Role Of Smad3 Research Articles

(S035) Evaluating the Role of SMAD 4 as a Predictor of Clinical Outcomes in Pancreatic Cancer

(S035) Evaluating the Role of SMAD 4 as a Predictor of Clinical Outcomes in Pancreatic Cancer

Roles of Smads in regulating epithelial mesenchymal transition mediated by TGF-β1/Smads pathway

Roles of Smads in regulating epithelial mesenchymal transition mediated by TGF-β1/Smads pathway

Smad4 in T cells plays a protective role in the development of autoimmune Sjögren's syndrome in the nonobese diabetic mouse.

We investigated the role of Smad4, a signaling molecule of the TGF-beta pathway, in T cells on the pathology of Sjögren's syndrome (SS) in nonobese diabetic (NOD) mice, an animal model of SS. T cell-specific Smad4-deleted (Smad4fl/fl,CD4-Cre; Smad4 tKO) NOD mice had accelerated development of SS compared with wild-type (Smad4+/+,CD4-Cre; WT) NOD mice, including increased lymphocyte infiltration into exocrine glands, decreased tear and saliva production, and increased levels of autoantibodies at 12 weeks of age. Activated/memory T cells and cytokine (IFN-γ, IL-17)-producing T cells were increased in Smad4 tKO NOD mice, however the proportion and function of regulatory T (Treg) cells were not different between Smad4 tKO and WT NOD mice. Effector T (Teff) cells from Smad4 tKO NOD mice were less sensitive than WT Teff cells to suppression by Treg cells. Th17 differentiation capability of Teff cells was similar between Smad4 tKO and WT NOD mice, but IL-17 expression was increased under inducible Treg skewing conditions in T cells from Smad4 tKO NOD mice. Our results demonstrate that disruption of the Smad4 pathway in T cells of NOD mice increases Teff cell activation resulting in upregulation of Th17 cells, indicating that Smad4 in T cells has a protective role in the development of SS in NOD mice.

Smad2 and Smad3 Regulate Chondrocyte Proliferation and Differentiation in the Growth Plate

TGFβs act through canonical and non-canonical pathways, and canonical signals are transduced via Smad2 and Smad3. However, the contribution of canonical vs. non-canonical pathways in cartilage is unknown because the role of Smad2 in chondrogenesis has not been investigated in vivo. Therefore, we analyzed mice in which Smad2 is deleted in cartilage (Smad2CKO), global Smad3-/- mutants, and crosses of these strains. Growth plates at birth from all mutant strains exhibited expanded columnar and hypertrophic zones, linked to increased proliferation in resting chondrocytes. Defects were more severe in Smad2CKO and Smad2CKO;Smad3-/- (Smad2/3) mutant mice than in Smad3-/- mice, demonstrating that Smad2 plays a role in chondrogenesis. Increased levels of Ihh RNA, a key regulator of chondrocyte proliferation and differentiation, were seen in prehypertrophic chondrocytes in the three mutant strains at birth. In accordance, TGFβ treatment decreased Ihh RNA levels in primary chondrocytes from control (Smad2fx/fx) mice, but inhibition was impaired in cells from mutants. Consistent with the skeletal phenotype, the impact on TGFβ-mediated inhibition of Ihh RNA expression was more severe in Smad2CKO than in Smad3-/- cells. Putative Smad2/3 binding elements (SBEs) were identified in the proximal Ihh promoter. Mutagenesis demonstrated a role for three of them. ChIP analysis suggested that Smad2 and Smad3 have different affinities for these SBEs, and that the repressors SnoN and Ski were differentially recruited by Smad2 and Smad3, respectively. Furthermore, nuclear localization of the repressor Hdac4 was decreased in growth plates of Smad2CKO and double mutant mice. TGFβ induced association of Hdac4 with Smad2, but not with Smad3, on the Ihh promoter. Overall, these studies revealed that Smad2 plays an essential role in the development of the growth plate, that both Smads 2 and 3 inhibit Ihh expression in the neonatal growth plate, and suggested they accomplish this by binding to distinct SBEs, mediating assembly of distinct repressive complexes.

Smad3 Sensitizes Hepatocelluar Carcinoma Cells to Cisplatin by Repressing Phosphorylation of AKT.

