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Myeloid cells play an essential role in the maintenance of liver homeostasis, as well as the initiation and termination of innate and adaptive immune responses. In chronic hepatic inflammation, the production of transforming growth factor beta (TGF-β) is pivotal for scarring and fibrosis induction and progression. TGF-β signalling is tightly regulated via the Smad protein family. Smad7 acts as an inhibitor of the TGF-β-signalling pathway, rendering cells that express high levels of it resistant to TGF-β-dependent signal transduction. In hepatocytes, the absence of Smad7 promotes liver fibrosis. Here, we examine whether Smad7 expression in myeloid cells affects the extent of liver inflammation, injury and fibrosis induction during chronic liver inflammation. Using the well-established model of chronic carbon tetrachloride (CCl 4 )-mediated liver injury, we investigated the role of Smad7 in myeloid cells in LysM-Cre Smad fl/fl mice that harbour a myeloid-specific knock-down of Smad7. We found that the chronic application of CCl 4 induces severe liver injury, with elevated serum alanine transaminase (ALT)/aspartate transaminase (AST) levels, centrilobular and periportal necrosis and immune-cell infiltration. However, the myeloid-specific knock-down of Smad7 did not influence these and other parameters in the CCl 4 -treated animals. In summary, our results suggest that, during long-term application of CCl 4 , Smad7 expression in myeloid cells and its potential effects on the TGF-β-signalling pathway are dispensable for regulating the extent of chronic liver injury and inflammation.

To maintain iron homoeostasis, the iron regulatory hormone hepcidin is tightly controlled by BMP-Smad signalling pathway, but the physiological role of Smad7 in hepcidin regulation remains elusive. We generated and characterized hepatocyte-specific Smad7 knockout mice (Smad7 Alb/Alb ), which showed decreased serum iron, tissue iron, haemoglobin concentration, up-regulated hepcidin and increased phosphor-Smad1/5/8 levels in both isolated primary hepatocytes and liver tissues. Increased levels of hepcidin lead to reduced expression of intestinal ferroportin and mild iron deficiency anaemia. Interestingly, we found no difference in hepcidin expression or phosphor-Smad1/5/8 levels between iron-challenged Smad7 Alb/Alb and Smad7 flox/flox , suggesting other factors assume the role of iron-induced hepcidin regulation in Smad7 deletion. We performed RNA-seq to identify differentially expressed genes in the liver. Significantly up-regulated genes were then mapped to pathways, revealing TGF-β signalling as one of the most relevant pathways, including the up-regulated genes Smad6, Bambi and Fst (Follistatin). We found that Smad6 and Bambi-but not Follistatin-are controlled by the iron-BMP-Smad pathway. Overexpressing Smad6, Bambi or Follistatin in cells significantly reduced hepcidin expression. Smad7 functions as a key regulator of iron homoeostasis by negatively controlling hepcidin expression, and Smad6 and Smad7 have non-redundant roles. Smad6, Bambi and Follistatin serve as additional inhibitors of hepcidin in the liver., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Transforming growth factor (TGF)-β1 exerts inhibitory effects on keratinocyte proliferation., Objectives: To examine whether Smad7, a known inhibitor of TGF-β1 signalling, is involved in psoriasis-associated keratinocyte hyperproliferation., Methods: Smad7 was evaluated in skin sections of patients with psoriasis and healthy controls and in mice with Aldara-induced skin pathology by real-time polymerase chain reaction and immunohistochemistry. To assess whether Smad7 positively regulates in vivo keratinocyte growth, mice treated with Aldara received daily cutaneous administration of Smad7 antisense oligonucleotide (AS). Keratin (K)6 and K16, cell-cycle-associated factors, cell-cycle and cell proliferation were evaluated in HaCaT cells either treated with Smad7 AS or transfected with Smad7 plasmid and in mice given Smad7 AS., Results: Smad7 was highly expressed in keratinocytes of patients with psoriasis and of mice treated with Aldara. In HaCaT cells, Smad7 knockdown inhibited cell growth, reduced K6 and K16 expression and promoted accumulation of cells in the S-phase of the cell cycle. Smad7-deficient keratinocytes exhibited reduced levels of CDC25A protein, a phosphatase that facilitates progression of cells through the S-phase, and hyperphosphorylation of eukaryotic initiation factor 2 (eIF2)α, a negative regulator of CDC25 protein translation. Consistently, Smad7 overexpression in HaCaT cells was followed by induction of K6 and K16 and increased cell proliferation. Topical application of Smad7 AS to Aldara-treated mice reduced epidermal thickness., Conclusions: Our data show that Smad7 is overexpressed in human and murine psoriasis and suggest a key role of this molecule in the control of keratinocyte proliferation., (© 2017 British Association of Dermatologists.)

