Smad3 Deficiency Research Articles

Activation of PPARα Ameliorates Cardiac Fibrosis in Dsg2-Deficient Arrhythmogenic Cardiomyopathy.

HighlightsCardiac-specific Dsg2 deletion induces excessive cardiac fibrosis in mice.Fenofibrate alleviates cardiac fibrosis in CS-Dsg2−/− mice.Cardiac-specific activation of PPARα ameliorates cardiac fibrosis in CS-Dsg2−/− mice.The inhibitory effect of PPARα on cardiac fibrosis is mediated by STAT3 and TGF-β /SMAD3 signaling.PPARα is a promising target for the intervention of ACM by ameliorating cardiac fibrosis.Background: Arrhythmogenic cardiomyopathy (ACM) is a genetic heart muscle disease characterized by progressive fibro-fatty replacement of cardiac myocytes. Up to now, the existing therapeutic modalities for ACM are mostly palliative. About 50% of ACM is caused by mutations in genes encoding desmosomal proteins including Desmoglein-2 (Dsg2). In the current study, the cardiac fibrosis of ACM and its underlying mechanism were investigated by using a cardiac-specific knockout of Dsg2 mouse model. Methods: Cardiac-specific Dsg2 knockout (CS-Dsg2−/−) mice and wild-type (WT) mice were respectively used as the animal model of ACM and controls. The myocardial collagen volume fraction was determined by histological analysis. The expression levels of fibrotic markers such as α-SMA and Collagen I as well as signal transducers such as STAT3, SMAD3, and PPARα were measured by Western blot and quantitative real-time PCR. Results: Increased cardiac fibrosis was observed in CS-Dsg2−/− mice according to Masson staining. PPARα deficiency and hyperactivation of STAT3 and SMAD3 were observed in the myocardium of CS-Dsg2−/− mice. The biomarkers of fibrosis such as α-SMA and Collagen I were upregulated after gene silencing of Dsg2 in HL-1 cells. Furthermore, STAT3 gene silencing by Stat3 siRNA inhibited the expression of fibrotic markers. The activation of PPARα by fenofibrate or AAV9-Pparα improved the cardiac fibrosis and decreased the phosphorylation of STAT3, SMAD3, and AKT in CS-Dsg2−/− mice. Conclusions: Activation of PPARα alleviates the cardiac fibrosis in ACM.

Smad3 deficiency improves islet-based therapy for diabetes and diabetic kidney injury by promoting β cell proliferation via the E2F3-dependent mechanism.

Rationale: Poor β cell proliferation is one of the detrimental factors hindering islet cell replacement therapy for patients with diabetes. Smad3 is an important transcriptional factor of TGF-β signaling and has been shown to promote diabetes by inhibiting β cell proliferation. Therefore, we hypothesize that Smad3-deficient islets may be a novel cell replacement therapy for diabetes.Methods: We examined this hypothesis in streptozocin-induced type-1 diabetic mice and type-2 diabetic db/db mice by transplanting Smad3 knockout (KO) and wild type (WT) islets under the renal capsule, respectively. The effects of Smad3KO versus WT islet replacement therapy on diabetes and diabetic kidney injury were examined. In addition, RNA-seq was applied to identify the downstream target gene underlying Smad3-regulated β cell proliferation in Smad3KO-db/db versus Smad3WT-db/db mouse islets.Results: Compared to Smad3WT islet therapy, treatment with Smad3KO islets produced a much better therapeutic effect on both type-1 and type-2 diabetes by significantly lowering serum levels of blood glucose and HbA1c and protected against diabetic kidney injuries by preventing an increase in serum creatinine and the development of proteinuria, mesangial matrix expansion, and fibrosis. These were associated with a significant increase in grafted β cell proliferation and blood insulin levels, resulting in improved glucose intolerance. Mechanistically, RNA-seq revealed that compared with Smad3WT-db/db mouse islets, deletion of Smad3 from db/db mouse islets markedly upregulated E2F3, a pivotal regulator of cell cycle G1/S entry. Further studies found that Smad3 could bind to the promoter of E2F3, and thus inhibit β cell proliferation via an E2F3-dependent mechanism as silencing E2F3 abrogated the proliferative effect on Smad3KO β cells.Conclusion: Smad3-deficient islet replacement therapy can significantly improve both type-1 and type-2 diabetes and protect against diabetic kidney injury, which is mediated by a novel mechanism of E2F3-dependent β cell proliferation.

Smad3 deficiency promotes beta cell proliferation and function in db/db mice via restoring Pax6 expression.

