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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.


Identification of the effector pathway downstream of mevalonate.(a) Cardiomyocyte differentiation in EBs treated with DMSO (control) or 10 μM ATV, zaragozic acid (ZA), GGTI-2133 (GGTI) or FTI-277 (FTI) during days 3–6. Results were analysed as in Fig. 1c. (b) Real-time PCR of T expression in EBs treated with DMSO or 10 μM ATV, ZA, GGTI or FTI during days 3–4 and collected on day 4. Results were analysed as in Fig. 1b. *P < 0.05, **P < 0.001. *Zaragonic acid increased T and Lhx1 expression, which might be due to upregulation of HMGCR levels on day 3 and 4 (Supplementary Figure 3e). (c) Real-time PCR of T levels in EBs treated with DMSO or 10 μM ATV, with/without 12.5 μM farnesol (FOH), during days 3–5 and collected on day 5. Results were analysed as in Fig. 1b. (d) Representative 2D-PAGE images of farnesylated proteins detected with biotin-phosphine from ES cells and EBs that were left untreated (left) or treated with 10 μM FTI-277 (right) and analysed on day 4. Results are representative of three cultures. Black arrowheads, ~20 kDa farnesylated proteins; white arrowhead, EB-specific farnesylated protein. (e) Immunostaining to detect the localisation of Flag-Myc-tagged Lamin B1 (WT) or Lamin B1 (C-S mutant) in ES cells. Nuclei were visualised by Hoechst 33342 staining. Scale bars, 5 μm. (f) Real-time PCR of the indicated genes in the Lamin B1 WT, KO and REV EBs. EBs were collected at the indicated times and analysed as in Fig. 1b. *P < 0.05.
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f4: Identification of the effector pathway downstream of mevalonate.(a) Cardiomyocyte differentiation in EBs treated with DMSO (control) or 10 μM ATV, zaragozic acid (ZA), GGTI-2133 (GGTI) or FTI-277 (FTI) during days 3–6. Results were analysed as in Fig. 1c. (b) Real-time PCR of T expression in EBs treated with DMSO or 10 μM ATV, ZA, GGTI or FTI during days 3–4 and collected on day 4. Results were analysed as in Fig. 1b. *P < 0.05, **P < 0.001. *Zaragonic acid increased T and Lhx1 expression, which might be due to upregulation of HMGCR levels on day 3 and 4 (Supplementary Figure 3e). (c) Real-time PCR of T levels in EBs treated with DMSO or 10 μM ATV, with/without 12.5 μM farnesol (FOH), during days 3–5 and collected on day 5. Results were analysed as in Fig. 1b. (d) Representative 2D-PAGE images of farnesylated proteins detected with biotin-phosphine from ES cells and EBs that were left untreated (left) or treated with 10 μM FTI-277 (right) and analysed on day 4. Results are representative of three cultures. Black arrowheads, ~20 kDa farnesylated proteins; white arrowhead, EB-specific farnesylated protein. (e) Immunostaining to detect the localisation of Flag-Myc-tagged Lamin B1 (WT) or Lamin B1 (C-S mutant) in ES cells. Nuclei were visualised by Hoechst 33342 staining. Scale bars, 5 μm. (f) Real-time PCR of the indicated genes in the Lamin B1 WT, KO and REV EBs. EBs were collected at the indicated times and analysed as in Fig. 1b. *P < 0.05.

Mentions: To identify the signalling pathways underlying statin-induced inhibition of primitive streak formation, we analysed the role of cholesterol, geranylgeranyl diphosphate and farnesyl diphosphate, which are all downstream of the mevalonate pathway (Supplementary Figure 3a). Similar to ATV treatment, a farnesyltransferase inhibitor (FTI-277) almost completely abolished cardiomyogenesis in EBs. In contrast, a squalene synthetase inhibitor (zaragozic acid), which blocks the biosynthesis of cholesterol, and a geranylgeranyltransferase inhibitor (GGTI-2133) did not suppress cardiomyogenesis (Fig. 4a, Supplementary Figure 3b and c). Consistent with the effect of ATV on primitive streak formation, real-time PCR confirmed that expression of the primitive streak genes T and Lhx1 were also markedly suppressed by FTI-277 (Fig. 4b, Supplementary Figure 3d). Moreover, FTI-277 inhibited primitive streak formation and induced Sox2 expression (ectodermal differentiation) in a dose-dependent manner (Supplementary Figure 3f). Farnesyltransferase is an enzyme transferring farnesyl diphosphate to the cysteines at the C-terminus of proteins. Importantly, farnesol rescued T expression in ATV-treated EBs, confirming the involvement of farnesyl diphosphate in primitive streak formation (Fig. 4c). These data indicate that the inhibition of farnesyl diphosphate inhibits primitive streak formation, similar to statin treatment.


