Endoglin mediates fibronectin/α5β1 integrin and TGF-β pathway crosstalk in endothelial cells

Both the transforming growth factor β (TGF-β) and integrin signalling pathways have well-established roles in angiogenesis. However, how these pathways integrate to regulate angiogenesis is unknown. Here, we show that the extracellular matrix component, fibronectin, and its cellular receptor, α5β1 integrin, specifically increase TGF-β1- and BMP-9-induced Smad1/5/8 phosphorylation via the TGF-β superfamily receptors endoglin and activin-like kinase-1 (ALK1). Fibronectin and α5β1 integrin increase Smad1/5/8 signalling by promoting endoglin/ALK1 cell surface complex formation. In a reciprocal manner, TGF-β1 activates α5β1 integrin and downstream signalling to focal adhesion kinase (FAK) in an endoglin-dependent manner. α5β1 integrin and endoglin form a complex on the cell surface and co-internalize, with their internalization regulating α5β1 integrin activation and signalling. Functionally, endoglin-mediated fibronectin/α5β1 integrin and TGF-β pathway crosstalk alter the responses of endothelial cells to TGF-β1, switching TGF-β1 from a promoter to a suppressor of migration, inhibiting TGF-β1-mediated apoptosis to promote capillary stability, and partially mediating developmental angiogenesis in vivo. These studies provide a novel mechanism for the regulation of TGF-β superfamily signalling and endothelial function through crosstalk with integrin signalling pathways.     

The Interaction of Protein-tyrosine Phosphatase α (PTPα) and RACK1 Protein Enables Insulin-like Growth Factor 1 (IGF-1)-stimulated Abl-dependent and -independent Tyrosine Phosphorylation of PTPα

Protein tyrosine phosphatase α (PTPα) promotes integrin-stimulated cell migration in part through the role of Src-phosphorylated PTPα-Tyr(P)-789 in recruiting and localizing p130Cas to focal adhesions. The growth factor IGF-1 also stimulates PTPα-Tyr-789 phosphorylation to positively regulate cell movement. This is in contrast to integrin-induced PTPα phosphorylation, that induced by IGF-1 can occur in cells lacking Src family kinases (SFKs), indicating that an unknown kinase distinct from SFKs can target PTPα. We show that this IGF-1-stimulated tyrosine kinase is Abl. We found that PTPα binds to the scaffold protein RACK1 and that RACK1 coordinates the IGF-1 receptor, PTPα, and Abl in a complex to enable IGF-1-stimulated and Abl-dependent PTPα-Tyr-789 phosphorylation. In cells expressing SFKs, IGF-1-stimulated phosphorylation of PTPα is mediated by RACK1 but is Abl-independent. Furthermore, expressing the SFKs Src and Fyn in SFK-deficient cells switches IGF-1-induced PTPα phosphorylation to occur in an Abl-independent manner, suggesting that SFK activity dominantly regulates IGF-1/IGF-1 receptor signaling to PTPα. RACK1 is a molecular scaffold that integrates growth factor and integrin signaling, and our identification of PTPα as a RACK1 binding protein suggests that RACK1 may coordinate PTPα-Tyr-789 phosphorylation in these signaling networks to promote cell migration.     

Novel Protein Interactions with Endoglin and Activin Receptor-like Kinase 1: Potential Role in Vascular Networks

