Chun-Lin Chen

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.

Cellular Heparan Sulfate Negatively Modulates Transforming Growth Factor-β1 (TGF-β1) Responsiveness in Epithelial Cells

Cell-surface proteoglycans have been shown to modulate transforming growth factor (TGF)-β responsiveness in epithelial cells and other cell types. However, the proteoglycan (heparan sulfate or chondroitin sulfate) involved in modulation of TGF-β responsiveness and the mechanism by which it modulates TGF-β responsiveness remain unknown. Here we demonstrate that TGF-β1 induces transcriptional activation of plasminogen activator inhibitor-1 (PAI-1) and growth inhibition more potently in CHO cell mutants deficient in heparan sulfate (CHO-677 cells) than in wild-type CHO-K1 cells. 125I-TGF-β1 affinity labeling analysis of cell-surface TGF-β receptors reveals that CHO-K1 and CHO-677 cells exhibit low (<1) and high (>1) ratios of 125I-TGF-β1 binding to TβR-II and TβR-I, respectively. Receptor-bound 125I-TGF-β1 undergoes nystatin-inhibitable rapid degradation in CHO-K1 cells but not in CHO-677 cells. In Mv1Lu cells (which, like CHO-K1 cells, exhibit nystatin-inhibitable rapid degradation of receptor-bound 125I-TGF-β1), treatment with heparitinase or a heparan sulfate biosynthesis inhibitor results in a change from a low (<1) to a high (>1) ratio of 125I-TGF-β1 binding to TβR-II and TβR-I and enhanced TGF-β1-induced transcriptional activation of PAI-1. Sucrose density gradient analysis indicates that a significant fraction of TβR-I and TβR-II is localized in caveolae/lipid-raft fractions in CHO-K1 and Mv1Lu cells whereas the majority of the TGF-β receptors are localized in non-lipid-raft fractions in CHO-677 cells. These results suggest that heparan sulfate negatively modulates TGF-β1 responsiveness by decreasing the ratio of TGF-β1 binding to TβR-II and TβR-I, facilitating caveolae/lipid-raft-mediated endocytosis and rapid degradation of TGF-β1, thus diminishing non-lipid-raft-mediated endocytosis and signaling of TGF-β1 in these epithelial cells.

Signaling mechanisms for chemotaxis

Cells recognize external chemical gradients and translate these environmental cues into amplified intracellular signaling that results in elongated cell shape, actin polymerization toward the leading edge, and movement along the gradient. Mechanisms underlying chemotaxis are conserved evolutionarily from Dictyostelium amoeba to mammalian neutrophils. Recent studies have uncovered several parallel intracellular signaling pathways that crosstalk in chemotaxing cells. Here, we review these signaling mechanisms in Dictyostelium discoideum.

Myosin I links PIP3 signaling to remodeling of the actin cytoskeleton in chemotaxis.

A subset of myosin motor proteins binds the membrane phospholipid PIP3 to link membrane and cytoskeletal dynamics. Linking Membrane Lipids to Cytoskeletal Dynamics The phospholipid PIP3 (phosphatidylinositol 3,4,5-trisphosphate) can be generated at the plasma membrane in cells orienting their movement according to an external chemical gradient (a process called chemotaxis) or in cells engulfing particles or other cells (a process called phagocytosis). Both chemotaxis and phagocytosis require reorganization of the cytoskeleton. Various type I myosin isoforms (specifically, ID, IE, and IF) are motor proteins that interact with the actin cytoskeleton as PIP3-binding proteins. Here, Chen et al. showed that myosins ID, IE, and IF were required for PIP3-induced changes in the cytoskeleton. Dictyostelium cells lacking these three myosin I isoforms showed defects in chemotaxis and in phagocytosis of yeast cells. Furthermore, these mutant Dictyostelium cells failed to remodel the cytoskeleton in response to a chemoattractant. Thus, specific myosin I isoforms couple stimuli that generate PIP3 to changes in the cytoskeleton during chemotaxis and phagocytosis. Class I myosins participate in various interactions between the cell membrane and the cytoskeleton. Several class I myosins preferentially bind to acidic phospholipids, such as phosphatidylserine and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], through a tail homology 1 (TH1) domain. Here, we show that the second messenger lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3) binds to the TH1 domain of a subset of Dictyostelium class I myosins (ID, IE, and IF) and recruits them to the plasma membrane. The PIP3-regulated membrane recruitment of myosin I promoted chemotaxis and induced chemoattractant-stimulated actin polymerization. Similarly, PIP3 recruited human myosin IF to the plasma membrane upon chemotactic stimulation in a neutrophil cell line. These data suggest a mechanism through which the PIP3 signal is transmitted through myosin I to the actin cytoskeleton.

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.

Cyclic stretch stimulates vascular smooth muscle cell alignment by redox-dependent activation of Notch3

Mice deficient in Notch3 have defects in arterial vascular smooth muscle cell (VSMC) mechanosensitivity, including impaired myogenic responses and autoregulation, and inappropriate VMSC orientation. Experiments were performed to determine if Notch3 is activated by mechanical stimulation and contributes to mechanosensitive responses of VSMCs, including cell realignment. Cyclic, uniaxial stretch (10%, 1 Hz) of human VSMCs caused Notch3 activation, demonstrated by a stretch-induced increase in hairy and enhancer of split 1/hairy-related transcription factor-1 expression, translocation of Notch3 to the nucleus, and a decrease in the Notch3 extracellular domain. These effects were prevented by inhibiting the expression [small interfering (si)RNA] or proteolytic activation of Notch3 {N-(R)-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-l-naphthylalanyl-l-alanine-2-aminoethyl amide (TAPI-1; 50 μmol/l) to inhibit TNF-α-converting enzyme (TACE) or N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT; 20 μmol/l) to inhibit γ-secretase}. Stretch increased the activity of ROS within VSMCs, determined using dichlorodihydrofluorescein fluorescence. Catalase (1,200 U/ml), which degrades H₂O₂, inhibited the stretch-induced activation of Notch3, whereas in nonstretched cells, increasing H₂O₂ activity [H₂O₂ or manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin] caused activation of Notch3. Stretch increased the activity of TACE, which was prevented by catalase. Stretch-induced activation of p38 MAPK in VSMCs was inhibited either by catalase or by inhibiting Notch3 expression (siRNA). Stretch caused VSMCs to realign perpendicular to the direction of the mechanical stimulus, which was significantly inhibited by catalase or by inhibiting the expression (siRNA) or activation of Notch3 (TAPI-1 or DAPT). Therefore, cyclic uniaxial stretch activates Notch3 signaling through a ROS-mediated mechanism, and the presence of Notch3 is necessary for proper stretch-induced cell alignment in VSMCs. This mechanism may contribute to the physiological role of Notch3 in mediating developmental maturation of VSMCs.

Cellular growth inhibition by TGF-β1 involves IRS proteins ☆

In Mv1Lu cells, insulin partially reverses transforming growth factor‐β1 (TGF‐β1) growth inhibition in the presence of α5β1 integrin antagonists. TGF‐β1 appears to induce phosphorylation of IRS‐2 in these cells; this is inhibited by a TGF‐β antagonist known to reverse TGF‐β growth inhibition. Stable transfection of 32D myeloid cells (which lack endogenous IRS proteins and are insensitive to growth inhibition by TGF‐β1) with IRS‐1 or IRS‐2 cDNA confers sensitivity to growth inhibition by TGF‐β1; this IRS‐mediated growth inhibition can be partially reversed by insulin in 32D cells stably expressing IRS‐2 and the insulin receptor (IR). These results suggest that growth inhibition by TGF‐β1 involves IRS proteins.