Kit Contents:

Component	Specification	Concentration (total volume)
SMAD Reporter	A mixture of inducible SMAD-responsive firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).	(100 ng/µl; 500 µl)*
Negative control	A mixture of non-inducible firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).	(100 ng/µl; 500 µl)
Positive control	A mixture of constitutively expressing GFP, constitutively expressing firefly luciferase, and constitutively expressing Renilla luciferase constructs (40:1:1).	(100 ng/µl; 250 µl)

* Supplied material provides sufficient reporter for 500 assays, using recommended 96-well plate transfection protocol. The number of assays per kit is a function of the assay plate format used (refer to Cignal Reporter Assay User Manual).

Storage Conditions: The Cignal reporter assay constructs are shipped ambient. Store all tubes at -20  ºC.

1. Dilute transfection-ready reporter, negative control, and positive control construct formulations.
2. Dilute relevant test nucleic acids (siRNA, shRNA, miRNA, expression vector).
3. Prepare appropriate combinations of reporter constructs, controls, and test nucleic acids.
4. Transfect plasmid mixtures separately into replicate wells of your cell line of interest using an optimized transfection procedure for the cell line under study.
5. If applicable, 16 to 24 hours post-transfection, treat the transfected cells with test proteins, peptides, or compounds of interest.
6. Two (2) to three (3) days post-transfection, assay the activities of the signaling pathways under study, utilizing the dual luciferase assay.

The Cignal Reporter Assays include pre-formulated, transfection-ready reporter, negative control, and positive control. The transcription factor reporter and negative control are transfected and subjected to experimental treatments, in parallel. Dual-luciferase results are calculated for each transfectant. The impact of the experimental treatments is determined by comparing the normalized luciferase activities of the reporter to the identically treated negative control, across the complete treatment regimen. The positive control serves as a control for transfection efficiency, by monitoring GFP expression, as well as a positive control for both the firefly and Renilla luciferase assays.

General performance

Average maximum response rate = 29.3
Average Z' factor at maximum response rate = 0.9
Average coefficient of variation (CV%) = 5.6%

Excellent signal to noise ratio

Cignal SMAD reporter assay can measure increase in TGFβ signaling pathway activity: 293-H cells were transfected with SMAD reporter, negative control and positive control (for transfection protocol refer our user manual) . After 24 hours of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin) and cells were treated with different doses of recombinant human transforming growth factor β 1 protein (hTGFβ1) for 18 hours. Dual Luciferase assay was performed, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated.

Within the vasculature, TGF-Beta (Transforming Growth Factor-Beta) 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-Beta, 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-Beta 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-Beta and Activin receptors phosphorylate SMAD2 and SMAD3, and BMP receptors phosphorylate SMAD1, SMAD5 and SMAD8. To initiate a particular TGF-Beta response, dimeric ligands of the TGF-Beta 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-Beta Receptor Type-I Associated Protein-1), which assist positively in R-SMAD/Co-SMAD oligomerisation (Ref.4). Phosphorylated SMAD3 associates with Importin-Beta1 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-Beta 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-Beta 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-Beta 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-Gamma (Interferon-Gamma), 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-Beta, 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.

References:

1. Wu G, Chen YG, Ozdamar B, Gyuricza CA, Chong PA, Wrana JL, Massague J, Shi Y.
Structural basis of SMAD2 recognition by the SMAD anchor for receptor activation.
Science. 2000 Jan 7; 287(5450): 92-7.
PubMed ID: 10615055

2. Cook RW, Thompson TB, Kurup SP, Jardetzky TS, Wookdruff TK.
Structural basis for a functional antagonist in the transforming growth factor beta superfamily.
J. Biol. Chem. 2005 Dec 2;280(48):40177-86. Epub 2005 Sep 26.
PubMed ID: 16186117

3. Keah HH, Hearn MT.
A molecular recognition paradigm: promiscuity associated with the ligand-receptor interactions of the activin members of the TGF-beta superfamily.
J. Mol. Recognit. 2005 Sep-Oct;18(5):385-403.
PubMed ID: 15948132

4. Moustakas A, Souchelnytskyi S, Heldin CH.
SMAD regulation in TGF-beta signal transduction.
J. Cell Sci. 2001 Dec; 114(Pt 24): 4359-69.
PubMed ID: 11792802

5. Moustakas A.
SMAD signalling network.
J. Cell Sci. 2002 Sep 1; 115(Pt 17): 3355-6.
PubMed ID: 12154066

6. Sebestyen A, Barna G, Nagy K, Janosi J, Paku S, Kohut E, Berczi L, Mihalik R, Kopper L.
Smad signal and TGFbeta induced apoptosis in human lymphoma cells.
Cytokine. 2005 Jun 7;30(5):228-35. Epub 2005 Mar 17.
PubMed ID: 15927846

7. Fuchs O, Provaznikova D, Peslova G.
Promyelocytic leukaemia protein and defect in transforming growth factor-beta signal pathway in acute promyelocytic leukaemia
Cas. Lek. Cesk. 2005;144(2):90-4.
PubMed ID: 15807293

8. Pessah M, Prunier C, Marais J, Ferrand N, Mazars A, Lallemand F, Gauthier JM, Atfi A.
c-Jun interacts with the corepressor TG-interacting factor (TGIF) to suppress SMAD2 transcriptional activity.
Proc. Natl. Acad. Sci. U S A. 2001 May 22; 98(11): 6198-203.
PubMed ID: 11371641

9. Lo RS, Wotton D, Massague J.
Epidermal growth factor signaling via Ras controls the SMAD transcriptional co-repressor TGIF.
EMBO. J. 2001 Jan 15; 20(1-2): 128-36.
PubMed ID: 11226163

10. Wicks SJ, Lui S, Abdel-Wahab N, Mason RM, Chantry A.
Inactivation of SMAD-transforming growth factor beta signaling by Ca (2+)-calmodulin-dependent protein kinase II.
Mol. Cell Biol. 2000 Nov; 20(21): 8103-11.
PubMed ID: 11027280

11. Xu J, Attisano L.
Mutations in the tumor suppressors SMAD2 and SMAD4/DPC4 inactivate transforming growth factor beta signaling by targeting SMADs to the ubiquitin-proteasome pathway.
Proc. Natl. Acad. Sci. U S A. 2000 Apr 25; 97(9): 4820-5.
PubMed ID: 10781087



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