The Snail signaling branch downstream of the TGF-ß/Smad3 pathway mediates Rho activation and subsequent stress fiber formation.

Cancer cells acquire malignant phenotypes through an epithelial-mesenchymal transition, which is induced by environmental factors or extracellular signaling molecules, including transforming growth factor-ß (TGF-ß). Among epithelial-mesenchymal transition-associated cell responses, cell morphological changes and cell motility are closely associated with remodeling of the actin stress fibers. Here, we examined the TGF-ß signaling pathways leading to these cell responses. Through knockdown experiments in A549 lung adenocarcinoma cells, we found that Smad3-mediated induction of Snail, but not that of Slug, is indispensable for morphological changes, stress fiber formation, and enhanced motility in cells stimulated with TGF-ß. Ectopic expression of Snail in SMAD3-knockout cells rescued the defect in morphological changes and stress fiber formation by TGF-ß, indicating that the role of Smad3 in these responses is to upregulate Snail expression. Mechanistically, Snail is required for TGF-ß-induced upregulation of Wnt5b, which in turn activates RhoA and subsequent stress fiber formation in cooperation with phosphoinositide 3-kinase. However, ectopic expression of Snail in SMAD3-knockout cells failed to rescue the defect in cell motility enhancement by TGF-ß, indicating that activation of the Smad3/Snail/Wnt5b axis is indispensable but not sufficient for enhancing cell motility; a Smad3-dependent but Snail-independent pathway to activate Rac1 is additionally required. Therefore, the Smad3-dependent pathway leading to enhanced cell motility has two branches: a Snail-dependent branch to activate RhoA and a Snail-independent branch to activate Rac1. Coordinated activation of these branches, together with activation of non-Smad signaling pathways, mediates enhanced cell motility induced by TGF-ß.

Interactions of Segmented Filamentous Bacteria (Candidatus Savagella) and bacterial drivers in colitis-associated colorectal cancer development.

Colorectal cancer (CRC) risk is influenced by host genetics, sex, and the gut microbiota. Using a genetically susceptible mouse model of CRC induced via inoculation with pathobiont Helicobacter spp. and demonstrating variable tumor incidence, we tested the ability of the Th17-enhancing commensal Candidatus Savagella, more commonly denoted as Segmented Filamentous Bacteria (SFB), to influence the incidence and severity of colitis-associated CRC in male and female mice. To document the composition of the gut microbiota during CRC development and identify taxa associated with disease, fecal samples were collected before and throughout disease development and characterized via 16S rRNA sequencing. While there were no significant SFB-dependent effects on disease incidence or severity, SFB was found to exert a sex-dependent protective effect in male mice. Furthermore, SFB stabilized the GM against Helicobacter-induced changes post-inoculation, resulting in a shift in disease association from Helicobacter spp. to Escherichia coli. These data support sex-dependent SFB-mediated effects on CRC risk, and highlight the complex community dynamics within the GM during exposure to inflammatory pathobionts.

Targeting the E3 Ubiquitin Ligase PJA1 Enhances Tumor-Suppressing TGFß Signaling.

RING-finger E3 ligases are instrumental in the regulation of inflammatory cascades, apoptosis, and cancer. However, their roles are relatively unknown in TGFß/SMAD signaling. SMAD3 and its adaptors, such as ß2SP, are important mediators of TGFß signaling and regulate gene expression to suppress stem cell-like phenotypes in diverse cancers, including hepatocellular carcinoma (HCC). Here, PJA1, an E3 ligase, promoted ubiquitination and degradation of phosphorylated SMAD3 and impaired a SMAD3/ß2SP-dependent tumor-suppressing pathway in multiple HCC cell lines. In mice deficient for SMAD3 (Smad3 +/-), PJA1 overexpression promoted the transformation of liver stem cells. Analysis of genes regulated by PJA1 knockdown and TGFß1 signaling revealed 1,584 co-upregulated genes and 1,280 co-downregulated genes, including many implicated in cancer. The E3 ligase inhibitor RTA405 enhanced SMAD3-regulated gene expression and reduced growth of HCC cells in culture and xenografts of HCC tumors, suggesting that inhibition of PJA1 may be beneficial in treating HCC or preventing HCC development in at-risk patients.Significance: These findings provide a novel mechanism regulating the tumor suppressor function of TGFß in liver carcinogenesis.

