Sarah McLean

The gene expression profile induced by Wnt 3a in NIH 3T3 fibroblasts

Wnt proteins play important roles in regulating cell differentiation, proliferation and polarity. Wnts have been proposed to play roles in tissue repair and fibrosis, yet the gene expression profile of fibroblasts exposed to Wnts has not been examined. We use Affymetrix genome-wide expression profiling to show that a 6-h treatment of fibroblasts of Wnt3a results in the induction of mRNAs encoding known Wnt targets such as the fibrogenic pro-adhesive molecule connective tissue growth factor (CTGF, CCN2). Wnt3a also induces mRNAs encoding potent pro-fibrotic proteins such as TGFβ and endothelin-1 (ET-1). Moreover, Wnt3a promotes genes associated with cell adhesion and migration, vasculature development, cell proliferation and Wnt signaling. Conversely, Wnt3a suppresses gene associated with skeletal development, matrix degradation and cell death. Results were confirmed using real-time polymerase chain reaction of cells exposed to Wnt3a and Wnt10b. These results suggest that Wnts induce genes promoting fibroblast differentiation towards angiogenesis and matrix remodeling, at the expense of skeletal development.

TGFβ (transforming growth factor β) receptor type III directs clathrin-mediated endocytosis of TGFβ receptor types I and II

The TGFbeta (transforming growth factor beta) pathway is an essential cell signalling pathway that is implicated in both normal developmental processes, such as organogenesis, and pathological disorders, such as cancer and fibrosis. There are three prototypical TbetaRs (TGFbeta receptors): TbetaRI (TbetaR type I), TGbetaRII (TbetaR type II) and TGFbetaRIII (TbetaR type III, also known as betaglycan). Whereas the role of TbetaRII and TbetaRI in TGFbeta signal propagation has been established, the contribution of TbetaRIII to TGFbeta signalling is less well understood. At the cell surface, TbetaRI and TbetaRII receptors can be internalized by clathrin-mediated endocytosis and clathrin-independent membrane-raft-dependent endocytosis. Interestingly, the endocytic route of the receptors plays a direct role in TGFbeta-dependent Smad signal transduction; receptors endocytosed via clathrin-mediated endocytosis activate Smad signalling, whereas receptors endocytosed via membrane rafts are targeted for degradation. The objective of the present study was to evaluate the contribution of TbetaRIII to TbetaRII and TbetaRI membrane partitioning, receptor half-life and signalling. Using sucrose-density ultracentrifugation to isolate membrane-raft fractions, we show that TbetaRIII recruits both TbetaRII and TbetaRI to non-raft membrane fractions. Immunofluorescence microscopy analysis demonstrated that overexpression of TbetaRIII affects intracellular trafficking of TbetaRII by recruiting TbetaRII to EEA1 (early endosome antigen 1)- and Rab5-positive early endosomes. Using 125I-labelled TGFbeta1 to follow cell-surface receptor degradation we show that overexpression of TbetaRIII also extends the receptor half-life of the TbetaRII-TbetaRI complex. Interestingly, we also show, using a luciferase reporter assay, that TbetaRIII increases basal TGFbeta signalling. As numerous pathologies show aberrant activation of TGFbeta signalling, the present study illustrates that TbetaRIII may represent a novel therapeutic target.

The extracellular domain of the TGFβ type II receptor regulates membrane raft partitioning

Cell-surface TGFbeta (transforming growth factor beta) receptors partition into membrane rafts and the caveolin-positive endocytic compartment by an unknown mechanism. In the present study, we investigated the determinant in the TGFbeta type II receptor (TbetaRII) that is necessary for membrane raft/caveolar targeting. Using subcellular fractionation and immunofluorescence microscopy techniques, we demonstrated that the extracellular domain of TbetaRII mediates receptor partitioning into raft and caveolin-positive membrane domains. Pharmacological perturbation of glycosylation using tunicamycin or the mutation of Mgat5 [mannosyl(alpha-1,6)-glycoprotein beta-1,6-N-acetylglucosaminyltransferase V] activity interfered with the raft partitioning of TbetaRII. However, this was not due to the glycosylation state of TbetaRII, as a non-glycosylated TbetaRII mutant remained enriched in membrane rafts. This suggested that other cell-surface glycoproteins associate with the extracellular domain of TbetaRII and direct their partitioning in membrane raft domains. To test this we analysed a GMCSF (granulocyte/macrophage colony-stimulating factor)-TbetaRII chimaeric receptor, which contains a glycosylated GMCSF extracellular domain fused to the transmembrane and intracellular domains of TbetaRII. This chimaeric receptor was found to be largely excluded from membrane rafts and caveolin-positive structures. Our results indicate that the extracellular domain of TbetaRII mediates receptor partitioning into membrane rafts and efficient entrance into caveolin-positive endosomes.

