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Background: Loeys-Dietz syndrome (LDS) is a rare autosomal-dominant connective tissue disorder caused by genetic mutations in the transforming growth factor-β (TGFβ) signaling pathway. In addition to vascular malformations, patients with LDS commonly present with bone and tendon abnormalities, including joint laxity. While TGFβ signaling dysregulation has been implicated in many of these clinical manifestations, the degree to which it influences the tendinopathy and tendon healing issues in LDS has not been determined., Methods: Wound healing after patellar tendon transection was compared between wild-type (WT) and Tgfbr2-mutant (LDS) mice (7 mice per group). In all mice, the right patellar tendon was transected at midsubstance, while the left was untouched to serve as a control. Mice were euthanized 6 weeks after surgery. Tendon specimens were harvested for histopathologic grading according to a previously validated scoring metric, and gene expression levels of Mmp2, Tgfb2, and other TGFβ-signaling genes were assayed. Between-group comparisons were made using 1-way analysis of variance with post hoc Tukey honestly significant difference testing., Results: Expression levels of assayed genes were similar between LDS and WT tendons at baseline; however, at 6 weeks after patellar tendon transection, LDS tendons showed sustained elevations in Mmp2 and Tgfb2 compared with baseline values; these elevations were not seen in normal tendons undergoing the same treatments. Histologically, untreated LDS tendons had significantly greater cellularity and cell rounding compared with untreated WT tendons, and both WT and LDS tendons had significantly worse histologic scores after surgery., Conclusion: We present the first mechanistic insight into the effect of LDS on tendons and tendon healing. The morphologic differences between LDS and WT tendons at baseline may help explain the increased risk of tendon/ligament dysfunction in patients with LDS, and the differential healing response to injury in LDS may account for the delayed healing and weaker repair tissue., Level of Evidence: Level V., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Opsin-3 (OPN3) is a potential key regulator of human melanocyte melanogenesis. How OPN3-mediated regulation of melanocyte melanogenesis is triggered is largely unclear. TGFβ can inhibit the growth of human melanocytes and reduce melanin synthesis in melanocytes. However, whether TGFβ2 can modulate pigmentation in normal human primary melanocytes through OPN3 is entirely unknown. In this study, we constructed a coculture model with human epidermal melanocytes and keratinocytes. OPN3, tyrosinase (TYR), tyrosinase-related protein (TRP)-1, and TRP-2 expression and TYR activity were detected to be higher in cocultured cells than in monocultured cells. Moreover, elevated levels of TGFβ2 were detected in the culture supernatant of melanocytes cocultured with keratinocytes. OPN3 inhibition in melanocytes decreased TYR, TRP-1, and TRP-2 expression and downregulated TYR activity. Our findings indicate that TGFβ2 upregulates TYR activity and TRP-1 and TRP-2 expression in human melanocytes through OPN3 and downstream calcium-dependent G-protein coupled signaling pathways to induce melanogenesis. Interestingly, treatment with the TGFβ2 receptor inhibitor LY2109761 (10 μM) did not inhibit TGFβ2-induced melanocyte melanogenesis though OPN3. Collectively, our data suggest that TGFβ2 upregulates TYR activity through OPN3 through a TGFβ2 receptor-independent and calcium-dependent G-protein coupled signaling pathway., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Conflict of interest T. F., None; M. I.-M., None; S. I., None; H. T., Alcon Japan (F), Kowa (F, C), Merck Sharp & Dohme Corp (C), Otsuka Pharmaceutical (F), Pfizer Japan (F), Santen Pharmaceutical (F), Senju Pharmaceutical (F); T. I., Alcon Japan (F), Kowa (F), Novartis Pharmaceutical (F), Otsuka Pharmaceutical (F), Pfizer Japan (F), Santen Pharmaceutical (F), Senju Pharmaceutical (F)., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Elevated transforming growth factor (TGF)-β2 in aqueous humor (AH) has been suggested to contribute to trabecular meshwork (TM) fibrosis and intraocular pressure (IOP) regulation in primary open-angle glaucoma (POAG), but TGF-β2 is downregulated in secondary open-angle glaucoma (SOAG). Because autotaxin (ATX) is upregulated in SOAG, we investigated the relationships and trans-signaling interactions of these mediators., Methods: The level of ATX in AH was determined using a two-site immunoenzymetric assay, and TGF-β levels were measured using the Bio-Plex Pro TGF-β Assay. RNA scope was used to assess the expression of ATX and TGF-β2 in human's eye specimen. And in vitro studies were performed using hTM cells to explore if trans-signaling of TGF-β2 regulates ATX expressions., Results: TGF-β2/ATX ratio was significantly high in AH of control or POAG compared with SOAG, and negatively correlated with IOP. RNA scope revelated positive expressions of both TGF-β2 and ATX in ciliary body (CB) and TM in control, but ATX expressions was significantly enhanced in SOAG. In hTM cells, ATX expressions were regulated by TGF-β2 with concentration-dependent manner. In counter, ATX also induced TGF-β1, TGF-β2 and TGFBI upregulations and activation of the Smad-sensitive promoter, as well as upregulation of fibrotic markers, and these upregulation was significantly suppressed by both TGF-β and ATX inhibition., Conclusions: Trans-signaling of TGF-β2 regulates ATX expressions and thereby induced upregulations of TGF-βs or fibrosis of hTM. TGF-β2 trans-signaling potently regulate ATX transcription and signaling in hTM cells, which may reflect different profile of these mediators in glaucoma subtypes. Trial Registration This prospective observational study was approved by the Institutional Review Board of the University of Tokyo and was registered with the University Hospital Medical Information Network Clinical Trials Registry of Japan (ID: UMIN000027137). All study procedures conformed to the Declaration of Helsinki. Written informed consent was obtained from each patient.

The TGF beta-1, -2 and -3 isoforms are transcribed from different genes but bind to the same receptors and signal through the same canonical and non-canonical signal transduction pathways. There are numerous regulatory mechanisms controlling the action of each isoform that include the organ-specific cells producing latent TGF beta growth factors, multiple effectors that activate the isoforms, ECM-associated SLRPs and basement membrane components that modulate the activity and localization of the isoforms, other interactive cytokine-growth factor receptor systems, such as PDGF and CTGF, TGF beta receptor expression on target cells, including myofibroblast precursors, receptor binding competition, positive and negative signal transduction effectors, and transcription and translational regulatory mechanisms. While there has long been the view that TGF beta-1and TGF beta-2 are pro-fibrotic, while TGF beta-3 is anti-fibrotic, this review suggests that view is too simplistic, at least in adult tissues, since TGF beta-3 shares far more similarities in its modulation of fibrotic gene expression with TGF beta-1 and TGF beta-2, than it does differences, and often the differences are subtle. Rather, TGF beta-3 should be seen as a fibro-modulatory partner to the other two isoforms that modulates a nuanced and better controlled response to injury. The complex interplay between the three isoforms and numerous interactive proteins, in the context of the cellular milieu, controls regenerative non-fibrotic vs. fibrotic healing in a response to injury in a particular organ, as well as the resolution of fibrosis, when that occurs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Human colostrum (HC) is a rich source of immune mediators that play a role in immune defences of a newly born infant. The mediators include transforming growth factor β (TGF-β) which exists in three isoforms that regulate cellular homeostasis and inflammation, can induce or suppress immune responses, limit T helper 1 cells (Th1) reactions and stimulate secretory immunoglobulin A (IgA) production. Human milk TGF-β also decreases apoptosis of intestinal cells and suppresses macrophage cytokine expression. The aim of the study was to determine the concentration of TGF-β2 in HC obtained from the mothers who delivered vaginally (VD) or by caesarean section (CS), and to compare the concentrations in HC from mothers who delivered at term (TB) or preterm (PB). In this study, 56% of preterm pregnancies were delivered via CS. The concentrations of TGF-β2 were measured in HC from 299 women who delivered in the 1st Department of Obstetrics and Gynaecology, Medical University of Warsaw: 192 (VD), 107 (CS), 251 (TB), and 48 (PB). The colostrum samples were collected within 5 days post-partum. TGF-β2 levels in HC were measured by the enzyme-linked immunosorbent assay (ELISA) test with the Quantikine ELISA Kit-Human TGF-β2 (cat.no. SB250). Statistical significance between groups was calculated by the Student t -test using StatSoft Statistica 13 software. The mean TGF-β2 concentration in patients who delivered at term or preterm were comparable. The levels of TGF-β2 in HC were higher after preterm than term being 4648 vs. 3899 ng/mL ( p = 0.1244). The delivery via CS was associated with higher HC concentrations of TGF-β2. The levels of TGF-β2 were significantly higher in HC after CS than VD (7429 vs. 5240 ng/mL; p = 0.0017). The data from this study suggest: caesarean section was associated with increased levels of TGF-β2 in HC. The increased levels of TGF-β2 in HC of women who delivered prematurely require further research. Early and exclusive breast-feeding by mothers after caesarean section and premature births with colostrum containing high TGF-β2 levels may prevent the negative impact of pathogens which often colonize the gastrointestinal tract and may reduce the risk of chronic diseases in this group of patients., Competing Interests: The authors declare no conflict of interest.

