ALK1 regulates the internalization of endoglin and the type III TGF-ß receptor.

Complex formation and endocytosis of transforming growth factor-ß (TGF-ß) receptors play important roles in signaling. However, their interdependence remained unexplored. Here, we demonstrate that ALK1, a TGF-ß type I receptor prevalent in endothelial cells, forms stable complexes at the cell surface with endoglin and with type III TGF-ß receptors (TßRIII). We show that ALK1 undergoes clathrin-mediated endocytosis (CME) faster than ALK5, type II TGF-ß receptor (TßRII), endoglin, or TßRIII. These complexes regulate the endocytosis of the TGF-ß receptors, with a major effect mediated by ALK1. Thus, ALK1 enhances the endocytosis of TßRIII and endoglin, while ALK5 and TßRII mildly enhance endoglin, but not TßRIII, internalization. Conversely, the slowly endocytosed endoglin has no effect on the endocytosis of either ALK1, ALK5, or TßRII, while TßRIII has a differential effect, slowing the internalization of ALK5 and TßRII, but not ALK1. Such effects may be relevant to signaling, as BMP9-mediated Smad1/5/8 phosphorylation is inhibited by CME blockade in endothelial cells. We propose a model that links TGF-ß receptor oligomerization and endocytosis, based on which endocytosis signals are exposed/functional in specific receptor complexes. This has broad implications for signaling, implying that complex formation among various receptors regulates their surface levels and signaling intensities.

TGF-beta and TNF-alpha cooperatively induce mesenchymal transition of lymphatic endothelial cells via activation of Activin signals.

Lymphatic systems play important roles in the maintenance of fluid homeostasis and undergo anatomical and physiological changes during inflammation and aging. While lymphatic endothelial cells (LECs) undergo mesenchymal transition in response to transforming growth factor-ß (TGF-ß), the molecular mechanisms underlying endothelial-to-mesenchymal transition (EndMT) of LECs remain largely unknown. In this study, we examined the effect of TGF-ß2 and tumor necrosis factor-α (TNF-α), an inflammatory cytokine, on EndMT using human skin-derived lymphatic endothelial cells (HDLECs). TGF-ß2-treated HDLECs showed increased expression of SM22α, a mesenchymal cell marker accompanied by increased cell motility and vascular permeability, suggesting HDLECs to undergo EndMT. Our data also revealed that TNF-α could enhance TGF-ß2-induced EndMT of HDLECs. Furthermore, both cytokines induced the production of Activin A while decreasing the expression of its inhibitory molecule Follistatin, and thus enhancing EndMT. Finally, we demonstrated that human dermal lymphatic vessels underwent EndMT during aging, characterized by double immunostaining for LYVE1 and SM22α. These results suggest that both TGF-ß and TNF-α signals play a central role in EndMT of LECs and could be potential targets for senile edema.

Alteration of Transforming Growth Factor ß Signaling Pathway Predicts Worse Prognosis in Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: Transforming growth factor ß (TGF-ß) signaling pathway is one of the core pathways in pancreatic ductal adenocarcinoma (PDAC). Prognostic value of TGF-ß pathway genes as a functionally related group in PDAC is rarely studied. METHODS: Seventy-two PDAC patients who underwent surgery between November 30, 2015, and September 13, 2017, in West China Hospital, Sichuan University, were identified and included in this study. Whole-exome sequencing or targeted next-generation sequencing was performed with tumor tissue. Clinicopathologic characteristics and survival data were retrospectively collected and analyzed. RESULTS: Genetic alterations were detected in 71 patients (98.6%). Although 1 patient (1.4%) had one genetic alteration, 33 patients (45.8%) had 2 to 4 alterations and 37 patients (51.4%) had 5 or more alterations. Twenty-five patients with TGF-ß pathway alteration were identified as TGF-ßm+ group. Other 47 patients were TGF-ßm- group. Mutation of TGF-ß pathway was independently associated with inferior survival (hazard ratio, 2.22, 95% confidence interval, 1.05-4.70, P = 0.04), especially in patients accepting radical surgery (hazard ratio, 3.25, 95% confidence interval, 1.01-10.49, P = 0.04). CONCLUSIONS: Inferior prognosis was observed in PDACs with mutations of TGF-ß pathway. Genomic information could help screen out patients at risk after surgery, and adjuvant therapy might benefit this subgroup of PDACs.