Rationale: Transforming Growth Factor-beta (TGF-β) /Smad3 signaling has been shown to play important roles in fibrotic and inflammatory diseases, but its role in beta cell function and type 2 diabetes is unknown.Methods: The role of Smad3 in beta cell function under type 2 diabetes condition was investigated by genetically deleting Smad3 from db/db mice. Phenotypic changes of pancreatic islets and beta cell function were compared between Smad3 knockout db/db (Smad3KO-db/db) mice and Smad3 wild-type db/db (Smad3WT-db/db) mice, and other littermate controls. Islet-specific RNA-sequencing was performed to identify Smad3-dependent differentially expressed genes associated with type 2 diabetes. In vitro beta cell proliferation assay and insulin secretion assay were carried out to validate the mechanism by which Smad3 regulates beta cell proliferation and function.Results: The results showed that Smad3 deficiency completely protected against diabetes-associated beta cell loss and dysfunction in db/db mice. By islet-specific RNA-sequencing, we identified 8160 Smad3-dependent differentially expressed genes associated with type 2 diabetes, where Smad3 deficiency markedly prevented the down-regulation of those genes. Mechanistically, Smad3 deficiency preserved the expression of beta cell development mediator Pax6 in islet, thereby enhancing beta cell proliferation and function in db/db mice in vivo and in Min6 cells in vitro.Conclusions: Taken together, we discovered a pathogenic role of Smad3 in beta cell loss and dysfunction via targeting the protective Pax6. Thus, Smad3 may represent as a novel therapeutic target for type 2 diabetes prevention and treatment.

Deletion of Smad3 protects against C-reactive protein-induced renal fibrosis and inflammation in obstructive nephropathy.

Introduction and Aims: Elevated plasma levels of C-reactive protein (CRP) are closely associated with progressive renal injury in patients with chronic kidney disease (CKD). Here, we tested a hypothesis that CRP may promote renal fibrosis and inflammation via a TGF-β/Smad3-dependent mechanism.Methods: Role and mechanisms of TGF-β/Smad3 in CRP-induced renal fibrosis and inflammation were examined in a mouse model of unilateral ureteral obstruction (UUO) induced in CRP Tg/Smad3 KO mice and in a rat tubular epithelial cell line in which Smad3 gene is stably knocked down (S3KD-NRK52E).Results: We found that mice overexpressing the human CRP gene were largely promoted renal inflammation and fibrosis as evidenced by increasing IL-1β, TNF-α, MCP-1 expression, F4/80+ macrophages infiltration, and marked accumulation of α-smooth muscle actin (α-SMA), collagen I and fibronectin in the UUO kidney, which were blunted when Smad3 gene was deleted in CRPtg-Smad3KO. Mechanistically, we found that the protection of renal inflammation and fibrosis in the UUO kidney of CRPtg-Smad3KO mice was associated with the inactivation of CD32-NF-κB and TGF-β/Smad3 signaling.Conclusion: In conclusion, Smad3 deficiency protects against CRP-mediated renal inflammation and fibrosis in the UUO kidney by inactivating CD32-NF-κB and TGF-β/Smad3 signaling.

Identification of Smad3-related transcriptomes in type-2 diabetic nephropathy by whole transcriptome RNA sequencing.

Smad3 deficiency prevents the development of type 2 diabetic nephropathy; however, the underlying molecular mechanisms remain unknown. In this study, we aimed to identify Smad3‐related genes involved in the pathogenesis of diabetic kidney disease. High‐throughput RNA sequencing was performed to profile the whole transcriptome in the diabetic kidney of Smad3 WT‐db/db, Smad3 KO‐db/db, Smad3+/− db/db and their littermate control db/m mice at 20 weeks. The gene ontology, pathways and alternative splicing of differentially expressed protein‐coding genes and long non‐coding RNAs related to Smad3 in diabetic kidney were analysed. Compared to Smad3 WT‐db/db mice, Smad3 KO‐db/db mice exhibited an alteration of genes associated with RNA splicing and metabolism, whereas heterozygosity deletion of Smad3 (Smad3+/− db/db mice) significantly altered genes related to cell division and cell cycle. Notably, three protein‐coding genes (Upk1b, Psca and Gdf15) and two lncRNAs (NONMMUG023520.2 and NONMMUG032975.2) were identified to be Smad3‐dependent and to be associated with the development of diabetic nephropathy. By using whole transcriptome RNA sequencing, we identified novel Smad3 transcripts related to the development of diabetic nephropathy. Thus, targeting these transcripts may represent a novel and effective therapy for diabetic nephropathy.

