Limits...
Transforming growth factor beta blocks Tec kinase phosphorylation, Ca2+ influx, and NFATc translocation causing inhibition of T cell differentiation.

Chen CH, Seguin-Devaux C, Burke NA, Oriss TB, Watkins SC, Clipstone N, Ray A - J. Exp. Med. (2003)

Bottom Line: Here we show that TGF-beta inhibits T cell differentiation at a more proximal step.We show that in stimulated CD4+ T cells, TGF-beta inhibits phosphorylation and activation of the Tec kinase Itk, increase in intracellular Ca2+ levels, NFATc translocation, and activation of the mitogen-activated protein kinase ERK that together regulate T cell differentiation.Our studies suggest that by inhibiting Itk, and consequently Ca2+ influx, TGF-beta limits T cell differentiation along both the Th1 and Th2 lineages.

View Article: PubMed Central - PubMed

Affiliation: Vion Pharmaceuticals, Incorporated, New Haven, CT 06511, USA.

ABSTRACT
Transforming growth factor (TGF)-beta inhibits T cell proliferation and differentiation. TGF-beta has been shown to inhibit the expression of transcription factors such as GATA-3 and T-bet that play important roles in T cell differentiation. Here we show that TGF-beta inhibits T cell differentiation at a more proximal step. An early event during T cell activation is increased intracellular calcium levels. Calcium influx in activated T cells and the subsequent activation of transcription factors such as NFATc, events essential for T cell differentiation, are modulated by the Tec kinases that are downstream of the T cell receptor and CD28. We show that in stimulated CD4+ T cells, TGF-beta inhibits phosphorylation and activation of the Tec kinase Itk, increase in intracellular Ca2+ levels, NFATc translocation, and activation of the mitogen-activated protein kinase ERK that together regulate T cell differentiation. Our studies suggest that by inhibiting Itk, and consequently Ca2+ influx, TGF-beta limits T cell differentiation along both the Th1 and Th2 lineages.

Show MeSH

Related in: MedlinePlus

TGF-β interferes with signals essential for NFATc translocation but not gene expression. In all experiments (A) whole cell lysates were prepared from CD4+ T cells stimulated under Th2 differentiating conditions in the presence or absence of TGF-β or CsA. The expression of NFATc was assessed by immunoblotting. (B) CD4+ T cells were incubated with or without leptomycin B (20 ng/ml) for 30 min and then stimulated with anti-CD3 and anti-CD28 under Th2 differentiating conditions ± TGF-β (50 pM). Nuclear extracts were prepared after 4 h of stimulation and analyzed for the presence of NFATc. (C) CD4+ T cells were stimulated with PMA and ionomycin under Th2 differentiating conditions with or without the addition of TGF-β (50 pM). Nuclear extracts were prepared and analyzed for the presence of NFATc and GATA-3. It is to be noted that while stimulation by Ag + APC or anti-CD3 + anti-CD28 maintains nuclear presence of NFATc up to 5 d, very little active NFATc is detected at 5 d after stimulation with a combination of PMA and ionomycin. (D) Expression of constitutively active NFATc (caNFATc1) in CD4+ T cells restores the expression of GATA-3 and IL-13 inhibited by TGF-β. Activated CD4+ T cells were infected with recombinant retrovirus expressing caNFATc1 and or control virus. Infected CD4+ T cells were subsequently incubated under Th2-differentiating condition with or without TGF-β (50 pM). Nuclear extracts were prepared at 5 d of culture and analyzed by immunoblotting for the presence of GATA-3. The same blot was then stripped and reprobed with anti-Oct-1 antibody. Culture supernatants were analyzed for the presence of IL-13.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2193945&req=5

fig3: TGF-β interferes with signals essential for NFATc translocation but not gene expression. In all experiments (A) whole cell lysates were prepared from CD4+ T cells stimulated under Th2 differentiating conditions in the presence or absence of TGF-β or CsA. The expression of NFATc was assessed by immunoblotting. (B) CD4+ T cells were incubated with or without leptomycin B (20 ng/ml) for 30 min and then stimulated with anti-CD3 and anti-CD28 under Th2 differentiating conditions ± TGF-β (50 pM). Nuclear extracts were prepared after 4 h of stimulation and analyzed for the presence of NFATc. (C) CD4+ T cells were stimulated with PMA and ionomycin under Th2 differentiating conditions with or without the addition of TGF-β (50 pM). Nuclear extracts were prepared and analyzed for the presence of NFATc and GATA-3. It is to be noted that while stimulation by Ag + APC or anti-CD3 + anti-CD28 maintains nuclear presence of NFATc up to 5 d, very little active NFATc is detected at 5 d after stimulation with a combination of PMA and ionomycin. (D) Expression of constitutively active NFATc (caNFATc1) in CD4+ T cells restores the expression of GATA-3 and IL-13 inhibited by TGF-β. Activated CD4+ T cells were infected with recombinant retrovirus expressing caNFATc1 and or control virus. Infected CD4+ T cells were subsequently incubated under Th2-differentiating condition with or without TGF-β (50 pM). Nuclear extracts were prepared at 5 d of culture and analyzed by immunoblotting for the presence of GATA-3. The same blot was then stripped and reprobed with anti-Oct-1 antibody. Culture supernatants were analyzed for the presence of IL-13.

