Insights into morphology and disease from the dog genome project.

Although most modern dog breeds are less than 200 years old, the symbiosis between man and dog is ancient. Since prehistoric times, repeated selection events have transformed the wolf into man's guardians, laborers, athletes, and companions. The rapid transformation from pack predator to loyal companion is a feat that is arguably unique among domesticated animals. How this transformation came to pass remained a biological mystery until recently: Within the past decade, the deployment of genomic approaches to study population structure, detect signatures of selection, and identify genetic variants that underlie canine phenotypes is ushering into focus novel biological mechanisms that make dogs remarkable. Ironically, the very practices responsible for breed formation also spurned morbidity; today, many diseases are correlated with breed identity. In this review, we discuss man's best friend in the context of a genetic model to understand paradigms of heritable phenotypes, both desirable and disadvantageous.

Fluid shear stress generates a unique signaling response by activating multiple TGFß family type I receptors in osteocytes.

Bone is a dynamic tissue that constantly adapts to changing mechanical demands. The transforming growth factor beta (TGFß) signaling pathway plays several important roles in maintaining skeletal homeostasis by both coupling the bone-forming and bone-resorbing activities of osteoblasts and osteoclasts and by playing a causal role in the anabolic response of bone to applied loads. However, the extent to which the TGFß signaling pathway in osteocytes is directly regulated by fluid shear stress (FSS) is unknown, despite work suggesting that fluid flow along canaliculi is a dominant physical cue sensed by osteocytes following bone compression. To investigate the effects of FSS on TGFß signaling in osteocytes, we stimulated osteocytic OCY454 cells cultured within a microfluidic platform with FSS. We find that FSS rapidly upregulates Smad2/3 phosphorylation and TGFß target gene expression, even in the absence of added TGFß. Indeed, relative to treatment with TGFß, FSS induced a larger increase in levels of pSmad2/3 and Serpine1 that persisted even in the presence of a TGFß receptor type I inhibitor. Our results show that FSS stimulation rapidly induces phosphorylation of multiple TGFß family R-Smads by stimulating multimerization and concurrently activating several TGFß and BMP type I receptors, in a manner that requires the activity of the corresponding ligand. While the individual roles of the TGFß and BMP signaling pathways in bone mechanotransduction remain unclear, these results implicate that FSS activates both pathways to generate a downstream response that differs from that achieved by either ligand alone.

SMAD2 and SMAD3 differentially regulate adiposity and the growth of subcutaneous white adipose tissue.

Adipose tissue is the primary site of energy storage, playing important roles in health. While adipose research largely focuses on obesity, fat also has other critical functions, producing adipocytokines and contributing to normal nutrient metabolism, which in turn play important roles in satiety and total energy homeostasis. SMAD2/3 proteins are downstream mediators of activin signaling, which regulate critical preadipocyte and mature adipocyte functions. Smad2 global knockout mice exhibit embryonic lethality, whereas global loss of Smad3 protects mice against diet-induced obesity. The direct contributions of Smad2 and Smad3 in adipose tissues, however, are unknown. Here, we sought to determine the primary effects of adipocyte-selective reduction of Smad2 or Smad3 on diet-induced adiposity using Smad2 or Smad3 "floxed" mice intercrossed with Adiponectin-Cre mice. Additionally, we examined visceral and subcutaneous preadipocyte differentiation efficiency in vitro. Almost all wild type subcutaneous preadipocytes differentiated into mature adipocytes. In contrast, visceral preadipocytes differentiated poorly. Exogenous activin A suppressed differentiation of preadipocytes from both depots. Smad2 conditional knockout (Smad2cKO) mice did not exhibit significant effects on weight gain, irrespective of diet, whereas Smad3 conditional knockout (Smad3cKO) male mice displayed a trend of reduced body weight on high-fat diet. On both diets, Smad3cKO mice displayed an adipose depot-selective phenotype, with a significant reduction in subcutaneous fat mass but not visceral fat mass. Our data suggest that Smad3 is an important contributor to the maintenance of subcutaneous white adipose tissue in a sex-selective fashion. These findings have implications for understanding SMAD-mediated, depot selective regulation of adipocyte growth and differentiation.

