Role Of TGF-beta Signaling Research Articles

Abstract P2112: Mechanism Of Bag3's Degradation Of Tgfbr2 In Cardiac Fibroblasts

BAG3 levels are diminished in patients with heart failure and several mutations have been identified to cause dilated cardiomyopathy with cardiac fibrosis. BAG3’s four binding domains are critical to its function to facilitate proteostasis. BAG3’s role has been extensively studied in CMs, where it aids sarcomere protein turnover through chaperone-assisted selective autophagy, through BAG3’s complex with HSP70, HSPB8, and E3 ligase CHIP. However, BAG3 is ubiquitously expressed and its role in non-myocytes has been overlooked. Cardiac fibroblasts are responsible for the excessive matrix deposition leading to fibrosis, mostly through TGF-beta signaling. Previously, we have shown the importance of BAG3 in cardiac fibroblasts through its role in TGF-beta signaling and identified BAG3 to facilitate the degradation of the type II TGF-beta receptor (TGFBR2). As BAG3 does not have inherent E3 activity, this study aimed to identify the specific E3 ligase of TGFBR2 and the binding domains of BAG3 that are crucial to this interaction to gain insight into the effects of the clinical mutations of BAG3. Immunoprecipitation (IP) of endogenous BAG3 in cardiac fibroblasts revealed five E3 ligases as high-confidence interactors of BAG3. To find the specific E3 ligase of TGFBR2, we performed an IP of a V5-tagged TGFBR2 and identified HSP70, HSPB8 and CHIP as binding partners. To delineate the critical domains of BAG3 that impact the TGFBR2 degradation, we overexpressed 3x-FLAG-tagged BAG3 proteins lacking each domain respectively (WW, IPV, PxxP, and BAG) and show that the BAG domain is imperative in TGFBR2 degradation. This has clinical implications for the common DCM-causing E455K mutation in the BAG domain. The ubiquitination of TGFBR2 was decreased upon overexpression of a BAG3 plasmid containing the E455K mutation. Lastly, the drug JG-98, which disrupts the binding of HSP70 to BAG3, was used to show the importance of the HSP70-BAG3 axis in the degradation of TGFBR2. In conclusion, we have uncovered the molecular mechanism of TGFBR2 degradation in cardiac fibroblasts mediated by the BAG3-HSP70-HSPB8 complex, with clinical implications for the loss of BAG3 and several common DCM-causing mutations in the development of cardiac fibrosis.

Local TGF-beta sequestration by fibrillin-1 regulates vascular wall homeostasis in the thoracic aorta

