Limits...
The mevalonate pathway regulates primitive streak formation via protein farnesylation

View Article: PubMed Central - PubMed

ABSTRACT

The primitive streak in peri-implantation embryos forms the mesoderm and endoderm and controls cell differentiation. The metabolic cues regulating primitive streak formation remain largely unknown. Here we utilised a mouse embryonic stem (ES) cell differentiation system and a library of well-characterised drugs to identify these metabolic factors. We found that statins, which inhibit the mevalonate metabolic pathway, suppressed primitive streak formation in vitro and in vivo. Using metabolomics and pharmacologic approaches we identified the downstream signalling pathway of mevalonate and revealed that primitive streak formation requires protein farnesylation but not cholesterol synthesis. A tagging-via-substrate approach revealed that nuclear lamin B1 and small G proteins were farnesylated in embryoid bodies and important for primitive streak gene expression. In conclusion, protein farnesylation driven by the mevalonate pathway is a metabolic cue essential for primitive streak formation.

No MeSH data available.


Related in: MedlinePlus

Metabolomic analysis of statin-treated mouse EBs.(a) Heat map showing differences in the profiles of 147 metabolites in EBs that were treated with or without ATV during days 3–5 and subjected to metabolomic analysis on days 4 and 5. (b) Principal component analysis of the EBs in (a). Principal components 1 and 2 account for 36.9% and 29.5% of total variance, respectively. (c) Intracellular levels of the indicated metabolites in the EBs in (a). Data are expressed as the relative area of signal peaks corresponding to each metabolite and represent the mean ± SD (n = 3). (d) Intracellular levels of the indicated free FAs and acylcarnitines in the EBs in (a) analysed as in (c). (e) Intracellular cholesterol levels in the EBs in (a) analysed as in (c). *P < 0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5121603&req=5

f3: Metabolomic analysis of statin-treated mouse EBs.(a) Heat map showing differences in the profiles of 147 metabolites in EBs that were treated with or without ATV during days 3–5 and subjected to metabolomic analysis on days 4 and 5. (b) Principal component analysis of the EBs in (a). Principal components 1 and 2 account for 36.9% and 29.5% of total variance, respectively. (c) Intracellular levels of the indicated metabolites in the EBs in (a). Data are expressed as the relative area of signal peaks corresponding to each metabolite and represent the mean ± SD (n = 3). (d) Intracellular levels of the indicated free FAs and acylcarnitines in the EBs in (a) analysed as in (c). (e) Intracellular cholesterol levels in the EBs in (a) analysed as in (c). *P < 0.005.

Mentions: The mevalonate pathway is important for the biosynthesis of terpenes and steroids such as cholesterol56. Therefore, we performed a metabolomic analysis to determine the effect of ATV on intracellular metabolite composition during primitive streak formation in EBs. Control and ATV-treated EBs were collected on days 4 and 5, and 147 metabolites were identified followed by unsupervised clustering and heat map visualisation (Fig. 3a). ATV-treated EBs had a strikingly different metabolomic profile compared to control EBs; ATV specifically downregulated 46 metabolites and upregulated 37 metabolites on days 4 and 5. We performed principal component analysis to graphically visualise the relationship between ATV-treated and vehicle-treated EB populations and days of differentiation (Fig. 3b). The first principal component distinguished ATV-treated EBs from controls, while the second principal component characterised the differences in differentiation between days 4 and 5. Sphingomyelins (d18:1/16:0), sphingosine and sphingomyelins (d18:1/18:0) were the main contributors to the first principal component (Fig. 3c, Supplementary Table 3). Interestingly, the free fatty acids (FAs) palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) were increased following ATV treatment, whereas intermediates of FA biosynthesis from acetyl-CoA, namely palmitoylcarnitine (16:0), acylcarnitine (18:0) and acylcarnitine (18:1) were decreased (Fig. 3d). Statins also enhanced the expression of the FA synthase (FAS) gene (Supplementary Table 4), together suggesting that statins enhance lipogenesis but suppress the catabolic β-oxidation of FAs. It has been reported that FAS-dependent de novo synthesis of FA is enhanced in active neural stem and progenitor cells (NSPCs) and is crucial for NSPC proliferation89. These observations support the idea that a statin-dependent increase in free FA and FAS activity would promote neurogenesis. On the other hand, statins markedly decreased amino acids and dipeptides such as threonine, arginine and Gly-Asp (Supplementary Table 3), which might contribute to the inhibition of the primitive streak formation. Surprisingly, given the major role of statins in inhibiting cholesterol production, the intracellular cholesterol level in ATV-treated EBs was actually higher than in control EBs (Fig. 3e). Cholesterol homeostasis is maintained by a balance of de novo synthesis and incorporation10. Thus, the observed elevation in intracellular cholesterol might be due to enhanced expression of the low-density lipoprotein receptor (Supplementary Table 4), which takes up extracellular cholesterol and is induced by statins11. Together, our results indicate that the inhibition of statin-mediated primitive streak formation is not caused by a decrease in intracellular cholesterol but is associated with other metabolic anomalies.


