Molecular Mechanisms of Transforming Growth Factor-β Signaling

I. Introduction II. Ligands and Receptors A. Ligands B. Receptors C. Receptor kinase signaling III. Smads A. Cloning of the Smads B. Smad regulation C. Smad nuclear function D. Smad structure E. Smads as negative regulators F. Smad-deficient mice G. Smads: an emerging model IV. TGF-β and the Cell Cycle A. TGF-β induction of the CKIs B. TGF-β-mediated decrease in Cdc25A levels in the breast epithelial cell line MCF10A C. TGF-β-mediated cyclin-CDK inhibition may be common strategy to arrest cells in G1 V. TGF-β and Cancer A. Receptor mutants B. Smad mutants C. Cell cycle mutants D. Multilevel resistance to TGF-β is important in multistep model of carcinogenesis VI. Conclusions THE transforming growth factor-βs (TGF-βs)1 are a family of potent multifunctional cytokines that modulate a wide variety of cellular activities (1–4). Originally identified as a factor that induced the growth of rat kidney fibroblasts in soft agar, TGF-β was later shown to be an inhibitor of cellular growth for many cell types, including cells derived from epithelial, endothelial, neuronal, hematopoietic, and lymphoid origins. This ability of TGF-β to cause growth inhibition is thought to play a critical role in its ability to influence many aspects of cellular functions. In addition, TGF-β-mediated growth inhibition may also play a more global regulatory role in complex physiological processes such as in the immune response and development (5). Other effects of TGF-β include its ability to modulate wound healing, extracellular matrix deposition, cellular adhesion and migration, and most recently, synaptic facilitation (1–4, 6). Deregulation of TGF-β signaling is implicated in the pathogenesis of many diseases including arthritis, atherosclerosis, glomerulonephritis, human hereditary telangiectasia, and carcinogenesis. Loss of cellular sensitivity to TGF-β-mediated growth inhibition may contribute directly to these pathological states, specifically carcinogenesis. With the demonstrated importance of TGF-β signaling in a variety of biological processes, and loss of TGF-β responsiveness as an important correlate of certain diseases, a tremendous effort has been undertaken in the last decade to elucidate the mechanisms by which TGF-β propagates its signal. An important step in understanding TGF-β signaling came with the identification of three cell surface proteins that bind to TGF-β ligand with high affinity. These were called type I, II, and III receptors based on their molecular weight. The type I and type II receptors belong to a large family of receptor serine/threonine kinases. Upon TGF-β ligand binding to type II receptor, type I receptor is recruited into a complex containing both receptors and ligand. This causes the phosphorylation and subsequent kinase activation of type I receptor by the constitutively active type II receptor kinase. Currently, activated type I receptor kinase is thought to be sufficient to modulate most TGF-β downstream signals, although it is possible that type II receptor may also contribute to downstream signaling. The type III receptor is not for may to TGF-β ligand to the type I and II In cells with signaling receptor for a TGF-β the of a large of type III receptor on the cell surface may to a TGF-β signal. The of is to on the of of downstream signaling from TGF-β and of the TGF-β receptor been identified a and be the of The TGF-β and receptors for be as an of the and receptors be in the (1–4). on family of TGF-β receptor the and the in understanding of Smad signaling in to the importance of TGF-β-mediated growth inhibition on a wide variety of cell types, in the molecular mechanisms by which TGF-β growth of the TGF-β signaling that been to be in human in the to and that in the The TGF-β are of a large of that are important in many biological of the include proteins proteins growth and and derived factor TGF-β been These are as The of a of the with the of the active the a high of with The of and been of the the an with the of the The of the TGF-β structure is the from three of the that for the from the This may for the of from of its TGF-β by binding to receptors on the cell in the came with the identification in and of these receptors receptors been I, II, of the type II and type I been to TGF-β signaling The type II receptor is a to of a a extracellular a and a large of of in kinases. of the to was to its with The type I receptor is a to of a extracellular a and a the type II receptor, the type I receptor kinases. The to was to its with the type II receptor, the type I receptor not bind to ligand. The and identification of both type II and type I TGF-β receptors are in in The type II receptor is a receptor kinase that with type I receptor on binding to Upon type I receptor kinase is by the constitutively active type II receptor in a as the of the the type I receptor kinase. constitutively active type I receptor has been by to the of This receptor