Signaling Mechanisms of Transforming Growth Factor-β (TGF-β) in Cancer: TGF-β Induces Apoptosis in Lung Cells by a Smad-Dependent Mechanism

Introduction1.1 TGF-β ligands, receptors and smads Transforming Growth Factor-beta (TGF-β), a cytokine that is expressed in a variety of normal tissues, including the lung (Bartram & Spear, 2004;Jakowlew et al., 1995Jakowlew et al., , 1998;;Kang et al., 2000;Montuenga et al., 1998), exerts diverse effects on a wide variety of cellular processes, including proliferation, differentiation, and apoptosis (Elliot & Blobe, 2005;Massagué, 1998).More than sixty different TGF-β family members have been identified in various oraganisms, with at least 29 of these proteins being encoded in humans.Among the many proteins in the TGF-β superfamily are four TGF-β ligands, five activins, eight bone morphogenetic proteins (BMP), and 15 growth and differentiation factors (GDF).Three TGF-β isoforms have been identified in humans, including TGF-β1, TGF-β2, and TGF-β3, with each being a homodimeric polypeptide with a molecular weight of 25-kDa.All three TGF-β isoforms are initially synthesized as 55-kDa proproteins that consist of an amino-terminal pro-region and a carboxy-terminal mature region (Gentry et al., 1988).The pro-region facilitates necessary dimerization of the proproteins for future activity.TGF-β is secreted in a latent, inactive form in which the 12.5-kDa carboxyl-terminal 112 amino acid-long mature form is non-covalently associated with the 80-kDa Latency-Associated Peptide (LAP) amino-terminal remainder (Barcellos-Hoff, 1996;Barcellos-Hoff & Ewan, 2000).The LAP forms a complex with the 12.5-kDa TGF-β to keep it inactive (Arndjelovic et al., 2003;Stander et al., 1999).This complex is referred to as the small latent TGF-β complex.The small latent TGF-β complex may associate with members of the latent TGF-β-binding protein (LTBP) family to form the large latent TGF-β complex (Öklü & Hesketh, 2000).The liberation of TGF-β from the latent complexes is referred to as activation (Annes et al., 2003).The precise steps that are involved in liberation of the bioactive dimer are not completely understood, but may involve cleavage of the LTBP or LAP or both (Hyytiäinen et al., 2004).Active TGF-β exerts its effects with specific high affinity receptors.In mammals, five TGF-β superfamily type I receptors and seven type II receptors have been identified (Derynck et al., www.intechopen.comTumor Suppressor Genes 146 2001; Massagué, 2000).The TGF-β type I and type II receptors are structurally related transmembrane glycoproteins that consist of an extracellular N-terminal ligand-binding domain with more than ten cysteine residues that regulate the dimeric structure, a transmembrane region, and a C-terminal serine/threonine kinase domain.The type I receptors, but not type II receptors, have a highly conserved region that is rich in glycine and serine residues, referred to as the GS domain, in the juxtamembrane domain next to the N-terminus of the kinase domain.The GS domain is a target for the type II receptor kinase, and upon its phosphorylation on specific serine and threonine residues, the type I receptor becomes activated (Heldin et al., 1997;Massagué, 2000).Being downstream of the type II receptor, the type I receptor plays an important role in determining the specifity of intracellular signals.The type I and II receptors exist as homodimers at the cell surface in the absence of ligands, but have an inherent heteromeric affinity for each other.Only select combinations of type I and II receptors act as ligand-binding signaling complexes.The molecular basis of the selectivity of the type I-type II receptor interactions remains poorly understood, but the structural complement at the interface may help define the selectivity of the receptor combinations.Most of the TGF-β ligands bind with high affinity to the type I receptor, also known as activin receptor-like kinase (ALK), or to the type II receptor, while others bind efficiently only to heteromeric receptor combinations.The intracellular signal transduction triggered by the kinase activity of TGF-β involves the phosphorylation of Smad family proteins and in turn, complex changes in the transcriptional regulation of various response genes.The Smad family proteins include Smad 1, 2, 3, 4, 5, 7, and 8.The Smads are divided into three subclasses depending on their structure and function: the receptor-regulated Smads (R-Smads), common-mediator Smad (Co-Smad), and inhibitory Smads (I-Smads).In general, the R-Smads, Smads 2 and 3, function downstream of the TGF-β ligands, while Smads 1, 5, and 8 are downstream of members of the BMP and GDF subfamilies of ligands.Smads 1, 2, 3, 5, and 8 are direct substrates for the TGF-β type I receptor kinase, whereas Co-Smad, Smad 4, participates in Smad complex formation.Smads 6 and 7, the I-Smads, interfere with TGF-β-induced Smaddependent signal transduction (Park, 2005;Whitman, 1997).Activation of cell surface receptors by ligands leads to phosphorylation of the R-Smads at