Objective- Abdominal aortic aneurysm is caused by the accumulation of inflammatory cells in the aortic wall. Our recent studies demonstrated that inhibition of Notch signaling attenuates abdominal aortic aneurysm formation by shifting the macrophage balance towards anti-inflammatory (M2) phenotype. Using IL12p40 -/- (interleukin 12 p40) mice, we investigated the effects of M2-predominant macrophages on the development of abdominal aortic aneurysm. Approach and Results- Male (8-10 week-old) wild-type and IL12p40 -/- mice (n=15) on C57BL/6 background were infused with Ang II (angiotensin II, 1000 ng/kg per minute) by implanting osmotic pumps subcutaneously for 28 days. In the IL12p40 -/- mice, Ang II significantly increased the maximal intraluminal diameter (9/15) as determined by transabdominal ultrasound imaging. In addition, IL12p40-deletion significantly increased aortic stiffness in response to Ang II as measured by pulse wave velocity and atomic force microscopy. Histologically, IL12p40 -/- mice exhibited increased maximal external diameter of aorta and aortic lesions associated with collagen deposition and increased elastin fragmentation compared with wild-type mice infused with Ang II. Mechanistically, IL12p40 deficiency by siRNA (small interfering RNA) augmented the Tgfβ2-mediated Mmp2 expression in wild-type bone marrow-derived macrophages without affecting the expression of Mmp9. No such effects of IL12p40 deficiency on MMP2/MMP9 was observed in human aortic smooth muscle cells or fibroblasts. Depletion of macrophages in IL12p40 -/- mice by clodronate liposomes significantly decreased the maximal external diameter of aorta and aortic stiffness in response to Ang II as determined by imaging and atomic force microscopy. Conclusions- IL12p40 depletion promotes the development of abdominal aortic aneurysm, in part, by facilitating recruitment of M2-like macrophages and potentiating aortic stiffness and fibrosis mediated by Tgfβ2.

Tenogenic differentiation of stem cells is needed for tendon tissue engineering approaches. A current challenge is the limited information on the cellular-level changes during tenogenic induction. Tendon cells in embryonic and adult tendons possess an array of cell-cell junction proteins that include cadherins and connexins, but how these proteins are impacted by tenogenic differentiation is unknown. Our objective was to explore how tenogenic induction of mesenchymal stem cells (MSCs) using the transforming growth factor (TGF)β2 impacted protein markers of tendon differentiation and protein levels of N-cadherin, cadherin-11 and connexin-43. MSCs were treated with TGFβ2 for 21 days. At 3 days, TGFβ2-treated MSCs developed a fibroblastic morphology and significantly decreased levels of N-cadherin protein, which were maintained through 21 days. Similar decreases in protein levels were found for cadherin-11. Connexin-43 protein levels significantly increased at 3 days, but then decreased below control levels, though not significantly. Protein levels of scleraxis and tenomodulin were significantly increased at day 14 and 21, respectively. Taken together, our results indicate that TGFβ2 is an inducer of tendon marker proteins (scleraxis and tenomodulin) in MSCs and that tenogenesis alters the protein levels of N-cadherin, cadherin-11 and connexin-43. These findings suggest a role for connexin-43 early in tenogenesis, and show that early-onset and sustained decreases in N-cadherin and cadherin-11 may be novel markers of tenogenesis in MSCs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Metanephric kidney development is orchestrated by the iterative branching morphogenesis of the ureteric bud. We describe an underlying patterning associated with the ramification of this structure and show that this pattern is conserved between developing kidneys, in different parts of the organ and across developmental time. This regularity is associated with a highly reproducible branching asymmetry that is consistent with locally operative growth mechanisms. We then develop a class of tip state models to represent elaboration of the ureteric tree and describe rules for 'half-delay' branching morphogenesis that describe almost perfectly the patterning of this structure. Spatial analysis suggests that the observed asymmetry may arise from mutual suppression of bifurcation, but not extension, between the growing ureteric tips, and demonstrates that disruption of patterning occurs in mouse mutants in which the distribution of tips on the surface of the kidney is altered. These findings demonstrate that kidney development occurs by way of a highly conserved reiterative pattern of asymmetric bifurcation that is governed by intrinsic and locally operative mechanisms., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

In multiple sclerosis, the pathological interaction between autoreactive Th cells and mononuclear phagocytes in the CNS drives initiation and maintenance of chronic neuroinflammation. Here, we found that intrathecal transplantation of neural stem/precursor cells (NPCs) in mice with experimental autoimmune encephalomyelitis (EAE) impairs the accumulation of inflammatory monocyte-derived cells (MCs) in the CNS, leading to improved clinical outcome. Secretion of IL-23, IL-1, and TNF-α, the cytokines required for terminal differentiation of Th cells, decreased in the CNS of NPC-treated mice, consequently inhibiting the induction of GM-CSF-producing pathogenic Th cells. In vivo and in vitro transcriptome analyses showed that NPC-secreted factors inhibit MC differentiation and activation, favoring the switch toward an antiinflammatory phenotype. Tgfb2-/- NPCs transplanted into EAE mice were ineffective in impairing MC accumulation within the CNS and failed to drive clinical improvement. Moreover, intrathecal delivery of TGF-β2 during the effector phase of EAE ameliorated disease severity. Taken together, these observations identify TGF-β2 as the crucial mediator of NPC immunomodulation. This study provides evidence that intrathecally transplanted NPCs interfere with the CNS-restricted inflammation of EAE by reprogramming infiltrating MCs into antiinflammatory myeloid cells via secretion of TGF-β2.

Because human corneal endothelial cells (HCECs) do not proliferate once the endothelial monolayer has formed, corneal wound healing is believed to be mediated by cell enlargement or migration, rather than by proliferation. However, the cellular mechanisms involved in wound healing by HCECs have not been fully determined. In this review, we focus on the effects of promyelocytic leukemia zinc finger (PLZF), a DNA-binding transcription factor, and transforming growth factor (TGF)-β2 on the proliferation and migration of cultured HCECs. Involvement of the mitogen-activated protein kinase (MAPK) signaling pathway in the migration of HCECs was also investigated. Expression of PLZF mRNA decreased as cell-cell contact was disrupted and returned to the original level as cell-cell contact was re-formed. Assessment with a real-time cell electronic sensing system revealed that proliferation of cultured HCECs was inhibited after infection with Ad-PLZF and exposure to TGF-β2. Migration of cultured HCECs was increased by TGF-β2 through p38 MAPK activation. We conclude that PLZF expression in cultured HCECs is closely related to the formation of cell-cell contact and that TGF-β2 suppresses proliferation of cultured HCECs, while promoting their migration through p38 MAPK activation.