Abstract Background Aortic dissection and rupture is the main cause of early cardiovascular mortality in patients with Marfan syndrome (MFS). MFS is caused by a defect in fibrillin-1, a building block of microfibrils in the extracellular matrix which binds transforming growth factor beta (TGF-beta) via interaction with latent TGF-beta binding proteins (LTBPs). Multiple mouse models, both pharmaceutically induced and genetically manipulated, have been used to investigate the pathophysiology and biomechanical aspects of thoracic aortic aneurysms and dissections. However, the role of TGF-beta in MFS has been controversial, with earlier studies suggesting that excess release of TGF-beta due to decreased interaction with dysfunctional fibrillin-1 leads to aortic dilation and vascular damage, while other studies have shown an important protective effect of TGF-beta. Studying dedicated mouse models for MFS, with defects interfering with TGF-beta binding and -function may help resolve these discrepancies. Purpose This study aimed to reveal insights in the role of TGF-beta signaling in aneurysm formation and dissection in MFS. Methods Mice lacking the fibrillin-1 binding site for LTBPs (Fbn1H1Δ/+ and Fbn1H1Δ/H1Δ), mice with a truncated fibrillin-1 (Fbn1GT-8/+), and mice with a combination of both alleles (Fbn1GT-8/ H1Δ) were subjected to in vivo cardiac ultrasound analysis. Ex vivo phase-contrast synchrotron X-ray imaging was performed at the Paul Scherrer Institute to visualize the elastic lamellae architecture in the vascular wall of the entire excised thoracic aorta in a subset of mice from each group. Results Fbn1GT-8/+, Fbn1 H1Δ/+ and Fbn1H1Δ/H1Δ mice had a normal life span, but Fbn1GT-8/ H1Δ mice showed increased mortality due to aortic rupture starting at 4–5 months of age. The aortic root was dilated both in Fbn1GT-8/+ and Fbn1GT-8/ H1Δ mice at 6 months of age, but not in Fbn1H1D/+ or Fbn1H1Δ/H1Δ mice. Synchrotron images showed significant elastic lamellae fragmentation in the thoracic aortic wall of Fbn1GT-8/+ mice, and to a larger extent in Fbn1GT-8/ H1Δ mice. Surprisingly, localized elastin fragmentation was also found in the ascending thoracic aorta of Fbn1 H1Δ/+ and Fbn1H1Δ/H1Δ mice, despite a lack of aortic aneurysm formation. Moreover, Fbn1H1Δ/H1Δ mice displayed more severe aortic wall damage. The localized microdissections found in these mouse models were characterized by a severe alteration of the elastic fiber organization, cellular influx and increased collagen deposition, as confirmed by histological analysis. Conclusions Our data suggest that loss of LTBP binding to fibrillin-1 leads to the development of localized microdissections in the aorta in the absence of aortic aneurysm, and exacerbates the aortic wall morphology abnormalities in mice with truncated fibrillin-1. We therefore hypothesize that local TGF-beta sequestration is required to maintain aortic homeostasis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Foundation for Cardiac Surgery (“VZW Fonds voor Hartchirurgie”), grant No. 489644Baillet-Latour Grant for Medical Research

Role of TGF-Beta Signaling in Beta Cell Proliferation and Function in Diabetes.

Beta (β) cell dysfunction or loss is the common pathological feature in all types of diabetes mellitus (diabetes). Resolving the underlying mechanism may facilitate the treatment of diabetes by preserving the β cell population and function. It is known that TGF-β signaling plays diverse roles in β cell development, function, proliferation, apoptosis, and dedifferentiation. Inhibition of TGF-β signaling expands β cell lineage in the development. However, deletion of Tgfbr1 has no influence on insulin demand-induced but abolishes inflammation-induced β cell proliferation. Among canonical TGF-β signaling, Smad3 but not Smad2 is the predominant repressor of β cell proliferation in response to systemic insulin demand. Deletion of Smad3 simultaneously improves β cell function, apoptosis, and systemic insulin resistance with the consequence of eliminated overt diabetes in diabetic mouse models, revealing Smad3 as a key mediator and ideal therapeutic target for type-2 diabetes. However, Smad7 shows controversial effects on β cell proliferation and glucose homeostasis in animal studies. On the other hand, overexpression of Tgfb1 prevents β cells from autoimmune destruction without influence on β cell function. All these findings reveal the diverse regulatory roles of TGF-β signaling in β cell biology.

Abstract 1714A: TGF-beta signaling on myeloid cells regulates ECM deposition in mammary carcinoma via adenosine dependent mechanisms

Abstract TGF-beta plays a crucial role in tumor microenvironment by regulating cell-cell and cell-stroma interactions. We previously demonstrated that TGF-beta signaling on myeloid cells regulates expression of CD73, a key enzyme for production of adenosine, a pro-tumorigenic metabolite implicated in regulation of tumor cell behaviors, immune response and angiogenesis. Using MMTV-PyMT mouse mammary tumor model, we discovered that deletion of TGF-beta signaling on myeloid cells (PyMT/TGFbRII-LysM) affects ECM formation in tumor tissue, specifically increases collagen and decreases fibronectin deposition, and these changes associate with mitigated tumor growth and reduced metastases. Using multiplexed five-color immunofluorescent staining and spatial analysis on a single-cell level, we discovered that reduced TGF-beta signaling on fibroblasts associates with their proximity to CD73 positive myeloid cells in tumor tissue. Consistent with these findings, in vitro gel contraction assay and Western blotting for Collagen I and pSMAD proteins confirmed that adenosine significantly downregulates TGF-beta signaling on fibroblasts. Using in vitro pharmacological approach, we found that this effect is regulated by A2a and A2b adenosine receptors. TCGA data base analysis revealed that patients with triple negative breast cancer and basal type have similar signature of adenosine and ECM profiles: high expression of A2b adenosine receptors correlates with decreased expression of Col1 and is associates with poor outcome. Taken together, our studies reveal a new role for TGF-beta signaling on myeloid cells in tumorigenesis. Discovered crosstalk between TGF-beta/CD73 on myeloid cells and TGF-beta signaling on fibroblasts can contribute to ECM remodeling and pro-tumorigenic actions of CAFs. Citation Format: Georgii Vasiukov, Tatiana Novitskaya, Samantha Schwager, Harold Moses, Timothy Blackwell, Igor Feoktistov, Sergey Novitskiy. TGF-beta signaling on myeloid cells regulates ECM deposition in mammary carcinoma via adenosine dependent mechanisms [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1714A.

