Nuclear receptor subfamily 1 group H member 2 (LXRB) is the predominant liver X receptor subtype regulating transcription of 2 major lipogenic genes in goat primary mammary epithelial cells

H. B. Shi, C. H. Zhang, Z. A. Xu, X. F. Xu, Z. B. Lv, J. Luo, J. J. Loor

Research output: Contribution to journalArticle

Abstract

In ruminants, recent research has identified a crucial role for liver X receptors (LXR) in regulating lipid metabolism in mammary cells. However, the differences between LXR subtypes in regulating ruminant lipid metabolism are unknown. We used overexpression and knockdown of LXRA and LXRB in goat primary mammary epithelial cells to distinguish subtype-specific regulation of sterol regulatory element binding protein 1c (SREBP1c) and fatty acid synthase (FASN) mRNA expression and their promoter activity. Incubation with siRNA targeting LXR decreased expression of LXRA and LXRB. Knockdown of LXRA and LXRB in cells incubated with dimethyl sulfoxide (DMSO, control) had no effect on the expression of SREBP1c or FASN. Knockdown of LXRB in cells incubated with LXR ligand T0901317 (T09) led to decreased expression of FASN, but not SREBP1c. Overexpression of LXRB plus T09 dramatically upregulated SREBP1c and FASN to levels higher than overexpression of LXRA with T09. Luciferase reporter assays in cells with site-directed mutagenesis of LXR response elements (LXRE; LXRE1 from −286 to −251 bp or LXRE2 from −235 to −219 bp) revealed that the SREBP1c promoter with the wild type or either LXRE mutation in cells supplemented with T09 decreased markedly only when LXRB was knocked down. Knockdown of LXRA and LXRB had no effect on the SREBP1c promoter when cells had a double LXRE mutation. Overexpression of LXRA only in cells incubated with T09 increased the activity of the SREBP1c promoter with the wild type and the LXRE2 mutation. In contrast, compared with each control group, overexpression of LXRB dramatically increased SREBP1c promoter activity, regardless of LXRE mutation. Furthermore, in cells stimulated with T09, knockdown of either LXRA or LXRB did not alter wild-type FASN promoter activity. Knockdown of LXRA increased wild-type and LXRE-site-mutated (LXRE from −677 to −662 bp) FASN promoter activity. Overexpression of LXRB increased wild-type and LXRE-site-mutated FASN promoter activity regardless of treatment with DMSO or T09, but overexpression of LXRA altered LXRE-site-mutated FASN promoter activity only in cells treated with DMSO. Increased activation of SREBP1c or FASN promoters containing LXRE mutations after overexpression of LXRB suggested that LXRB activates endogenous SREBP1c, which can then bind to the promoter of SREBP1c via an auto-loop circuit regulatory mechanism. Collectively, these results highlight an important role for LXRB in the transcriptional regulation of SREBP1c and FASN in goat mammary epithelial cells. Activation of this nuclear receptor controls lipogenesis via different mechanisms.

Original languageEnglish (US)
Pages (from-to)6743-6752
Number of pages10
JournalJournal of Dairy Science
Volume100
Issue number8
DOIs
StatePublished - Aug 2017

Fingerprint

Sterol Regulatory Element Binding Protein 1
Fatty Acid Synthases
major genes
fatty-acid synthase
Goats
sterols
breasts
binding proteins
Breast
epithelial cells
transcription (genetics)
Epithelial Cells
goats
promoter regions
liver
receptors
Genes
Dimethyl Sulfoxide
mutation
cells

Keywords

  • gene expression
  • mammary cells
  • promoter activation
  • ruminant

ASJC Scopus subject areas

  • Food Science
  • Animal Science and Zoology
  • Genetics

Cite this

Nuclear receptor subfamily 1 group H member 2 (LXRB) is the predominant liver X receptor subtype regulating transcription of 2 major lipogenic genes in goat primary mammary epithelial cells. / Shi, H. B.; Zhang, C. H.; Xu, Z. A.; Xu, X. F.; Lv, Z. B.; Luo, J.; Loor, J. J.

In: Journal of Dairy Science, Vol. 100, No. 8, 08.2017, p. 6743-6752.

