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. [5] [6]
SMAD3 is a member of the SMAD family of proteins. It acts as a mediator of the signals initiated by the transforming growth factor beta (TGF-β) superfamily of cytokines, which regulate cell proliferation, differentiation and death. [7] [8] Based on its essential role in TGF beta signaling pathway, SMAD3 has been related with tumor growth in cancer development.
The human SMAD3 gene is located on chromosome 15 on the cytogenic band at 15q22.33. The gene is composed of 9 exons over 129,339 base pairs. [9] It is one of several human homologues of a gene that was originally discovered in the fruit fly Drosophila melanogaster .
The expression of SMAD3 has been related to the mitogen-activated protein kinase (MAPK/ERK pathway), particularly to the activity of mitogen-activated protein kinase kinase-1 (MEK1). [10] Studies have demonstrated that inhibition of MEK1 activity also inhibits SMAD3 expression in epithelial cells and smooth muscle cells, two cell types highly responsive to TGF-β1. [10]
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.
Once SMAD3 is phosphorylated at the C-terminus, it dissociates from SARA and forms a heterodimeric complex with SMAD4, which is required for the transcriptional regulation of many target genes. [11] The complex of two SMAD3 (or of two SMAD2) and one SMAD4 binds directly to DNA though interactions of the MH1 domain. These complexes are recruited to sites throughout the genome by cell lineage-defining transcription factors (LDTFs) that determine the context-dependent nature of TGF-β action. The DNA binding sites in promoters and enhancers are known as the SMAD-binding elements (SBEs). These sites contain the CAG(AC)|(CC) and GGC(GC)|(CG) consensus sequences, the latter also known as 5GC sites. [12] The 5GC-motifs are highly represented as clusters of sites, in SMAD-bound regions genome-wide. These clusters can also contain CAG(AC)|(CC) sites. SMAD3/SMAD4 complex also binds to the TPA-responsive gene promoter elements, which have the sequence motif TGAGTCAG. [13]
Transcriptional coregulators, such as WWTR1 (TAZ), interact with SMAD3 to promote their function.
The X-ray structures of the SMAD3 MH1 domain bound to the GTCT DNA reveal characteristic features of the fold. The MH1 structure consists of four-helices and three sets of antiparallel β-hairpins, one of which is used to interact with DNA. It also revealed the presence of a bound Zn2+, coordinated by His126, Cys64, Cys109 and Cys121 residues. [11] [12] The main DNA binding region of the MH1 domain comprises the loop following the β1 strand, and the β2-β3 hairpin. In the complex with a member of the 5GC DNAs, the GGCGC motif, the convex face of the DNA-binding hairpin dives into the concave major groove of the duplex DNA containing five base pairs (GGCGC /'GCGCC'). In addition, the three residues strictly conserved in all R-SMADS and in SMAD4 (Arg74 and Gln76 located in β2 and Lys81 in β3 in SMAD3) participate in a network of specific hydrogen bonds with the dsDNA. Several tightly bound water molecules at the protein-DNA interface that contribute to the stabilization of the interactions have also been detected. The SMAD3 complex with the GGCGC site reveals that the protein-DNA interface is highly complementary and that one MH1 protein covers a DNA binding site of six base pairs.
The MH2 domain mediates the interaction of R-SMADS with activated TGF-β receptors, and with SMAD4 after receptor-mediated phosphorylation of the Ser-X-Ser motif present in R-SMADS. The MH2 domain is also a binding platform for cytoplasmic anchors, DNA-binding cofactors, histone modifiers, chromatin readers, and nucleosome- positioning factors. The structure of the complex of SMAD3 and SMAD4 MH2 domains has been determined. [14] The MH2 fold is defined by two sets of antiparallel β-strands (six and five strands respectively) arranged as a β-sandwich flanked by a triple-helical bundle on one side and by a set of large loops and a helix on the other.