Transforming Growth Factor-ß Signaling in Immunity and Cancer.

Transforming growth factor (TGF)-ß is a crucial enforcer of immune homeostasis and tolerance, inhibiting the expansion and function of many components of the immune system. Perturbations in TGF-ß signaling underlie inflammatory diseases and promote tumor emergence. TGF-ß is also central to immune suppression within the tumor microenvironment, and recent studies have revealed roles in tumor immune evasion and poor responses to cancer immunotherapy. Here, we present an overview of the complex biology of the TGF-ß family and its context-dependent nature. Then, focusing on cancer, we discuss the roles of TGF-ß signaling in distinct immune cell types and how this knowledge is being leveraged to unleash the immune system against the tumor.

Embryonic Stem Cell-Derived mmu-miR-291a-3p Inhibits Cellular Senescence in Human Dermal Fibroblasts Through the TGF-ß Receptor 2 Pathway.

Senescent cells accumulate in various tissues over time and contribute to tissue dysfunction and aging-associated phenotypes. Accumulating evidence suggests that cellular senescence can be inhibited through pharmacological intervention, as well as through treatment with soluble factors derived from embryonic stem cells (ESCs). In an attempt to investigate the anti-senescence factors secreted by ESCs, we analyzed mouse ESC-derived extracellular microRNAs in conditioned medium via microRNA array analysis. We selected mmu-miR-291a-3p as a putative anti-senescence factor via bioinformatics analysis. We validated its inhibitory effects on replicative, Adriamycin-induced, and ionizing radiation-induced senescence in human dermal fibroblasts. Treatment of senescent cells with mmu-miR-291a-3p decreased senescence-associated ß-galactosidase activity, enhanced proliferative potential, and reduced mRNA and protein expression of TGF-ß receptor 2, p53, and p21. mmu-miR-291a-3p in conditioned medium was enclosed in ESC-derived exosomes and exosomes purified from ESC conditioned medium inhibited cellular senescence. The inhibitory effects of mmu-miR-291a-3p were mediated through the TGF-ß receptor 2 signaling pathway. Hsa-miR-371a-3p and hsa-miR-520e, the human homologs of mmu-miR-291a-3p, showed similar anti-senescence activity. Furthermore, mmu-miR-291a-3p accelerated the excisional skin wound healing process in aged mice. Our results indicate that the ESC-derived mmu-miR-291a-3p is a novel candidate agent that can be utilized for cell-free therapeutic intervention against aging and aging-related diseases.

Betaglycan (TGFBR3) Functions as an Inhibin A, but Not Inhibin B, Coreceptor in Pituitary Gonadotrope Cells in Mice.

Inhibins are gonadal hormones that act on pituitary gonadotrope cells to suppress FSH synthesis and secretion. Inhibin A and B are heterodimers of the inhibin ⍺-subunit disulfide-linked to one of two inhibin ß-subunits. Homodimers or heterodimers of the inhibin ß-subunits form the activins, which stimulate FSH production. Activins signal through complexes of type I and II receptor serine/threonine kinases to increase transcription of the FSHß subunit gene. According to in vitro observations, inhibins impair FSH synthesis by competitively binding to activin type II receptors, particularly in the presence of the TGFß type III receptor (TGFBR3, or betaglycan). The role of TGFBR3 in inhibin action in vivo has not been determined. Here, we ablated Tgfbr3 specifically in murine gonadotropes. Conditional knockout females were supra-fertile, exhibiting enhanced folliculogenesis, numbers of ovulated eggs per cycle, and litter sizes relative to control mice. Despite these phenotypes, FSH levels appeared to be unaltered in knockout mice, and the mechanisms underlying their enhanced fertility remain unexplained. Inhibin B is the predominant form of the hormone in males and in females during most stages of the estrous cycle. Remarkably, inhibin A, but not inhibin B, suppression of FSH synthesis was impaired in cultured pituitaries of knockout mice, which may explain the absence of discernible changes in FSH levels in vivo. Collectively, these data challenge current dogma by demonstrating that TGFBR3 (betaglycan) functions as an inhibin A, but not an inhibin B, coreceptor in gonadotrope cells in vivo. Mechanisms of inhibin B action merit further investigation.