SMAD family member 3 (SMAD3) and SMAD4 repress HIF2α-dependent iron-regulatory genes.

Hypoxia-inducible factor 2α (HIF2α) directly regulates a battery of genes essential for intestinal iron absorption. Interestingly, iron deficiency and overload disorders do not result in increased intestinal expression of glycolytic or angiogenic HIF2α target genes. Similarly, inflammatory and tumor foci can induce a distinct subset of HIF2α target genes in vivo These observations indicate that different stimuli activate distinct subsets of HIF2α target genes via mechanisms that remain unclear. Here, we conducted a high-throughput siRNA-based screen to identify genes that regulate HIF2α's transcriptional activity on the promoter of the iron transporter gene divalent metal transporter-1 (DMT1). SMAD family member 3 (SMAD3) and SMAD4 were identified as potential transcriptional repressors. Further analysis revealed that SMAD4 signaling selectively represses iron-absorptive gene promoters but not the inflammatory or glycolytic HIF2α or HIF1α target genes. Moreover, the highly homologous SMAD2 did not alter HIF2α transcriptional activity. During iron deficiency, SMAD3 and SMAD4 expression was significantly decreased via proteasomal degradation, allowing for derepression of iron target genes. Several iron-regulatory genes contain a SMAD-binding element (SBE) in their proximal promoters; however, mutation of the putative SBE on the DMT1 promoter did not alter the repressive function of SMAD3 or SMAD4. Importantly, the transcription factor forkhead box protein A1 (FOXA1) was critical in SMAD4-induced DMT1 repression, and DNA binding of SMAD4 was essential for the repression of HIF2α activity, suggesting an indirect repressive mechanism through DNA binding. These results provide mechanistic clues to how HIF signaling can be regulated by different cellular cues.

Aberrant Smad3 phosphoisoforms in cyst-lining epithelial cells in the cpk mouse, a model of autosomal recessive polycystic kidney disease.

Cystic epithelia acquire mesenchymal-like features in polycystic kidney disease (PKD). In this phenotypic alteration, it is well known that transforming growth factor (TGF)-ß/Smad3 signaling is involved; however, there is emerging new data on Smad3 phosphoisoforms: Smad3 phosphorylated at linker regions (pSmad3L), COOH-terminal regions (pSmad3C), and both (pSmad3L/C). pSmad3L/C has a pathological role in colorectal cancer. Mesenchymal phenotype-specific cell responses in the TGF-ß/Smad3 pathway are implicated in carcinomas. In this study, we confirmed mesenchymal features and examined Smad3 phosphoisoforms in the cpk mouse, a model of autosomal recessive PKD. Kidney sections were stained with antibodies against mesenchymal markers and domain-specific phospho-Smad3. TGF-ß, pSmad3L, pSmad3C, JNK, cyclin-dependent kinase (CDK) 4, and c-Myc were evaluated by Western blotting. Cophosphorylation of pSmad3L/C was assessed by immunoprecipitation. α-Smooth muscle actin, which indicates mesenchymal features, was expressed higher in cpk mice. pSmad3L expression was increased in cpk mice and was predominantly localized in the nuclei of tubular epithelial cells in cysts; however, pSmad3C was equally expressed in both cpk and control mice. Levels of pSmad3L, JNK, CDK4, and c-Myc protein in nuclei were significantly higher in cpk mice than in controls. Immunoprecipitation showed that Smad3 was cophosphorylated (pSmad3L/C) in cpk mice. Smad3 knockout/cpk double-mutant mice revealed amelioration of cpk abnormalities. These findings suggest that upregulating c-Myc through the JNK/CDK4-dependent pSmad3L pathway may be key to the pathophysiology in cpk mice. In conclusion, a qualitative rather than a quantitative abnormality of the TGF-ß/Smad3 pathway is involved in PKD and may be a target for disease-specific intervention.