Targeted DNA oxidation by LSD1-SMAD2/3 primes TGF-ß1/ EMT genes for activation or repression.

The epithelial-to-mesenchymal transition (EMT) is a complex transcriptional program induced by transforming growth factor ß1 (TGF-ß1). Histone lysine-specific demethylase 1 (LSD1) has been recognized as a key mediator of EMT in cancer cells, but the precise mechanism that underlies the activation and repression of EMT genes still remains elusive. Here, we characterized the early events induced by TGF-ß1 during EMT initiation and establishment. TGF-ß1 triggered, 30-90 min post-treatment, a nuclear oxidative wave throughout the genome, documented by confocal microscopy and mass spectrometry, mediated by LSD1. LSD1 was recruited with phosphorylated SMAD2/3 to the promoters of prototypic genes activated and repressed by TGF-ß1. After 90 min, phospho-SMAD2/3 downregulation reduced the complex and LSD1 was then recruited with the newly synthesized SNAI1 and repressors, NCoR1 and HDAC3, to the promoters of TGF-ß1-repressed genes such as the Wnt soluble inhibitor factor 1 gene (WIF1), a change that induced a late oxidative burst. However, TGF-ß1 early (90 min) repression of transcription also required synchronous signaling by reactive oxygen species and the stress-activated kinase c-Jun N-terminal kinase. These data elucidate the early events elicited by TGF-ß1 and the priming role of DNA oxidation that marks TGF-ß1-induced and -repressed genes involved in the EMT.

Long noncoding RNA ZFAS1 suppresses chondrocytes apoptosis via miR-302d-3p/SMAD2 in osteoarthritis.

Osteoarthritis (OA) seriously affects people's quality of life due to joint pain, stiffness, disability, and dyskinesia worldwide. Long noncoding RNA zinc finger antisense 1 (ZFAS1) is downregulated and tightly associated with proliferation, migration, apoptosis, and matrix synthesis of chondrocyte in OA. However, the molecular mechanisms of ZFAS1 in OA remain unknown. The expression correlation between ZFAS1, miR-302d-3p, and SMAD2 in OA tissues was analyzed by Pearson correlation analysis. ZFAS1 was a lower expression, and expedited proliferation and repressed apoptosis of chondrocytes. MiR-302d-3p was a direct target of ZFAS1. MiR-302d-3p hindered proliferation and facilitated apoptosis of chondrocytes. MiR-302d-3p partially reversed the effect of ZFAS1 on proliferation and apoptosis of chondrocytes. SMAD2 was positively regulated by the ZFAS1/miR-302d-3p. MiR-302d-3p-mediated proliferation and apoptosis were partly abrogated by targeting SMAD2. ZFAS1 promoted chondrocytes proliferation and repressed apoptosis possibly by regulating miR-302d-3p/SMAD2 axis, providing a potential target for OA treatment.

Excess TGF-ß1 Drives Cardiac Mesenchymal Stromal Cells to a Pro-Fibrotic Commitment in Arrhythmogenic Cardiomyopathy.

Arrhythmogenic Cardiomyopathy (ACM) is characterized by the replacement of the myocardium with fibrotic or fibro-fatty tissue and inflammatory infiltrates in the heart. To date, while ACM adipogenesis is a well-investigated differentiation program, ACM-related fibrosis remains a scientific gap of knowledge. In this study, we analyze the fibrotic process occurring during ACM pathogenesis focusing on the role of cardiac mesenchymal stromal cells (C-MSC) as a source of myofibroblasts. We performed the ex vivo studies on plasma and right ventricular endomyocardial bioptic samples collected from ACM patients and healthy control donors (HC). In vitro studies were performed on C-MSC isolated from endomyocardial biopsies of both groups. Our results revealed that circulating TGF-ß1 levels are significantly higher in the ACM cohort than in HC. Accordingly, fibrotic markers are increased in ACM patient-derived cardiac biopsies compared to HC ones. This difference is not evident in isolated C-MSC. Nevertheless, ACM C-MSC are more responsive than HC ones to TGF-ß1 treatment, in terms of pro-fibrotic differentiation and higher activation of the SMAD2/3 signaling pathway. These results provide the novel evidence that C-MSC are a source of myofibroblasts and participate in ACM fibrotic remodeling, being highly responsive to ACM-characteristic excess TGF-ß1.