Specification of Drosophila neuropeptidergic neurons by the splicing component brr2.

During embryonic development, a number of genetic cues act to generate neuronal diversity. While intrinsic transcriptional cascades are well-known to control neuronal sub-type cell fate, the target cells can also provide critical input to specific neuronal cell fates. Such signals, denoted retrograde signals, are known to provide critical survival cues for neurons, but have also been found to trigger terminal differentiation of neurons. One salient example of such target-derived instructive signals pertains to the specification of the Drosophila FMRFamide neuropeptide neurons, the Tv4 neurons of the ventral nerve cord. Tv4 neurons receive a BMP signal from their target cells, which acts as the final trigger to activate the FMRFa gene. A recent FMRFa-eGFP genetic screen identified several genes involved in Tv4 specification, two of which encode components of the U5 subunit of the spliceosome: brr2 (l(3)72Ab) and Prp8. In this study, we focus on the role of RNA processing during target-derived signaling. We found that brr2 and Prp8 play crucial roles in controlling the expression of the FMRFa neuropeptide specifically in six neurons of the VNC (Tv4 neurons). Detailed analysis of brr2 revealed that this control is executed by two independent mechanisms, both of which are required for the activation of the BMP retrograde signaling pathway in Tv4 neurons: (1) Proper axonal pathfinding to the target tissue in order to receive the BMP ligand. (2) Proper RNA splicing of two genes in the BMP pathway: the thickveins (tkv) gene, encoding a BMP receptor subunit, and the Medea gene, encoding a co-Smad. These results reveal involvement of specific RNA processing in diversifying neuronal identity within the central nervous system.

Identification and functional analysis of transforming growth factor-ß type I receptor (TßR1) from Scylla paramamosain: The first evidence of TßR1 involved in development and innate immunity in crustaceans.

The transforming growth factor-ß (TGF-ß) receptor-mediated TGF-ß signaling cascade plays important roles in diverse cellular processes, including cell proliferation, differentiation, growth, apoptosis and inflammation in vertebrates. In the present study, the type I TGF-ß receptor (TßR1) was firstly identified and characterized in mud crab Scylla paramamosain. The full-length cDNA of SpTßR1 was 1, 986 bp with a 1, 608 bp open reading frame, which encoded a putative protein of 535 amino acids including a typical transmembrane region, a conserved glycine-serine (GS) motif and a S_TKc domain (Serine/Threonine protein kinases, catalytic domain). Real-time PCR analysis showed that SpTßR1 was predominantly expressed at early embryonic development stage and was highly expressed at postmolt stages during molt cycle, suggesting its participation in development and growth. Moreover, the expression levels of SpTßR1 in hepatopancreas and hemocytes were positively induced after the challenges of Vibro alginolyticus and Poly (I:C), indicating the involvement of SpTßR1 in responding to both bacterial and viral infections. The in vivo RNA interference assays demonstrated that the expression levels of two NF-κB members (SpRelish and SpDorsal) and six antimicrobial peptide (AMP) genes (SpCrustin and SpALF2-6) were significantly suppressed when the SpTßR1 was silenced. Additionally, the expression levels of SpTßR1, SpRelish, SpDorsal and AMPs were consistently down-regulated or up-regulated when the primary cultured hemocytes were treated with TßR1 antagonist or agonist for 24 h. These results indicated that TßR1 not only contributed to the crabs' development and growth but also played vital role in the innate immunity of S. paramamosain, and it also provided new insights into the origin or evolution of TGF-ß receptors in crustacean species and even in invertebrates.

Transcriptional and translational abundance of Bone morphogenetic protein (BMP) 2, 4, 6, 7 and their receptors BMPR1A, 1B and BMPR2 in buffalo ovarian follicle and the role of BMP4 and BMP7 on estrogen production and survival of cultured granulosa cells.

