“Ultimately, in the world of cellular physiology, TGF-β should not be regarded as a ‘thing’, but rather as an element of a complex biological signaling language, which is used for both intercellular and intracellular communication. Like a symbol or a letter of an alphabet in a code or a language, the meaning of TGF-β always needs to be considered in the context of all the other signals present.” – M. Sporn (1)
Transforming Growth Factor Beta (TGF-β) constitutes a superfamily of cytokines – a class of intercellular signaling molecules in some ways similar to hormones – deployed across the animal kingdom by most cell types to facilitate cellular communication. Under normal physiological conditions, members of the TGF-β family control early embryonic development, motility (cell movement) and apoptosis (programmed cell death), and play important roles in immune system function and tumor suppression (2). They play important roles in cell fate decisions, including differentiation – a highly controlled set of modifications in gene expression that leads to higher cellular specialization via changes in phenotype and behavior – and are central to proliferative inhibition across diverse cell types, though transformed cells often escape this influence (3). TGF-β family members are also integral to a number of disease states, including cancer, heart disease, and diabetes, among others. For a sense of its importance, estimates suggest that nearly all pancreatic and colon cancer cell lines possess mutations in one or more of the TGF-β pathway genes (3).
During the 1970s, in the midst of a broad attempt to identify proteins involved in tumorigenesis – what was at the time described as “transformation” and defined as in vitro anchorage-independent growth (1) – TGF-β was identified as one of two polypeptides, along with TGF-α, responsible for inducing rat kidney fibroblast culture growth in soft agar conditions (4). These “transforming growth factors“, originally thought to function only as growth factors, were described as 25-kDa homodimers (1). However, this simple picture was overturned in 1984 when TGF-β, unlike its TGF-α twin, was identified as a potent growth inhibitor that mediated epidermal growth factor (EGF) effects on cellular transformation as well (1, 4, 5), leading to the contemporary view of TGF-β as robust, multifunctional signaling molecules.
Based on phylogenetic gene sequence analysis of the multigene families involved in TGF-β signaling, Newfeld et al. established TGF-β evolution among metazoa, after their divergence from plants and fungi (6). More specifically, they placed its emergence as a fully functioning pathway at approximately 1 billion years ago (6) between the separation events of arthropods and nematodes and of arthropods and vertebrates. Since then, the TGF-β signaling pathway has been highly conserved across species (5) [Figure 1 (7), Consurf Jmol, left]. The mature cytokine ligands are characteristically constituted, in part, by nine C-terminus cysteines, seven of which observe a defined spacing pattern in all TGF-β subfamily members (8). In humans, thirty-four members of the TGF-β family – including three major isoforms called TGF-β1, TGF-β2 and TGF-β3 – utilize the same serine/threonine kinase receptors (5), further demonstrating the functional conservation within the family.
The superfamily, including nearly forty distinct members (2), can best be grouped by sequence similarity into four main subfamilies: TGF-β1-3, bone morphogenic proteins (BMP), activin/inhibins, and a group including divergent members (6). More broadly, the superfamily is associated with the specialized high-affinity cell surface receptors with which its members interact, as well as the downstream cellular components of the Smad and MAPK molecular pathways through which its members ultimately regulate gene transcription [Figure 5 (9)].
TGF-β STRUCTURE AND FUNCTION
The genes associated with TGF-β isoforms encode 390-412 amino acids-long precursor proteins constituting three distinct domains: a N-terminal signal domain, which associates the full precursor molecule to the proper cellular secretory pathways; a propeptide domain, which may support folding or dimerization of the mature cytokine; and an approximately 100-114 amino acids-long C-terminal “TGF-β-like” domain – the functional autocrine signaling molecule – which is highly conserved across the superfamily (2, 7). The secreted precursors are proteolytically processed in the Golgi apparatus, which then releases the mature C-terminal protein fragment as a disulfide-bonded homodimer. This homodimer is constituted by two identical subunits linked by four internal disulfide bonds and a single cross-linking disulfide bond (5), and is further stabilized by hydrophobic effects. Structurally, each monomer of the dimer pair is comprised of several β strands (9).
Isoform Structural Distinctions
The three isoforms of TGF-β currently recognized were first identified from three peaks of activity seen during some of the earliest attempts to purify TGF-β. The N-linked glycosylation sites, nine cysteine residues, the hydrophobic dimer interface and the protein backbone are highly conserved features across all three isoforms (5). However, the chromosomal location of the gene associated with each isoform is distinct. In humans, the TGF-β1 gene is located at 19q13; the TGF-β2 gene at 1q41; and the TGF-β3 gene at 14q23-4 (5).