Endoglin and activin receptor-like kinase 1 are specialized transforming growth factor-beta (TGF-β) superfamily receptors, primarily expressed in endothelial cells. Mutations in the corresponding ENG or ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT1 and HHT2 respectively). To discover proteins interacting with endoglin, ACVRL1 and TGF-β receptor type 2 and involved in TGF-β signaling, we applied LUMIER, a high-throughput mammalian interactome mapping technology. Using stringent criteria, we identified 181 novel unique and shared interactions with ACVRL1, TGF-β receptor type 2, and endoglin, defining potential novel important vascular networks. In particular, the regulatory subunit B-beta of the protein phosphatase PP2A (PPP2R2B) interacted with all three receptors. Interestingly, the PPP2R2B gene lies in an interval in linkage disequilibrium with HHT3, for which the gene remains unidentified. We show that PPP2R2B protein interacts with the ACVRL1/TGFBR2/endoglin complex and recruits PP2A to nitric oxide synthase 3 (NOS3). Endoglin overexpression in endothelial cells inhibits the association of PPP2R2B with NOS3, whereas endoglin-deficient cells show enhanced PP2A-NOS3 interaction and lower levels of endogenous NOS3 Serine 1177 phosphorylation. Our data suggest that endoglin regulates NOS3 activation status by regulating PPP2R2B access to NOS3, and that PPP2R2B might be the HHT3 gene. Furthermore, endoglin and ACVRL1 contribute to several novel networks, including TGF-β dependent and independent ones, critical for vascular function and potentially defective in HHT.Transforming growth factor-β (TGF-β)¹ superfamily ligands, including TGF-βs, activins and bone morphogenic proteins (BMPs), regulate several pathways essential for vascular development and function (1). Responses to these ligands are controlled by type I and II serine kinase receptors, coreceptors and signaling SMAD intermediates. Endothelial cells express the coreceptor, endoglin, and the specialized type I receptor, ACVRL1 (activin receptor-like kinase 1 or ALK1); both molecules are critical for regulation of angiogenesis and vasomotor function by TGF-β superfamily ligands (2, 3).Mutations in ENG and ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT), types 1 and 2, respectively (4). HHT affects 1 in 5000–8000 people worldwide and is characterized by arteriovenous malformations (AVMs) in multiple organs, potentially leading to severe hemorrhages and strokes (4). Haploinsufficiency is the underlying cause of HHT, indicating that reduced levels of functional endoglin or ACVRL1 (ALK1) proteins predispose to endothelial dysfunction and AVMs (5). Although the mechanisms responsible for AVMs remain unclear, the elucidation of how members of the TGF-β superfamily and their molecular networks regulate vascular integrity is vital for future treatments of HHT.We have demonstrated that endoglin interacts with endothelial nitric oxide synthase (NOS3 or eNOS) and regulates its activation (2). NOS3 is a Ca⁺² and calmodulin-regulated enzyme that produces NO● in response to humoral and mechanical stimuli via dynamic interactions with various allosteric regulators such as heat shock protein 90 (HSP90). NOS3 is also regulated by dynamic changes in its phosphorylation status. For example, effects of the vascular endothelial growth factor (VEGF) on angiogenesis, vascular permeability and vasomotor tone are mediated in part through Akt-dependent phosphorylation of NOS3 Ser1177 and by increased NOS3-HSP90 association (6). Although phosphorylation of NOS3 Ser1177 is indicative of agonist-induced activation, it is preceded by dephosphorylation at Thr495. TGF-β1 and -β3 but not -β2 responses can sensitize NOS3 for activation by inducing dephosphorylation at Thr495, and therefore contribute to NOS3 activation and NO-dependent vasorelaxation (7). Endoglin regulates TGF-β1 and -β3 but not -β2 responses, and is required for their induction of NOS3 Thr495 dephosphorylation (7, 8).In the vascular endothelium of HHT patients and in Eng and Alk1 heterozygous mice, impaired association of NOS3 with HSP90 renders the enzyme uncoupled, causing production of superoxide (●O₂⁻) instead of NO● (2, 3, 9) and leading to endothelial damage. Interestingly, TGF-β1 and -β3 do not induce phosphorylation at NOS3 Ser1177, yet NOS3 activation in response to TGF-β1 is abolished in endoglin-deficient cells, impairing vasomotor function (3). ACVRL1 (or ALK1) also interacts with NOS3, and its reduced levels in endothelial cells similarly cause NOS3-derived oxidative stress (3, 9).In view of the crucial roles of endoglin and ACVRL1 in the development and maintenance of the normal vasculature and the definite contribution of their mutated state to HHT, we used the LUMIER high-throughput technology (10) to identify novel protein interactions and molecular networks for these predominantly endothelial receptors. We included TGFBR2 to further define TGF-β protein networks potentially important for vascular function, and attempt to distinguish the TGF-β networks from those associated with BMP9/BMP10 and mediated by ACVRL1 in a complex with BMPR2 and endoglin (11, 12).One of identified proteins interacting with all three receptors was protein phosphatase 2A (PP2A), implicated in multiple pathways. PP2A is a holoenzyme with one structural subunit (PPP2R1A or PPP2R1B) associated with one catalytic subunit (PPP2CA or PPP2CB) and one of 19 regulatory B subunits, the latter conferring specificity to the enzyme by recruiting interacting proteins (13, 14). Of interest, PP2A interacts with NOS3 to regulate Ser1177 phosphorylation and NO● production (15). However, the mechanisms governing recruitment of PP2A to NOS3 and the contribution of TGF-β/BMP receptor complexes are unknown. Recently, the human PPP2R2B gene coding for PPP2R2B protein (also known as PP2A-Bβ regulatory subunit) was mapped to chromosome 5q31-q32, in an interval in linkage disequilibrium with the HHT3 locus (16, 17). We now report that PPP2R2B interacts with the ACVRL1/TGFBR2/endoglin complex and that endoglin governs NOS3 phosphorylation and activation status by hindering PP2A access to NOS3 via the PPP2R2B subunit. Loss of endoglin leads to constitutive reduction in NOS3 phosphorylation and likely to changes in several networks with consequent endothelial dysfunction.     

