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Regulation of adipose tissue stromal cells behaviors by endogenic Oct4 expression control.

Kim JH, Jee MK, Lee SY, Han TH, Kim BS, Kang KS, Kang SK - PLoS ONE (2009)

Bottom Line: Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage.Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1.The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Biotechnology, Seoul National University, Seoul, Republic of Korea.

ABSTRACT

Background: To clarify the role of the POU domain transcription factor Oct4 in Adipose Tissue Stromal Cells (ATSCs), we investigated the regulation of Oct4 expression and other embryonic genes in fully differentiated cells, in addition to identifying expression at the gene and protein levels. The ATSCs and several immature cells were routinely expressing Oct4 protein before and after differentiating into specific lineages.

Methodology/principal findings and conclusions: Here, we demonstrated the role of Oct4 in ATSCs on cell proliferation and differentiation. Exogenous Oct4 improves adult ATSCs cell proliferation and differentiation potencies through epigenetic reprogramming of stemness genes such as Oct4, Nanog, Sox2, and Rex1. Oct4 directly or indirectly induces ATSCs reprogramming along with the activation of JAK/STAT3 and ERK1/2. Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage. Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1. The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes. Finally we have demonstrated the therapeutic utility of Oct4/ATSCs were introduced into the mouse traumatic brain, engrafted cells was more effectively induces regeneration activity with high therapeutic modality than that of control ATSCs. Engrafted Oct4/ATSCs efficiently migrated and transdifferentiated into action potential carrying, functionally neurons in the hippocampus and promoting the amelioration of lesion cavities.

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Exogenic Oct4 induced cell proliferation and survival related signal proteins expression in Oct4/ATSCs.(A) The involvement of JAK/STAT3, ERK1/2, MEK, and inhibition and activation resulted in prominent cell growth attenuation. Oct4 downregulation prominently induces inactivation of JAK/STAT3, AKT, MAPK, mTOR, and c-myc. When we treated with PI3K and ERK1/2 inhibitors, associated cell growth signal mediators and cell proliferation was decreased profoundly. For Western blotting, equal amounts of protein extracts were subjected to 10% SDS-PAGE analysis and transferred to nitrocellulose membranes. Optimally diluted antibodies were incubated with the membranes. The relative band intensities were determined using Quality-one 1-D Analysis software (B) Inhibition of Oct4 expression induced P38, SAPK/JUNK and mitochondria involving apoptotic ATSC death. Treatment of siOct4 actively induced apoptotic mediators such as caspases 8 and 9 and prominently increased apoptotic cell death along with highly increased Bax, PARP, and Cytochrome C expression or activation and Bcl2 downregulation.
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pone-0007166-g002: Exogenic Oct4 induced cell proliferation and survival related signal proteins expression in Oct4/ATSCs.(A) The involvement of JAK/STAT3, ERK1/2, MEK, and inhibition and activation resulted in prominent cell growth attenuation. Oct4 downregulation prominently induces inactivation of JAK/STAT3, AKT, MAPK, mTOR, and c-myc. When we treated with PI3K and ERK1/2 inhibitors, associated cell growth signal mediators and cell proliferation was decreased profoundly. For Western blotting, equal amounts of protein extracts were subjected to 10% SDS-PAGE analysis and transferred to nitrocellulose membranes. Optimally diluted antibodies were incubated with the membranes. The relative band intensities were determined using Quality-one 1-D Analysis software (B) Inhibition of Oct4 expression induced P38, SAPK/JUNK and mitochondria involving apoptotic ATSC death. Treatment of siOct4 actively induced apoptotic mediators such as caspases 8 and 9 and prominently increased apoptotic cell death along with highly increased Bax, PARP, and Cytochrome C expression or activation and Bcl2 downregulation.

Mentions: The control ATSCs underwent a progressive reduction in proliferation potential, and finally underwent senescence after passage 25–30 (78–90 days in culture). As shown in Figure 2, after 3 days of in vitro culture, the Oct4/ATSCs expressed several stemness genes with extended cell growth. And Oct4/ATSCs overexpressed the oncogenic gene, c-myc with prominently extended the S phase in cell cycles (Fig. 1C). Oct4-overexpressing ATSCs induced a 1.5-fold increase in colony formation with increased synthetic DNA and telomerase activity along with slightly extended telomere lengths (Fig. 1D). The Oct4-overexpressed ATSC cells showed prominent effects on upregulation of a variety of proliferation-associated genes, including RUNX3, CDK2 and CDK4, and telomere reverse transcriptase (TERT; Fig. 1D). We evaluated the expression of Oct4 (POU5F1) to determine whether exogenic Oct4 induced the expression of early developmental genes KLF4, Sox-2, Rex-1, Utf1, Dapp5, FGF4, ERas, and Nanog in cultured ATSCs (Fig. 1E). Moreover, most of the Oct4 target genes were upregulated including Rex1, Nanog, and Sox2 (Fig. 1E). When Oct4/ATSCs were engrafted in the postnatal mouse brain, engrafted cells showed a strong tumorigenic potency in vivo (Fig. 1F). Engrafted Oct4/ATSCs presented highly neurogenic behavior in the fetal mouse brain 2 months after engraftment (Fig. 1F). Moreover, engrafted Oct4/ATSC cells in SCID/NOD mice formed the traditional teratoma morphology after 2–3 (n = 4) months. Oct4/ATSC-derived teratoma tissue developed into the three germs layers that tissues or organs such as endocrine gland tissue, muscle, and neural cells (Fig. 1F).


