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Olfactomedin 2, a novel regulator for transforming growth factor-β-induced smooth muscle differentiation of human embryonic stem cell-derived mesenchymal cells.

Shi N, Guo X, Chen SY - Mol. Biol. Cell (2014)

Bottom Line: Olfm2 also inhibited HERP1 expression.Moreover, blockade of Olfm2 expression inhibited TGF-β-induced SRF binding to SM gene promoters in a chromatin setting, whereas overexpression of Olfm2 dose dependently enhanced SRF binding.These results demonstrate that Olfm2 mediates TGF-β-induced SM gene transcription by empowering SRF binding to CArG box in SM gene promoters.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, University of Georgia, Athens, GA 30602.

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Olfm2 inhibited HERP1 expression and its binding to SRF. (A, B) TGF-β suppressed HERP1 mRNA expression. Serum-starved hES-MCs were treated with TGF-β for the time indicated. qPCR was performed to examine Olfm2 (A) and HERP1 (B) mRNA expression. The mRNA expression was normalized to cyclophilin. *p < 0.01 compared with the vehicle-treated group (0 h). (C) Olfm2 inhibited HERP1 mRNA expression. hES-MCs were transfected with control or Olfm2 plasmid, followed by serum starvation for 24 h. qPCR was performed to detect HERP1 expression. *p < 0.01 compared with the control plasmid–transfected group. (D, E) Knockdown of Olfm2 attenuated TGF-β-induced down-regulation of HERP1. hES-MCs were transduced with Ad-GFP or Ad-shOlfm2 (shOlfm2), followed by vehicle (–) or TGF-β (+) treatment for 24 h. qPCR was performed to detect Olfm2 (D) and HERP1 (E) mRNA expression. *p < 0.01 compared with Ad-GFP–transduced group with vehicle treatment. #p < 0.01 compared with Ad-GFP-transduced group with TGF-β treatment. (F) Olfm2 was essential for TGF-β–inhibited HERP1 binding to SRF. hES-MCs were transduced with Ad-GFP or shOlfm2, followed by vehicle or TGF-β treatment for 24 h. Cell lysates were immunoprecipitated with normal IgG or SRF antibody. The immunoprecipitates were blotted (IB) with HERP1, Olfm2, and SRF antibody as indicated. (G) Quantification of HERP1 protein immunoprecipitated with SRF by normalized to α-tubulin as shown in F. *p < 0.01 compared with Ad-GFP-transduced and SRF-immunoprecipitated group with vehicle treatment; #p < 0.01 compared with Ad-GFP–transduced and SRF-immunoprecipitated group with TGF-β treatment (n = 3).
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Figure 7: Olfm2 inhibited HERP1 expression and its binding to SRF. (A, B) TGF-β suppressed HERP1 mRNA expression. Serum-starved hES-MCs were treated with TGF-β for the time indicated. qPCR was performed to examine Olfm2 (A) and HERP1 (B) mRNA expression. The mRNA expression was normalized to cyclophilin. *p < 0.01 compared with the vehicle-treated group (0 h). (C) Olfm2 inhibited HERP1 mRNA expression. hES-MCs were transfected with control or Olfm2 plasmid, followed by serum starvation for 24 h. qPCR was performed to detect HERP1 expression. *p < 0.01 compared with the control plasmid–transfected group. (D, E) Knockdown of Olfm2 attenuated TGF-β-induced down-regulation of HERP1. hES-MCs were transduced with Ad-GFP or Ad-shOlfm2 (shOlfm2), followed by vehicle (–) or TGF-β (+) treatment for 24 h. qPCR was performed to detect Olfm2 (D) and HERP1 (E) mRNA expression. *p < 0.01 compared with Ad-GFP–transduced group with vehicle treatment. #p < 0.01 compared with Ad-GFP-transduced group with TGF-β treatment. (F) Olfm2 was essential for TGF-β–inhibited HERP1 binding to SRF. hES-MCs were transduced with Ad-GFP or shOlfm2, followed by vehicle or TGF-β treatment for 24 h. Cell lysates were immunoprecipitated with normal IgG or SRF antibody. The immunoprecipitates were blotted (IB) with HERP1, Olfm2, and SRF antibody as indicated. (G) Quantification of HERP1 protein immunoprecipitated with SRF by normalized to α-tubulin as shown in F. *p < 0.01 compared with Ad-GFP-transduced and SRF-immunoprecipitated group with vehicle treatment; #p < 0.01 compared with Ad-GFP–transduced and SRF-immunoprecipitated group with TGF-β treatment (n = 3).