The role of TGF-beta signaling in dendritic cell tolerance.

Transforming growth factor beta (TGF-β) is a pleiotropic cytokine present in vertebrate and invertebrate organisms that functions in numerous physiological and pathological processes. TGF-β impacts all the cells of the immune system, and of the three known TGF-β isoforms, TGF-β1 is the predominant isoform expressed in immune cells. TGF-β1 is known to play a pivotal role in the function of all immune cells especially in the regulation of T cell development and in the induction of immunological tolerance in dendritic cells (DCs). Based on the importance of DCs in regulation of the innate and adaptive arms of the immune system, in this review we explore the regulatory functions of TGF-β required for establishment and maintenance of DC-mediated immune tolerance.

127 : Role of TGF-beta signaling in circulating and tissue resident memory CD8+ T cells

Tissue resident memory (Trm) CD8 + T cells are a newly described memory T cell populations that are distinct from circulating memory T cells. The Trm CD8 + T cell responses are critical for protection against pathogens that invade the host via skin and mucosal tissues. Transforming growth factor- β eta (TGF- β eta) is thought to be important for controlling the numbers of KLRG + effector T cells and promoting the residence of the Trm CD8 + T cells in several nonlymhpoid tissues including intestine. We will present data on how TGF- β eta signaling differentially regulates circulating and tissue resident memory CD8 + T cells after systemic infection with Listeria monocytogenes in mice.

Abstract 3764: Loss of β2SP (ELF) enhances the stem-like traits and invasiveness of HCC cells.

Abstract β2SP(ELF) is one of adapter proteins for Smad3/Smad4 complex formation during TGF-β signal transduction. 40% of β2SP+/- mice spontaneously develop hepatocellular carcinoma (HCC) and most cases of human HCC, gastric cancer, and lung cancer demonstrate significant reductions in β2SP expression. In our study, we investigated the possible mechanism for loss of β2SP induced HCC tumorigenesis. Recent studies have highlighted the involvement of cancer stem cells during the formation and progression of various types of cancers, epithelial cells that have undergone EMT (epithelial mesenchymal transition) acquire stem-cell properties during cancer development. Giving the role of TGF-beta signaling in stem cell differentiation and EMT during embryonic development, we hypothesize loss of β2SP results in alteration of liver stem cell that may contribute to the development of HCC in β2SP+/− mice. It has been shown that EpCAM-positive adult liver cells has stem cell property and can differentiate into functional hepatocytes in xenograft model. We found that EpCAM-positive hepatocyte number doubled in β2SP+/− mouse compared to wild type by fluorescence-activated cell sorter (FACS). Furthermore EpCAM level in β2SP+/− mouse liver was three times higher than that in the liver of wild type mouse. To study the mechanism by which Epcam is regulated by β2SP, HCC cells were transfected by β2SP siRNA. Inhibition of β2SP increased the expression of Epcam, and EMT markers vimentin was increased and E-cadherin expression was decreased in PLC/PRF/5 and SNU449 cells, while TGF-β1 and Smad3 rescued the expression of Epcam and E-cadherin, but not vimentin. We also found that suppression of β2SP expression promotes adhesion, migration and cloning formation of PLC/PRF/5 and SNU449 cells. Our data suggests loss of β2SP enhances the stem-like traits and invasiveness of HCC cells. Citation Format: Xiuling Zhi, Ling Lin, Narayan Shivapurkar, Wilma Jogunoori, Lopa Mishra, Aiwu R He. Loss of β2SP (ELF) enhances the stem-like traits and invasiveness of HCC cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3764. doi:10.1158/1538-7445.AM2013-3764

Alterations in the Smad pathway in human cancers.