Background: Heptocelluar carcinoma (HCC) is insensitive to chemotherapy due to limited bioavailability and acquired drug resistance. Smad3 plays dual roles by inhibiting cell growth initially and promoting the progression of advanced tumors in HCC. However, the role of smad3 in chemosensitivity of HCC remains elusive. Methods: The role of smad3 in chemosensitivity of HCC was measured by cell viability, apoptosis, plate colony formation assays and xenograft tumor models. Non-smad signaling was detected by Western blotting to search for the underlying mechanisms. Results: Smad3 enhanced the chemosensitivity of HCC cells to cisplatin. Smad3 upregulated p21Waf1/Cip1 and downregulated c-myc and bcl2 with the treatment of cisplatin. Moreover, overexpression of smad3 repressed the phosphorylation of AKT, and vice versa. Inhibition of PI3K/AKT pathway by LY294002 restored chemosensitivity of smad3-deficiency cells to cisplatin in HCC. Conclusion: Smad3 sensitizes HCC cells to the effects of cisplatin by repressing phosphorylation of AKT and combination of inhibitor of AKT pathway and conventional chemotherapy may be a potential way to solve drug resistance in HCC.

Smad7 and its Potential as Therapeutic Target in Inflammatory Bowel Diseases.

The etiology of Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease (IBD) in humans, is still unknown, but evidence suggests that genetic and environmental factors interact to promote an excessive immune response that leads to tissue damage. Defects in the counter-regulatory mechanisms are also supposed to make a major contribution to the amplification and maintenance of the IBD-related inflammatory response. One such a mechanism involves TGF-β1, a cytokine synthesized by both immune and non-immune cells in the gut, which is essential in the maintenance of immune homeostasis. In both CD and UC, active inflammation occurs in areas characterized by enhanced production of TGF-β1 and reduced ability of this cytokine to activate Smad-associated signaling and suppress inflammatory pathways. The defective TGF-β1 activity is due to elevated levels of Smad7, an intracellular protein that inhibits TGF-β1-associated Smad signaling. Data from original studies and reviews were selected through an accurate research of the literature using the terms "IBD", "colitis", "Crohn's disease", "Smad7", "TGF-β1", "antisense oligonucleotide" and "Mongersen". Twenty-fours studies describing the most accredited hypothesis about IBD pathogenesis and the role of Smad7 in the negative control of TGF-β1 were discussed in the review. Additionally, we reported data from original work illustrating the generation and the in vitro and in vivo effect of a specific Smad7 antisense oligonucleotide on intestinal inflammatory signals. We also discussed the results of phase 1 and phase 2 studies assessing the safety profile and clinical efficacy of Mongersen, an oral Smad7 antisense oligonucleotidecontaining drug, in patients with active CD. Data indicate that, in IBD, high Smad7 contributes to sustain detrimental immune responses and knockdown of this molecule can help attenuate the ongoing mucosal inflammation in patients with such disorders.

Smad2 increases the apoptosis of activated human hepatic stellate cells induced by TRAIL

The activation of hepatic stellate cells (HSCs) plays a critical role in the development of liver fibrosis. The induction of apoptosis in activated HSCs during the recovery phase of hepatic fibrosis represents a potential anti-fibrotic therapy. We have previously shown that Smad2 protects against hepatic fibrogenesis; however, the role of Smad2 in the regulation of activated HSC apoptosis remains unknown. We hypothesized that Smad2 regulates the apoptosis of activated HSCs, leading to the resolution of liver fibrosis. To test this hypothesis, the livers of rats were harvested at 0 and 4weeks after hepatic fibrosis was established by CCl4 injection. Furthermore, TGF-β1-activated HSCs were treated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) following the silencing or overexpression of Smad2. Both the phosphorylation of Smad2 and TRAIL were detected in fibrotic liver tissues. The results of TUNEL and α-SMA double-staining showed an increase in the apoptosis of activated HSCs during the spontaneous recovery phase. The knockdown of Smad2 reduced TRAIL-induced apoptosis in TGF-β1-activated human LX-2 cells and resulted in an increased expression of α-SMA and collagen I (Col. I). In contrast, the overexpression of Smad2 increased TRAIL-induced HSC apoptosis and reduced the expression of α-SMA and Col. I. The mechanisms underlying these findings were associated with the Smad2-mediated down-regulation of X-linked inhibitor of apoptosis protein (XIAP), resulting in enhanced caspase-3 activity and apoptosis. In conclusion, Smad2 enhances TRAIL-induced apoptosis in activated HSCs, which facilitates the resolution of hepatic fibrosis.

