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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

p38 and p28 are related SEL1L variants.A. Western blot analysis: Lysates (50 ug) from different cell lines, including 293FT (embryo kidney), MCF10A (non-tumorigenic breast), MCF7, SKBr3 (breast cancer) and KMS11 (multiple myeloma), were resolved by SDS-PAGE (10%) and probed with monoclonal to SEL1L N-terminus. Vinculin was used as a loading control. In addition to SEL1LA (95 KDa), the N-terminal SEL1L antibody recognized two smaller encoded products, at approximately 38 and 28 KDa (designated p38 and p28). P38 was more abundant than SEL1LA and both were up-modulated in the cancer cell lines relative to MCF10A. P28 was detectable only in SKBr3. Bands above p38, probably corresponding to immature precursors or post-translationally modified products, were occasionally seen in the tested cell lines. The blot is representative of four independent experiments. B. RT-PCR analysis: RNAs from KMS11, MCF10A and SKBr3 cells were analyzed by RT-PCR using primers specific for SEL1LA. Signals shown were obtained with 27 cycles for SEL1LA. HPRT was used as a loading control. SEL1LA was up-modulated in the cancer cell lines relative to the non-tumorigenic MCF10A line. The image is representative of three different assays based on independent treatments. C. Intra/inter-molecular disulfide bonds analysis of p38. 293FT cell lysates were resolved by SDS-PAGE (10%) under reducing (R) and non-reducing (NR) conditions and blotted with monoclonal anti-SEL1L N-terminus. P38 migrated as a doublet under non-reducing conditions (the lanes comparing p38 migration under reducing and non-reducing conditions are from the same gel). D. Down-modulation of SEL1LA, p28 and p38 by SEL1L small interfering RNA (siRNA): Left panel: SKBr3 cells (3×105) were treated with scrambled siRNA or siRNA specific to SEL1L (siRNA SEL1L) for 48 hrs, followed by a second siRNA treatment for further 48 hrs. Silencing efficiency was verified by Western blot. SEL1LA, p28 and p38 protein levels decreased close to 55%, 30% and 16% respectively compared to cells treated with scrambled siRNA. Vinculin was used as a loading control. Right panel: 293FT cells (6×105) were treated with scrambled siRNA or siRNA-SEL1L for 48 hrs. SEL1LA and p38 protein levels decreased close to 45% and 23% respectively compared to cells treated with scrambled siRNA. Vinculin was used as a loading control. The histograms show values normalized relative to housekeeping signals and expressed as fold modulation relative to controls, densitometric analysis was determined using the Scion imaging program. (www.scioncorp.com). The data are the averages of three independent experiments, ±SD; Student's t-test was used to determine statistical significance *p<0.1; **p<0.05. E. Analysis of p38 and SEL1LA stability: 293 FT cells (6×105) were treated for 3, 6 and 18 hours with cycloheximide (CHX, 200 µg/ml). Aliquots of lysates (50 µg) were resolved by SDS-PAGE (10%) and probed with monoclonal anti-SEL1L and anti-vinculin antibodies. Unlike SEL1LA, which progressively decreased during cycloheximide exposure up to about 90%, p38 levels did not change significantly. The histogram shows the densitometric quantifications obtained through Scion imaging program (www.scioncorp.com). Values were normalized relative to housekeeping signals and expressed as fold modulation relative to untreated samples. The data are averages of two independent experiments, ±SD.