246-LB: Smad3 Deficiency Promotes Beta-Cell Proliferation by Augmenting Glucose-Stimulated UPR

Understanding the mechanism governing β cell proliferation is essential for the in vitro expansion of β cells and the replacement therapy for patients with diabetes. We recently found that deletion of Smad3, a downstream effector of TGF-β signaling, promoted β cell proliferation, while the underlying mechanisms remain elusive. It’s reported that high glucose stimulates β cell proliferation through unfold protein response (UPR). In this study, we tested the hypothesis that whether deficiency of Smad3 promotes β cell proliferation by regulating UPR. As a result, we found that knockout of Smad3 promoted β cell proliferation in diabetic db/db mice, but not in healthy db/m mice. Interestingly, islets from db/db mice of Smad3 knockout presented increased UPR compared with db/m mice of Smad3 knockout. In cultured islet cells, deletion of Smad3 increased β cell proliferation under high glucose (20mM) conditions, which was associated with up-regulated and non-decompensated UPR. Further studies showed that inhibition of UPR with an inhibitor 4-PBA abolished the pro-proliferative effect of Smad3 deficiency in β cells. In conclusion, Smad3 deficiency increases β cell proliferation by augmenting UPR. Therefore, targeting Smad3 may be an effective therapy for diabetes. Disclosure H. Wang: None. H. He: None. X. Huang: None. L. Wang: None. R.C. Ma: Advisory Panel; Self; AstraZeneca. Stock/Shareholder; Self; GemVCare. Other Relationship; Self; AstraZeneca, Bayer Healthcare Pharmaceuticals Inc., Merck & Co., Inc., Novo Nordisk A/S, Pfizer Inc., Sanofi-Aventis, Tricida Inc. H. Lan: None. Funding Research Grants Council of Hong Kong (C7018-16G, R4012-18); Health and Medical Research Fund of Hong Kong (14152321, 05161326, 06173986)

In Vitro Lineage-Specific Differentiation of Vascular Smooth Muscle Cells in Response to SMAD3 Deficiency: Implications for SMAD3-Related Thoracic Aortic Aneurysm.

SMAD3 pathogenic variants are associated with the development of thoracic aortic aneurysms. We sought to determine the role of SMAD3 in lineage-specific vascular smooth muscle cells (VSMCs) differentiation and function. Approach and Results: SMAD3 c.652delA, a frameshift mutation and nonsense-mediated decay, was introduced in human-induced pluripotent stem cells using CRISPR-Cas9. The wild-type and SMAD3-/- (c.652delA) human-induced pluripotent stem cells were differentiated into cardiovascular progenitor cells or neural crest stem cells and then to lineage-specific VSMCs. Differentiation, contractility, extracellular matrix synthesis, and TGF-β (transforming growth factor-β) signaling of the differentiated VSMCs were analyzed. The homozygous frameshift mutation resulted in SMAD3 deficiency and was confirmed in human-induced pluripotent stem cells by Sanger sequencing and immunoblot analysis. In cardiovascular progenitor cell-VSMCs, SMAD3 deletion significantly disrupted canonical TGF-β signaling and decreased gene expression of VSMC markers, including SM α-actin, myosin heavy chain 11, calponin-1, SM22α, and key controlling factors, SRF and myocardin, but increased collagen expression. The loss of SMAD3 significantly decreased VSMC contractility. In neural crest stem cells-VSMCs, SMAD3 deficiency did not significantly affect the VSMC differentiation but decreased ELN (elastin) expression and increased phosphorylated SMAD2. Expression of mir-29 was increased in SMAD3-/- VSMCs, and inhibition of mir-29 partially rescued ELN expression. SMAD3-dependent TGF-β signaling was essential for the differentiation of cardiovascular progenitor cell-VSMCs but not for the differentiation of neural crest stem cell-VSMCs. The lineage-specific TGF-β responses in human VSMCs may potentially contribute to the development of aortic root aneurysms in patients with SMAD3 mutations.

SAT-177 KLF11 DEFICIENCY EXACERBATES RENAL INJURY IN EXPERIMENTAL UNILATERAL URETERIC OBSTRUCTION

Kruppel-like factors (KLFs) are a family of zinc-finger transcription factors that play a critical role in regulation of growth and development, proliferation, and regeneration. Members of this family have been linked to podocyte dysfunction and renal fibrosis. In other systems, KLF11 has been shown to regulate TGF-beta signaling and fibrosis. Although KLF deficiency has been linked to endometriosis-related fibrosis, the role of KLF11 in mediating renal injury has not been previously established. We sought to test the hypothesis that KLF11 deficiency exacerbates renal damage in the murine unilateral ureteral obstruction (UUO) of renal fibrosis. We generated KLF11 knockout (KO), Smad 3 wild type (WT) mice, KLF11 WT Smad 3 KO, KLF11 KO Smad 3 KO, and KLF11/Smad3 WT mice and performed unilateral ureteral obstruction. Kidneys were harvested after 9 days (n=4 animals per group). Semiquantitative histopathologic analysis of renal atrophy, fibrosis, and inflammation was performed in a blinded fashion. Gene expression analysis was performed on renal cortex using Pathway Detect RNA Arrays. Renal atrophy (Figure 1) and inflammation (data not shown) were increased in KLF11 KO Smad3 WT mice compared to WT/WT mice subjected to UUO. As expected, Smad3 deficiency was associated with significantly less renal injury (Figure 1). Compared to WT/WT mice, KLF11 KO Smad3 WT mice showed marked upregulation of pro-inflammatory chemokines including CCL7 (250-fold compared to sham), Cxcl 11 (170-fold), Ccl12 (143-fold), Csf1 (117-fold), Ccl5 (96-fold), and Ccl2 (76-fold), inductions that were 2-5 fold higher than those observed in WT/WT mice. KLF11 deficiency was associated with modest upregulation of pro-fibrotic genes including Col1a2, Col1a1, and TGF-beta 1-3. Smad3 deficiency was associated with significant reduction of both pro-inflammatory and pro-fibrotic genes, both in the presence and absence of Klf11 deficiency. KLF11 deficiency exacerbates renal injury in the UUO model of renal fibrosis through upregulation of pro-inflammatory chemokine genes. To our knowledge, this is the first report linking KLF11 signaling to renal injury. Future studies will focus on potential mechanisms whereby KLF11 mediated chemokine signaling intersects with TGF-beta/Smad signaling to produce renal injury.

