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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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Effects of TGFβ on differentiation, barrier function and scratch wound repair in NHU cell cultures.(a) NHU cells were differentiated with or without TZ/PD or ABS/Ca2+ in the presence or absence of TGFβ; UPK2 expression was assessed by RTqPCR after 4 days and normalised against GAPDH. Controls included SB431542, vehicle (0.1% DMSO), PD153035 and TZ alone. Bars represent means ± SD of triplicate PCR determinations. (b) Barrier function was assessed by TER measurement after 7 days culture of NHU cells seeded at 0.5×106 cells/cm2 onto SnapWell™ membranes and treated with or without ABS/Ca2+ in the presence of DMSO (0.1%), TGFβ1 (2 ng/ml) or SB431542 (3 µM). Bars represent mean ± SD of 3 independent replicate cultures. (c–g) After 7 days pre-culture with or without ABS/Ca2+ to induce differentiation, confluent cultures were incubated with DMSO (0.1%, vehicle control), TGFβ1 (2 ng/ml), SB431542 (10 µM) or PD153035 (1 µM) for 3 h and then scratch-wounded. Cultures were maintained in respective treatments in an environmental chamber and images were taken every 10 minutes by time-lapse microscopy until the wounds healed. The experiment was repeated on 3 independent NHU cell lines with the same results. Differential interference contrast micrographs are shown for (c) non-differentiated (6 h) and (d) differentiated cultures (8 h) post-wounding; scale bar 500 µm. The time-course of wound repair was quantified from timelapse micrographs at 2 hour intervals and expressed against the original wound as the percentage healed for confluent (e) non-differentiated and (f) differentiated cultures. Points represent means of triplicate cultures ± SD. Phase contrast micrographs (g) of repaired scratch wounds in differentiated NHU cell cultures after 16 h in the presence of DMSO (0.1%), TGFβ1 (2 ng/ml), PD153035 (1 µM) or SB431542 (3 µM). Scale bar: 500 µm.
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pone-0051404-g005: Effects of TGFβ on differentiation, barrier function and scratch wound repair in NHU cell cultures.(a) NHU cells were differentiated with or without TZ/PD or ABS/Ca2+ in the presence or absence of TGFβ; UPK2 expression was assessed by RTqPCR after 4 days and normalised against GAPDH. Controls included SB431542, vehicle (0.1% DMSO), PD153035 and TZ alone. Bars represent means ± SD of triplicate PCR determinations. (b) Barrier function was assessed by TER measurement after 7 days culture of NHU cells seeded at 0.5×106 cells/cm2 onto SnapWell™ membranes and treated with or without ABS/Ca2+ in the presence of DMSO (0.1%), TGFβ1 (2 ng/ml) or SB431542 (3 µM). Bars represent mean ± SD of 3 independent replicate cultures. (c–g) After 7 days pre-culture with or without ABS/Ca2+ to induce differentiation, confluent cultures were incubated with DMSO (0.1%, vehicle control), TGFβ1 (2 ng/ml), SB431542 (10 µM) or PD153035 (1 µM) for 3 h and then scratch-wounded. Cultures were maintained in respective treatments in an environmental chamber and images were taken every 10 minutes by time-lapse microscopy until the wounds healed. The experiment was repeated on 3 independent NHU cell lines with the same results. Differential interference contrast micrographs are shown for (c) non-differentiated (6 h) and (d) differentiated cultures (8 h) post-wounding; scale bar 500 µm. The time-course of wound repair was quantified from timelapse micrographs at 2 hour intervals and expressed against the original wound as the percentage healed for confluent (e) non-differentiated and (f) differentiated cultures. Points represent means of triplicate cultures ± SD. Phase contrast micrographs (g) of repaired scratch wounds in differentiated NHU cell cultures after 16 h in the presence of DMSO (0.1%), TGFβ1 (2 ng/ml), PD153035 (1 µM) or SB431542 (3 µM). Scale bar: 500 µm.

Mentions: The changes observed in TGFβ-associated transcript and protein expression suggested that modulation of TGFβ/activin signalling was involved in the differentiation of human urothelial cells. To test this hypothesis, the effect of exogenous TGFβ1 was assessed on expression of UPK2 transcript by quantitative RT-PCR (Fig. 5A). As a positive control, NHU cultures were induced to differentiate using the TZ/PD and ABS/Ca2+ protocols. TGFβ1 alone or in combination with PD153035 or TZ did not induce UPK2 expression, showing that TGFβ1 alone does not induce or promote differentiation in NHU cell cultures. Inclusion of TGFβ1 into the TZ/PD protocol actually reduced expression of UPK2 transcript, implying an inhibitory effect on differentiation by that exogenous TGFβ1 (Fig. 5A). Inclusion of SB431542 to inhibit TGFβ signalling did not significantly affect UPK2 transcript expression (not shown).