Mentions: The effects of TGF-β on NFATc could be either on suppression of NFATc expression or on inhibition of NFATc translocation. As shown in Fig. 3 A, CD4+ T cells stimulated by a combination of anti-CD3 and anti-CD28 under Th2-differentiating conditions displayed a net increase in NFATc levels which was not inhibited by either TGF-β or CsA as determined by immunoblotting of whole cell lysates. There are three splice variants of NFATc and the slightly slower migration pattern of the bands in TGF-β– and CsA-treated cultures reflects inhibition of dephosphorylation of NFATc that is normally effected by calcineurin. These observations showed that although TGF-β, like CsA, inhibits processing of NFATc, it does not interfere with net NFATc accumulation in the cell. In a recent study, Feske et al. showed that the duration of nuclear presence of NFAT influences the pattern of expression of several cytokines in human T cells (35). This observation led us to suspect that TGF-β may interfere with the nuclear residence of NFATc either by inhibiting its import (by suppressing dephosphorylation) or by expediting its export (by enhancing rephosphorylation). To investigate this, we used the drug leptomycin B (LMB), which inhibits Crm-1–mediated export of NFATc causing accumulation of NFATc in the nucleus (36). Thus, if TGF-β interfered with NFATc import, LMB would be unable to cause nuclear accumulation of NFATc. On the other hand, if TGF-β did not interfere with import of NFATc but only expedited its export, similar levels of NFATc should be observed in control and TGF-β-treated cells in the presence of LMB. As shown in Fig. 3 B, cells stimulated under Th2-differentiating conditions in the presence of both TGF-β and LMB showed very little evidence of nuclear trapping by LMB compared with control stimulated cells that were treated with LMB suggesting that TGF-β interfered with a signaling event that controls NFATc import (dephosphorylation).


Transforming growth factor beta blocks Tec kinase phosphorylation, Ca2+ influx, and NFATc translocation causing inhibition of T cell differentiation.

Chen CH, Seguin-Devaux C, Burke NA, Oriss TB, Watkins SC, Clipstone N, Ray A - J. Exp. Med. (2003)

TGF-β interferes with signals essential for NFATc translocation but not gene expression. In all experiments (A) whole cell lysates were prepared from CD4+ T cells stimulated under Th2 differentiating conditions in the presence or absence of TGF-β or CsA. The expression of NFATc was assessed by immunoblotting. (B) CD4+ T cells were incubated with or without leptomycin B (20 ng/ml) for 30 min and then stimulated with anti-CD3 and anti-CD28 under Th2 differentiating conditions ± TGF-β (50 pM). Nuclear extracts were prepared after 4 h of stimulation and analyzed for the presence of NFATc. (C) CD4+ T cells were stimulated with PMA and ionomycin under Th2 differentiating conditions with or without the addition of TGF-β (50 pM). Nuclear extracts were prepared and analyzed for the presence of NFATc and GATA-3. It is to be noted that while stimulation by Ag + APC or anti-CD3 + anti-CD28 maintains nuclear presence of NFATc up to 5 d, very little active NFATc is detected at 5 d after stimulation with a combination of PMA and ionomycin. (D) Expression of constitutively active NFATc (caNFATc1) in CD4+ T cells restores the expression of GATA-3 and IL-13 inhibited by TGF-β. Activated CD4+ T cells were infected with recombinant retrovirus expressing caNFATc1 and or control virus. Infected CD4+ T cells were subsequently incubated under Th2-differentiating condition with or without TGF-β (50 pM). Nuclear extracts were prepared at 5 d of culture and analyzed by immunoblotting for the presence of GATA-3. The same blot was then stripped and reprobed with anti-Oct-1 antibody. Culture supernatants were analyzed for the presence of IL-13.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193945&req=5