In their mature forms, TGF-β1 and TGF-β2 are 71% identical; within the 29% of residue variation are several important differences, for example: TGF-β2 has a distinct amino terminal sequence that is not recognized by antibodies in TGF-β1. TGF-β2 also has a unique interchain disulfide bond formed at cysteine 77. Further, the absolute magnitude of the hydrophobic area is larger in TGF-β1 than in TGF-β2, and there are a number of divergent amino acids along their surfaces and loops (residues 8-14), further distinguishing each isoform from another [Figure 2 (10)]. TGF-β3, which is the most abundant isoform in chick embryos and plays an important role in the fusion of the palatal shelves in birds during development, is 76% identical to TGF-β2 and 71% identical to TGF-β1 (5) [Figure 4 (10)]. Although the secondary crystalline structures do not differ greatly, some proteins have higher affinities for certain isoforms, demonstrating the functional consequences of these small structural differences among them (7) [Figure 3 (10)].
FUNCTIONAL CHARACTERISTICS IN NORMAL TISSUES
The Functional Pathway
TGF-β family members play central roles in metazoan developmental processes, including initiation of appendage formation in adult flies, establishment of the mammalian left-right body plane (11), and regulation of nematode morphology (12). TGF-β signaling is initiated through highly specific binding and complex formation between the active ligand molecule and its corresponding cell surface receptors [Figure 6a, Figure 6b (9)]. The TGF-β receptors, types I and II, are serine/threonine kinases and are brought into proximity such that the TGF-β receptor II phosphorylates the receptor I kinase domain, which subsequently phosphorylates R-Smad proteins – members of the Smad tumor suppressing protein family (13). Once phosphorylated, R-Smad proteins undergo homotrimerization to form heteromeric oligomer complexes with the Co-Smad protein, Smad4. Once in the nucleus, these Smad complexes regulate target gene transcription (9, 13). This regulation is not explicit or rigid, but rather variable depending on the cell’s genetic status, functional identity, environmental constraints, and concurrent signaling, which collectively determine the specific genes affected and the outputs modulated (3) [Figure 5 (9)].
Diverse Functional Roles
During human development, homeostasis is maintained across the embryo’s rapidly expanding networks of tissues and organ systems through carefully balanced molecular signaling pathways (14). In this context, from embryogenesis to organogenesis and continuing throughout the life-cycle, TGF-β superfamily cytokines play critical roles. For example, bone morphogenic protein-4 (BMP4), a member of the superfamily, is active during early inner cell mass proliferation, and later in development among neural, bone and dermal cell types (3). Other family members are involved in left-right axis symmetry and asymmetry formation, vascular development, and in cardiac, lung, craniofacial, and urogenital development (3). Mutations in these critical TGF-β systems, whether in the genes encoding the cytokines, their receptors, or members of the downstream intercellular signaling pathways, are responsible for a wide spectrum of developmental disorders, as well as many adult disease states (3).
TGF-β IN CANCER
TGF-β signaling not only acts as a tumor suppressor, but has been shown, in vitro and in vivo, to act as a powerful stimulator of tumor progression, driving malignancy toward increasing invasiveness and metastases. Early in tumorigenesis, TGF-β, through its normal pathways, actively suppresses cell growth in general and tumor growth particularly (4). However, if a tumor population is established, such as in carcinomas for example, biochemical and genetic alterations reinterpret TGF-β pathway signaling activity to instead differentially promote proliferation and ultimately invasion, partially through alteration of the cellular microenvironment (4, 5). Further, TGF-β activity levels correlate with clinical tumor stage and have been used to predict tumor progression (15), during which, transformed cells typically develop progressive insensitivity to TGF-β growth suppression signals. Concurrently, these tumor cells begin to secrete TGF-β at elevated levels, which in turn differentially increases tumor cell signaling while restricting the normal cellular environment surrounding the tumor growth (13) through normal growth restriction pathways and by restraining immune surveillance. Since most tumor cells that have selectively lost responsiveness to TGF-β growth-inhibitory influence retain their sensitivity to other TGF-β signaling pathway effects, TGF-β may promote tumor migration / invasion behavior (3) [Figure 7 (15)].
TGF-β also induces epithelial mesenchymal transition (EMT) – a process vital to cellular proliferation, growth and development – leading to a loss of cell junctions, which increases cell motility by releasing tumor cells into their local environment, causing invasion and metastasis (5).
TGF-β emerged early in metazoan evolution and, perhaps driven by ligand duplication and subsequent sequence diversification, was quickly established as a powerful and ubiquitous family of cytokine signaling molecules, associated receptor families and transcription factors. Various dating techniques have placed TGF-β genesis at around 1 billion years ago, after the divergence of nematodes from arthropods and the other Animal phyla (6).
Progress in our understanding of the TGF-β superfamily in general and the TGF-β subfamily in particular has been steady. We now know that TGF-β evolved as a critical component in the biochemical physiology of multicellular animals, and that mutations in genes associated with it are major factors in human disease. The discernment of its subtle but pervasive mechanistic interactions has progressed dramatically in key areas, but there remains much yet to discover. For example, the connection between TGF-β and cellular microenvironmental changes, as well as the mechanisms of control over those connections (including posttranslational modifications such as phosphorylation, acylation, alkylation, isophrenylation, etc.) are leading research questions. Efforts to fully characterize the structural and functional distinctions among TGF-β isoforms, particularly in inflammatory and immune systems – Including their gene level regulatory elements – constitutes another among the many lines currently being pursued in TGF-β research (1).
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