Statins are cholesterol lowering drugs and have shown beneficial effects on glaucoma. With regard to the mechanism of statin action on glaucoma, we investigated the effects of statins on transforming growth factor-beta 2 (TGF-β2)-induced expression of extracellular matrix (ECM) proteins in human astrocytes of the optic nerve head (ONH) lamina cribrosa (LC). By using primary human ONH astrocytes, we found that both simvastatin and lovastatin inhibited TGF-β2-mediated expression of ECM proteins such as connective tissue growth factor, collagen I, fibronectin, and plasminogen activator inhibitor-1. Suppression of ECM related proteins is due to inhibition of Smad2/3 activation as statins inhibit TGF-β2-induced Smad2 phosphorylation and Smad2/3 nuclear accumulation. In ONH astrocytes, TGF-β2 does not induce MAPK activation. In this study we found an anti-fibrotic effect of statins in human astrocytes of the ONH and identified TGF-β2 as a mediator of statin action, which may support a beneficial role for statins in blocking glaucomatous axonal damage induced by ECM remodeling., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Tartrate-resistant acid phosphatase (TRAP/ACP5), a metalloenzyme that is characteristic for its expression in activated osteoclasts and in macrophages, has recently gained considerable focus as a driver of metastasis and was associated with clinically relevant parameters of cancer progression and cancer aggressiveness., Methods: MDA-MB-231 breast cancer cells with different TRAP expression levels (overexpression and knockdown) were generated and characterized for protein expression and activity levels. Functional cell experiments, such as proliferation, migration and invasion assays were performed as well as global phosphoproteomic and proteomic analysis was conducted to connect molecular perturbations to the phenotypic changes., Results: We identified an association between metastasis-related properties of TRAP-overexpressing MDA-MB-231 breast cancer cells and a TRAP-dependent regulation of Transforming growth factor (TGFβ) pathway proteins and Cluster of differentiation 44 (CD44). Overexpression of TRAP increased anchorage-independent and anchorage-dependent cell growth and proliferation, induced a more elongated cellular morphology and promoted cell migration and invasion. Migration was increased in the presence of the extracellular matrix (ECM) proteins osteopontin and fibronectin and the basement membrane proteins collagen IV and laminin I. TRAP-induced properties were reverted upon shRNA-mediated knockdown of TRAP or treatment with the small molecule TRAP inhibitor 5-PNA. Global phosphoproteomics and proteomics analyses identified possible substrates of TRAP phosphatase activity or signaling intermediates and outlined a TRAP-dependent regulation of proteins involved in cell adhesion and ECM organization. Upregulation of TGFβ isoform 2 (TGFβ2), TGFβ receptor type 1 (TβR1) and Mothers against decapentaplegic homolog 2 (SMAD2), as well as increased intracellular phosphorylation of CD44 were identified upon TRAP perturbation. Functional antibody-mediated blocking and chemical inhibition demonstrated that TRAP-dependent migration and proliferation is regulated via TGFβ2/TβR, whereas proliferation beyond basal levels is regulated through CD44., Conclusion: Altogether, TRAP promotes metastasis-related cell properties in MDA-MB-231 breast cancer cells via TGFβ2/TβR and CD44, thereby identifying a potential signaling mechanism associated to TRAP action in breast cancer cells.

The study established enhanced expression of vascular endothelial growth factor (VEGF) in the subpopulation of osteoblasts located in the regeneration region of femoral bone fracture near the titanium implants with bioactive calcium phosphate and hydroxyapatite coatings and suppressed activity of transforming growth factor-β2 (TGF-β2) in chondroblasts during the two weeks after surgery. In the delayed posttraumatic period, the distribution of TGF-β2 inversely related to its maximal activity. The data revealed the up-regulating effect of bioresorbable coatings on expression of VEGF and TGF-β2 and their implication in the control over various stages of reparative osteogenesis.

Purpose: Increased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor β2 (TGFβ2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFβ2 signaling pathways in CLAN formation., Methods: Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGFβ2, with or without the inhibitors of TGFβ receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFβ2 plus inhibitors for 10 days or pretreated with TGFβ2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4',6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett's post hoc test., Results: TGFβ2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFβ receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFβ receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption., Conclusions: TGFβ2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers.

The secondary palate separates the oral from the nasal cavity and its closure during embryonic development is sensitive to genetic perturbations. Mice with deleted Foxf2, encoding a forkhead transcription factor, are born with cleft palate, and an abnormal tongue morphology has been proposed as the underlying cause. Here, we show that Foxf2(-/-) maxillary explants cultured in vitro, in the absence of tongue and mandible, failed to close the secondary palate. Proliferation and collagen content were decreased in Foxf2(-/-) palatal shelf mesenchyme. Phosphorylation of Smad2/3 was reduced in mutant palatal shelf, diagnostic of attenuated canonical Tgfβ signaling, whereas phosphorylation of p38 was increased. The amount of Tgfβ2 protein was diminished, whereas the Tgfb2 mRNA level was unaltered. Expression of several genes encoding extracellular proteins important for Tgfβ signaling were reduced in Foxf2(-)(/)(-) palatal shelves: a fibronectin splice-isoform essential for formation of extracellular Tgfβ latency complexes; Tgfbr3 - or betaglycan - which acts as a co-receptor and an extracellular reservoir of Tgfβ; and integrins αV and β1, which are both Tgfβ targets and required for activation of latent Tgfβ. Decreased proliferation and reduced extracellular matrix content are consistent with diminished Tgfβ signaling. We therefore propose that gene expression changes in palatal shelf mesenchyme that lead to reduced Tgfβ signaling contribute to cleft palate in Foxf2(-)(/)(-) mice., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Transforming growth factor-β induces an epithelial to mesenchymal transition (EMT) in the lens, presented as an aberrant growth and differentiation of lens epithelial cells. Studies in other models of EMT have shown that TGF-β-driven EMT is dependent on the expression of the reactive oxygen species (ROS)-producing enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase-4 (Nox4). We investigate the role of this enzyme in TGF-β-induced lens EMT and determine whether it is required for this pathologic process., Methods: Rat lens epithelial explants were used to investigate the role of Nox4 in TGF-β-driven lens EMT. Nox1-4 expression and localization was determined by immunolabeling and/or RT-PCR. NADPH-oxidase-produced ROS were visualized microscopically using the fluorescent probe, dihydroethidium (DHE). VAS2870, a pan-NADPH oxidase inhibitor, was used to determine the specificity of Nox4 expression and its role in ROS production, and subsequently TGF-β-driven EMT., Results: We demonstrate, for the first time to our knowledge, in rat lens epithelial explants that TGF-β treatment induces Nox4 (but not Nox1-3) expression and activity. Increased Nox4 expression was first detected at 6 to 8 hours following TGF-β treatment and was maintained in explants up to 48 hours. At 8 hours after TGF-β treatment, Nox4 was observed in cell nuclei, while at later stages in the EMT process (at 48 hours), Nox4 was predominately colocalized with α-smooth muscle actin. The inhibition of Nox4 expression and activity using VAS2870 inhibited EMT progression., Conclusions: Transforming growth factor-β drives the expression of the ROS-producing enzyme Nox4 in rat lens epithelial cells and Nox4 inhibition can impede the EMT process.

Primary Open Angle Glaucoma (POAG) is an irreversible, vision-threatening disease that affects millions worldwide. The principal risk factor of POAG is increased intraocular pressure (IOP) due to pathological changes in the trabecular meshwork (TM). The TGFβ signaling pathway activator TGFβ2 and the Wnt signaling pathway inhibitor secreted frizzled-related protein 1 (sFRP1) are elevated in the POAG TM. In this study, we determined whether there is a crosstalk between the TGFβ/Smad pathway and the canonical Wnt pathway using luciferase reporter assays. Lentiviral luciferase reporter vectors for studying the TGFβ/Smad pathway or the canonical Wnt pathway were transduced into primary human non-glaucomatous TM (NTM) cells. Cells were treated with or without a combination of 5 μg/ml TGFβ2 and/or 100 ng/ml Wnt3a recombinant proteins, and luciferase levels were measured using a plate reader. We found that TGFβ2 inhibited Wnt3a-induced canonical Wnt pathway activation, while Wnt3a inhibited TGFβ2-induced TGFβ/Smad pathway activation (n = 6, p < 0.05) in 3 NTM cell strains. We also found that knocking down of Smad4 or β-catenin using siRNA in HTM5 cells transfected with similar luciferase reporter plasmids abolished the inhibitory effect of TGFβ2 and/or Wnt3a on the other pathway (n = 6). Our results suggest the existence of a cross-inhibition between the TGFβ/Smad and canonical Wnt pathways in the TM, and this cross-inhibition may be mediated by Smad4 and β-catenin., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Several growth factors (GFs) have been implicated in long-term memory (LTM), but no single GF can support all of the plastic changes that occur during memory formation. Because GFs engage highly convergent signaling cascades that often mediate similar functional outcomes, the relative contribution of any particular GF to LTM is difficult to ascertain. To explore this question, we determined the unique contribution of distinct GF families (signaling via TrkB and TGF-βr-II) to LTM formation in Aplysia. We demonstrate that TrkB and TGF-βr-II signaling are differentially recruited during two-trial training in both time (by trial 1 or 2, respectively) and space (in distinct subcellular compartments). These GFs independently regulate MAPK activation and synergistically regulate gene expression. We also show that trial 1 TrkB and trial 2 TGF-βr-II signaling are required for LTM formation. These data support the view that GFs engaged in LTM formation are interactive components of a complex molecular network., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cleft palate is a common birth defect affecting 1 in 700 births. Transforming growth factor-βs (TGF-βs) are important signaling molecules, and their functions in murine palate development have received great attention. TGF-β3 is expressed exclusively in palatal epithelial cells and mediates epithelial fusion, whereas the importance of TGF-β1 and 2 in palate have not yet been demonstrated in vivo, since inactivation of Tgf-β1 or Tgf-β2 genes in mice did not reveal significant palate defects. We hypothesized that TGF-β1 and TGF-β2 can compensate each other during palate formation. To test this, we generated Tgf-β1 and Tgf-β2 compound mutant mice and found that approximately 40% of [Tgf-β1(+/-); Tgf-β2(-/-)] compound mutant embryos display cleft palate on C57 background. In addition, 26% of Tgf-β2(-/-) embryos on 129 background, but not in C57 or Black Swiss, displayed cleft palate. TGF-β1 and 2 functions are required for murine palate development in strain-dependent manner., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Endothelial-mesenchymal transition (EndoMT) has been recognized as a key determinant of tumor microenvironment in cancer progression and metastasis. Endothelial cells undergoing EndoMT lose their endothelial markers, acquire the mesenchymal phenotype, and become more invasive with increased migratory abilities. Early stages of esophageal adenocarcinoma (EAC) are characterized by strong microvasculature whose impact in tumor progression remains undefined. Our aim was to determine the role of EndoMT in EAC by investigating the impact of tumor cells on normal primary human esophageal microvascular endothelial cells (HEMEC). HEMEC were either cocultured with OE33 adenocarcinoma cells or treated with IL-1β and transforming growth factor-β2 (TGF-β2) for indicated periods and analyzed for EndoMT-associated changes by real-time PCR, Western blotting, immunofluorescence staining, and functional assays. Additionally, human EAC tissues were investigated for detection of EndoMT-like cells. Our results demonstrate an increased expression of mesenchymal markers [fibroblast-specific protein 1 (FSP1), collagen1α2, vimentin, α-smooth muscle actin (α-SMA), and Snail], decreased expression of endothelial markers [CD31, von Willebrand factor VIII (vWF), and VE-cadherin], and elevated migration ability in HEMEC following coculture with OE33 cells. The EndoMT-related changes were inhibited by IL-1β and TGF-β2 gene silencing in OE33 cells. Recombinant IL-1β and TGF-β2 induced EndoMT in HEMEC. Although the level of VEGF expression was elevated in EndoMT cells, the angiogenic property of these cells was diminished. In vivo, by immunostaining EndoMT-like cells were detected at the invasive front of EAC. Our findings underscore a significant role for EndoMT in EAC and provide new insights into the mechanisms and significance of EndoMT in the context of tumor progression., (Copyright © 2014 the American Physiological Society.)

