Long-chain unsaturated fatty acids reduce the transcriptional activity of the rat follicle-stimulating hormone β-subunit gene

Abstract

Here, we assessed the effects of long-chain fatty acids (LCFAs) and the LCFA receptor agonist GW9508 on the transcription of the gonadotropin subunit genes Cga, Lhb and Fshb because LCFA receptor GPR120 was observed in mouse gonadotropes in our recent study. A transcription assay using LβT2 cells demonstrated that LCFAs, oleic acid, α-linolenic acid, docosahexaenoic acid and palmitate, repressed the expression of Cga, Lhb, and Fshb at concentrations between 50 and 100 µM. On the other hand, treatment with 10 µM unsaturated LCFAs, oleic acid, α-linolenic acid and docosahexaenoic acid, repressed only Fshb expression, while the same dose of a saturated LCFA, palmitate, had no effect on the expression of gonadotropin subunit genes. Furthermore, GW9508 did not affect promoter activity. Next, we examined deletion mutants of the upstream region of Fshb and found that the upstream regulatory region (-2824 to -2343 bp) of Fshb was responsible for the notable repression by 10 µM unsaturated LCFAs. Our results suggest that the upstream region of Fshb is susceptible to unsaturated LCFAs. In addition, unsaturated LCFAs play a role in repressing Fshb expression through the distal -2824 to -2343 bp region, which might be independent of the LCFA receptor GPR120 pathway.

Fig. 1.

Transient transfection assay of the rat gonadotropin subunit gene promoters in LβT2 cells with or without treatment with oleic acid, α-linolenic acid, docosahexaenoic acid (DHA), palmitic acid or the long-chain fatty acid (LCFA) receptor agonist GW9508. Reporter constructs containing Cga (-3793/+37 b), Lhb (-2930/+17 b) or Fshb (-2824 to +28 b) promoters fused with the secreted alkaline phosphatase (SEAP) gene in the pSEAP-Basic vector were transfected into LβT2 cells. The cells were exposed to oleic acid, α-linolenic acid, DHA, palmitic acid, or GW9508 (1, 10, 50 or 100 µM) 7–8 h after transfection. An aliquot of the culture medium was used for the SEAP assay. SEAP activities are presented as the activity relative to that of the basic vector. Values are the mean ± SEM of four independent experiments. * P < 0.05 vs. pSEAP2-Basic (Dunnett’s method).

Fig. 2.

Deletion analysis of the rat Fshb promoter region (-2824 to +28 b regions) in LβT2 cells with or without treatment with 10 µM unsaturated LCFAs oleic acid, α-linolenic acid, or docosahexaenoic acid (DHA). On the left, reporter constructs are shown containing serial deletion mutants of the Fshb promoter fused with the secreted alkaline phosphatase (SEAP) gene in the pSEAP2-Basic vector that were transfected into LβT2 cells. On the right, SEAP activities are represented as the activity relative to that of the basic vector. Values are the mean ± SEM for four independent experiments. * P < 0.05 vs. pSEAP2-Basic (Dunnett’s method).

Fig. 3.

Locations of gene transcription factor binding sites in the -2824 to -2343 b region upstream of the Fshb promoter that was responsive to unsaturated LCFAs. Abbreviations of transcription factors: Forkhead box protein J1 (FOXJ1), glioma-associated oncogene homolog 1 (GLI1), hairy and enhancer of split (HES)/split-related with YRPW motif (HEY), Msh homeobox 1-like protein (MSX), paired-like homeodomain 1 (PITX1), paired related homeobox 2 (PRRX2), runt-related transcription factor 1 (RUNX1), sex-determining region Y-box 2 (SOX2)/sex-determining region Y-box 8 (SOX8), and TEA domain family member 2 (TEAD2).