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Secretion of novel SEL1L endogenous variants is promoted by ER stress/UPR via endosomes and shed vesicles in human cancer cells.

Cattaneo M, Lotti LV, Martino S, Alessio M, Conti A, Bachi A, Mariani-Costantini R, Biunno I - PLoS ONE (2011)

Bottom Line: We describe here two novel endogenous variants of the human endoplasmic reticulum (ER) cargo receptor SEL1LA, designated p38 and p28.Biochemical and RNA interference studies in tumorigenic and non-tumorigenic cells indicate that p38 and p28 are N-terminal, ER-anchorless and more stable relative to the canonical transmembrane SEL1LA.P28 is detected only in the poorly differentiated SKBr3 cell line, where it is secreted after ER stress.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Technologies, National Research Council, Milan, Italy.

ABSTRACT
We describe here two novel endogenous variants of the human endoplasmic reticulum (ER) cargo receptor SEL1LA, designated p38 and p28. Biochemical and RNA interference studies in tumorigenic and non-tumorigenic cells indicate that p38 and p28 are N-terminal, ER-anchorless and more stable relative to the canonical transmembrane SEL1LA. P38 is expressed and constitutively secreted, with increase after ER stress, in the KMS11 myeloma line and in the breast cancer lines MCF7 and SKBr3, but not in the non-tumorigenic breast epithelial MCF10A line. P28 is detected only in the poorly differentiated SKBr3 cell line, where it is secreted after ER stress. Consistently with the presence of p38 and p28 in culture media, morphological studies of SKBr3 and KMS11 cells detect N-terminal SEL1L immunolabeling in secretory/degradative compartments and extracellularly-released membrane vesicles. Our findings suggest that the two new SEL1L variants are engaged in endosomal trafficking and secretion via vesicles, which could contribute to relieve ER stress in tumorigenic cells. P38 and p28 could therefore be relevant as diagnostic markers and/or therapeutic targets in cancer.

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Localizations of SEL1L in DTT-treated SKBr3 cells.After DTT treatment for 3 hrs, gold particles for N-terminal SEL1L (10 nm) are found by cryoimmunogold electron microscopy along the microvilli (panel A) and on membrane-bound vesicles in the extracellular space (panels A, A1) or emerging from the plasma membrane (panels C, F). Similarly, labeling for the tetraspan protein CD63 is detected in multivesicular bodies and in vesicles at the cell surface (panels B, D, G, I). By double immunolabeling (panels E, H), N-terminal SEL1L (5–10 nm gold, arrowheads) localizes in CD63-positive (15 nm gold, arrows) exosomes, while C-terminal SEL1L (panel J, arrowhead) is confined in the endoplasmic reticulum. Bars: 0.1 µm; E: er: endoplasmic reticulum; Ly: lysosomes; MVB: multivesicular body; PM: plasma membrane.
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pone-0017206-g004: Localizations of SEL1L in DTT-treated SKBr3 cells.After DTT treatment for 3 hrs, gold particles for N-terminal SEL1L (10 nm) are found by cryoimmunogold electron microscopy along the microvilli (panel A) and on membrane-bound vesicles in the extracellular space (panels A, A1) or emerging from the plasma membrane (panels C, F). Similarly, labeling for the tetraspan protein CD63 is detected in multivesicular bodies and in vesicles at the cell surface (panels B, D, G, I). By double immunolabeling (panels E, H), N-terminal SEL1L (5–10 nm gold, arrowheads) localizes in CD63-positive (15 nm gold, arrows) exosomes, while C-terminal SEL1L (panel J, arrowhead) is confined in the endoplasmic reticulum. Bars: 0.1 µm; E: er: endoplasmic reticulum; Ly: lysosomes; MVB: multivesicular body; PM: plasma membrane.

Mentions: To define the subcellular localizations of the endogenous SEL1L products, SKBr3 and KMS11 cells labeled with the monoclonal or polyclonal antibodies against SEL1L were analyzed by high-resolution immunoelectron microscopy (IEM). Ultrathin cryosections of untreated SKBr3 cells revealed that, in addition to the ER, the monoclonal and polyclonal antibodies against the SEL1L N-terminus [34], [35] labeled peripheral cytoplasmic vesicles, often associated with structures morphologically consistent with late endosomes/multivesicular bodies (MVBs), organelles containing membrane vesicles that can be released extracellularly as exosomes [48] (Figure 3A, B). In agreement, immunofluorescence showed that the immunolabeling obtained with the monoclonal antibody to the SEL1L N-terminus co-localized with the ER marker calreticulin and, in few dots only, with the Golgi marker giantin, but was uniquely present in the peripheral cytoplasm (Figure 3D–I). Double immunolabeling by IEM confirmed that SEL1L codistributed with calreticulin along the ER, while only SEL1L was present in endosomes/MVBs (Figure S3G). In DTT-treated SKBr3 cells the IEM labeling was more evident along the plasma membrane (PM), particularly in association with microvilli, and in 80–200 nm vesicles apparently emerging from the PM and shed extracellularly (Figure 4A–A1, C, F; Figure S3H–I), as observed for the exogenous myc-tagged SEL1LB protein in SEL1LBmyc-transfected 293 FT cells (Figure S3J–K) [26].