βarrestin2 interacts with TβRII to regulate Smad-dependent and Smad-independent signal transduction.

The Transforming Growth Factor beta (TGFβ) signaling pathway is necessary for a variety of normal cellular processes. However, the distinct mechanisms involved in TGFβ receptor turnover and the effect on signal transduction have yet to be fully elucidated. We have previously shown that TβRIII is able to interact with the TβRII/TβRI complex to increase clathrin-dependent endocytosis and receptor half-life. Others have shown that βarrestin2 binds TβRIII to mediate TβRII/TβRIII endocytosis. To further understand the mechanism regulating TGFβ receptor signaling, we evaluated the role of βarrestin2 in TGFβ receptor signal transduction, half-life and trafficking. We have found that TβRII binds βarrestin2 in the absence of TβRIII. Furthermore, using immunofluorescence microscopy we show that βarrestin2 traffics to the early endosome with TβRII. We investigated the effect of loss of βarrestin2 on TβRII dynamics and found that loss of βarrestin2 increases steady-state levels of TβRII at the cell surface. The interaction of TβRII with βarrestin2 is involved in modulating TGFβ signal transduction, as loss of βarrestin2 increases the phosphorylation of p38 and modestly affects pSmad levels. Using a luciferase assay to assess TGFβ-dependent transcription we show that loss of βarrestin2 decreases Smad-dependent TGFβ-stimulated transcription. Furthermore, loss of βarrestin2 increases p38 signal transduction, which correlated with increased cell death via apoptosis. Overall, our results suggest a role for βarrestin2 in the regulation of Smad-dependent and independent TGFβ pathways.

Modulation of Type II TGF-β Receptor Degradation by Integrin-Linked Kinase

Cutaneous responses to injury, infection, and tumor formation involve the activation of resident dermal fibroblasts and subsequent transition to myofibroblasts. The key for induction of myofibroblast differentiation is the activation of transforming growth factor-β (TGF-β) receptors and stimulation of integrins and their associated proteins, including integrin-linked kinase (ILK). Cross-talk processes between TGF-β and ILK are crucial for myofibroblast formation, as ILK-deficient dermal fibroblasts exhibit impaired responses to TGF-β receptor stimulation. We now show that ILK associates with type II TGF-β receptors (TβRII) in ligand- and receptor kinase activity-independent manners. In cells with targeted Ilk gene inactivation, cellular levels of TβRII are decreased, through mechanisms that involve enhanced ubiquitination and proteasomal degradation. Partitioning of TGF-β receptors into membrane has been linked to proteasome-dependent receptor degradation. We found that interfering with membrane raft formation in ILK-deficient cells restored TβRII levels and signaling. These observations support a model whereby ILK functions in fibroblasts to direct TβRII away from degradative pathways during their differentiation into myofibroblasts.

Sage or guide? Student perceptions of the role of the instructor in a flipped classroom

Higher education has begun to shift from a teacher-centred instructional approach to a student-centred learning approach; many instructors have embraced this change by developing flipped classrooms...

Medical myth busting to engage physiology students in scientific literature

teaching students to critically evaluate scientific literature can be an onerous task for students and the instructor alike. To overcome this challenge and to add intrigue, creativity, and critical thinking to literature analysis, medical myth busting was developed as an assignment for a senior

TGFβ in endosomal signaling.

