Evidence for a Role of MSK1 in Transforming Growth Factor-β-mediated Responses through p38α and Smad Signaling Pathways

Smad proteins are central mediators of the transforming growth factor-β (TGF-β) superfamily signaling. The mitogen-activated protein kinase (MAPK) p38 is also one of the downstream targets required for TGF-β-mediated responses. Although the interplay between the p38 and Smad signaling pathways might allow cells to display diverse patterns of responses to TGF-β, the mechanism of this cross-talk is not well established. We report here that inhibition of the p38α isoform suppressed the ability of Smad3 to mediate TGF-β-induced transcriptional responses. The inhibition of p38 activity blocked TGF-β-mediated phosphorylation of the MSK1 kinase, a substrate of p38 that plays an important role in the remodeling of chromatin. Moreover, we observed that expression of dominant-interfering mutants of MSK1 blocked the binding of Smad3 to the coactivator p300 in response to TGF-β signaling. These data reveal a new mechanism whereby the Smad signaling pathway and the p38 cascade are integrated in the nucleus to activate gene expression. Smad proteins are central mediators of the transforming growth factor-β (TGF-β) superfamily signaling. The mitogen-activated protein kinase (MAPK) p38 is also one of the downstream targets required for TGF-β-mediated responses. Although the interplay between the p38 and Smad signaling pathways might allow cells to display diverse patterns of responses to TGF-β, the mechanism of this cross-talk is not well established. We report here that inhibition of the p38α isoform suppressed the ability of Smad3 to mediate TGF-β-induced transcriptional responses. The inhibition of p38 activity blocked TGF-β-mediated phosphorylation of the MSK1 kinase, a substrate of p38 that plays an important role in the remodeling of chromatin. Moreover, we observed that expression of dominant-interfering mutants of MSK1 blocked the binding of Smad3 to the coactivator p300 in response to TGF-β signaling. These data reveal a new mechanism whereby the Smad signaling pathway and the p38 cascade are integrated in the nucleus to activate gene expression. The transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; AF, dominant negative p38 mutant that cannot be phosphorylated; CBP, CREB-binding protein; CKD, C-terminal kinase dead; CREB, cAMP-response element-binding protein; EGFP, enhanced green fluorescent protein; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; JNK, c-Jun N-terminal kinase; MAPK, mitogenactivated protein kinase; MDCK, Madin-Darby canine kidney; MKK, mitogen-activated protein kinase kinase; MSK, mitogen- and stress-activated protein kinase; MOPS, 4-morpholinepropanesulfonic acid; NKD, N-terminal kinase dead; PAI, plasminogen activator inhibitor; TβRI and TβRII, TGF-β and abbreviations used are: TGF-β, transforming growth factor-β; AF, dominant negative p38 mutant that cannot be phosphorylated; CBP, CREB-binding protein; CKD, C-terminal kinase dead; CREB, cAMP-response element-binding protein; EGFP, enhanced green fluorescent protein; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; JNK, c-Jun N-terminal kinase; MAPK, mitogenactivated protein kinase; MDCK, Madin-Darby canine kidney; MKK, mitogen-activated protein kinase kinase; MSK, mitogen- and stress-activated protein kinase; MOPS, 4-morpholinepropanesulfonic acid; NKD, N-terminal kinase dead; PAI, plasminogen activator inhibitor; TβRI and TβRII, TGF-β and is a that a of extracellular and of TGF-β to the TGF-β and of the TGF-β The is phosphorylation of and the The to the the expression of a of binding proteins The ability of to in response to TGF-β also a of that Smad signaling cannot for the of of TGF-β, signaling pathways downstream of the TGF-β that to the of TGF-β The of are the mitogen-activated protein the extracellular signal-regulated c-Jun N-terminal and p38 The of TGF-β ERK, JNK, and p38 in and the of are not to TGF-β, the p38 pathway is that are in the of we of p38 and in of and is in p38α to and activate p38 in downstream of p38 The MSK1 kinase of an N-terminal and a C-terminal and the activity of is required for of the protein MSK1 is in the the the of for the transcriptional of is well the mechanism of of well the of pathways in is not well the we that the p38α isoform is TGF-β and that this isoform is required for gene also that this of p38 