Jung San Huang

The TGF‐β type V receptor (TβR‐V) mediates growth inhibition by IGFBP‐3 and TGF‐β in epithelial cells and loss of TβR‐V expression in these cells leads to development of carcinoma. The mechanisms by which TβR‐V mediates growth inhibition (tumor suppressor) signaling remain elusive. Previous studies revealed that IGFBP‐3 and TGF‐β inhibit growth in epithelial cells by stimulating TβR‐V‐mediated IRS‐1/2‐dependent activation and cytoplasm‐to‐nucleus translocation of IGFBP‐3‐ or TGF‐β‐stimulated protein phosphatase (PPase), resulting in dephosphorylation of pRb‐related proteins (p107, p130) or pRb, and growth arrest. To define the signaling, we characterized/identified the IGFBP‐3‐ and TGF‐β‐stimulated PPases in cell lysates and nucleus fractions in Mv1Lu cells treated with IGFBP‐3 and TGF‐β, using a cell‐free assay with ³²P‐labeled casein as a substrate. Both IGFBP‐3‐ and TGF‐β‐stimulated PPase activities in cell lysates are abolished when cells are co‐treated with TGF‐β/IGFBP‐3 antagonist or RAP (LRP‐1/TβR‐V antagonist). However, the IGFBP‐3‐stimulated PPase activity, but not TGF‐β‐stimulated PPase activity, is sensitive to inhibition by okadaic acid (OA). In addition, OA or PP2A_c siRNA reverses IGFBP‐3 growth inhibition, but not TGF‐β growth inhibition, in Mv1Lu and 32D cells. These suggest that IGFBP‐3‐ and TGF‐β‐stimulated PPases are identical to PP2A and PP1, respectively. By Western blot/phosphorimager/immunofluorescence‐microscopy analyses, IGFBP‐3 and TGF‐β stimulate TβR‐V‐mediated IRS‐2‐dependent activation and cytoplasm‐to‐nucleus translocation of PP2A_c and PP1_c, resulting in dephosphorylation of p130/p107 and pRb, respectively, and growth arrest. Small molecule TGF‐β enhancers, which potentiate TGF‐β growth inhibition by enhancing TβR‐I–TβR‐II‐mediated canonical signaling and thus activating TβR‐V‐mediated tumor suppressor signaling cascade (TβR‐V/IRS‐2/PP1/pRb), could be used to prevent and treat carcinoma.

CRSBP‐1 (mammalian LYVE‐1) is a membrane glycoprotein highly expressed in lymphatic endothelial cells (LECs). It has multiple ligands, including hyaluronic acid (HA) and growth factors/cytokines (e.g., PDGF‐BB and VEGF‐A) containing CRS motifs (clusters of basic amino‐acid residues). The ligand binding activities are mediated by Link module and acidic‐amino‐acid‐rich (AAAR) domains, respectively. These CRSBP‐1/LYVE‐1 ligands have been shown to induce opening of lymphatic intercellular junctions in LEC monolayers and in lymphatic vessels in wild‐type mice. We hypothesize that CRSBP‐1/LYVE‐1 ligands, particularly CRS‐containing growth factors/cytokines, are secreted by immune and cancer cells for lymphatic entry during adaptive immune responses and lymphatic metastasis. We have looked into the origin of the Link module and AAAR domain of LYVE‐1 in evolution and its association with the development of lymph nodes and efficient adaptive immunity. Lymph nodes represent the only major recent innovation of the adaptive immune systems in evolution particularly to mammals and bird. Here we demonstrate that the development of the LYVE‐1 gene with the AAAR domain in evolution is associated with acquisition of lymph nodes and adaptive immunity. LYVE‐1 from other species, which have no lymph nodes, lack the AAAR domain and efficient adaptive immunity. Synthetic CRSBP‐1 ligands PDGF and VEGF peptides, which contain the CRS motifs of PDGF‐BB and VEGF‐A, respectively, specifically bind to CRSBP‐1 but do not interact with either PDGFβR or VEGFR2. These peptides function as adjuvants by enhancing adaptive immunity of pseudorabies virus (PRV) vaccine in pigs. These results support the notion that LYVE‐1 is involved in adaptive immunity in mammals.