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pone-0017206-g001: p38 and p28 are related SEL1L variants.A. Western blot analysis: Lysates (50 ug) from different cell lines, including 293FT (embryo kidney), MCF10A (non-tumorigenic breast), MCF7, SKBr3 (breast cancer) and KMS11 (multiple myeloma), were resolved by SDS-PAGE (10%) and probed with monoclonal to SEL1L N-terminus. Vinculin was used as a loading control. In addition to SEL1LA (95 KDa), the N-terminal SEL1L antibody recognized two smaller encoded products, at approximately 38 and 28 KDa (designated p38 and p28). P38 was more abundant than SEL1LA and both were up-modulated in the cancer cell lines relative to MCF10A. P28 was detectable only in SKBr3. Bands above p38, probably corresponding to immature precursors or post-translationally modified products, were occasionally seen in the tested cell lines. The blot is representative of four independent experiments. B. RT-PCR analysis: RNAs from KMS11, MCF10A and SKBr3 cells were analyzed by RT-PCR using primers specific for SEL1LA. Signals shown were obtained with 27 cycles for SEL1LA. HPRT was used as a loading control. SEL1LA was up-modulated in the cancer cell lines relative to the non-tumorigenic MCF10A line. The image is representative of three different assays based on independent treatments. C. Intra/inter-molecular disulfide bonds analysis of p38. 293FT cell lysates were resolved by SDS-PAGE (10%) under reducing (R) and non-reducing (NR) conditions and blotted with monoclonal anti-SEL1L N-terminus. P38 migrated as a doublet under non-reducing conditions (the lanes comparing p38 migration under reducing and non-reducing conditions are from the same gel). D. Down-modulation of SEL1LA, p28 and p38 by SEL1L small interfering RNA (siRNA): Left panel: SKBr3 cells (3×105) were treated with scrambled siRNA or siRNA specific to SEL1L (siRNA SEL1L) for 48 hrs, followed by a second siRNA treatment for further 48 hrs. Silencing efficiency was verified by Western blot. SEL1LA, p28 and p38 protein levels decreased close to 55%, 30% and 16% respectively compared to cells treated with scrambled siRNA. Vinculin was used as a loading control. Right panel: 293FT cells (6×105) were treated with scrambled siRNA or siRNA-SEL1L for 48 hrs. SEL1LA and p38 protein levels decreased close to 45% and 23% respectively compared to cells treated with scrambled siRNA. Vinculin was used as a loading control. The histograms show values normalized relative to housekeeping signals and expressed as fold modulation relative to controls, densitometric analysis was determined using the Scion imaging program. (www.scioncorp.com). The data are the averages of three independent experiments, ±SD; Student's t-test was used to determine statistical significance *p<0.1; **p<0.05. E. Analysis of p38 and SEL1LA stability: 293 FT cells (6×105) were treated for 3, 6 and 18 hours with cycloheximide (CHX, 200 µg/ml). Aliquots of lysates (50 µg) were resolved by SDS-PAGE (10%) and probed with monoclonal anti-SEL1L and anti-vinculin antibodies. Unlike SEL1LA, which progressively decreased during cycloheximide exposure up to about 90%, p38 levels did not change significantly. The histogram shows the densitometric quantifications obtained through Scion imaging program (www.scioncorp.com). Values were normalized relative to housekeeping signals and expressed as fold modulation relative to untreated samples. The data are averages of two independent experiments, ±SD.

Mentions: A monoclonal antibody raised against the N-terminus of the SEL1L protein [34] was used to screen a panel of whole lysates from five human cell lines, including MCF7 and SKBr3 (breast cancer), KMS11 (Ig-K-secreting multiple myeloma), 293FT (embryo kidney) and the non-tumorigenic epithelial breast cell line MCF10A. Besides the well-known 95 KDa SEL1LA protein, two additive bands, designated as p38 and p28, were visualized at approximately 38 KDa and 28 KDa (Figure 1A). While p28 was exclusively detected in the SKBr3 line, p38 was strongly expressed, at levels higher than SEL1LA, in all the cancer cell lines tested, with lower levels in MCF10A, where SEL1LA expression (protein and RNA) was also lower (Figure 1A–B). A polyclonal antibody raised against the SEL1L C-terminus, which encompasses the SEL1LA tail anchor for insertion into the ER membrane, detected the 95 KDa SEL1LA protein, but not p38 and p28, although an additional band was evidenced at about 55 KDa, which could indicate the carboxy-terminal fragment resulting from cleavage of the native protein (Figure S1A). Notably the p38 variant was strongly expressed also in other tested cancer cell lines, including Namalwa (lymphoma), MDAMB453 (breast cancer), HeLa (cervical cancer), and G144, G166, G179 (glioblastoma), all of which resulted negative for p28 (Figure S1B). Analysis of SEL1L protein profile in the non-tumorigenic human fetal brain cell line CB660 compared to the three glioblastoma cell lines (Figure S1B) provided further in vitro evidence that p38 was indeed expressed at higher levels in tumor cells.