Members of the TGF-beta superfamily exhibit various biological activities, and perturbations of their signaling are linked to certain clinical disorders including cancer. The role of TGF-beta signaling as a tumor suppressor pathway is best illustrated by the presence of inactivating mutations in genes encoding TGF-beta receptors and Smads in human carcinomas. This perspective is further supported by studies of tumor development in mouse models after modulation of receptors and Smads. TGF-beta also controls processes such as cell invasion, immune regulation, and microenvironment alterations that cancer cells may exploit to their advantage for their progression. Consequently, the output of a TGF-beta response is highly situation dependent, across different tissues, and also in cancer in general. Understanding the mechanisms of TGF-beta superfamily signaling is thus important for the development of new ways to treat various types of cancer. This review focuses on recent advances in understanding the Smad dependent TGF-beta pathway as it relates to human carcinogenesis.

Abstract 1482: Role of TGF-beta signaling and mechanism of osteoblastic tumorigenesis in prostate cancer

Abstract Prostate cancer is the second leading cause of cancer death in men with 80% of deaths occurring due to bone metastasis. Transforming growth factor-beta (TGFβ) signaling is important for the regulation of bone mass and matrix properties. The role of TGFβ signaling in prostate cancer-induced bone metastasis is unclear due in part to a lack of prostate cancer cell lines that can efficiently induce bone metastasis and osteoblastic lesions in animal models. We have previously shown that a newly established human prostate cancer cell line, PacMetUT1, produces a large amount of active TGFβ1 and induces extensive bone metastases in calvaria, rib, femur, and tibia when inoculated in the left cardiac ventricle of male nude mice. Experimental data from our lab demonstrated that stable knockdown of TGFβ1 using a short hairpin-RNA resulted in a decreased tumor incidence and bone formation when the cells were directly injected into the right tibias of male nude mice. Systemic administration of either a small inhibitor of TGFβ type I receptor kinase or a pan-TGFβ binding protein also decreased osteoblastic intratibial tumor lesions. However, the mechanism by which prostate cancer cells metastasize to bone and how they induce formation of osteoblastic lesions remains to be intensively investigated. We hypothesize that TGFβ and other growth factors released from tumor cells can induce aromatase gene expression and activity in bone cells leading to an enhanced estradiol synthesis which would lead to an increase in bone formation as estrogen is a known osteogenic factor. We examined the effect of TGFβ1 and BMP-4/7 treatment on aromatase expression in pre-osteoblastic cells in vitro. In vitro treatment with 5ng/ml of TGFβ1 and 25ng/ml of BMP-4/7 resulted in an increase in aromatase expression in 2T3, a mouse mesenchymal stem cell and in 63 year old male mesenchymal stem cells (MSC) at the protein level. Furthermore, TGFβ resulted in an induction of Smad-1/5 signaling pathway in these pre-osteoblastic cells. Transient knockdown of Smad-1/5 expression in male MSC by siRNA, resulted in a decreased aromatase expression. Our results demonstrate TGFβ to be a major driver of osteoblastic tumorigenesis in PacMetUT1 cells, and Smad-1/5-dependent aromatase upregulation by TGFβ superfamily ligands in pre-osteoblastic cells. Future studies will delineate whether the induction of aromatase mediates osteoblastic bone metastasis in vivo, and if combination treatment of TGFβ and aromatase inhibitors can be used as a novel therapeutic strategy for bone metastatic prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1482. doi:10.1158/1538-7445.AM2011-1482

Administration of Pigment Epithelium-Derived Factor Inhibits Left Ventricular Remodeling and Improves Cardiac Function in Rats with Acute Myocardial Infarction