Deletion of Smad3 prevents renal fibrosis and inflammation in type 2 diabetic nephropathy

BackgroundTransforming growth factor (TGF)-β/Smad3 signaling is highly activated in kidneys of patients with type 2 diabetic nephropathy (T2DN), however, the precise role of Smad3 in the pathogenesis of diabetic nephropathy remains unclear. MethodsSmad3 knockout (KO)-db/db mice were generated by intercrossing of male and female double-heterozygous Smad3+/− db/m mice. Renal functions including urinary albumin excretion and serum creatinine were determined. Renal histological injury including renal fibrosis and inflammation were examined by periodic acid Schiff (PAS), periodic acid-silver methenamine (PASM), and immunohistochemistry (IHC) staining. ResultsSmad3 knockout (KO)-db/db mice were protected from the development of diabetic kidney injury, characterized by the normal levels of urinary albumin excretion and serum creatinine without any evidence for renal fibrosis and inflammation. In contrast, Smad3 wild-type (WT) db/db and Smad3+/− db/db mice developed progressively decline in renal function over the 12 to 32-week time course, including increased microalbuminuria and elevated levels of serum creatinine. Pathologically, Smad3 WT db/db and Smad3+/− db/db mice exhibited a marked deposition of collagen-I (colI), collagen-IV(col-IV), and an increased infiltration of F4/80+ macrophages in kidney. Mechanistically, Smad3 deficiency decreased the lncRNA Erbb4-IR transcription, while increased miR-29b transcription and therefore protected the kidney from progressive renal injury in db/db mice. ConclusionResults from this study imply that Smad3 may represent as a novel and effective therapeutic target for T2DN.

Increased Smad3 and reduced Smad2 levels mediate the functional switch of TGF-β from growth suppressor to growth and metastasis promoter through TMEPAI/PMEPA1 in triple negative breast cancer.

Screening of several TNBC cell lines showed altered Smad2 and Smad3 protein levels compared to normal mammary epithelial cells, suggesting the possibility that it could play an important role in the escape of cancer cells from TGF-β mediated growth inhibition. To assess the functional relevance of these endogenous molecules, Smad2 or Smad3 expression was knocked down individually and assessed their effects on pro-oncogenic properties of TGF-β. Smad3 deficiency reduced growth and invasion capacity of breast cancer cells in comparison to Smad2 which had no effect. Smad3 deficiency was also found to be associated with a reduction in the expressions of TMEPAI/PMEPA1 and EMT inducing transcription factors, E-Cadherin and increased expression of cell cycle inhibitors and Vimentin. On the other hand, Smad2 deficiency had opposite effect on these regulators. Interestingly, the decreased growth, invasion and associated gene expressions were largely reversed by overexpressing TMEPAI in Smad3 knockdown cells, suggesting that Smad3-TMEPAI axis may be involved in subverting growth suppressive effects of TGF-β into growth promotion. Similarly, altered levels of Smad proteins and TMEPAI were also noted in primary TNBC tumor tissues. Analysis of the existing databases provided additional support in terms of TMEPAI and Smad2 expression impacting the survival of TNBC patients. Taken together, our data demonstrate a novel role for Smad3 in cancer transformation and cancer progression through TMEPAI and further suggest that selective targeting of TGF-β-Smad3-TMEPAI axis may be beneficial in triple negative breast cancer therapy and prevention.