Distinct roles of myofibroblast-specific Smad2 and Smad3 signaling in repair and remodeling of the infarcted heart.

TGF-ßs regulate fibroblast responses, by activating Smad2 or Smad3 signaling, or via Smad-independent pathways. We have previously demonstrated that myofibroblast-specific Smad3 is critically implicated in repair of the infarcted heart. However, the role of fibroblast Smad2 in myocardial infarction remains unknown. This study investigates the role of myofibroblast-specific Smad2 signaling in myocardial infarction, and explores the mechanisms responsible for the distinct effects of Smad2 and Smad3. In a mouse model of non-reperfused myocardial infarction, Smad2 activation in infarct myofibroblasts peaked 7 days after coronary occlusion. In vitro, TGF-ß1, -ß2 and -ß3, but not angiotensin 2 and bone morphogenetic proteins-2, -4 and -7, activated fibroblast Smad2. Myofibroblast-specific Smad2 and Smad3 knockout mice (FS2KO, FS3KO) and corresponding control littermates underwent non-reperfused infarction. In contrast to the increase in rupture rates and adverse remodeling in FS3KO mice, FS2KO animals had mortality comparable to Smad2 fl/fl controls, and exhibited a modest but transient improvement in dysfunction after 7 days of coronary occlusion. At the 28 day timepoint, FS2KO and Smad2 fl/fl mice had comparable adverse remodeling. Although both FS3KO and FS2KO animals had increased myofibroblast density in the infarct, only FS3KO mice exhibited impaired scar organization, associated with perturbed alignment of infarct myofibroblasts. In vitro, Smad3 but not Smad2 knockdown downmodulated fibroblast α2 and α5 integrin expression. Moreover, Smad3 knockdown reduced expression of the GTPase RhoA, whereas Smad2 knockdown markedly increased fibroblast RhoA levels. Smad3-dependent integrin expression may be important for fibroblast activation, whereas RhoA may transduce planar cell polarity pathway signals, essential for fibroblast alignment. Myofibroblast-specific Smad3, but not Smad2 is required for formation of aligned myofibroblast arrays in the infarct. The distinct in vivo effects of myofibroblast Smad2 and Smad3 may involve Smad3-dependent integrin synthesis, and contrasting effects of Smad2 and Smad3 on RhoA expression.

Activin/Smad2 and Wnt/ß-catenin up-regulate HAS2 and ALDH3A2 to facilitate mesendoderm differentiation of human embryonic stem cells.

Activin and Wnt signaling are necessary and sufficient for mesendoderm (ME) differentiation of human embryonic stem cells (ESCs). In this study, we report that during ME differentiation induced by Activin and Wnt, Activin/Smad2 induces a decrease of the repressive histone modification of H3K27me3 by promoting the proteasome-dependent degradation of enhancer of zeste 2 polycomb (EZH2)-repressive complex 2 subunit. As a result, recruitment of the forkhead protein FOXH1 on open chromatin regions integrates the signals of Activin/Smad2 and Wnt/ß-catenin to activate the expression of the ME genes including HAS2 and ALDH3A2 Consistently, H3K27me3 decrease is enriched on open chromatin around regulatory regions. Furthermore, knockdown of HAS2 or ALDH3A2 greatly attenuates ME differentiation. These findings unveil a pathway from extracellular signals to epigenetic modification-mediated gene activation during ME commitment.

Hydrogen sulfide attenuates paraquat-induced epithelial-mesenchymal transition of human alveolar epithelial cells through regulating transforming growth factor-ß1/Smad2/3 signaling pathway.

