Shuan Shian Huang

Synthetic TGF-β antagonist accelerates wound healing and reduces scarring

Wound healing consists of re‐epithelialization, contraction and formation of granulation and scar tissue. TGF‐β is involved in these events, but its exact roles are not well understood. Here we demonstrate that topical application of a synthetic TGF‐β antagonist accelerates re‐epithelialization in pig burn wounds (100% re‐epithelialization in antagonist‐treated wounds vs. ~ 70% reepithelialization in control wounds on postburn day 26) and reduces wound contraction and scarring in standard pig skin burn, pig skin excision and rabbit skin excision wounds. These results support the distinct roles of TGF‐β in the complex process of wound healing and demonstrate the feasibility of manipulating wound healing by TGF‐β antagonist.

Inhibitors of clathrin-dependent endocytosis enhance TGFβ signaling and responses

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.

Cholesterol suppresses cellular TGF-β responsiveness: implications in atherogenesis

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.

Cholesterol modulates cellular TGF‐β responsiveness by altering TGF‐β binding to TGF‐β receptors

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.

A mechanism by which dietary trans fats cause atherosclerosis

Dietary trans fats (TFs) have been causally linked to atherosclerosis, but the mechanism by which they cause the disease remains elusive. Suppressed transforming growth factor (TGF)-β responsiveness in aortic endothelium has been shown to play an important role in the pathogenesis of atherosclerosis in animals with hypercholesterolemia. We investigated the effects of a high TF diet on TGF-β responsiveness in aortic endothelium and integration of cholesterol in tissues. Here, we show that normal mice fed a high TF diet for 24 weeks exhibit atherosclerotic lesions and suppressed TGF-β responsiveness in aortic endothelium. The suppressed TGF-β responsiveness is evidenced by markedly reduced expression of TGF-β type I and II receptors and profoundly decreased levels of phosphorylated Smad2, an important TGF-β response indicator, in aortic endothelium. These mice exhibit greatly increased integration of cholesterol into tissue plasma membranes. These results suggest that dietary TFs cause atherosclerosis, at least in part, by suppressing TGF-β responsiveness. This effect is presumably mediated by the increased deposition of cholesterol into cellular plasma membranes in vascular tissue, as in hypercholesterolemia.

Identification of insulin receptor substrate proteins as key molecules for the TβR-V/LRP-1-mediated growth inhibitory signaling cascade in epithelial and myeloid cells

The type V TGF‐β receptor (TβR‐V) mediates IGF‐independent growth inhibition by IGFBP‐3 and mediates growth inhibition by TGF‐β 1 in concert with the other TGF‐β receptor types. TβR‐V was recently found to be identical to LRP‐1. Here we find that insulin and (Q3A4Y15L16) IGF‐I (an IGF‐I analog that has a low affinity for IGFBP‐3) antagonize growth inhibition by IGFBP‐3 in mink lung epithelial cells (Mv1Lu cells) stimulated by serum. In these cells, IGFBP‐3 induces serine‐specific dephosphorylation of IRS‐1 and IRS‐2. The IGFBP‐3‐induced dephosphorylation of IRS‐2 is prevented by cotreatment of cells with insulin, (Q3A4Y15L16) IGF‐I, or TβR‐V/LRP‐1 antagonists. The magnitude of the IRS‐2 dephosphorylation induced by IGFBP‐3 positively correlates with the degree of growth inhibition by IGFBP‐3 in Mv1Lu cells and mutant cells derived from Mv1Lu cells. Stable transfection of murine 32D myeloid cells (which lack endogenous IRS proteins and are insensitive to growth inhibition by IGFBP‐3) with IRS‐1 or IRS‐2 cDNA confers sensitivity to growth inhibition by IGFBP‐3; this IRS‐mediated growth inhibition can be completely reversed by insulin in 32D cells stably expressing IRS‐2 and the insulin receptor. These results suggest that IRS‐1 and IRS‐2 are key molecules for the TβR‐V/LRP‐1‐mediated growth inhibitory signaling cascade.

CRSBP-1/LYVE-1 ligands disrupt lymphatic intercellular adhesion by inducing tyrosine phosphorylation and internalization of VE-cadherin

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.

CRSBP‐1/LYVE‐1 ligands stimulate contraction of the CRSBP‐1‐associated ER network in lymphatic endothelial cells

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.

DMSO Enhances TGF-β Activity by Recruiting the Type II TGF-β Receptor From Intracellular Vesicles to the Plasma Membrane.

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 by 125I‐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.

7‐Dehydrocholesterol (7‐DHC), but not Cholesterol, Causes Suppression of Canonical TGF‐β Signaling and Is Likely Involved in the Development of Atherosclerotic Cardiovascular Disease (ASCVD)

For several decades, cholesterol has been thought to cause ASCVD. Limiting dietary cholesterol intake has been recommended to reduce the risk of the disease. However, several recent epidemiological studies do not support a relationship between dietary cholesterol and/or blood cholesterol and ASCVD. Consequently, the role of cholesterol in atherogenesis is now uncertain. Much evidence indicates that TGF‐β, an anti‐inflammatory cytokine, protects against ASCVD and that suppression of canonical TGF‐β signaling (Smad2‐dependent) is involved in atherogenesis. We had hypothesized that cholesterol causes ASCVD by suppressing canonical TGF‐β signaling in vascular endothelium. To test this hypothesis, we determine the effects of cholesterol, 7‐dehydrocholesterol (7‐DHC; the biosynthetic precursor of cholesterol), and other sterols on canonical TGF‐β signaling. We use Mv1Lu cells (a model cell system for studying TGF‐β activity) stably expressing the Smad2‐dependent luciferase reporter gene. We demonstrate that 7‐DHC (but not cholesterol or other sterols) effectively suppresses the TGF‐β‐stimulated luciferase activity. We also demonstrate that 7‐DHC suppresses TGF‐β‐stimulated luciferase activity by promoting lipid raft/caveolae formation and subsequently recruiting cell‐surface TGF‐β receptors from non‐lipid raft microdomains to lipid rafts/caveolae where TGF‐β receptors become inactive in transducing canonical signaling and undergo rapid degradation upon TGF‐β binding. We determine this by cell‐surface 125I‐TGF‐β‐cross‐linking and sucrose density gradient ultracentrifugation. We further demonstrate that methyl‐β‐cyclodextrin (MβCD), a sterol‐chelating agent, reverses 7‐DHC‐induced suppression of TGF‐β‐stimulated luciferase activity by extrusion of 7‐DHC from resident lipid rafts/caveolae. These results suggest that 7‐DHC, but not cholesterol, promotes lipid raft/caveolae formation, leading to suppression of canonical TGF‐β signaling and atherogenesis. J. Cell. Biochem. 118: 1387–1400, 2017.