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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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Related in: MedlinePlus

Olfm2 induced SM marker expression in an SRF/CArG-dependent manner. (A) Knockdown of SRF blocked Olfm2-induced SM marker expression. hES-MCs were cotransfected with control (–) or Olfm2 plasmid and control (shCtrl) or SRF shRNA (shSRF) plasmid as indicated, followed by serum starvation for 24 h. Western blotting was performed to detect the expression of proteins indicated. (B–E) Quantification of protein expression shown in A by normalizing to α-tubulin. *p < 0.01 compared with shCtrl-transfected group without Olfm2. #p < 0.01 compared with Olfm2-transfected group with shCtrl (n = 3). (F, G) CArG box mutation diminished the SM marker promoter activity activated by Olfm2. (F) α-SMA promoter (from −2.6 to +2.8 kb) constructs with wild-type (WT) or mutant CArG box either in the first intron (CArGm intron) or in the promoter region (CArGm (A+B)) were cotransfected with the control (Ctrl) or Olfm2 plasmid into hES-MCs for 48 h. (G) SM22α promoter constructs with wild-type (WT) or mutant CArG box in the two SRF-binding sites (CArGmnear or CArGmfar) were cotransfected with the control or Olfm2 plasmid into hES-MCs for 48 h. Luciferase assay was performed. Fold induction of promoter activity was calculated relative to the WT promoter cotransfected with the control plasmid (set as 1). *p < 0.01 compared with control plasmid–transfected WT promoter. #p < 0.01 compared with Olfm2 plasmid–transfected WT promoter (n = 3).
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Figure 4: Olfm2 induced SM marker expression in an SRF/CArG-dependent manner. (A) Knockdown of SRF blocked Olfm2-induced SM marker expression. hES-MCs were cotransfected with control (–) or Olfm2 plasmid and control (shCtrl) or SRF shRNA (shSRF) plasmid as indicated, followed by serum starvation for 24 h. Western blotting was performed to detect the expression of proteins indicated. (B–E) Quantification of protein expression shown in A by normalizing to α-tubulin. *p < 0.01 compared with shCtrl-transfected group without Olfm2. #p < 0.01 compared with Olfm2-transfected group with shCtrl (n = 3). (F, G) CArG box mutation diminished the SM marker promoter activity activated by Olfm2. (F) α-SMA promoter (from −2.6 to +2.8 kb) constructs with wild-type (WT) or mutant CArG box either in the first intron (CArGm intron) or in the promoter region (CArGm (A+B)) were cotransfected with the control (Ctrl) or Olfm2 plasmid into hES-MCs for 48 h. (G) SM22α promoter constructs with wild-type (WT) or mutant CArG box in the two SRF-binding sites (CArGmnear or CArGmfar) were cotransfected with the control or Olfm2 plasmid into hES-MCs for 48 h. Luciferase assay was performed. Fold induction of promoter activity was calculated relative to the WT promoter cotransfected with the control plasmid (set as 1). *p < 0.01 compared with control plasmid–transfected WT promoter. #p < 0.01 compared with Olfm2 plasmid–transfected WT promoter (n = 3).

Mentions: Although Olfm2 is a nuclear factor likely involving the SM gene transcription, analysis of Olfm2 protein structure using DNA-Binder software revealed that Olfm2 lacks the DNA-binding domain, suggesting that Olfm2 may serve as a coactivator for a key transcription factor that regulates SM differentiation. SRF is a key nuclear transcription factor regulating the expression of most SM marker genes by binding to highly conserved CArG box present within nearly all SM-specific promoters (Miano, 2003; Mack, 2011). Because Olfm2 is critical for SM marker expression, we sought to determine whether Olfm2 can induce SM marker expression in the absence of SRF. As shown in Figure 4, A–E, knockdown of SRF by shRNA blocked Olfm2-induced SM marker expression, suggesting that SRF was essential for Olfm2-induced SM differentiation. Moreover, CArG box mutations significantly inhibited Olfm2 induction of α-SMA and SM22α promoter activity (Figure 4, F and G), suggesting that CArG box is indispensable for Olfm2 function. These data demonstrate that Olfm2 mediates SM differentiation in a SRF/CArG-dependent manner.


