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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 facilitated and promoted SRF binding to the promoters of SMC marker genes in a chromatin setting. (A–C) Knockdown of Olfm2 blocked TGF-β-induced SRF binding to SM22α and SMMHC promoters. hES-MCs were transduced with Ad-GFP or shOlfm2, followed by vehicle or TGF-β treatment (1 ng/ml) as indicated for 24 h. ChIP assay were performed to detect binding of SRF to CArG box in the SM22α and SMMHC promoters as indicated. Representative semiquantitative PCR (A) and qPCR (B, C) results. *p < 0.01 compared with the vehicle-treated groups; #p < 0.01 compared with Ad-GFP–transduced group treated with TGF-β for each corresponding gene. (D–F) Olfm2 enhanced SRF binding to CArG box in the SM22α and SMMHC promoters. hES-MCs were transfected with control (+; 20 μg) or Olfm2 plasmid (10–20 μg) in 10-cm cell culture dishes. ChIP assay was performed to detect binding of SRF to CArG box in the SM22α and SMMHC promoters as indicated. Representative semiquantitative PCR (D) and qPCR (E, F) results. *p < 0.01 compared with the control plasmid–transfected group for each corresponding gene (n = 3).
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Figure 8: Olfm2 facilitated and promoted SRF binding to the promoters of SMC marker genes in a chromatin setting. (A–C) Knockdown of Olfm2 blocked TGF-β-induced SRF binding to SM22α and SMMHC promoters. hES-MCs were transduced with Ad-GFP or shOlfm2, followed by vehicle or TGF-β treatment (1 ng/ml) as indicated for 24 h. ChIP assay were performed to detect binding of SRF to CArG box in the SM22α and SMMHC promoters as indicated. Representative semiquantitative PCR (A) and qPCR (B, C) results. *p < 0.01 compared with the vehicle-treated groups; #p < 0.01 compared with Ad-GFP–transduced group treated with TGF-β for each corresponding gene. (D–F) Olfm2 enhanced SRF binding to CArG box in the SM22α and SMMHC promoters. hES-MCs were transfected with control (+; 20 μg) or Olfm2 plasmid (10–20 μg) in 10-cm cell culture dishes. ChIP assay was performed to detect binding of SRF to CArG box in the SM22α and SMMHC promoters as indicated. Representative semiquantitative PCR (D) and qPCR (E, F) results. *p < 0.01 compared with the control plasmid–transfected group for each corresponding gene (n = 3).

Mentions: Because SRF binding to CArG box is crucial for SM gene transcription and Olfm2 mediated SRF release from HERP1, we sought to determine whether Olfm2 plays a role in SRF binding to SM marker gene promoter. Previous studies showed that SRF binding to CArG box is functionally important for SM22α promoter activity both in vitro and in vivo (Miano, 2003; McDonald et al., 2006). We thus performed chromatin immunoprecipitation (ChIP) assays to determine whether Olfm2 affects SRF binding to this CArG box. As shown in Figure 8A, SRF weakly bound to the CArG box of SM22α promoter in a basal state. TGF-β treatment significantly enhanced the binding (Figure 8, A and B), consistent with previous studies (Hautmann et al., 1999). Knockdown of Olfm2, however, significantly diminished TGF-β–enhanced SRF binding to the promoter (Figure 8, A and B), suggesting that Olfm2 is essential for SRF binding to SM marker promoter. Moreover, ectopic expression of Olfm2 in TGF-β–untreated cells dose dependently enhanced SRF binding to SM22α promoter (Figure 8, D and E). To determine whether this is a common mechanism for Olfm2 to regulate SRF targets in SM differentiation, we tested the Olfm2 effect on SRF binding to another SM promoter, SMMHC promoter, and observed similar results as with SM22α promoter (Figure 8, A–F). These data demonstrate that Olfm2 is a novel factor facilitating SRF binding to SM gene promoter, a key event for SM differentiation.