Smad3 Suppresses Epithelial Cell Migration and Proliferation via the Clock Gene Dec1, Which Negatively Regulates the Expression of Clock Genes Dec2 and Per1

Smad3 has circadian expression; however, whether Smad3 affects the expression of clock genes is poorly understood. Here, we investigated the regulatory mechanisms between Smad3 and the clock genes Dec1, Dec2, and Per1. In Smad3 knockout mice, the amplitude of locomotor activity was decreased, and Dec1 expression was decreased in the suprachiasmatic nucleus, liver, kidney, and tongue compared with control mice. Conversely, Dec2 and Per1 expression was increased compared with that of control mice. In Smad3 knockout mice, immunohistochemical staining revealed that Dec1 expression decreased, whereas Dec2 and Per1 expression increased in the endothelial cells of the kidney and liver. In NIH3T3 cells, Smad3 overexpression increased Dec1 expression, but decreased Dec2 and Per1 expression. In a wound-healing experiment that used Smad3 knockout mice, Dec1 expression decreased in the basal cells of squamous epithelium, promoting wound healing of the mucosa. Finally, the migration and proliferation of Smad3 knockdown squamous carcinoma cells was suppressed by Dec1 overexpression but was promoted by Dec2 overexpression. Dec1 overexpression decreased E-cadherin and proliferating cell nuclear antigen expression, whereas these expression levels were increased by Dec2 overexpression. These results suggest Smad3 is relevant to circadian rhythm and regulates cell migration and proliferation through Dec1, Dec2, and Per1 expression.

Identification of SMAD3 as a Novel Mediator of Inflammation in Human Myometrium In Vitro.

Preterm birth remains the primary cause of early neonatal death and is a major determinant for long-term health consequences. Aberrant intrauterine inflammation and infection are known to augment the synthesis of proinflammatory cytokines and induce uterine contractions, which can subsequently lead to preterm birth. The transforming growth factor-β (TGF-β) superfamily members regulate numerous cellular processes through the activation of intracellular mediators known as mothers against decapentaplegic homolog (SMADs). Studies in nongestational tissues have shown that SMAD3 plays a role in immune regulation and inflammation; however, its role in human labour remains unknown. Thus, the present study aimed at (i) characterising the expression of SMAD3 in the human myometrium; (ii) determining the effect of bacterial and viral products and proinflammatory cytokines on SMAD3 transcriptional activity in primary human myometrial cells; and (iii) investigating the effect of SMAD3 siRNA knockdown on the production of prolabour mediators in primary human myometrial cells. Phosphorylated (i.e., active) SMAD3 protein expression was lower in the myometrium after spontaneous term labour compared to the myometrium from nonlabouring women. Using a luciferase assay, the proinflammatory cytokines IL-1β and TNF, and viral analogue polyinosinic : polycytidylic acid (poly(I : C)) significantly reduced SMAD3 transcriptional activity in human primary myometrial cells. Loss-of-function studies found that SMAD3 knockdown in myometrial cells significantly increased IL-1β- and poly(I : C)-induced proinflammatory cytokines (IL-1A, IL-6), chemokines (IL-8, MCP-1), the adhesion molecule ICAM-1, COX-2 mRNA expression, and subsequent PGF2α release. In conclusion, SMAD3 deficiency is associated with increased production of proinflammatory and prolabour mediators in the human myometrium.

Abstract 698: Functional Analysis of Smad3 Deficiency in VSMCs Derived From Crispr/cas9-modified Human Induced Pluripotent Stem Cells

Introduction: Transcription factor Smad3 plays key roles in both TGF-β signaling transduction and vascular smooth muscle cells(VSMCs) differentiation. SMAD3 gene mutations cause aneurysms-osteoarthritis syndrome(AOS), an autosomal dominant genetic disease with high rate occurrence of thoracic aortic aneurysm(TAA) and early-onset osteoarthritis. The production of patient-derived hiPSCs and genomic-modified ihPSCs by using Crispr/Cas9 technique provide a promise for in vitro disease modeling of SMAD3 mutation in VSMCs as well as drug screening for potential therapeutic targets. Hypothesis: We hypothesis that introducing SMAD3 mutations into hIPSCs that mimic the identified genotypic alteration in AOS patients could recapture the phenotypic defects of differentiation, contractibility and extracellular matrix(ECM) of VSMCs derived from hIPSCs-differentiated neural crest stem cells(NCSCs), the embryonic origin of VSMCs for both aorta root and ascending aorta. Method: hIPSCs were generated from non-TAA patients’ peripheral blood mononuclear cells by overexpression of Yamanaka factors. The indel mutation targeting SMAD3 exon5 were induced by using Crispr/Cas9 technique, which form frameshift mutation close to one in identified AOS patients. Selected cell clones of genomic modified hIPSCs were differentiated into NCSCs-derived VSMCs. Comparing analysis of VSMCs function were proceed in both hIPSCs lines with or without SMAD3 mutation. Result: We generated a homozygote with SMAD3 frameshift mutation. It contains a 7-base pair deletion(c.633_639delGATGTCC), which is similar with a pathogenic Smad3 mutation (c.652 delA). This mutation leads to a premature stop codon and remove the Smad3 protein. The qRCR and WB result showed that the expression of ACTA2 decreased in the differentiated SMC with SMAD3 mutation. Also, the expression of elastin, collagen type III were significantly down-regulated in the SMAD3 mutation cell line while the MMP9 was up-regulated. Conclusion: SMAD3 mutation may cause defects of differentiation, contractibility and ECM synthesis of VSMCs derived from NCSC.