Mentions: HERP1 is a transcriptional repressor abundantly expressed in the developing vascular system (Nakagawa et al., 2000; Iso et al., 2001a, b). HERP1 has been shown to physically associate with SRF and inhibit SM differentiation by interfering with SRF binding to CArG box (Doi et al., 2005). Because both HERP1 and Olfm2 interact with SRF with opposite effects on SM differentiation, we hypothesize that TGF-β induction of Olfm2 promotes SM differentiation through inhibition of HERP1 expression or HERP1 binding to SRF. Indeed, TGF-β induced time-dependent suppression of HERP1 but up-regulated Olfm2 expression (Figure 7, A and B). Of importance, Olfm2 overexpression decreased HERP1 mRNA expression (Figure 7C), whereas knockdown of Olfm2 effectively attenuated TGF-β–induced blockade of HERP1 expression (Figure 7, D and E). These data suggest that Olfm2 mediated suppression of HERP1 by TGF-β. To determine whether Olfm2 affects HERP1 interaction with SRF, we performed CoIP assay in cells where Olfm2 expression was blocked. We found that TGF-β treatment inhibited HERP1-SRF interaction. However, knockdown of Olfm2 restored the HERP1 binding to SRF that was suppressed by TGF-β, indicating that Olfm2 mediated the TGF-β–induced dissociation of HERP1 from SRF (Figure 7, F and G). These data demonstrate that Olfm2 regulates TGF-β–induced SM differentiation by removing the inhibitory effect of HERP1, that is, releasing SRF from HERP1.


Olfactomedin 2, a novel regulator for transforming growth factor-β-induced smooth muscle differentiation of human embryonic stem cell-derived mesenchymal cells.

Shi N, Guo X, Chen SY - Mol. Biol. Cell (2014)