kinase the TGF-β and certain that type I receptor is a of TGF-β signaling. it is not type II has physiological type I receptor with which it Other TGF-β also a type II and type I receptor kinase and a type II and type I receptors are to both the and by as the TGF-β receptors were type II receptors extracellular and type I In addition, receptor type certain that the not such is the that receptors containing be as type I receptors receptors that bind ligand a be as type II in the identification of for TGF-β type I receptor include the that Smads may as for the TGF-β type I The Smad family of proteins has been identified as of the TGF-β from the to the Smad been and the TGF-β and signals, and the signal. to be a for these Smads by the Smads into the the of is shown in In the most identified of the Smad and receptor In a of the role of Smads in TGF-β signaling. the of the Smads from their in and that a model for Smad activation TGF-β nuclear for Smads as both a factor and be on the structure and possible function of the Smad that to be to elucidate the role of Smads in TGF-β be the of signaling in C. and The signaling is shown from ligand to Smad of the Smad is also shown for and as of the TGF-β signal. to and to and activation Other of TGF-β receptor that been in the include and it is physiological role of these proteins in TGF-β signaling. these not be in Smad were identified a in and C. in which a signaling is In signaling proteins that are to their TGF-β is the ligand (5). The receptor are and type In a to of a was in in that with of and a constitutively active is by of that is in the signaling In C. the is The receptor are and to and and were identified as that are downstream of in an that that of the The that for of a that may play a role in the These are of the importance of these in TGF-β signaling. With of these proteins in both and C. in C. from such as and were for on that is in human was to a called in identified as a These of the and been called the of the a model has as to Smads are Upon phosphorylation by type I TGF-β receptor, to its to a complex and into the in the the Smads the of important in the TGF-β such may be the This model is the of of that be in by that certain Smads are with TGF-β and are and are TGF-β This phosphorylation is induced and to levels by on on the kinase for the The on the possible kinase that Smads TGF-β came from a in which not was to be and with the receptor that may as a for the receptor kinase of the type I receptor in cells to phosphorylation of in the of type II of three in the of the phosphorylation of by type I receptor kinase was it was shown that these three are by type I receptor The be with type I receptor, that is an in for the type I receptor kinase. of the of is for its nuclear the by which nuclear phosphorylation is may the of that it a complex with to the and its nuclear Smads as in understanding of Smad nuclear function came with the that was in a complex with the factor Upon a induced complex on the response of the This is the of the response factor is in that a role as a of the signal. of the was sufficient to of the not cause an in an role for in activation of the is as to binding ability is although phosphorylation and subsequent with may be important in binding to The that is also in the of of with and a to that directly with a containing of the that with the of the negative into the of the of and the of This that the Smad complex may be directly in Smads bind development to the came with the that Smads binding ability The of bind to a in the in cells that also bind in a as the Smads an binding it is as to the of Smad binding on function for binding be to with to of these with the in the activation of This of may in in a which also the binding the to activation was to an the role that Smad binding may play in Smads Smad phosphorylation and to the are thought to regulation of of which may be the for a important in extracellular matrix a the of the it was of TGF-β activation of in a cell line and of with was sufficient to the in a were with from of and in the activation of the in human breast cell line The is an of a of binding and a of the Smads may by as a factor of binding to as a This is by the of a in which was to from a by of a derived from the was shown to bind directly to on a to the the of Smad binding and on activation of by of were The of was a of from the induced by the the was activated by TGF-β This that although of Smads may an in the ability of TGF-β to levels of Smads may not be sufficient to a activation may the of a binding to the of complex with the a of was to the with the the TGF-β and with the induction as in the The from was the of the Smad binding the the of the binding of Smads and their ability to the of the be in Smad complex binding may cause effects that be in these with the of a large of as the of Smad binding may play a role in the of to in of structure to of to their binding that an in the may be to The of Smad nuclear and binding