Mice Lacking Endoglin in Macrophages Show an Impaired Immune Response

Endoglin is an auxiliary receptor for members of the TGF-β superfamily and plays an important role in the homeostasis of the vessel wall. Mutations in endoglin gene (ENG) or in the closely related TGF-β receptor type I ACVRL1/ALK1 are responsible for a rare dominant vascular dysplasia, the Hereditary Hemorrhagic Telangiectasia (HHT), or Rendu-Osler-Weber syndrome. Endoglin is also expressed in human macrophages, but its role in macrophage function remains unknown. In this work, we show that endoglin expression is triggered during the monocyte-macrophage differentiation process, both in vitro and during the in vivo differentiation of blood monocytes recruited to foci of inflammation in wild-type C57BL/6 mice. To analyze the role of endoglin in macrophages in vivo, an endoglin myeloid lineage specific knock-out mouse line (Eng^fl/flLysMCre) was generated. These mice show a predisposition to develop spontaneous infections by opportunistic bacteria. Eng^fl/flLysMCre mice also display increased survival following LPS-induced peritonitis, suggesting a delayed immune response. Phagocytic activity is impaired in peritoneal macrophages, altering one of the main functions of macrophages which contributes to the initiation of the immune response. We also observed altered expression of TGF-β1 target genes in endoglin deficient peritoneal macrophages. Overall, the altered immune activity of endoglin deficient macrophages could help to explain the higher rate of infectious diseases seen in HHT1 patients.     

C18 ORF1, a Novel Negative Regulator of Transforming Growth Factor-β Signaling

Transforming growth factor (TGF)-β signaling is deliberately regulated at multiple steps in its pathway from the extracellular microenvironment to the nucleus. However, how TGF-β signaling is activated or attenuated is not fully understood. We recently identified transmembrane prostate androgen-induced RNA (TMEPAI), which is involved in a negative feedback loop of TGF-β signaling. When we searched for a family molecule(s) for TMEPAI, we found C18ORF1, which, like TMEPAI, possesses two PY motifs and one Smad-interacting motif (SIM) domain. As expected, C18ORF1 could block TGF-β signaling but not bone morphogenetic protein signaling. C18ORF1 bound to Smad2/3 via its SIM and competed with the Smad anchor for receptor activation for Smad2/3 binding to attenuate recruitment of Smad2/3 to the TGF-β type I receptor (also termed activin receptor-like kinase 5 (ALK5)), in a similar fashion to TMEPAI. Knockdown of C18ORF1 prolonged duration of TGF-β-induced Smad2 phosphorylation and concomitantly potentiated the expression of JunB, p21, and TMEPAI mRNAs induced by TGF-β. Consistently, TGF-β-induced cell migration was enhanced by the knockdown of C18ORF1. These results indicate that the inhibitory function of C18ORF1 on TGF-β signaling is similar to that of TMEPAI. However, in contrast to TMEPAI, C18ORF1 was not induced upon TGF-β signaling. Thus, we defined C18ORF1 as a surveillant of steady state TGF-β signaling, whereas TMEPAI might help C18ORF1 to inhibit TGF-β signaling in a coordinated manner when cells are stimulated with high levels of TGF-β.     

Role of TGF-β1 and MAP Kinases in the Antiproliferative Effect of Aspirin in Human Vascular Smooth Muscle Cells

Background

We aimed to test the antiproliferative effect of acetylsalicylic acid (ASA) on vascular smooth muscle cells (VSMC) from bypass surgery patients and the role of transforming growth factor beta 1 (TGF-β1).