is the kinase the of the in response to TGF-β signaling. These reveal a mechanism for the p38 kinase and and the p38 and and The The of dominant negative of and The are p38 mutants that cannot be phosphorylation to The expression MSK1 and MSK1 kinase C-terminal kinase and MSK1 N-terminal kinase for the a for and and The expression the of the and and The expression the and Smad3 and cells in the a and cells in the the the of expression activity a the to the The of of of and downstream the and in and in the of of for and for to and and activator and and and a the the in the of TGF-β for for the of the kinase activity cells MSK1 in a and MSK1 the to in and in of kinase of and for kinase an cells in and to the for to protein for the and of and cells and the p38 TGF-β in for and in the role of p38 kinase in TGF-β-mediated of we in the of a p38 and The the The the and a the in TGF-β a in activity the These the of the p38 is the that p38 kinase plays an important role in TGF-β-mediated the to of Smad3 and and to be TGF-β and data that inhibition of p38 kinase might the ability of to mediate TGF-β transcriptional responses. the of signaling we also the of and the that and activate p38 the of the this we cells mutants of the the data the p38 we that of and the TGF-β-induced expression in the to TGF-β-induced expression of observed the cells used of cells for and expression of the of JNK, TGF-β-mediated that p38 plays a role in TGF-β-mediated gene expression Smad we the of the p38 the expression of in We to the a of Smad signaling of the TGF-β the of a that the that of cells the p38 blocked TGF-β-mediated expression of of in cells to in we in and cells the expression of the expression of is TGF-β a mechanism to that of in of and cells to TGF-β the of in response to TGF-β that inhibition of p38 might the ability of to mediate transcriptional of p38 the transcriptional of cells a in the of the of a between Smad and p38 and of Smad3 of phosphorylation of Smad3 the TGF-β and and to the nucleus the for a Smad proteins to TGF-β the to the nucleus the mechanism p38 the of the Smad we the p38 the ability of the to in Smad3 phosphorylation of cells to TGF-β, and phosphorylation not of cells the inhibition of p38 the of Smad3 in response to TGF-β cells and to of TGF-β signaling. in Smad3 the in the of TGF-β an of cells TGF-β-mediated of Smad3 in inhibition of p38 not of Smad and not Smad phosphorylation and cells the expression and the p38 the of TGF-β TGF-β for and Smad3 phosphorylation a The an cells the the p38 for and to TGF-β for the cells for Smad to is the of in the of the p38 and well of p38α and the the p38 the cells We to the p38 that in this cells dominant negative p38 the We observed that the p38α isoform to TGF-β-mediated the dominant negative mutant of this isoform this The and not a TGF-β-mediated expression expression of we to the p38α isoform a in cells that p38α for TGF-β-mediated isoform is for Smad3 cells of and of and in dominant negative of cells the and not TGF-β and for activity and activity to the The are of a protein and protein of the p38 an The and an MSK1 in a that p38 and activate the kinase MSK1 in to of that the of the to the mechanism p38 to we the ability of TGF-β signaling to the phosphorylation of cells MSK1 in the in the of the and the of MSK1 phosphorylation an that cells phosphorylation of MSK1 be in the of TGF-β signaling of MSK1 the in a phosphorylation of the of TGF-β signaling of the of the to MSK1 phosphorylation TGF-β the phosphorylation of cells TGF-β for and the of phosphorylation of MSK1 that the of cells to TGF-β a and phosphorylation of We also the ability of TGF-β to the of MSK1 an kinase the phosphorylation of we observed an in the activity of MSK1 in response to TGF-β signaling and of MSK1 are p38 cells TβRI and MSK1 phosphorylation The expression of the cells TGF-β for of and MSK1 phosphorylation in a The an cells not TGF-β for and an MSK1 activity in a kinase The a the cells TβRI TβRI not and MSK1 a The expression of the cells the p38 the of TGF-β TGF-β for and MSK1 phosphorylation a The an the role of p38 kinase in the of we the of the p38 the ability of TGF-β to mediate the phosphorylation of in in cells MSK1 the of the TGF-β the of the of inhibition observed cells TGF-β is the that p38 might of MSK1 in the TGF-β signaling in the the that MSK1 to the of gene expression TGF-β, we the ability of the of MSK1 