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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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Related in: MedlinePlus

Analysis of p38 and p28 secretion in SKBr3, KMS11 and MCF10A cells exposed to chemical and pharmacological treatments.A. Western blot analysis of untreated and DTT-treated MCF10A cells: MCF10A cells were exposed to DTT (2 mM) for 3 hours and successively maintained for 24 hrs in OPTIMEM. Secreted protein (50 µg) extracted from the culture medium by TCA precipitation, and aliquots of cell lysates (50 µg) were resolved by SDS-PAGE (10%) and blotted with monoclonal anti-SEL1L and anti-vinculin antibodies. P38 was not detectable in the culture medium, both in presence and in absence of DTT. In addition to p38, MCF10A cells showed a higher band, probably corresponding to an immature precursor or post-translationally-modified product. The image is representative of two independent experiments. A1. RT-PCR analysis of untreated and DTT-treated MCF10A cells: RNA was extracted from the samples described in panel A and analyzed by RT-PCR for the UPR response. The histogram shows expression values normalized relative to housekeeping signals and expressed as fold modulation relative to the untreated samples; densitometric analysis was performed by Scion imaging program. UPR activation upon DTT treatment is indicated by up-modulation of BIP and CHOP and XBP-1 splicing, concomitantly SEL1LA is incremented (gray bar). The corresponding images are shown in Figure S2A. The data are the averages of two different assays based on independent treatments, ±SD. B. Western blot analysis of untreated and DTT-treated SKBr3 cells: Secretion of p38 and p28 was evaluated in untreated and DTT-treated SKBr3 cells. Cells exposed to DTT (2 mM) for 3 hrs or not exposed were maintained for 24 hrs in OPTIMEM. Secreted protein (50 µg) extracted from the culture medium by TCA precipitation, and aliquots of cell lysates (50 µg) were resolved by SDS-PAGE (12%) and blotted with anti-SEL1L and anti-vinculin antibodies. ER stress/UPR strongly promoted secretion of p38 and, to a lesser extent, p28 in the culture medium. The image is representative of five independent experiments. C. Western blot analysis of KMS11 cells treated with DTT and MG132: KMS11 cells were exposed to DTT (2 mM) or MG132 (10 µM) for 3 hrs and successively maintained for 24 hrs in OPTIMEM. Secreted protein (30 µg), extracted from the culture medium by TCA precipitation, and aliquots of cell lysates (50 µg) were resolved by SDS-PAGE (10%) and blotted with anti-SEL1L and anti-vinculin antibodies. Both treatments markedly induced p38 secretion in the culture medium. The image is representative of five independent experiments. C1. RT-PCR analysis of KMS11 cells treated with DTT and MG132: RNA was extracted from the same samples described in panel C and analyzed by RT-PCR for the UPR response. The histogram shows expression values normalized relative to housekeeping signals and expressed as fold modulation relative to untreated samples; densitometric analysis was determined by Scion imaging program. UPR activation upon DTT treatment is indicated by the up-modulation of ATF6, BIP, and CHOP and by XBP-1 splicing (see Figure S2C), concomitantly the expression of SEL1LA, HRD1 and GADD45β is incremented (gray bar). The corresponding images are shown in Figure S2C. MG132 treatment did not trigger UPR activation, as indicated by the absence of ATF6 and BIP un-modulation and lack of XBP-1 splicing (see Figure S2C), nevertheless, CHOP and GADD45β were markedly up-modulated and SEL1LA and HRD1 down-modulated (black bars). The data are averages of four different assays based on independent treatments, ±SD. C2. SEL1LA protein expression in KMS11 cells treated with DTT and MG132: The histogram shows SEL1LA protein expression values obtained from the samples described in panel C, normalized relative to housekeeping signals and expressed as fold modulation relative to the untreated sample; densitometric analysis was performed using the Scion imaging program. MG132 determined SEL1LA protein accumulation up to 2 times relative to the control level (black bar). The data are the averages of four independent experiments, ±SD.