Smad3 and Bmal1 regulate p21 and S100A4 expression in myocardial stromal fibroblasts via TNF-α.

Bmal1, a clock gene, is associated with depression, hypertrophy, metabolic syndrome and diabetes. Smad3, which is involved in the TGF-ß signaling pathway, plays an important role in the regulation of tumor progression, fibrosis, obesity and diabetes. Our previous report showed that Smad3 has circadian expression in mouse livers. In the current study, we focused on the heart, especially on the myocardial stromal fibroblasts because the roles of Bmal1 and Smad3 in this tissue are poorly understood. Bmal1 and Smad3 have circadian expression in mouse hearts, and their circadian expression patterns were similar. Bmal1 expression decreased in the hearts of whole-body Smad3 knockout mice, whereas Smad3 expression had little effect on heart-specific Bmal1 knockout mice. Both Smad3 knockout and heart-specific Bmal1 knockout mice showed increases in p21, S100A4, CD206 and TNF-α expression in the myocardial stromal fibroblasts and macrophage compared to control mice. We also examined Smad3, Bmal1 and Dec1 expression in human tissue from old myocardial infarctions. Expression of Smad3, Bmal1 and Dec1 decreased in the stromal fibroblasts of tissue from old myocardial infarctions compared to control cases. On the other hand, p21, S100A4 and TNF-α increased in the stromal fibroblasts of tissue from old myocardial infarctions. Furthermore, expression of Smad3, Bmal1 and Dec1 decreased in TNF-α treated-NIH3T3 cells but expression of p21 and S100A4 increased. This new evidence suggests that Smad3 and Bmal1 regulate p21 and S100A4 expression in myocardial stromal fibroblasts through TNF-α.

A Smad3-PTEN regulatory loop controls proliferation and apoptotic responses to TGF-ß in mouse endometrium.

The TGF-ß/Smad and the PI3K/AKT signaling pathways are important regulators of proliferation and apoptosis, and their alterations lead to cancer development. TGF-ß acts as a tumor suppressor in premalignant cells, but it is a tumor promoter for cancerous cells. Such dichotomous actions are dictated by different cellular contexts. Here, we have unveiled a PTEN-Smad3 regulatory loop that provides a new insight in the complex cross talk between TGF-ß/Smad and PI3K/AKT signaling pathways. We demonstrate that TGF-ß triggers apoptosis of wild-type polarized endometrial epithelial cells by a Smad3-dependent activation of PTEN transcription, which results in the inhibition of PI3K/AKT signaling pathway. We show that specific Smad3 knockdown or knockout reduces basal and TGF-ß-induced PTEN expression in endometrial cells, resulting in a blockade of TGF-ß-induced apoptosis and an enhancement of cell proliferation. Likewise Smad3 deletion, PTEN knockout prevents TGF-ß-induced apoptosis and increases cell proliferation by increasing PI3K/AKT/mTOR signaling. In summary, our results demonstrate that Smad3-PTEN signaling axis determine cellular responses to TGF-ß.

Functions of Smad Transcription Factors in TGF-ß1-Induced Selectin Ligand Expression on Murine CD4 Th Cells.

