Pin1 Promotes Transforming Growth Factor-��-induced Migration and Invasion

Transforming growth factor-β (TGF-β) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-β responses. TGF-β binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-β-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-β, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-β-induced growth-inhibitory responses and a number of TGF-β/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-β-mediated migration and invasion. Accordingly, TGF-β induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-β-induced migration and invasion of cancer cells.     

Diacylglycerol Kinase �� and Protein Kinase C�� Modulate Epidermal Growth Factor Receptor Abundance and Degradation through Ubiquitin-specific Protease 8

Many human epithelial cancers are characterized by abnormal activation of the epidermal growth factor receptor (EGFR), which is often caused by its excessive expression in tumor cells. The abundance of EGFR is modulated, in part, by its ubiquitination, which targets it for degradation. The components responsible for adding ubiquitin to EGFR are well characterized, but this is a reversible process, and the mechanisms that modulate the removal of ubiquitin from the EGFR are not well known. We found that de-ubiquitination of EGFR was regulated by diacylglycerol kinase δ (DGKδ), a lipid kinase that terminates diacylglycerol signaling. In DGKδ-deficient cells, ubiquitination of EGFR was enhanced, which attenuated the steady-state levels of EGFR and promoted its ligand-induced degradation. These effects were not caused by changes in the ubiquitinating apparatus, but instead were due to reduced expression of the de-ubiquitinase, ubiquitin-specific protease 8 (USP8). Depletion of protein kinase Cα (PKCα), a target of diacylglycerol, rescued the levels of USP8 and normalized EGFR degradation in DGKδ-deficient cells. Moreover, the effects of PKCα were caused by its inhibition of Akt, which stabilizes USP8. Our data indicate a novel mechanism where DGKδ and PKCα modulate the levels of ubiquitinated EGFR through Akt and USP8.     

Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Epiphyseal Cartilage Development through β-Catenin-dependent and -independent Pathways

The epidermal growth factor receptor (EGFR) is an essential player in the development of multiple organs during embryonic and postnatal stages. To understand its role in epiphyseal cartilage development, we generated transgenic mice with conditionally inactivated EGFR in chondrocytes. Postnatally, these mice exhibited a normal initiation of cartilage canals at the perichondrium, but the excavation of these canals into the cartilage was strongly suppressed, resulting in a delay in the formation of the secondary ossification center (SOC). This delay was accompanied by normal chondrocyte hypertrophy but decreased mineralization and apoptosis of hypertrophic chondrocytes and reduced osteoclast number at the border of marrow space. Immunohistochemical analyses demonstrated that inactivation of chondrocyte-specific EGFR signaling reduced the amounts of matrix metalloproteinases (MMP9, -13, and -14) and RANKL (receptor activator of NF-κB ligand) in the hypertrophic chondrocytes close to the marrow space and decreased the cartilage matrix degradation in the SOC. Analyses of EGFR downstream signaling pathways in primary epiphyseal chondrocytes revealed that up-regulation of MMP9 and RANKL by EGFR signaling was partially mediated by the canonical Wnt/β-catenin pathway, whereas EGFR-enhanced MMP13 expression was not. Further biochemical studies suggested that EGFR signaling stimulates the phosphorylation of LRP6, increases active β-catenin level, and induces its nuclear translocation. In line with these in vitro studies, deficiency in chondrocyte-specific EGFR activity reduced β-catenin amount in hypertrophic chondrocytes in vivo. In conclusion, our work demonstrates that chondrocyte-specific EGFR signaling is an important regulator of cartilage matrix degradation during SOC formation and epiphyseal cartilage development and that its actions are partially mediated by activating the β-catenin pathway.     

Vascular Endothelial Growth Factor (VEGF) Receptor-2 Tyrosine 1175 Signaling Controls VEGF-induced von Willebrand Factor Release from Endothelial Cells via Phospholipase C-��1- and Protein Kinase A-dependent Pathways