Exogenous H2 S donor, sodium hydrosulfide (NaHS), can influence the bleomycin-induced pulmonary fibrosis by attenuating the epithelial-mesenchymal transition (EMT) of alveolar epithelial cells, but whether NaHS affects paraquat (PQ)-induced EMT and the molecular mechanisms remain unclarified. The aim of the present study is to examine the effect of exogenous NaHS on PQ-induced EMT in human alveolar epithelial cells (A549 cells) and assess if this effect occurs through regulating transforming growth factor (TGF)-ß1/Smad2/3 signaling pathway. The expressions of endogenous H2 S producing enzymes, namely cystathionine ß-synthase, cystathionine γ-lyase and 3-mercaptopyruvate sulfur transferase, were detected by reverse transcription-polymerase chain reaction and western blotting. The induced EMT was assessed by morphological and phenotypic characterizations, and the protein level of E-cadherin and vimentin were detected by western blotting. To investigate the effect of NaHS on PQ-induced EMT and potential mechanism, A549 cells were pretreated with NaHS before incubating with PQ and then evaluated by morphological changes, cell migration ability, the expression of EMT markers and TGF-ß1/Smad2/3 signaling pathway related proteins. PQ significantly downregulated the expression levels of cystathionine ß-synthase and cystathionine γ-lyase, but not 3-mercaptopyruvate sulfur transferase, in a time-dependent manner in A549 cells. Exogenous NaHS could significantly retard PQ-induced morphological changes and cell migration ability. Furthermore, exogenous NaHS significantly upregulated the expression of E-cadherin, whereas it downregulated the expression of vimentin. In addition, exogenous NaHS could also significantly attenuates PQ-induced TGF-ß1, phosphorylated Smad2/3 proteins expression, which induced by PQ in a time-dependent manner. This study provides the first evidence that exogenous NaHS attenuates PQ-induced EMT and migration of human alveolar epithelial cells through regulating the TGF-ß1/Smad2/3 signaling pathway.

Is Growth Differentiation Factor 11 a Realistic Therapeutic for Aging-Dependent Muscle Defects?

This "Controversies in Cardiovascular Research" article evaluates the evidence for and against the hypothesis that the circulating blood level of growth differentiation factor 11 (GDF11) decreases in old age and that restoring normal GDF11 levels in old animals rejuvenates their skeletal muscle and reverses pathological cardiac hypertrophy and cardiac dysfunction. Studies supporting the original GDF11 hypothesis in skeletal and cardiac muscle have not been validated by several independent groups. These new studies have either found no effects of restoring normal GDF11 levels on cardiac structure and function or have shown that increasing GDF11 or its closely related family member growth differentiation factor 8 actually impairs skeletal muscle repair in old animals. One possible explanation for what seems to be mutually exclusive findings is that the original reagent used to measure GDF11 levels also detected many other molecules so that age-dependent changes in GDF11 are still not well known. The more important issue is whether increasing blood [GDF11] repairs old skeletal muscle and reverses age-related cardiac pathologies. There are substantial new and existing data showing that GDF8/11 can exacerbate rather than rejuvenate skeletal muscle injury in old animals. There is also new evidence disputing the idea that there is pathological hypertrophy in old C57bl6 mice and that GDF11 therapy can reverse cardiac pathologies. Finally, high [GDF11] causes reductions in body and heart weight in both young and old animals, suggestive of a cachexia effect. Our conclusion is that elevating blood levels of GDF11 in the aged might cause more harm than good.

FoxH1 mediates a Grg4 and Smad2 dependent transcriptional switch in Nodal signaling during Xenopus mesoderm development.