BMPs and their receptors modulate the granulosa cell (GC) function in the follicle of domestic animals. Since little is known on BMPs in the buffalo, the present study was aimed to investigate the expression of BMP2, 4, 6, 7 and their receptors BMPR1A, BMPR1B, BMPR2 in the GC and theca cells (TC) of ovarian follicles and the role of BMP4 and BMP7 on buffalo GC. Follicles were classified into four groups based on size and E2 level in the follicular fluid as follows: (i) Group1(4-6 mm; <0.5 ng/mL) (ii) Group 2 (7-9 mm; 0.5-5 ng/mL) (iii) Group 3 (10-13 mm; 5-40 ng/mL) and (iv) Group 4 (dominant follicle) (>13 mm; >180 ng/mL). The results revealed that except BMP6, BMP2, 4 7 and receptors BMPR1A, BMPR1B and BMPR2 showed a minimum of 1.5-2 fold increase in mRNA expression in the GC of dominant follicle as compared to other follicle classes. In the dominant follicle, a two-fold increase in BMP4 and BMP7 expression was observed in the TC. At 100 ng/mL, the BMP4 and BMP7 either alone or in combination maximally down-regulated CASPASE3 and stimulated the transcripts of PCNA, FSHR and CYP19A1 that was supported by E2 secretion in the granulosa cell culture suggesting their role in cell survival and E2 production. In conclusion, GC and TC of dominant follicles express BMP 2, 4, 6, 7 and their receptors BMPR1A, BMPR1B and BMPR2. BMP4 and BMP7 stimulate E2 production and promote GC survival.

Pericyte ALK5/TIMP3 Axis Contributes to Endothelial Morphogenesis in the Developing Brain.

The murine embryonic blood-brain barrier (BBB) consists of endothelial cells (ECs), pericytes (PCs), and basement membrane. Although PCs are critical for inducing vascular stability, signaling pathways in PCs that regulate EC morphogenesis during BBB development remain unexplored. Herein, we find that murine embryos lacking the transforming growth factor ß (TGF-ß) receptor activin receptor-like kinase 5 (Alk5) in brain PCs (mutants) develop gross germinal matrix hemorrhage-intraventricular hemorrhage (GMH-IVH). The germinal matrix (GM) is a highly vascularized structure rich in neuronal and glial precursors. We show that GM microvessels of mutants display abnormal dilation, reduced PC coverage, EC hyperproliferation, reduced basement membrane collagen, and enhanced perivascular matrix metalloproteinase activity. Furthermore, ALK5-depleted PCs downregulate tissue inhibitor of matrix metalloproteinase 3 (TIMP3), and TIMP3 administration to mutants improves endothelial morphogenesis and attenuates GMH-IVH. Overall, our findings reveal a key role for PC ALK5 in regulating brain endothelial morphogenesis and a substantial therapeutic potential for TIMP3 during GMH-IVH.

γ-Secretase inhibitor reduces immunosuppressive cells and enhances tumour immunity in head and neck squamous cell carcinoma.

Immune evasion is a hallmark feature of cancer, and it plays an important role in tumour initiation and progression. In addition, tumour immune evasion severely hampers the desired antitumour effect in multiple cancers. In this study, we aimed to investigate the role of the Notch pathway in immune evasion in the head and neck squamous cell carcinoma (HNSCC) microenvironment. We first demonstrated that Notch1 signaling was activated in a Tgfbr1/Pten-knockout HNSCC mouse model. Notch signaling inhibition using a γ-secretase inhibitor (GSI-IX, DAPT) decreased tumour burden in the mouse model after prophylactic treatment. In addition, flow cytometry analysis indicated that Notch signaling inhibition reduced the sub-population of myeloid-derived suppressor cells (MDSCs), tumour-associated macrophages (TAMs) and regulatory T cells (Tregs), as well as immune checkpoint molecules (PD1, CTLA4, TIM3 and LAG3), in the circulation and in the tumour. Immunohistochemistry (IHC) of human HNSCC tissues demonstrated that elevation of the Notch1 downstream target HES1 was correlated with MDSC, TAM and Treg markers and with immune checkpoint molecules. These results suggest that modulating the Notch signaling pathway may decrease MDSCs, TAMs, Tregs and immune checkpoint molecules in HNSCC.

The Cytokine TGF-ß Promotes the Development and Homeostasis of Alveolar Macrophages.