Olfm2 inhibited HERP1 expression and its binding to SRF. (A, B) TGF-β suppressed HERP1 mRNA expression. Serum-starved hES-MCs were treated with TGF-β for the time indicated. qPCR was performed to examine Olfm2 (A) and HERP1 (B) mRNA expression. The mRNA expression was normalized to cyclophilin. *p < 0.01 compared with the vehicle-treated group (0 h). (C) Olfm2 inhibited HERP1 mRNA expression. hES-MCs were transfected with control or Olfm2 plasmid, followed by serum starvation for 24 h. qPCR was performed to detect HERP1 expression. *p < 0.01 compared with the control plasmid–transfected group. (D, E) Knockdown of Olfm2 attenuated TGF-β-induced down-regulation of HERP1. hES-MCs were transduced with Ad-GFP or Ad-shOlfm2 (shOlfm2), followed by vehicle (–) or TGF-β (+) treatment for 24 h. qPCR was performed to detect Olfm2 (D) and HERP1 (E) mRNA expression. *p < 0.01 compared with Ad-GFP–transduced group with vehicle treatment. #p < 0.01 compared with Ad-GFP-transduced group with TGF-β treatment. (F) Olfm2 was essential for TGF-β–inhibited HERP1 binding to SRF. hES-MCs were transduced with Ad-GFP or shOlfm2, followed by vehicle or TGF-β treatment for 24 h. Cell lysates were immunoprecipitated with normal IgG or SRF antibody. The immunoprecipitates were blotted (IB) with HERP1, Olfm2, and SRF antibody as indicated. (G) Quantification of HERP1 protein immunoprecipitated with SRF by normalized to α-tubulin as shown in F. *p < 0.01 compared with Ad-GFP-transduced and SRF-immunoprecipitated group with vehicle treatment; #p < 0.01 compared with Ad-GFP–transduced and SRF-immunoprecipitated group with TGF-β treatment (n = 3).
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Figure 7: Olfm2 inhibited HERP1 expression and its binding to SRF. (A, B) TGF-β suppressed HERP1 mRNA expression. Serum-starved hES-MCs were treated with TGF-β for the time indicated. qPCR was performed to examine Olfm2 (A) and HERP1 (B) mRNA expression. The mRNA expression was normalized to cyclophilin. *p < 0.01 compared with the vehicle-treated group (0 h). (C) Olfm2 inhibited HERP1 mRNA expression. hES-MCs were transfected with control or Olfm2 plasmid, followed by serum starvation for 24 h. qPCR was performed to detect HERP1 expression. *p < 0.01 compared with the control plasmid–transfected group. (D, E) Knockdown of Olfm2 attenuated TGF-β-induced down-regulation of HERP1. hES-MCs were transduced with Ad-GFP or Ad-shOlfm2 (shOlfm2), followed by vehicle (–) or TGF-β (+) treatment for 24 h. qPCR was performed to detect Olfm2 (D) and HERP1 (E) mRNA expression. *p < 0.01 compared with Ad-GFP–transduced group with vehicle treatment. #p < 0.01 compared with Ad-GFP-transduced group with TGF-β treatment. (F) Olfm2 was essential for TGF-β–inhibited HERP1 binding to SRF. hES-MCs were transduced with Ad-GFP or shOlfm2, followed by vehicle or TGF-β treatment for 24 h. Cell lysates were immunoprecipitated with normal IgG or SRF antibody. The immunoprecipitates were blotted (IB) with HERP1, Olfm2, and SRF antibody as indicated. (G) Quantification of HERP1 protein immunoprecipitated with SRF by normalized to α-tubulin as shown in F. *p < 0.01 compared with Ad-GFP-transduced and SRF-immunoprecipitated group with vehicle treatment; #p < 0.01 compared with Ad-GFP–transduced and SRF-immunoprecipitated group with TGF-β treatment (n = 3).
Mentions: HERP1 is a transcriptional repressor abundantly expressed in the developing vascular system (Nakagawa et al., 2000; Iso et al., 2001a, b). HERP1 has been shown to physically associate with SRF and inhibit SM differentiation by interfering with SRF binding to CArG box (Doi et al., 2005). Because both HERP1 and Olfm2 interact with SRF with opposite effects on SM differentiation, we hypothesize that TGF-β induction of Olfm2 promotes SM differentiation through inhibition of HERP1 expression or HERP1 binding to SRF. Indeed, TGF-β induced time-dependent suppression of HERP1 but up-regulated Olfm2 expression (Figure 7, A and B). Of importance, Olfm2 overexpression decreased HERP1 mRNA expression (Figure 7C), whereas knockdown of Olfm2 effectively attenuated TGF-β–induced blockade of HERP1 expression (Figure 7, D and E). These data suggest that Olfm2 mediated suppression of HERP1 by TGF-β. To determine whether Olfm2 affects HERP1 interaction with SRF, we performed CoIP assay in cells where Olfm2 expression was blocked. We found that TGF-β treatment inhibited HERP1-SRF interaction. However, knockdown of Olfm2 restored the HERP1 binding to SRF that was suppressed by TGF-β, indicating that Olfm2 mediated the TGF-β–induced dissociation of HERP1 from SRF (Figure 7, F and G). These data demonstrate that Olfm2 regulates TGF-β–induced SM differentiation by removing the inhibitory effect of HERP1, that is, releasing SRF from HERP1.

Bottom Line: Olfm2 also inhibited HERP1 expression.Moreover, blockade of Olfm2 expression inhibited TGF-β-induced SRF binding to SM gene promoters in a chromatin setting, whereas overexpression of Olfm2 dose dependently enhanced SRF binding.These results demonstrate that Olfm2 mediates TGF-β-induced SM gene transcription by empowering SRF binding to CArG box in SM gene promoters.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, University of Georgia, Athens, GA 30602.

Show MeSH
Related in: MedlinePlus