In the vertebrate blastula and gastrula the Nodal pathway is essential for formation of the primary germ layers and the organizer. Nodal autoregulatory feedback potentiates signaling activity, but mechanisms limiting embryonic Nodal ligand transcription are poorly understood. Here we describe a transcriptional switch mechanism mediated by FoxH1, the principle effector of Nodal autoregulation. FoxH1 contains a conserved engrailed homology (EH1) motif that mediates direct binding of groucho-related gene 4 (Grg4), a Groucho family corepressor. Nodal-dependent gene expression is suppressed by FoxH1, but enhanced by a FoxH1 EH1 mutant, indicating that the EH1 motif is necessary for repression. Grg4 blocks Nodal-induced mesodermal gene expression and Nodal autoregulation, suggesting that Grg4 limits Nodal pathway activity. Conversely, blocking Grg4 function in the ectoderm results in ectopic expression of Nodal target genes. FoxH1 and Grg4 occupy the Xnr1 enhancer, and Grg4 occupancy is dependent on the FoxH1 EH1 motif. Grg4 occupancy at the Xnr1 enhancer significantly decreases with Nodal activation or Smad2 overexpression, while FoxH1 occupancy is unaffected. These results suggest that Nodal-activated Smad2 physically displaces Grg4 from FoxH1, an essential feature of the transcriptional switch mechanism. In support of this model, when FoxH1 is unable to bind Smad2, Grg4 occupancy is maintained at the Xnr1 enhancer, even in the presence of Nodal signaling. Our findings reveal that FoxH1 mediates both activation and repression of Nodal gene expression. We propose that this transcriptional switch is essential to delimit Nodal pathway activity in vertebrate germ layer formation.

Foxf2 is required for secondary palate development and Tgfß signaling in palatal shelf mesenchyme.

The secondary palate separates the oral from the nasal cavity and its closure during embryonic development is sensitive to genetic perturbations. Mice with deleted Foxf2, encoding a forkhead transcription factor, are born with cleft palate, and an abnormal tongue morphology has been proposed as the underlying cause. Here, we show that Foxf2(-/-) maxillary explants cultured in vitro, in the absence of tongue and mandible, failed to close the secondary palate. Proliferation and collagen content were decreased in Foxf2(-/-) palatal shelf mesenchyme. Phosphorylation of Smad2/3 was reduced in mutant palatal shelf, diagnostic of attenuated canonical Tgfß signaling, whereas phosphorylation of p38 was increased. The amount of Tgfß2 protein was diminished, whereas the Tgfb2 mRNA level was unaltered. Expression of several genes encoding extracellular proteins important for Tgfß signaling were reduced in Foxf2(-)(/)(-) palatal shelves: a fibronectin splice-isoform essential for formation of extracellular Tgfß latency complexes; Tgfbr3 - or betaglycan - which acts as a co-receptor and an extracellular reservoir of Tgfß; and integrins αV and ß1, which are both Tgfß targets and required for activation of latent Tgfß. Decreased proliferation and reduced extracellular matrix content are consistent with diminished Tgfß signaling. We therefore propose that gene expression changes in palatal shelf mesenchyme that lead to reduced Tgfß signaling contribute to cleft palate in Foxf2(-)(/)(-) mice.

Astaxanthin attenuated pressure overload-induced cardiac dysfunction and myocardial fibrosis: Partially by activating SIRT1.

BACKGROUND: Myocardial fibrosis contributes to cardiac dysfunction. Astaxanthin (AST), a member of the carotenoid family, is a well-known antioxidant, but its effect on and underlying mechanisms in myocardial fibrosis are poorly understood. METHODS: In vivo, myocardial fibrosis and cardiac dysfunction were induced using transverse aortic constriction (TAC). AST was administered to mice for 12weeks post-surgery. In vitro, transforming growth factor ß1 (TGF-ß1) was used to stimulate human cardiac fibroblasts (HCFs). EX-527 (6-chloro-2, 3, 4, 9-tetrahydro-1H-carbazole-1-carboxamide) and SIRT1 siRNA were used to inhibit SIRT1 in vivo and in vitro, respectively. The effects of AST on cardiac function and fibrosis were determined. SIRT1 expression and activity were measured to explore the mechanisms underlying its effects. RESULTS: AST improved cardiac function and attenuated fibrosis. Receptor activated-SMADs (R-SMADs), including SMAD2 and SMAD3, played important roles in these processes. The TAC surgery-induced increases in the expression of phosphorylated and acetylated R-SMADs were attenuated by treatment with AST, the translocation and transcriptional activity of R-SMADs were also restrained. These effects were accompanied by an increase in the expression and activity of SIRT1. Inhibiting SIRT1 attenuated the acetylation and transcriptional activity of R-SMADs, but not their phosphorylation and translocation. CONCLUSIONS: Our data demonstrate that AST improves cardiac function and attenuates fibrosis by decreasing phosphorylation and deacetylation of R-SMADs. SIRT1 contributes to AST's protective function by reducing acetylation of R-SMADs. GENERAL SIGNIFICANCE: These data suggest that AST may be useful as a preventive/therapeutic agent for cardiac dysfunction and myocardial fibrosis.