The transforming growth factor beta (TGFβ) signaling pathway is important for normal cell homeostasis and has critical roles in apoptosis, cell-cycle arrest, and cellular differentiation (reviewed in Massague, 2008). In the classical TGFβ pathway, the endosomal trafficking of receptors has a direct outcome on signal transduction-receptors internalized via clathrin-mediated endocytosis enter the early endosome and propagate signaling, while those internalized via membrane rafts are targeted for degradation. Recently, there have been a number of articles that have identified TGFβ receptor-binding proteins that direct receptor endocytosis and/or intracellular trafficking and affect signal output (Atfi et al., 2007; Bauge, Girard, Leclercq, Galera, & Boumediene, 2012; Bizet et al., 2011, 2012; Chen et al., 2007; Gunaratne, Benchabane, & Di Guglielmo, 2012; Hao et al., 2011; McLean, Bhattacharya, & Di Guglielmo, 2013; Zhao et al., 2012). Given the importance of TGFβ receptor trafficking to signaling outcome, this chapter will focus on strategies to isolate membrane rafts and techniques to follow the trafficking of cell-surface TGFβ receptors and provide examples of functional readouts to assess TGFβ signal transduction.

Abstract 3019: TGFβ3 is a less potent inducer of TGFβ signaling than TGFβ1 in non-small cell lung cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Transforming growth factor beta (TGFβ) is an essential cytokine for tissue homeostasis. Its signalling pathway is under intense study as it is commonly dysregulated in many cancers, including pancreatic cancer, non-small cell lung cancer, and colorectal cancer. The TGFβ signalling cascade is propagated by the binding of ligand to cell-surface serine-threonine kinase receptors, which leads to the phosphorylation of receptor regulated Smad (R-Smad) proteins. There are three main ligands capable of activating the classical TGFβ pathway: TGFβ1, TGFβ2 and TGFβ3. These ligands show distinct spatial and temporal expression patterns and have non-overlapping functions in vivo. Extensive studies have been conducted on the role of TGFβ1 in the tumour microenvironment, and the role of TGFβ3 has largely been thought to be the same as TGFβ1 though there are few studies to support this claim. Interestingly in the wound microenvironment, TGFβ1 and TGFβ3 have vastly different signalling outcomes: TGFβ1 promotes the formation of a scar while TGFβ3 induces scar-free wound resolution. The objective of the present study was to evaluate the signalling capacity of TGFβ1 and TGFβ3 in non-small cell lung cancer cells. Our studies show that TGFβ3 is less potent at initiating Smad2 phosphorylation than TGFβ1, resulting in reduced transcriptional activity, as assessed by microarray analysis. We also observed that TGFβ3 is less effective than TGFβ1 at altering cell-cell adhesions. In order to assess the differences in signalling potential, we investigated TGFβ receptor engagement at the cell surface using radiolabelled TGFβ ligands. We observed that the ratio of activated receptors in signalling complexes is altered in the presence of TGFβ3, which may lead to a decreased signalling capacity. Future studies will evaluate the capacity of TGFβ3 to induce cancer cell migration and invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3019. doi:1538-7445.AM2012-3019

Abstract 1967: Ligand-dependent TGFβ signalling potential

Transforming growth factor beta (TGFβ) is a cytokine which regulates many normal biological processes such as organogenesis and cell cycle control. Its signaling pathway is under intense study as it is commonly dysregulated in pathophysiological conditions such as cancer and fibrosis. The TGFβ signalling cascade is initiated by the binding of ligand to cell-surface serine-threonine kinase receptors. Binding of ligand to the TGFβ receptors initiates phosphorylation of intracellular signalling proteins called Smads, which can then enter the nucleus and initiate specific transcriptional programs. There are three TGFβ ligands which can activate canonical Smad signalling: TGFβ1, TGFβ2 and TGFβ3. These ligands share significant sequence homology (70-80%) but have different spatial and temporal patterns of expression in development. Although TGFβ1 and TGFβ3 are highly expressed in the tumour microenvironment, the role of TGFβ3 has been largely over-looked and thought to be the same as TGFβ1. The objective of the present study is to evaluate the signalling capacity of TGFβ1 and TGFβ3 in non-small cell lung cancer cells. Using dose response studies, we show that TGFβ1 ligand induces Smad2 phosphorylation to a greater extent than TGFβ3. We also show that TGFβ3 induces a lower reduction in steady-state levels of E-cadherin, a protein involved in cell-cell adhesion, compared to TGFβ1. To evaluate the transcriptional programs of both ligands, we used microarray technology to assess gene transcription in non-small cell lung cancer cells treated with TGFβ1 or TGFβ3. Consistent with our signalling data, we found that TGFβ3 induces far fewer genes than TGFβ1. Future studies will evaluate the differential roles of TGFβ1 and TGFβ3 in cancer cell migration and epithelial-to-mesenchymal transition. 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 1967. doi:10.1158/1538-7445.AM2011-1967