Background: Transforming growth factor beta 1 (TGFβ1) is a pleiotropic cytokine that contributes to reparative skeletal remodeling by inducing osteoblast proliferation, migration, and angiogenesis. Organic bone matrix is the largest bodily reservoir for latent TGFβ1, and active osteoblasts express cognate receptors for TGFβ1 (TGFβRI and TGFβRII). During malignant osteolysis, TGFβ1 is liberated from eroded bone matrix and promotes local progression of osteotropic solid tumors by its mitogenic and prosurvival activities., Hypothesis: Canine osteosarcoma (OS) cells will possess TGFβ1 signaling machinery. Blockade of TGFβ1 signaling will attenuate pro-tumorigenic activities in OS cells. Naturally occurring primary OS samples will express cognate TGFβ1 receptors; and in dogs with OS, focal malignant osteolysis will contribute to circulating TGFβ1 concentrations., Animals: Thirty-three dogs with appendicular OS., Methods: Expression of TGFβ1 and its cognate receptors, as well as the biologic effects of TGFβ1 blockade, was characterized in OS cells. Ten spontaneous OS samples were characterized for TGFβRI/II expressions by immunohistochemistry. In 33 dogs with OS, plasma TGFβ1 concentrations were quantified and correlated with bone resorption., Results: Canine OS cells secrete TGFβ1, express cognate receptors, and TGFβ1 signaling blockade decreases proliferation, migration, and vascular endothelial growth factor secretion. Naturally occurring OS samples abundantly and uniformly express TGFβRI/II, and in OS-bearing dogs, circulating TGFβ1 concentrations correlate with urine N-telopeptide excretion., Conclusions and Clinical Importance: Canine OS cells possess TGFβ1 signaling machinery, potentially allowing for the establishment of an autocrine and paracrine pro-tumorigenic signaling loop. As such, TGFβ1 inhibitors might impede localized OS progression in dogs., (Copyright © 2014 by the American College of Veterinary Internal Medicine.)

In patients, non-proliferative disseminated tumour cells (DTCs) can persist in the bone marrow (BM) while other organs (such as lung) present growing metastasis. This suggested that the BM might be a metastasis 'restrictive soil' by encoding dormancy-inducing cues in DTCs. Here we show in a head and neck squamous cell carcinoma (HNSCC) model that strong and specific transforming growth factor-β2 (TGF-β2) signalling in the BM activates the MAPK p38α/β, inducing an (ERK/p38)(low) signalling ratio. This results in induction of DEC2/SHARP1 and p27, downregulation of cyclin-dependent kinase 4 (CDK4) and dormancy of malignant DTCs. TGF-β2-induced dormancy required TGF-β receptor-I (TGF-β-RI), TGF-β-RIII and SMAD1/5 activation to induce p27. In lungs, a metastasis 'permissive soil' with low TGF-β2 levels, DTC dormancy was short-lived and followed by metastatic growth. Importantly, systemic inhibition of TGF-β-RI or p38α/β activities awakened dormant DTCs, fuelling multi-organ metastasis. Our work reveals a 'seed and soil' mechanism where TGF-β2 and TGF-β-RIII signalling through p38α/β regulates DTC dormancy and defines restrictive (BM) and permissive (lung) microenvironments for HNSCC metastasis.

Background: Induction of epithelial-mesenchymal transition (EMT) is essential for the metastasis of tumor cells and maintaining their stemness. This study aimed to examine whether endothelial cells, which are most closely located to tumor cells in vivo, play a role in inducing EMT in tumor cells or not., Methods: Concentrated culture medium of bovine aortic endothelial cells (BAECs) was applied to tumor cell lines (A549 and PANC-1) and epithelial cell line (NMuMg). Cadherin conversion, expressions of α-smooth muscle actin and ZO-1, actin fiber formation and cell migration were examined as hallmarks of the induction of EMT in these cell lines. Transforming growth factor β (TGFβ) antibodies were used to neutralize TGFβ1, TGFβ2 and TGFβ3. Expression and release of TGFβ proteins in BAECs as well as in porcine and human endothelial cells were assessed by Western blotting and ELISA, respectively., Results: Conditioned medium of BAEC induced EMT in the examined cell lines. All endothelial cells from various species and locations expressed TGFβ1 and TGFβ2 proteins and much lower level of TGFβ3 protein. Conditioned medium from these endothelial cells contained TGFβ1 and TGFβ2, but TGFβ3 could not be detected. Neutralizing antibody against each of TGFβ1 or TGFβ2 did not reverse endothelium-dependent EMT, but simultaneous neutralization of both TGFβ1 and TGFβ2 completely abolished it., Conclusions: Endothelial cells may play a role in the induction and maintenance of EMT in tumor cells by constitutively releasing TGFβ1 and TGFβ2., General Significance: The present results provide a novel strategy of the inhibition of tumor metastasis by targeting vascular endothelium., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: Posthemorrhagic hydrocephalus (pHC) is a serious complication following subarachnoid hemorrhage (SAH) and intraventricular hemorrhage (IVH). Besides known clinical predictors, different cytokines have drawn attention to the development of chronic hydrocephalus. Transforming growth factor (TGF) β1 and TGF β2 are involved in fibrogenesis, scar formation, cell survival, and tissue differentiation and may play a role in the occurrence of pHC. TGF β1 is stored in platelets in large amount and is released in the cerebrospinal fluid (CSF) after SAH and IVH. Both TGF β1 and TGF β2 can be expressed by various intracranial cells., Methods: TGF β1 and β2 were measured in CSF and blood samples of 42 patients with SAH or IVH with acute hydrocephalus during the first 10 days after ictus. Furthermore, albumin was measured in CSF as an indicator for the amount of blood. Patients were categorized as developing pHC requiring shunt treatment or not-developing pHC within 6 months., Results: After adjusting for age, SAH resulted significantly more often in pHC than did IVH. Plasma levels of TGF β1 showed a marked increase over time, whereas CSF levels of TGF β1 constantly decreased. The time course of TGF β1 and albumin in CSF was paralleled and did not correlate with the development of shunt dependent pHC. Also, TGF β1 plasma concentrations did not correlate with shunt dependent pHC. TGF β2 concentrations in plasma showed stable values over time without any variations. TGF β2 in CSF described a parabolic course with a peak at day 6 after ictus. No correlation was found concerning TGF β2 in plasma or CSF and shunt dependent pHC., Conclusion: TGF β1 in CSF is derived by platelets from the cisternal or ventricular clot. TGF β2 in CSF is derived as a general reaction of traumatized brain tissue. These data do not confirm a crucial role of TGF β1 and TGF β2 release in the development of pHC., (Georg Thieme Verlag KG Stuttgart · New York.)