Role of Smad3 in platelet-derived growth factor-C-induced liver fibrosis.

Chronic liver injury leads to fibrosis and cirrhosis. Cirrhosis, the end stage of chronic liver disease, is a leading cause of death worldwide and increases the risk of developing hepatocellular carcinoma. Currently, there is a lack of effective antifibrotic therapies to treat fibrosis and cirrhosis. Development of antifibrotic therapies requires an in-depth understanding of the cellular and molecular mechanisms involved in inflammation and fibrosis after hepatic injury. Two growth factor signaling pathways that regulate liver fibrosis are transforming growth factor-β (TGFβ) and platelet-derived growth factor (PDGF). However, their specific contributions to fibrogenesis are not well understood. Using a genetic model of liver fibrosis, we investigated whether the canonical TGFβ signaling pathway was necessary for fibrogenesis. PDGF-C transgenic (PDGF-C Tg) mice were intercrossed with mice that lack Smad3, and molecular and histological fibrosis was analyzed. PDGF-C Tg mice that also lacked Smad3 had less fibrosis and improved liver lobule architecture. Loss of Smad3 also reduced expression of collagen genes, which were induced by PDGF-C, but not the expression of genes frequently associated with hepatic stellate cell (HSC) activation. In vitro HSCs isolated from Smad3-null mice proliferated more slowly than cells from wild-type mice. Taken together, these findings indicate that PDGF-C activates TGFβ/Smad3 signaling pathways to regulate HSC proliferation, collagen production and ultimately fibrosis. In summary, these results suggest that inhibition of both PDGF and TGFβ signaling pathways may be required to effectively attenuate fibrogenesis in patients with chronic liver disease.

Down-regulation of osteopontin mediates a novel mechanism underlying the cytostatic activity of TGF-β.

Loss of a cytostatic response to TGF-β has been implicated in multiple hyper-proliferative disorders, including cancer. Although several key genes involved in the cytostatic activity of TGF-β have in the past been identified, its exact mode of action is yet to be elucidated. A comprehensive understanding of the mechanisms underlying the cytostatic activity of TGF-β may open up new avenues for the development of therapeutic strategies. Quantitative real-time RT-PCR was used to assess osteopontin (OPN) gene expression in human hepatoma-derived Huh-7 and lung adenocarcinoma-derived A549 cells. Reporter assays using an OPN promoter-luciferase construct and its mutated counterparts were performed to assess its transcriptional activity. Binding of Smad4 to the OPN gene promoter was investigated using chromatin immunoprecipitation (CHIP). The putative role of Smad4 in OPN gene expression down-regulation was also assessed using a shRNA-mediated knockdown strategy. The anti-proliferative effect of TGF-β on different cancer-derived cell lines was determined using the cell proliferation reagent WST-1. We found that the OPN expression levels dose-dependently decreased in TGF-β-treated Huh-7 and A549 cells. Our reporter assays indicated that this TGF-β-induced repression occurred at the transcriptional level, and could largely be abrogated by disruption of an element (TIE2) similar to the TGF-β inhibitory element found in other TGF-β-repressed genes. Our CHIP assay revealed that the Smad protein complex specifically binds to the OPN gene promoter, and that the TGF-β-mediated inhibition of OPN was lost upon shRNA-mediated knockdown of Smad4. Moreover, we found that the deregulation of OPN gene expression by TGF-β occurred concomitantly with loss of the TGF-β anti-proliferative response, whereas a neutralizing anti-OPN antibody partially restored this response. Our results indicate that the OPN gene is a direct target of Smad-mediated TGF-β signaling, implying that OPN expression inhibition serves as a novel mechanism underlying the cytostatic activity of TGF-β.