Research output: Contribution to journalArticle

@article{dc7dd53e7fc84d73ac042d921097abd3,
title = "Nuclear receptor subfamily 1 group H member 2 (LXRB) is the predominant liver X receptor subtype regulating transcription of 2 major lipogenic genes in goat primary mammary epithelial cells",
abstract = "In ruminants, recent research has identified a crucial role for liver X receptors (LXR) in regulating lipid metabolism in mammary cells. However, the differences between LXR subtypes in regulating ruminant lipid metabolism are unknown. We used overexpression and knockdown of LXRA and LXRB in goat primary mammary epithelial cells to distinguish subtype-specific regulation of sterol regulatory element binding protein 1c (SREBP1c) and fatty acid synthase (FASN) mRNA expression and their promoter activity. Incubation with siRNA targeting LXR decreased expression of LXRA and LXRB. Knockdown of LXRA and LXRB in cells incubated with dimethyl sulfoxide (DMSO, control) had no effect on the expression of SREBP1c or FASN. Knockdown of LXRB in cells incubated with LXR ligand T0901317 (T09) led to decreased expression of FASN, but not SREBP1c. Overexpression of LXRB plus T09 dramatically upregulated SREBP1c and FASN to levels higher than overexpression of LXRA with T09. Luciferase reporter assays in cells with site-directed mutagenesis of LXR response elements (LXRE; LXRE1 from −286 to −251 bp or LXRE2 from −235 to −219 bp) revealed that the SREBP1c promoter with the wild type or either LXRE mutation in cells supplemented with T09 decreased markedly only when LXRB was knocked down. Knockdown of LXRA and LXRB had no effect on the SREBP1c promoter when cells had a double LXRE mutation. Overexpression of LXRA only in cells incubated with T09 increased the activity of the SREBP1c promoter with the wild type and the LXRE2 mutation. In contrast, compared with each control group, overexpression of LXRB dramatically increased SREBP1c promoter activity, regardless of LXRE mutation. Furthermore, in cells stimulated with T09, knockdown of either LXRA or LXRB did not alter wild-type FASN promoter activity. Knockdown of LXRA increased wild-type and LXRE-site-mutated (LXRE from −677 to −662 bp) FASN promoter activity. Overexpression of LXRB increased wild-type and LXRE-site-mutated FASN promoter activity regardless of treatment with DMSO or T09, but overexpression of LXRA altered LXRE-site-mutated FASN promoter activity only in cells treated with DMSO. Increased activation of SREBP1c or FASN promoters containing LXRE mutations after overexpression of LXRB suggested that LXRB activates endogenous SREBP1c, which can then bind to the promoter of SREBP1c via an auto-loop circuit regulatory mechanism. Collectively, these results highlight an important role for LXRB in the transcriptional regulation of SREBP1c and FASN in goat mammary epithelial cells. Activation of this nuclear receptor controls lipogenesis via different mechanisms.",
keywords = "gene expression, mammary cells, promoter activation, ruminant",
author = "Shi, {H. B.} and Zhang, {C. H.} and Xu, {Z. A.} and Xu, {X. F.} and Lv, {Z. B.} and J. Luo and Loor, {J. J.}",
year = "2017",
month = "8",
doi = "10.3168/jds.2016-12510",
language = "English (US)",
volume = "100",
pages = "6743--6752",
journal = "Journal of Dairy Science",
issn = "0022-0302",
publisher = "Elsevier Limited",
number = "8",

}

TY - JOUR

T1 - Nuclear receptor subfamily 1 group H member 2 (LXRB) is the predominant liver X receptor subtype regulating transcription of 2 major lipogenic genes in goat primary mammary epithelial cells

AU - Shi, H. B.

AU - Zhang, C. H.

AU - Xu, Z. A.

AU - Xu, X. F.

AU - Lv, Z. B.

AU - Luo, J.

AU - Loor, J. J.

