Gonadotropin-releasing hormone modulates cholesterol synthesis and steroidogenesis in SH-SY5Y cells.

TLDR: It is found that GnRH up-regulates the expression of key genes of cholesterol and steroid synthesis when used in a narrow range around 1.0 nM, suggesting a new physiological role for GnRH in the brain.

About: This article is published in The Journal of Steroid Biochemistry and Molecular Biology.The article was published on 2011-04-01 and is currently open access. It has received 26 citations till now. The article focuses on the topics: Gonadotropin-releasing hormone & Steroid biosynthesis.

Figures

Fig. 3. LH expression after GnRH stimulus. A: LH mRNA in SH-SY5Y cells stimulatedwith 1.0nMGnRH. Values are normalized using GAPDH as housekeeping gene and reported as x-fold variation vs control. Values for each sample represent the mean± SE of the RT triplicate measured in duplicate (6 measures in total). Three experiments were performed. B: PCR amplification of LH subunit in untreated SH-SY5Y cells. The size of the PCR product is 320-bp.

Fig. 2. StAR and P450scc expression after GnRH stimulus. StAR mRNA levels (A) and western blotting analysis for StAR protein (B) in SH-SY5Y cells stimulated with 1.0nM GnRH. P450scc mRNA levels (C) in SH-SY5Y cells stimulated with 1.0nM GnRH. For both target genes, values are normalized using GAPDH as housekeeping gene and reported as x-fold variation vs control. Values for each sample represent themean± SE of the RT triplicatemeasured in duplicate (6measures in total). Three experiments were performed.

Fig. 1. Seladin-1/DHCR24 expression after GnRH stimulus. Seladin-1/DHCR24 mRNA in SH-SY5Y cells (A) and in FNC cells (B) stimulatedwith 1.0nMGnRH. Values are normalized using GAPDH as housekeeping gene and reported as x-fold variation vs control. Each sample represents the mean± SE of the RT triplicate measured in duplicate (6 measures in total). Five experiments were performed.

Fig. 6. Effect of GnRH on steroidogenesis in SH-SY5Y cells. Summary of themodulation of seladin-1/DHCR24, StAR, P450scc and LH expression, cell cholesterol amount and E2 production after treatment of SH-SY5Y cells with 1.0nM GnRH. For each target the variation is represented by a smoothed curve connecting the experimental points.

Fig. 4. Seladin-1/DHCR24, StAR and P450scc expression after GnRH stimulus and LH-Receptor blockage. Seladin-1/DHCR24 mRNA (A), StAR mRNA (B) and P450scc mRNA (C) in SH-SY5Y cells stimulated with 1.0nM GnRH after the blockage of LHReceptorwithAb-LHR (1:200). Values arenormalizedusingGAPDHashousekeeping gene and reported as x-fold variation vs control. Values for each sample represent themean± SE of the RT triplicatemeasured in duplicate (6measures in total). Three experiments were performed.

Fig. 5. Cell Cholesterol and E2 production after GnRH stimulus. A: Total cell cholesterol in SH-SY5Y cells stimulated with 1.0nM GnRH. Values are reported as fold variation vs control. The absolute amount of cholesterol measured for the control is 19.51±0.62ng cholesterol/ g protein. Each point is performed in triplicate and the results represent the mean± SE of five experiments. B: E2 amount released in the medium of SH-SY5Y cells stimulated with 1.0nM GnRH. The absolute amount of E2 measured for the control is 1.75±0.19pg/ml. Values are reported as fold variation vs control. Each point is performed in quadruplicate and the results represent the mean± SE of two experiments.

Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "Gonadotropin-releasing hormone modulates cholesterol synthesis and steroidogenesis in sh-sy5y cells" ?

Danza et al. this paper found that GnRH upregulated the expression of key genes of cholesterol and steroid synthesis when used in a narrow range around 1.0 nM. 

Q2. What is the role of StAR protein in the steroidogenesis process?

StAR protein is responsible for cholesterol transport into the inner mitochondrial membrane and this represents the rate limiting step of steroidogenesis. 

Q3. What is the effect of GnRH on the steroidogenesis?

GnRH is active only at in a narrow range around 1 nM while lower or higher concentrations are unable to stimulate steroidogenesis. 

Q4. How many cells were plated in a six-well plate?

Cells were plated at a density of 2 × 105 cells in six-wells plates, maintained in serum free medium 24 h and than treated with various concentrations of GnRH for the indicated periods. 

Q5. What is the effect of the antibody used to block the LH-receptor?

The antibody used to block the LH-receptor completely reverts the GnRH effect strongly suggesting that, similar to the HPG axis, GnRH action is mediated by LH. 

Q6. What is the role of GnRH secreting cells in the hypothalamus?

The physiological role of GnRH secreting cells is to respond to circulating levels of sex steroids and regulate GnRH secretion for a correct functionality of the HPG axis. 

Q7. How many ng of cDNA were used in the PCR mixture?

PCR mixture (25 l final volume) included 1× final concentration of QuantiTect Primer assay mix, 1× final concentration of Power SYBR Green Master Mix (Applied Biosystems) and 50 ng cDNA. 

Q8. How did the authors incubate cells with GnRH?

To assess whether the effect of GnRH is mediated by LH, the authors incubated SH-SY5Y cells with 1.0 nM GnRH in the presence of an antibody directed towards an extracellular domain of the LHreceptor. 

Q9. How many micrograms of total protein were loaded for each sample?

Twenty micrograms of total protein was loaded for each sample, resolved by 10% SDS polyacrylamide gel electrophoresis at 100 V and transferred to polyvinylidene difluoride (PVDF) membrane from Millipore (Billerica, MA, USA). 

Q10. How was the total RNA transcribed into cDNA?

1–2 g of total RNA was reverse transcribed into cDNA by using TaqMan reverse transcription reagents with random examers (Applied Biosystems Inc., Foster City, CA, USA). 

Q11. What is the role of GnRH in the regulation of cholesterol and estrogen?

This hormone could act as a common modulator of cholesterol and estrogen biosynthesis by an autocrine-paracrine mechanism, strongly supporting the presence of a local miniature endocrine axis. 

Q12. What is the effect of GnRH on cell cholesterol?

Their data add new interesting information demonstrating that GnRH action, in a human neuronal-like cell model, is mediated by LH and modulates also the last step of cholesterol biosynthesis, enhancing cell cholesterol. 

Q13. How was the RNA isolated from SH-SY5Y cells?

SH-SY5Y cells were incubated for 1 h at 37 ◦C with the LHR(H-50) antibody diluted 1:200 in serum-free medium before the hormone treatment. 

Q14. How long did the cells remain in the serum-free medium?

The following day, cultures were placed in serum-free medium for 24 h prior to treatment with 0.01–1000 nM GnRH or 100 mIU/ml LH at 37 ◦C for the incubation times indicated in Section 3. 

Q15. How did the authors quantify the mRNA levels of seladin-1/DHCR24?

To verify whether the modulation of the expression of seladin1/DHCR24 operated by 1.0 nM GnRH induces a variation of cell cholesterol the authors quantified this sterol by GC/MS in total cell lysates after incubating SH-SY5Y for 90 min, 3, 6, 24, 48 and 72 h.