two serine residues in a SSXS motif at their extreme C-termini.This phosphorylation allows the R-Smads to form both homomeric and heteromeric complexes with Smad4 that accumulate in the nucleus.There, they are directly involved in transcriptional regulation of target genes in cooperation with other transcription factors.Signaling by TGF-β is mediated by a ligand-induced heteromeric complex of two types of transmembrane serine/threonine kinase receptors designated as TGF-β type I receptor (TGF-β RI) and type II receptor (TGF-β RII).Initial ligand binding to constitutively active TGF-β RII is followed by recruitment of TGF-β RI into the heteromeric complex.Subsequent phosphorylation of TGF-β RI at its GS-domain and activation is mediated by TGF-β RII and leads to activation of TGF-β RI.Upon this activating phosphorylation, TGF-β RI phosphorylates the receptor-activated Smad proteins (R-Smads), Smad2 and Smad3, which form a heteromeric complex with the co-Smad, Smad4, and enter the nucleus.In the nucleus, the Smad complex associates with other transcription factors for transcriptional activation of specific target genes (Massagué and Wotton, 2000; ten Dijke et al., 2000;Wrana and Attisano, 2000). www.intechopen.comSignaling Mechanisms of Transforming Growth Factor-β (TGF-β) in Cancer: TGF-β Induces Apoptosis in Lung Cells by a Smad-Dependent Mechanism 147 1.2 Tumor suppressor activity of TGF-β TGF-β was originally called one of the most potent polypeptide growth inhibitors isolated from natural sources (Moses et al., 1985;Tucker et al., 1984).When it was demonstrated that TGF-β could act as an autocrine negative growth regulator in the several different epithelial cell lines, it was hypothesized that TGF-β may act as an inhibitor of tumor progression, a tumor suppressor (Artega et al., 1990;Glick et al., 1989).The identification and characterization of the intermediates in the TGF-β signaling pathway, comprised of the genes and proteins for the TGF-β receptors and Smads, has increased our understanding of the role of TGF-β as a tumor suppressor.The involvement of the TGF-β signaling pathway in tumor suppression is shown by mutations in the genes that encode the TGF-β receptors and Smad proteins in human tumors.The gene for TGF-β RII is frequently mutated in colon carcinoma cells from patients with hereditary non-polyposis colorectal cancer that also show microsatellite instability, as well as in gastric cancers and gliomas (Chung et al., 1996;Izumoto et al., 1997;Markowitz et al., 1995).A specific region of adenine nucleotides in the coding region of TGF-β RII is prone to mutation in these patients from germline defects in their capacity for DNA mismatch repair.The nucleotide deletions or additions result in a shortened version of TGF-β RII that cannot participate in signaling transduction (Lu et al., 1996).However, the TGF-β RII gene is not mutated in other types of carcinoma with microsatellite instability, including breast, liver, pancreatic, and endometrial carcinoma (Abe et al., 1996;Kawate et al., 1999;Vincent et al., 1996), while, a somatic frameshift mutation in the polyadenine tract of the TGF-β RII gene does occur in some endometrial cancer patients (Parekh et al., 2002).Missense and inactivating mutations in TGF-β RII have also been detected in colon cancers that do not exhibit microsatellite instability (Grady et al., 1999).Expression of TGF-β RII can be decreased in some cases of carcinoma, including head and neck squamous carcinoma, breast carcinoma, and laryngeal carcinoma (Eisma et al., 1996;Franchi et al., 2001;Gobbi et al., 1999).Re-expression of TGF-β RII in carcinoma cells that have either lost expression of TGFβ RII or show reduced TGF-β RII expression can inhibit the ability to become malignant.Although less common than in TGF-β RII, mutations in TGF-β RI also occur in patients with a variety of cancers, including ovarian cancers, metastatic breast cancers, T-cell lymphomas, and head and neck cancer metastases (Chen et al., 1998(Chen et al., , 2001;;Goggins, 1998;Schiemann et al., 1999).Patients with ovarian cancer show a high frequency of mutations of TGF-β RI (Chen et al., 2001), while expression of TGF-β RI is transcriptionally repressed by DNA methylation in cells from patients with gastric cancer (Kang et al., 1999).Over-expression of TGF-β RI in colon carcinoma cells with low levels of TGF-β R I a l s o i n h i b i t s t u m o r progression as with TGF-β RII (Wang et al., 1996).Mutations in TGF-β RI do not appear to be associated with TGF-β RII mutations; such mutations suggest that these TGF-β receptors may function as tumor suppressors.Decreased TGF-β receptor expression or availability of TGF-β receptors at the cell surface may allow tumor cells to escape the growth inhibitory function of TGF-β (Kim et al., 2000).Expression of the TGF-β receptors in tumor cells may also be reduced by altered levels or activities of transcription factors that are required for expression of TGF-β RII, such as the Ets transcription factor.Hypermethylation of CpG isla