Oxidative stress and inflammation are involved in cardiac remodeling after acute myocardial infarction (AMI). We have found that pigment epithelium-derived factor (PEDF) inhibits vascular inflammation through its anti-oxidative properties. However, effects of PEDF on cardiac remodeling after AMI remain unknown. We investigated whether PEDF could inhibit left ventricular remodeling and improve cardiac function in rats with AMI. AMI was induced in 8-week-old Sprague-Dawley rats by ligation of the left ascending coronary artery. Rats were treated intravenously with vehicle or 10 μg PEDF/100 g b.wt. every day for up to 2 weeks after AMI. Each rat was followed until 16 weeks of age. PEDF levels in infarcted areas and serum were significantly decreased at 1 week after AMI and remained low during the observational periods. PEDF administration inhibited apoptotic cell death and oxidative stress generation around the infarcted areas at 2 and 8 weeks after AMI. Further, PEDF injection suppressed cardiac fibrosis by reducing transforming growth factor-β and type III collagen expression, improved left ventricular ejection fraction, ameliorated diastolic dysfunction, and inhibited the increase in left ventricular mass index at 8 weeks after AMI. The present study demonstrated that PEDF could inhibit tissue remodeling and improve cardiac function in AMI rats. Substitution of PEDF may be a novel therapeutic strategy for cardiac remodeling after AMI.

Pathway analysis of gene lists associated with platinum-based chemotherapy resistance in ovarian cancer: The big picture

Objective Ovarian cancer is the leading cause of death from gynecological cancers in the Western world (Parkin et al., 2005). The overall 5-year survival is only 30% (Moss and Kaye, 2002), which is for a significant part due to platinum-based chemotherapy resistance. In this study, we performed a pathway analysis on nine published gene sets associated with platinum resistance in ovarian cancer, including a study by us. With this exploratory study, we aim to identify overlapping pathways associated with platinum-based chemotherapy resistance mechanisms in ovarian cancer. Methods Gene Ontology (GO) analysis and Ingenuity Pathway Analysis (IPA) were performed to determine which functional processes were differentially represented in the combined gene lists of nine studies (457 genes) compared to all Unigene identifiers or the Ingenuity knowledge base. Results The GO and IPA analysis resulted in the generation of 23 gene networks, and showed that 13 GO processes (≥2 times enriched), 71 canonical pathways ( p < 0.05,), eight toxicity pathways ( p < 0.05) and 74 biological functions ( p < 0.005) are significantly associated with the 9-study gene set. Conclusion and recommendations Several pathways identified have previously been shown to be associated with therapy resistance: these include ‘oxidative stress response mediated by Nrf2,’ ‘TP53 signaling’ and ‘TGFbeta signaling.’ The role of TGFbeta signaling and related miRNAs identified in the network analysis in epithelial-to-mesenchymal transition (EMT) and stemness as well as the possible relation with platin-based chemotherapy resistance are further discussed in detail. We propose that future international cooperation should aim at a uniform pooled analysis of the wealth of ovarian cancer array data already available. This will enhance the power of each separate ovarian cancer study and can lead to promising results.

Investigating the role of TGFbeta signaling in asymmetric morphogenesis of the zebrafish heart

Investigating the role of TGFbeta signaling in asymmetric morphogenesis of the zebrafish heart

Endocytic regulation of TGF-β signaling

Transforming growth factor-beta (TGF-beta) signaling is tightly regulated to ensure its proper physiological functions in different cells and tissues. Like other cell surface receptors, TGF-beta receptors are internalized into the cell, and this process plays an important regulatory role in TGF-beta signaling. It is well documented that TGF-beta receptors are endocytosed via clathrin-coated vesicles as TGF-beta endocytosis can be blocked by potassium depletion and the GTPase-deficient dynamin K44A mutant. TGF-beta receptors may also enter cells via cholesterol-rich membrane microdomain lipid rafts/caveolae and are found in caveolin-1-positive vesicles. Although receptor endocytosis is not essential for TGF-beta signaling, clathrin-mediated endocytosis has been shown to promote TGF-beta-induced Smad activation and transcriptional responses. Lipid rafts/caveolae are widely regarded as signaling centers for G protein-coupled receptors and tyrosine kinase receptors, but they are indicated to facilitate the degradation of TGF-beta receptors and therefore turnoff of TGF-beta signaling. This review summarizes current understanding of TGF-beta receptor endocytosis, the possible mechanisms underlying this process, and the role of endocytosis in modulation of TGF-beta signaling.