Secretion of novel SEL1L endogenous variants is promoted by ER stress/UPR via endosomes and shed vesicles in human cancer cells.

Cattaneo M, Lotti LV, Martino S, Alessio M, Conti A, Bachi A, Mariani-Costantini R, Biunno I - PLoS ONE (2011)

Localizations of SEL1L in DTT-treated SKBr3 cells.After DTT treatment for 3 hrs, gold particles for N-terminal SEL1L (10 nm) are found by cryoimmunogold electron microscopy along the microvilli (panel A) and on membrane-bound vesicles in the extracellular space (panels A, A1) or emerging from the plasma membrane (panels C, F). Similarly, labeling for the tetraspan protein CD63 is detected in multivesicular bodies and in vesicles at the cell surface (panels B, D, G, I). By double immunolabeling (panels E, H), N-terminal SEL1L (5–10 nm gold, arrowheads) localizes in CD63-positive (15 nm gold, arrows) exosomes, while C-terminal SEL1L (panel J, arrowhead) is confined in the endoplasmic reticulum. Bars: 0.1 µm; E: er: endoplasmic reticulum; Ly: lysosomes; MVB: multivesicular body; PM: plasma membrane.
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Related In: Results  -  Collection

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pone-0017206-g004: Localizations of SEL1L in DTT-treated SKBr3 cells.After DTT treatment for 3 hrs, gold particles for N-terminal SEL1L (10 nm) are found by cryoimmunogold electron microscopy along the microvilli (panel A) and on membrane-bound vesicles in the extracellular space (panels A, A1) or emerging from the plasma membrane (panels C, F). Similarly, labeling for the tetraspan protein CD63 is detected in multivesicular bodies and in vesicles at the cell surface (panels B, D, G, I). By double immunolabeling (panels E, H), N-terminal SEL1L (5–10 nm gold, arrowheads) localizes in CD63-positive (15 nm gold, arrows) exosomes, while C-terminal SEL1L (panel J, arrowhead) is confined in the endoplasmic reticulum. Bars: 0.1 µm; E: er: endoplasmic reticulum; Ly: lysosomes; MVB: multivesicular body; PM: plasma membrane.
Mentions: To define the subcellular localizations of the endogenous SEL1L products, SKBr3 and KMS11 cells labeled with the monoclonal or polyclonal antibodies against SEL1L were analyzed by high-resolution immunoelectron microscopy (IEM). Ultrathin cryosections of untreated SKBr3 cells revealed that, in addition to the ER, the monoclonal and polyclonal antibodies against the SEL1L N-terminus [34], [35] labeled peripheral cytoplasmic vesicles, often associated with structures morphologically consistent with late endosomes/multivesicular bodies (MVBs), organelles containing membrane vesicles that can be released extracellularly as exosomes [48] (Figure 3A, B). In agreement, immunofluorescence showed that the immunolabeling obtained with the monoclonal antibody to the SEL1L N-terminus co-localized with the ER marker calreticulin and, in few dots only, with the Golgi marker giantin, but was uniquely present in the peripheral cytoplasm (Figure 3D–I). Double immunolabeling by IEM confirmed that SEL1L codistributed with calreticulin along the ER, while only SEL1L was present in endosomes/MVBs (Figure S3G). In DTT-treated SKBr3 cells the IEM labeling was more evident along the plasma membrane (PM), particularly in association with microvilli, and in 80–200 nm vesicles apparently emerging from the PM and shed extracellularly (Figure 4A–A1, C, F; Figure S3H–I), as observed for the exogenous myc-tagged SEL1LB protein in SEL1LBmyc-transfected 293 FT cells (Figure S3J–K) [26].

Bottom Line: We describe here two novel endogenous variants of the human endoplasmic reticulum (ER) cargo receptor SEL1LA, designated p38 and p28.Biochemical and RNA interference studies in tumorigenic and non-tumorigenic cells indicate that p38 and p28 are N-terminal, ER-anchorless and more stable relative to the canonical transmembrane SEL1LA.P28 is detected only in the poorly differentiated SKBr3 cell line, where it is secreted after ER stress.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Technologies, National Research Council, Milan, Italy.

ABSTRACT
We describe here two novel endogenous variants of the human endoplasmic reticulum (ER) cargo receptor SEL1LA, designated p38 and p28. Biochemical and RNA interference studies in tumorigenic and non-tumorigenic cells indicate that p38 and p28 are N-terminal, ER-anchorless and more stable relative to the canonical transmembrane SEL1LA. P38 is expressed and constitutively secreted, with increase after ER stress, in the KMS11 myeloma line and in the breast cancer lines MCF7 and SKBr3, but not in the non-tumorigenic breast epithelial MCF10A line. P28 is detected only in the poorly differentiated SKBr3 cell line, where it is secreted after ER stress. Consistently with the presence of p38 and p28 in culture media, morphological studies of SKBr3 and KMS11 cells detect N-terminal SEL1L immunolabeling in secretory/degradative compartments and extracellularly-released membrane vesicles. Our findings suggest that the two new SEL1L variants are engaged in endosomal trafficking and secretion via vesicles, which could contribute to relieve ER stress in tumorigenic cells. P38 and p28 could therefore be relevant as diagnostic markers and/or therapeutic targets in cancer.

Show MeSH
Related in: MedlinePlus