Regulation of adipose tissue stromal cells behaviors by endogenic Oct4 expression control.

Kim JH, Jee MK, Lee SY, Han TH, Kim BS, Kang KS, Kang SK - PLoS ONE (2009)

Exogenic Oct4 induced cell proliferation and survival related signal proteins expression in Oct4/ATSCs.(A) The involvement of JAK/STAT3, ERK1/2, MEK, and inhibition and activation resulted in prominent cell growth attenuation. Oct4 downregulation prominently induces inactivation of JAK/STAT3, AKT, MAPK, mTOR, and c-myc. When we treated with PI3K and ERK1/2 inhibitors, associated cell growth signal mediators and cell proliferation was decreased profoundly. For Western blotting, equal amounts of protein extracts were subjected to 10% SDS-PAGE analysis and transferred to nitrocellulose membranes. Optimally diluted antibodies were incubated with the membranes. The relative band intensities were determined using Quality-one 1-D Analysis software (B) Inhibition of Oct4 expression induced P38, SAPK/JUNK and mitochondria involving apoptotic ATSC death. Treatment of siOct4 actively induced apoptotic mediators such as caspases 8 and 9 and prominently increased apoptotic cell death along with highly increased Bax, PARP, and Cytochrome C expression or activation and Bcl2 downregulation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747014&req=5

pone-0007166-g002: Exogenic Oct4 induced cell proliferation and survival related signal proteins expression in Oct4/ATSCs.(A) The involvement of JAK/STAT3, ERK1/2, MEK, and inhibition and activation resulted in prominent cell growth attenuation. Oct4 downregulation prominently induces inactivation of JAK/STAT3, AKT, MAPK, mTOR, and c-myc. When we treated with PI3K and ERK1/2 inhibitors, associated cell growth signal mediators and cell proliferation was decreased profoundly. For Western blotting, equal amounts of protein extracts were subjected to 10% SDS-PAGE analysis and transferred to nitrocellulose membranes. Optimally diluted antibodies were incubated with the membranes. The relative band intensities were determined using Quality-one 1-D Analysis software (B) Inhibition of Oct4 expression induced P38, SAPK/JUNK and mitochondria involving apoptotic ATSC death. Treatment of siOct4 actively induced apoptotic mediators such as caspases 8 and 9 and prominently increased apoptotic cell death along with highly increased Bax, PARP, and Cytochrome C expression or activation and Bcl2 downregulation.
Mentions: The control ATSCs underwent a progressive reduction in proliferation potential, and finally underwent senescence after passage 25–30 (78–90 days in culture). As shown in Figure 2, after 3 days of in vitro culture, the Oct4/ATSCs expressed several stemness genes with extended cell growth. And Oct4/ATSCs overexpressed the oncogenic gene, c-myc with prominently extended the S phase in cell cycles (Fig. 1C). Oct4-overexpressing ATSCs induced a 1.5-fold increase in colony formation with increased synthetic DNA and telomerase activity along with slightly extended telomere lengths (Fig. 1D). The Oct4-overexpressed ATSC cells showed prominent effects on upregulation of a variety of proliferation-associated genes, including RUNX3, CDK2 and CDK4, and telomere reverse transcriptase (TERT; Fig. 1D). We evaluated the expression of Oct4 (POU5F1) to determine whether exogenic Oct4 induced the expression of early developmental genes KLF4, Sox-2, Rex-1, Utf1, Dapp5, FGF4, ERas, and Nanog in cultured ATSCs (Fig. 1E). Moreover, most of the Oct4 target genes were upregulated including Rex1, Nanog, and Sox2 (Fig. 1E). When Oct4/ATSCs were engrafted in the postnatal mouse brain, engrafted cells showed a strong tumorigenic potency in vivo (Fig. 1F). Engrafted Oct4/ATSCs presented highly neurogenic behavior in the fetal mouse brain 2 months after engraftment (Fig. 1F). Moreover, engrafted Oct4/ATSC cells in SCID/NOD mice formed the traditional teratoma morphology after 2–3 (n = 4) months. Oct4/ATSC-derived teratoma tissue developed into the three germs layers that tissues or organs such as endocrine gland tissue, muscle, and neural cells (Fig. 1F).

Bottom Line: Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage.Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1.The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Biotechnology, Seoul National University, Seoul, Republic of Korea.

ABSTRACT

Background: To clarify the role of the POU domain transcription factor Oct4 in Adipose Tissue Stromal Cells (ATSCs), we investigated the regulation of Oct4 expression and other embryonic genes in fully differentiated cells, in addition to identifying expression at the gene and protein levels. The ATSCs and several immature cells were routinely expressing Oct4 protein before and after differentiating into specific lineages.

Methodology/principal findings and conclusions: Here, we demonstrated the role of Oct4 in ATSCs on cell proliferation and differentiation. Exogenous Oct4 improves adult ATSCs cell proliferation and differentiation potencies through epigenetic reprogramming of stemness genes such as Oct4, Nanog, Sox2, and Rex1. Oct4 directly or indirectly induces ATSCs reprogramming along with the activation of JAK/STAT3 and ERK1/2. Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage. Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1. The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes. Finally we have demonstrated the therapeutic utility of Oct4/ATSCs were introduced into the mouse traumatic brain, engrafted cells was more effectively induces regeneration activity with high therapeutic modality than that of control ATSCs. Engrafted Oct4/ATSCs efficiently migrated and transdifferentiated into action potential carrying, functionally neurons in the hippocampus and promoting the amelioration of lesion cavities.

Show MeSH
Related in: MedlinePlus