YAP/TAZ Are Mechanoregulators of TGF-ß-Smad Signaling and Renal Fibrogenesis.

Like many organs, the kidney stiffens after injury, a process that is increasingly recognized as an important driver of fibrogenesis. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are related mechanosensory proteins that bind to Smad transcription factors, the canonical mediators of profibrotic TGF-ß responses. Here, we investigated the role of YAP/TAZ in the matrix stiffness dependence of fibroblast responses to TGF-ß In contrast to growth on a stiff surface, fibroblast growth on a soft matrix led to YAP/TAZ sequestration in the cytosol and impaired TGF-ß-induced Smad2/3 nuclear accumulation and transcriptional activity. YAP knockdown or treatment with verteporfin, a drug that was recently identified as a potent YAP inhibitor, elicited similar changes. Furthermore, verteporfin reduced YAP/TAZ levels and decreased the total cellular levels of Smad2/3 after TGF-ß stimulation. Verteporfin treatment of mice subjected to unilateral ureteral obstruction similarly reduced YAP/TAZ levels and nuclear Smad accumulation in the kidney, and attenuated renal fibrosis. Our data suggest that organ stiffening cooperates with TGF-ß to induce fibrosis in a YAP/TAZ- and Smad2/3-dependent manner. Interference with this YAP/TAZ and TGF-ß/Smad crosstalk likely underlies the antifibrotic activity of verteporfin. Finally, through repurposing of a clinically used drug, we illustrate the therapeutic potential of a novel mechanointerference strategy that blocks TGF-ß signaling and renal fibrogenesis.

Inhibition of Activin Signaling Slows Progression of Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD), characterized by the formation of numerous kidney cysts, is caused by PKD1 or PKD2 mutations and affects 0.1% of the population. Although recent clinical studies indicate that reduction of cAMP levels slows progression of PKD, this finding has not led to an established safe and effective therapy for patients, indicating the need to find new therapeutic targets. The role of TGF-ß in PKD is not clearly understood, but nuclear accumulation of phosphorylated SMAD2/3 in cyst-lining cells suggests the involvement of TGF-ß signaling in this disease. In this study, we ablated the TGF-ß type 1 receptor (also termed activin receptor-like kinase 5) in renal epithelial cells of PKD mice, which had little to no effect on the expression of SMAD2/3 target genes or the progression of PKD. Therefore, we investigated whether alternative TGF-ß superfamily ligands account for SMAD2/3 activation in cystic epithelial cells. Activins are members of the TGF-ß superfamily and drive SMAD2/3 phosphorylation via activin receptors, but activins have not been studied in the context of PKD. Mice with PKD had increased expression of activin ligands, even at early stages of disease. In addition, treatment with a soluble activin receptor IIB fusion (sActRIIB-Fc) protein, which acts as a soluble trap to sequester activin ligands, effectively inhibited cyst formation in three distinct mouse models of PKD. These data point to activin signaling as a key pathway in PKD and a promising target for therapy.

Serum Glucocorticoid-Regulated Kinase 1 Blocks CKD-Induced Muscle Wasting Via Inactivation of FoxO3a and Smad2/3.