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)

p38 and p28 are related SEL1L variants.A. Western blot analysis: Lysates (50 ug) from different cell lines, including 293FT (embryo kidney), MCF10A (non-tumorigenic breast), MCF7, SKBr3 (breast cancer) and KMS11 (multiple myeloma), were resolved by SDS-PAGE (10%) and probed with monoclonal to SEL1L N-terminus. Vinculin was used as a loading control. In addition to SEL1LA (95 KDa), the N-terminal SEL1L antibody recognized two smaller encoded products, at approximately 38 and 28 KDa (designated p38 and p28). P38 was more abundant than SEL1LA and both were up-modulated in the cancer cell lines relative to MCF10A. P28 was detectable only in SKBr3. Bands above p38, probably corresponding to immature precursors or post-translationally modified products, were occasionally seen in the tested cell lines. The blot is representative of four independent experiments. B. RT-PCR analysis: RNAs from KMS11, MCF10A and SKBr3 cells were analyzed by RT-PCR using primers specific for SEL1LA. Signals shown were obtained with 27 cycles for SEL1LA. HPRT was used as a loading control. SEL1LA was up-modulated in the cancer cell lines relative to the non-tumorigenic MCF10A line. The image is representative of three different assays based on independent treatments. C. Intra/inter-molecular disulfide bonds analysis of p38. 293FT cell lysates were resolved by SDS-PAGE (10%) under reducing (R) and non-reducing (NR) conditions and blotted with monoclonal anti-SEL1L N-terminus. P38 migrated as a doublet under non-reducing conditions (the lanes comparing p38 migration under reducing and non-reducing conditions are from the same gel). D. Down-modulation of SEL1LA, p28 and p38 by SEL1L small interfering RNA (siRNA): Left panel: SKBr3 cells (3×105) were treated with scrambled siRNA or siRNA specific to SEL1L (siRNA SEL1L) for 48 hrs, followed by a second siRNA treatment for further 48 hrs. Silencing efficiency was verified by Western blot. SEL1LA, p28 and p38 protein levels decreased close to 55%, 30% and 16% respectively compared to cells treated with scrambled siRNA. Vinculin was used as a loading control. Right panel: 293FT cells (6×105) were treated with scrambled siRNA or siRNA-SEL1L for 48 hrs. SEL1LA and p38 protein levels decreased close to 45% and 23% respectively compared to cells treated with scrambled siRNA. Vinculin was used as a loading control. The histograms show values normalized relative to housekeeping signals and expressed as fold modulation relative to controls, densitometric analysis was determined using the Scion imaging program. (www.scioncorp.com). The data are the averages of three independent experiments, ±SD; Student's t-test was used to determine statistical significance *p<0.1; **p<0.05. E. Analysis of p38 and SEL1LA stability: 293 FT cells (6×105) were treated for 3, 6 and 18 hours with cycloheximide (CHX, 200 µg/ml). Aliquots of lysates (50 µg) were resolved by SDS-PAGE (10%) and probed with monoclonal anti-SEL1L and anti-vinculin antibodies. Unlike SEL1LA, which progressively decreased during cycloheximide exposure up to about 90%, p38 levels did not change significantly. The histogram shows the densitometric quantifications obtained through Scion imaging program (www.scioncorp.com). Values were normalized relative to housekeeping signals and expressed as fold modulation relative to untreated samples. The data are averages of two independent experiments, ±SD.