Dimethyl sulfoxide (DMSO) is used to treat many diseases/symptoms. The molecular basis of the pharmacological actions of DMSO has been unclear. We hypothesized that DMSO exerts some of these actions by enhancing TGF‐β activity. Here we show that DMSO enhances TGF‐β activity by ∼3–4‐fold in Mv1Lu and NMuMG cells expressing Smad‐dependent luciferase reporters. In Mv1Lu cells, DMSO enhances TGF‐β‐stimulated expression of P‐Smad2 and PAI‐1. It increases cell‐surface expression of TGF‐β receptors (TβR‐I and/or TβR‐II) by ∼3–4‐fold without altering their cellular levels as determined by¹²⁵I‐labeled TGF‐β‐cross‐linking/Western blot analysis, suggesting the presence of large intracellular pools in these cells. Sucrose density gradient ultracentrifugation/Western blot analysis reveals that DMSO induces recruitment of TβR‐II (but not TβR‐I) from its intracellular pool to plasma‐membrane microdomains. It induces more recruitment of TβR‐II to non‐lipid raft microdomains than to lipid rafts/caveolae. Mv1Lu cells transiently transfected with TβR‐II‐HA plasmid were treated with DMSO and analyzed by indirect immunofluoresence staining using anti‐HA antibody. In these cells, TβR‐II‐HA is present as a vesicle‐like network in the cytoplasm as well as in the plasma membrane. DMSO causes depletion of TβR‐II‐HA‐containing vesicles from the cytoplasm and co‐localization of TβR‐II‐HA and cveolin‐1 at the plasma membrane. These results suggest that DMSO, a fusogenic substance, enhances TGF‐β activity presumably by inducing fusion of cytoplasmic vesicles (containing TβR‐II) and the plasma membrane, resulting in increased localization of TβR‐II to non‐lipid raft microdomains where canonical signaling occurs. Fusogenic activity of DMSO may play a pivotal role in its pharmacological actions involving membrane proteins with large cytoplasmic pools. J. Cell. Biochem. 117: 1568–1579, 2016.

Regular consumption of moderate amounts of ethanol has important health benefits on atherosclerotic cardiovascular disease (ASCVD). Overindulgence can cause many diseases, particularly alcoholic liver disease (ALD). The mechanisms by which ethanol causes both beneficial and harmful effects on human health are poorly understood. Here we demonstrate that ethanol enhances TGF‐β‐stimulated luciferase activity with a maximum of 0.5–1% (v/v) in Mv1Lu cells stably expressing a luciferase reporter gene containing Smad2‐dependent elements. In Mv1Lu cells, 0.5% ethanol increases the level of P‐Smad2, a canonical TGF‐β signaling sensor, by ∼2‐3‐fold. Ethanol (0.5%) increases cell‐surface expression of the type II TGF‐β receptor (TβR‐II) by ∼2‐3‐fold from its intracellular pool, as determined by I¹²⁵‐TGF‐β‐cross‐linking/Western blot analysis. Sucrose density gradient ultracentrifugation and indirect immunofluorescence staining analyses reveal that ethanol (0.5% and 1%) also displaces cell‐surface TβR‐I and TβR‐II from lipid rafts/caveolae and facilitates translocation of these receptors to non‐lipid raft microdomains where canonical signaling occurs. These results suggest that ethanol enhances canonical TGF‐β signaling by increasing non‐lipid raft microdomain localization of the TGF‐β receptors. Since TGF‐β plays a protective role in ASCVD but can also cause ALD, the TGF‐β enhancer activity of ethanol at low and high doses appears to be responsible for both beneficial and harmful effects. Ethanol also disrupts the location of lipid raft/caveolae of other membrane proteins (e.g., neurotransmitter, growth factor/cytokine, and G protein‐coupled receptors) which utilize lipid rafts/caveolae as signaling platforms. Displacement of these membrane proteins induced by ethanol may result in a variety of pathologies in nerve, heart and other tissues. J. Cell. Biochem. 117: 860–871, 2016.

CRSBP‐l/LYVE‐1 ligands (PDGF‐BB, VEGF‐A¹⁶⁵ and hyaluronic acid) have been shown to induce opening of lymphatic intercellular junctions in vitro and in vivo by stimulating contraction of lymphatic endothelial cells (LECs). The mechanism by which CRSBP‐1 ligands stimulate contraction of LECs is not understood. Here we demonstrate that CRSBP‐1 is localized to the plasma membrane as well as intracellular fibrillar structures in LECs, including primary human dermal LECs and SVEC4‐10 cells. CRSBP‐1‐associated fibrillar structures are identical to the ER network as evidenced by the co‐localization of CRSBP‐1 and BiP in these cells. CRSBP‐1 ligands stimulate contraction of the ER network in a CRSBP‐1‐dependent and paclitaxel (a microtubule‐stabilizing agent)‐sensitive manner. These results suggest that ligand‐stimulated ER contraction is associated with ligand‐stimulated contraction in LECs.