Muscle proteolysis in CKD is stimulated when the ubiquitin-proteasome system is activated. Serum glucocorticoid-regulated kinase 1 (SGK-1) is involved in skeletal muscle homeostasis, but the role of this protein in CKD-induced muscle wasting is unknown. We found that, compared with muscles from healthy controls, muscles from patients and mice with CKD express low levels of SGK-1. In mice, SGK-1-knockout (SGK-1-KO) induced muscle loss that correlated with increased expression of ubiquitin E3 ligases known to facilitate protein degradation by the ubiquitin-proteasome, and CKD substantially aggravated this response. SGK-1-KO also altered the phosphorylation levels of transcription factors FoxO3a and Smad2/3. In C2C12 muscle cells, expression of dominant negative FoxO3a or knockdown of Smad2/3 suppressed the upregulation of E3 ligases induced by loss of SGK-1. Additionally, SGK-1 overexpression increased the level of phosphorylated N-myc downstream-regulated gene 1 protein, which directly interacted with and suppressed the phosphorylation of Smad2/3. Overexpression of SGK-1 in wild-type mice with CKD had similar effects on the phosphorylation of FoxO3a and Smad2/3 and prevented CKD-induced muscle atrophy. Finally, mechanical stretch of C2C12 muscle cells or treadmill running of wild-type mice with CKD stimulated SGK-1 production, and treadmill running inhibited proteolysis in muscle. These protective responses were absent in SGK-1-KO mice. Thus, SGK-1 could be a mechanical sensor that mediates exercise-induced improvement in muscle wasting stimulated by CKD.

Endoglin Regulation of Smad2 Function Mediates Beclin1 Expression and Endothelial Autophagy.

Autophagy is the targeted degradation of proteins and organelles critical for homeostasis and cell survival. Transforming growth factor ß (TGF-ß) differentially regulates autophagy in a context-specific manner, although the precise intracellular mechanisms remain less clear. Importantly, how TGF-ß controls autophagic responses in endothelial cells (EC) during angiogenesis is unknown. Here we identified endoglin, an EC-specific TGF-ß co-receptor essential for angiogenesis, as a key determinant of autophagy. Among the two opposing TGF-ß Smad pathways in the EC system (Smad1/5/8 and Smad2/3), we found Smad2 as the major transcriptional regulator of autophagy that targets beclin1 (BECN1) gene expression. Smad2, but not Smad3, acts as a repressor upstream of the BECN1 promoter region. Overall, endoglin promotes autophagy by impeding Smad2 transcriptional repressor activity. Notably, increased beclin1 levels upon Smad2 knockdown directly correlated with enhanced autophagy during angiogenesis. Taken together, these results establish endoglin as a critical mediator of autophagy and demonstrate a new transcriptional mechanism by which Smad2 inhibits angiogenesis.

The SMAD2/3 pathway is involved in hepaCAM-induced apoptosis by inhibiting the nuclear translocation of SMAD2/3 in bladder cancer cells.

The aim of this study was to explore the correlation between hepatocyte cell adhesion molecule (hepaCAM) and SMAD family member 2/3 (SMAD2/3) in bladder carcinoma, and the involvement of the SMAD2/3 pathway in hepaCAM-induced tumor apoptosis. Immunohistochemistry was used to measure hepaCAM and p-SMAD2/3 protein levels in bladder cancer tissues. Flow cytometry and Hoechst staining were used to study the effect of hepaCAM on cellular apoptosis. Western blot was employed to determine the expression of hepaCAM and SMAD2/3/caspase pathway molecules using a hepaCAM overexpression adenovirus, a caspase inhibitor (Z-VAD-FMK), and a SMAD2/3 activator (transforming growth factor (TGF)-ß1), respectively. Translocation of p-SMAD2/3 was measured by immunofluorescence and western blot. HepaCAM proteins were significantly decreased (P < 0.05), while p-SMAD2/3 proteins were remarkably increased (P < 0.05) in bladder carcinoma compared to adjacent tissues. However, the low hepaCAM and high p-SMAD2/3 were not statistically associated with clinicopathological characteristics of the patients. A negative linear correlation between hepaCAM and p-SMAD2/3 was observed according to Pearson analysis (r = -0.712/-0.724, P = 0.008/0.011). Overexpression of hepaCAM activated caspase 3/8/9 and downregulated poly-ADP ribose polymerase (PARP) and p-SMAD2/3. Treatment of bladder cancer cells with Z-VAD-FMK + hepaCAM significantly downregulated procaspase 3/8/9 and PARP and induced cellular apoptosis, compared with that using Z-VAD-FMK alone. Similarly, combined treatment of TGF-ß1 + hepaCAM significantly downregulated p-SMAD2/3, procaspase 3/8/9, and PARP and induced apoptosis of bladder cancer cells, compared with TGF-ß1 alone. Overexpression of hepaCAM prevented the p-SMAD2/3 translocation from the cytoplasm to the nucleus in bladder cancer cells BIU-87 and T24. Our findings uncover that the p-SMAD2/3 pathway is critical for hepaCAM-induced cancer cell apoptosis and provide valuable insights for current and future Ad-hepaCAM and p-SMAD2/3 clinical trials.

