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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

TGF-β induced Olfm2 nuclear accumulation. (A) TGF-β promoted the nuclear localization of Olfm2 in hES-MCs. Serum-starved hES-MCs were treated with vehicle or TGF-β (1 ng/ml) for 24 h. Immunostaining was performed to detect Olfm2 localization. 4′,6-Diamidino-2-phenylindole was used to stain nuclei. (B) TGF-β enhanced nuclear Olfm2 level. Cell lysates of hES-MCs treated with vehicle or TGF-β (24 h) were fractionated into nuclear and cytoplasmic portions and subjected to Western blot. Lamin B and α-tubulin were used as loading controls for the nuclear and cytoplasmic fractions, respectively. (C) Quantification of nuclear Olfm2 protein shown in B. *p < 0.01 compared with vehicle-treated group (n = 3).
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Figure 3: TGF-β induced Olfm2 nuclear accumulation. (A) TGF-β promoted the nuclear localization of Olfm2 in hES-MCs. Serum-starved hES-MCs were treated with vehicle or TGF-β (1 ng/ml) for 24 h. Immunostaining was performed to detect Olfm2 localization. 4′,6-Diamidino-2-phenylindole was used to stain nuclei. (B) TGF-β enhanced nuclear Olfm2 level. Cell lysates of hES-MCs treated with vehicle or TGF-β (24 h) were fractionated into nuclear and cytoplasmic portions and subjected to Western blot. Lamin B and α-tubulin were used as loading controls for the nuclear and cytoplasmic fractions, respectively. (C) Quantification of nuclear Olfm2 protein shown in B. *p < 0.01 compared with vehicle-treated group (n = 3).

Mentions: To investigate how Olfm2 regulates TGF-β–induced SM differentiation, we first detected the subcellular location of Olfm2. Olfm2 immunostaining showed that Olfm2 was located in both cytoplasm and nuclei of TGF-β–untreated hES-MCs. TGF-β stimulation appeared to cause a majority of Olfm2 to accumulate in the nuclei (Figure 3A), suggesting that Olfm2 may serve as a nuclear factor to regulate transcriptional activation of SM markers. To confirm the Olfm2 nuclear location, we fractionated hES-MC protein extracts into nuclear and cytoplasmic portions for Olfm2 Western blotting analysis. As shown in Figure 3, B and C, before TGF-β treatment, only 38% of Olfm2 was located in the nuclei. TGF-β induction, however, increased the nuclear Olfm2 to 81% of the total protein, consistent with the immunostaining results in Figure 3A. These results indicate that TGF-β induces Olfm2 translocation to nuclei of hES-MCs.


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)

TGF-β induced Olfm2 nuclear accumulation. (A) TGF-β promoted the nuclear localization of Olfm2 in hES-MCs. Serum-starved hES-MCs were treated with vehicle or TGF-β (1 ng/ml) for 24 h. Immunostaining was performed to detect Olfm2 localization. 4′,6-Diamidino-2-phenylindole was used to stain nuclei. (B) TGF-β enhanced nuclear Olfm2 level. Cell lysates of hES-MCs treated with vehicle or TGF-β (24 h) were fractionated into nuclear and cytoplasmic portions and subjected to Western blot. Lamin B and α-tubulin were used as loading controls for the nuclear and cytoplasmic fractions, respectively. (C) Quantification of nuclear Olfm2 protein shown in B. *p < 0.01 compared with vehicle-treated group (n = 3).
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Related In: Results  -  Collection

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Figure 3: TGF-β induced Olfm2 nuclear accumulation. (A) TGF-β promoted the nuclear localization of Olfm2 in hES-MCs. Serum-starved hES-MCs were treated with vehicle or TGF-β (1 ng/ml) for 24 h. Immunostaining was performed to detect Olfm2 localization. 4′,6-Diamidino-2-phenylindole was used to stain nuclei. (B) TGF-β enhanced nuclear Olfm2 level. Cell lysates of hES-MCs treated with vehicle or TGF-β (24 h) were fractionated into nuclear and cytoplasmic portions and subjected to Western blot. Lamin B and α-tubulin were used as loading controls for the nuclear and cytoplasmic fractions, respectively. (C) Quantification of nuclear Olfm2 protein shown in B. *p < 0.01 compared with vehicle-treated group (n = 3).
Mentions: To investigate how Olfm2 regulates TGF-β–induced SM differentiation, we first detected the subcellular location of Olfm2. Olfm2 immunostaining showed that Olfm2 was located in both cytoplasm and nuclei of TGF-β–untreated hES-MCs. TGF-β stimulation appeared to cause a majority of Olfm2 to accumulate in the nuclei (Figure 3A), suggesting that Olfm2 may serve as a nuclear factor to regulate transcriptional activation of SM markers. To confirm the Olfm2 nuclear location, we fractionated hES-MC protein extracts into nuclear and cytoplasmic portions for Olfm2 Western blotting analysis. As shown in Figure 3, B and C, before TGF-β treatment, only 38% of Olfm2 was located in the nuclei. TGF-β induction, however, increased the nuclear Olfm2 to 81% of the total protein, consistent with the immunostaining results in Figure 3A. These results indicate that TGF-β induces Olfm2 translocation to nuclei of hES-MCs.

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