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Related In: Results  -  Collection

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pone-0017206-g001: p38 and p28 are related SEL1L variants.A. Western blot analysis: Lysates (50 ug) from different cell lines, including 293FT (embryo kidney), MCF10A (non-tumorigenic breast), MCF7, SKBr3 (breast cancer) and KMS11 (multiple myeloma), were resolved by SDS-PAGE (10%) and probed with monoclonal to SEL1L N-terminus. Vinculin was used as a loading control. In addition to SEL1LA (95 KDa), the N-terminal SEL1L antibody recognized two smaller encoded products, at approximately 38 and 28 KDa (designated p38 and p28). P38 was more abundant than SEL1LA and both were up-modulated in the cancer cell lines relative to MCF10A. P28 was detectable only in SKBr3. Bands above p38, probably corresponding to immature precursors or post-translationally modified products, were occasionally seen in the tested cell lines. The blot is representative of four independent experiments. B. RT-PCR analysis: RNAs from KMS11, MCF10A and SKBr3 cells were analyzed by RT-PCR using primers specific for SEL1LA. Signals shown were obtained with 27 cycles for SEL1LA. HPRT was used as a loading control. SEL1LA was up-modulated in the cancer cell lines relative to the non-tumorigenic MCF10A line. The image is representative of three different assays based on independent treatments. C. Intra/inter-molecular disulfide bonds analysis of p38. 293FT cell lysates were resolved by SDS-PAGE (10%) under reducing (R) and non-reducing (NR) conditions and blotted with monoclonal anti-SEL1L N-terminus. P38 migrated as a doublet under non-reducing conditions (the lanes comparing p38 migration under reducing and non-reducing conditions are from the same gel). D. Down-modulation of SEL1LA, p28 and p38 by SEL1L small interfering RNA (siRNA): Left panel: SKBr3 cells (3×105) were treated with scrambled siRNA or siRNA specific to SEL1L (siRNA SEL1L) for 48 hrs, followed by a second siRNA treatment for further 48 hrs. Silencing efficiency was verified by Western blot. SEL1LA, p28 and p38 protein levels decreased close to 55%, 30% and 16% respectively compared to cells treated with scrambled siRNA. Vinculin was used as a loading control. Right panel: 293FT cells (6×105) were treated with scrambled siRNA or siRNA-SEL1L for 48 hrs. SEL1LA and p38 protein levels decreased close to 45% and 23% respectively compared to cells treated with scrambled siRNA. Vinculin was used as a loading control. The histograms show values normalized relative to housekeeping signals and expressed as fold modulation relative to controls, densitometric analysis was determined using the Scion imaging program. (www.scioncorp.com). The data are the averages of three independent experiments, ±SD; Student's t-test was used to determine statistical significance *p<0.1; **p<0.05. E. Analysis of p38 and SEL1LA stability: 293 FT cells (6×105) were treated for 3, 6 and 18 hours with cycloheximide (CHX, 200 µg/ml). Aliquots of lysates (50 µg) were resolved by SDS-PAGE (10%) and probed with monoclonal anti-SEL1L and anti-vinculin antibodies. Unlike SEL1LA, which progressively decreased during cycloheximide exposure up to about 90%, p38 levels did not change significantly. The histogram shows the densitometric quantifications obtained through Scion imaging program (www.scioncorp.com). Values were normalized relative to housekeeping signals and expressed as fold modulation relative to untreated samples. The data are averages of two independent experiments, ±SD.
Mentions: A monoclonal antibody raised against the N-terminus of the SEL1L protein [34] was used to screen a panel of whole lysates from five human cell lines, including MCF7 and SKBr3 (breast cancer), KMS11 (Ig-K-secreting multiple myeloma), 293FT (embryo kidney) and the non-tumorigenic epithelial breast cell line MCF10A. Besides the well-known 95 KDa SEL1LA protein, two additive bands, designated as p38 and p28, were visualized at approximately 38 KDa and 28 KDa (Figure 1A). While p28 was exclusively detected in the SKBr3 line, p38 was strongly expressed, at levels higher than SEL1LA, in all the cancer cell lines tested, with lower levels in MCF10A, where SEL1LA expression (protein and RNA) was also lower (Figure 1A–B). A polyclonal antibody raised against the SEL1L C-terminus, which encompasses the SEL1LA tail anchor for insertion into the ER membrane, detected the 95 KDa SEL1LA protein, but not p38 and p28, although an additional band was evidenced at about 55 KDa, which could indicate the carboxy-terminal fragment resulting from cleavage of the native protein (Figure S1A). Notably the p38 variant was strongly expressed also in other tested cancer cell lines, including Namalwa (lymphoma), MDAMB453 (breast cancer), HeLa (cervical cancer), and G144, G166, G179 (glioblastoma), all of which resulted negative for p28 (Figure S1B). Analysis of SEL1L protein profile in the non-tumorigenic human fetal brain cell line CB660 compared to the three glioblastoma cell lines (Figure S1B) provided further in vitro evidence that p38 was indeed expressed at higher levels in tumor cells.

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