There is increasing evidence that vascular endothelial growth factor (VEGF) contributes to inflammation independent of its angiogenic functions. Targeting some of the components in endothelial Weibel-Palade bodies (WPBs) effectively inhibits VEGF-induced inflammation, but little is known about how VEGF regulates WPB exocytosis. In this study, we showed that VEGF receptor-2 (VEGFR2), but not VEGFR1, is responsible for VEGF-induced release of von Willebrand factor (vWF), a major marker of WPBs. This is in good contrast to VEGF-stimulated interleukin-6 release from endothelium, which is selectively mediated through VEGFR1. We further demonstrated that VEGFR2-initiated phospholipase C-γ1 (PLCγ1)/calcium signaling is important but insufficient for full vWF release, suggesting the possible participation of another effector pathway. We found that cAMP/protein kinase A (PKA) signaling is required for full vWF release. Importantly, a single mutation of Tyr¹¹⁷⁵ in the C terminus of VEGFR2, a tyrosine residue crucial for embryonic vasculogenesis, abolished vWF release, concomitant with defective activations of both PLCγ1 and PKA. These data suggest that Tyr¹¹⁷⁵ mediates both PLCγ1-dependent and PKA-dependent signaling pathways. Taken together, our results not only reveal a novel Tyr¹¹⁷⁵-mediated signaling pathway but also highlight a potentially new therapeutic target for the management of vascular inflammation.Vascular endothelial growth factor (VEGF)² is a crucial regulator of vasculogenesis, angiogenesis, and vascular permeability (1–5). A number of studies have suggested that VEGF promotes proliferation, migration, and survival of endothelial cells (1, 4). VEGF (also termed VEGF-A) is a member of the growth factor subfamily that includes VEGF-B, -C, -D, and -E and placental growth factor (PlGF). VEGF binds to two high affinity tyrosine kinase receptors, VEGFR1 (also known as Flt-1) and VEGFR2 (also known as KDR/Flk-1), whereas VEGF-E binds to VEGFR2 alone, and PlGF binds to VEGFR1 alone. Within the vessel wall, VEGFR2 is selectively expressed in endothelium. In contrast, VEGFR1 is present on both endothelial cells and monocytes (1, 2).In addition to its role in promoting angiogenesis, there is increasing evidence that VEGF contributes to inflammation independent of its angiogenic functions, although the molecular basis for this effect is incompletely understood (6–8). VEGF is well expressed in the chronic inflammatory skin disease, psoriasis, and in synovial fluid in rheumatoid arthritis (9–12). In addition, previous studies found an association between human severe sepsis/septic shock with elevated circulating levels of VEGF and PlGF (13, 14). Using an in vitro monocyte migration assay and in vivo mouse models of arthritis, several groups, including ours, have suggested that one mechanism by which VEGF causes inflammation is by modulating the infiltration and secretion of monocytes/macrophages via the activation of VEGFR1 (11, 12, 15). On the other hand, emerging evidence suggests that endothelial activation is also important for VEGF-induced inflammation (6, 8, 9). In a mouse model of sepsis, it was demonstrated that the inhibition of VEGFR2, but not VEGFR1, attenuates sepsis mortality, possibly at least in part by suppressing vascular inflammation associated with endothelial activation (9). Consistent with this, ectopic VEGF-A expression in mice enhances leukocyte rolling and adhesion in venules mediated through the P-selectin on the surface of endothelial cells (6). These studies indicate that endothelial activation is another mechanism for VEGF-induced inflammation.P-selectin and von Willebrand factor (vWF) are the best characterized constituents of Weibel-Palade bodies (WPBs), endothelial storage granules that also contain various inflammatory mediators (16–18). As a major component in WPBs, vWF is also involved in their biogenesis and thus is used as a marker of WPBs (18, 19). WPB exocytosis, which gives rise to rapid release of vWF and other mediators such as interleukin-8 (IL-8) (17), and translocation of P-selectin from within granules to the endothelial surfaces triggering leukocyte rolling, are critical early events in endothelial activation and vascular inflammation (16). It has been reported that VEGF regulates vWF/WPB release (20), but the precise roles of VEGF receptors and their downstream effectors in this process have not been defined. In this study, we sought to dissect the signaling pathway by which VEGF induces vWF/WPB release.     

TGF-β1 Up-Regulates Connective Tissue Growth Factor Expression in Human Granulosa Cells through Smad and ERK1/2 Signaling Pathways

Connective tissue growth factor (CTGF), which is also called CCN2, is a secreted matricellular protein. CTGF regulates various important cellular functions by interacting with multiple molecules in the microenvironment. In the ovary, CTGF is mainly expressed in granulosa cells and involved in the regulation of follicular development, ovulation and luteinization. TGF-β1 has been shown to up-regulate CTGF expression in rat and hen granulosa cells. However, the underlying molecular mechanisms of this up-regulation remain undefined. More importantly, whether the stimulatory effect of TGF-β1 on CTGF expression can be observed in human granulosa cells remains unknown. In the present study, our results demonstrated that TGF-β1 treatment up-regulates CTGF expression in both immortalized human granulosa cells and primary human granulosa cells. Using a siRNA-mediated knockdown approach and a pharmacological inhibitor, we demonstrated that the inhibition of Smad2, Smad3 or ERK1/2 attenuates the TGF-β1-induced up-regulation of CTGF. This study provides important insights into the molecular mechanisms that mediate TGF-β1-up-regulated CTGF expression in human granulosa cells.     