to of the the of cells to in expression of MSK1 in cells the of cells to TGF-β, that MSK1 might a of the TGF-β signaling this we observed that expression of a dominant negative mutant of MSK1 TGF-β-mediated of the we that MSK1 kinase in the TGF-β signaling the and is in cells MSK1 the dominant negative MSK1 TGF-β for and activity and activity to the the of cells and in the of N-terminal kinase C-terminal kinase and TGF-β a to and The expression of of the of Smad3 and the protein CBP, in the TGF-β signaling pathway the and MSK1 the activity of we to MSK1 might to the of in response to TGF-β signaling. this cells and expression p300 the of the of the cells also MSK1 dominant negative mutants of this kinase MSK1 a a of the of Smad3 p300 the this of the of MSK1 MSK1 the of in response to TGF-β signaling. is that MSK1 plays an important role in TGF-β signaling of the between Smad3 and the coactivator Smad proteins to ability to c-Jun and that are of binding and to an binding activity the that the in the in response to to the Smad signaling a of and the of pathways downstream of TGF-β of the of is the p38 of to in TGF-β signaling of the of Smad proteins and the transcriptional The in the reveal a mechanism for cross-talk between and p38 signaling pathways that a downstream of the of Smad3 and the coactivator p300 in response to TGF-β p38 pathway to be TGF-β in of p38 and in a p38α and in TGF-β also that is p38α to activate the the we observed that of not TGF-β to a in the that p38α might an in signaling. this we observed that expression of a dominant-interfering of p38α blocked the of TGF-β-mediated gene expression. the observed the dominant negative mutants of the p38 expression of the expression of the p38 between is to that the expression of p38 might the of transcriptional to and p38 also a kinase that plays an important role in remodeling in ability to and to the this we that TGF-β the phosphorylation of MSK1 the in a we observed that this MSK1 in response to TGF-β signaling is for of the of MSK1 a dominant negative mutant of this kinase an we that expression of dominant negative mutants of MSK1 TGF-β-mediated of Smad3 is the that of MSK1 TGF-β might allow the of transcriptional the mechanism the kinase MSK1 to the of to be MSK1 to and to activate one is that of TGF-β signaling the of in the of p300 and Smad in an of transcriptional not is that the of MSK1 TGF-β might to the of MSK1 the the that we to a of for the of downstream targets of MSK1 in the of and the of targets the transcriptional responses. The transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; AF, dominant negative p38 mutant that cannot be phosphorylated; CBP, CREB-binding protein; CKD, C-terminal kinase dead; CREB, cAMP-response element-binding protein; EGFP, enhanced green fluorescent protein; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; JNK, c-Jun N-terminal kinase; MAPK, mitogenactivated protein kinase; MDCK, Madin-Darby canine kidney; MKK, mitogen-activated protein kinase kinase; MSK, mitogen- and stress-activated protein kinase; MOPS, 4-morpholinepropanesulfonic acid; NKD, N-terminal kinase dead; PAI, plasminogen activator inhibitor; TβRI and TβRII, TGF-β and abbreviations used are: TGF-β, transforming growth factor-β; AF, dominant negative p38 mutant that cannot be phosphorylated; CBP, CREB-binding protein; CKD, C-terminal kinase dead; CREB, cAMP-response element-binding protein; EGFP, enhanced green fluorescent protein; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; JNK, c-Jun N-terminal kinase; MAPK, mitogenactivated protein kinase; MDCK, Madin-Darby canine kidney; MKK, mitogen-activated protein kinase kinase; MSK, mitogen- and stress-activated protein kinase; MOPS, 4-morpholinepropanesulfonic acid; NKD, N-terminal kinase dead; PAI, plasminogen activator inhibitor; TβRI and TβRII, TGF-β and is a that a of extracellular and of TGF-β to the TGF-β and of the TGF-β The is phosphorylation of and the The to the the expression of a of binding proteins The ability of to in response to TGF-β also a of that Smad signaling cannot for the of of TGF-β, signaling pathways downstream of the TGF-β that to the of TGF-β The of are the mitogen-activated protein the extracellular signal-regulated c-Jun N-terminal and p38 The of TGF-β ERK, JNK, and p38 in and the of are not to TGF-β, the p38 pathway is that are in the of we of p38 and in of and is in p38α to and