Tendon formation and repair rely on specific combinations of transcription factors, growth factors, and mechanical parameters that regulate the production and spatial organization of type I collagen. Here, we investigated the function of the zinc finger transcription factor EGR1 in tendon formation, healing, and repair using rodent animal models and mesenchymal stem cells (MSCs). Adult tendons of Egr1-/- mice displayed a deficiency in the expression of tendon genes, including Scx, Col1a1, and Col1a2, and were mechanically weaker compared with their WT littermates. EGR1 was recruited to the Col1a1 and Col2a1 promoters in postnatal mouse tendons in vivo. Egr1 was required for the normal gene response following tendon injury in a mouse model of Achilles tendon healing. Forced Egr1 expression programmed MSCs toward the tendon lineage and promoted the formation of in vitro-engineered tendons from MSCs. The application of EGR1-producing MSCs increased the formation of tendon-like tissues in a rat model of Achilles tendon injury. We provide evidence that the ability of EGR1 to promote tendon differentiation is partially mediated by TGF-β2. This study demonstrates EGR1 involvement in adult tendon formation, healing, and repair and identifies Egr1 as a putative target in tendon repair strategies.

Purpose: There are limited studies on the factors that regulate the processing of TGF-β2 and extracellular matrix (ECM) proteins into their mature form. Bone morphogenic protein 1 (BMP1) is an enzyme responsible for the cleavage and maturation of growth factors and ECM proteins. The purpose of our study was to determine whether cultured human trabecular meshwork (TM) cells express BMP1, BMP1 expression is regulated by TGF-β2, BMP1 is biologically active, and BMP1 regulates LOX activity., Methods: Primary human TM cells were isolated and subjected to quantitative PCR (qPCR) and Western immunoblotting (WB) for BMP1. BMP1 immunolocalization was performed in TM tissues. qPCR was used to determine BMP1 mRNA expression and WB results were used to determine BMP1 protein expression. BMP1 activity was measured in TM cells treated with TGF-β2 or with a combination of TGF-β2/UK383367. Lysyl oxidase (LOX) enzyme activity was evaluated by WB in TM cells treated with BMP1 or with a combination of BMP1/β-aminoprorionitrile (BAPN)., Results: Human TM cells expressed mRNA and protein for BMP1. Exogenous TGF-β2 increased mRNA expression compared to their controls (P<0.05). An ELISA showed TGF-β2-induced BMP1 secretion compared to their controls in all cell strains (P<0.05). Secreted BMP1 stimulated LOX enzymatic activity in TM cells., Conclusions: BMP1 is expressed in the human TM. TGF-β2 induction of BMP1 may be responsible for increased processing of growth factors and ECM proteins into their mature forms, resulting in TM stiffness and resistance to ECM degradation.

Fibrosarcoma is a rare malignant tumor originating from fibroblasts. Transforming growth factor beta 2 (TGFβ2) has been established to regulate processes correlated to fibrosarcoma tumorigenesis. In this study, we investigated the possible participation of syndecan-2 (SDC-2), a cell membrane heparan sulfate (HS) proteoglycan on these TGFβ2 functions. Our results demonstrate that the inhibition of SDC-2 expression by short interfering RNA (siSDC2) abolished TGFβ2-dependent HT1080 cell adhesion (P ≤ 0.01). In parallel, the downregulation of SDC-2 significantly inhibited TGFβ2-induced Smad2 phosphorylation (P ≤ 0.01). The immunoflourescence signal of TGF receptor III as well as its protein expression was decreased in SDC-2-deficient cells. The enhancement of adhesion molecules integrin β1 (P ≤ 0.01) and focal adhesion kinase expression, induced by TGFβ2 treatment (P ≤ 0.001), was markedly inhibited in SDC-2-defficient cells (P ≤ 0.01). Conclusively, the obtained data suggest that SDC-2 modulates TGFβ2 transcriptional regulation via Smad signaling to facilitate fibrosarcoma cell adhesion., (Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.)

Glioblastoma are aggressive astrocytic brain tumours characterized by microvascular proliferation and an abnormal vasculature, giving rise to brain oedema and increased patient morbidity. Here, we have characterized the transcriptome of tumour-associated blood vessels and describe a gene signature clearly associated with pleomorphic, pathologically altered vessels in human glioblastoma (grade IV glioma). We identified 95 genes differentially expressed in glioblastoma vessels, while no significant differences in gene expression were detected between vessels in non-malignant brain and grade II glioma. Differential vascular expression of ANGPT2, CD93, ESM1, ELTD1, FILIP1L and TENC1 in human glioblastoma was validated by immunohistochemistry, using a tissue microarray. Through qPCR analysis of gene induction in primary endothelial cells, we provide evidence that increased VEGF-A and TGFβ2 signalling in the tumour microenvironment is sufficient to invoke many of the changes in gene expression noted in glioblastoma vessels. Notably, we found an enrichment of Smad target genes within the distinct gene signature of glioblastoma vessels and a significant increase of Smad signalling complexes in the vasculature of human glioblastoma in situ. This indicates a key role of TGFβ signalling in regulating vascular phenotype and suggests that, in addition to VEGF-A, TGFβ2 may represent a new target for vascular normalization therapy., (Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Epithelial ovarian cancers (EOCs) often exhibit morphologic features of embryonic Müllerian duct-derived tissue lineages and colonize peritoneal surfaces that overlie connective and adipose tissues. However, the mechanisms that enable EOC cells to readily adapt to the peritoneal environment are poorly understood. In this study, we show that expression of HOXA9, a Müllerian-patterning gene, is strongly associated with poor outcomes in patients with EOC and in mouse xenograft models of EOC. Whereas HOXA9 promoted EOC growth in vivo, HOXA9 did not stimulate autonomous tumor cell growth in vitro. On the other hand, expression of HOXA9 in EOC cells induced normal peritoneal fibroblasts to express markers of cancer-associated fibroblasts (CAFs) and to stimulate growth of EOC and endothelial cells. Similarly, expression of HOXA9 in EOC cells induced normal adipose- and bone marrow-derived mesenchymal stem cells (MSCs) to acquire features of CAFs. These effects of HOXA9 were due in substantial part to its transcriptional activation of the gene encoding TGF-β2 that acted in a paracrine manner on peritoneal fibroblasts and MSCs to induce CXCL12, IL-6, and VEGF-A expression. These results indicate that HOXA9 expression in EOC cells promotes a microenvironment that is permissive for tumor growth.

Purpose: Analysis of aqueous humor from patients with primary open-angle glaucoma (POAG) revealed marked increases in the content of endothelin-1 (ET-1) and transforming growth factor-beta (TGF-β). We determined the consequences of TGF-β signaling on ET-1 expression and secretion by human trabecular meshwork (TM) cells., Methods: Primary or transformed (NTM5 and GTM3) human TM cells conditioned in serum-free media were incubated in the absence or presence of TGF-β1 or -β2. Relative changes in preproendothelin (ppET)-1 mRNA content and secreted ET-1 peptide were quantified by real-time PCR and ELISA, respectively. In some experiments, TGF-β or ET-1 receptor antagonists, or Rho G-protein inhibitors, were evaluated for effects on TGF-β signaling. Filamentous actin organization was visualized by phalloidin., Results: Primary or transformed human TM cells cultured in the presence of TGF-β1 or -β2 exhibit a marked (>8-fold) increase in ppET-1 mRNA content compared to vehicle controls. Coincubation with SB-505124, an inhibitor of TGFβRI/ALK-5 signaling, prevented TGF-β-mediated ppET-1 mRNA expression. In contrast, coincubation with ET(A) (BQ-123) or ET(B) (BQ-788) receptor antagonists had no effect on TGF-β-mediated ppET-1 mRNA expression. TGF-β1 and -β2 each elicited a robust (>7-fold) secretion of ET-1 while enhancing stress fiber organization. Inhibition of Rho signaling attenuated TGF-β-mediated increases in ppET-1 mRNA content, ET-1 secretion, and stress fiber organization., Conclusions: TGF-β, signaling through the TGFβRI/ALK-5 receptor, elicits marked increases in ET-1 mRNA content and ET-1 secretion from cultured primary or transformed human TM cells. Elevated levels of TGF-β2 present in AH of POAG patients may elevate intraocular pressure, in part, by eliciting aberrant Rho G-protein dependent cell contraction, and increasing ET-1 synthesis and secretion, in human TM cells.

