Smad family members have been found only in animals. Within the animal kingdom they have been identified in eumetazoans (multicellular organisms with many types of cells) but not yet in metazoans such as sponges (multicellular organisms with very few cell types). However, several transmembrane receptors with similarity to both type I and type II TGF-β receptors have been identified in a freshwater sponge. A phylogenetic analysis showed that the sponge receptors are very similar to the unusual C. elegans receptors DAF-1 and SMA-6 that also fall between receptor types. The similarity between sponge and nematode receptors suggests that Smad-like proteins will eventually be found in sponges. Thus, ancestral TGF-β family members and their signaling pathways predate the metazoan/eumetazoan divergence roughly 1.5 billion years ago.
Members of SMAD family
Table 1. SMAD family related products
Mothers against decapentaplegic homolog 1 also known as SMAD family member 1 or SMAD1 is a protein that in humans is encoded by the SMAD1 gene. The Smad1/Mad subfamily contains signal transducing R-Smads dedicated to DPP/BMP subfamily ligands. This protein mediates the signals of the bone morphogenetic proteins (BMPs), which are involved in a range of biological activities including cell growth, apoptosis, morphogenesis, development and immune responses.
Figure 1. Structure of the SMAD1 protein.
Mothers against decapentaplegic homolog 2 also known as SMAD family member 2 or SMAD2 is a protein that in humans is encoded by the SMAD2 gene. SMAD2 mediates the signal of the transforming growth factor (TGF)-beta, and thus regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation. This protein is recruited to the TGF-beta receptors through its interaction with the SMAD anchor for receptor activation (SARA) protein.
Mothers against decapentaplegic homolog 3 also known as SMAD family member 3 or SMAD3 is a protein that in humans is encoded by the SMAD3 gene. SMAD3 is a polypeptide with a molecular weight of 48,080 Da. It belongs to the SMAD family of proteins. SMAD3 is recruited by SARA (SMAD Anchor for Receptor Activation) to the membrane, where the TGF-β receptor is located. The receptors for TGF-β, (including nodal, activin, myostatin and other family members) are membrane serine/threonine kinases that preferentially phosphorylate and activate SMAD2 and SMAD3. Based on its essential role in TGF beta signaling pathway, SMAD3 has been related with tumor growth in cancer development.
SMAD4, also called SMAD family member 4, Mothers against decapentaplegic homolog 4, or DPC4 (Deleted in Pancreatic Cancer-4) is a highly-conserved protein present in all metazoans. It belongs to the SMAD family of transcription factor proteins, which act as mediators of TGF-β signal transduction. The TGF-β family of cytokines regulates critical processes during the lifecycle of metazoans, with important roles during embryo development, tissue homeostasis, regeneration, and immune regulation. SMAD 4 belongs to the co-SMAD group, the second class of the SMAD family. SMAD4 is the only known co-SMAD in most metazoans. It also belongs to the Darwin family of proteins that modulate members of the TGF-β protein superfamily, a family of proteins that all play a role in the regulation of cellular responses.
Figure 2. Structure of the SMAD4 protein.
Mothers against decapentaplegic homolog 5 also known as SMAD5 is a protein that in humans is encoded by the SMAD5 gene. It belongs to the SMAD family of proteins, which belong to the TGF-β superfamily of modulators. Like many other TGF-β family members SMAD5 is involved in cell signalling and modulates signals of bone morphogenetic proteins (BMP's). The binding of ligands causes the oligomerization and phosphorylation of the SMAD5 protein. SMAD5 is a receptor regulated SMAD (R-SMAD) and is activated by bone morphogenetic protein type 1 receptor kinase. It may play a role in the pathway where TGF-β is an inhibitor of hematopoietic progenitor cells.
Figure 3. Structure of the SMAD5 protein.
SMAD family member 6, also known as SMAD6, is a protein that in humans is encoded by the SMAD6 gene. It belongs to the SMAD family of proteins, which belong to the TGF-β superfamily of modulators. Like many other TGF-β family members SMAD6 is involved in cell signalling. It acts as a regulator of TGFβ family (such as bone morphogenetic proteins) activity by competing with SMAD4 and preventing the transcription of SMAD4's gene products. There are two known isoforms of this protein. Heterozygous, damaging mutations in SMAD6 are the most frequent genetic cause of non-syndromic craniosynostosis identified to date.
Mothers against decapentaplegic homolog 7 or SMAD7 is a protein that in humans is encoded by the SMAD7 gene. SMAD7 is involved in cell signalling. It is a TGF-β type 1 receptor antagonist. It blocks TGF-β1 and activin associating with the receptor, blocking access to SMAD2. It is an inhibitory SMAD (I-SMAD) and is enhanced by SMURF2. SMAD7 enhances muscle differentiation. Smad proteins contain two conserved domains. The Mad Homology domain 1 (MH1 domain) is at the N-terminal and the Mad Homology domain 2 (MH2 domain) is at the C-terminal. Between them there is a linker region which is full of regulatory sites. The MH1 domain has DNA binding activity while the MH2 domain has transcriptional activity. The linker region contains important regulatory peptide motifs including potential phosphorylation sites for mitogen-activated protein kinases(MAPKs), Erk-family MAP kinases, the Ca2+/calmodulin-dependent protein kinase II (CamKII) and protein kinase C (PKC).
