Mathematical modeling identifies Smad nucleocytoplasmic shuttling as a dynamic signal-interpreting system

TGF-beta-induced Smad signal transduction from the membrane into the nucleus is not linear and unidirectional, but rather a dynamic network that couples Smad phosphorylation and dephosphorylation through continuous nucleocytoplasmic shuttling of Smads. To understand the quantitative behavior of this network, we have developed a tightly constrained computational model, exploiting the interplay between mathematical modeling and experimental strategies. The model simultaneously reproduces four distinct datasets with excellent accuracy and provides mechanistic insights into how the network operates. We use the model to make predictions about the outcome of fluorescence recovery after photobleaching experiments and the behavior of a functionally impaired Smad2 mutant, which we then verify experimentally. Successful model performance strongly supports the hypothesis of a dynamic maintenance of Smad nuclear accumulation during active signaling. The presented work establishes Smad nucleocytoplasmic shuttling as a dynamic network that flexibly transmits quantitative features of the extracellular TGF-beta signal, such as its duration and intensity, into the nucleus.     

Activin B receptor ALK7 is a negative regulator of pancreatic beta-cell function

All major cell types in pancreatic islets express the transforming growth factor (TGF)-beta superfamily receptor ALK7, but the physiological function of this receptor has been unknown. Mutant mice lacking ALK7 showed normal pancreas organogenesis but developed an age-dependent syndrome involving progressive hyperinsulinemia, reduced insulin sensitivity, liver steatosis, impaired glucose tolerance, and islet enlargement. Hyperinsulinemia preceded the development of any other defect, indicating that this may be one primary consequence of the lack of ALK7. In agreement with this, mutant islets showed enhanced insulin secretion under sustained glucose stimulation, indicating that ALK7 negatively regulates glucose-stimulated insulin release in beta-cells. Glucose increased expression of ALK7 and its ligand activin B in islets, but decreased that of activin A, which does not signal through ALK7. The two activins had opposite effects on Ca(2+) signaling in islet cells, with activin A increasing, but activin B decreasing, glucose-stimulated Ca(2+) influx. On its own, activin B had no effect on WT cells, but stimulated Ca(2+) influx in cells lacking ALK7. In accordance with this, mutant mice lacking activin B showed hyperinsulinemia comparable with that of Alk7(-/-) mice, but double mutants showed no additive effects, suggesting that ALK7 and activin B function in a common pathway to regulate insulin secretion. These findings uncover an unexpected antagonism between activins A and B in the control of Ca(2+) signaling in beta-cells. We propose that ALK7 plays an important role in regulating the functional plasticity of pancreatic islets, negatively affecting beta-cell function by mediating the effects of activin B on Ca(2+) signaling.     

Growth differentiation factor 9:bone morphogenetic protein 15 heterodimers are potent regulators of ovarian functions

The TGF-β superfamily is the largest family of secreted proteins in mammals, and members of the TGF-β family are involved in most developmental and physiological processes. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), oocyte-secreted paralogs of the TGF-β superfamily, have been shown genetically to control ovarian physiology. Although previous studies found that GDF9 and BMP15 homodimers can modulate ovarian pathways in vitro, the functional species-specific significance of GDF9:BMP15 heterodimers remained unresolved. Therefore, we engineered and produced purified recombinant mouse and human GDF9 and BMP15 homodimers and GDF9:BMP15 heterodimers to compare their molecular characteristics and physiological functions. In mouse granulosa cell and cumulus cell expansion assays, mouse GDF9 and human BMP15 homodimers can up-regulate cumulus expansion-related genes (Ptx3, Has2, and Ptgs2) and promote cumulus expansion in vitro, whereas mouse BMP15 and human GDF9 homodimers are essentially inactive. However, we discovered that mouse GDF9:BMP15 heterodimer is ∼10- to 30-fold more biopotent than mouse GDF9 homodimer, and human GDF9:BMP15 heterodimer is ∼1,000- to 3,000-fold more bioactive than human BMP15 homodimer. We also demonstrate that the heterodimers require the kinase activities of ALK4/5/7 and BMPR2 to activate SMAD2/3 but unexpectedly need ALK6 as a coreceptor in the signaling complex in granulosa cells. Our findings that GDF9:BMP15 heterodimers are the most bioactive ligands in mice and humans compared with homodimers explain many puzzling genetic and physiological data generated during the last two decades and have important implications for improving female fertility in mammals.Ligands of the TGF-β superfamily, the largest family of secreted proteins in mammals, are synthesized as dimers and bind transmembrane type 1 and type 2 serine-threonine kinase receptors to activate downstream signaling cascades (e.g., the SMADs) in many developmental, physiological, and pathophysiological processes (1, 2). Growth differentiation factor 9 (GDF9) and bone morphogenic protein 15 (BMP15) are key oocyte-secreted members of the TGF-β superfamily and can regulate female fertility in several mammals (2, 3). Although GDF9 and BMP15 are closely related paralogs, they have been shown in vitro to signal through divergent SMAD2/3 and SMAD1/5/8 pathways, respectively (4–6).By studying gene knockouts and mutant models, putative roles of GDF9 and BMP15 in female reproduction have been described in mice, sheep, and humans. Our group previously discovered that Gdf9-null female mice are sterile (7), and Gdf9^+/−Bmp15^−/− double-mutant mice had more severe fertility defects than subfertile Bmp15^−/− mice (8, 9). BMP15 or GDF9 heterozygous mutant sheep have increased litter size, whereas homozygous mutants are sterile and phenocopy Gdf9^−/− mice (10, 11). In humans, mutations in GDF9 and BMP15 have been associated with premature ovarian failure and dizygotic twinning (12–14). These data suggest synergistic functions of the two gene products and potential species-specific differences in the bioactivity of these proteins. Although an in vitro study has detected the GDF9:BMP15 heterodimer by immunoprecipitation (15), and cooperative effects of the two homodimers were studied by other groups (16–18), the functions of GDF9:BMP15 heterodimers in any species remain largely unknown.In the present study, we demonstrate that GDF9:BMP15 heterodimers are the most bioactive ligands in the regulation of cumulus expansion genes. These heterodimers signal through a unique BMP receptor type 2 (BMPR2)-ALK4/5/7-ALK6 receptor complex to induce the phosphorylation of SMAD2/3 in human and mouse granulosa cells. Our findings open up prospects for the understanding of the synergistic roles of GDF9 and BMP15 proteins in ovarian functions and have important implications for improving female reproductive productivity in mammals.     