Cell-surface retention sequence (CRS) binding protein (CRSBP-1) is a membrane glycoprotein identified by its ability to bind PDGF-BB and VEGF-A via their CRS motifs (clusters of basic amino acid residues). CRSBP-1 is identical to LYVE-1 and exhibits dual ligand (CRS-containing proteins and hyaluronic acid) binding activity, suggesting the importance of CRSBP-1 ligands in lymphatic function. Here, we show that CRSBP-1 ligands induce disruption of VE-cadherin-mediated intercellular adhesion and opening of intercellular junctions in lymphatic endothelial cell (LEC) monolayers as determined by immunofluorescence microscopy and Transwell permeability assay. This occurs by interaction with CRSBP-1 in the CRSBP-1–PDGFβR–β-catenin complex, resulting in tyrosine phosphorylation of the complex, dissociation of β-catenin and p120-catenin from VE-cadherin, and internalization of VE-cadherin. Pretreatment of LECs with a PDGFβR kinase inhibitor abolishes ligand-stimulated tyrosine phosphorylation of VE-cadherin, halts the ligand-induced disruption of VE-cadherin intercellular adhesion and blocks the ligand-induced opening of intercellular junctions. These CRSBP-1 ligands also induce opening of lymphatic intercellular junctions that respond to PDGFβR kinase inhibitor in wild-type mice (but not in Crsbp1-null mice) as evidenced by increased transit of injected FITC–dextran and induced edema fluid from the interstitial space into lymphatic vessels. These results disclose a novel mechanism involved in the opening of lymphatic intercellular junctions.

Clathrin-dependent endocytosis is believed to be involved in TGFβ-stimulated cellular responses, but the subcellular locus at which TGFβ induces signaling remains unclear. Here, we demonstrate that inhibitors of clathrin-dependent endocytosis, which are known to arrest the progression of endocytosis at coated-pit stages, inhibit internalization of cell-surface-bound TGFβ and promote colocalization and accumulation of TβR-I and SARA at the plasma membrane. These inhibitors enhance TGFβ-induced signaling and cellular responses (Smad2 phosphorylation/nuclear localization and expression of PAI-1). Dynasore, a newly identified inhibitor of dynamin GTPase activity, is one of the most potent inhibitors among those tested and, furthermore, is a potent enhancer of TGFβ. Dynasore ameliorates atherosclerosis in the aortic endothelium of hypercholesterolemic ApoE-null mice by counteracting the suppressed TGFβ responsiveness caused by the hypercholesterolemia, presumably acting through its effect on TGFβ endocytosis and signaling in vascular cells.

Transforming growth factor‐β (TGF‐β) responsiveness in cultured cells can be modulated by TGF‐β partitioning between lipid raft/caveolae‐ and clathrin‐mediated endocytosis pathways. The TβR‐II/TβR‐I binding ratio of TGF‐β on the cell surface has recently been found to be a signal that controls TGF‐β partitioning between these pathways. Since cholesterol is a structural component in lipid rafts/caveolae, we have studied the effects of cholesterol on TGF‐β binding to TGF‐β receptors and TGF‐β responsiveness in cultured cells and in animals. Here we demonstrate that treatment with cholesterol, alone or complexed in lipoproteins, decreases the TβR‐II/TβR‐I binding ratio of TGF‐β while treatment with cholesterol‐lowering or cholesterol‐depleting agents increases the TβR‐II/TβR‐I binding ratio of TGF‐β in all cell types studied. Among cholesterol derivatives and analogs examined, cholesterol is the most potent agent for decreasing the TβR‐II/TβR‐I binding ratio of TGF‐β. Cholesterol treatment increases accumulation of the TGF‐β receptors in lipid rafts/caveolae as determined by sucrose density gradient ultracentrifugation analysis of cell lysates. Cholesterol/LDL suppresses TGF‐β responsiveness and statins/β‐CD enhances it, as measured by the levels of P‐Smad2 and PAI‐1 expression in cells stimulated with TGF‐β. Furthermore, the cholesterol effects observed in cultured cells are also found in the aortic endothelium of atherosclerotic ApoE‐null mice fed a high cholesterol diet. These results indicate that high plasma cholesterol levels may contribute to the pathogenesis of certain diseases (e.g., atherosclerosis) by suppressing TGF‐β responsiveness. J. Cell. Physiol. 215: 223–233, 2008.

Hypercholesterolemia is a major causative factor for atherosclerotic cardiovascular disease. The molecular mechanisms by which cholesterol initiates and facilitates the process of atherosclerosis are not well understood. Here, we demonstrate that cholesterol treatment suppresses or attenuates TGF-β responsiveness in all cell types studied as determined by measuring TGF-β-induced Smad2 phosphorylation and nuclear translocation, TGF-β-induced PAI-1 expression, TGF-β-induced luciferase reporter gene expression and TGF-β-induced growth inhibition. Cholesterol, alone or complexed in lipoproteins (LDL, VLDL), suppresses TGF-β responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-β receptors and facilitating rapid degradation of TGF-β and thus suppressing TGF-β-induced signaling. Conversely, cholesterol-lowering agents (fluvastatin and lovastatin) and cholesterol-depleting agents (β-cyclodextrin and nystatin) enhance TGF-β responsiveness by increasing non-lipid raft microdomain accumulation of TGF-β receptors and facilitating TGF-β-induced signaling. Furthermore, the effects of cholesterol on the cultured cells are also found in the aortic endothelium of ApoE-null mice fed a high-cholesterol diet. These results suggest that high cholesterol contributes to atherogenesis, at least in part, by suppressing TGF-β responsiveness in vascular cells.