proteins including mCherry/LC3, RFP/Rab7, and GFP/NEDD4‐2. LAMP3 mRNA levels in these cells were comparable to those that induced protein expression, but the proteins appeared to be rapidly degraded by the cell's quality control system as demonstrated by the appearance of LAMP3 protein expression following autophagy and proteasome inhibition assays.CONCLUSIONThese results confirm, in vitro, the specific nature of LAMP3 expression in certain cells types and demonstrate its dependency on stimulation by distinct proteins, paracrine signaling, and disruption of cell quality control.Support or Funding InformationNIH HL 129150 (SM), NIH HL 119436 (MFB), VA Merit Award BX001176 (MFB)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.