Ester Piek

Functional characterization of transforming growth factor beta signaling in Smad2- and SMAD3-deficient fibroblasts

A prominent pathway of transforming growth factor (TGF)-β signaling involves receptor-dependent phosphorylation of Smad2 and Smad3, which then translocate to the nucleus to activate transcription of target genes. To investigate the relative importance of these two Smad proteins in TGF-β1 signal transduction, we have utilized a loss of function approach, based on analysis of the effects of TGF-β1 on fibroblasts derived from mouse embryos deficient in Smad2 (S2KO) or Smad3 (S3KO). TGF-β1 caused 50% inhibition of cellular proliferation in wild-type fibroblasts as assessed by [3H]thymidine incorporation, whereas the growth of S2KO or S3KO cells was only weakly inhibited by TGF-β1. Lack of Smad2 or Smad3 expression did not affect TGF-β1-induced fibronectin synthesis but resulted in markedly suppressed induction of plasminogen activator inhibitor-1 by TGF-β1. Moreover, TGF-β1-mediated induction of matrix metalloproteinase-2 was selectively dependent on Smad2, whereas induction of c-fos, Smad7, and TGF-β1 autoinduction relied on expression of Smad3. Investigation of transcriptional activation of TGF-β-sensitive reporter genes in the different fibroblasts showed that activation of the (Smad binding element)4-Lux reporter by TGF-β1 was dependent on expression of Smad3, but not Smad2, whereas activation of the activin response element-Lux reporter was strongly suppressed in S2KO fibroblasts but, on the contrary, enhanced in S3KO cells. Our findings indicate specific roles for Smad2 and Smad3 in TGF-β1 signaling.

Hierarchical model of gene regulation by transforming growth factor β

Transforming growth factor βs (TGF-βs) regulate key aspects of embryonic development and major human diseases. Although Smad2, Smad3, and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) have been proposed as key mediators in TGF-β signaling, their functional specificities and interactivity in controlling transcriptional programs in different cell types and (patho)physiological contexts are not known. We investigated expression profiles of genes controlled by TGF-β in fibroblasts with ablations of Smad2, Smad3, and ERK MAPK. Our results suggest that Smad3 is the essential mediator of TGF-β signaling and directly activates genes encoding regulators of transcription and signal transducers through Smad3/Smad4 DNA-binding motif repeats that are characteristic for immediate-early target genes of TGF-β but absent in intermediate target genes. In contrast, Smad2 and ERK predominantly transmodulated regulation of both immediate-early and intermediate genes by TGF-β/Smad3. These results suggest a previously uncharacterized hierarchical model of gene regulation by TGF-β in which TGF-β causes direct activation by Smad3 of cascades of regulators of transcription and signaling that are transmodulated by Smad2 and/or ERK.

Suppressor and oncogenic roles of transforming growth factor-β and its signaling pathways in tumorigenesis

Transforming growth factor-beta (TGF-beta) has been implicated in oncogenesis since the time of its discovery almost 20 years ago. The complex, multifunctional activities of TGF-beta endow it with both tumor suppressor and tumor promoting activities, depending on the stage of carcinogenesis and the responsivity of the tumor cell. Dysregulation or alteration of TGF-beta signaling in tumorigenesis can occur at many different levels, including activation of the ligand, mutation or transcriptional suppression of the receptors, or alteration of downstream signal transduction pathways resulting from mutation or changes in expression patterns of signaling intermediates or from changes in expression of other proteins which modulate signaling. New insights into signaling from the TGF-beta receptors, including the identification of Smad signaling pathways and their interaction with mitogen-activated protein (MAP) kinase pathways, are providing an understanding of the changes involved in the change from tumor suppressor to tumor promoting activities of TGF-beta. It is now appreciated that loss of sensitivity to inhibition of growth by TGF-beta by most tumor cells is not synonymous with complete loss of TGF-beta signaling but rather suggests that tumor cells gain advantage by selective inactivation of the tumor suppressor activities of TGF-beta with retention of its tumor promoting activities, especially those dependent on cross talk with MAP kinase pathways and AP-1.

TGF-β Signaling in Mammary Gland Development and Tumorigenesis

Ligands of the TGF-β superfamily are unique in that they signal through transmembrane receptor serine-threonine kinases, rather than tyrosine kinases. The receptor complex couples to a signal transduction pathway involving a novel family of proteins, the Smads. On phosphorylation, Smads translocate to the nucleus where they modulate transcriptional responses. However, TGF-βs can also activate the mitogen-activated protein kinase (MAPK)4 pathway, and the different biological responses to TGF-β depend to varying degrees on activation of either or both of these two pathways. The Smad pathway is a nexus for cross-talk with other signal transduction pathways and for modulation by many different interacting proteins. Despite compelling evidence that TGF-β has tumor suppressor activity in the mammary gland, neither TGF-β receptors nor Smads are genetically inactivated in human breast cancer, though receptor expression is reduced. Possible reasons are discussed in relation to the dual role of TGF-β as tumor suppressor and oncogene.

Is Smad3 a Major Player in Signal Transduction Pathways Leading to Fibrogenesis

Transforming growth factor (TGF)-beta plays a central role in fibrosis, contributing both to the influx and activation of inflammatory cells, as well as to activation of fibroblasts to elaborate extracellular matrix. In the past few years, new insight has been gained into signal transduction pathways downstream of the TGF-beta receptor serine-threonine kinases with the identification of a family of evolutionarily conserved Smad proteins. Two receptor-activated Smad proteins, Smad2 and Smad3, are phosphorylated by the activated TGF-beta type I receptor kinase, after which they partner with the common mediator, Smad4, and are translocated to the nucleus to where they participate in transcriptional complexes to control expression of target genes. We have shown in wound healing studies of mice null for Smad3, that loss of this key signaling intermediate interferes with the chemotaxis of inflammatory cells to TGF-beta as well as with their ability to autoinduce TGF-beta. Moreover, studies with mouse embryo fibroblasts null for Smad3 show that TGF-beta-dependent induction of c-Jun and c-Fos, important in induction of collagen as well as in autoinduction of TGF-beta, is mediated by Smad3. Based on these observations, we hypothesize that loss of Smad3 will confer resistance to fibrosis and result in reduced inflammatory cell infiltrates, reduced autoinduction of TGF-beta, important to sustain the process, and reduced elaboration of collagen. Preliminary observations in a model of radiation-induced fibrosis confirm this hypothesis and suggest that inhibitors of Smad3 might have clinical application both to improve wound healing and to reduce fibrosis.

Genetic/genomic analysis of signal transduction pathways using cDNA microarray technology

cDNA microarray technology provides a novel approach to identify individual target genes and survey global genetic programs under the control of specific signalling pathways in mammalian systems. Smad2 and Smad3 are highly homologous members of the receptor-regulated subfamily of Smad proteins with a central role in TGF- signalling and target gene regulation.