Alveolar macrophages (AMs) derive from fetal liver monocytes, which colonize the lung during embryonic development and give rise to fully mature AMs perinatally. AM differentiation requires granulocyte macrophage colony-stimulating factor (GM-CSF), but whether additional factors are involved in AM regulation is not known. Here we report that AMs, in contrast to most other tissue macrophages, were also dependent on transforming growth factor-ß receptor (TGF-ßR) signaling. Conditional deletion of TGF-ßR in mice at different time points halted the development and differentiation of AMs. In adult mice, TGF-ß was also critical for AM homeostasis. The source of TGF-ß was AMs themselves, indicative of an autocrine loop that promotes AM self-maintenance. Mechanistically, TGF-ßR signaling resulted in upregulation of PPAR-γ, a signature transcription factor essential for the development of AMs. These findings reveal an additional layer of complexity regarding the guidance cues, which govern the genesis, maturation, and survival of AMs.

Cbfß2 deficiency preserves Langerhans cell precursors by lack of selective TGFß receptor signaling.

The mouse Langerhans cell (LC) network is established through the differentiation of embryonic LC precursors. BMP7 and TGFß1 initiate cellular signaling that is essential for inducing LC differentiation and preserving LCs in a quiescent state, respectively. Here we show that loss of Cbfß2, one of two RNA splice variants of the Cbfb gene, results in long-term persistence of embryonic LC precursors after their developmental arrest at the transition into the EpCAM+ stage. This phenotype is caused by selective loss of BMP7-mediated signaling essential for LC differentiation, whereas TGFßR signaling is intact, maintaining cells in a quiescent state. Transgenic Cbfß2 expression at the neonatal stage, but not at the adult stage, restored differentiation from Cbfß2-deficient LC precursors. Loss of developmental potential in skin-residential precursor cells was accompanied by diminished BMP7-BMPR1A signaling. Collectively, our results reveal an essential requirement for the Cbfß2 variant in LC differentiation and provide novel insight into how the establishment and homeostasis of the LC network is regulated.

Overactivation of hedgehog signaling in the developing Müllerian duct interferes with duct regression in males and causes subfertility.

The influence of the hedgehog signaling pathway on reproduction was studied in transgenic mice in which a dominant active allele of the hedgehog signal transducer, smoothened (Smo), was conditionally expressed in the developing Müllerian duct and gonads through recombination mediated by anti-Müllerian hormone receptor 2-cre (Amhr2cre ). Previous studies showed that development of the oviduct and uterus are abnormal in female Amhr2cre/+SmoM2 mice. In the current study, focusing on mutant males, litter size was reduced 53% in crosses with wild-type females. An extra band of undifferentiated tissue extended along each epididymis and vas deferens, a position suggesting derivation from Müllerian ducts that failed to regress fully. Hedgehog signaling was elevated in this tissue, based on mRNA levels of target genes. Amhr2 mRNA was dramatically reduced in the uterus of mutant females and in the extra tissue in the tract of mutant males, suggesting that AMHR2 signaling was inadequate for complete Müllerian duct regression. Spermatogenesis and sperm motility were normal, but testis weight was reduced 37% and epididymal sperm number was reduced 36%. The number of sperm recovered from the uteri of wild-type females after mating with mutant males was reduced 78%. This suggested that sperm transport through the male tract was reduced, resulting in fewer sperm in the ejaculate. Consistent with this, mutant males had unusually tortuous vas deferentia with constrictions within the lumen. We concluded that persistence of a relatively undifferentiated remnant of Müllerian tissue is sufficient to cause subtle changes in the male reproductive tract that reduce fertility.

Transforming growth factor beta 1 increases collagen content, and stimulates procollagen I and tissue inhibitor of metalloproteinase-1 production of dental pulp cells: Role of MEK/ERK and activin receptor-like kinase-5/Smad signaling.

BACKGROUND/PURPOSE: In order to clarify the role of transforming growth factor beta 1 (TGF-ß1) in pulp repair/regeneration responses, we investigated the differential signaling pathways responsible for the effects of TGF-ß1 on collagen turnover, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinase-1 (TIMP-1) production in human dental pulp cells. METHODS: Pulp cells were exposed to TGF-ß1 with/without pretreatment and coincubation by 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenyl mercapto)butadiene (U0126; a mitogen-activated protein kinase kinase [MEK]/extracellular signal-regulated kinase [ERK] inhibitor) and 4-(5-benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H- imidazol-2-yl)-benzamide hydrate (SB431542; an activin receptor-like kinase-5/Smad signaling inhibitor). Sircol collagen assay was used to measure cellular collagen content. Culture medium procollagen I, TIMP-1, and MMP-3 levels were determined by enzyme-linked immunosorbent assay. RESULTS: TGF-ß1 increased the collagen content, procollagen I, and TIMP-1 production, but slightly decreased MMP-3 production of pulp cells. SB431542 and U0126 prevented the TGF-ß1-induced increase of collagen content and TIMP-1 production of dental pulp cells. CONCLUSION: These results indicate that TGF-ß1 may be involved in the healing/regeneration processes of dental pulp in response to injury by stimulation of collagen and TIMP-1 production. These events are associated with activin receptor-like kinase-5/Smad2/3 and MEK/ERK signaling.