While low nephron number is associated with increased risk of developing cardiovascular and renal disease, the functional consequences of a high nephron number are unknown. We tested the hypothesis that a high nephron number provides protection against hypertensive and renal insults. Mean arterial pressure (MAP) and renal function were characterized in male wild-type (WT) and transforming growth factor-β2 heterozygous (Tgfb2(+/-)) mice under basal conditions and following a chronic high-salt diet. Kidneys were collected for unbiased stereological analysis. Baseline MAP and renal function were indistinguishable between genotypes. The chronic high-salt diet (5% NaCl for 4 wk followed by 8% NaCl for 4 wk) led to similar step-wise increases in urine volume, Na(+) excretion, and albuminuria in the genotypes. The 5% NaCl diet induced modest and similar increases in MAP (3.5 ± 1.6 and 3.4 ± 0.8 mmHg in WT and Tgfb2(+/-), respectively). After the step up to the 8% NaCl diet, MAP increased further in WT (+15.9 ± 5.1 mmHg), but not Tgfb2(+/-) (-0.1 ± 1.0 mmHg), mice. Nephron number was 30% greater in Tgfb2(+/-) than WT mice and was not affected by the chronic high-salt diet. Mean glomerular volume was lower in Tgfb2(+/-) than WT mice, and the chronic high-salt diet induced significant glomerular hypertrophy. In a separate cohort of mice, an acute, 7-day, 8% NaCl diet induced similar rises in MAP in the genotypes. This is the first study to examine the physiological characteristics of a model of high nephron number, and the findings are consistent with this phenotype providing protection against chronic, but not acute, hypertensive insults.

The cervix is central to the female genital tract immune response to pathogens and foreign male Ags introduced at coitus. Seminal fluid profoundly influences cervical immune function, inducing proinflammatory cytokine synthesis and leukocyte recruitment. In this study, human Ect1 cervical epithelial cells and primary cervical cells were used to investigate agents in human seminal plasma that induce a proinflammatory response. TGF-β1, TGF-β2, and TGF-β3 are abundant in seminal plasma, and Affymetrix microarray revealed that TGF-β3 elicits changes in Ect1 cell expression of several proinflammatory cytokine and chemokine genes, replicating principal aspects of the Ect1 response to seminal plasma. The differentially expressed genes included several induced in the physiological response of the cervix to seminal fluid in vivo. Notably, all three TGF-β isoforms showed comparable ability to induce Ect1 cell expression of mRNA and protein for GM-CSF and IL-6, and TGF-β induced a similar IL-6 and GM-CSF response in primary cervical epithelial cells. TGF-β neutralizing Abs, receptor antagonists, and signaling inhibitors ablated seminal plasma induction of GM-CSF and IL-6, but did not alter IL-8, CCL2 (MCP-1), CCL20 (MIP-3α), or IL-1α production. Several other cytokines present in seminal plasma did not elicit Ect1 cell responses. These data identify all three TGF-β isoforms as key agents in seminal plasma that signal induction of proinflammatory cytokine synthesis in cervical cells. Our findings suggest that TGF-β in the male partner's seminal fluid may influence cervical immune function after coitus in women, and potentially be a determinant of fertility, as well as defense from infection.

An early event in heart valve formation is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endothelial cells in specific regions of the heart tube, the endocardial cushions. The Type III TGFβ receptor (TGFβR3) is required for TGFβ2- or BMP-2-stimulated EMT in atrioventricular endocardial cushion (AVC) explants in vitro but the mediators downstream of TGFβR3 are not well described. Using AVC and ventricular explants as an in vitro assay, we found an absolute requirement for specific TGFβR3 cytoplasmic residues, GAIP-interacting protein, C terminus (GIPC), and specific Activin Receptor-Like Kinases (ALK)s for TGFβR3-mediated EMT when stimulated by TGFβ2 or BMP-2. The introduction of TGFβR3 into nontransforming ventricular endocardial cells, followed by the addition of either TGFβ2 or BMP-2, results in EMT. TGFβR3 lacking the entire cytoplasmic domain, or only the 3C-terminal amino acids that are required to bind GIPC, fails to support EMT in response to TGFβ2 or BMP-2. Overexpression of GIPC in AVC endocardial cells enhanced EMT while siRNA-mediated silencing of GIPC in ventricular cells overexpressing TGFβR3 significantly inhibited EMT. Targeting of specific ALKs by siRNA revealed that TGFβR3-mediated EMT requires ALK2 and ALK3, in addition to ALK5, but not ALK4 or ALK6. Taken together, these data identify GIPC, ALK2, ALK3, and ALK5 as signaling components required for TGFβR3-mediated endothelial cell EMT., (Copyright © 2011. Published by Elsevier Inc.)

Purpose: The aqueous humor (AH) contains numerous immunosuppressive molecules that contribute to the ocular immune privilege. Here, we mimic an inflammatory environment to analyze the inhibitory effects of the AH on lipopolysaccharide (LPS)-induced maturation of dendritic cells (DC)., Methods: Different concentrations of AH were added to dendritic cell cultures together with LPS. Dendritic cell surface markers CD80, CD86, and MHC-II were assessed by use of flow cytometry. Endocytic capability and mixed lymphocyte reaction were measured as functional maturation., Results: AH inhibited LPS-induced DC maturation, resulting in down-regulated expression of CD80, CD86, MHC-II, enhancement of endocytic capacity, and reduced T cell activation. Neutralizing transforming growth factor beta 2 (TGF-β(2)) in AH can totally reverse the inhibitory effect. Treatment with prostaglandin E2 (PGE(2)) antagonist alone had no effect on DC maturation. However, blocking of both TGF-β(2) and PGE(2) in the AH resulted in synergistic suppression of the inhibiting effect of AH., Conclusions: These results reveal that TGF-β(2) in the AH is of crucial importance in maintaining DC in the immature state. Further experiments will clarify the immune role of PGE(2) in AH.

In patients with primary open angle glaucoma (POAG), the optic nerve head (ONH) shows characteristic cupping correlated with visual field defects. The progressive optic neuropathy is characterized by irreversible loss of retinal ganglion cells (RGC). The critical risk factor for axonal damage at the ONH is an elevated intraocular pressure (IOP). The increase in IOP correlates with axonal loss in the ONH, which might be due to an impaired axoplasmatic flow leading to the loss of RGCs. Damage to the optic nerve is thought to occur in the lamina cribrosa (LC) region of the ONH, which is composed of characteristic sieve-like connective tissue cribriform plates through which RGC axons exit the eye. The cupping of the optic disc, and the compression and excavation of LC are characteristic signs of glaucomatous ONH remodelling. In ONH of POAG patients a disorganized distribution and deposition of elastic fibers and a typical pronounced thickening of the connective tissue septae surrounding the optic nerve fibers is found. Transforming growth factor (TGF)-β2 could be one of the pathogenic factors responsible for the structural alterations in POAG patients as the TGF-β2 levels in the ONH of glaucomatous eyes are elevated as well as in the aqueous homour. TGF-β2 leads to an increased synthesis of extracellular matrix (ECM) molecules mediated by connective tissue growth factor and to an impaired ECM degradation in cultured ONH astrocytes. Bone morphogenetic protein (BMP)-4 effectively antagonizes the effects of TGF-β2 on matrix deposition. The BMP antagonist gremlin blocks this inhibition, allowing TGF-β2 stimulation of ECM synthesis. Overall, the ECM in the ONH is kept in balance in the OHN by factors that augment or block the activity of TGF-β2., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Renal dysplasia, defined by defective ureteric branching morphogenesis and nephrogenesis, is the major cause of renal failure in infants and children. Here, we define a pathogenic role for a β-catenin-activated genetic pathway in murine renal dysplasia. Stabilization of β-catenin in the ureteric cell lineage before the onset of kidney development increased β-catenin levels and caused renal aplasia or severe hypodysplasia. Analysis of gene expression in the dysplastic tissue identified downregulation of genes required for ureteric branching and upregulation of Tgfβ2 and Dkk1. Treatment of wild-type kidney explants with TGFβ2 or DKK1 generated morphogenetic phenotypes strikingly similar to those observed in mutant kidney tissue. Stabilization of β-catenin after the onset of kidney development also caused dysplasia and upregulation of Tgfβ2 and Dkk1 in the epithelium. Together, these results demonstrate that elevation of β-catenin levels during kidney development causes dysplasia., (Copyright © 2011 by the American Society of Nephrology)

TGFβ signaling cascade plays a vital role in neoplastic transformation, but the function of betaglycan, which is a TGFβ accessory receptor, is still unknown in particular cancer. Evaluation of betaglycan expression both at mRNA (real-time PCR) and protein (ELISA) level in the context of TGFβ canonical signaling components, i.e., TGFβ1, TGFβ2, and TGFβRII, in endometrial carcinomas was performed. Betaglycan mRNA expression level was significantly (p < .001) downregulated with simultaneous betaglycan protein level upregulation in cancer samples. Obtained results suggest that endometrial cancer is associated with disruption of accessory receptor betaglycan expression, what may alter TGFβ2-induced signaling.