Tyrosine Phosphorylation of β-Catenin and Plakoglobin Enhanced by Hepatocyte Growth Factor and Epidermal Growth Factor in Human Carcinoma Cells

The effect of hepatocyte growth factor /scatter factor (HGF/SF) and epidermal growth factor (EGF) on cadherin-mediated adhesion of human carcinoma cells was studied. HGF/SF induced scattering of colonic adenocarcinoma HT29 and gastric adenocarcinomas MKN7 and MKN74 cells. Likewise, EGF induced scattering of HT29 and MKN7 cells. These cells expressed E-cadherin, which was concentrated at cell-cell contact sites. When the scattering of these cells was induced by HGF/SF or EGF, the E-cadherin concentration at cell-cell boundaries tended to decrease. Irnmunoblotting analyses, however, demonstrated that these growth factor treatments did not alter the expression of E-cadherin and E-cadherin-associated proteins, α- and β-catenin and plakoglobin. β-Catenin, plakoglobin and an unidentified 115-kDa molecule associated with E-cadherin were found to be phosphorylated at tyrosine residues, and these phosphorylations were enhanced by the growth factor treatments. These results suggest that HGF/SF and EGF may modulate the function of the cadherin-catenin system via tyrosine phosphorylation of cadherin-associated proteins.     

Stimulation of Platelet-derived Growth Factor Receptor β (PDGFRβ) Activates ADAM17 and Promotes Metalloproteinase-dependent Cross-talk between the PDGFRβ and Epidermal Growth Factor Receptor (EGFR) Signaling Pathways

Binding of the platelet-derived growth factor (PDGF)-B to its receptor PDGFRβ promotes proliferation, migration, and recruitment of pericytes and smooth muscle cells to endothelial cells, serving to stabilize developing blood vessels. The main goals of this study were to determine whether the extracellular domain of the PDGFRβ can be proteolytically released from cell membranes and, if so, to identify the responsible sheddase and determine whether activation of the PDGFRβ stimulates its shedding and potentially that of other membrane proteins. We found that the PDGFRβ is shed from cells by a metalloproteinase and used loss-of-function experiments to identify ADAM10 as the sheddase responsible for constitutive and ionomycin-stimulated processing of the PDGFRβ. Moreover, we showed that ligand-dependent activation of the PDGFRβ does not trigger its own shedding by ADAM10, but instead it stimulates ADAM17 and shedding of substrates of ADAM17, including tumor necrosis factor α and transforming growth factor α. Finally, we demonstrated that treatment of mouse embryonic fibroblasts with PDGF-B triggers a metalloproteinase-dependent cross-talk between the PDGFRβ and the epidermal growth factor receptor (EGFR)/ERK1/2 signaling axis that is also critical for PDGF-B-stimulated cell migration, most likely via ADAM17-dependent release and activation of ligands of the EGFR. This study identifies the principal sheddase for the PDGFRβ and provides new insights into the mechanism of PDGFRβ-dependent signal transduction and cross-talk with the EGFR.     

Proteinase-activated Receptor-2 Transactivation of Epidermal Growth Factor Receptor and Transforming Growth Factor-β Receptor Signaling Pathways Contributes to Renal Fibrosis

Chronic kidney diseases cause significant morbidity and mortality in the population. During renal injury, kidney-localized proteinases can signal by cleaving and activating proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor involved in inflammation and fibrosis that is highly expressed in renal tubular cells. Following unilateral ureteric obstruction, PAR2-deficient mice displayed reduced renal tubular injury, fibrosis, collagen synthesis, connective tissue growth factor (CTGF), and α-smooth muscle actin gene expression at 7 days, compared with wild-type controls. In human proximal tubular epithelial cells in vitro, PAR2 stimulation with PAR2-activating peptide (PAR2-AP) alone significantly up-regulated the expression of CTGF, a potent profibrotic cytokine. The induction of CTGF by PAR2-AP was synergistically increased when combined with transforming growth factor-β (TGF-β). Consistent with these findings, treating human proximal tubular epithelial cells with PAR2-AP induced Smad2/3 phosphorylation in the canonical TGF-β signaling pathway. The Smad2 phosphorylation and CTGF induction required signaling via both the TGFβ-receptor and EGF receptor suggesting that PAR2 utilizes transactivation mechanisms to initiate fibrogenic signaling. Taken together, our data support the hypothesis that PAR2 synergizes with the TGFβ signaling pathway to contribute to renal injury and fibrosis.     

