SMAD Signaling Network

Within the vasculature, TGF-β (Transforming Growth Factor-β) superfamily of secreted polypeptide growth factors play an important role in a variety of pathophysiologic processes, including angiogenesis, vascular remodeling, atherogenesis and in regulating cellular responses such as growth, proliferation, differentiation, migration, adhesion, survival, and specification of developmental fate. Apart from TGF-β, the superfamily also includes the Activins and the BMPs (Bone Morphogenetic Proteins). These factors signal through heteromeric complexes of Type-II and Type-I serine-threonine kinase receptors, which activate the downstream SMAD (Sma and Mad Related Family) signal transduction pathway (Ref.1, 2 & 3).

Based on their structures and known functional roles, the mammalian SMAD family members (Mad-homologues, MADH) fall into at least three broad classes: (i) the Co-SMADs (Co-mediator SMADs), SMAD4/DPC4 and SMAD10, participate in signaling by diverse TGF-β family members; (ii) the R-SMADs (Receptor-regulated SMADs), including SMAD1, SMAD2, SMAD3, SMAD5, and SMAD8, which are each involved in a specific signaling pathways; and (iii) the antagonistic SMADs, including SMAD6 and SMAD7, which negatively regulate these pathways (Ref.1). TGF-β and Activin receptors phosphorylate SMAD2 and SMAD3, and BMP receptors phosphorylate SMAD1, SMAD5 and SMAD8. To initiate a particular TGF-β response, dimeric ligands of the TGF-β superfamily bind with high affinity to Type-II receptor and trans-phosphorylate Type-I receptor serine/threonine kinases on the cell surface. Activated receptors recruit adaptor proteins such as DAB2 (Disabled-2) and SNX6 (Sortin Nexin-6) that positively affect signal transduction (Ref.5). Small GTPases such as Rab5 catalyze movement of activated receptor complexes to early endosomal compartments (Ref.6), where they encounter phospholipid-bound carriers such as SARA (SMAD Anchor for Receptor Activation) that assist in recruitment of the SMADs to the Type-I receptor kinase. SARA does not interact with either SMAD1 or SMAD5 (Ref.7).

The binding of R-SMADs, SMAD2 and SMAD3, to the phosphorylated GS domain via their phosphoserine-binding MH2 (Mad Homology-2) domain leads to its rapid dissociation from the receptor and SARA. These phosphoserines are recognized by the MH2 domain of another SMAD leading to homo-oligomerisation of R-SMADs or hetero-oligomerisation with the unique Co-SMAD (SMAD4/DPC4 in mammals). SMAD4/DPC4 is anchored to the cytoplasm by scaffolding proteins such as TRAP1 (TGF-β Receptor Type-I Associated Protein-1), which assist positively in R-SMAD/Co-SMAD oligomerisation (Ref.4). Phosphorylated SMAD3 associates with Importin-β1 and is imported to the nucleus. The Ran GTPase catalyses the transport and release of the SMAD3 complex in the nucleoplasm. In contrast, phosphorylated SMAD2 fails to bind to Importins and is autonomously imported to the nucleus. In the ground state, SMAD4/DPC4 enters the nucleus constitutively and is immediately exported back to the cytoplasm by the Exportin CRM1/XPO1. But, after TGF-β stimulation, SMAD4/DPC4 enters the nucleus in complex with R-SMADs (R-SMAD/Co-SMAD complexes) and regulates gene expression. Both SMAD3 and SMAD4/DPC4 bind the SBE (SMAD-Binding Elements) to DNA sequences. In contrast, SMAD2 fails to bind to SBEs but it participates in DNA-bound complexes via its interaction with SMAD4/DPC4, and activates expression of specific genes through cooperative interactions with DNA-binding proteins, including members of the winged-helix family of TFs (Transcription Factors), FAST1 and FAST2 (Forkhead Activin Signal Transducers) (Ref.6). In addition, both R-SMADs and the Co-SMAD interact with many general and tissue-specific TFs via their MH1 or MH2 domains. The transcriptional activity of nuclear SMAD complexes within the nucleus is modulated by DNA-binding protein TGIF (TGF-β Induced Factor), proto-oncogene Ski and SnoN, which act as SMAD transcriptional co-repressors (Ref. 7 & 9). Non-DNA-binding TF also associate with nuclear SMADs and recruit co-activators such as CBP (CREB-Binding Protein)/p300 that lead to acetylation of nucleosomal histones and/or associated TF, which are crucial for transcriptional induction. Alternatively, the nuclear SMADs also recruit co-repressors that associate with HDACs (Histone Deacetylases) such as CTBP (C-Terminal Binding Protein) and Sin3, thus leading to transcriptional repression of target genes. R-SMADs 1, 2, and 3, can move independently into the nucleus, but SMAD4 must first complex with one of these SMADs to become localized in the nucleus. Nuclear SMADs also participate in ubiquitination reactions that lead to downregulation of the pathway itself or degradation of other TFs. Phosphorylated nuclear SMAD3 is ubiquitinated by the Roc1/SCF E3 Lligase after completion of its transcriptional role and is exported to the cytoplasm for proteasomal degradation (Ref.4). Cytoplasmic R-SMADs in the ground cell state is attacked by a SMAD-specific E3 Lligase family, the SMURFs (SMAD Ubiquitin Regulatory Factor-2), which also lead to proteasomal degradation of R-SMADs, and thus keep the available R-SMAD pools low. Alternatively, nuclear R-SMAD-SMURF complexes attack transcriptional repressors SnoN, and thus downregulate the repressor (Ref.5).

Third classes of SMADs, the I-SMADs, such as SMAD7 inhibit the recruitment and phosphorylation of R-SMADs. It also associates with SMURFs to form the SMAD7-SMURF complex after TGF-β stimulation and ubiquitinates the receptors on the cell surface or endosomal membranes; these are then targeted for degradation in proteasomes and lysosomes (Ref.4). Another adaptor protein, STRAP1, also binds to both Type-I receptors and SMAD7, and enhances the inhibitory activity of SMAD7 (Ref.5). Microtubules serve as tracks for intracellular SMAD movement. Filamin, an actin crosslinking factor and scaffolding protein, also associates with SMADs and positively regulates transduction of SMAD signals. SMAD signaling can be regulated by the Ras-ERK-MAPK pathway in response to receptor tyrosine kinase activation and also by the functional interaction of SMAD2 with Ca2+-Calmodulin (Ref.8). In addition, the expression of SMAD6 and SMAD7 is enhanced by multiple signals including EGF (Epidermal Growth Factor), stimulation of AP-1 (Activator Protein-1) by phorbol ester 12-O Tetradecanoylphorbol-13 Acetate, and IFN-γ (Interferon-γ), which provide an important mechanism whereby these pathways negatively regulate SMAD activation (Ref.9).

SMADs are ubiquitously expressed throughout development and in all adult tissues and many of them (SMAD2, SMAD4/DPC4, SMAD5, SMAD6 and SMAD8) are produced from alternatively spliced mRNAs (Ref.4). SMAD2 and SMAD4/DPC4 are important for transcriptional and antiproliferative responses to TGF-β, and their inactivation in human cancers indicates that they are tumor suppressors (Ref. 10 & 11). Deletion of SMAD3 results in slow follicular growth, increased atresia, and infertility by either affecting selected hormone levels, by altering the expression of selected receptors in the ovary and/or by altering genes that regulate cell survival in the ovary.

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