PY - 2017/8

Y1 - 2017/8

N2 - In ruminants, recent research has identified a crucial role for liver X receptors (LXR) in regulating lipid metabolism in mammary cells. However, the differences between LXR subtypes in regulating ruminant lipid metabolism are unknown. We used overexpression and knockdown of LXRA and LXRB in goat primary mammary epithelial cells to distinguish subtype-specific regulation of sterol regulatory element binding protein 1c (SREBP1c) and fatty acid synthase (FASN) mRNA expression and their promoter activity. Incubation with siRNA targeting LXR decreased expression of LXRA and LXRB. Knockdown of LXRA and LXRB in cells incubated with dimethyl sulfoxide (DMSO, control) had no effect on the expression of SREBP1c or FASN. Knockdown of LXRB in cells incubated with LXR ligand T0901317 (T09) led to decreased expression of FASN, but not SREBP1c. Overexpression of LXRB plus T09 dramatically upregulated SREBP1c and FASN to levels higher than overexpression of LXRA with T09. Luciferase reporter assays in cells with site-directed mutagenesis of LXR response elements (LXRE; LXRE1 from −286 to −251 bp or LXRE2 from −235 to −219 bp) revealed that the SREBP1c promoter with the wild type or either LXRE mutation in cells supplemented with T09 decreased markedly only when LXRB was knocked down. Knockdown of LXRA and LXRB had no effect on the SREBP1c promoter when cells had a double LXRE mutation. Overexpression of LXRA only in cells incubated with T09 increased the activity of the SREBP1c promoter with the wild type and the LXRE2 mutation. In contrast, compared with each control group, overexpression of LXRB dramatically increased SREBP1c promoter activity, regardless of LXRE mutation. Furthermore, in cells stimulated with T09, knockdown of either LXRA or LXRB did not alter wild-type FASN promoter activity. Knockdown of LXRA increased wild-type and LXRE-site-mutated (LXRE from −677 to −662 bp) FASN promoter activity. Overexpression of LXRB increased wild-type and LXRE-site-mutated FASN promoter activity regardless of treatment with DMSO or T09, but overexpression of LXRA altered LXRE-site-mutated FASN promoter activity only in cells treated with DMSO. Increased activation of SREBP1c or FASN promoters containing LXRE mutations after overexpression of LXRB suggested that LXRB activates endogenous SREBP1c, which can then bind to the promoter of SREBP1c via an auto-loop circuit regulatory mechanism. Collectively, these results highlight an important role for LXRB in the transcriptional regulation of SREBP1c and FASN in goat mammary epithelial cells. Activation of this nuclear receptor controls lipogenesis via different mechanisms.

AB - In ruminants, recent research has identified a crucial role for liver X receptors (LXR) in regulating lipid metabolism in mammary cells. However, the differences between LXR subtypes in regulating ruminant lipid metabolism are unknown. We used overexpression and knockdown of LXRA and LXRB in goat primary mammary epithelial cells to distinguish subtype-specific regulation of sterol regulatory element binding protein 1c (SREBP1c) and fatty acid synthase (FASN) mRNA expression and their promoter activity. Incubation with siRNA targeting LXR decreased expression of LXRA and LXRB. Knockdown of LXRA and LXRB in cells incubated with dimethyl sulfoxide (DMSO, control) had no effect on the expression of SREBP1c or FASN. Knockdown of LXRB in cells incubated with LXR ligand T0901317 (T09) led to decreased expression of FASN, but not SREBP1c. Overexpression of LXRB plus T09 dramatically upregulated SREBP1c and FASN to levels higher than overexpression of LXRA with T09. Luciferase reporter assays in cells with site-directed mutagenesis of LXR response elements (LXRE; LXRE1 from −286 to −251 bp or LXRE2 from −235 to −219 bp) revealed that the SREBP1c promoter with the wild type or either LXRE mutation in cells supplemented with T09 decreased markedly only when LXRB was knocked down. Knockdown of LXRA and LXRB had no effect on the SREBP1c promoter when cells had a double LXRE mutation. Overexpression of LXRA only in cells incubated with T09 increased the activity of the SREBP1c promoter with the wild type and the LXRE2 mutation. In contrast, compared with each control group, overexpression of LXRB dramatically increased SREBP1c promoter activity, regardless of LXRE mutation. Furthermore, in cells stimulated with T09, knockdown of either LXRA or LXRB did not alter wild-type FASN promoter activity. Knockdown of LXRA increased wild-type and LXRE-site-mutated (LXRE from −677 to −662 bp) FASN promoter activity. Overexpression of LXRB increased wild-type and LXRE-site-mutated FASN promoter activity regardless of treatment with DMSO or T09, but overexpression of LXRA altered LXRE-site-mutated FASN promoter activity only in cells treated with DMSO. Increased activation of SREBP1c or FASN promoters containing LXRE mutations after overexpression of LXRB suggested that LXRB activates endogenous SREBP1c, which can then bind to the promoter of SREBP1c via an auto-loop circuit regulatory mechanism. Collectively, these results highlight an important role for LXRB in the transcriptional regulation of SREBP1c and FASN in goat mammary epithelial cells. Activation of this nuclear receptor controls lipogenesis via different mechanisms.

KW - gene expression

KW - mammary cells

KW - promoter activation

KW - ruminant

UR - http://www.scopus.com/inward/record.url?scp=85020723115&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85020723115&partnerID=8YFLogxK

U2 - 10.3168/jds.2016-12510

DO - 10.3168/jds.2016-12510

M3 - Article

C2 - 28624275

AN - SCOPUS:85020723115

VL - 100

SP - 6743

EP - 6752

JO - Journal of Dairy Science

JF - Journal of Dairy Science

SN - 0022-0302

IS - 8

ER -