Abstract 15568: Selective Deletion of Smad4 in Smooth Muscle Cells Causes Thoracic Aortic Aneurysm in Mice

TGFβ signaling have been implicated in the onset of thoracic aortic aneurysms (TAAs). Recently, it has been described that mutations in SMAD4, an intracellular transducer required in the TGFβ canonical pathway, present aortic disease in humans. Although transgenic models recapitulating mutations of TGFβ pathway have been studied, the role of Smad4 in the onset of TAA has never been explored. Smad4 homozygotes mice are embryonically lethal, thus, we postnatally targeted the gene, using a tamoxifen (TAM)-inducible Cre recombinase under the smooth muscle myosin heavy chain promoter. Activation by TAM suppressed Smad4 in aorta SMCs of Smadf/f;Myh11-CreERT2 mice. Most of these mice died 4/6 months later from hemothorax due to rupture of thoracic aorta, while untreated animals survived. Echocardiography showed a progressive dilatation of aorta and TAA formation. Histological analysis revealed progressive fragmentation of elastic lamellae with increased inflammatory infiltrates at sites of disarrangement, rich in macrophages. At the molecular level, we found that lack of Smad4 was followed by increased levels of P-Smad2 and P-Smad1/5/8 early after TAM. This finding suggested an enhancement of TGFβ receptors activity, a condition that could increase non-canonical TGFβ signaling, the canonical one being certainly depressed. We actually found a significant increase in one of the non-canonical TGFβ signaling, i.e. the p65 subunit of NFkB, a protein complex involved in regulating responses to different stress stimuli, including cytokines. Given the particular relevance that the latter molecules may have in TAA, we tested expression of some of them, finding a selective IL-1β upregulation, produced and stored as an inactive precursor and released in its active form by caspase-1 cleavage. In turn, caspase-1 requires the function of NLRP3 inflammasome, a multiprotein platform that assembles in response to infection and tissue damage and is responsible for activation of inflammatory caspases. Therefore, we also tested NLRP3 mRNA levels and found them to be significantly upregulated. Overall, we demonstrated that selective Smad4 deletion in the SMC of adult mice is sufficient to induce ultrastructural damage and immune reaction culminating in the onset of TAA.

Protective Role of Smad7 in Acute Kidney Injury (AKI)

Protective Role of Smad7 in Acute Kidney Injury (AKI)

Smad7 Modulates Epidermal Growth Factor Receptor Turnover through Sequestration of c-Cbl.

Epidermal growth factor (EGF) regulates various cellular events, including proliferation, differentiation, migration, and tumorigenesis. For the maintenance of homeostasis, EGF signaling should be tightly regulated to prevent the aberrant activation. Smad7 has been known as inhibitory Smad that blocks the signal transduction of transforming growth factor β. In the process of cell proliferation or transformation, Smad7 has been shown the opposite activities as a promoter or suppressor depending on cell types or microenvironments. We found that the overexpression of Smad7 in human HaCaT keratinocyte cells and mouse skin tissues elevated EGF receptor (EGFR) activity by impairing ligand-induced ubiquitination and degradation of activated receptor, which is induced by the E3 ubiquitin ligase c-Cbl. The C-terminal MH2 region but not MH1 region of Smad7 is critical for interaction with c-Cbl to inhibit the ubiquitination of EGFR. Interestingly, wild-type Smad7, but not Smad6 or mutant Smad7, destabilized the EGF-induced complex formation of c-Cbl and EGFR. These data suggest a novel role for Smad7 as a promoter for prolonging the EGFR signal in keratinocyte and skin tissue by reducing its ligand-induced ubiquitination and degradation.