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pone-0017206-g002: Analysis of p38 and p28 secretion in SKBr3, KMS11 and MCF10A cells exposed to chemical and pharmacological treatments.A. Western blot analysis of untreated and DTT-treated MCF10A cells: MCF10A cells were exposed to DTT (2 mM) for 3 hours and successively maintained for 24 hrs in OPTIMEM. Secreted protein (50 µg) extracted from the culture medium by TCA precipitation, and aliquots of cell lysates (50 µg) were resolved by SDS-PAGE (10%) and blotted with monoclonal anti-SEL1L and anti-vinculin antibodies. P38 was not detectable in the culture medium, both in presence and in absence of DTT. In addition to p38, MCF10A cells showed a higher band, probably corresponding to an immature precursor or post-translationally-modified product. The image is representative of two independent experiments. A1. RT-PCR analysis of untreated and DTT-treated MCF10A cells: RNA was extracted from the samples described in panel A and analyzed by RT-PCR for the UPR response. The histogram shows expression values normalized relative to housekeeping signals and expressed as fold modulation relative to the untreated samples; densitometric analysis was performed by Scion imaging program. UPR activation upon DTT treatment is indicated by up-modulation of BIP and CHOP and XBP-1 splicing, concomitantly SEL1LA is incremented (gray bar). The corresponding images are shown in Figure S2A. The data are the averages of two different assays based on independent treatments, ±SD. B. Western blot analysis of untreated and DTT-treated SKBr3 cells: Secretion of p38 and p28 was evaluated in untreated and DTT-treated SKBr3 cells. Cells exposed to DTT (2 mM) for 3 hrs or not exposed were maintained for 24 hrs in OPTIMEM. Secreted protein (50 µg) extracted from the culture medium by TCA precipitation, and aliquots of cell lysates (50 µg) were resolved by SDS-PAGE (12%) and blotted with anti-SEL1L and anti-vinculin antibodies. ER stress/UPR strongly promoted secretion of p38 and, to a lesser extent, p28 in the culture medium. The image is representative of five independent experiments. C. Western blot analysis of KMS11 cells treated with DTT and MG132: KMS11 cells were exposed to DTT (2 mM) or MG132 (10 µM) for 3 hrs and successively maintained for 24 hrs in OPTIMEM. Secreted protein (30 µg), extracted from the culture medium by TCA precipitation, and aliquots of cell lysates (50 µg) were resolved by SDS-PAGE (10%) and blotted with anti-SEL1L and anti-vinculin antibodies. Both treatments markedly induced p38 secretion in the culture medium. The image is representative of five independent experiments. C1. RT-PCR analysis of KMS11 cells treated with DTT and MG132: RNA was extracted from the same samples described in panel C and analyzed by RT-PCR for the UPR response. The histogram shows expression values normalized relative to housekeeping signals and expressed as fold modulation relative to untreated samples; densitometric analysis was determined by Scion imaging program. UPR activation upon DTT treatment is indicated by the up-modulation of ATF6, BIP, and CHOP and by XBP-1 splicing (see Figure S2C), concomitantly the expression of SEL1LA, HRD1 and GADD45β is incremented (gray bar). The corresponding images are shown in Figure S2C. MG132 treatment did not trigger UPR activation, as indicated by the absence of ATF6 and BIP un-modulation and lack of XBP-1 splicing (see Figure S2C), nevertheless, CHOP and GADD45β were markedly up-modulated and SEL1LA and HRD1 down-modulated (black bars). The data are averages of four different assays based on independent treatments, ±SD. C2. SEL1LA protein expression in KMS11 cells treated with DTT and MG132: The histogram shows SEL1LA protein expression values obtained from the samples described in panel C, normalized relative to housekeeping signals and expressed as fold modulation relative to the untreated sample; densitometric analysis was performed using the Scion imaging program. MG132 determined SEL1LA protein accumulation up to 2 times relative to the control level (black bar). The data are the averages of four independent experiments, ±SD.

Mentions: To explore the behavior and secretion of p38 and p28 in cells under ER stress/UPR, we analyzed by SDS-PAGE and immunoblot cell lysates and culture supernatants of MCF10A, SKBr3 and KMS11 cells under normal conditions and after treatment with DTT, which causes protein misfolding by reducing disulfide bonds [45] (Figure 2A–C). In all the tested cells DTT triggered ER stress/UPR, assessed by XBP-1 splicing combined with BIP, ATF6 and CHOP up-modulation, and boosted SEL1LA mRNA expression (Figure 2A1,C1; Figure S2A–C), while the SEL1LA protein level did not increase significantly (Figure 2A–C, C2). P38, but not SEL1LA, was readily detectable in SKBr3 and KMS11 supernatants, increasing close to three- and five-fold respectively after DTT (Figure 2B–C). No evidence of p38 was detected in MCF10A supernatants, under both normal and DTT-stressed conditions (Figure 2A). P28 was exclusively found after DTT treatment and only in the supernatant of the SKBr3 cell line, which expresses this variant (Figure 2B).