Selectins are carbohydrate-binding adhesion molecules that control leukocyte traffic. Induction of selectin ligands on T cells is controlled primarily by cytokines, including TGF-ß1, and requires p38α MAPK, but transcriptional mechanisms that underlie cytokine-driven selectin ligand expression are poorly understood. In this study, we show, using mice with conditional deletions of the TGF-ß1-responsive transcription factors Smad2, Smad3, or Smad4, that induction of selectin ligands on CD4 cells in response to TGF-ß1 requires Smad4 plus either Smad2 or Smad3. Analysis of CD4 cells from mice with only one functional Smad4 allele revealed a sharp gene dosage effect, suggesting the existence of a threshold of TGF-ß1 signal strength required for selectin ligand induction. Both Smad4 plus either Smad2 or Smad3 were selectively required for induction of Fut7 and Gcnt1, glycosyltransferases critical for selectin ligand biosynthesis, but they were not required for St3gal4 or St3gal6 induction. Smad4 plus either Smad2 or Smad3 were also required for induction of Runx transcription factors by TGF-ß1. Enforced expression of Runx2, but not Runx1 or Runx3, in Smad2/Smad3 doubly deficient CD4 cells restored selectin ligand expression to wild-type levels. In contrast, enforced expression of Runx1, Runx2, or Runx3 failed to restore differentiation of TGF-ß1-dependent Th cell lineages, including Th17, Th9, and induced regulatory T cells. These results show that Smads are directly required for Th cell differentiation independent of Runx induction but only indirectly required via Runx2 for TGF-ß1-induced selectin ligand induction on murine CD4 T cells.

Anti-fibrotic effect of wogonin in renal tubular epithelial cells via Smad3-dependent mechanisms.

Renal fibrosis, a common feature and leading cause for End Stage Renal Disease, still lacks effective therapy. In the current study, we detected and compared the anti-fibrotic effects of wogonin and wogonoside, two major components of Scutellaria baicalensis Georgi, in TGF-ß1-treated tubular epithelial cells of human and murine origins. Results consistently showed that compared with wogonoside, wogonin inhibits TGF-ß1-induced upregulated mRNA and protein levels of collagen I and α-SMA with more efficiency, which was further confirmed by the immunofluorescence results that wogonin decreased the percentage of collagen I and α-SMA positive cells in TGF-ß1-treated tubular epithelial cells. Mechanistically, wogonin mainly decreased Smad3 phosphorylation, but had marginal effect on non-canonical TGF-ß signaling pathways, such as p38 and ERK MAP Kinase. Furthermore, in the cells deficient for TGF-ß signaling or downstream Smad3, results demonstrated that even high concentration of wogonin failed to further decrease the level of collagen I and α-SMA, indicating the essential role of TGF-ß/Smad3 signaling inhibition in the therapeutic action of wogonin in TGF-ß1-stimulated tubular epithelial cells. Collectively, our results indicated that wogonin may be utilized as a potential anti-fibrotic Traditional Chinese Medicine monomer in the treatment of renal fibrosis.

Inhibition of development of laser-induced choroidal neovascularization with suppression of infiltration of macrophages in Smad3-null mice.

We evaluated the effects of the loss of Smad3 on the development of experimental argon laser-induced choroidal neovascularization (CNV) in mice. An in vitro angiogenesis model was also used to examine the role of transforming growth factor-ß1 (TGFß1)/Smad3 signaling in vessel-like tube formation by human umbilical vein endothelial cells (HUVECs). CNV was induced in eyes of 8-12-week-old B6.129-background Smad3-deficient (KO) mice (n=47) and wild-type (WT) mice (n=47) by argon laser irradiation. Results showed that the size of the CNV induced was significantly smaller in KO mice as compared with WT mice at day 14 as revealed by high-resolution angiography with fluorescein isothiocyanate-dextran. Immunohistochemistry and real-time reverse transcription-polymerase chain reaction of RNA extracted from laser-irradiated choroidal tissues were conducted on specimens at specific timepoints. Invasion of macrophages (F4/80+), but not neutrophils (myeloperoxidase+), and appearance of myofibroblasts (α-smooth muscle actin+) were suppressed in laser-irradiated KO tissues. mRNA expression of inflammation-related factors, that is, vascular endothelial growth factor (VEGF), macrophage-chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and TGFß1 in choroidal tissues was suppressed by the loss of Smad3. We then examined the effects of adding a Smad3 inhibitor, SIS3, or an ALK5 inhibitor, SB431542, on tube formation promoted by TGFß1 or VEGF in HUVECs cocultured with fibroblast feeder. Further addition of SIS3 or SB431542 augmented vessel-like tube formation by HUVECs in the presence of TGFß1 or VEGF. In conclusion, lack of Smad3 attenuated the growth of laser-induced CNV with suppression of inflammation by macrophages in mice. Because blocking TGFß1/Smad3 signal stimulated the activity of angiogenesis of HUVECs in vitro, the reduction of CNV in vivo in KO mice is attributed to a decrease in growth factor levels in the tissue by the loss of Smad3.