Abstract P1-01-22: TGFβ-Smad3-TMEPAI axis drives the tumor progression of triple negative breast cancer

Abstract Background: In the absence of mutations and/or deletions in the TGF-β signaling molecules, how triple negative breast cancer (TNBC) cells become resistant to growth inhibitory signaling of TGF-β was addressed by screening of several TNBC cell lines for Smad proteins involved in TGF-β signaling. Unexpectedly, altered Smad2 and Smad3 protein levels were detected in breast cancer cells relative to normal mammary epithelial cells, suggesting a novel mechanism to escape from TGF-β mediated growth inhibition. In order to reflect the true functional status of the endogenous molecules, Smad2 or Smad3 were individually knocked down and tested its effect on pro-oncogenic behavior of TGF-β. Materials and Methods: All cell lines were cultured according to the recommended standard procedures. TMEPAI knockdown was achieved by using lentiviral vectors and knockdown of Smad2 and Smad3 was achieved by using retroviral vectors. DNA transfections and luciferase assays were performed according to vendor instructions. Cell proliferation was measured by quantitation of total cellular DNA. Immunoblotting, invasion and immunohistochemical assays were performed using standard methods. Bioinformatics: Triple negative breast cancer patient dataset (GSE58812) was used to compare Smad2, Smad3, Smad4 and TMEPAI/PMEPA1 expressions correlated with overall survival using the PROGgene tool by using median gene expression value as a dividing point. Results: While Smad2 deficiency has no effect on breast cancer cell behavior, Smad3 deficiency reduced growth and invasion capacity of breast cancer cells. Interestingly, Smad3 deficiency was associated with reduced gene expressions of transmembrane prostate androgen induced (TMEPAI or PMEPA1) gene and EMT inducing transcription factors and increased expression of cell cycle inhibitors. In contrast, Smad2 deficiency had opposite effect on these regulators. Importantly, the decreased growth and invasion and their associated gene expressions in Smad3 knockdown cells were largely reversed by overexpression of TMEPAI. Meta-analysis of Jezequel dataset of triple negative breast cancer patients suggested higher TMEPAI/PMEPA1 and lower Smad2 expressions are significantly associated with decreased survival. Our results support the idea that development of several triple negative breast cancers may involve expansion of cell populations with altered Smad2 and Smad3 levels resulting in Smad3 dependent expression of TMEPAI, which provides a competitive advantage for cancer cells to grow and metastasize in presence of TGF-β. Moreover, following meta-analysis of published microarray datasets in lung and gastric cancers also revealed that increased TMEPAI and Smad3 expression and decreased Smad2 expressions were significantly associated with poorer patient prognosis in non-small cell lung adenocarcinomas and gastric cancers suggesting universality of this phenomenon. Conclusion: We identified TGF-β-Smad3-TMEPAI signaling axis as a driver of tumor progression in triple negative breast cancer. Our results suggest that novel therapeutics targeting TMEPAI will selectively inhibit oncogenic activity of TGF-β and promote its tumor suppressive activity in treating TNBC. Citation Format: Saikumar P, Singha PK, Pandeswara S, Manjeri VA. TGFβ-Smad3-TMEPAI axis drives the tumor progression of triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-22.

Cardiovascular phenotype in Smad3 deficient mice with renovascular hypertension.

Renovascular hypertension (RVH) has deleterious effects on both the kidney and the heart. TGF-β signaling through Smad3 directs tissue fibrosis in chronic injury models. In the 2-kidney 1-clip (2K1C) model of RVH, employing mice on the 129 genetic background, Smad3 deficiency (KO) protects the stenotic kidney (STK) from development of interstitial fibrosis. However, these mice have an increased incidence of sudden cardiac death following 2K1C surgery. The purpose of this study was to characterize the cardiovascular phenotype of these mice. Renal artery stenosis (RAS) was established in Wild-type (WT) and Smad3 KO mice (129 genetic background) by placement of a polytetrafluoroethylene cuff on the right renal artery. Mortality was 25.5% for KO mice with RAS, 4.1% for KO sham mice, 1.2% for WT with RAS, and 1.8% for WT sham mice. Myocardial tissue of mice studied at 3 days following surgery showed extensive myocyte necrosis in KO but not WT mice. Myocyte necrosis was associated with a rapid induction of Ccl2 expression, macrophage influx, and increased MMP-9 activity. At later time points, both KO and WT mice developed myocardial fibrosis. No aortic aneurysms or dissections were observed at any time point. Smad3 KO mice were backcrossed to the C57BL/6J strain and subjected to RAS. Sudden death was observed at 10–14 days following surgery in 62.5% of mice; necropsy revealed aortic dissections as the cause of death. As observed in the 129 mice, the STK of Smad3 KO mice on the C57BL/6J background did not develop significant chronic renal damage. We conclude that the cardiovascular manifestations of Smad3 deficient mice are strain-specific, with myocyte necrosis in 129 mice and aortic rupture in C57BL/6J mice. Future studies will define mechanisms underlying this strain-specific effect on the cardiovascular system.