TGFßR1 Blockade with Galunisertib (LY2157299) Enhances Anti-Neuroblastoma Activity of the Anti-GD2 Antibody Dinutuximab (ch14.18) with Natural Killer Cells.

PURPOSE: Immunotherapy of high-risk neuroblastoma using the anti-GD2 antibody dinutuximab induces antibody-dependent cell-mediated cytotoxicity (ADCC). Galunisertib, an inhibitor of TGFßR1, was examined for its ability to enhance the efficacy of dinutuximab in combination with human ex vivo activated NK (aNK) cells against neuroblastoma. EXPERIMENTAL DESIGN: TGFB1 and TGFBR1 mRNA expression was determined for 249 primary neuroblastoma tumors by microarray analysis. The ability of galunisertib to inhibit SMAD activity induced by neuroblastoma patient blood and bone marrow plasmas in neuroblastoma cells was tested. The impact of galunisertib on TGFß1-induced inhibition of aNK cytotoxicity and ADCC in vitro and on anti-neuroblastoma activity in NOD-scid gamma (NSG) mice was determined. RESULTS: Neuroblastomas express TGFB1 and TGFBR1 mRNA. Galunisertib suppressed SMAD activation in neuroblastoma cells induced by exogenous TGFß1 or by patient blood and bone marrow plasma, and suppressed SMAD2 phosphorylation in human neuroblastoma cells growing in NSG mice. In NK cells treated in vitro with exogenous TGFß1, galunisertib suppressed SMAD2 phosphorylation and restored the expression of DNAM-1, NKp30, and NKG2D cytotoxicity receptors and the TRAIL death ligand, the release of perforin and granzyme A, and the direct cytotoxicity and ADCC of aNK cells against neuroblastoma cells. Addition of galunisertib to adoptive cell therapy with aNK cells plus dinutuximab reduced tumor growth and increased survival of mice injected with two neuroblastoma cell lines or a patient-derived xenograft. CONCLUSIONS: Galunisertib suppresses activation of SMAD2 in neuroblastomas and aNK cells, restores NK cytotoxic mechanisms, and increases the efficacy of dinutuximab with aNK cells against neuroblastoma tumors. Clin Cancer Res; 23(3); 804-13. ©2016 AACRSee related commentary by Zenarruzabeitia et al., p. 615.

TGF-ß receptor maintains CD4 T helper cell identity during chronic viral infections.

Suppression of CD8 and CD4 T cells is a hallmark in chronic viral infections, including hepatitis C and HIV. While multiple pathways are known to inhibit CD8 T cells, the host molecules that restrict CD4 T cell responses are less understood. Here, we used inducible and CD4 T cell-specific deletion of the gene encoding the TGF-ß receptor during chronic lymphocytic choriomeningitis virus infection in mice, and determined that TGF-ß signaling restricted proliferation and terminal differentiation of antiviral CD4 T cells. TGF-ß signaling also inhibited a cytotoxic program that includes granzymes and perforin expression at both early and late stages of infection in vivo and repressed the transcription factor eomesodermin. Overexpression of eomesodermin was sufficient to recapitulate in great part the phenotype of TGF-ß receptor-deficient CD4 T cells, while SMAD4 was necessary for CD4 T cell accumulation and differentiation. TGF-ß signaling also restricted accumulation and differentiation of CD4 T cells and reduced the expression of cytotoxic molecules in mice and humans infected with other persistent viruses. These data uncovered an eomesodermin-driven CD4 T cell program that is continuously suppressed by TGF-ß signaling. During chronic viral infection, this program limits CD4 T cell responses while maintaining CD4 T helper cell identity.

Geometric analysis of pathways dynamics: Application to versatility of TGF-ß receptors.

