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Differentiation-associated reprogramming of the transforming growth factor β receptor pathway establishes the circuitry for epithelial autocrine/paracrine repair.

Fleming JM, Shabir S, Varley CL, Kirkwood LA, White A, Holder J, Trejdosiewicz LK, Southgate J - PLoS ONE (2012)

Bottom Line: Exogenous TGFβ enhanced the repair and resulted in hyperplastic scarring, indicating a feedback loop implicit in an autocrine pathway.Our study demonstrates that the circuitry of the TGFβR pathway is defined transcriptionally within a tissue-specific differentiation programme.The findings provide evidence for re-evaluating the role of TGFβR signalling in epithelial homeostasis as an autocrine-regulated pathway that suppresses differentiation and promotes tissue repair.

View Article: PubMed Central - PubMed

Affiliation: Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York, United Kingdom.

ABSTRACT
Transforming growth factor (TGF) β has diverse and sometimes paradoxical effects on cell proliferation and differentiation, presumably reflecting a fundamental but incompletely-understood role in regulating tissue homeostasis. It is generally considered that downstream activity is modulated at the ligand:receptor axis, but microarray analysis of proliferative versus differentiating normal human bladder epithelial cell cultures identified unexpected transcriptional changes in key components of the canonical TGFβ R/activin signalling pathway associated with cytodifferentiation. Changes included upregulation of the transcriptional modulator SMAD3 and downregulation of inhibitory modulators SMURF2 and SMAD7. Functional analysis of the signalling pathway revealed that non-differentiated normal human urothelial cells responded in paracrine mode to TGFβ by growth inhibition, and that exogenous TGFβ inhibited rather than promoted differentiation. By contrast, in differentiated cell cultures, SMAD3 was activated upon scratch-wounding and was involved in promoting tissue repair. Exogenous TGFβ enhanced the repair and resulted in hyperplastic scarring, indicating a feedback loop implicit in an autocrine pathway. Thus, the machinery for autocrine activation of the SMAD3-mediated TGFβR pathway is established during urothelial differentiation, but signalling occurs only in response to a trigger, such as wounding. Our study demonstrates that the circuitry of the TGFβR pathway is defined transcriptionally within a tissue-specific differentiation programme. The findings provide evidence for re-evaluating the role of TGFβR signalling in epithelial homeostasis as an autocrine-regulated pathway that suppresses differentiation and promotes tissue repair. This provides a new paradigm to help unravel the apparently diverse and paradoxical effect of TGFβ signalling on cell proliferation and differentiation.

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Effect of differentiation on SMAD3 and pSMAD3 protein expression in NHU cells.(a) Protein lysates were prepared from NHU cells treated with 0.1% DMSO (control), TZ/PD or ABS/Ca2+ for 1, 3 and 6 days. Cell extracts (25 µg) were resolved on 4–12% Bis-Tris polyacrylamide gels and transferred onto PDVF membranes. Membranes were incubated with titrated primary antibodies for 16 h at 4°C to SMAD3 and pSMAD3, as indicated. Bound antibody was detected with Alexa Fluor® 680 and LI-COR IRDye™ 800 conjugated secondary antibodies and visualised using the Odyssey™ Imaging System. β-actin was used as an internal loading control. HeLa cells treated with TGFβ1 (2 ng/ml) for 24 h was used as a positive control. L is the molecular size ladder. (b) NHU cells were seeded at 500 cells/cm2 onto glass slides, allowed to adhere and treated with or without TZ/PD or ABS/Ca2+ for 6 days and fixed in formalin. Media were replaced every 3 days with fresh treatments. Indirect immunofluorescence was performed with anti-SMAD3 and anti-pSMAD 3 antibodies as indicated and detected with Alexa 594-conjugated secondary antibodies. Picture inserts show the respective Hoescht 33258 nuclear stain. Scale bar 90 µm.
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pone-0051404-g002: Effect of differentiation on SMAD3 and pSMAD3 protein expression in NHU cells.(a) Protein lysates were prepared from NHU cells treated with 0.1% DMSO (control), TZ/PD or ABS/Ca2+ for 1, 3 and 6 days. Cell extracts (25 µg) were resolved on 4–12% Bis-Tris polyacrylamide gels and transferred onto PDVF membranes. Membranes were incubated with titrated primary antibodies for 16 h at 4°C to SMAD3 and pSMAD3, as indicated. Bound antibody was detected with Alexa Fluor® 680 and LI-COR IRDye™ 800 conjugated secondary antibodies and visualised using the Odyssey™ Imaging System. β-actin was used as an internal loading control. HeLa cells treated with TGFβ1 (2 ng/ml) for 24 h was used as a positive control. L is the molecular size ladder. (b) NHU cells were seeded at 500 cells/cm2 onto glass slides, allowed to adhere and treated with or without TZ/PD or ABS/Ca2+ for 6 days and fixed in formalin. Media were replaced every 3 days with fresh treatments. Indirect immunofluorescence was performed with anti-SMAD3 and anti-pSMAD 3 antibodies as indicated and detected with Alexa 594-conjugated secondary antibodies. Picture inserts show the respective Hoescht 33258 nuclear stain. Scale bar 90 µm.

