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Pluripotency gene expression and growth control in cultures of peripheral blood monocytes during their conversion into programmable cells of monocytic origin (PCMO): evidence for a regulatory role of autocrine activin and TGF-β.

Ungefroren H, Hyder A, Hinz H, Groth S, Lange H, El-Sayed KM, Ehnert S, Nüssler AK, Fändrich F, Gieseler F - PLoS ONE (2015)

Bottom Line: Inhibition of autocrine activin signaling by either SB431542 or follistatin reduced both Smad2 activation and Oct4A/Nanog upregulation.Inhibition of autocrine TGF-β signaling by either SB431542 or anti-TGF-β antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells.Our data show that during PCMO generation pluripotency marker expression is controlled positively by activin/Smad2 and negatively by TGF-β/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-β/Smad3, and to a lesser extent, activin/Smad2 signaling.

View Article: PubMed Central - PubMed

Affiliation: Clinic for Applied Cellular Medicine, UKSH, Kiel, Germany.

ABSTRACT
Previous studies have shown that peripheral blood monocytes can be converted in vitro to a stem cell-like cell termed PCMO as evidenced by the re-expression of pluripotency-associated genes, transient proliferation, and the ability to adopt the phenotype of hepatocytes and insulin-producing cells upon tissue-specific differentiation. However, the regulatory interactions between cultured cells governing pluripotency and mitotic activity have remained elusive. Here we asked whether activin(s) and TGF-β(s), are involved in PCMO generation. De novo proliferation of PCMO was higher under adherent vs. suspended culture conditions as revealed by the appearance of a subset of Ki67-positive monocytes and correlated with down-regulation of p21WAF1 beyond day 2 of culture. Realtime-PCR analysis showed that PCMO express ActRIIA, ALK4, TβRII, ALK5 as well as TGF-β1 and the βA subunit of activin. Interestingly, expression of ActRIIA and ALK4, and activin A levels in the culture supernatants increased until day 4 of culture, while levels of total and active TGF-β1 strongly declined. PCMO responded to both growth factors in an autocrine fashion with intracellular signaling as evidenced by a rise in the levels of phospho-Smad2 and a drop in those of phospho-Smad3. Stimulation of PCMO with recombinant activins (A, B, AB) and TGF-β1 induced phosphorylation of Smad2 but not Smad3. Inhibition of autocrine activin signaling by either SB431542 or follistatin reduced both Smad2 activation and Oct4A/Nanog upregulation. Inhibition of autocrine TGF-β signaling by either SB431542 or anti-TGF-β antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells. Furthermore, anti-TGF-β antibody moderately enhanced Oct4A/Nanog expression. Our data show that during PCMO generation pluripotency marker expression is controlled positively by activin/Smad2 and negatively by TGF-β/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-β/Smad3, and to a lesser extent, activin/Smad2 signaling.

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Kinetics of Smad activation during PCMO culture.The activation state of Smad2 (left) and Smad3 (right) in adherent (a) and suspended (s) monocytes/PCMO was assessed at various time points by immunoblotting and ELISA (graphs below blots) for p-Smad2(Ser465/467) and p-Smad3(Ser423/425). Alpha-tubulin was used for normalization of p-Smad2 and p-Smad3 signal intensities from blots and for the respective ELISA data (means ± SD). The p-Smad3 blots had to be exposed for a much longer time than the p-Smad2 blots due to the low expression of Smad3. Numbers below the blots indicate the densitometric values for phospho-Smads normalized to those for α-tubulin. Data are representative of four different donors.
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pone.0118097.g004: Kinetics of Smad activation during PCMO culture.The activation state of Smad2 (left) and Smad3 (right) in adherent (a) and suspended (s) monocytes/PCMO was assessed at various time points by immunoblotting and ELISA (graphs below blots) for p-Smad2(Ser465/467) and p-Smad3(Ser423/425). Alpha-tubulin was used for normalization of p-Smad2 and p-Smad3 signal intensities from blots and for the respective ELISA data (means ± SD). The p-Smad3 blots had to be exposed for a much longer time than the p-Smad2 blots due to the low expression of Smad3. Numbers below the blots indicate the densitometric values for phospho-Smads normalized to those for α-tubulin. Data are representative of four different donors.

Mentions: Previous data from our group have shown that during conversion to PCMO the monocytes’ response towards exogenous TGF-β1 is changing with respect to Smad2 and Smad3 activation in such a way that they become sensitive to Smad2C and insensitive to Smad3C phosphorylation [32]. Given the presence of activin A and TGF-β1 in the culture supernatants and thus the existence of a self-stimulatory/autocrine signaling loop in these cells, we hypothesised that the sensitivity of PCMO to endogenous Smad-activating agents is altered in a similar way. In accordance with this assumption, we observed a strong and selective upregulation of phospho-Smad2C in adherent cells until day 4 as measured by both phosphoimmunoblotting and ELISA (Fig. 4, left panel). This increase in phospho-Smad2C may have been stimulated by endogenous activin secretion in combination with upregulation of activin receptor expression rather than TGF-β(s), the levels of which declined between day 2 and 4 (see above).