Imaging colon cancer development in mice: IL-6 deficiency prevents adenoma in azoxymethane-treated Smad3 knockouts.

The development of colorectal cancer in the azoxymethane-induced mouse model can be observed by using a miniaturized optical coherence tomography (OCT) imaging system. This system is uniquely capable of tracking disease development over time, allowing for the monitoring of morphological changes in the distal colon due to tumor development and the presence of lymphoid aggregates. By using genetically engineered mouse models deficient in Interleukin 6 (IL-6) and Smad family member 3 (Smad3), the role of inflammation on tumor development and the immune system can be elucidated. Smad3 knockout mice develop inflammatory response, wasting, and colitis associated cancer while deficiency of proinflammatory cytokine IL-6 confers resistance to tumorigenesis. We present pilot data showing that the Smad3 knockout group had the highest tumor burden, highest spleen weight, and lowest thymus weight. The IL-6 deficiency in Smad3 knockout mice prevented tumor development, splenomegaly, and thymic atrophy. This finding suggests that agents that inhibit IL-6 (e.g. anti-IL-6 antibody, non-steroidal anti-inflammatory drugs [NSAIDs], etc.) could be used as novel therapeutic agents to prevent disease progression and increase the efficacy of anti-cancer agents. OCT can also be useful for initiating early therapy and assessing the benefit of combination therapy targeting inflammation.

A tight control of Rif1 by Oct4 and Smad3 is critical for mouse embryonic stem cell stability.

Prolonged culture of embryonic stem cells (ESCs) leads them to adopt embryonal carcinoma cell features, creating enormous dangers for their further application. The mechanism involved in ESC stability has not, however, been extensively studied. We previously reported that SMAD family member 3 (Smad3) has an important role in maintaining mouse ESC stability, as depletion of Smad3 results in cancer cell-like properties in ESCs and Smad3-/- ESCs are prone to grow large, malignant teratomas. To understand how Smad3 contributes to ESC stability, we performed microarray analysis to compare the transcriptome of wild-type and Smad3-/- ESCs. We found that Rif1 (RAP1-associated protein 1), a factor important for genomic stability, is significantly upregulated in Smad3-/- ESCs. The expression level of Rif1 needs to be tightly controlled in ESCs, as a low level of Rif1 is associated with ESC differentiation, but a high level of Rif1 is linked to ESC transformation. In ESCs, Oct4 activates Rif1, whereas Smad3 represses its expression. Oct4 recruits Smad3 to bind to Rif1 promoter, but Smad3 joining facilitates the loading of a polycomb complex that generates a repressive epigenetic modification on Rif1 promoter, and thus maintains the expression of Rif1 at a proper level in ESCs. Interestingly, Rif1 short hairpin RNA (shRNA)-transduced Smad3-/- ESCs showed less malignant properties than the control shRNA-transduced Smad3-/- ESCs, suggesting a critical role of Rif1 in maintaining the stability of ESCs during proliferation.

Smad3-deficient mice have reduced esophageal fibrosis and angiogenesis in a model of egg-induced eosinophilic esophagitis.

