Limits...
Erlins restrict SREBP activation in the ER and regulate cellular cholesterol homeostasis.

Huber MD, Vesely PW, Datta K, Gerace L - J. Cell Biol. (2013)

Bottom Line: Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins.Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery.We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037.

ABSTRACT
Cellular cholesterol levels are controlled by endoplasmic reticulum (ER) sterol sensing proteins, which include Scap and Insig-1. With cholesterol sufficiency, Insig inhibits the activation of sterol regulatory element binding proteins (SREBPs), key transcription factors for cholesterol and fatty acid biosynthetic genes, by associating with Scap-SREBP complexes to promote their ER retention. Here we show that the multimeric ER proteins erlins-1 and -2 are additional SREBP regulators. Depletion of erlins from cells grown with sterol sufficiency led to canonical activation of SREBPs and their target genes. Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins. Erlins bound cholesterol with specificity and strong cooperativity and responded to ER cholesterol changes with altered diffusional mobility, suggesting that erlins themselves may be regulated by cholesterol. Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery. We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.

Show MeSH
SREBP target gene activation and lipid accumulation in cells with erlin depletion. (A) Silencing of erlin-1 and -2 with siRNA. HeLa cells were transfected with erlin-targeting or control (ctrl) siRNAs and incubated in complete medium or with LD, and were analyzed by q-RT-PCR (left and middle) and Western blotting (right) for erlin mRNA and protein levels. (B) q-RT-PCR of SREBP target genes. P-values for all comparisons to control <0.031; n = 5. (C) Oil Red O (ORO) staining of siRNA-transfected cells. Overlays of phase contrast (blue) and red fluorescence (left) and quantification of Oil Red O staining (right). *, P < 10−4; n = 3. (D) Quantification of cellular lipids in erlin-depleted cells. (left) Esterified cholesterol (gray sections) is the difference between total cholesterol and free cholesterol (black). Values were normalized to phosphoglyceride levels and expressed relative to control. **, P < 10−4; *, P < 4 × 10−4; #, P > 0.36; n = 3. Error bars indicate standard deviations.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3824017&req=5

fig1: SREBP target gene activation and lipid accumulation in cells with erlin depletion. (A) Silencing of erlin-1 and -2 with siRNA. HeLa cells were transfected with erlin-targeting or control (ctrl) siRNAs and incubated in complete medium or with LD, and were analyzed by q-RT-PCR (left and middle) and Western blotting (right) for erlin mRNA and protein levels. (B) q-RT-PCR of SREBP target genes. P-values for all comparisons to control <0.031; n = 5. (C) Oil Red O (ORO) staining of siRNA-transfected cells. Overlays of phase contrast (blue) and red fluorescence (left) and quantification of Oil Red O staining (right). *, P < 10−4; n = 3. (D) Quantification of cellular lipids in erlin-depleted cells. (left) Esterified cholesterol (gray sections) is the difference between total cholesterol and free cholesterol (black). Values were normalized to phosphoglyceride levels and expressed relative to control. **, P < 10−4; *, P < 4 × 10−4; #, P > 0.36; n = 3. Error bars indicate standard deviations.

Mentions: We analyzed whether siRNA-mediated knockdown of erlin-1 and/or erlin-2 in HeLa cells affects the activity of SREBPs under conditions of cholesterol sufficiency (Fig. 1). As a positive control for SREBP activation, lipid-depleted cell cultures were analyzed in parallel (Sakai et al., 1996). This condition strongly activates both SREBP-2, which controls genes governing cholesterol biosynthesis, and SREBP-1a, which regulates genes for both cholesterol and fatty acid biosynthesis (Hannah et al., 2001). Whereas erlin levels were strongly reduced with targeting siRNAs, they were not affected by lipid depletion (LD; Fig. 1 A).


Erlins restrict SREBP activation in the ER and regulate cellular cholesterol homeostasis.

Huber MD, Vesely PW, Datta K, Gerace L - J. Cell Biol. (2013)

SREBP target gene activation and lipid accumulation in cells with erlin depletion. (A) Silencing of erlin-1 and -2 with siRNA. HeLa cells were transfected with erlin-targeting or control (ctrl) siRNAs and incubated in complete medium or with LD, and were analyzed by q-RT-PCR (left and middle) and Western blotting (right) for erlin mRNA and protein levels. (B) q-RT-PCR of SREBP target genes. P-values for all comparisons to control <0.031; n = 5. (C) Oil Red O (ORO) staining of siRNA-transfected cells. Overlays of phase contrast (blue) and red fluorescence (left) and quantification of Oil Red O staining (right). *, P < 10−4; n = 3. (D) Quantification of cellular lipids in erlin-depleted cells. (left) Esterified cholesterol (gray sections) is the difference between total cholesterol and free cholesterol (black). Values were normalized to phosphoglyceride levels and expressed relative to control. **, P < 10−4; *, P < 4 × 10−4; #, P > 0.36; n = 3. Error bars indicate standard deviations.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3824017&req=5

fig1: SREBP target gene activation and lipid accumulation in cells with erlin depletion. (A) Silencing of erlin-1 and -2 with siRNA. HeLa cells were transfected with erlin-targeting or control (ctrl) siRNAs and incubated in complete medium or with LD, and were analyzed by q-RT-PCR (left and middle) and Western blotting (right) for erlin mRNA and protein levels. (B) q-RT-PCR of SREBP target genes. P-values for all comparisons to control <0.031; n = 5. (C) Oil Red O (ORO) staining of siRNA-transfected cells. Overlays of phase contrast (blue) and red fluorescence (left) and quantification of Oil Red O staining (right). *, P < 10−4; n = 3. (D) Quantification of cellular lipids in erlin-depleted cells. (left) Esterified cholesterol (gray sections) is the difference between total cholesterol and free cholesterol (black). Values were normalized to phosphoglyceride levels and expressed relative to control. **, P < 10−4; *, P < 4 × 10−4; #, P > 0.36; n = 3. Error bars indicate standard deviations.
Mentions: We analyzed whether siRNA-mediated knockdown of erlin-1 and/or erlin-2 in HeLa cells affects the activity of SREBPs under conditions of cholesterol sufficiency (Fig. 1). As a positive control for SREBP activation, lipid-depleted cell cultures were analyzed in parallel (Sakai et al., 1996). This condition strongly activates both SREBP-2, which controls genes governing cholesterol biosynthesis, and SREBP-1a, which regulates genes for both cholesterol and fatty acid biosynthesis (Hannah et al., 2001). Whereas erlin levels were strongly reduced with targeting siRNAs, they were not affected by lipid depletion (LD; Fig. 1 A).

Bottom Line: Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins.Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery.We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037.

ABSTRACT
Cellular cholesterol levels are controlled by endoplasmic reticulum (ER) sterol sensing proteins, which include Scap and Insig-1. With cholesterol sufficiency, Insig inhibits the activation of sterol regulatory element binding proteins (SREBPs), key transcription factors for cholesterol and fatty acid biosynthetic genes, by associating with Scap-SREBP complexes to promote their ER retention. Here we show that the multimeric ER proteins erlins-1 and -2 are additional SREBP regulators. Depletion of erlins from cells grown with sterol sufficiency led to canonical activation of SREBPs and their target genes. Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins. Erlins bound cholesterol with specificity and strong cooperativity and responded to ER cholesterol changes with altered diffusional mobility, suggesting that erlins themselves may be regulated by cholesterol. Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery. We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.

Show MeSH