Mentions: To determine whether changes in SMAD3 transcript translated to changes in protein expression, differentiating NHU cell cultures were analysed by immunoblotting (Fig. 2A). Total SMAD3 protein was increased following both differentiation treatments. The relative amount of phosphorylated SMAD3 (pSMAD3) decreased in control cells from days 1–6 as cells reached confluence. Differentiated cells expressed very low amounts of pSMAD3 indicating that although basal SMAD3 expression increased, actual activity based on phosphorylation decreased, possibly as a reflection of other changes to the pathway (Table 2). These findings were confirmed by immunofluorescence (Fig. 2B): SMAD3 immunolabelling was most intense in differentiated cells, but pSMAD3 labelling was low or absent in the majority of cells in all culture conditions, with intense nuclear labelling observed in occasional cells only.


Differentiation-associated reprogramming of the transforming growth factor β receptor pathway establishes the circuitry for epithelial autocrine/paracrine repair.

Fleming JM, Shabir S, Varley CL, Kirkwood LA, White A, Holder J, Trejdosiewicz LK, Southgate J - PLoS ONE (2012)

Effect of differentiation on SMAD3 and pSMAD3 protein expression in NHU cells.(a) Protein lysates were prepared from NHU cells treated with 0.1% DMSO (control), TZ/PD or ABS/Ca2+ for 1, 3 and 6 days. Cell extracts (25 µg) were resolved on 4–12% Bis-Tris polyacrylamide gels and transferred onto PDVF membranes. Membranes were incubated with titrated primary antibodies for 16 h at 4°C to SMAD3 and pSMAD3, as indicated. Bound antibody was detected with Alexa Fluor® 680 and LI-COR IRDye™ 800 conjugated secondary antibodies and visualised using the Odyssey™ Imaging System. β-actin was used as an internal loading control. HeLa cells treated with TGFβ1 (2 ng/ml) for 24 h was used as a positive control. L is the molecular size ladder. (b) NHU cells were seeded at 500 cells/cm2 onto glass slides, allowed to adhere and treated with or without TZ/PD or ABS/Ca2+ for 6 days and fixed in formalin. Media were replaced every 3 days with fresh treatments. Indirect immunofluorescence was performed with anti-SMAD3 and anti-pSMAD 3 antibodies as indicated and detected with Alexa 594-conjugated secondary antibodies. Picture inserts show the respective Hoescht 33258 nuclear stain. Scale bar 90 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3526617&req=5

