Limits...
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.

Show MeSH

Related in: MedlinePlus

Analysis of differentiating NHU cells.NHU cells were differentiated by (a) TZ/PD or (b) ABS/Ca2+ protocols, as described in Materials and Methods, for the times indicated. Following RNA extraction, cDNA was generated and absolute quantitative PCR for UPK2 was performed. The VIC-labelled UKP2 product was normalised against the internal control (FAM-labelled GAPDH). Bars represent mean ± SD of triplicate PCR determinations. NHU cells transduced with UPK2-eGFP lentivirus were (c) left untreated, or differentiated by (d) TZ/PD or (e) ABS/Ca2+ protocols and assessed by epifluorescence; 6 day timepoint shown. Scale bar 80 µm. To verify changes in TGFβ target gene expression during differentiation, cultures from an independent NHU cell line were induced to differentiate with TZ/PD or ABS/Ca2+ for 72 h (f). RTqPCR using comparative quantification by cycle threshold (CT) with SYBRgreen was performed in triplicate and normalised against the internal control (GAPDH). Data show TGFβ pathway-associated gene expression in response to treatment with TZ/PD or ABS/Ca2+ versus vehicle control. Bars represent mean ± SD.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3526617&req=5

pone-0051404-g001: Analysis of differentiating NHU cells.NHU cells were differentiated by (a) TZ/PD or (b) ABS/Ca2+ protocols, as described in Materials and Methods, for the times indicated. Following RNA extraction, cDNA was generated and absolute quantitative PCR for UPK2 was performed. The VIC-labelled UKP2 product was normalised against the internal control (FAM-labelled GAPDH). Bars represent mean ± SD of triplicate PCR determinations. NHU cells transduced with UPK2-eGFP lentivirus were (c) left untreated, or differentiated by (d) TZ/PD or (e) ABS/Ca2+ protocols and assessed by epifluorescence; 6 day timepoint shown. Scale bar 80 µm. To verify changes in TGFβ target gene expression during differentiation, cultures from an independent NHU cell line were induced to differentiate with TZ/PD or ABS/Ca2+ for 72 h (f). RTqPCR using comparative quantification by cycle threshold (CT) with SYBRgreen was performed in triplicate and normalised against the internal control (GAPDH). Data show TGFβ pathway-associated gene expression in response to treatment with TZ/PD or ABS/Ca2+ versus vehicle control. Bars represent mean ± SD.

Mentions: Expression of urothelium differentiation-restricted uroplakin 2 (UPK2) transcript was used as a marker to monitor the differentiation status of NHU cell cultures. Whereas there was minimal UPK2 expression in control cultures maintained in standard growth medium, both differentiation-inducing protocols (TZ/PD and ABS/Ca2+) resulted in time-dependent increases in UPK2 transcript (Figs. 1A–B). NHU cultures stably transduced with UPK2-eGFP lentivirus contained few eGFP-expressing cells even at confluence (Fig. 1C). Following induction of differentiation, a majority of cells became fluorescent within 6 days. Cells differentiated pharmacologically by TZ/PD remained discrete and rounded, whereas those differentiated by ABS/Ca2+ formed integrated, overlying cell sheets (Figs. 1D–E). To identify gene expression changes common to both differentiation strategies, gene arrays were performed on cRNA derived from parallel cultures harvested at 6 days post induction of differentiation. The array data for the two differentiation-inducing protocols was validated by examining changes in expression of marker genes associated with differentiated urothelium, including UPK2 (Table S1).


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)

Analysis of differentiating NHU cells.NHU cells were differentiated by (a) TZ/PD or (b) ABS/Ca2+ protocols, as described in Materials and Methods, for the times indicated. Following RNA extraction, cDNA was generated and absolute quantitative PCR for UPK2 was performed. The VIC-labelled UKP2 product was normalised against the internal control (FAM-labelled GAPDH). Bars represent mean ± SD of triplicate PCR determinations. NHU cells transduced with UPK2-eGFP lentivirus were (c) left untreated, or differentiated by (d) TZ/PD or (e) ABS/Ca2+ protocols and assessed by epifluorescence; 6 day timepoint shown. Scale bar 80 µm. To verify changes in TGFβ target gene expression during differentiation, cultures from an independent NHU cell line were induced to differentiate with TZ/PD or ABS/Ca2+ for 72 h (f). RTqPCR using comparative quantification by cycle threshold (CT) with SYBRgreen was performed in triplicate and normalised against the internal control (GAPDH). Data show TGFβ pathway-associated gene expression in response to treatment with TZ/PD or ABS/Ca2+ versus vehicle control. Bars represent mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3526617&req=5

pone-0051404-g001: Analysis of differentiating NHU cells.NHU cells were differentiated by (a) TZ/PD or (b) ABS/Ca2+ protocols, as described in Materials and Methods, for the times indicated. Following RNA extraction, cDNA was generated and absolute quantitative PCR for UPK2 was performed. The VIC-labelled UKP2 product was normalised against the internal control (FAM-labelled GAPDH). Bars represent mean ± SD of triplicate PCR determinations. NHU cells transduced with UPK2-eGFP lentivirus were (c) left untreated, or differentiated by (d) TZ/PD or (e) ABS/Ca2+ protocols and assessed by epifluorescence; 6 day timepoint shown. Scale bar 80 µm. To verify changes in TGFβ target gene expression during differentiation, cultures from an independent NHU cell line were induced to differentiate with TZ/PD or ABS/Ca2+ for 72 h (f). RTqPCR using comparative quantification by cycle threshold (CT) with SYBRgreen was performed in triplicate and normalised against the internal control (GAPDH). Data show TGFβ pathway-associated gene expression in response to treatment with TZ/PD or ABS/Ca2+ versus vehicle control. Bars represent mean ± SD.
Mentions: Expression of urothelium differentiation-restricted uroplakin 2 (UPK2) transcript was used as a marker to monitor the differentiation status of NHU cell cultures. Whereas there was minimal UPK2 expression in control cultures maintained in standard growth medium, both differentiation-inducing protocols (TZ/PD and ABS/Ca2+) resulted in time-dependent increases in UPK2 transcript (Figs. 1A–B). NHU cultures stably transduced with UPK2-eGFP lentivirus contained few eGFP-expressing cells even at confluence (Fig. 1C). Following induction of differentiation, a majority of cells became fluorescent within 6 days. Cells differentiated pharmacologically by TZ/PD remained discrete and rounded, whereas those differentiated by ABS/Ca2+ formed integrated, overlying cell sheets (Figs. 1D–E). To identify gene expression changes common to both differentiation strategies, gene arrays were performed on cRNA derived from parallel cultures harvested at 6 days post induction of differentiation. The array data for the two differentiation-inducing protocols was validated by examining changes in expression of marker genes associated with differentiated urothelium, including UPK2 (Table S1).

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