Plasminogen Activator Inhibitor-1 Regulates Integrin ��v��3 Expression and Autocrine Transforming Growth Factor �� Signaling

Fibrosis is characterized by elevated transforming growth factor β (TGFβ) signaling, resulting in extracellular matrix accumulation and increased PAI-1 (plasminogen activator inhibitor) expression. PAI-1 induces the internalization of urokinase plasminogen activator/receptor and integrin αvβ3 from the cell surface. Since increased αvβ3 expression correlates with increased TGFβ signaling, we hypothesized that aberrant PAI-1-mediated αvβ3 endocytosis could initiate an autocrine loop of TGFβ activity. We found that in PAI-1 knock-out (KO) mouse embryonic fibroblasts), αvβ3 endocytosis was reduced by ∼75%, leaving αvβ3 in enlarged focal adhesions, similar to wild type cells transfected with PAI-1 small interfering RNA. TGFβ signaling was significantly enhanced in PAI-1 KO cells, as demonstrated by a 3-fold increase in SMAD2/3-containing nuclei and a 2.9-fold increase in TGFβ activity that correlated with an increase in αvβ3 and TGFβ receptor II expression. As expected, PAI-1 KO cells had unregulated plasmin activity, which was only partially responsible for TGFβ activation, as evidenced by a mere 25% reduction in TGFβ activity when plasmin was inhibited. Treatment of cells with an αvβ3-specific cyclic RGD peptide (GpenGRGD) led to a more profound (59%) TGFβ inhibition; a nonspecific RGD peptide (GRGDNP) inhibited TGFβ by only 23%. Human primary fibroblasts were used to confirm that PAI-1 inhibition and β3 overexpression led to an increase in TGFβ activity. Consistent with a fibrotic phenotype, PAI-1 KO cells were constitutively myofibroblasts that had a 1.6-fold increase in collagen deposition over wild type cells. These data suggest that PAI-1-mediated regulation of αvβ3 integrin is critical for the control of TGFβ signaling and the prevention of fibrotic disease.Fibrotic disorders can result from environmental toxins, persistent infection, autoimmune disease, or mechanical injury, leading to the hardening and scarring of tissues. In fibrotic diseases, such as liver cirrhosis, renal fibrosis, and idiopathic lung fibrosis, or in pathological wound healing, such as hypertrophic scarring, scleroderma, and Dupuytren disease, the persistence of myofibroblasts contributes to disease progression by overproduction of extracellular matrix (ECM)² and by excessive contraction (1–3). A shift in the balance of growth factors and cytokines that promote ECM deposition and proteases that degrade matrix often contributes to fibrotic disease (4, 5). Plasmin, a broad spectrum protease that is generated from plasminogen by uPA, is one of the proteases that degrades matrix and activates growth factors and other proteases (6). Since uPA activity is inhibited by PAI-1, the overexpression of PAI-1 results in matrix accumulation. For this reason, PAI-1 is a key prognostic marker for fibrotic disease. PAI-1 exerts its inhibitory activity on uPA by stimulating the endocytosis of the cell surface uPA·uPAR complex through the low density lipoprotein receptor-related protein (7). Integrin αvβ3 is also internalized with the uPA·uPAR·low density lipoprotein receptor-related protein complex (8). After endocytosis, uPAR and integrins are recycled back to the cell surface for another round of binding (8, 9). uPAR and αvβ3 promote cellular attachment and spreading, since they are receptors for the extracellular matrix molecule, vitronectin (10). Thus, cycling of the complex is thought to stimulate the attachment and detachment that is necessary for cell migration (8). Consequently, a shift in the expression of any of these components (PAI-1/uPA/uPAR/αvβ3) can result in either aggressive migration, as seen in cancer invasion, or a persistent increase in cell adhesion and cell tension, as seen in myofibroblasts in fibrotic tissue.The family of TGFβ growth factors has been intensively studied for their role in fibrotic wound healing. Up-regulation of TGFβ results in amplified and persistent overproduction of molecules, such as integrins and PAI-1 and other protease inhibitors (e.g. TIMPs) (2, 3). Up-regulated integrins continue the cycle of TGFβ signaling by participating in the sustained activation of TGFβ from its latent form. To date, studies have found that various αv integrins participate in the activation of TGFβ (αvβ3, αvβ5, αvβ6, and αvβ8), but the mechanism differs (11–15). Integrins can serve as docking proteins to localize proteases that cleave and activate latent TGFβ in the ECM, or they can directly activate latent TGFβ in a protease-independent manner. Recently, it was discovered that latent TGFβ is also activated by mechanical stress generated from an integrin-mediated interaction between myofibroblasts and the ECM, primarily involving αvβ5. The mechanical stress promotes a conformational change that activates the latent TGFβ complex (15). αv integrins also modulate TGFβ signaling through the binding of αvβ3 to TGFβ receptor II (TGFβRII) in the presence of TGFβ. This interaction was shown to promote a dramatic increase in the proliferation of lung fibroblasts and induce invasion of epithelial breast cancer cells (16, 17).Our data establish a role for the PAI-1-mediated control of αvβ3 expression and support a significant role for αvβ3 in TGFβ signaling. Using PAI-1 KO cells, we tested the hypothesis that the absence of PAI-1 would result in the accumulation of αvβ3 on the cell surface, since PAI-1 promotes the endocytosis of uPA·uPAR·αvβ3. PAI-1-mediated endocytosis of β3 was significantly reduced in the PAI-1 KO cells. Correspondingly, we report that β3 accumulated at the cell surface in enlarged β3-containing focal adhesions. Thus, we explored whether the accumulation of αvβ3 on the cell surface had fibrogenic effects even in the absence of profibrotic PAI-1. Our results demonstrate dramatically increased TGFβ activity and an increase in collagen expression in PAI-1 KO cells. Together, these findings suggest that PAI-1 modulates β3 expression and localization and, in turn, TGFβ signaling. Our data reveal that maintaining precise levels of PAI-1 is a key to preventing fibrosis. Understanding the consequence of regulating PAI-1 activity is critical in light of the many clinical therapies currently under development that target PAI-1 (18, 19).     