pone-0051404-g002: Effect of differentiation on SMAD3 and pSMAD3 protein expression in NHU cells.(a) Protein lysates were prepared from NHU cells treated with 0.1% DMSO (control), TZ/PD or ABS/Ca2+ for 1, 3 and 6 days. Cell extracts (25 µg) were resolved on 4–12% Bis-Tris polyacrylamide gels and transferred onto PDVF membranes. Membranes were incubated with titrated primary antibodies for 16 h at 4°C to SMAD3 and pSMAD3, as indicated. Bound antibody was detected with Alexa Fluor® 680 and LI-COR IRDye™ 800 conjugated secondary antibodies and visualised using the Odyssey™ Imaging System. β-actin was used as an internal loading control. HeLa cells treated with TGFβ1 (2 ng/ml) for 24 h was used as a positive control. L is the molecular size ladder. (b) NHU cells were seeded at 500 cells/cm2 onto glass slides, allowed to adhere and treated with or without TZ/PD or ABS/Ca2+ for 6 days and fixed in formalin. Media were replaced every 3 days with fresh treatments. Indirect immunofluorescence was performed with anti-SMAD3 and anti-pSMAD 3 antibodies as indicated and detected with Alexa 594-conjugated secondary antibodies. Picture inserts show the respective Hoescht 33258 nuclear stain. Scale bar 90 µm.
Mentions: To determine whether changes in SMAD3 transcript translated to changes in protein expression, differentiating NHU cell cultures were analysed by immunoblotting (Fig. 2A). Total SMAD3 protein was increased following both differentiation treatments. The relative amount of phosphorylated SMAD3 (pSMAD3) decreased in control cells from days 1–6 as cells reached confluence. Differentiated cells expressed very low amounts of pSMAD3 indicating that although basal SMAD3 expression increased, actual activity based on phosphorylation decreased, possibly as a reflection of other changes to the pathway (Table 2). These findings were confirmed by immunofluorescence (Fig. 2B): SMAD3 immunolabelling was most intense in differentiated cells, but pSMAD3 labelling was low or absent in the majority of cells in all culture conditions, with intense nuclear labelling observed in occasional cells only.

Bottom Line: Exogenous TGFβ enhanced the repair and resulted in hyperplastic scarring, indicating a feedback loop implicit in an autocrine pathway.Our study demonstrates that the circuitry of the TGFβR pathway is defined transcriptionally within a tissue-specific differentiation programme.The findings provide evidence for re-evaluating the role of TGFβR signalling in epithelial homeostasis as an autocrine-regulated pathway that suppresses differentiation and promotes tissue repair.

View Article: PubMed Central - PubMed

Affiliation: Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York, United Kingdom.

ABSTRACT
Transforming growth factor (TGF) β has diverse and sometimes paradoxical effects on cell proliferation and differentiation, presumably reflecting a fundamental but incompletely-understood role in regulating tissue homeostasis. It is generally considered that downstream activity is modulated at the ligand:receptor axis, but microarray analysis of proliferative versus differentiating normal human bladder epithelial cell cultures identified unexpected transcriptional changes in key components of the canonical TGFβ R/activin signalling pathway associated with cytodifferentiation. Changes included upregulation of the transcriptional modulator SMAD3 and downregulation of inhibitory modulators SMURF2 and SMAD7. Functional analysis of the signalling pathway revealed that non-differentiated normal human urothelial cells responded in paracrine mode to TGFβ by growth inhibition, and that exogenous TGFβ inhibited rather than promoted differentiation. By contrast, in differentiated cell cultures, SMAD3 was activated upon scratch-wounding and was involved in promoting tissue repair. Exogenous TGFβ enhanced the repair and resulted in hyperplastic scarring, indicating a feedback loop implicit in an autocrine pathway. Thus, the machinery for autocrine activation of the SMAD3-mediated TGFβR pathway is established during urothelial differentiation, but signalling occurs only in response to a trigger, such as wounding. Our study demonstrates that the circuitry of the TGFβR pathway is defined transcriptionally within a tissue-specific differentiation programme. The findings provide evidence for re-evaluating the role of TGFβR signalling in epithelial homeostasis as an autocrine-regulated pathway that suppresses differentiation and promotes tissue repair. This provides a new paradigm to help unravel the apparently diverse and paradoxical effect of TGFβ signalling on cell proliferation and differentiation.

Show MeSH
Related in: MedlinePlus