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)

Analysis of p38 and p28 secretion in SKBr3, KMS11 and MCF10A cells exposed to chemical and pharmacological treatments.A. Western blot analysis of untreated and DTT-treated MCF10A cells: MCF10A cells were exposed to DTT (2 mM) for 3 hours and successively maintained for 24 hrs in OPTIMEM. Secreted protein (50 µg) extracted from the culture medium by TCA precipitation, and aliquots of cell lysates (50 µg) were resolved by SDS-PAGE (10%) and blotted with monoclonal anti-SEL1L and anti-vinculin antibodies. P38 was not detectable in the culture medium, both in presence and in absence of DTT. In addition to p38, MCF10A cells showed a higher band, probably corresponding to an immature precursor or post-translationally-modified product. The image is representative of two independent experiments. A1. RT-PCR analysis of untreated and DTT-treated MCF10A cells: RNA was extracted from the samples described in panel A and analyzed by RT-PCR for the UPR response. The histogram shows expression values normalized relative to housekeeping signals and expressed as fold modulation relative to the untreated samples; densitometric analysis was performed by Scion imaging program. UPR activation upon DTT treatment is indicated by up-modulation of BIP and CHOP and XBP-1 splicing, concomitantly SEL1LA is incremented (gray bar). The corresponding images are shown in Figure S2A. The data are the averages of two different assays based on independent treatments, ±SD. B. Western blot analysis of untreated and DTT-treated SKBr3 cells: Secretion of p38 and p28 was evaluated in untreated and DTT-treated SKBr3 cells. Cells exposed to DTT (2 mM) for 3 hrs or not exposed were maintained for 24 hrs in OPTIMEM. Secreted protein (50 µg) extracted from the culture medium by TCA precipitation, and aliquots of cell lysates (50 µg) were resolved by SDS-PAGE (12%) and blotted with anti-SEL1L and anti-vinculin antibodies. ER stress/UPR strongly promoted secretion of p38 and, to a lesser extent, p28 in the culture medium. The image is representative of five independent experiments. C. Western blot analysis of KMS11 cells treated with DTT and MG132: KMS11 cells were exposed to DTT (2 mM) or MG132 (10 µM) for 3 hrs and successively maintained for 24 hrs in OPTIMEM. Secreted protein (30 µg), extracted from the culture medium by TCA precipitation, and aliquots of cell lysates (50 µg) were resolved by SDS-PAGE (10%) and blotted with anti-SEL1L and anti-vinculin antibodies. Both treatments markedly induced p38 secretion in the culture medium. The image is representative of five independent experiments. C1. RT-PCR analysis of KMS11 cells treated with DTT and MG132: RNA was extracted from the same samples described in panel C and analyzed by RT-PCR for the UPR response. The histogram shows expression values normalized relative to housekeeping signals and expressed as fold modulation relative to untreated samples; densitometric analysis was determined by Scion imaging program. UPR activation upon DTT treatment is indicated by the up-modulation of ATF6, BIP, and CHOP and by XBP-1 splicing (see Figure S2C), concomitantly the expression of SEL1LA, HRD1 and GADD45β is incremented (gray bar). The corresponding images are shown in Figure S2C. MG132 treatment did not trigger UPR activation, as indicated by the absence of ATF6 and BIP un-modulation and lack of XBP-1 splicing (see Figure S2C), nevertheless, CHOP and GADD45β were markedly up-modulated and SEL1LA and HRD1 down-modulated (black bars). The data are averages of four different assays based on independent treatments, ±SD. C2. SEL1LA protein expression in KMS11 cells treated with DTT and MG132: The histogram shows SEL1LA protein expression values obtained from the samples described in panel C, normalized relative to housekeeping signals and expressed as fold modulation relative to the untreated sample; densitometric analysis was performed using the Scion imaging program. MG132 determined SEL1LA protein accumulation up to 2 times relative to the control level (black bar). The data are the averages of four independent experiments, ±SD.