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 induced SM marker expression in an SRF/CArG-dependent manner. (A) Knockdown of SRF blocked Olfm2-induced SM marker expression. hES-MCs were cotransfected with control (–) or Olfm2 plasmid and control (shCtrl) or SRF shRNA (shSRF) plasmid as indicated, followed by serum starvation for 24 h. Western blotting was performed to detect the expression of proteins indicated. (B–E) Quantification of protein expression shown in A by normalizing to α-tubulin. *p < 0.01 compared with shCtrl-transfected group without Olfm2. #p < 0.01 compared with Olfm2-transfected group with shCtrl (n = 3). (F, G) CArG box mutation diminished the SM marker promoter activity activated by Olfm2. (F) α-SMA promoter (from −2.6 to +2.8 kb) constructs with wild-type (WT) or mutant CArG box either in the first intron (CArGm intron) or in the promoter region (CArGm (A+B)) were cotransfected with the control (Ctrl) or Olfm2 plasmid into hES-MCs for 48 h. (G) SM22α promoter constructs with wild-type (WT) or mutant CArG box in the two SRF-binding sites (CArGmnear or CArGmfar) were cotransfected with the control or Olfm2 plasmid into hES-MCs for 48 h. Luciferase assay was performed. Fold induction of promoter activity was calculated relative to the WT promoter cotransfected with the control plasmid (set as 1). *p < 0.01 compared with control plasmid–transfected WT promoter. #p < 0.01 compared with Olfm2 plasmid–transfected WT promoter (n = 3).
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Figure 4: Olfm2 induced SM marker expression in an SRF/CArG-dependent manner. (A) Knockdown of SRF blocked Olfm2-induced SM marker expression. hES-MCs were cotransfected with control (–) or Olfm2 plasmid and control (shCtrl) or SRF shRNA (shSRF) plasmid as indicated, followed by serum starvation for 24 h. Western blotting was performed to detect the expression of proteins indicated. (B–E) Quantification of protein expression shown in A by normalizing to α-tubulin. *p < 0.01 compared with shCtrl-transfected group without Olfm2. #p < 0.01 compared with Olfm2-transfected group with shCtrl (n = 3). (F, G) CArG box mutation diminished the SM marker promoter activity activated by Olfm2. (F) α-SMA promoter (from −2.6 to +2.8 kb) constructs with wild-type (WT) or mutant CArG box either in the first intron (CArGm intron) or in the promoter region (CArGm (A+B)) were cotransfected with the control (Ctrl) or Olfm2 plasmid into hES-MCs for 48 h. (G) SM22α promoter constructs with wild-type (WT) or mutant CArG box in the two SRF-binding sites (CArGmnear or CArGmfar) were cotransfected with the control or Olfm2 plasmid into hES-MCs for 48 h. Luciferase assay was performed. Fold induction of promoter activity was calculated relative to the WT promoter cotransfected with the control plasmid (set as 1). *p < 0.01 compared with control plasmid–transfected WT promoter. #p < 0.01 compared with Olfm2 plasmid–transfected WT promoter (n = 3).
Mentions: Although Olfm2 is a nuclear factor likely involving the SM gene transcription, analysis of Olfm2 protein structure using DNA-Binder software revealed that Olfm2 lacks the DNA-binding domain, suggesting that Olfm2 may serve as a coactivator for a key transcription factor that regulates SM differentiation. SRF is a key nuclear transcription factor regulating the expression of most SM marker genes by binding to highly conserved CArG box present within nearly all SM-specific promoters (Miano, 2003; Mack, 2011). Because Olfm2 is critical for SM marker expression, we sought to determine whether Olfm2 can induce SM marker expression in the absence of SRF. As shown in Figure 4, A–E, knockdown of SRF by shRNA blocked Olfm2-induced SM marker expression, suggesting that SRF was essential for Olfm2-induced SM differentiation. Moreover, CArG box mutations significantly inhibited Olfm2 induction of α-SMA and SM22α promoter activity (Figure 4, F and G), suggesting that CArG box is indispensable for Olfm2 function. These data demonstrate that Olfm2 mediates SM differentiation in a SRF/CArG-dependent manner.

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