We propose a new geometric approach to describe the qualitative dynamics of chemical reactions networks. By this method we identify metastable regimes, defined as low dimensional regions of the phase space close to which the dynamics is much slower compared to the rest of the phase space. These metastable regimes depend on the network topology and on the orders of magnitude of the kinetic parameters. Benchmarking of the method on a computational biology model repository suggests that the number of metastable regimes is sub-exponential in the number of variables and equations. The dynamics of the network can be described as a sequence of jumps from one metastable regime to another. We show that a geometrically computed connectivity graph restricts the set of possible jumps. We also provide finite state machine (Markov chain) models for such dynamic changes. Applied to signal transduction models, our approach unravels dynamical and functional capacities of signalling pathways, as well as parameters responsible for specificity of the pathway response. In particular, for a model of TGFß signalling, we find that the ratio of TGFBR2 to TGFBR1 receptors concentrations can be used to discriminate between metastable regimes. Using expression data from the NCI60 panel of human tumor cell lines, we show that aggressive and non-aggressive tumour cell lines function in different metastable regimes and can be distinguished by measuring the relative concentrations of receptors of the two types.

Effects of transforming growth factor on the developing embryonic ureter: An in-vitro megaureter model in mice.

INTRODUCTION: It is generally agreed that the cause of a megaureter is narrowing at the vesicoureteral junction, with a functional obstruction arising from an aperistaltic, juxtavesical segment that is unable to transport urine at an acceptable rate. Histological examinations of megaureter specimens have reported several histological analyses, and the pathogenic role of transforming growth factor is still a matter of speculation. OBJECTIVE: To evaluate whether transforming growth factor-beta (TGF-ß) and its receptors (TGFRs) are expressed during ureterovesical junction (UVJ) and lower ureter development in mice, and whether exogenous TGF-ß might postpone the maturation of smooth muscle cells, in the pathogenesis of megaureter using an embryonic organ-culture model. METHODS: Expression of TGF-ß and TGFRs on the lower ureter and UVJ were determined at different embryonic days (E) (E16, 18, 20 and postnatal day 1). The functional studies were performed by harvesting ureters from wild-type mice at embryonic day 16 (E16), which were grown in serum-free organ-culture; some cultures were supplemented with TGF-ß (2 and 20 ng/ml) and/or with soluble TGFR, which blocks bioactivity. Organs were harvested after 6 days and the expression of CD31 and Ki67 were assessed using immunohistochemistry. The muscle content of the UVJ and ureter were analyzed by flowcytometry. RESULTS: The TGF-ß and TGFR positive cells were immune detected in embryonic ureters. The TGF-ß expression was highest on E18 and decreased postnatally. Exogenous TGF-ß decreased ureterovesical (UV) muscle differentiation and proliferation. The longitudinal muscle fibers were significantly less in TGF-ß explants. The TGF-ß also decreased the proportions of cells expressing α smooth muscle actin (α-SMA). Soluble TGFR blocked the effects of exogenous TGF-ß. CONCLUSIONS: In organ culture, exogenous TGF-ß postpones the UV smooth muscle proliferation and affects the muscular structure. Whether the effects of TGF-ß are direct or indirect, these form an in-vitro megaureter model. The finding that TGF-ß is highest in embryonic ureters in vivo and decreased postnatally suggests that a pathological persistence might potentially explain the pathogenesis of primary megaureters.

Foxf2 is required for secondary palate development and Tgfß signaling in palatal shelf mesenchyme.

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 Tgfß signaling, whereas phosphorylation of p38 was increased. The amount of Tgfß2 protein was diminished, whereas the Tgfb2 mRNA level was unaltered. Expression of several genes encoding extracellular proteins important for Tgfß signaling were reduced in Foxf2(-)(/)(-) palatal shelves: a fibronectin splice-isoform essential for formation of extracellular Tgfß latency complexes; Tgfbr3 - or betaglycan - which acts as a co-receptor and an extracellular reservoir of Tgfß; and integrins αV and ß1, which are both Tgfß targets and required for activation of latent Tgfß. Decreased proliferation and reduced extracellular matrix content are consistent with diminished Tgfß signaling. We therefore propose that gene expression changes in palatal shelf mesenchyme that lead to reduced Tgfß signaling contribute to cleft palate in Foxf2(-)(/)(-) mice.