The transforming growth factor-β (TGF-β) pathway contributes to maintain the quiescence of adult neural stem and progenitor cells in the brain. In the retina, Müller cells are discussed to represent a glial cell population with progenitor-like characteristics. Here, we aimed to investigate if elevated TGF-β signaling modulates the proliferation of Müller cells during retinal development. We generated mutant mice with a systemic, heterozygous up-regulation of TGF-β signaling by deleting its inhibitor SMAD7. We investigated apoptosis, proliferation, and differentiation of Müller cells in the developing retina. We show that a heterozygous deletion of SMAD7 results in an increased proliferation of Müller cell progenitors in the central retina at postnatal day 4, the time window when Müller cells differentiate in the mouse retina. This in turn results in a thickened retina and inner nuclear layer and a higher number of differentiated Müller cells in the more developed retina. Müller cells in mutant mice contain higher amounts of nestin than those of control animals which indicates that the increase in TGF-β signaling activity during retinal development contribute to maintain some progenitor-like characteristics in Müller cells even after their differentiation period. We conclude that TGF-β signaling influences Müller cell proliferation and differentiation during retinal development.

Chronic pancreatitis is the most common disease of the exocrine pancreas, characterized by progressive inflammation, acinar atrophy and fibrosis. Transforming growth factor-β signaling (TGFβ) is the most potent fibrogenic cytokine known, and its increased expression is a common denominator for fibrosis in chronic pancreatitis. Smad7 is induced by the TGFβ superfamily members as an intracellular inhibitory feedback antagonizing TGFβ signaling. To investigate the functional role of Smad7 in vivo, we induced chronic pancreatitis by repeated administration of cerulein in mice that are deficient in exon-I of Smad7. The response to chronic pancreatitis induction was significantly more severe in Smad7 mutant mice as indicated by a stronger accumulation of extracellular matrix, increased levels of inflammatory cells and an elevated number of mesenchymal cells/myofibroblasts in Smad7 mutant pancreata. Taken together, we conclude that lack of a functional Smad7 gene results in more severe damage in chronic pancreatitis. Therefore, Smad7 could be envisaged as a promising target in antifibrotic therapy of the pancreas., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background and Aim: In Crohn's disease [CD], the pathological process is driven by an excessive immune response that is poorly counterbalanced by regulatory mechanisms. One such a mechanism involves aryl hydrocarbon receptor [AhR], a transcription factor that delivers protective signals in the gut. Expression of AhR is reduced in CD lamina propria mononuclear cells [LPMC] even though factors accounting for such a defect remain unknown. Since CD LPMC express elevated levels of Smad7, an inhibitor of transforming growth factor beta 1 [TGF-β1] activity, and TGF-β1 regulates AhR in other systems, we examined the link between AhR and Smad7 in the gut., Methods: AhR and interleukin [IL]-22 were evaluated in normal LPMC stimulated with TGF-β1 and 6-formylindolo[3,2-b]carbazole [Ficz], an activator of AhR, and in CD LPMC incubated with a Smad7 antisense oligonucleotide and then stimulated with Ficz and TGF-β1. AhR and IL-22 expression was evaluated in LPMC of Smad7-transgenic mice. Finally, we evaluated the protective effect of Ficz on colitis in RAG1 mice injected with naïve or Smad7-overexpressing T cells., Results: In normal LPMC, TGF-β1 induced AhR and this event was associated with increased production of IL-22 following stimulation with Ficz. Treatment of CD LPMC with Smad7 antisense oligonucleotide enabled TGF-β1 to enhance AhR expression. Consistently, AhR expression and Ficz-induced IL-22 production were markedly reduced in T cells of