be with type of role in Smad binding may regulatory that function in the of The may be to the the Smads and the in the of the Smad binding may not be in the of Smad binding may for In the in the that the a binding an to in of the binding and into these important Smad ability by extracellular In a cellular Smad may also be by extracellular that its for TGF-β-mediated such of be the phosphorylation in of these are to an the in the with growth factor which a kinase in phosphorylation of the in the to nuclear with both and in the These that the phosphorylation of Smads in may their responsiveness to TGF-β signaling. The activation of the by Smads is by a of that the structure of Smads to their The Smads of high and with and with both and C. these signaling the of of the Smad with their C. and In addition, the Smads in a TGF-β signaling are with as to possible for the of the In these the of and a to a This the model in which Smads are recruited to by binding and to the is be activated This a model for Smad regulation such that the to the of the of the Smad Upon ligand the from its the to modulate and of and and the important for and of and were In the in both the and are The and are their This with the that and is sufficient to of of The of the of on the importance of the as an for the An of its structure has into the by which with Smads and the is by The has a of the importance of for Smad function a of from Smads identified in both and human on the the was shown to of a of and These are on by a and a on the The and may play an important role in Smad function in these with In addition, mutants from human in these were to in the of the ability of and Smads to was a three proteins an of the of is from it was that in the the from is on the of the The is in both and in in of the and both and of important for of TGF-β on an in that is important in TGF-β activation of the In the cell TGF-β responsiveness as with the a of and an in for of TGF-β was This of is from and in the the and Smad family been and these proteins were identified was identified of and was identified from proteins induced These proteins with the Smad family an a The proteins the three regulatory the in the Smads a of regulation by TGF-β and its family was shown to be induced of TGF-β in both and epithelial cells and may with such as induction In addition, by TGF-β in a by of be by of the for The of and to with and by the receptor complex were In was and the type I and type II receptor complex was the complex of the type I receptor kinase although the of type II receptor kinase be This that and with the to the ability of the type I receptor to and the ability of the type I receptor to and may directly bind to the receptor complex to with the complex of is to TGF-β-mediated induction of not with the receptor, and not with the receptor both the and it is that these Smad family and are by TGF-β and its and the the that be activated by TGF-β in both epithelial cells and human it is possible that may play a role as an of effects of TGF-β in to its possible role in regulation as a negative of signaling. these an of for Smad and the importance of Smad family as of the TGF-β signaling mice were The mice were The that include to an and a were with that is for a as of the and which a in and to mice were the of loss on TGF-β signaling in not be Other Smad-deficient mice that are include and is to these model has as to Smads are activated by TGF-β family in the a Smad signaling and and of signaling. Upon phosphorylation by the receptor the which in and to the bind to and a model for TGF-β activation of Smads from the to the TGF-β causes the of type II I II receptor the type I receptor the I receptor, in its Smad a with This most of the Smad complex into the the Smads with as as of the to modulate This model is for a of the it is the Smads are by type I receptor in a phosphorylation a with into the is also In the more are in to the the of as a the Smads may Smad into the Smads bind to of the With the Smads These are of the more important the role of Smads in TGF-β signaling. In addition, and may a of Smads with in TGF-β signaling. of Smad-deficient mice a of into the physiological role Smads play in TGF-β signaling. model for the TGF-β Upon binding of ligand to the II receptor I receptor is recruited into the complex and its by II This the I receptor which Upon the with the to a that into the The Smad complex of an factor by binding to the phosphorylation of and the phosphorylation of and of the and most important of TGF-β is its ability to arrest certain cells in the G1 of the cell This may be to of the of TGF-β on a wide variety of The of the ability of TGF-β to a G1 arrest may contribute to diseases such as human hereditary and The of many cells to to TGF-β that negative signaling may the cell step in the development and of In the by which TGF-β causes cell cycle The three cell in epithelial human and human breast epithelial the cell cycle in a a common strategy by