Methodology/Principal Findings

VSMC were isolated from remaining internal mammary artery from patients who underwent bypass surgery. Cell proliferation and DNA fragmentation were assessed by ELISA. Protein expression was assessed by Western blot. ASA inhibited BrdU incorporation at 2 mM. Anti-TGF-β1 was able to reverse this effect. ASA (2 mM) induced TGF-β1 secretion; however it was unable to induce Smad activation. ASA increased p38^MAPK phosphorylation in a TGF-β1-independent manner. Anti-CD105 (endoglin) was unable to reverse the antiproliferative effect of ASA. Pre-surgical serum levels of TGF-β1 in patients who took at antiplatelet doses ASA were assessed by ELISA and remained unchanged.

Conclusions/Significance

In vitro antiproliferative effects of aspirin (at antiinflammatory concentration) on human VSMC obtained from bypass patients are mediated by TGF-β1 and p38^MAPK. Pre-surgical serum levels of TGF- β1 from bypass patients who took aspirin at antiplatelet doses did not change.     

Wiskott-Aldrich Syndrome Protein (WASp) Controls the Delivery of Platelet Transforming Growth Factor-β1

Platelets are immunologically competent cells containing cytokines such as TGF-β1 that regulate cell-mediated immunity. However, the mechanisms underlying cytokine secretion from platelets are undefined. The Wiskott-Aldrich syndrome protein (WASp) regulates actin polymerization in nucleated hematopoietic cells but has other role(s) in platelets. WASp-null (WASp^−/−) platelets stimulated with a PAR-4 receptor agonist had increased TGF-β1 release compared with WT platelets; inhibiting WASp function with wiskostatin augmented TRAP-induced TGF-β1 release in human platelets. TGF-β1 release is dissociated from α-granule secretion (P-selectin up-regulation) and occurs more gradually, with ∼10–15% released after 30–60 min. Blockade of Src family kinase-mediated WASp Tyr-291/Tyr-293 phosphorylation increased TGF-β1 release, with no additive effect in WASp^−/− platelets, signifying that phosphorylation is critical for WASp-limited TGF-β1 secretion. Inhibiting F-actin assembly with cytochalasin D enhanced secretion in WT platelets and further increased TGF-β1 release in WASp^−/− platelets, indicating that WASp and actin assembly independently regulate TGF-β1 release. A permeabilized platelet model was used to test the role of upstream small GTPases in TGF-β1 release. N17Cdc42, but not Rac1 mutants, increased TGF-β1 secretion and abrogated WASp phosphorylation. We conclude that WASp function restricts TGF-β1 secretion in a Cdc42- and Src family kinase-dependent manner and independently of actin assembly.     

Suberoylanilide hydroxamic acid (SAHA) inhibits transforming growth factor-beta 2-induced increases in aqueous humor outflow resistance

Transforming growth factor-beta 2 (TGF-β2) is highly concentrated in the aqueous humor of primary open-angle glaucoma patients. TGF-β2 causes fibrosis of outflow tissues, such as the trabecular meshwork (TM), and increases intraocular pressure by increasing resistance to aqueous humor outflow. Recently, histone deacetylase (HDAC) activity was investigated in fibrosis in various tissues, revealing that HDAC inhibitors suppress tissue fibrosis. However, the effect of HDAC inhibitors on fibrosis in the eye was not determined. Here, we investigated the effect of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on TGF-β2-induced increased resistance to aqueous humor outflow. We found that SAHA suppressed TGF-β2-induced outflow resistance in perfused porcine eyes. Moreover, SAHA cotreatment suppressed TGF-β2-induced ocular hypertension in rabbits. The permeability of monkey TM (MTM) and Schlemm’s canal (MSC) cell monolayers was decreased by TGF-β2 treatment. SAHA inhibited the effects of TGF-β2 on the permeability of these cells. TGF-β2 also increased the expression of extracellular matrix proteins (fibronectin and collagen type I or IV) in MTM, MSC, and human TM (HTM) cells, while SAHA inhibited TGF-β2-induced extracellular matrix protein expression in these cells. SAHA also inhibited TGF-β2-induced phosphorylation of Akt and ERK, but did not inhibit Smad2/3 phosphorylation, the canonical pathway of TGF-β signaling. Moreover, SAHA induced the expression of phosphatase and tensin homolog, a PI3K/Akt signaling factor, as well as bone morphogenetic protein 7, an endogenous antagonist of TGF-β. These results imply that SAHA prevents TGF-β2-induced increases in outflow resistance and regulates the non-Smad pathway of TGF-β signaling in TM and MSC cells.     

Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

The transforming growth factor-beta (TGF-β) superfamily is one of the most diversified cell signaling pathways and regulates many physiological and pathological processes. Recently, neuropilin-1 (NRP-1) was reported to bind and activate the latent form of TGF-β1 (LAP-TGF-β1). We investigated the role of NRP-1 on Smad signaling in stromal fibroblasts upon TGF-β stimulation. Elimination of NRP-1 in stromal fibroblast cell lines increases Smad1/5 phosphorylation and downstream responses as evidenced by up-regulation of inhibitor of differentiation (Id-1). Conversely, NRP-1 loss decreases Smad2/3 phosphorylation and its responses as shown by down-regulation of α-smooth muscle actin (α-SMA) and also cells exhibit more quiescent phenotypes and growth arrest. Moreover, we also observed that NRP-1 expression is increased during the culture activation of hepatic stellate cells (HSCs), a liver resident fibroblast. Taken together, our data suggest that NRP-1 functions as a key determinant of the diverse responses downstream of TGF-β1 that are mediated by distinct Smad proteins and promotes myofibroblast phenotype.     

Transforming Growth Factor-β1 Inhibits Trophoblast Cell Invasion by Inducing Snail-mediated Down-regulation of Vascular Endothelial-cadherin Protein

Human trophoblast cells express transforming growth factor-β (TGF-β) and TGF-β receptors. It has been shown that TGF-β1 treatment decreases the invasiveness of trophoblast cells. However, the molecular mechanisms underlying TGF-β1-decreased trophoblast invasion are still not fully understood. In the current study, we demonstrated that treatment of HTR-8/SVneo human trophoblast cells with TGF-β1 decreased cell invasion and down-regulated the expression of vascular endothelial cadherin (VE-cadherin). In addition, the inhibitory effect of TGF-β1 on VE-cadherin was confirmed in primary cultures of human trophoblast cells. Moreover, knockdown of VE-cadherin using siRNA decreased the invasiveness of HTR-8/SVneo cells and primary cultures of trophoblast cells. Treatment with TGF-β1 induced the activation of Smad-dependent signaling pathways and the expression of Snail and Slug. Knockdown of Smads attenuated TGF-β1-induced up-regulation of Snail and Slug and down-regulation of VE-cadherin. Interestingly, depletion of Snail, but not Slug, attenuated TGF-β1-induced down-regulation of VE-cadherin. Furthermore, overexpression of Snail suppressed VE-cadherin expression. Chromatin immunoprecipitation analyses showed the direct binding of Snail to the VE-cadherin promoter. These results provide evidence that Snail mediates TGF-β1-induced down-regulation of VE-cadherin, which subsequently contributed to TGF-β1-decreased trophoblast cell invasion.     

Soluble endoglin specifically binds bone morphogenetic proteins 9 and 10 via its orphan domain, inhibits blood vessel formation, and suppresses tumor growth

Endoglin (CD105), a transmembrane protein of the transforming growth factor β superfamily, plays a crucial role in angiogenesis. Mutations in endoglin result in the vascular defect known as hereditary hemorrhagic telangiectasia (HHT1). The soluble form of endoglin was suggested to contribute to the pathogenesis of preeclampsia. To obtain further insight into its function, we cloned, expressed, purified, and characterized the extracellular domain (ECD) of mouse and human endoglin fused to an immunoglobulin Fc domain. We found that mouse and human endoglin ECD-Fc bound directly, specifically, and with high affinity to bone morphogenetic proteins 9 and 10 (BMP9 and BMP10) in surface plasmon resonance (Biacore) and cell-based assays. We performed a function mapping analysis of the different domains of endoglin by examining their contributions to the selectivity and biological activity of the protein. The BMP9/BMP10 binding site was localized to the orphan domain of human endoglin composed of the amino acid sequence 26-359. We established that endoglin and type II receptors bind to overlapping sites on BMP9. In the in vivo chick chorioallantoic membrane assay, the mouse and the truncated human endoglin ECD-Fc both significantly reduced VEGF-induced vessel formation. Finally, murine endoglin ECD-Fc acted as an anti-angiogenic factor that decreased blood vessel sprouting in VEGF/FGF-induced angiogenesis in in vivo angioreactors and reduced the tumor burden in the colon-26 mouse tumor model. Together our findings indicate an important role of soluble endoglin ECD in the regulation of angiogenesis and highlight efficacy of endoglin-Fc as a potential anti-angiogenesis therapeutic agent.     