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pone-0017206-g002: Analysis of p38 and p28 secretion in SKBr3, KMS11 and MCF10A cells exposed to chemical and pharmacological treatments.A. Western blot analysis of untreated and DTT-treated MCF10A cells: MCF10A cells were exposed to DTT (2 mM) for 3 hours and successively maintained for 24 hrs in OPTIMEM. Secreted protein (50 µg) extracted from the culture medium by TCA precipitation, and aliquots of cell lysates (50 µg) were resolved by SDS-PAGE (10%) and blotted with monoclonal anti-SEL1L and anti-vinculin antibodies. P38 was not detectable in the culture medium, both in presence and in absence of DTT. In addition to p38, MCF10A cells showed a higher band, probably corresponding to an immature precursor or post-translationally-modified product. The image is representative of two independent experiments. A1. RT-PCR analysis of untreated and DTT-treated MCF10A cells: RNA was extracted from the samples described in panel A and analyzed by RT-PCR for the UPR response. The histogram shows expression values normalized relative to housekeeping signals and expressed as fold modulation relative to the untreated samples; densitometric analysis was performed by Scion imaging program. UPR activation upon DTT treatment is indicated by up-modulation of BIP and CHOP and XBP-1 splicing, concomitantly SEL1LA is incremented (gray bar). The corresponding images are shown in Figure S2A. The data are the averages of two different assays based on independent treatments, ±SD. B. Western blot analysis of untreated and DTT-treated SKBr3 cells: Secretion of p38 and p28 was evaluated in untreated and DTT-treated SKBr3 cells. Cells exposed to DTT (2 mM) for 3 hrs or not exposed were maintained for 24 hrs in OPTIMEM. Secreted protein (50 µg) extracted from the culture medium by TCA precipitation, and aliquots of cell lysates (50 µg) were resolved by SDS-PAGE (12%) and blotted with anti-SEL1L and anti-vinculin antibodies. ER stress/UPR strongly promoted secretion of p38 and, to a lesser extent, p28 in the culture medium. The image is representative of five independent experiments. C. Western blot analysis of KMS11 cells treated with DTT and MG132: KMS11 cells were exposed to DTT (2 mM) or MG132 (10 µM) for 3 hrs and successively maintained for 24 hrs in OPTIMEM. Secreted protein (30 µg), extracted from the culture medium by TCA precipitation, and aliquots of cell lysates (50 µg) were resolved by SDS-PAGE (10%) and blotted with anti-SEL1L and anti-vinculin antibodies. Both treatments markedly induced p38 secretion in the culture medium. The image is representative of five independent experiments. C1. RT-PCR analysis of KMS11 cells treated with DTT and MG132: RNA was extracted from the same samples described in panel C and analyzed by RT-PCR for the UPR response. The histogram shows expression values normalized relative to housekeeping signals and expressed as fold modulation relative to untreated samples; densitometric analysis was determined by Scion imaging program. UPR activation upon DTT treatment is indicated by the up-modulation of ATF6, BIP, and CHOP and by XBP-1 splicing (see Figure S2C), concomitantly the expression of SEL1LA, HRD1 and GADD45β is incremented (gray bar). The corresponding images are shown in Figure S2C. MG132 treatment did not trigger UPR activation, as indicated by the absence of ATF6 and BIP un-modulation and lack of XBP-1 splicing (see Figure S2C), nevertheless, CHOP and GADD45β were markedly up-modulated and SEL1LA and HRD1 down-modulated (black bars). The data are averages of four different assays based on independent treatments, ±SD. C2. SEL1LA protein expression in KMS11 cells treated with DTT and MG132: The histogram shows SEL1LA protein expression values obtained from the samples described in panel C, normalized relative to housekeeping signals and expressed as fold modulation relative to the untreated sample; densitometric analysis was performed using the Scion imaging program. MG132 determined SEL1LA protein accumulation up to 2 times relative to the control level (black bar). The data are the averages of four independent experiments, ±SD.
Mentions: To explore the behavior and secretion of p38 and p28 in cells under ER stress/UPR, we analyzed by SDS-PAGE and immunoblot cell lysates and culture supernatants of MCF10A, SKBr3 and KMS11 cells under normal conditions and after treatment with DTT, which causes protein misfolding by reducing disulfide bonds [45] (Figure 2A–C). In all the tested cells DTT triggered ER stress/UPR, assessed by XBP-1 splicing combined with BIP, ATF6 and CHOP up-modulation, and boosted SEL1LA mRNA expression (Figure 2A1,C1; Figure S2A–C), while the SEL1LA protein level did not increase significantly (Figure 2A–C, C2). P38, but not SEL1LA, was readily detectable in SKBr3 and KMS11 supernatants, increasing close to three- and five-fold respectively after DTT (Figure 2B–C). No evidence of p38 was detected in MCF10A supernatants, under both normal and DTT-stressed conditions (Figure 2A). P28 was exclusively found after DTT treatment and only in the supernatant of the SKBr3 cell line, which expresses this variant (Figure 2B).

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