SMAD3 functions as a transcriptional modulator, binding the TRE (TPA responsive element) in the promoter region of many genes that are regulated by TGF-β. SMAD3 and SMAD4 can also form a complex with c-Fos and c-jun at the AP-1/SMAD site to regulate TGF-β-inducible transcription. [13] The genes regulated by SMAD3-mediated TGFβ signaling affect differentiation, growth and death. TGF-β/SMAD signaling pathway has been shown to have a critical role in the expression of genes controlling differentiation of embryonic stem cells. [15] Some of the developmental genes regulated by this pathway include FGF1, NGF, and WNT11 as well as stem/progenitor cell associated genes CD34 and CXCR4. [16] The activity of this pathway as a regulator of pluripotent cell states requires the TRIM33-SMAD2/3 chromatin reading complex. [15]
Besides the activity of TGF-β in the up-regulation of genes, this signaling molecule also induces the repression of target genes containing the TGF-β inhibitory element (TIE). [17] [18] SMAD3 plays also a critical role in TGF-β-induced repression of target genes, specifically it is required for the repression of c-myc. The transcriptional repression of c-myc is dependent on direct SMAD3 binding to a repressive SMAD binding element (RSBE), within TIE of the c-myc promoter. The c-myc TIE is a composite element, composed of an overlapping RSBE and a consensus E2F site, which is capable of binding at least SMAD3, SMAD4, E2F4, and p107. [18]
Increased SMAD3 activity has, however, been implicated in the pathogenesis of scleroderma.
SMAD3 is also a multifaceted regulator in adipose physiology and the pathogenesis of obesity and type 2 diabetes. SMAD3-knockout mice have diminished adiposity, [19] with improved glucose tolerance and insulin sensitivity. Despite their reduced physical activity arising from muscle atrophy, [20] these SMAD3-knockout mice are resistant to high-fat-diet induced obesity. SMAD3-knockout mouse is a legitimate animal model of human aneurysms‐osteoarthritis syndrome (AOS), also named Loeys-Dietz Syndrome (type 3). SMAD3 deficiency promotes inflammatory aortic aneurysms in angiotensin II-infused mice via the activation of iNOS. Macrophage depletion and inhibition of iNOS activity prevent aortic aneurysms related to SMAD3 gene mutation [21]
The role of SMAD3 in the regulation of genes important for cell fate, such as differentiation, growth and death, implies that an alteration in its activity or repressing of its activity can lead to the formation or development of cancer. Also several studies have proven the bifunctional tumor suppressor/oncogene role of TGF beta signaling pathway in carcinogenesis. [22]
One way in which SMAD3 transcriptional activator function is repressed, is by the activity of EVI-1. [23] EVI-1 encodes a zinc-finger protein that may be involved in leukaemic transformation of haematopoietic cells. The zinc-finger domain of EVI-1 interacts with SMAD3, thereby suppressing the transcriptional activity of SMAD3. EVI-1 is thought to be able to promote growth and to block differentiation in some cell types by repressing TGF-β signalling and antagonizing the growth-inhibitory effects of TGF-β. [23]
The activity of SMAD3 in prostate cancer is related with the regulation of angiogenic molecules expression in tumor vascularization and cell-cycle inhibitor in tumor growth. [24] [25] The progressive growth of primary tumors and metastases in prostate cancer depends on an adequate blood supply provided by tumor angiogenesis. Studies analyzing SMAD3 levels of expression in prostate cancer cell lines found that the two androgen-independent and androgen receptor-negative cell lines (PC-3MM2 and DU145) have high expression levels of SMAD3. Analysis of the relation between SMAD3 and the regulation of angiogenic molecules suggest that SMAD3 may be one of key components as a repressor of the critical angiogenesis switch in prostate cancer. [25] The pituitary tumor-transforming gene 1 (PTTG1) has also an impact in SMAD3-mediated TGFβ signaling. PTTG1 has been associated with various cancer cells including prostate cancer cells. Studies showed that the overexpression of PTTG1 induces a decrease in SMAD3 expression, promoting the proliferation of prostate cancer cells via the inhibition of SMAD3. [24]
In mice, mutation of SMAD3 has been linked to colorectal adenocarcinoma,[3] increased systemic inflammation, and accelerated wound healing.[4] Studies have shown that mutations in SMAD3 gene promote colorectal cancer in mice. [26] [27] [28] The altered activity of SMAD3 was linked to chronic inflammation and somatic mutations that contribute to chronic colitis and the development of colorectal cancer. [28] The results generated on mice helped identify SMAD3 like a possible player in human colorectal cancer. The impact of SMAD3 has also been analyzed in colorectal cancer human cell lines, using single-nucleotide polymorphism (SNP) microarray analysis. The results showed reductions in SMAD3 transcriptional activity and SMAD2-SMAD4 complex formation, underlining the critical roles of these three proteins within the TGF-β signaling pathway and the impact of this pathway in colorectal cancer development. [29]