Smad7-transgenic mice. In RAG1 mice, Ficz ameliorated colitis induced by wild type T cells but did not affect colitis induced by transfer of Smad7-overexpressing T cells., Conclusions: The inverse correlation between Smad7 and AhR expression helps to propagate inflammatory signals in the gut., (Copyright © 2016 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Smad7 is an important negative modulator that regulates the duration and strength of TGF-β signaling. Dysregulation of Smad7 is associated with the pathogenesis of many human diseases. Various mouse models are developed to facilitate addressing the physiological functions of Smad7. We have recently demonstrated that loss of Smad7 function by deletion in its MH2 domain leads to multiple cardiac defects and aggravates ethanol-induced liver injury. Here, we describe the procedure to construct and characterize the Smad7 conditional knockout mice.

Hepatic fibrosis in nonalcoholic steatohepatitis (NASH) is the common pathophysiological process resulting from chronic liver inflammation and oxidative stress. Although significant research has been carried out on the role of leptin-induced NADPH oxidase in fibrogenesis, the molecular mechanisms that connect the leptin-NADPH oxidase axis in upregulation of transforming growth factor (TGF)-β signaling have been unclear. We aimed to investigate the role of leptin-mediated upregulation of NADPH oxidase and its subsequent induction of micro-RNA 21 (miR21) in fibrogenesis. Human NASH livers and a high-fat (60% kcal) diet-fed chronic mouse model, where hepatotoxin bromodichloromethane was used to induce NASH, were used for this study. To prove the role of the leptin-NADPH oxidase-miR21 axis, mice deficient in genes for leptin, p47phox, and miR21 were used. Results showed that wild-type mice and human livers with NASH had increased oxidative stress, increased p47phox expression, augmented NF-κB activation, and increased miR21 levels. These mice and human livers showed increased TGF-β, SMAD2/3-SMAD4 colocalizations in the nucleus, increased immunoreactivity against Col1α, and α-SMA with a concomitant decrease in protein levels of SMAD7. Mice that were deficient in leptin or p47phox had decreased activated NF-κB and miR21 levels, suggesting the role of leptin and NADPH oxidase in inducing NF-κB-mediated miR21 expression. Further miR21 knockout mice had decreased colocalization events of SMAD2/3-SMAD4 in the nucleus, increased SMAD7 levels, and decreased fibrogenesis. Taken together, the studies show the novel role of leptin-NADPH oxidase induction of miR21 as a key regulator of TGF-β signaling and fibrogenesis in experimental and human NASH., (Copyright © 2015 the American Physiological Society.)

Genomic instability is one of the representative causes in genetic disorder, where the proper cellular response to DNA damage is essential in maintaining genomic stability. ATM and the Mre11-Rad50-Nbs1 (MRN) complex play critical roles in the cellular response to DNA damage such as DNA double-strand break (DSB). In this study, we report that Smad7 is indispensible in DNA damage response as a novel component of MRN complex. Smad7 enhances cell survival against DNA damage by accelerating ATM dependent DNA repair signaling. In Smad7-deficient mouse embryonic fibroblast cells, the loss of Smad7 decreases ATM activation and inhibits recruitment of ATM to the sites of DSBs. Smad7 interacts with Nbs1, a member of MRN complex, and enhances the interaction between ATM and Nbs1 upon DNA damage response, leading to phosphorylation of downstream substrates. Ectopic expression of Smad7 in the skin of mice enhances the phosphorylation of ATM upon X-irradiation. We found that effect of Smad7 on enhancing DNA repair is independent of its inhibitory activity of TGF-β signaling. Taken together, our results highlight a critical function of Smad7 in DSB response and establish the novel mechanism in which Smad7 facilitates the recruitment of ATM to the MRN complex through direct interaction with Nbs1.