Aqueous humor (AqH) has been shown to have significant immunosuppressive effects on APCs in animal models. We wanted to establish whether, in humans, AqH can regulate dendritic cell (DC) function and to identify the dominant mechanism involved. Human AqH inhibited the capacity of human peripheral blood monocyte-derived DC to induce naive CD4(+) T cell proliferation and cytokine production in vitro, associated with a reduction in DC expression of the costimulatory molecule CD86. This was seen both for DC cultured under noninflammatory conditions (immature DC) and for DC stimulated by proinflammatory cytokines (mature DC). DC expression of MHC classes I/II and CD83 was reduced (mature DC only). Myeloid DC from peripheral blood were similarly sensitive to the effects of human AqH, but only under inflammatory conditions. The addition of α-melanocyte stimulating hormone and vasoactive intestinal peptide did not cause significant inhibition at physiological levels. However, the addition of exogenous cortisol at physiological levels recapitulated the AqH-induced reduction in CD86 and inhibition of DC-induced T cell proliferation, and blockade of cortisol in AqH partially reversed its suppressive effects. TGF-β2 had an additional effect with cortisol, and although simultaneous blockade of cortisol and TGF-β2 in AqH reduced its effectiveness, there was still a cortisol- and TGF-β-independent component. In humans, AqH regulates DC maturation and function by the combined actions of cortisol and TGF-β2, a pathway that is likely to contribute to the maintenance of immune privilege in the eye.

Cardiac valve formation is crucial for embryonic and adult heart function. Valve malformations constitute the most common congenital cardiac defect, but little is known about the molecular mechanisms regulating valve formation and homeostasis. Here, we show that endocardial Notch1 and myocardial Bmp2 signal integration establish a valve-forming field between 2 chamber developmental domains. Patterning occurs through the activation of endocardial epithelial-to-mesenchymal transition (EMT) exclusively in prospective valve territories. Mice with constitutive endocardial Notch1 activity ectopically express Hey1 and Heyl. They also display an activated mesenchymal gene program in ventricles and a partial (noninvasive) EMT in vitro that becomes invasive upon BMP2 treatment. Snail1, TGF-β2, or Notch1 inhibition reduces BMP2-induced ventricular transformation and invasion, whereas BMP2 treatment inhibits endothelial Gsk3β, stabilizing Snail1 and promoting invasiveness. Integration of Notch and Bmp2 signals is consistent with Notch1 signaling being attenuated after myocardial Bmp2 deletion. Notch1 activation in myocardium extends Hey1 expression to nonchamber myocardium, represses Bmp2, and impairs EMT. In contrast, Notch deletion abrogates endocardial Hey gene transcription and extends Bmp2 expression to the ventricular endocardium. This embryonic Notch1-Bmp2-Snail1 relationship may be relevant in adult valve disease, in which decreased NOTCH signaling causes valve mesenchyme cell formation, fibrosis, and calcification.

The mechanisms underlying age-associated thymic involution are unknown. In mice, thymic involution shows mouse strain-dependent genetic variation. Identification of the underlying genes would provide mechanistic insight into this elusive process. We previously showed that responsiveness of hematopoietic stem and progenitor cells (HSPCs) to transforming growth factor-beta 2, a positive regulator of HSPC proliferation, is regulated by a quantitative trait locus (QTL) on chr. 4, Tb2r1. Interestingly, Tgfb2(+/-) mice have delayed thymic involution. Therefore, we tested the hypothesis that a QTL on chr. 4 might regulate thymic involution. Aged, but not young, B6.D2-chr.4 congenic mice, where the telomeric region of chr. 4 was introgressed from DBA/2 to C57BL/6 mice, had larger thymi, and better maintenance of early thymic precursors than C57BL/6 control mice. These observations unequivocally demonstrate that the telomeric region of chr. 4 contains a QTL, Ti1 (thymic involution 1) that regulates thymic involution, and suggest the possibility that Ti1 may be identical to Tb2r1.

Reproductive biomedicine has made significant advances in the area of assisted reproductive technologies in the last two and half decades. However, embryo implantation remains a major obstacle in securing high pregnancy rates. Various non-human primate models including rhesus, marmoset and baboon have been employed to elucidate in vivo mechanisms underlying the uterine events that initiate, sustain and complete implantation. This review collates the information available on the molecular profile of gestational endometrium in primates. Collectively, these studies reveal dynamic spatio-temporal changes in the expression of cytokines, growth factors, cell-adhesion molecules, cytoskeleton elements and other factors in the endometrium during the post-implantation phase of pregnancy. Considering that the endometrial events during the pre-implantation stages of pregnancy may dictate implantation success, we have developed a bonnet monkey (Macaca radiata) model where pregnancy can be detected at the pre-implantation stage. Using this model, we investigated some of the endometrial events that occur before the completion of implantation. Remarkable changes were observed in endometrial expression of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFalpha), as well as expression of immunosuppressive factors such as transforming growth factor beta-2 (TGFbeta2), interleukin-6 (IL-6) and placental protein-14 (PP-14), even before the embryo starts invading the endometrium. This highlights the super-imposition of endometrial receptivity by embryonic stimuli, marked by differential expression and/or localization of the factors that regulate endometrial transformation for embryo survival, growth and development.

Increased abundance of mucin secretory cells is a characteristic feature of the epithelium in asthma and other chronic airway diseases. We showed previously that the mechanical stresses of airway constriction, both in the intact mouse lung and a cell culture model, activate the epidermal growth factor receptor (EGFR), a known modulator of mucin expression in airway epithelial cells. Here we tested whether chronic, intermittent, short-duration compressive stress (30 cm H(2)O) is sufficient to increase the abundance of MUC5AC-positive cells and intracellular mucin levels in human bronchial epithelial cells cultured at an air-liquid interface. Compressive stress applied for 1 hour per day for 14 days significantly increased the percentage of cells staining positively for MUC5AC protein (22.0 +/- 3.8%, mean +/- SD) relative to unstimulated controls (8.6 +/- 2.6%), and similarly changed intracellular MUC5AC protein levels measured by Western and slot blotting. The effect of compressive stress was gradual, with significant changes in MUC5AC-positive cell numbers evident by Day 7, but required as little as 10 minutes of compressive stress daily. Daily treatment of cells with an EGFR kinase inhibitor (AG1478, 1 muM) significantly but incompletely attenuated the response to compressive stress. Complete attenuation could be accomplished by simultaneous treatment with the combination of AG1478 and a transforming growth factor (TGF)-beta(2) (1 microg/ml)-neutralizing antibody, or with anti-TGF-beta(2) alone. Our findings demonstrate that short duration episodes of mechanical stress, representative of those occurring during bronchoconstriction, are sufficient to increase goblet cell number and MUC5AC protein expression in bronchial epithelial cells in vitro. We propose that the mechanical environment present in asthma may fundamentally bias the composition of airway epithelial lining in favor of mucin secretory cells.