which TGF-β may on the cell that the TGF-β into the are not and the of Smads in has not been The nuclear by which TGF-β the cell cycle is and be by with to the is a cell cycle that is the cell of the the it from a to a that is the cell cycle the cell from are to TGF-β are in the of G1 the the the cell is to the cell cycle TGF-β growth arrest was to be with an of although the of was not that in the cell cycle that the kinase of was for G1 to the kinase of these was in a The regulatory of these are as and their are the In to the kinase of the be in three the be and has an phosphorylation on and phosphorylation on and The and that phosphorylation also be In the of the kinase is and the is a of molecular as the kinase been shown to with and was that of the for cyclin-CDK phosphorylation was certain cyclin-CDK The phosphorylation of its with the binding to for in the of the G1 to The step is by the ability of to in including to The step is by the ability of to a of in which may include proteins in to and family of the cyclin-CDK are not arrest cells in TGF-β to the activities of both the by which be as TGF-β was shown to be to the of kinase in the These CKIs be into based on and The of proteins include and The of and The of CKIs bind to cyclin-CDK and complex the of CKIs bind to which the from their regulatory In the epithelial cell line The to be implicated in TGF-β G1 arrest was was to be with in epithelial cells and by both and the in to and the of that it a negative regulatory role in the cell the structure of the complex and has been with both and that bind to the active of In of arrest is in the TGF-β to its ability to TGF-β not its In epithelial TGF-β the of This inhibitor by and The model for TGF-β growth arrest in epithelial cells is that TGF-β is This causes an in to their and of which with is and to its The of both and with a decrease in phosphorylation and arrest in In the human cell line The by which TGF-β the of the CKIs and is more in human TGF-β causes a in and levels to an in an with and and a decrease in the of cyclin-CDK The of with is thought to on that the family of and function by the of the in its of and kinase the by which TGF-β the of and a of the TGF-β response in the of both and was TGF-β response to a that family bind to although in binding were for of the TGF-β with that proteins TGF-β These in to with the TGF-β response is also important in the TGF-β-mediated although TGF-β not its of The inhibitor not ability to that the of regulation by TGF-β is phosphorylation of not TGF-β possible the of an which may to of the and the of the to be a of and the large of TGF-β-mediated growth to the ability of both of these proteins to bind and the of The of to important for the ability of and large to bind to be sufficient to TGF-β cellular with a which is in its ability to bind that it is to TGF-β-mediated growth arrest in epithelial cells the phosphorylation of the ability of a cell to it was that be to function a of of such as the of the This model was the of TGF-β-mediated induction of the CKIs the of and with an by which to TGF-β and an for This model to also in epithelial has been shown to bind directly to not In that levels are not induced TGF-β cells are with the proteins in of TGF-β were with an of an ability to TGF-β-mediated This implicated proteins to the of such as as of the signaling with with demonstrated that of the inhibition of response to TGF-β by In to a function for by these directly to as a of TGF-β activation of the and in of with the of which by is sufficient to of containing the that to and family is also of both of were to in soft the three of which are a and a nuclear a which also to both of the both and the a role for as and been implicated in nuclear receptor signaling signaling signaling and the by which TGF-β to of the and to into are by TGF-β that it to and its cellular The that and are in TGF-β signaling an to its signaling the Smads may be to as of the the from the activated receptor complex directly to the the and the of Smads to the cell cycle on a be which of the may to the TGF-β to a complex with to activation of the and with induction of the a and model in which a the proteins be induced by The Smads may a complex with to into the This is by that of their with the and in a in the that the Smads common the model to be important of in the that of the Smads not to the activation of the and the with the activation of by the of Smads as in the last is possible that Smads with are to with not a signaling also the receptor may be in with Smads to the and in in the which the and are by a for cell cycle arrest was in a epithelial MCF10A In these a decrease in levels of the cell cycle is TGF-β levels for decrease by is thought to on an model with and the in the of an on by is thought to be for cell cycle In the MCF10A the correlate a decrease in with an in phosphorylation of and may be by which TGF-β cause an arrest in also in possible of regulation by which TGF-β growth inhibition