Smad7 is well demonstrated as a negative regulator of TGF-β signaling. Its alteration in expression often results in diseases such as cancer and fibrosis. However, the exact role of Smad7 in regulating bone remodeling during mammalian development has not been properly delineated. In this study we performed experiments to clarify the involvement of Smad7 in regulating osteogenesis and osteoclastogenesis both invivo and invitro. Genetically engineered Smad7(ΔE1) (KO) mice were used, whereby partial functional of Smad7 is lost by deleting exon I of the Smad7 gene and the truncated proteins cause a hypomorphic allele. Analysis with μCT imagery and bone histomorphometry showed that the KO mice had lower TbN, TbTh, higher TbSp in the metaphysic region of the femurs at 6, 12, 24weeks from birth, as well as decreased MAR and increased osteoclast surface compared with the WT mice. In vitro BM-MSC multi-lineage differentiation evaluation showed that the KO group had reduced osteogenic potential, fewer mineralized nodules, lower ALP activity, and reduced gene expression of Col1A1, Runx2 and OCN. The adipogenic potential was elevated in the KO group with more formation of lipid droplets, and increased gene expression of Adipsin and C/EBPα. The osteoclastogenic potential of KO mice BMMs was elevate, with emergence of more osteoclasts, larger resorptive areas, and increased gene expression of TRAP and CTR. Our results indicate that partial loss of Smad7 function in mice leads to compromised bone formation and enhanced bone resorption. Thus, Smad7 is acknowledged as a novel key regulator between osteogenesis and osteoclastogenesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Smad7 is a principal inhibitor of the TGFβ-Smad signalling pathway. We have investigated the functional significance of Smad7 in hepatocellular carcinoma (HCC). Smad7 knockout (KO) and wild-type (WT) mice were injected with diethylnitrosamine (DEN) to induce HCC. The effects of Smad7 on cellular features were examined in HCC cells, using a Smad7 over-expression or deletion approach. Signalling pathway components modulated by Smad7 in HCC were evaluated using luciferase reporter assay and co-immunoprecipitation. Smad7 was down-regulated in human HCCs compared with the adjacent normal tissues (p < 0.001). Smad7 KO mice were more susceptible to DEN-induced HCC than WT mice (78% versus 22%, p < 0.05). HCCs from KO mice displayed a greater proliferation activity (p < 0.05) and a reduced apoptotic index compared with WT littermates (p < 0.05). Deletion of Smad7 promoted cell proliferation in primary cultured HCC cells. In addition, over-expression of Smad7 in HCC cell lines markedly suppressed cell growth (p < 0.0001) and colony formation (p < 0.01). Cell cycle analysis revealed an increase in the G1 phase and a reduction in the S-phase populations, accompanied by up-regulation of p27(Kip1) and down-regulation of cyclin D1. Smad7 increased cell apoptosis (p < 0.01) by mediating an intrinsic [caspase-9, caspase-3 and poly(ADP-ribose) polymerase] apoptotic pathway. Moreover, Smad7 inhibited NF-κB signalling by interacting with TAB2, an upstream activator of NF-κB, and inhibited TGFβ signalling by suppressing phosphorylation of Smad3. In conclusion, loss of Smad7 enhances susceptibility to HCC. Smad7 suppresses HCC cell growth by inhibiting proliferation and G1 -S phase transition and inducing apoptosis through attenuation of NF-κB and TGFβ signalling. Smad7 acts as a potential tumour suppressor in liver., (Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Adaptive immunity has a major impact on atherosclerosis, with pro- and anti-atherosclerotic effects exerted by different subpopulations of T cells. Transforming growth factor-β (TGF-β) may promote development either of anti-atherosclerotic regulatory T cells or of T helper 17 (TH17) cells, depending on factors in the local milieu. We have addressed the effect on atherosclerosis of enhanced TGF-β signaling in T cells. Bone marrow from mice with a T cell-specific deletion of Smad7, a potent inhibitor of TGF-β signaling, was transplanted into