TGF-β-induced SMAD3 transcriptional regulation response has been associated with breast cancer bone metastasis by its effects on tumor angiogenesis, and epithelial-mesenchymal transition (EMT). There have been identified diverse molecules that act over the TGF-β/SMAD signaling pathway, affecting primarily the SMAD2/3 complex, which have been associated with the development of breast cancer. [30]
FOXM1 (forkhead box M1) is a molecule that binds with SMAD3 to sustain activation of the SMAD3/SMAD4 complex in the nucleus. The research over FOXM1 suggested that it prevents the E3 ubiquitin-protein ligase transcriptional intermediary factor 1 γ (TIF1γ) from binding SMAD3 and monoubiquitinating SMAD4, which stabilized the SMAD3/SMAD4 complex. FOXM1 is a key player in the activity of the SMAD3/SMAD4 complex, promoting SMAD3 modulator transcriptional activity, and also plays an important role in the turnover of the activity of SMAD3/SMAD4 complex. Based on the importance of this molecule, studies have found that FOXM1 is overexpressed in highly aggressive human breast cancer tissues. The results from these studies also found that the FOXM1/SMAD3 interaction was required for TGF-β-induced breast cancer invasion, which was the result of SMAD3/SMAD4-dependent upregulation of the transcription factor SLUG. [31]
MED15 is a mediator molecule that promotes the activity of the TGF-β/SMAD signaling. The deficiency of this molecule attenuates the activity of the TGF-β/SMAD signaling pathway over the genes required for induction of epithelial-mesenchymal transition. The action of MED15 is related with the phosphorylation of SMAD2/3 complex. The knockdown of MED15 reduces the amount of SMAD3 phosphorylated, therefore reducing its activity as transcription modulator. However, in cancer, MED15 is also highly expressed in clinical breast cancer tissues correlated with hyperactive TGF-β signaling, as indicated by SMAD3 phosphorylation. The studies suggest that MED15 increases the metastatic potential of a breast cancer cell line by increasing TGF-β-induced epithelial–mesenchymal transition. [32]
Smad3 activation plays a role in the pathogenesis of renal fibrosis, [33] probably by inducing activation of bone marrow-derived fibroblasts. [34]
The SMAD proteins are homologs of both the Drosophila protein "mothers against decapentaplegic" (MAD) and the C. elegans protein SMA. The name is a combination of the two. During Drosophila research, it was found that a mutation in the gene MAD in the mother repressed the gene decapentaplegic in the embryo. The phrase "Mothers against" was inspired by organizations formed by mothers to oppose social problems, such as Mothers Against Drunk Driving (MADD); and based on a tradition of such unusual naming within the gene research community. [35]
A reference assembly of SMAD3 is available.
In cellular biology, paracrine signaling is a form of cell signaling, a type of cellular communication in which a cell produces a signal to induce changes in nearby cells, altering the behaviour of those cells. Signaling molecules known as paracrine factors diffuse over a relatively short distance, as opposed to cell signaling by endocrine factors, hormones which travel considerably longer distances via the circulatory system; juxtacrine interactions; and autocrine signaling. Cells that produce paracrine factors secrete them into the immediate extracellular environment. Factors then travel to nearby cells in which the gradient of factor received determines the outcome. However, the exact distance that paracrine factors can travel is not certain.
Transforming growth factor beta (TGF-β) is a multifunctional cytokine belonging to the transforming growth factor superfamily that includes three different mammalian isoforms and many other signaling proteins. TGFB proteins are produced by all white blood cell lineages.
Bone morphogenetic protein 4 is a protein that in humans is encoded by BMP4 gene. BMP4 is found on chromosome 14q22-q23.
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. MAD homolog 2 belongs to the SMAD, a family of proteins similar to the gene products of the Drosophila gene 'mothers against decapentaplegic' (Mad) and the C. elegans gene Sma. SMAD proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways.
SMAD4, also called SMAD family member 4, Mothers against decapentaplegic homolog 4, or DPC4 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 TGFβ family of cytokines regulates critical processes during the lifecycle of metazoans, with important roles during embryo development, tissue homeostasis, regeneration, and immune regulation.
SMAD family member 6, also known as SMAD6, is a protein that in humans is encoded by the SMAD6 gene.