fig3: TGF-β interferes with signals essential for NFATc translocation but not gene expression. In all experiments (A) whole cell lysates were prepared from CD4+ T cells stimulated under Th2 differentiating conditions in the presence or absence of TGF-β or CsA. The expression of NFATc was assessed by immunoblotting. (B) CD4+ T cells were incubated with or without leptomycin B (20 ng/ml) for 30 min and then stimulated with anti-CD3 and anti-CD28 under Th2 differentiating conditions ± TGF-β (50 pM). Nuclear extracts were prepared after 4 h of stimulation and analyzed for the presence of NFATc. (C) CD4+ T cells were stimulated with PMA and ionomycin under Th2 differentiating conditions with or without the addition of TGF-β (50 pM). Nuclear extracts were prepared and analyzed for the presence of NFATc and GATA-3. It is to be noted that while stimulation by Ag + APC or anti-CD3 + anti-CD28 maintains nuclear presence of NFATc up to 5 d, very little active NFATc is detected at 5 d after stimulation with a combination of PMA and ionomycin. (D) Expression of constitutively active NFATc (caNFATc1) in CD4+ T cells restores the expression of GATA-3 and IL-13 inhibited by TGF-β. Activated CD4+ T cells were infected with recombinant retrovirus expressing caNFATc1 and or control virus. Infected CD4+ T cells were subsequently incubated under Th2-differentiating condition with or without TGF-β (50 pM). Nuclear extracts were prepared at 5 d of culture and analyzed by immunoblotting for the presence of GATA-3. The same blot was then stripped and reprobed with anti-Oct-1 antibody. Culture supernatants were analyzed for the presence of IL-13.
Mentions: The effects of TGF-β on NFATc could be either on suppression of NFATc expression or on inhibition of NFATc translocation. As shown in Fig. 3 A, CD4+ T cells stimulated by a combination of anti-CD3 and anti-CD28 under Th2-differentiating conditions displayed a net increase in NFATc levels which was not inhibited by either TGF-β or CsA as determined by immunoblotting of whole cell lysates. There are three splice variants of NFATc and the slightly slower migration pattern of the bands in TGF-β– and CsA-treated cultures reflects inhibition of dephosphorylation of NFATc that is normally effected by calcineurin. These observations showed that although TGF-β, like CsA, inhibits processing of NFATc, it does not interfere with net NFATc accumulation in the cell. In a recent study, Feske et al. showed that the duration of nuclear presence of NFAT influences the pattern of expression of several cytokines in human T cells (35). This observation led us to suspect that TGF-β may interfere with the nuclear residence of NFATc either by inhibiting its import (by suppressing dephosphorylation) or by expediting its export (by enhancing rephosphorylation). To investigate this, we used the drug leptomycin B (LMB), which inhibits Crm-1–mediated export of NFATc causing accumulation of NFATc in the nucleus (36). Thus, if TGF-β interfered with NFATc import, LMB would be unable to cause nuclear accumulation of NFATc. On the other hand, if TGF-β did not interfere with import of NFATc but only expedited its export, similar levels of NFATc should be observed in control and TGF-β-treated cells in the presence of LMB. As shown in Fig. 3 B, cells stimulated under Th2-differentiating conditions in the presence of both TGF-β and LMB showed very little evidence of nuclear trapping by LMB compared with control stimulated cells that were treated with LMB suggesting that TGF-β interfered with a signaling event that controls NFATc import (dephosphorylation).

Bottom Line: Here we show that TGF-beta inhibits T cell differentiation at a more proximal step.We show that in stimulated CD4+ T cells, TGF-beta inhibits phosphorylation and activation of the Tec kinase Itk, increase in intracellular Ca2+ levels, NFATc translocation, and activation of the mitogen-activated protein kinase ERK that together regulate T cell differentiation.Our studies suggest that by inhibiting Itk, and consequently Ca2+ influx, TGF-beta limits T cell differentiation along both the Th1 and Th2 lineages.

View Article: PubMed Central - PubMed

Affiliation: Vion Pharmaceuticals, Incorporated, New Haven, CT 06511, USA.

ABSTRACT
Transforming growth factor (TGF)-beta inhibits T cell proliferation and differentiation. TGF-beta has been shown to inhibit the expression of transcription factors such as GATA-3 and T-bet that play important roles in T cell differentiation. Here we show that TGF-beta inhibits T cell differentiation at a more proximal step. An early event during T cell activation is increased intracellular calcium levels. Calcium influx in activated T cells and the subsequent activation of transcription factors such as NFATc, events essential for T cell differentiation, are modulated by the Tec kinases that are downstream of the T cell receptor and CD28. We show that in stimulated CD4+ T cells, TGF-beta inhibits phosphorylation and activation of the Tec kinase Itk, increase in intracellular Ca2+ levels, NFATc translocation, and activation of the mitogen-activated protein kinase ERK that together regulate T cell differentiation. Our studies suggest that by inhibiting Itk, and consequently Ca2+ influx, TGF-beta limits T cell differentiation along both the Th1 and Th2 lineages.

Show MeSH
Related in: MedlinePlus