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)

Effects of TGFβ on differentiation, barrier function and scratch wound repair in NHU cell cultures.(a) NHU cells were differentiated with or without TZ/PD or ABS/Ca2+ in the presence or absence of TGFβ; UPK2 expression was assessed by RTqPCR after 4 days and normalised against GAPDH. Controls included SB431542, vehicle (0.1% DMSO), PD153035 and TZ alone. Bars represent means ± SD of triplicate PCR determinations. (b) Barrier function was assessed by TER measurement after 7 days culture of NHU cells seeded at 0.5×106 cells/cm2 onto SnapWell™ membranes and treated with or without ABS/Ca2+ in the presence of DMSO (0.1%), TGFβ1 (2 ng/ml) or SB431542 (3 µM). Bars represent mean ± SD of 3 independent replicate cultures. (c–g) After 7 days pre-culture with or without ABS/Ca2+ to induce differentiation, confluent cultures were incubated with DMSO (0.1%, vehicle control), TGFβ1 (2 ng/ml), SB431542 (10 µM) or PD153035 (1 µM) for 3 h and then scratch-wounded. Cultures were maintained in respective treatments in an environmental chamber and images were taken every 10 minutes by time-lapse microscopy until the wounds healed. The experiment was repeated on 3 independent NHU cell lines with the same results. Differential interference contrast micrographs are shown for (c) non-differentiated (6 h) and (d) differentiated cultures (8 h) post-wounding; scale bar 500 µm. The time-course of wound repair was quantified from timelapse micrographs at 2 hour intervals and expressed against the original wound as the percentage healed for confluent (e) non-differentiated and (f) differentiated cultures. Points represent means of triplicate cultures ± SD. Phase contrast micrographs (g) of repaired scratch wounds in differentiated NHU cell cultures after 16 h in the presence of DMSO (0.1%), TGFβ1 (2 ng/ml), PD153035 (1 µM) or SB431542 (3 µM). Scale bar: 500 µm.
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pone-0051404-g005: Effects of TGFβ on differentiation, barrier function and scratch wound repair in NHU cell cultures.(a) NHU cells were differentiated with or without TZ/PD or ABS/Ca2+ in the presence or absence of TGFβ; UPK2 expression was assessed by RTqPCR after 4 days and normalised against GAPDH. Controls included SB431542, vehicle (0.1% DMSO), PD153035 and TZ alone. Bars represent means ± SD of triplicate PCR determinations. (b) Barrier function was assessed by TER measurement after 7 days culture of NHU cells seeded at 0.5×106 cells/cm2 onto SnapWell™ membranes and treated with or without ABS/Ca2+ in the presence of DMSO (0.1%), TGFβ1 (2 ng/ml) or SB431542 (3 µM). Bars represent mean ± SD of 3 independent replicate cultures. (c–g) After 7 days pre-culture with or without ABS/Ca2+ to induce differentiation, confluent cultures were incubated with DMSO (0.1%, vehicle control), TGFβ1 (2 ng/ml), SB431542 (10 µM) or PD153035 (1 µM) for 3 h and then scratch-wounded. Cultures were maintained in respective treatments in an environmental chamber and images were taken every 10 minutes by time-lapse microscopy until the wounds healed. The experiment was repeated on 3 independent NHU cell lines with the same results. Differential interference contrast micrographs are shown for (c) non-differentiated (6 h) and (d) differentiated cultures (8 h) post-wounding; scale bar 500 µm. The time-course of wound repair was quantified from timelapse micrographs at 2 hour intervals and expressed against the original wound as the percentage healed for confluent (e) non-differentiated and (f) differentiated cultures. Points represent means of triplicate cultures ± SD. Phase contrast micrographs (g) of repaired scratch wounds in differentiated NHU cell cultures after 16 h in the presence of DMSO (0.1%), TGFβ1 (2 ng/ml), PD153035 (1 µM) or SB431542 (3 µM). Scale bar: 500 µm.
Mentions: The changes observed in TGFβ-associated transcript and protein expression suggested that modulation of TGFβ/activin signalling was involved in the differentiation of human urothelial cells. To test this hypothesis, the effect of exogenous TGFβ1 was assessed on expression of UPK2 transcript by quantitative RT-PCR (Fig. 5A). As a positive control, NHU cultures were induced to differentiate using the TZ/PD and ABS/Ca2+ protocols. TGFβ1 alone or in combination with PD153035 or TZ did not induce UPK2 expression, showing that TGFβ1 alone does not induce or promote differentiation in NHU cell cultures. Inclusion of TGFβ1 into the TZ/PD protocol actually reduced expression of UPK2 transcript, implying an inhibitory effect on differentiation by that exogenous TGFβ1 (Fig. 5A). Inclusion of SB431542 to inhibit TGFβ signalling did not significantly affect UPK2 transcript expression (not shown).

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