activate p38 in downstream of p38 The MSK1 kinase of an N-terminal and a C-terminal and the activity of is required for of the protein MSK1 is in the the Although the of for the transcriptional of is well the mechanism of of well the of pathways in is not well the we that the p38α isoform is TGF-β and that this isoform is required for gene also that this of p38 is the kinase the of the in response to TGF-β signaling. These reveal a mechanism for the p38 kinase and and the p38 and and The The of dominant negative of and The are p38 mutants that cannot be phosphorylation to The expression MSK1 and MSK1 kinase C-terminal kinase and MSK1 N-terminal kinase for the a for and and The expression the of the and and The expression the and Smad3 and cells in the a and cells in the the the of expression activity a the to the The of of of and downstream the and in and in the of of for and for to and and activator and and and a the the in the of TGF-β for for the of the kinase activity cells MSK1 in a and MSK1 the to in and in of kinase of and for kinase an cells in and to the for to protein for the and of and cells and the p38 TGF-β in for and and and the p38 and and The The of dominant negative of and The are p38 mutants that cannot be phosphorylation to The expression MSK1 and MSK1 kinase C-terminal kinase and MSK1 N-terminal kinase for the a for and and The expression the of the and and The expression the and Smad3 and cells in the a and cells in the the the of expression activity a the to the The of of of and downstream the and in and in the of of for and for to and and activator and and and a the the in the of TGF-β for for the of the kinase activity cells MSK1 in a and MSK1 the to in and in of kinase of and for kinase an cells in and to the for to protein for the and of and cells and the p38 TGF-β in for and in the role of p38 kinase in TGF-β-mediated of we in the of a p38 and The the The the and a the in TGF-β a in activity the These the of the p38 is the that p38 kinase plays an important role in TGF-β-mediated the to of Smad3 and and to be TGF-β and data that inhibition of p38 kinase might the ability of to mediate TGF-β transcriptional responses. the of signaling we also the of and the that and activate p38 the of the this we cells mutants of the the data the p38 we that of and the TGF-β-induced expression in the to TGF-β-induced expression of observed the cells used of cells for and expression of the of JNK, TGF-β-mediated that p38 plays a role in TGF-β-mediated gene expression Smad we the of the p38 the expression of in We to the a of Smad signaling of the TGF-β the of a that the that of cells the p38 blocked TGF-β-mediated expression of of in cells to in we in and cells the expression of the expression of is TGF-β a mechanism to that of in of and cells to TGF-β the of in response to TGF-β that inhibition of p38 might the ability of to mediate transcriptional of p38 the transcriptional of cells a in the of the of a between Smad and p38 and of Smad3 of phosphorylation of Smad3 the TGF-β and and to the nucleus the for a Smad proteins to TGF-β the to the nucleus the mechanism p38 the of the Smad we the p38 the ability of the to in Smad3 phosphorylation of cells to TGF-β, and phosphorylation not of cells the inhibition of p38 the of Smad3 in response to TGF-β cells and to of TGF-β signaling. in Smad3 the in the of TGF-β an of cells TGF-β-mediated of Smad3 in inhibition of p38 not of Smad and for Smad to is the of in the of the p38 and well of p38α and the the p38 the cells We to the p38 that in this cells dominant negative p38 the We observed that the p38α isoform to TGF-β-mediated the dominant negative mutant of this isoform this The and not a TGF-β-mediated expression expression of we to the p38α isoform a in cells that p38α for TGF-β-mediated isoform is for Smad3 cells of and of and in dominant negative of cells the and not TGF-β and for activity and activity to the The are of a protein and protein of the p38 an The and an MSK1 in a that p38 and activate the kinase MSK1 in to of that the of the to the mechanism p38 to we the ability of TGF-β signaling to the phosphorylation of cells MSK1 in the in the of the and the of MSK1 phosphorylation an that cells phosphorylation of MSK1 be in the of TGF-β signaling of MSK1 the in a phosphorylation of the of TGF-β signaling of the of the to MSK1 phosphorylation TGF-β the phosphorylation of cells TGF-β for and