TGF-β to cell cycle in a cell This may the that cell type from the cell may from cell type to cell is the TGF-β-mediated induction of epithelial cells and human In TGF-β may of the three for to a G1 cell cycle The breast epithelial cell is such is a the by which TGF-β In of the by which TGF-β on the cell cycle to an arrest in G1 on cell Other cell that are also to the TGF-β such as may not these that to arrest the cells in TGF-β may cyclin-CDK activities to the growth inhibition cyclin-CDK kinase may be a common by which TGF-β arrest cells in G1 of the cell TGF-β causes G1 cell cycle arrest by cyclin-CDK In the human TGF-β the CKIs and which to the of by and the of and by In epithelial is to TGF-β induced and it to bind and its In MCF10A TGF-β the of the from The ability of TGF-β to cyclin-CDK to be a common strategy by which it causes growth and negative growth regulators in a In be in the from the growth regulators the from the negative growth regulators are proteins downstream of such as on the function as to that the cell cycle is the cell is with growth such as TGF-β to a variety of such as In on the of the TGF-β that are for the TGF-β to such as the are in of TGF-β signaling may cellular the cell growth that in In more in type II receptor that been in The of be on the of Smad family in In addition, cell cycle in the TGF-β growth arrest that are in be In an of that loss of of the type II receptor are in a of In most of the cell type II receptor was not with an to the TGF-β signal. in the type II receptor of mutants the importance of the type II receptor on receptor II was into cells and the cells were for and in soft In the receptor a in and a in were not that the in TGF-β signaling directly with an in that the downstream signaling were also been in cell and breast that in receptor type II are not for These the that type II receptor is an important in type I receptor are of the downstream of TGF-β signaling been shown to be in of these are and that a on that to is in This is for and not the in which is to The was from and to a human to the of In was in of in are for that on may also contribute to The that to from that may be the to on of Smad it is an model to that is to their physiological role in TGF-β signaling their is for the loss of in has to be were to in three not be by phosphorylation of their to the these may the and subsequent with to nuclear it was that the of Smad and Smad bind to and the from signaling. of the may the from with its of the of and of the was not by the of the the may be from with of Smads in with Smad mutants the ability of the Smads to the In to also been in and and been in and in cell cycle are a of a in a may a growth for the and also been in and and and loss of on is in both of these the is of in and the of to these is Other downstream of TGF-β include and and in these been in wide variety of In to of the negative regulators in cell cycle regulators also the cell a tremendous growth In certain been in and the family of The of both of cell cycle mutants has been in the to TGF-β growth inhibition may levels in a Receptor an and in downstream of the may also the in cell cycle regulators such as and may negative growth regulators in to TGF-β from cell These may also with such as to cellular it is the Smads are of the cell cycle their not to an in CKIs and the Smads are in extracellular matrix as their in the induction of in to in that may a growth to the These levels of in an that is to negative growth Upon TGF-β type I receptor is recruited to the type II complex on the cell surface and is activated by the type II receptor kinase phosphorylation of its type I receptor kinase its This phosphorylation is with the nuclear of and and is thought to by with In the and of certain to modulate a certain of TGF-β such as This activation may Smad binding to in the by binding to such as levels are by TGF-β and the ability of the Smad to the by the phosphorylation of and by type I This may a negative by which the cell the of Smad signaling. The of the last is the of tremendous from the The of the Smad from the to the is the for TGF-β signaling. it is to on a possible role for the Smads as for TGF-β-mediated growth Smad to a of cells arrest is to the TGF-β-mediated growth by an in levels a decrease in levels to decrease cyclin-CDK to be Smad not sufficient to cause an in and the of TGF-β-mediated to growth inhibition in Smads are for TGF-β-mediated growth inhibition also to be the molecular by which the and are activated the and be the of In not are the in the cell cycle in extracellular matrix such as may a on the cell surface such that these cells a growth are more to This may be the with which has been shown to be in many the mice not more to with their it is possible that are also for The loss of may a in carcinogenesis that the of a more and of In to its in diseases such as TGF-β may cause more global effects in such as in the immune wound healing, and synaptic Smad signaling TGF-β growth