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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 did not affect the expression of SRF, Myocd, and their interaction. (A–C) Olfm2 did not alter SRF and Myocd mRNA expression. hES-MCs were transfected with control (Ctrl) or Olfm2 plasmid, followed by serum starvation for 24 h. qPCR was performed to detect the mRNA expression of Olfm2, SRF, and Myocd as indicated. (D) Olfm2 did not affect SRF-Myocd interaction. hES-MCs were cotransfected with control (−; 20 μg) or Olfm2 plasmid (+; 10–20 μg) and FLAG-Myocd plasmid (Myocd, 20 μg) in 10-cm dishes. Cell lysates were immunoprecipitated with normal IgG or SRF antibody. The immunoprecipitates were blotted (IB) with FLAG, Olfm2, or SRF antibody. (E) Quantification of Myocd protein coimmunoprecipitated with SRF by normalizing to Myocd input level shown in D. N.S., not significant.
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Figure 6: Olfm2 did not affect the expression of SRF, Myocd, and their interaction. (A–C) Olfm2 did not alter SRF and Myocd mRNA expression. hES-MCs were transfected with control (Ctrl) or Olfm2 plasmid, followed by serum starvation for 24 h. qPCR was performed to detect the mRNA expression of Olfm2, SRF, and Myocd as indicated. (D) Olfm2 did not affect SRF-Myocd interaction. hES-MCs were cotransfected with control (−; 20 μg) or Olfm2 plasmid (+; 10–20 μg) and FLAG-Myocd plasmid (Myocd, 20 μg) in 10-cm dishes. Cell lysates were immunoprecipitated with normal IgG or SRF antibody. The immunoprecipitates were blotted (IB) with FLAG, Olfm2, or SRF antibody. (E) Quantification of Myocd protein coimmunoprecipitated with SRF by normalizing to Myocd input level shown in D. N.S., not significant.

Mentions: Myocardin (Myocd) is a transcriptional coactivator of SRF, and Myocd-SRF interaction is crucial for the differentiation of SM lineage (Wang et al., 2001, 2002). Therefore we tested whether Olfm2 affects SRF and Myocd expression as well as their interaction. We found that the ectopic expression of Olfm2 had no effect on mRNA expression of either SRF or Myocd (Figure 6, A–C). To determine whether Olfm2 affects the interaction between SRF and Myocd, we transfected hES-MCs with FLAG-tagged Myocd expression plasmid and performed CoIP assays with endogenous SRF. As shown in Figure 6, D and E, Olfm2 did not alter the interaction between SRF and Myocd. These data suggest that mechanisms other than modulation of SRF/Myocd binding are involved in Olfm2-mediated 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 did not affect the expression of SRF, Myocd, and their interaction. (A–C) Olfm2 did not alter SRF and Myocd mRNA expression. hES-MCs were transfected with control (Ctrl) or Olfm2 plasmid, followed by serum starvation for 24 h. qPCR was performed to detect the mRNA expression of Olfm2, SRF, and Myocd as indicated. (D) Olfm2 did not affect SRF-Myocd interaction. hES-MCs were cotransfected with control (−; 20 μg) or Olfm2 plasmid (+; 10–20 μg) and FLAG-Myocd plasmid (Myocd, 20 μg) in 10-cm dishes. Cell lysates were immunoprecipitated with normal IgG or SRF antibody. The immunoprecipitates were blotted (IB) with FLAG, Olfm2, or SRF antibody. (E) Quantification of Myocd protein coimmunoprecipitated with SRF by normalizing to Myocd input level shown in D. N.S., not significant.
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Related In: Results  -  Collection

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Figure 6: Olfm2 did not affect the expression of SRF, Myocd, and their interaction. (A–C) Olfm2 did not alter SRF and Myocd mRNA expression. hES-MCs were transfected with control (Ctrl) or Olfm2 plasmid, followed by serum starvation for 24 h. qPCR was performed to detect the mRNA expression of Olfm2, SRF, and Myocd as indicated. (D) Olfm2 did not affect SRF-Myocd interaction. hES-MCs were cotransfected with control (−; 20 μg) or Olfm2 plasmid (+; 10–20 μg) and FLAG-Myocd plasmid (Myocd, 20 μg) in 10-cm dishes. Cell lysates were immunoprecipitated with normal IgG or SRF antibody. The immunoprecipitates were blotted (IB) with FLAG, Olfm2, or SRF antibody. (E) Quantification of Myocd protein coimmunoprecipitated with SRF by normalizing to Myocd input level shown in D. N.S., not significant.
Mentions: Myocardin (Myocd) is a transcriptional coactivator of SRF, and Myocd-SRF interaction is crucial for the differentiation of SM lineage (Wang et al., 2001, 2002). Therefore we tested whether Olfm2 affects SRF and Myocd expression as well as their interaction. We found that the ectopic expression of Olfm2 had no effect on mRNA expression of either SRF or Myocd (Figure 6, A–C). To determine whether Olfm2 affects the interaction between SRF and Myocd, we transfected hES-MCs with FLAG-tagged Myocd expression plasmid and performed CoIP assays with endogenous SRF. As shown in Figure 6, D and E, Olfm2 did not alter the interaction between SRF and Myocd. These data suggest that mechanisms other than modulation of SRF/Myocd binding are involved in Olfm2-mediated 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