the of phosphorylation of MSK1 that the of cells to TGF-β a and phosphorylation of We also the ability of TGF-β to the of MSK1 an kinase the phosphorylation of we observed an in the activity of MSK1 in response to TGF-β signaling and of MSK1 are p38 cells TβRI and MSK1 phosphorylation The expression of the cells TGF-β for of and MSK1 phosphorylation in a The an cells not TGF-β for and an MSK1 activity in a kinase The a the cells TβRI TβRI not and MSK1 a The expression of the cells the p38 the of TGF-β TGF-β for and MSK1 phosphorylation a The an the role of p38 kinase in the of we the of the p38 the ability of TGF-β to mediate the phosphorylation of in in cells MSK1 the of the TGF-β the of the of inhibition observed cells TGF-β is the that p38 might of MSK1 in the TGF-β signaling in the the that MSK1 to the of gene expression TGF-β, we the ability of the of MSK1 to of the the of cells to in expression of MSK1 in cells the of cells to TGF-β, that MSK1 might a of the TGF-β signaling this we observed that expression of a dominant negative mutant of MSK1 TGF-β-mediated of the we that MSK1 kinase in the TGF-β signaling the and is in cells MSK1 the dominant negative MSK1 TGF-β for and activity and activity to the the of cells and in the of N-terminal kinase C-terminal kinase and TGF-β a to and The expression of of the of Smad3 and the protein CBP, in the TGF-β signaling pathway the and MSK1 the activity of we to MSK1 might to the of in response to TGF-β signaling. this cells and expression p300 the of the of the cells also MSK1 dominant negative mutants of this kinase MSK1 a a of the of Smad3 p300 the this of the of MSK1 MSK1 the of in response to TGF-β signaling. is that MSK1 plays an important role in TGF-β signaling of the between Smad3 and the coactivator p38 in the role of p38 kinase in TGF-β-mediated of we in the of a p38 and The the The the and a the in TGF-β a in activity the These the of the p38 is the that p38 kinase plays an important role in TGF-β-mediated the to of Smad3 and and to be TGF-β and data that inhibition of p38 kinase might the ability of to mediate TGF-β transcriptional responses. the of signaling we also the of and the that and activate p38 the of the this we cells mutants of the the data the p38 we that of and the TGF-β-induced expression in the to TGF-β-induced expression of observed the cells used of cells for and expression of the of JNK, TGF-β-mediated that p38 plays a role in TGF-β-mediated gene expression Smad we the of the p38 the expression of in We to the a of Smad signaling of the TGF-β the of a that the that of cells the p38 blocked TGF-β-mediated expression of of in cells to in we in and cells the expression of the expression of is TGF-β a mechanism to that of in of and cells to TGF-β the of in response to TGF-β that inhibition of p38 might the ability of to mediate transcriptional of p38 the transcriptional of cells a in the of the of a between Smad and p38 TGF-β-mediated and of Smad3 of phosphorylation of Smad3 the TGF-β and and to the nucleus the for a Smad proteins to TGF-β the to the nucleus the mechanism p38 the of the Smad we the p38 the ability of the to in Smad3 phosphorylation of cells to TGF-β, and phosphorylation not of cells the inhibition of p38 the of Smad3 in response to TGF-β cells and to of TGF-β signaling. in Smad3 the in the of TGF-β an of cells TGF-β-mediated of Smad3 in inhibition of p38 not of Smad and p38α for Smad to is the of in the of the p38 and well of p38α and the the p38 the cells We to the p38 that in this cells dominant negative p38 the We observed that the p38α isoform to TGF-β-mediated the dominant negative mutant of this isoform this The and not a TGF-β-mediated expression expression of we to the p38α isoform a in cells that p38α for TGF-β-mediated TGF-β MSK1 in a that p38 and activate the kinase MSK1 in to of that the of the to the mechanism p38 to we the ability of TGF-β signaling to the phosphorylation of cells MSK1 in the in the of the and the of MSK1 phosphorylation an that cells phosphorylation of MSK1 be in the of TGF-β signaling of MSK1 the in a phosphorylation of the of TGF-β signaling of the of the to MSK1 phosphorylation TGF-β the phosphorylation of cells TGF-β for and the of phosphorylation of MSK1 that the of cells to TGF-β a and phosphorylation of We also the ability of TGF-β to the of MSK1 an kinase the phosphorylation of we observed an in the activity of MSK1 