OBJECTIVES: Eosinophilic esophagitis (EoE) is a food-triggered disease associated with esophageal fibrosis and stricture formation in a subset of patients. In the present study we used a murine model of egg (ovalbumin [OVA])-induced EoE to determine whether inhibiting transforming growth factor-ß1 (TGF-ß1) signaling through the Smad3 pathway would inhibit features of esophageal remodeling including fibrosis, angiogenesis, and basal zone hyperplasia. METHODS: Wild-type (WT) and Smad3-deficient (KO [knockout]) mice were sensitized intraperitoneally and then challenged chronically with intraesophageal OVA for 1 month. Levels of esophageal eosinophils, esophageal TGF-ß1+ and vascular endothelial growth factor (VEGF)+ cells, and features of esophageal remodeling (fibrosis, angiogenesis, basal zone hyperplasia) were quantitated by immunohistochemistry and image analysis. RESULTS: OVA challenge induced a similar increase in the levels of esophageal major basic protein (MBP)+ eosinophils and esophageal TGF-ß1+ cells in WT and Smad3 KO mice. Smad3 KO mice challenged with OVA had significantly less esophageal fibrosis and esophageal angiogenesis compared with OVA-challenged WT mice. The reduced esophageal angiogenesis in Smad3 KO mice was associated with reduced numbers of VEGF+ cells in the esophagus. There was a trend toward OVA-challenged Smad3 KO to have reduced basal zone hyperplasia, but this was not statistically significant. CONCLUSIONS: In a mouse model of egg-induced EoE, Smad3-deficient mice have significantly less esophageal remodeling, especially fibrosis and angiogenesis that is associated with reduced expression of VEGF. Targeting the TGF-ß1/Smad3 pathway may be a novel strategy to reduce esophageal fibrosis and its associated complications such as esophageal strictures in EoE.

Myostatin augments muscle-specific ring finger protein-1 expression through an NF-kB independent mechanism in SMAD3 null muscle.

Smad (Sma and Mad-related protein) 2/3 are downstream signaling molecules for TGF-ß and myostatin (Mstn). Recently, Mstn was shown to induce reactive oxygen species (ROS) in skeletal muscle via canonical Smad3, nuclear factor-κB, and TNF-α pathway. However, mice lacking Smad3 display skeletal muscle atrophy due to increased Mstn levels. Hence, our aims were first to investigate whether Mstn induced muscle atrophy in Smad3(-/-) mice by increasing ROS and second to delineate Smad3-independent signaling mechanism for Mstn-induced ROS. Herein we show that Smad3(-/-) mice have increased ROS levels in skeletal muscle, and inactivation of Mstn in these mice partially ablates the oxidative stress. Furthermore, ROS induction by Mstn in Smad3(-/-) muscle was not via nuclear factor-κB (p65) signaling but due to activated p38, ERK MAPK signaling and enhanced IL-6 levels. Consequently, TNF-α, nicotinamide adenine dinucleotide phosphate oxidase, and xanthine oxidase levels were up-regulated, which led to an increase in ROS production in Smad3(-/-) skeletal muscle. The exaggerated ROS in the Smad3(-/-) muscle potentiated binding of C/EBP homology protein transcription factor to MuRF1 promoter, resulting in enhanced MuRF1 levels leading to muscle atrophy.

Suppression of malignancy by Smad3 in mouse embryonic stem cell formed teratoma.

Disease associated gene deficient embryonic stem cells can serve as valuable in vitro models to study disease mechanisms and screen drugs. Smad3 mediated TGF-ß/Activin/Nodal signaling plays important roles in many biological processes. Despite numerous studies regarding Smad3 function, the role of Smad3 in mouse ES cells is not well studied. To understand the function of Smad3 in mouse ES cells, we derived Smad3-/- ES cells and wild type ES cells. Smad3-/- ES cells display no defect on self-renewal. They express similar level of pluripotent genes and lineage genes compared to wild type ES cells. However, Smad3 ablation results in transient difference in germ layer marker expression during embryoid body formation. Mesoderm lineage marker expression is significantly reduced in the embryoid body formed by Smad3-/- ES cells compared to wild type ES cells. Intriguingly, subcutaneous injection of Smad3-/- ES cells into nude mice leads to formation of malignant immature teratomas, whilst wild type ES cells tend to form mature teratomas. Smad3-/- ES cell formed teratomas can therefore provide a new model for the study of the mechanism of malignant teratomas.

GM-CSF contributes to aortic aneurysms resulting from SMAD3 deficiency.