Mothers against decapentaplegic homolog 9 also known as SMAD9, SMAD8, and MADH6 is a protein that in humans is enocoded by the SMAD9 gene. Like many other TGF-β family members, SMAD9 is involved in cell signalling. When a bone morphogenetic protein binds to a receptor (BMP type 1 receptor kinase) it causes SMAD9 to interact with SMAD anchor for receptor activation (SARA). The binding of ligands causes the phosphorylation of the SMAD9 protein and the dissociation from SARA and the association with SMAD4. It is subsequently transferred to the nucleus where it forms complexes with other proteins and acts as a transcription factor. SMAD9 is a receptor regulated SMAD (R-SMAD) and is activated by bone morphogenetic protein type 1 receptor kinase. There are two isoforms of the protein. Confusingly, it is also sometimes referred to as SMAD8 in the literature.
One mechanism by which Smads facilitate TGF-β induced cytostasis is by downregulating Myc, which is a transcription factor that promotes cell growth. Myc is also represses p15(Ink4b) and p21(Cip1), which are inhibitors of Cdk4 and Cdk2 respectively. When there is no TGF-β present, a repressor complex composed of Smad3, and the transcription factors E2F4 and p107 exist in the cytoplasm. However, when TGF-β signal is present, this complex localizes to the nucleus, where it associates with Smad4 and binds to the TGF-β inhibitory element (TIE) of the Myc promoter to repress its transcription.
In addition to Myc, Smads are also involved in the downregulation of Inhibitor of DNA Binding (ID) proteins. IDs are transcription factors that regulate genes involved in cell differentiation, maintaining multi-potency in stem cells, and promoting continuous cell cycling. Therefore, downregulating ID proteins is a pathway by which TGF-β signaling could arrest the cell cycle. In a DNA microarray screen, Id2 and Id3 were found to be repressed by TGF-β, but induced by BMP signaling. Knocking out Id2 and Id3 genes in epithelial cells enhances cell cycle inhibition by TGF-β, showing that they are important in mediating this cytostatic effect.
Receptor-regulated Smads (R-Smads), Smad1, 2, 3, 5 and 8, are the only Smads directly phosphorylated and activated by the kinase domain of type I receptors. Phosphorylation of R-Smads results in a conformational change, allowing complex-formation with the common-Smad (Co-Smad), Smad4. Activated complexes subsequently accumulate in the nucleus where they cooperate with other transcriptional co-regulators to modify target gene transcription. The third class of Smads includes the inhibitory Smads (I-Smads), Smad6 and Smad7, which function in a negative feedback loop to inhibit TGF-β superfamily signaling.
Role in disease
Research has shown that phosphorylated Smad2 is ectopically localized to cytoplasmic granules rather than the nucleus, in hippocampal neurons of patients with Alzheimer’s disease. Specifically, the ectopically located phosphorylated Smad2s were found within amyloid plaques, and attached to neurofibrillary tangles. These data suggest that Smad2 is involved in the development of Alzheimer’s disease. Recent studies show that the peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) is involved in promoting the abnormal localization of Smad2. Pin1 was found to co-localize with Smad2/3 and phosphorylated tau proteins within the cytoplasmic granules, suggesting a possible interaction. Transfecting Smad2 expressing cells with Pin1 causes proteasome-mediated Smad2 degradation, as well as increased association of Smad2 with phosphorylated tau. This feedback loop is bidirectional; Smad2 is also capable of increasing Pin1 mRNA synthesis. Thus, the two proteins could be caught in a “vicious cycle” of regulation. Pin1 causes both itself and Smad2 to be associated in insoluble neurofibrillary tangles, resulting in low levels of both soluble proteins. Smad2 then promotes Pin1 RNA synthesis to try and compensate, which only drives more Smad2 degradation and association with neurofibrillary tangles.
Defects in Smad signaling can result in TGF-β resistance, causing dysregulation of cell growth. Deregulation of TGF-β signaling has been implicated in many cancer types, including pancreatic, colon, breast, lung, and prostate cancer. Smad4 is most commonly mutated in human cancers, particularly pancreatic and colon cancer. Smad4 is inactivated in nearly half of all pancreatic cancers. As a result, Smad4 was first termed Deleted in Pancreatic Cancer Locus 4 (DPC4) upon its discovery. Germline Smad4 mutations are partially responsible for genetic disposition for human familial juvenile polyposis, which puts a person at high risk of developing potentially cancerous gastrointestinal polyps.
Despite evidence showing that Smad3 is more critical than Smad2 in TGF-β signaling, the rate of Smad3 mutations in cancer is lower than that of Smad2. Choriocarcinoma tumor cells are TGF-β signaling resistant, as well as lacking Smad3 expression. Studies show that reintroducing Smad3 into choriocarcinoma cells is sufficient to increase TIMP-1 (tissue inhibitor of metalloprotease-1) levels, a mediator of TGF-β’s anti-invasive effect, and thus restore TGF-β signaling. However, reintroducing Smad3 was not sufficient to rescue the anti-invasive effect of TGF-β. This suggests that other signaling mechanisms in addition to Smad3 are defective in TGF-β resistant choriocarcinoma.
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