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 facilitated and promoted SRF binding to the promoters of SMC marker genes in a chromatin setting. (A–C) Knockdown of Olfm2 blocked TGF-β-induced SRF binding to SM22α and SMMHC promoters. hES-MCs were transduced with Ad-GFP or shOlfm2, followed by vehicle or TGF-β treatment (1 ng/ml) as indicated for 24 h. ChIP assay were performed to detect binding of SRF to CArG box in the SM22α and SMMHC promoters as indicated. Representative semiquantitative PCR (A) and qPCR (B, C) results. *p < 0.01 compared with the vehicle-treated groups; #p < 0.01 compared with Ad-GFP–transduced group treated with TGF-β for each corresponding gene. (D–F) Olfm2 enhanced SRF binding to CArG box in the SM22α and SMMHC promoters. hES-MCs were transfected with control (+; 20 μg) or Olfm2 plasmid (10–20 μg) in 10-cm cell culture dishes. ChIP assay was performed to detect binding of SRF to CArG box in the SM22α and SMMHC promoters as indicated. Representative semiquantitative PCR (D) and qPCR (E, F) results. *p < 0.01 compared with the control plasmid–transfected group for each corresponding gene (n = 3).
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Figure 8: Olfm2 facilitated and promoted SRF binding to the promoters of SMC marker genes in a chromatin setting. (A–C) Knockdown of Olfm2 blocked TGF-β-induced SRF binding to SM22α and SMMHC promoters. hES-MCs were transduced with Ad-GFP or shOlfm2, followed by vehicle or TGF-β treatment (1 ng/ml) as indicated for 24 h. ChIP assay were performed to detect binding of SRF to CArG box in the SM22α and SMMHC promoters as indicated. Representative semiquantitative PCR (A) and qPCR (B, C) results. *p < 0.01 compared with the vehicle-treated groups; #p < 0.01 compared with Ad-GFP–transduced group treated with TGF-β for each corresponding gene. (D–F) Olfm2 enhanced SRF binding to CArG box in the SM22α and SMMHC promoters. hES-MCs were transfected with control (+; 20 μg) or Olfm2 plasmid (10–20 μg) in 10-cm cell culture dishes. ChIP assay was performed to detect binding of SRF to CArG box in the SM22α and SMMHC promoters as indicated. Representative semiquantitative PCR (D) and qPCR (E, F) results. *p < 0.01 compared with the control plasmid–transfected group for each corresponding gene (n = 3).
Mentions: Because SRF binding to CArG box is crucial for SM gene transcription and Olfm2 mediated SRF release from HERP1, we sought to determine whether Olfm2 plays a role in SRF binding to SM marker gene promoter. Previous studies showed that SRF binding to CArG box is functionally important for SM22α promoter activity both in vitro and in vivo (Miano, 2003; McDonald et al., 2006). We thus performed chromatin immunoprecipitation (ChIP) assays to determine whether Olfm2 affects SRF binding to this CArG box. As shown in Figure 8A, SRF weakly bound to the CArG box of SM22α promoter in a basal state. TGF-β treatment significantly enhanced the binding (Figure 8, A and B), consistent with previous studies (Hautmann et al., 1999). Knockdown of Olfm2, however, significantly diminished TGF-β–enhanced SRF binding to the promoter (Figure 8, A and B), suggesting that Olfm2 is essential for SRF binding to SM marker promoter. Moreover, ectopic expression of Olfm2 in TGF-β–untreated cells dose dependently enhanced SRF binding to SM22α promoter (Figure 8, D and E). To determine whether this is a common mechanism for Olfm2 to regulate SRF targets in SM differentiation, we tested the Olfm2 effect on SRF binding to another SM promoter, SMMHC promoter, and observed similar results as with SM22α promoter (Figure 8, A–F). These data demonstrate that Olfm2 is a novel factor facilitating SRF binding to SM gene promoter, a key event for SM differentiation.

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