Heterozygous loss-of-function SMAD3 (Mothers against decapentaplegic homolog 3) mutations lead to aneurysm-osteoarthritis syndrome (AOS). In the present study, we found that mice lacking Smad3 had a vascular phenotype similar to AOS, marked by the progressive development of aneurysms. These aneurysms were associated with various pathological changes in transmural inflammatory cell infiltration. Bone marrow transplants from Smad3-/- mice induced aortitis and aortic root dilation in irradiated WT recipient mice. Transplantation of CD4+ T cells from Smad3-/- mice also induced aortitis in Smad3+/+ recipient mice, while depletion of CD4+ T cells in Smad3-/- mice reduced the infiltration of inflammatory cells in the aortic root. Furthermore, IFN-γ deficiency increased, while IL-17 deficiency decreased, disease severity in Smad3+/- mice. Cytokine secretion was measured using a cytokine quantibody array, and Smad3-/- CD4+ T cells secreted more GM-CSF than Smad3+/+ CD4+ T cells. GM-CSF induced CD11b+Gr-1+Ly-6Chi inflammatory monocyte accumulation in the aortic root, but administration of anti-GM-CSF mAb to Smad3-/- mice resulted in significantly less inflammation and dilation in the aortic root. We also identified a missense mutation (c.985A>G) in a family of thoracic aortic aneurysms. Intense inflammatory infiltration and GM-CSF expression was observed in aortas specimens of these patients, suggesting that GM-CSF is potentially involved in the development of AOS.

p300 is elevated in systemic sclerosis and its expression is positively regulated by TGF-ß: epigenetic feed-forward amplification of fibrosis.

Fibrosis, the hallmark of systemic sclerosis (SSc), is characterized by persistent fibroblast activation triggered by transforming growth factor-ß (TGF-ß). As the acetyltransferase p300 has a key role in fibrosis and its availability governs the intensity of fibrotic responses, we investigated p300 expression in SSc and the molecular basis of its regulation. We found that expression of p300 was markedly elevated in SSc skin biopsies and was induced by TGF-ß in explanted normal skin fibroblasts. Stimulation of p300 by TGF-ß was independent of Smads and involved the early-immediate transcription factor Egr-1 (early growth response 1), a key regulator of profibrotic TGF-ß signaling. Indeed, Egr-1 was both sufficient and necessary for p300 regulation in vitro and in vivo. Increased p300 accumulation in TGF-ß-treated fibroblasts was associated with histone hyperacetylation, whereas p300 depletion, or selective pharmacological blockade of its acetyltransferase activity, attenuated TGF-ß-induced responses. Moreover, TGF-ß enhanced both p300 recruitment and in vivo histone H4 acetylation at the COL1A2 (collagen, type I, α2) locus. These findings implicate p300-mediated histone acetylation as a fundamental epigenetic mechanism in fibrogenesis and place Egr-1 upstream in TGF-ß-driven stimulation of p300 gene expression. The results establish a firm link between fibrosis with aberrant p300 expression and epigenetic activity that, to our knowledge, is previously unreported. Targeted disruption of p300-mediated histone acetylation might therefore represent a viable antifibrotic strategy.

Genetic partitioning of interleukin-6 signalling in mice dissociates Stat3 from Smad3-mediated lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease that is unresponsive to current therapies and characterized by excessive collagen deposition and subsequent fibrosis. While inflammatory cytokines, including interleukin (IL)-6, are elevated in IPF, the molecular mechanisms that underlie this disease are incompletely understood, although the development of fibrosis is believed to depend on canonical transforming growth factor (TGF)-ß signalling. We examined bleomycin-induced inflammation and fibrosis in mice carrying a mutation in the shared IL-6 family receptor gp130. Using genetic complementation, we directly correlate the extent of IL-6-mediated, excessive Stat3 activity with inflammatory infiltrates in the lung and the severity of fibrosis in corresponding gp130(757F) mice. The extent of fibrosis was attenuated in B lymphocyte-deficient gp130(757F);µMT(-/-) compound mutant mice, but fibrosis still occurred in their Smad3(-/-) counterparts consistent with the capacity of excessive Stat3 activity to induce collagen 1α1 gene transcription independently of canonical TGF-ß/Smad3 signalling. These findings are of therapeutic relevance, since we confirmed abundant STAT3 activation in fibrotic lungs from IPF patients and showed that genetic reduction of Stat3 protected mice from bleomycin-induced lung fibrosis.