The mevalonate pathway regulates primitive streak formation via protein farnesylation
Identification of the effector pathway downstream of mevalonate.(a) Cardiomyocyte differentiation in EBs treated with DMSO (control) or 10 μM ATV, zaragozic acid (ZA), GGTI-2133 (GGTI) or FTI-277 (FTI) during days 3–6. Results were analysed as in Fig. 1c. (b) Real-time PCR of T expression in EBs treated with DMSO or 10 μM ATV, ZA, GGTI or FTI during days 3–4 and collected on day 4. Results were analysed as in Fig. 1b. *P < 0.05, **P < 0.001. *Zaragonic acid increased T and Lhx1 expression, which might be due to upregulation of HMGCR levels on day 3 and 4 (Supplementary Figure 3e). (c) Real-time PCR of T levels in EBs treated with DMSO or 10 μM ATV, with/without 12.5 μM farnesol (FOH), during days 3–5 and collected on day 5. Results were analysed as in Fig. 1b. (d) Representative 2D-PAGE images of farnesylated proteins detected with biotin-phosphine from ES cells and EBs that were left untreated (left) or treated with 10 μM FTI-277 (right) and analysed on day 4. Results are representative of three cultures. Black arrowheads, ~20 kDa farnesylated proteins; white arrowhead, EB-specific farnesylated protein. (e) Immunostaining to detect the localisation of Flag-Myc-tagged Lamin B1 (WT) or Lamin B1 (C-S mutant) in ES cells. Nuclei were visualised by Hoechst 33342 staining. Scale bars, 5 μm. (f) Real-time PCR of the indicated genes in the Lamin B1 WT, KO and REV EBs. EBs were collected at the indicated times and analysed as in Fig. 1b. *P < 0.05.
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f4: Identification of the effector pathway downstream of mevalonate.(a) Cardiomyocyte differentiation in EBs treated with DMSO (control) or 10 μM ATV, zaragozic acid (ZA), GGTI-2133 (GGTI) or FTI-277 (FTI) during days 3–6. Results were analysed as in Fig. 1c. (b) Real-time PCR of T expression in EBs treated with DMSO or 10 μM ATV, ZA, GGTI or FTI during days 3–4 and collected on day 4. Results were analysed as in Fig. 1b. *P < 0.05, **P < 0.001. *Zaragonic acid increased T and Lhx1 expression, which might be due to upregulation of HMGCR levels on day 3 and 4 (Supplementary Figure 3e). (c) Real-time PCR of T levels in EBs treated with DMSO or 10 μM ATV, with/without 12.5 μM farnesol (FOH), during days 3–5 and collected on day 5. Results were analysed as in Fig. 1b. (d) Representative 2D-PAGE images of farnesylated proteins detected with biotin-phosphine from ES cells and EBs that were left untreated (left) or treated with 10 μM FTI-277 (right) and analysed on day 4. Results are representative of three cultures. Black arrowheads, ~20 kDa farnesylated proteins; white arrowhead, EB-specific farnesylated protein. (e) Immunostaining to detect the localisation of Flag-Myc-tagged Lamin B1 (WT) or Lamin B1 (C-S mutant) in ES cells. Nuclei were visualised by Hoechst 33342 staining. Scale bars, 5 μm. (f) Real-time PCR of the indicated genes in the Lamin B1 WT, KO and REV EBs. EBs were collected at the indicated times and analysed as in Fig. 1b. *P < 0.05.
Mentions: To identify the signalling pathways underlying statin-induced inhibition of primitive streak formation, we analysed the role of cholesterol, geranylgeranyl diphosphate and farnesyl diphosphate, which are all downstream of the mevalonate pathway (Supplementary Figure 3a). Similar to ATV treatment, a farnesyltransferase inhibitor (FTI-277) almost completely abolished cardiomyogenesis in EBs. In contrast, a squalene synthetase inhibitor (zaragozic acid), which blocks the biosynthesis of cholesterol, and a geranylgeranyltransferase inhibitor (GGTI-2133) did not suppress cardiomyogenesis (Fig. 4a, Supplementary Figure 3b and c). Consistent with the effect of ATV on primitive streak formation, real-time PCR confirmed that expression of the primitive streak genes T and Lhx1 were also markedly suppressed by FTI-277 (Fig. 4b, Supplementary Figure 3d). Moreover, FTI-277 inhibited primitive streak formation and induced Sox2 expression (ectodermal differentiation) in a dose-dependent manner (Supplementary Figure 3f). Farnesyltransferase is an enzyme transferring farnesyl diphosphate to the cysteines at the C-terminus of proteins. Importantly, farnesol rescued T expression in ATV-treated EBs, confirming the involvement of farnesyl diphosphate in primitive streak formation (Fig. 4c). These data indicate that the inhibition of farnesyl diphosphate inhibits primitive streak formation, similar to statin treatment.

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.