Figure 1. TGF-β/SMAD Signaling Pathway.
An overview of TGF-β/SMAD
The TGF-β superfamily includes a diverse range of structurally and functionally related proteins, such as bone morphogenetic proteins (BMPs), activins, inhibins, growth differentiation factors (GDFs), and glial-derived neurotrophic factors (GDNFs). These members multifunctionally regulate a wide range of biological processes, such as morphogenesis, embryonic development, adult stem cell differentiation, immune regulation, wound healing, inflammation, and cancer. These proteins signal by stimulating formation of specific heteromeric complexes of type I and type II serine/threonine kinase receptors. When bioavailable TGF-β reaches the surface of the target cell, it binds a homodimer of TGF-β type II receptors (TβRII). The TGF-β-TβRII complex provides a structural interface that facilitates stable complex formation with a homodimer of the TGF-β type I receptor (TβRI). As an active receptor complex, the TβRII, which is a constitutively active kinase, undergoes autophosphorylation, and catalyzes transphosphorylation of the TβRI. Transphosphorylation of the TβRI activates kinase activity.
SMADs, the substrates for TβRI kinases, which are known to have a signaling function, were first recognized as the products of the Drosophila Mad and C. elegans Sma genes, which are downstream of the BMP-analogous ligand-receptor systems in these organisms. SMADs are ubiquitously expressed throughout development and in all adult tissues and many of them are produced from alternatively spliced mRNAs. SMAD proteins involve intracellular TGF-β signaling. According to their functions, members of the SMAD family can be categorized into three groups: 1) the receptor-regulated SMADs (R-SMADs) including SMAD1, SMAD2, SMAD3, SMAD5, and SMAD8; 2) the common SMAD (Co-SMAD), of which there is only one member, SMAD4; 3) the inhibitory SMADs (I-SMADs), which include SMAD6 and SMAD7. The R-SMADs bind to membrane-bound serine/threonine receptors, and are activated by their kinase activity. As a co-factor, the Co-SMAD binds to the activated R-SMADs to form a complex that translocates into the nucleus. I-SMADs counteract the effects of R-SMADs, thus exerting an inhibitory effect on TGF-β superfamily signaling by various mechanisms.
SMAD-dependent signaling mediated by TGF-β
Upon ligand binding, the constitutively active type II receptor kinase phosphorylates the type I receptor which, in turn, activates the downstream signal transduction cascades, including SMAD pathways. TGF-βs, activin, and Nodal initiate intracellular signaling by binding to the TβRII. Then, TGF-β activates the TβRI kinase, resulting in phosphorylation of SMAD2 and SMAD3, while SMAD1, 5, and 8 can signal by BMPs in certain contexts. Subsequently, the activated SMAD2 and SMAD3 form oligomeric complexes with SMAD4. TGF-βs, activin, and Nodal signaling through type I receptors are known as activin receptor-like kinase (ALK)-4, -5, and -7, respectively. As an exception, ALK-1, preferentially expressed in ECs, binds TGF-β and activates SMAD1/5 pathways. Recently, BMP-9 and BMP-10 were reported to bind to ALK-1. Once activated, R-SMADs will be complex with the common mediator SMAD4 (Co-SMAD) and translocate to the nucleus, where SMAD complexes regulate transcription of target genes through their interaction with various transcription factors, including the induction of SMAD7.
SMAD-independent signaling mediated by TGF-β
TGF-β has been shown to activate diverse non-SMAD parallel downstream pathways, such as extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase. TβRII and TβRI signal through TRAF6 and TAK1 to activate the JNK, p38 and inhibitor of kappa B (IkB) kinase (IKK) pathways, and through ShcA to activate the ERK pathway. TRAF6 also promotes the cleavage of TβRI by TACE; the cleaved intracelluar domain (ICD) of TβRI then associates with transcription factors to activate gene expression. The TβRI can directly phosphorylate PAR6 through recruitment by Smurf1 and target RhoA for degradation, which leads to the dissociation of tight junctions. Furthermore, TGF-β activates the RhoA during EMT. The Rho family of small GTPases is comprised of RhoA, Rac1, and Cdc42 and regulates the formation of stress fibers, lamellipodia, and filopodia, respectively. In BMP-associated non-SMAD signaling, BMP type II receptors bind and activate LIMK1 to inhibit the actin-disassembling factor cofilin.
Cross talks with other signaling
Wnt signaling benefits from extensive cross talks with other signaling pathways, particularly TGF-β and BMP signaling, and the combinatorial signaling often works in early embryos to allow overlapping signaling pathways to specify different territories and cell fates. In early embryos, extensive mutual regulation and crosstalk between Wnt and Nodal/activin/BMP pathways and later between Wnt and BMP signaling exist at multiple levels, and these interactions are essential for embryonic patterning and development of multiple lineages.
Notch signaling is an evolutionarily conserved pathway that regulates stem-cell-fate determination and differentiation during embryonic development, tissue homeostasis, and carcinogenesis. Many developmental processes regulated by Notch signaling are also controlled by TGF-β family ligands including BMPs, thus setting the stage for frequently occurring cross talk between the two pathways. Similar to BMPs, TGF-β can also cooperate with Notch to induce Hes1, Hey1, and Jag1 expression in a SMAD3-dependent manner through a SMAD3–NICD interaction.
Extensive cross talk between TGF-β and PI3K pathways has been reported for various cell types including stem cells and cancer cells. The cross talk activities are often complex and can result in mutual activation or inhibition depending on the cellular context and biological processes involved. In hESCs, activin-induced SMAD2 and/or SMAD3 signaling can modulate cell-fate decisions depending on the status of PI3K activation. In the presence of robust PI3K signals, SMAD2 and SMAD3 activate the expression of the pluripotency gene Nanog to maintain self-renewal. However, low PI3K activity switches SMAD2/3 signaling to direct mesendoderm differentiation.
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