Abstract 1958: Opposing roles of Smad2 and Smad3 in the regulation of growth and invasion of TNBC through TMEPAI

Abstract The dependency of triple negative breast cancers (TNBC) on TGF-β signaling activity for their growth and metastasis is well documented. Breast tumors develop resistance to TGF-β-induced growth inhibition, which helps in tumor progression. Additionally, TGF-β is enriched in the tumor microenvironment and is a key inducer of epithelial to mesenchymal transition (EMT) in breast cancer. In an effort to understand the mechanisms by which TGF-β induces breast tumorigenesis, we sought to identify the relative individual contributions of Smad2 and Smad3 in mediating cancer cell growth and metastasis by TGF-β. Screening of several TNBC cell lines showed increased Smad3/Smad2 expression ratio compared to normal mammary epithelial cells, which suggested breast cancer cells may escape TGF-β mediated cell cycle arrest by altering their relative expressions of Smad3 and Smad2. Since overexpression of R-Smads may not always accurately reflect the functional status of the endogenous molecules, we individually knocked down each R-Smad and tested its effect on pro-oncogenic behavior of TGF-β. Although Smad2 deficiency has no effect on breast cancer cell behavior, Smad3 deficiency reduced growth and invasion capacity of breast cancer cells. Interestingly, while Smad3 deficiency was associated with reduced TMEPAI/PMEPA1 and EMT gene expressions and increased expression of cell cycle inhibitors, Smad2 deficiency had opposite effect on these regulators. Moreover, the decreased growth, invasion and associated gene expressions were largely reversed by overexpressing TMEPAI in Smad3 knockdown cells, suggesting that Smad3-TMEPAI axis may be involved in subverting growth suppressive effects of TGF-β into growth promotion. Compared to control and Smad2 deficient cells, Smad3 deficient cells had markedly elevated PTEN protein levels and suppressed Akt phosphorylation both under basal conditions and TGF-β stimulation. We identified that Smad3 but not Smad2 plays an important role in stimulating TMEPAI expression, growth and EMT in triple negative breast cancer cells by promoting TGF-β dependent non-canonical signaling by decreasing the cellular content of PTEN and p27 in a TMEPAI dependent manner. Taken together, the results demonstrate a novel role for Smad3 in cancer transformation and cancer progression through TMEPAI. Current therapeutic strategies are aiming at antagonizing the whole TGF-β signaling thereby completely blocking both Smad2 and Smad3 signaling pathways. Selective targeting of TGF-β-Smad3-TMEPAI axis may be more beneficial in triple negative breast cancer therapy and prevention. Citation Format: Prajjal K. Singha, Srilakshmi Pandeswara, Manjeri A. Venkatachalam, Pothana Saikumar. Opposing roles of Smad2 and Smad3 in the regulation of growth and invasion of TNBC through TMEPAI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1958. doi:10.1158/1538-7445.AM2017-1958

TGF-β Signaling: New Insights Into Aortic Aneurysms

Aneurysm-osteoarthritis syndrome (AOS) is an autosomal dominant SMAD3 mutation that is characterized by aneurysms within the arterial tree and early-onset arthritis of the joints (van der Linde et al., 2012, van der Linde et al., 2013). It is uniquely different from Marfan's syndrome (MFS) which is a disorder of the extracellular matrix, specifically fibrillin-1 (Judge and Dietz, 2005). In this issue of EBioMedicine, van der Pluijm et al. dissect out the features of a whole body SMAD3 deficient mouse and compares it to a Fibulin-4 deficient mouse model (extracellular matrix defect model) (Fig. 8 of paper) (van der Pluijm et al., 2016). It is extremely interesting that while the phenotypes in aneurysm formation are similar, the molecular mechanisms are quite distinct. The authors note that there were no changes in aortic stiffness or MMP activity within the vascular smooth muscle cells (VSMCs) of the SMAD3-deficient mice, but instead more inflammatory infiltration of immune cells into the adventitia. Likewise, while both models show elevation of phosphorylated SMAD2 and ERK, the downstream targets of TGF-β signaling are decreased with SMAD3 deficiency, but have no effect or increased signaling with Fibulin-4 deficient mice (van der Pluijm et al., 2016).

Smad3 deficiency inhibits dentate gyrus LTP by enhancing GABAA neurotransmission.