Background: During the estrous cycle, the rat uterine endometrium undergoes many changes such as cell proliferation and apoptosis. If implantation occurs, stromal cells differentiate into decidual cells and near the end of pregnancy, a second wave of apoptosis occurs. This process called decidual regression, is tightly regulated as is it crucial for successful pregnancy. We have previously shown that TGF-beta1, TGF-beta2 and TGF-beta3 are expressed in the endometrium during decidual basalis regression, but although we had demonstrated that TGF- beta1 was involved in the regulation of apoptosis in decidual cells, the ability of TGF- beta2 and TGF-beta3 isoforms to trigger apoptotic mechanisms in these cells remains unknown. Moreover, we hypothesized that the TGF-betas were also present and regulated in the non-pregnant endometrium during the estrous cycle. The aim of the present study was to determine and compare the specific effect of each TGF-beta isoform in the regulation of apoptosis in sensitized endometrial stromal cells in vitro, and to investigate the regulation of TGF-beta isoforms in the endometrium during the estrous cycle in vivo., Methods: Rats with regular estrous cycle (4 days) were killed at different days of estrous cycle (diestrus, proestrus, estrus and metestrus). Pseudopregnancy was induced with sex steroids in ovariectomized rats and rats were killed at different days (days 1-9). Uteri were collected and either fixed for immunohistochemical staining (IHC) or processed for RT-PCR and Western analyses. For the in vitro part of the study, rats were ovariectomized and decidualization was induced using sex steroids. Endometrial stromal decidual cells were purified, cultured and treated with different concentrations of TGF-beta isoforms., Results: Our results showed that all three TGF-beta isoforms are present, but are localized differently in the endometrium during the estrous cycle and their expression is regulated differently during pseudopregnancy. In cultured stromal cells, we found that TGF-beta3 isoform induced Smad2 phosphorylation, indicating that the Smad pathway is activated by TGF-beta3 in these cells. Furthermore, TGF-beta2 and TGF-beta3 induced a dose-dependant increase of apoptosis in cultured stromal cells, as demonstrated by Hoechst nuclear staining. Noteworthy, TGF-beta2 and TGF-beta3 reduced the level of the anti-apoptotic XIAP protein, as well as the level of phosphorylated/active Akt, a well known survival protein, in a dose-dependent manner., Conclusion: Those results suggest that TGF-beta might play an important role in the remodelling endometrium during the estrous cycle and in the regulation of apoptosis in rat decidual cells, in which inhibition of Akt survival pathway might be an important mechanism involved in the regulation of apoptosis.

Aim: To address the cellular components and the contractile mechanisms of the idiopathic epiretinal membrane (ERM)., Methods: Ten surgically removed ERMs were fixed in 4% paraformaldehyde and analysed by whole-mount immunohistochemistry with anti-glial fibrillar acidic protein (GFAP) and alpha smooth-muscle actin (alphaSMA) antibodies. Type I collagen gel contraction assay, an established wound-healing assay in vitro, was performed using cultured bovine hyalocytes or normal human astrocytes (NHA) to evaluate the contractile property of the cells in the presence of tissue growth factor (TGF)-beta2. The expression of alphaSMA was also analysed by western blot analysis to examine myofibroblastic transdifferentiation of the cells. Vitreous-induced collagen gel contraction was also evaluated., Results: All membranes were composed of alphaSMA immunopositive cells in contracted foci and GFAP immunopositive cells in the periphery. No apparent double positive cells were observed in any membranes examined. Cultured hyalocytes showed overexpression of alphaSMA and hypercontraction of collagen gels in response to TGF-beta2, while glial cells showed marginal change. The vitreous from ERM patients also caused overexpression of alphaSMA and hypercontraction of the gels embedding hyalocytes, which were almost completely inhibited in the presence of anti-TGF-beta2 neutralising antibody., Conclusions: Hyalocytes might be one of the critical components of ERM mediating its contractile property through the effect of TGF-beta2 in the vitreous fluid.

During human embryogenesis, neural crest cells migrate to the anterior chamber of the eye and then differentiate into the inner layers of the cornea, the iridocorneal angle, and the anterior portion of the iris. When proper development does not occur, this causes iridocorneal angle dysgenesis and intraocular pressure (IOP) elevation, which ultimately results in developmental glaucoma. Here, we show that heparan sulfate (HS) deficiency in mouse neural crest cells causes anterior chamber dysgenesis, including corneal endothelium defects, corneal stroma hypoplasia, and iridocorneal angle dysgenesis. These dysfunctions are phenotypes of the human developmental glaucoma, Peters anomaly. In the neural crest cells of mice embryos, disruption of the gene encoding exostosin 1 (Ext1), which is an indispensable enzyme for HS synthesis, resulted in disturbed TGF-beta2 signaling. This led to reduced phosphorylation of Smad2 and downregulated expression of forkhead box C1 (Foxc1) and paired-like homeodomain transcription factor 2 (Pitx2), transcription factors that have been identified as the causative genes for developmental glaucoma. Furthermore, impaired interactions between HS and TGF-beta2 induced developmental glaucoma, which was manifested as an IOP elevation caused by iridocorneal angle dysgenesis. These findings suggest that HS is necessary for neural crest cells to form the anterior chamber via TGF-beta2 signaling. Disturbances of HS synthesis might therefore contribute to the pathology of developmental glaucoma.

Microglia, the immune cells of the CNS, play essential roles in both physiological and pathological brain states. Here we have used an in vitro model to demonstrate neuroprotection of a 48 h-microglial conditioned medium (MCM) towards cerebellar granule neurons (CGNs) challenged with the neurotoxin 6-hydroxydopamine, which induces a Parkinson-like neurodegeneration, and to identify the protective factor(s). MCM nearly completely protects CGNs from 6-hydroxydopamine neurotoxicity and at least some of the protective factor(s) are peptidic in nature. While the fraction of the medium containing molecules < 30 kDa completely protects CGNs, fractions containing molecules < 10 kDa or > 10 kDa are not neuroprotective. We further demonstrate that microglia release high amounts of transforming growth factor-beta2 (TGF-beta2) and that its exogenous addition to the fraction of the medium not containing it (< 10 kDa) fully restores the neuroprotective action. Moreover, MCM neuroprotection is significantly counteracted by an inhibitor of TGF-beta2 transduction pathway. Our results identify TGF-beta2 as an essential neuroprotective factor released by microglia in its culture medium that requires to be fully effective the concomitant presence of other factor(s) of low molecular weight.

The Arf tumor suppressor (also known as Cdkn2a) acts as an oncogene sensor induced by ;abnormal' mitogenic signals in incipient cancer cells. It also plays a crucial role in embryonic development: newborn mice lacking Arf are blind due to a pathological process resembling severe persistent hyperplastic primary vitreous (PHPV), a human eye disease. The cell-intrinsic mechanism implied in the oncogene sensor model seems unlikely to explain Arf regulation during embryo development. Instead, transforming growth factor beta2 (Tgfbeta2) might control Arf expression, as we show that mice lacking Tgfbeta2 have primary vitreous hyperplasia similar to Arf(-/-) mice. Consistent with a potential linear pathway, Tgfbeta2 induces Arf transcription and p19(Arf) expression in cultured mouse embryo fibroblasts (MEFs); and Tgfbeta2-dependent cell cycle arrest in MEFs is maintained in an Arf-dependent manner. Using a new model in which Arf expression can be tracked by beta-galactosidase activity in Arf(lacZ/+) mice, we show that Tgfbeta2 is required for Arf transcription in the developing vitreous as well as in the cornea and the umbilical arteries, two previously unrecognized sites of Arf expression. Chemical and genetic strategies show that Arf promoter induction depends on Tgfbeta receptor activation of Smad proteins; the induction correlates with Smad2 phosphorylation in MEFs and Arf-expressing cells in vivo. Chromatin immunoprecipitation shows that Smads bind to genomic DNA proximal to Arf exon 1beta. In summary, Tgfbeta2 and p19(Arf) act in a linear pathway during embryonic development. We present the first evidence that p19(Arf) expression can be coupled to extracellular cues in normal cells and suggest a new mechanism for Arf control in tumor cells.

The ligand specificity of transforming growth factor beta (TGFbeta) in vivo in mouse cardiac cushion epithelial-to-mesenchymal transition (EMT) is poorly understood. To elucidate the function of TGFbeta in cushion EMT, we analyzed Tgfb1(-/-), Tgfb2(-/-), and Tgfb3(-/-) mice between embryonic day (E) 9.5 and E14.5 using both in vitro and in vivo approaches. Atrioventricular (AV) canal collagen gel assays at E9.5 indicated normal EMT in both Tgfb1(-/-) and Tgfb3(-/-) mice. However, analysis of Tgfb2(-/-) AV explants at E9.5 and E10.5 indicated that EMT, but not cushion cell proliferation, was initially delayed but later remained persistent. This was concordant with the observation that Tgfb2(-/-) embryos, and not Tgfb1(-/-) or Tgfb3(-/-) embryos, develop enlarged cushions at E14.5 with elevated levels of well-validated indicators of EMT. Collectively, these data indicate that TGFbeta2, and not TGFbeta1 or TGFbeta3, mediates cardiac cushion EMT by promoting both the initiation and cessation of EMT.