Tumor Suppressor PTEN Inhibits Integrin- and Growth Factor–mediated Mitogen-activated Protein (MAP) Kinase Signaling Pathways

The tumor suppressor PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. We demonstrate here that expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein resulted in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation were also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, we examined these components after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity were inhibited by expression of PTEN, whereas EGF receptor autophosphorylation was unaffected. The ability of cells to spread at normal rates was partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation was enhanced as indicated by paxillin staining. The phosphatase domain of PTEN was essential for all of these functions, because PTEN with an inactive phosphatase domain did not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression did not affect c-Jun NH₂-terminal kinase (JNK) or PDGF-stimulated Akt. Our data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading.     

��-Catenin/T-cell Factor Signaling Is Activated during Lung Injury and Promotes the Survival and Migration of Alveolar Epithelial Cells

The Wnt/β-catenin signaling cascade activates genes that allow cells to adopt particular identities throughout development. In adult self-renewing tissues like intestine and blood, activation of the Wnt pathway maintains a progenitor phenotype, whereas forced inhibition of this pathway promotes differentiation. In the lung alveolus, type 2 epithelial cells (AT2) have been described as progenitors for the type 1 cell (AT1), but whether AT2 progenitors use the same signaling mechanisms to control differentiation as rapidly renewing tissues is not known. We show that adult AT2 cells do not exhibit constitutive β-catenin signaling in vivo, using the AXIN2^+/LacZ reporter mouse, or after fresh isolation of an enriched population of AT2 cells. Rather, this pathway is activated in lungs subjected to bleomycin-induced injury, as well as upon placement of AT2 cells in culture. Forced inhibition of β-catenin/T-cell factor signaling in AT2 cultures leads to increased cell death. Cells that survive show reduced migration after wounding and reduced expression of AT1 cell markers (T1α and RAGE). These results suggest that AT2 cells may function as facultative progenitors, where activation of Wnt/β-catenin signaling during lung injury promotes alveolar epithelial survival, migration, and differentiation toward an AT1-like phenotype.