MP29-04 THE LOSS OF PTEN CONSEQUENT TO URETERAL OBSTRUCTION CONTRIBUTES TO RENAL FIBROSIS

INTRODUCTION AND OBJECTIVES: Phosphate Tensin Homologue on Chromosome 10 (PTEN), the principle negative regulator of PI3K pathway, is a known tumor suppressor that is mutated or lost in many cancer types. Recent studies highlight the emerging role of tumor suppressor PTEN in organ fibrosis including lung and skin where loss of PTEN expression is linked to progression of fibrosis via promoting epithelial plasticity in the lung and induction of myofibroblasts in the skin. However, potential involvement of this molecule in obstructive uropathies is not defined. METHODS: Obstruction induced renal fibrosis was modeled in vivo by unilateral ureteral obstruction (UUO) in mice and in vitro by gene silencing and pharmacological inhibition of PTEN in HK-2 human renal tubular epithelial cells and NRK-49F rat renal fibroblasts. RESULTS: PTEN expression is dramatically decreased in the obstructed (ligated) kidney, while profibrotic TGF-b1/SMAD3 and p53 signal transduction is increased compared to contralateral control or sham kidney. PTEN silencing induced phenotypic modifications from typical cuboidal to an elongated fibroblast-like shape (4-fold increase) and a 40% decrease in the relative cell number compared to control shRNA expressing HK-2 cultures (p<0.01). Stable gene depletion of PTEN in HK-2 cells long term (3-5 days) resulted in increased expression of the profibrotic markers CTGF, PAI-1 and fibronectin compared to control shRNA expressing cultures. Loss of PTEN resulted in activation of SMAD2/3 transcription factor. SMAD3 knockdown or pharmacological inhibition of SMAD3 in PTEN depleted HK-2 cells reversed growth inhibition and suppressed PAI-1 induction, confirming a critical role of SMAD3 downstream of PTEN in orchestrating fibrotic responses. CONCLUSIONS: Unilateral ureteral obstruction induces loss of PTEN expression, resulting in the increased expression of profibrotic cytokines in renal tubular epithelial cells that is dependent on the SMAD3 signaling pathway. PTEN is a potential therapeutic target in the treatment of ureteral obstruction and renal fibrosis. This model paves the way for further clinical studies in humans.

The intricate role of Smads in the regulation of FPGS gene expression

Antifolates are important components of treatment regimens of hematological malignancies and solid tumors. Following cellular uptake, antifolates undergo polyglutamylation, catalyzed by the enzyme folylpoly-γ-glutamate synthetase (FPGS), which is crucial for their intracellular retention and cytotoxic activity. Hence, loss of FPGS activity results in diminished antifolate polyglutamylation and antifolate resistance. Aberrant TGF-β/Smad signaling is thought to be a major contributor to leukemogenesis, as members of this pathway are negative regulators of hematopoiesis. Recently we characterized the human antifolate resistant leukemia cell line MTA C-3 which lost 97% of its cellular FPGS activity and harbors a heterozygous point mutation in exon12 of FPGS. This resulted in the loss of 99% of the expression of the wild type FPGS allele, while the expression of the mutant allele was retained. Research into the molecular mechanism underlying the selective silencing of the wild type allele in these cells established exon12 of FPGS as an intragenic transcriptional regulator, with the ability to drive transcription in vitro . We further showed that exon12 can be occupied by many transcription factors and chromatin remodeling proteins (e.g. Smad4/Ets-1, HP-1 and Brg1) in vivo and that the expression of FPGS is inversely correlated with the binding of a Smad4/Ets-1 complex to exon12, both in acute lymphoblastic leukemia cells and acute myeloid leukemia blast specimens. Here we further demonstrate the possible roles of Smads in the regulation of FPGS gene expression. We found that treatment of MTA C-3 cells with 5-aza-deoxycytidine and trichostatin A results in repression of the mutant allele as well as in decreased expression of FPGS in drug sensitive parental CCRF-CEM cells. This repression correlated with an increased expression of Smad3 and the inhibitory Smad7. These findings bear important implications for the rational overcoming of antifolate resistance in leukemia.

Smad7 suppresses HCC development in a DEN induced mouse model

Introduction: Hepatocellular carcinoma (HCC) is the most frequent primary liver tumor. The transforming growth factor-β (TGF-β) signaling pathway plays a critical role in cancer progression. Smad7, a TGF-β target gene, acts as physiological feedback inhibitor of TGF-β and has been found tumorigenic in several tumors. Knowledge about the role of Smad7 in HCC is limited so far. This study investigated the potential effects of Smad7 in a DEN induced HCC mouse model.

The role of Smad7 in oral mucositis.