Using RNA interference to identify the different roles of SMAD2 and SMAD3 in NIH/3T3 fibroblast cells

Smad proteins are principal intracellular signaling mediators of transforming growth factor beta (TGF-beta) that regulate a wide range of biological processes. However, the identities of Smad partners mediating TGF-beta signaling are not fully understood. We firstly examined the expression of Smad2 and Smad3 induced by TGF-beta 1 in normal NIH/3T3 cells. The expression of Smad2 and Smad3 was assessed by RT-PCR and Western blotting. The results showed that the expression of Smad2 was increased after treatment with TGF-betaI, but Smad3 was more sensitive to TGF-betaI than Smad2. RNA interference (RNAi) provides a new approach for elucidation of gene function. Use of hairpin siRNA expression vectors for RNAi has provided a rapid and versatile method for assessing gene function in mammalian cells. Here, we have constructed Smad2 and Smad3 hairpin siRNA expression plasmids, and then transfected them into mouse NIH/3T3 cells. Endogenous Smad2 and Smad3 proteins decreased significantly at 48 h after transfection. We found the expression of Smad3 in Smad2-depleted cells was increased, however, the expression of Smad2 in Smad3-depleted cells was not changed. Consistently, the expression of Smad4 mRNA was also attenuated in Smad3-depleted cells. From these data, we suggest that Smad3, but not Smad2, may play a key role in TGF-beta signaling.

Cyclic GMP/Protein Kinase G Phosphorylation of Smad3 Blocks Transforming Growth Factor-β–Induced Nuclear Smad Translocation

See related article, pages 185–192 In response to hemodynamic overload, the heart undergoes a complex adaptive remodeling process that involves cardiac myocyte hypertrophy, transformation of fibroblast into myofibroblast, high-level expression of extracellular matrix (ECM) proteins, interstitial fibrosis, and cell death.1 Differentiation of cardiac fibroblasts into myofibroblasts is critical to the production and deposition of collagens and plays a decisive role in myocardial fibrosis and morphological alterations during the progression of adaptive myocardial hypertrophy to decompensation and heart failure.1,2 Among the plethora of identified fibrogenic factors, transforming growth factor (TGF)-β3 plays a fundamental role in hypertrophic and fibrotic remodeling of the heart, where it regulates cardiomyocyte growth, fibroblast activation, and ECM deposition.4,5 The expression of ventricular TGF-β mRNA and protein is increased in numerous models of pathological cardiac hypertrophy and in cardiac cells in response to putative hypertrophic stimuli.6,7 In vitro, TGF-β activates myofibroblast transformation and increases ECM production.7 Blockade of TGF-β signaling is predicted to blunt fibrosis.5 Recent studies from Chen et al8 convincingly demonstrated that disruption of TGF-β signaling by inducible dominant-negative mutation of the TGF-β receptor type II (TβRII) gene significantly reduced the pressure overload–induced myofibroblast transformation and interstitial fibrosis in mouse heart. Thus, there is considerable interest in understanding how signaling by TGF-β receptors is transduced and how the inevitable damage produced could be mitigated. Of the 3 isoforms of TGF-β expressed in mammals,9,10 TGF-β1 is expressed in the adult heart, where it is secreted by cardiomyocytes and myofibroblasts and retained in significant amounts in ECM as a latent cytokine. Recent advances have led to clear description of downstream of TGF-β signal transduction pathways initiated by binding of TGF-β to membrane-bound heteromeric receptor kinases (TβRI and TβRII) that transduce intracellular signals via both Smad and non-Smad pathways …

Mechanotransduction of bovine articular cartilage superficial zone protein by transforming growth factor β signaling