in response to TGF-β signaling We the role of p38 kinase in the of we the of the p38 the ability of TGF-β to mediate the phosphorylation of in in cells MSK1 the of the TGF-β the of the of inhibition observed cells TGF-β is the that p38 might of MSK1 in the TGF-β signaling MSK1 in the the that MSK1 to the of gene expression TGF-β, we the ability of the of MSK1 to of the the of cells to in expression of MSK1 in cells the of cells to TGF-β, that MSK1 might a of the TGF-β signaling this we observed that expression of a dominant negative mutant of MSK1 TGF-β-mediated of the we that MSK1 kinase in the TGF-β signaling MSK1 the of Smad3 and the protein CBP, in the TGF-β signaling pathway the and MSK1 the activity of we to MSK1 might to the of in response to TGF-β signaling. this cells and expression p300 the of the of the cells also MSK1 dominant negative mutants of this kinase MSK1 a a of the of Smad3 p300 the this of the of MSK1 MSK1 the of in response to TGF-β signaling. is that MSK1 plays an important role in TGF-β signaling of the between Smad3 and the coactivator Smad proteins to ability to c-Jun and that are of binding and to an binding activity the that the in the in response to to the Smad signaling a of and the of pathways downstream of TGF-β of the of is the p38 of to in TGF-β signaling of the of Smad proteins and the transcriptional The in the reveal a mechanism for cross-talk between and p38 signaling pathways that a downstream of the of Smad3 and the coactivator p300 in response to TGF-β p38 pathway to be TGF-β in of p38 and in a p38α and in TGF-β also that is p38α to activate the the we observed that of not TGF-β to a in the that p38α might an in signaling. this we observed that expression of a dominant-interfering of p38α blocked the of TGF-β-mediated gene expression. the observed the dominant negative mutants of the p38 expression of the expression of the p38 between is to that the expression of p38 might the of transcriptional to and p38 also a kinase that plays an important role in remodeling in ability to and to the this we that TGF-β the phosphorylation of MSK1 the in a we observed that this MSK1 in response to TGF-β signaling is for of the of MSK1 a dominant negative mutant of this kinase an we that expression of dominant negative mutants of MSK1 TGF-β-mediated of Smad3 is the that of MSK1 TGF-β might allow the of transcriptional the mechanism the kinase MSK1 to the of to be MSK1 to and to activate one is that of TGF-β signaling the of in the of p300 and Smad in an of transcriptional not is that the of MSK1 TGF-β might to the of MSK1 the the that we to a of for the of downstream targets of MSK1 in the of and the of targets the transcriptional responses. The Smad proteins to ability to c-Jun and that are of binding and to an binding activity the that the in the in response to to the Smad signaling a of and the of pathways downstream of TGF-β of the of is the p38 of to in TGF-β signaling of the of Smad proteins and the transcriptional The in the reveal a mechanism for cross-talk between and p38 signaling pathways that a downstream of the of Smad3 and the coactivator p300 in response to TGF-β signaling. The p38 pathway to be TGF-β in of p38 and in a p38α and in TGF-β also that is p38α to activate the the we observed that of not TGF-β to a in the that p38α might an in signaling. this we observed that expression of a dominant-interfering of p38α blocked the of TGF-β-mediated gene expression. the observed the dominant negative mutants of the p38 expression of the expression of the p38 between is to that the expression of p38 might the of transcriptional to and p38 also a kinase that plays an important role in remodeling in ability to and to the this we that TGF-β the phosphorylation of MSK1 the in a we observed that this MSK1 in response to TGF-β signaling is for of the of MSK1 a dominant negative mutant of this kinase an we that expression of dominant negative mutants of MSK1 TGF-β-mediated of Smad3 is the that of MSK1 TGF-β might allow the of transcriptional the mechanism the kinase MSK1 to the of to be MSK1 to and to activate one is that of TGF-β signaling the of in the of p300 and Smad in an of transcriptional not is that the of MSK1 TGF-β might to the of MSK1 the the that we to a of for the of downstream targets of MSK1 in the of and the of targets the transcriptional responses. We and for expression We also for of the and for and