Cigarette smoke-induced alveolar epithelial-mesenchymal transition is mediated by Rac1 activation.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is the major pathophysiological process in lung fibrosis observed in chronic obstructive pulmonary disease (COPD) and lung cancer. Smoking is a risk factor for developing EMT, yet the mechanism remains largely unknown. In this study, we investigated the role of Rac1 in cigarette smoke (CS) induced EMT. METHODS: EMT was induced in mice and pulmonary epithelial cells by exposure of CS and cigarette smoke extract (CSE) respectively. RESULTS: Treatment of pulmonary epithelial cells with CSE elevated Rac1 expression associated with increased TGF-ß1 release. Blocking TGF-ß pathway restrained CSE-induced changes in EMT-related markers. Pharmacological inhibition or knockdown of Rac1 decreased the CSE exposure induced TGF-ß1 release and ameliorated CSE-induced EMT. In CS-exposed mice, pharmacological inhibition of Rac1 reduced TGF-ß1 release and prevented aberrations in expression of EMT markers, suggesting that Rac1 is a critical signaling molecule for induction of CS-stimulated EMT. Furthermore, Rac1 inhibition or knockdown abrogated CSE-induced Smad2 and Akt (PKB, protein kinase B) activation in pulmonary epithelial cells. Inhibition of Smad2, PI3K (phosphatidylinositol 3-kinase) or Akt suppressed CSE-induced changes in epithelial and mesenchymal marker expression. CONCLUSIONS AND GENERAL SIGNIFICANCE: Altogether, these data suggest that CS initiates EMT through Rac1/Smad2 and Rac1/PI3K/Akt signaling pathway. Our data provide new insights into the fundamental basis of EMT and suggest a possible new course of therapy for COPD and lung cancer.

Integrin beta 8 (ITGB8) regulates embryo implantation potentially via controlling the activity of TGF-B1 in mice.

Integrins (ITGs) are mediators of cell-cell and cell-matrix interactions, which are also associated with embryo implantation processes by controlling the interaction of blastocyst with endometrium. During early pregnancy, ITGbeta8 (ITGB8) has been shown to interact with latent transforming growth factor (TGF) beta 1 (TGFB1) at the fetomaternal interface. However, the precise role of ITGB8 in the uterus and its association with embryo implantation has not been elucidated. Therefore, we attempted to ascertain the role of ITGB8 during the window of embryo implantation process by inhibiting its function or protein expression. Uterine plasma membrane-anchored ITGB8 was augmented at peri-implantation and postimplantation stages. A similar pattern of mRNA expression was also found during the embryo implantation period. An immunolocalization study revealed the presence of ITGB8 on luminal epithelial cells along with mild expression on the stromal cells throughout the implantation period studied; however, an intense fluorescence was noted only during the peri- and postimplantation stages. Bioneutralization and mRNA silencing of the uterine Itgb8 at preimplantation stage reduced the rate/frequency of embryo implantation and subsequent pregnancy, suggesting its indispensable role during the embryo implantation period. ITGB8 can also regulate the liberation of active TGFB1 from its latent complex, which, in turn, acts on SMAD2/3 phosphorylation (activation) in the uterus during embryo implantation. This indicates involvement of ITGB8 in the embryo implantation process through regulation of activation of TGFB1.