Pluripotency gene expression and growth control in cultures of peripheral blood monocytes during their conversion into programmable cells of monocytic origin (PCMO): evidence for a regulatory role of autocrine activin and TGF-β.

Ungefroren H, Hyder A, Hinz H, Groth S, Lange H, El-Sayed KM, Ehnert S, Nüssler AK, Fändrich F, Gieseler F - PLoS ONE (2015)

Kinetics of Smad activation during PCMO culture.The activation state of Smad2 (left) and Smad3 (right) in adherent (a) and suspended (s) monocytes/PCMO was assessed at various time points by immunoblotting and ELISA (graphs below blots) for p-Smad2(Ser465/467) and p-Smad3(Ser423/425). Alpha-tubulin was used for normalization of p-Smad2 and p-Smad3 signal intensities from blots and for the respective ELISA data (means ± SD). The p-Smad3 blots had to be exposed for a much longer time than the p-Smad2 blots due to the low expression of Smad3. Numbers below the blots indicate the densitometric values for phospho-Smads normalized to those for α-tubulin. Data are representative of four different donors.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4338298&req=5

pone.0118097.g004: Kinetics of Smad activation during PCMO culture.The activation state of Smad2 (left) and Smad3 (right) in adherent (a) and suspended (s) monocytes/PCMO was assessed at various time points by immunoblotting and ELISA (graphs below blots) for p-Smad2(Ser465/467) and p-Smad3(Ser423/425). Alpha-tubulin was used for normalization of p-Smad2 and p-Smad3 signal intensities from blots and for the respective ELISA data (means ± SD). The p-Smad3 blots had to be exposed for a much longer time than the p-Smad2 blots due to the low expression of Smad3. Numbers below the blots indicate the densitometric values for phospho-Smads normalized to those for α-tubulin. Data are representative of four different donors.
Mentions: Previous data from our group have shown that during conversion to PCMO the monocytes’ response towards exogenous TGF-β1 is changing with respect to Smad2 and Smad3 activation in such a way that they become sensitive to Smad2C and insensitive to Smad3C phosphorylation [32]. Given the presence of activin A and TGF-β1 in the culture supernatants and thus the existence of a self-stimulatory/autocrine signaling loop in these cells, we hypothesised that the sensitivity of PCMO to endogenous Smad-activating agents is altered in a similar way. In accordance with this assumption, we observed a strong and selective upregulation of phospho-Smad2C in adherent cells until day 4 as measured by both phosphoimmunoblotting and ELISA (Fig. 4, left panel). This increase in phospho-Smad2C may have been stimulated by endogenous activin secretion in combination with upregulation of activin receptor expression rather than TGF-β(s), the levels of which declined between day 2 and 4 (see above).

Bottom Line: Inhibition of autocrine activin signaling by either SB431542 or follistatin reduced both Smad2 activation and Oct4A/Nanog upregulation.Inhibition of autocrine TGF-β signaling by either SB431542 or anti-TGF-β antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells.Our data show that during PCMO generation pluripotency marker expression is controlled positively by activin/Smad2 and negatively by TGF-β/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-β/Smad3, and to a lesser extent, activin/Smad2 signaling.

View Article: PubMed Central - PubMed

Affiliation: Clinic for Applied Cellular Medicine, UKSH, Kiel, Germany.

ABSTRACT
Previous studies have shown that peripheral blood monocytes can be converted in vitro to a stem cell-like cell termed PCMO as evidenced by the re-expression of pluripotency-associated genes, transient proliferation, and the ability to adopt the phenotype of hepatocytes and insulin-producing cells upon tissue-specific differentiation. However, the regulatory interactions between cultured cells governing pluripotency and mitotic activity have remained elusive. Here we asked whether activin(s) and TGF-β(s), are involved in PCMO generation. De novo proliferation of PCMO was higher under adherent vs. suspended culture conditions as revealed by the appearance of a subset of Ki67-positive monocytes and correlated with down-regulation of p21WAF1 beyond day 2 of culture. Realtime-PCR analysis showed that PCMO express ActRIIA, ALK4, TβRII, ALK5 as well as TGF-β1 and the βA subunit of activin. Interestingly, expression of ActRIIA and ALK4, and activin A levels in the culture supernatants increased until day 4 of culture, while levels of total and active TGF-β1 strongly declined. PCMO responded to both growth factors in an autocrine fashion with intracellular signaling as evidenced by a rise in the levels of phospho-Smad2 and a drop in those of phospho-Smad3. Stimulation of PCMO with recombinant activins (A, B, AB) and TGF-β1 induced phosphorylation of Smad2 but not Smad3. Inhibition of autocrine activin signaling by either SB431542 or follistatin reduced both Smad2 activation and Oct4A/Nanog upregulation. Inhibition of autocrine TGF-β signaling by either SB431542 or anti-TGF-β antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells. Furthermore, anti-TGF-β antibody moderately enhanced Oct4A/Nanog expression. Our data show that during PCMO generation pluripotency marker expression is controlled positively by activin/Smad2 and negatively by TGF-β/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-β/Smad3, and to a lesser extent, activin/Smad2 signaling.

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Related in: MedlinePlus