Transforming Growth Factor-β3 (TGF-β3) Knock-in Ameliorates Inflammation Due to TGF-β1 Deficiency While Promoting Glucose Tolerance

Three homologues of TGF-β exist in mammals as follows: TGF-β1, TGF-β2, and TGF-β3. All three proteins share high homology in their amino acid sequence, yet each TGF-β isoform has unique heterologous motifs that are highly conserved during evolution. Although these TGF-β proteins share similar properties in vitro, isoform-specific properties have been suggested through in vivo studies and by the unique phenotypes for each TGF-β knock-out mouse. To test our hypothesis that each of these homologues has nonredundant functions, and to identify such isoform-specific roles, we genetically exchanged the coding sequence of the mature TGF-β1 ligand with a sequence from TGF-β3 using targeted recombination to create chimeric TGF-β1/3 knock-in mice (TGF-β1^Lβ3/Lβ3). In the TGF-β1^Lβ3/Lβ3 mouse, localization and activation still occur through the TGF-β1 latent associated peptide, but cell signaling is triggered through the TGF-β3 ligand that binds to TGF-β receptors. Unlike TGF-β1^−/− mice, the TGF-β1^Lβ3/Lβ3 mice show neither embryonic lethality nor signs of multifocal inflammation, demonstrating that knock-in of the TGF-β3 ligand can prevent the vasculogenesis defects and autoimmunity associated with TGF-β1 deficiency. However, the TGF-β1^Lβ3/Lβ3 mice have a shortened life span and display tooth and bone defects, indicating that the TGF-β homologues are not completely interchangeable. Remarkably, the TGF-β1^Lβ3/Lβ3 mice display an improved metabolic phenotype with reduced body weight gain and enhanced glucose tolerance by induction of beneficial changes to the white adipose tissue compartment. These findings reveal both redundant and unique nonoverlapping functional diversity in TGF-β isoform signaling that has relevance to the design of therapeutics aimed at targeting the TGF-β pathway in human disease.     

TGF-β1 expression is associated with invasion and metastasis of intrahepatic cholangiocarcinoma

Background

Transforming growth factor (TGF)-β is involved in many physiologic processes, it often promotes metastasis, and its high expression is correlated with poor prognosis. In the present study, we analyzed the correlation between transforming growth factor beta 1 (TGF-β1) expression and prognosis in intrahepatic cholangiocarcinoma.

Results

We examined the expression of TGF-β1 in 78 intrahepatic cholangiocarcinomas by immunohistochemistry and correlated the expression with clinicopathological parameters. TGF-β1 was expressed in 37 of 78 (47.4 %) intrahepatic cholangiocarcinomas. The expression of TGF-β1 was significantly correlated with lymph node metastasis, distant metastasis, and tumour recurrence. Patients with TGF-β1-positive tumours had significantly shorter survival time. In a multivariant analysis, the expression of TGF-β1 and the tumour stage were independent prognostic factors.

Conclusions

Our data suggest that expression of TGF-β1 is a novel prognostic marker for intrahepatic cholangiocarcinoma.     

Endoglin in liver fibrogenesis: Bridging basic science and clinical practice

Endoglin, also known as cluster of differentiation CD105, was originally identified 25 years ago as a novel marker of endothelial cells. Later it was shown that endoglin is also expressed in pro-fibrogenic cells including mesangial cells, cardiac and scleroderma fibroblasts, and hepatic stellate cells. It is an integral membranebound disulfide-linked 180 kDa homodimeric receptor that acts as a transforming growth factor-β(TGF-β) auxiliary co-receptor. In humans, several hundreds of mutations of the endoglin gene are known that give rise to an autosomal dominant bleeding disorder that is characterized by localized angiodysplasia and arteriovenous malformation. This disease is termed hereditary hemorrhagic telangiectasia type Ⅰ and induces various vascular lesions, mainly on the face, lips, hands and gastrointestinal mucosa. Two variants of endoglin(i.e., S- and L-endoglin) are formed by alternative splicing that distinguishes from each other in the length of their cytoplasmic tails. Moreover, a soluble form of endoglin, i.e.,sol-Eng, is shedded by the matrix metalloprotease-14 that cleaves within the extracellular juxtamembrane region. Endoglin interacts with the TGF-β signaling receptors and influences Smad-dependent and-independent effects. Recent work has demonstrated that endoglin is a crucial mediator during liver fibrogenesis that critically controls the activity of the different Smad branches. In the present review, we summarize the present knowledge of endoglin expression and function, its involvement in fibrogenic Smad signaling, current models to investigate endoglin function, and the diagnostic value of endoglin in liver disease.     

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.