Transforming growth factor-β signaling through intracellular Smad3 has been implicated in Parkinson's disease (PD) and it fulfills an important role in the neurogenesis and synaptic plasticity that occurs in the adult dentate gyrus (DG). The long-term potentiation (LTP) induced in the DG by high-frequency stimulation of the medial perforant pathway is abolished in the DG of Smad3-deficient mice, but not in the CA1 hippocampal region. Here, we show that NMDA- and AMPA-type glutamate receptors do not participate in the inhibition of LTP associated with Smad3 deficiency. Moreover, there is no difference in the hippocampal GAD65 and GAD67 content, suggesting that GABA biosynthesis remains unaffected. Increased conductance and higher action potential firing thresholds were evident in intracellular recordings of granule cells from Smad3 deficient mice. Interestingly, phasic and tonic GABAA receptor (GABAA R)-mediated neurotransmission is enhanced in the DG of Smad3-deficient mice, and LTP induction can be rescued by inhibiting GABAA R with picrotoxin. Hence, Smad3 signaling in the DG appears to be necessary to induce LTP by regulating GABAA neurotransmission, suggesting a central role of this intracellular signaling pathway in the hippocampal brain plasticity related to learning and memory. Smad3 deficient mice represent a new and interesting model of Parkinson's disease, displaying hippocampal dysfunctions that include decreased neurogenesis and the failure to induce LTP in the dentate gyrus. Here we show that Smad3 deficiency inhibits LTP induction by enhancing phasic and tonic GABAA receptor-mediated neurotransmission, while LTP induction can be rescued with a GABAA receptor antagonist. Alteration of GABA neurotransmission is thought to produce hippocampal cognitive dysfunction in Down's syndrome or Alzheimer's disease, and here we provide new insights into the hippocampal changes in an animal model of Parkinson's disease.

Smad3 deficiency protects mice from obesity-induced podocyte injury that precedes insulin resistance

Signaling by TGF-β/Smad3 plays a key role in renal fibrosis. As obesity is one of the major risk factors of chronic and end-stage renal disease, we studied the role of Smad3 signaling in the pathogenesis of obesity-related renal disease. After switching to a high fat diet, the onset of Smad3 C-terminal phosphorylation, increase in albuminuria, and the early stages of peripheral and renal insulin resistance occurred at 1 day, and 4 and 8 weeks, respectively, in C57BL/6 mice. The loss of synaptopodin, a functional marker of podocytes, and phosphorylation of the Smad3 linker region (T179 and S213) appeared after 4 weeks of the high fat diet. This suggests a temporal pattern of Smad3 signaling activation leading to kidney injury and subsequent insulin resistance in the development of obesity-related renal disease. In vivo, Smad3 knockout attenuated the high fat diet-induced proteinuria, renal fibrosis, overall podocyte injury, and mitochondrial dysfunction in podocytes. In vitro palmitate caused a rapid activation of Smad3 in 30 min, loss of synaptopodin in 2 days, and impaired insulin signaling in 3 days in isolated mouse podocytes. Blockade of either Smad3 phosphorylation by SIS3 (a Smad3 inhibitor) or T179 phosphorylation by flavopiridol (a CDK9 inhibitor) prevented the palmitate-induced loss of synaptopodin and mitochondrial function in podocytes. Thus, Smad3 signaling plays essential roles in obesity-related renal disease and may be a novel therapeutic target.

SMAD3 deficiency promotes vessel wall remodeling, collagen fiber reorganization and leukocyte infiltration in an inflammatory abdominal aortic aneurysm mouse model

TGF-β signaling plays critical roles in the pathogenesis of aneurysms; however, it is still unclear whether its role is protective or destructive. In this study, we investigate the role of SMAD3 in the pathogenesis of calcium chloride (CaCl2)-induced abdominal aortic aneurysms (AAA) in Smad3−/−, Smad3+/− and Smad3+/+ mice. We find that loss of SMAD3 drastically increases wall thickening of the abdominal aorta. Histological analyses show significant vessel wall remodeling with elastic fiber fragmentation. Remarkably, under polarized light, collagen fibers in the hyperplastic adventitia of Smad3−/− mice show extensive reorganization accompanied by loosely packed thin and radial collagen fibers. The expressions of matrix metalloproteinases including MMP2, MMP9, and MMP12 and infiltration of macrophage/T cells are drastically enhanced in the vascular wall of Smad3−/− mice. We also observe marked increase of NF-κB and ERK1/2 signaling as well as the expression of nuclear Smad2, Smad4 and TGF-β1 in the vessel wall of Smad3−/− mice. In addition, we find that SMAD3 expression is reduced in the dedifferentiated medial smooth muscle-like cells of human AAA patients. These findings provide direct in vivo evidence to support the essential roles of SMAD3 in protecting vessel wall integrity and suppressing inflammation in the pathogenesis of AAAs.