hypercholesterolemic Ldlr(-/-) mice. Smad7-deficient mice had significantly larger atherosclerotic lesions that contained large collagen-rich caps, consistent with a more stable phenotype. The inflammatory cytokine interleukin-6 (IL-6) was expressed in the atherosclerotic aorta, and increased mRNA for IL-17A and the TH17-specific transcription factor RORγt were detected in draining lymph nodes. Treating Smad7-deficient chimeras with neutralizing IL-17A antibodies reversed stable cap formation. IL-17A stimulated collagen production by human vascular smooth muscle cells, and RORγt mRNA correlated positively with collagen type I and α-smooth muscle actin mRNA in a biobank of human atherosclerotic plaques. These data link IL-17A to induction of a stable plaque phenotype, could lead to new plaque-stabilizing therapies, and should prompt an evaluation of cardiovascular events in patients treated with IL-17 receptor blockade.

Rationale: Growth and remodeling of the pharyngeal arch arteries are vital for the development of a mature great vessel system. Dysmorphogenesis of the fourth arch arteries can result in interruption of the aortic arch type B, typically found in DiGeorge syndrome. Tbx1 haploinsufficient embryos, which model DiGeorge syndrome, display fourth arch artery defects during formation of the vessels. Recovery from such defects is a documented yet unexplained phenotype in Tbx1 haploinsufficiency., Objective: To understand the nature of fourth arch artery growth recovery in Tbx1 haploinsufficiency and its underlying genetic control., Methods and Results: We categorized vessel phenotypes of Tbx1 heterozygotes as hypoplastic or aplastic at the conclusion of pharyngeal artery formation and compared these against the frequency of vessel defects scored at the end of great vessel development. The frequency of hypoplastic vessels decreased during embryogenesis, whereas no reduction of vessel aplasia was seen, implying recovery is attributable to remodeling of hypoplastic vessels. We showed that Smad7, an inhibitory Smad within the transforming growth factor-β pathway, is regulated by Tbx1, is required for arch artery remodeling, and genetically interacts with Tbx1 in this process. Tbx1 and Tbx1;Smad7 haploinsufficiency affected several remodeling processes; however, concurrent haploinsufficiency particularly impacted on the earliest stage of vascular smooth muscle cell vessel coverage and subsequent fibronectin deposition. Conditional reconstitution of Smad7 with a Tbx1Cre driver indicated that the interaction between the 2 genes is cell autonomous., Conclusions: Tbx1 acts upstream of Smad7 controlling vascular smooth muscle and extracellular matrix investment of the fourth arch artery.

Smad7 is an inhibitory molecule induced by members of the transforming growth factor-β (TGF-β) family, including TGF-β, activin, nodal and bone morphogenetic proteins (BMPs). To elucidate the in vivo functions of Smad7, we generated conditional Smad7-knockout mice in which the Mad homology 2 (MH2) domain and the poly (A) signal sequence were flanked with loxP sites (floxed). The Smad7-floxed mice exhibited no obvious phenotype. Smad7 total-null mice on a C57BL/6 background died within a few days of birth, whereas mice with an ICR background developed to adulthood but were significantly smaller than wild-type mice. Unexpectedly, phospho-Smad2 and phospho-Smad3 were decreased in Smad7-deficient mouse embryonic fibroblast (MEF) cells, whereas phospho-Smad1/5/8 was similarly expressed in wild-type and Smad7-deficient MEF cells. Moreover, expression levels of TGF-β type I receptor (ALK5) were higher in Smad7-deficient MEF cells than in wild-type MEF cells. Plasminogen activator inhibitor-1 (PAI-1) and inhibitor of differentiation-1 (Id-1) mRNA were similarly expressed in wild-type and Smad7-deficient MEF cells. Some differences were observed in mitogen-activated protein kinase (MAPK)-signalling between wild-type and Smad7-deficient MEF cells. We demonstrated that Smad7 plays an important role in normal mouse growth and provide a useful tool for analysing Smad7 functions in vivo.