Mothers against decapentaplegic homolog 7 or SMAD7 is a protein that in humans is encoded by the SMAD7 gene.
R-SMADs are receptor-regulated SMADs. SMADs are transcription factors that transduce extracellular TGF-β superfamily ligand signaling from cell membrane bound TGF-β receptors into the nucleus where they activate transcription TGF-β target genes. R-SMADS are directly phosphorylated on their c-terminus by type 1 TGF-β receptors through their intracellular kinase domain, leading to R-SMAD activation.
Smads comprise a family of structurally similar proteins that are the main signal transducers for receptors of the transforming growth factor beta (TGF-B) superfamily, which are critically important for regulating cell development and growth. The abbreviation refers to the homologies to the Caenorhabditis elegans SMA and MAD family of genes in Drosophila.
The transforming growth factor beta (TGFB) signaling pathway is involved in many cellular processes in both the adult organism and the developing embryo including cell growth, cell differentiation, cell migration, apoptosis, cellular homeostasis and other cellular functions. The TGFB signaling pathways are conserved. In spite of the wide range of cellular processes that the TGFβ signaling pathway regulates, the process is relatively simple. TGFβ superfamily ligands bind to a type II receptor, which recruits and phosphorylates a type I receptor. The type I receptor then phosphorylates receptor-regulated SMADs (R-SMADs) which can now bind the coSMAD SMAD4. R-SMAD/coSMAD complexes accumulate in the nucleus where they act as transcription factors and participate in the regulation of target gene expression.
Endoglin (ENG) is a type I membrane glycoprotein located on cell surfaces and is part of the TGF beta receptor complex. It is also commonly referred to as CD105, END, FLJ41744, HHT1, ORW and ORW1. It has a crucial role in angiogenesis, therefore, making it an important protein for tumor growth, survival and metastasis of cancer cells to other locations in the body.
The SKI protein is a nuclear proto-oncogene that is associated with tumors at high cellular concentrations. SKI has been shown to interfere with normal cellular functioning by both directly impeding expression of certain genes inside the nucleus of the cell as well as disrupting signaling proteins that activate genes.
The upstream signaling pathway is triggered by the binding of a signaling molecule, a ligand, to a receiving molecule, a receptor. Receptors and ligands exist in many different forms, and only recognize/bond to particular molecules. Upstream extracellular signaling transduce a variety of intracellular cascades.
Upstream stimulatory factor 1 is a protein that in humans is encoded by the USF1 gene.
MDS1 and EVI1 complex locus protein EVI1 (MECOM) also known as ecotropic virus integration site 1 protein homolog (EVI-1) or positive regulatory domain zinc finger protein 3 (PRDM3) is a protein that in humans is encoded by the MECOM gene. EVI1 was first identified as a common retroviral integration site in AKXD murine myeloid tumors. It has since been identified in a plethora of other organisms, and seems to play a relatively conserved developmental role in embryogenesis. EVI1 is a nuclear transcription factor involved in many signaling pathways for both coexpression and coactivation of cell cycle genes.
Serine-threonine kinase receptor-associated protein is an enzyme that in humans is encoded by the STRAP gene.
Forkhead box protein H1 is a protein that in humans is encoded by the FOXH1 gene.
Transforming growth factor beta (TGF-β) is a potent cell regulatory polypeptide homodimer of 25kD. It is a multifunctional signaling molecule with more than 40 related family members. TGF-β plays a role in a wide array of cellular processes including early embryonic development, cell growth, differentiation, motility, and apoptosis.
The Nodal signaling pathway is a signal transduction pathway important in regional and cellular differentiation during embryonic development.
In Xenopus laevis, the specification of the three germ layers occurs at the blastula stage. Great efforts have been made to determine the factors that specify the endoderm and mesoderm. On the other hand, only a few examples of genes that are required for ectoderm specification have been described in the last decade. The first molecule identified to be required for the specification of ectoderm was the ubiquitin ligase Ectodermin ; later, it was found that the deubiquitinating enzyme, FAM/USP9x, is able to overcome the effects of ubiquitination made by Ectodermin in Smad4. Two transcription factors have been proposed to control gene expression of ectodermal specific genes: POU91/Oct3/4 and FoxIe1/Xema. A new factor specific for the ectoderm, XFDL156, has shown to be essential for suppression of mesoderm differentiation from pluripotent cells.