The mevalonate pathway regulates primitive streak formation via protein farnesylation
Metabolomic analysis of statin-treated mouse EBs.(a) Heat map showing differences in the profiles of 147 metabolites in EBs that were treated with or without ATV during days 3–5 and subjected to metabolomic analysis on days 4 and 5. (b) Principal component analysis of the EBs in (a). Principal components 1 and 2 account for 36.9% and 29.5% of total variance, respectively. (c) Intracellular levels of the indicated metabolites in the EBs in (a). Data are expressed as the relative area of signal peaks corresponding to each metabolite and represent the mean ± SD (n = 3). (d) Intracellular levels of the indicated free FAs and acylcarnitines in the EBs in (a) analysed as in (c). (e) Intracellular cholesterol levels in the EBs in (a) analysed as in (c). *P < 0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121603&req=5

f3: Metabolomic analysis of statin-treated mouse EBs.(a) Heat map showing differences in the profiles of 147 metabolites in EBs that were treated with or without ATV during days 3–5 and subjected to metabolomic analysis on days 4 and 5. (b) Principal component analysis of the EBs in (a). Principal components 1 and 2 account for 36.9% and 29.5% of total variance, respectively. (c) Intracellular levels of the indicated metabolites in the EBs in (a). Data are expressed as the relative area of signal peaks corresponding to each metabolite and represent the mean ± SD (n = 3). (d) Intracellular levels of the indicated free FAs and acylcarnitines in the EBs in (a) analysed as in (c). (e) Intracellular cholesterol levels in the EBs in (a) analysed as in (c). *P < 0.005.
Mentions: The mevalonate pathway is important for the biosynthesis of terpenes and steroids such as cholesterol56. Therefore, we performed a metabolomic analysis to determine the effect of ATV on intracellular metabolite composition during primitive streak formation in EBs. Control and ATV-treated EBs were collected on days 4 and 5, and 147 metabolites were identified followed by unsupervised clustering and heat map visualisation (Fig. 3a). ATV-treated EBs had a strikingly different metabolomic profile compared to control EBs; ATV specifically downregulated 46 metabolites and upregulated 37 metabolites on days 4 and 5. We performed principal component analysis to graphically visualise the relationship between ATV-treated and vehicle-treated EB populations and days of differentiation (Fig. 3b). The first principal component distinguished ATV-treated EBs from controls, while the second principal component characterised the differences in differentiation between days 4 and 5. Sphingomyelins (d18:1/16:0), sphingosine and sphingomyelins (d18:1/18:0) were the main contributors to the first principal component (Fig. 3c, Supplementary Table 3). Interestingly, the free fatty acids (FAs) palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) were increased following ATV treatment, whereas intermediates of FA biosynthesis from acetyl-CoA, namely palmitoylcarnitine (16:0), acylcarnitine (18:0) and acylcarnitine (18:1) were decreased (Fig. 3d). Statins also enhanced the expression of the FA synthase (FAS) gene (Supplementary Table 4), together suggesting that statins enhance lipogenesis but suppress the catabolic β-oxidation of FAs. It has been reported that FAS-dependent de novo synthesis of FA is enhanced in active neural stem and progenitor cells (NSPCs) and is crucial for NSPC proliferation89. These observations support the idea that a statin-dependent increase in free FA and FAS activity would promote neurogenesis. On the other hand, statins markedly decreased amino acids and dipeptides such as threonine, arginine and Gly-Asp (Supplementary Table 3), which might contribute to the inhibition of the primitive streak formation. Surprisingly, given the major role of statins in inhibiting cholesterol production, the intracellular cholesterol level in ATV-treated EBs was actually higher than in control EBs (Fig. 3e). Cholesterol homeostasis is maintained by a balance of de novo synthesis and incorporation10. Thus, the observed elevation in intracellular cholesterol might be due to enhanced expression of the low-density lipoprotein receptor (Supplementary Table 4), which takes up extracellular cholesterol and is induced by statins11. Together, our results indicate that the inhibition of statin-mediated primitive streak formation is not caused by a decrease in intracellular cholesterol but is associated with other metabolic anomalies.

View Article: PubMed Central - PubMed

ABSTRACT

The primitive streak in peri-implantation embryos forms the mesoderm and endoderm and controls cell differentiation. The metabolic cues regulating primitive streak formation remain largely unknown. Here we utilised a mouse embryonic stem (ES) cell differentiation system and a library of well-characterised drugs to identify these metabolic factors. We found that statins, which inhibit the mevalonate metabolic pathway, suppressed primitive streak formation in vitro and in vivo. Using metabolomics and pharmacologic approaches we identified the downstream signalling pathway of mevalonate and revealed that primitive streak formation requires protein farnesylation but not cholesterol synthesis. A tagging-via-substrate approach revealed that nuclear lamin B1 and small G proteins were farnesylated in embryoid bodies and important for primitive streak gene expression. In conclusion, protein farnesylation driven by the mevalonate pathway is a metabolic cue essential for primitive streak formation.

No MeSH data available.


Related in: MedlinePlus