Mechanical signals are key determinants in tissue morphogenesis, maintenance, and restoration strategies in regenerative medicine, although molecular mechanisms of mechanotransduction remain to be elucidated. This study was undertaken to investigate the mechanotransduction process of expression of superficial zone protein (SZP), a critical joint lubricant. Regional expression of SZP was first quantified in cartilage obtained from the femoral condyles of immature bovines, using immunoblotting, and visualized by immunohistochemistry. Contact pressure mapping in whole joints was accomplished using pressure-sensitive film and a load application system for joint testing. Friction measurements on cartilage plugs were acquired under boundary lubrication conditions using a pin-on-disk tribometer modified for reciprocating sliding. Direct mechanical stimulation by shear loading of articular cartilage explants was performed with and without inhibition of transforming growth factor beta (TGFbeta) signaling, and SZP content in media was quantified by enzyme-linked immunosorbent assay. An unexpected pattern of SZP localization in knee cartilage was initially identified, with anterior regions exhibiting high levels of SZP expression. Regional SZP patterns were regulated by mechanical signals and correlated with tribological behavior. Direct relationships were demonstrated between high levels of SZP expression, maximum contact pressures, and low friction coefficients. Levels of SZP expression and accumulation were increased by applying shear stress, depending on location within the knee, and were decreased to control levels with the use of a specific inhibitor of TGFbeta receptor type I kinase and subsequent phospho-Smad2/3 activity. These findings indicate a new role for TGFbeta signaling in the mechanism of cellular mechanotransduction that is especially significant for joint lubrication.

Abstract 1757: A 35-Year-Old Female with Anterior ST Segment Elevation and Cardiogenic Shock

A 35 year-old female with history of spontaneous pneumothorax presented with acute onset chest pain. She was hypotensive and tachycardic. Exam revealed tall stature, distended neck veins, and diffuse crackles. CXR showed pulmonary edema, no pneuomothorax and normal cardiac and mediastinal silhouettes. Electrocardiogram showed antero-lateral ST elevation. The patient was intubated and stabilized. Transthoracic echocardiogram showed anterior wall motion abnormality, no pericardial effusion, a normal aortic root with no obvious dissection or aortic insufficiency. CT angiogram of the chest showed no aortic dissection. As no dissection was identified she was taken for cardiac catheterization for acute MI. Angiography revealed a patent right coronary and an occluded left-main. The left-main was successfully treated with angioplasty and stenting. Subsequent TEE revealed an aortic intramural hematoma adjacent to the left-main ostium. Repeat CT angiography revealed non-flow limiting dissections of the left subclavian, the superficial mesenteric, and the left internal carotid arteries. Her body habitus and presentation suggested a malignant connective tissue disorder. Family history included maternal fatal aortic rupture at age 40 and sudden death of her maternal grandfather at age 45 of unknown etiology. In addition to Marfan Syndrome, her aortic dissection without aortic dilation suggested a possible TGF-B receptor mutation (Loeys-Dietz syndrome). Genetic testing revealed a novel Fibrillin-1 mutation at intron 40. The location at the splice acceptor site of the intron/exon boundary was likely to be pathogenic. Her daughter has tall stature and normal aortic root and is undergoing mutation confirmation testing. The phenotypic characteristics of patients with Marfan and related syndromes may vary and are associated with important morbidity and mortality. Basic science and clinical translational studies have advanced our understanding of disease pathogenesis and our approach to patient care. Genetic testing offers potential for definitive identification of family members at risk. Illumination of the role of TGF-beta signaling in the pathobiology of these syndromes has led to new possibilities in treatment and diagnosis.