Autoreactive CD4+ T lymphocytes play a vital role in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Since the discovery of T helper 17 cells, there is an ongoing debate whether T helper 1, T helper 17 or both subtypes of T lymphocytes are important for the initiation of autoimmune neuroinflammation. We examined peripheral blood CD4+ cells from patients with active and stable relapsing-remitting multiple sclerosis, and used mice with conditional deletion or over-expression of the transforming growth factor-beta inhibitor Smad7, to delineate the role of Smad7 in T cell differentiation and autoimmune neuroinflammation. We found that Smad7 is up-regulated in peripheral CD4+ cells from patients with multiple sclerosis during relapse but not remission, and that expression of Smad7 strongly correlates with T-bet, a transcription factor defining T helper 1 responses. Concordantly, mice with transgenic over-expression of Smad7 in T cells developed an enhanced disease course during experimental autoimmune encephalomyelitis, accompanied by elevated infiltration of inflammatory cells and T helper 1 responses in the central nervous system. On the contrary, mice with a T cell-specific deletion of Smad7 had reduced disease and central nervous system inflammation. Lack of Smad7 in T cells blunted T cell proliferation and T helper 1 responses in the periphery but left T helper 17 responses unaltered. Furthermore, frequencies of regulatory T cells were increased in the central nervous system of mice with a T cell-specific deletion and reduced in mice with a T cell-specific over-expression of Smad7. Downstream effects of transforming growth factor-beta on in vitro differentiation of naïve T cells to T helper 1, T helper 17 and regulatory T cell phenotypes were enhanced in T cells lacking Smad7. Finally, Smad7 was induced during T helper 1 differentiation and inhibited during T helper 17 differentiation. Taken together, the level of Smad7 in T cells determines T helper 1 polarization and regulates inflammatory cellular responses. Since a Smad7 deletion in T cells leads to immunosuppression, Smad7 may be a potential new therapeutic target in multiple sclerosis.

The members of the TGF-beta superfamily, i.e., TGF-beta isoforms, activins, and bone morphogenetic proteins, regulate growth, differentiation, and apoptosis, both during embryonic development and during postnatal life. Smad7 is induced by the TGF-beta superfamily members and negatively modulates their signaling, thus acting in a negative, autocrine feedback manner. In addition, Smad7 is induced by other stimuli. Thus, it can fine-tune and integrate TGF-beta signaling with other signaling pathways. To investigate the functional role(s) of Smad7 in vivo, we generated mice deficient in exon I of Smad7, leading to a partial loss of Smad7 function. Mutant animals are viable, but significantly smaller on the outbred CD-1 mouse strain background. Mutant B cells showed an overactive TGF-beta signaling measured as increase of phosphorylated Smad2-positive B cells compared with B cells from wild-type mice. In agreement with this expected increase in TGF-beta signaling, several changes in B cell responses were observed. Mutant B cells exhibited increased Ig class switch recombination to IgA, significantly enhanced spontaneous apoptosis in B cells, and a markedly reduced proliferative response to LPS stimulation. Interestingly, LPS treatment reverted the apoptotic phenotype in the mutant cells. Taken together, the observed phenotype highlights a prominent role for Smad7 in development and in regulating the immune system's response to TGF-beta.