Oral mucositis, a severe oral ulceration, is a common toxic effect of radio- or chemoradio-therapy and a limiting factor to using the maximum dose of radiation for effective cancer treatment. Among cancer patients, at least 40% and up to 70%, of individuals treated with standard chemotherapy regimens or upper-body radiation, develop oral mucositis. To date, there is no FDA approved drug to treat oral mucositis in cancer patients. The key challenges for oral mucositis treatment are to repair and protect ulcerated oral mucosa without promoting cancer cell growth. Oral mucositis is the result of complex, multifaceted pathobiology, involving a series of signaling pathways and a chain of interactions between the epithelium and submucosa. Among those pathways and interactions, the activation of nuclear factor-kappa B (NF-κB) is critical to the inflammation process of oral mucositis. We recently found that activation of TGFβ (transforming growth factor β) signaling is associated with the development of oral mucositis. Smad7, the negative regulator of TGFβ signaling, inhibits both NF-κB and TGFβ activation and thus plays a pivotal role in the prevention and treatment of oral mucositis by attenuating growth inhibition, apoptosis, and inflammation while promoting epithelial migration. The major objective of this review is to evaluate the known functions of Smad7, with a particular focus on its molecular mechanisms and its function in blocking multiple pathological processes in oral mucositis.

Smad4 suppresses the progression of renal cell carcinoma via the activation of forkhead box protein H1.

Smad4 has recently been identified as a tumor suppressor gene in a variety of cancers, yet the role of Smad4 in renal cell carcinoma (RCC) remained to be elusive. Therefore, the aim of the present study was to explore the function of Smad4 in RCC. The expression of Smad4 reduced the growth rate of RCC. The levels of Smad4 and forkhead box protein H1 (FOXH1) mRNA were reduced, while the levels of estrogen receptor were increased in RCC cells compared with those in human renal epithelial cells (P<0.01). Western blot analysis showed an identical trend among the three molecules. Glutathione S‑transferase pull‑down and immunoprecipitation assays proved the interaction between Smad4 and FOXH1. An immunofluorescence assay revealed that Smad4 and FOXH1 were colocalized in the nuclei of RCC cells. Smad4 interacts with Smad2 and migrates into the nucleus, where it interacts with FOXH1 to repress the protein expression of estrogen receptor. These results indicate that Smad4 acts as a tumor suppressor by activating FOXH1, and then suppressing the expression of estrogen receptor, in addition to tumor migration and invasion. Hence, Smad4 should be investigated as a potential target for the treatment for RCC.

Oocyte-derived Smad4 is not required for development of the oocyte or the preimplantation embryo

The generation of a competent egg requires complex molecular interactions between the oocyte and the ovary, and transforming growth factor β (TGF-β) is a major signaling pathway. Smad4 is a central regulator of the TGF-β signaling pathway as it mediates gene expression triggered by activation of TGF-β receptors. Deletion of Smad4 in granulosa cells disrupts follicle development; however, the role of Smad4 in the oocyte has not been confirmed. Furthermore, the role of Smad4 in embryo development has not been confirmed because previous studies of Smad4del/del embryos were generated from heterozygous parents, and thus it is possible that maternal transcripts rescue development before embryonic day 6.5 (E6.5) when Smad4del/del embryos die. To determine the role of TGF-β signaling in oocyte and embryo development, mice with oocyte-specific deletion of Smad4 were studied. Fertility was evaluated in Mutant (Smad4F/F:ZP3Cre) and Control (Smad4F/F) females mated continuously with control males during a 6-month period. Surprisingly, Mutant females were fertile with the same litter size (Mutants, 9.23 ± 0.4; Controls, 9.42 ± 0.4) and interlitter period as Controls. Ovulation rate induced using a superovulation regime did not differ between Controls and Mutants at both 6 weeks and 6 months. Embryo development was assessed at E6.5 using Control and Mutant females mated with heterozygous males. Development of Smad4del/del embryos at E6.5 was retarded consistent with previous studies of embryos generated from heterozygous parents indicating that there is no rescue of preimplantation development by maternal transcripts. The numbers of implanted embryos at 6.5 dpc also did not differ (Control: 9.1 ± 0.4; Mutant: 7.0 ± 0.9). However, only 26.3% of E6.5 embryos carried by Mutant females were Smad4del/del compared with the expected ratio of 50%. Since litter size was not decreased, this indicates that either the number of Smad4del sperm fertilizing the oocytes is reduced or implantation of Smad4del/del embryos is suboptimal. In summary, we have shown that Smad4 in the oocyte, and thus TGF-β signaling, is not required for oocyte or follicle development, ovulation, fertilization, preimplantation development, or implantation.