Gastric tumor development in Smad3-deficient mice initiates from forestomach/glandular transition zone along the lesser curvature.

SMAD proteins are downstream effectors of the TGF-ß signaling pathway. Smad3-null mice develop colorectal cancer by 6 months of age. In this study, we have examined whether the loss of Smad3 promotes gastric neoplasia in mice. The stomachs of Smad3⁻/⁻ mice were compared with age-matched Smad3 heterozygous and wild-type mice. E-cadherin, Ki-67, phosphoSTAT3, and TFF2/SP expression was analyzed by immunohistochemisty. The short hairpin RNA (ShRNA)-mediated knockdown of Smad3 in AGS and MKN28 cells was also performed. In addition, we examined alterations in DCLK1-expressing cells. Smad3⁻/⁻ mouse stomachs at 6 months of age revealed the presence of exophytic growths along the lesser curvature in the proximal fundus. Six-month-old Smad3⁻/⁻ mouse stomachs showed metaplastic columnar glands initiating from the transition zone junction between the forestomach and the glandular epithelium along the lesser curvature. Ten-month-old Smad3⁻/⁻ mice all exhibited invasive gastric neoplastic changes with increased Ki-67, phosphoSTAT3 expression, and aberrant cytosolic E-cadherin staining in papillary glands within the invading submucosal gland. The shRNA-mediated knockdown of Smad3 in AGS and MKN28 cells promoted the expression of phosphoSTAT3. DCLK1-expressing cells, which also stained for the tuft cell marker acetylated-α-tubulin, were observed in 10-month-old Smad3⁻/⁻ mice within tumors and in fundic invasive lesions. In conclusion, Smad3-null mice develop gastric tumors in the fundus, which arise from the junction between the forestomach and the glandular epithelium and progress to prominent invasive tumors over time. Smad3-null mice represent a novel model of fundic gastric tumor initiated from forestomach/glandular transition zone along the lesser curvature.

Genetic deficiency of Smad3 protects the kidneys from atrophy and interstitial fibrosis in 2K1C hypertension.

Although the two-kidney, one-clip (2K1C) model is widely used as a model of human renovascular hypertension, mechanisms leading to the development of fibrosis and atrophy in the cuffed kidney and compensatory hyperplasia in the contralateral kidney have not been defined. Based on the well-established role of the transforming growth factor (TGF)-ß signaling pathway in renal fibrosis, we tested the hypothesis that abrogation of TGF-ß/Smad3 signaling would prevent fibrosis in the cuffed kidney. Renal artery stenosis (RAS) was established in mice with a targeted disruption of exon 2 of the Smad3 gene (Smad3 KO) and wild-type (WT) controls by placement of a polytetrafluoroethylene cuff on the right renal artery. Serial pulse-wave Doppler ultrasound assessments verified that blood flow through the cuffed renal artery was decreased to a similar extent in Smad3 KO and WT mice. Two weeks after surgery, systolic blood pressure and plasma renin activity were significantly elevated in both the Smad3 KO and WT mice. The cuffed kidney of WT mice developed renal atrophy (50% reduction in weight after 6 wk, P < 0.0001), which was associated with the development of interstitial fibrosis, tubular atrophy, and interstitial inflammation. Remarkably, despite a similar reduction of renal blood flow, the cuffed kidney of the Smad3 KO mice showed minimal atrophy (9% reduction in weight, P = not significant), with no significant histopathological alterations (interstitial fibrosis, tubular atrophy, and interstitial inflammation). We conclude that abrogation of TGF-ß/Smad3 signaling confers protection against the development of fibrosis and atrophy in RAS.