TGF‐Beta Signaling in Breast Cancer

The antiestrogen tamoxifen is one of the most successful drugs in the endocrine treatment of breast cancer and significantly reduces the risk of recurrence and death. Antiestrogens act by inhibiting the production of growth-stimulatory factors as well as by activating peptides with growth-inhibitory effects like transforming growth factor- beta (TGF-beta). In hormone-responsive breast cancer cells treatment with antiestrogens leads to the conversion of TGF-beta1 into a biologically active form. Expression of TGF-beta2 and TGF-beta receptor (TbetaR) II is induced via a transcriptional mechanism involving p38 MAP kinase. Inhibition of p38 abolishes antiestrogen-dependent growth inhibition. However, the role of TGF-beta in breast cancer progression is ambiguous, as it was shown to display both tumor-suppressing and -enhancing effects. A polymorphism in the promoter of TGF-beta2 that enhances expression of the protein was associated with lymph node metastasis in breast cancer patients, pointing to a role of TGF-beta2 in the process of invasion. An immunohistochemical study on TbetaRI and TbetaRII expression in breast cancer tissues indicates that the estrogen receptor (ER) status of a tumor is an important marker and a potential mediator of the transition of TGF-beta from tumor suppressor to tumor promoter. In ER-negative tumors, expression of TbetaRII was associated with a subset of tumors that appeared to be highly aggressive, leading to strongly reduced overall survival times. Further characterization of the influence of ER expression on TGF-beta signal transduction shows that ER-alpha plays a crucial role in TGF-beta signaling.

The Transforming Growth Factor-β-Smad3/4 Signaling Pathway Acts as a Positive Regulator for TLR2 Induction by Bacteria via a Dual Mechanism Involving Functional Cooperation with NF-κB and MAPK Phosphatase 1-dependent Negative Cross-talk with p38 MAPK

The transforming growth factor beta (TGF-beta) pathway represents an important signaling pathway involved in the regulation of diverse biological processes, including cell proliferation, differentiation, and apoptosis. Despite the known role of TGF-betaR-mediated signaling in suppressing immune response, its role in regulating human Toll-like receptors (TLRs), key host defense receptors that recognize invading bacterial pathogens, however, remains unknown. Here, we show for the first time that TGF-betaR-Smad3/4 signaling pathway acts as a positive regulator for TLR2 induction by bacterium nontypeable Hemophilus influenzae (NTHi) in vitro and in vivo. The positive regulation of TLR2 induction by TGF-betaR is mediated via a dual mechanism involving distinct signaling pathways. One mechanism involves functional cooperation between the TGF-betaR-Smad3/4 pathway and NF-kappaB pathway. Another involves MAP kinase phosphatase 1 (MKP-1)-dependent inhibition of p38 MAPK, a known negative regulator for TLR2 induction. Moreover, we showed that TbetaR-mediated signaling is probably activated by NTHi-derived TGF-beta mimicry molecule via an autocrine-independent mechanism. Thus, our study provides new insights into the role of TGF-beta signaling in positively regulating host defense response by tightly controlling the expression level of TLR2 during bacterial infections and may lead to new therapeutic strategies for modulating host defense and inflammatory response.

Decreased expression of inhibitory SMAD6 and SMAD7 in keloid scarring

Keloids are benign skin tumours occurring during wound healing in genetically predisposed patients. They are characterised by an abnormal deposition of extracellular matrix components, in particular collagen. There is evidence that transforming growth factor-beta (TGFbeta) is involved in keloid formation. SMAD proteins play a crucial role in TGFbeta signaling and in terminating the TGFbeta signal by a negative feedback loop through SMAD6 and 7. It is unclear how TGFbeta signaling is connected to the pathogenesis of keloids. Therefore, we investigated the expression of SMAD mRNA and proteins in keloids, in normal skin and in normal scars. Dermal fibroblasts were obtained from punch-biopsies of keloids, normal scars and normal skin. Cells were stimulated with TGFbeta1 and the expression of SMAD2, 3, 4, 6 and 7 mRNA was analysed by real time RT-PCR. Protein expression was determined by Western blot analysis. Our data demonstrate a decreased mRNA expression of the inhibitory SMAD6 and 7 in keloid fibroblasts as compared to normal scar (p<0.01) and normal skin fibroblasts (p<0.05). SMAD3 mRNA was found to be lower in keloids (p<0.01) and in normal scar fibroblasts (p<0.001) compared to normal skin fibroblasts. Our data showed for the first time a decreased expression of the inhibitory SMAD6 and SMAD7 in keloid fibroblasts. This could explain why TGFbeta signaling is not terminated in keloids leading to overexpression of extracellularmatrix in keloids. These data support a possible role of SMAD6 and 7 in the pathogenesis of keloids.