Abstract 2963: Loss of Smad4 within the intestinal tract results in altered intestinal homeostasis and tumor development

Abstract Background: Inactivation of the TGF-β signaling occurs in 50-75% of all colorectal cancer cases. Down-regulation of Smad4, the central mediator of TGF-β signaling, occurs in &amp;gt;50% of stage III patients. How Smad4 contributes to normal intestinal homeostasis and functions as a tumor suppressor are incompletely understood. We reported that β-catenin mRNA expression is inhibited by BMP-mediated Smad4 signaling, and loss of Smad4 signaling results in biologically significant amplification of the Wnt/β-catenin signal. Here, we report on the consequences of Smad4 loss for intestinal homeostasis. Methods: We generated mice genetically engineered to undergo inducible, tissue-specific deletion of Smad4 within intestinal epithelial cells. K19-CreERT x Smad4 flx/flx and Lrig1-CreERT x Smad4 flx/flx mice were treated with vehicle or tamoxifen to induce recombination at the Smad4 locus in the intestinal stem cell compartment. Intestines from sacrificed mice were cleansed for enteroid isolation, Ussing chamber studies, RNA-seq analysis and preservation by formalin fixation and paraffin embedding (FFPE). We validated Smad4 status via immunohistochemistry and stained FFPE sections for different cell populations within the crypt. To evaluate inflammatory cytokines, we performed Luminex cytokine array analysis on colonic mucosa from control and tamoxifen treated animals. To determine the role for Smad4 in intestinal response to injury, we also treated control and tamoxifen treated animals with dextran sodium sulfate (DSS). We utilized RNA-Seq analysis to determine an intestinal Smad4 loss gene signature. Results: Smad4 deficient crypts exhibited an expansion of the Ki67 positive cells in the proliferative zone and fewer Carbonic anhydrase II staining cells suggesting fewer mature enterocytes. We found that loss of Smad4 in the intestinal crypt is accompanied by an accumulation of pericryptal CD3+ lymphocytes and F4/80+ macrophages and is associated with increased intestinal permeability as measured by low molecular weight FITC Dextran using the Ussing chamber. Cytokine analysis revealed Macrophage Inflammatory Protein 3a (MIP3a) to be up-regulated in Smad4 knockout mice. Guided by our RNA-Seq analysis, we observe increased levels of pErk in Smad4 deplete tissue. We also observed that mice with intestinal epithelial Smad4 depletion developed advanced mucinous adenocarcinoma after recovery from an inflammatory stimulus induced by DSS. Conclusions: These results suggest that Smad4 loss disrupts intestinal homeostasis by enabling persistently high levels of Erk signaling to interfere with cell maturation, thereby contributing to increased inflammation. We postulate that the increased inflammation observed in Smad4 knockout mice leads to a tumorigenic environment. Citation Format: Tanner J. Freeman, Jillian Pope, Josh Smith, Daniel Sharbel, Kay Washington, Xi Chen, Rupesh Chaturvedi, Keith Wilson, Noah Shroyer, Punita Dhawan, Anna Means, Natasha G. Deane, R. Daniel Beauchamp. Loss of Smad4 within the intestinal tract results in altered intestinal homeostasis and tumor development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2963. doi:10.1158/1538-7445.AM2014-2963

Analysis of the SMAD4 gene in asthma

Abstract Considering the importance of the TGF-β signaling pathway for normal lung function and especially its roles in inflammation and tissue remodeling, key features of asthma pathology, it can be assumed that these molecules may harbor mutations in asthmatics. The aim of this study was to analyze the SMAD4 gene in patients with asthma. Analysis has encompassed exons 10, 11, 12 and 13 encoding the carboxy-terminal (MH2) domain of the SMAD4 protein, where mutations most frequently occur. The study included 50 patients (20 men and 30 women) with asthma aged between 17 and 73 years (average age 45.2±15.6 years). Polymerase chain reaction (PCR) was used to amplify exons 10, 11, 12 and 13 of the SMAD4 gene and the obtained PCR products were subjected to direct DNA sequencing. No nucleotide changes were found in any of the analyzed exons in either of the subjects. Based on the results of this study, it seems that mutations in the carboxy-terminal (MH2) domain of the SMAD4 are not present in asthmatic patients. Future research should be directed at the analysis of the complete gene, including regulatory elements, in order to resolve the exact role of SMAD4 in asthma.