Perturbation of transforming growth factor (TGF)-ß1 association with latent TGF-β binding protein yields inflammation and tumors

Transforming growth factor-β (TGF-β) activity is controlled at many levels including the conversion of the latent secreted form to its active state. TGF-β is often released as part of an inactive tripartite complex consisting of TGF-β, the TGF-β propeptide, and a molecule of latent TGF-β binding protein (LTBP). The interaction of TGF-β and its cleaved propeptide renders the growth factor latent, and the liberation of TGF-β from this state is crucial for signaling. To examine the contribution of LTBP to TGF-β function, we generated mice in which the cysteines that link the propeptide to LTBP were mutated to serines, thereby blocking covalent association. Tgfb1^C33S/C33S mice had multiorgan inflammation, lack of skin Langerhans cells (LC), and a shortened lifespan, consistent with decreased TGF-β1 levels. However, the inflammatory response and decreased lifespan were not as severe as observed with Tgfb1^−/− animals. Tgfb1^C33S/C33S mice exhibited decreased levels of active TGF-β1, decreased TGF-β signaling, and tumors of the stomach, rectum, and anus. These data suggest that the association of LTBP with the latent TGF-β complex is important for proper TGF-β1 function and that Tgfb1^C33S/C33S mice are hypomorphs for active TGF-β1. Moreover, although mechanisms exist to activate latent TGF-β1 in the absence of LTBP, these mechanisms are not as efficient as those that use the latent complex containing LTBP.     

Stromal growth and epithelial cell proliferation in ventral prostates of liver X receptor knockout mice

With specific liver X receptor α and β (LXRα and LXRβ) antibodies, we found that LXRα is strongly expressed in the luminal and basal cells of prostatic epithelium. The ventral prostates (VP) of LXRα^−/− mice are characterized by the presence of smooth-muscle actin-positive stromal overgrowth around the prostatic ducts and by numerous fibrous nodules pushing into the ducts and causing obstruction, so that most of the ducts were extremely dilated. BrdU labeling and Ki67 staining revealed epithelial and stromal proliferation in the fibrous nodules. However, the dense stroma surrounding the ducts was not positive for proliferation markers. There was no detectable difference between WT and LXRα^−/− mice VP in the expression of the androgen receptor, but there was an increase in nuclear expression of Snail and Smad 2/3, indicating enhanced TGF-β signaling. Upon treatment of WT mice for 3 months with the LXR agonist T2320 or for 3 weeks with β-sitosterol, LXRα was downregulated, and a VP phenotype similar to that of LXRα^−/− mice resulted. We conclude that in rodents, LXRα seems to control VP stromal growth and that LXRα^−/− mice may be a useful model to study prostatic stromal hyperplasia. Because LXRα is expressed in the epithelium, the excessive stromal growth in LXRα^−/− mice indicates that LXRα is essential for epithelial stromal communication.     

Dissecting the involvement of tropomyosin-related kinase A and p75 neurotrophin receptor signaling in NGF deficit-induced neurodegeneration

NGF, the principal neurotrophic factor for basal forebrain cholinergic neurons (BFCNs), has been correlated to Alzheimer''s disease (AD) because of the selective vulnerability of BFCNs in AD. These correlative links do not substantiate a comprehensive cause–effect mechanism connecting NGF deficit to overall AD neurodegeneration. A demonstration that neutralizing NGF activity could have consequences beyond a direct interference with the cholinergic system came from studies in the AD11 mouse model, in which the expression of a highly specific anti-NGF antibody determines a neurodegeneration that encompasses several features of human AD. Because the transgenic antibody binds to mature NGF much more strongly than to proNGF and prevents binding of mature NGF to the tropomyosin-related kinase A (TrkA) receptor and to p75 neurotrophin receptor (p75NTR), we postulated that neurodegeneration in AD11 mice is provoked by an imbalance of proNGF/NGF signaling and, consequently, of TrkA/p75NTR signaling. To test this hypothesis, in this study we characterize the phenotype of two lines of transgenic mice, one in which TrkA signaling is inhibited by neutralizing anti-TrkA antibodies and a second one in which anti-NGF mice were crossed to p75NTR^{exonIII(−/−)} mice to abrogate p75NTR signaling. TrkA neutralization determines a strong cholinergic deficit and the appearance of β-amyloid peptide (Aβ) but no tau-related pathology. In contrast, abrogating p75NTR signaling determines a full rescue of the cholinergic and Aβ phenotype of anti-NGF mice, but tau hyperphosphorylation is exacerbated. Thus, we demonstrate that inhibiting TrkA signaling activates Aβ accumulation and that different streams of AD neurodegeneration are related in complex ways to TrkA versus p75NTR signaling.     

An engineered selenocysteine defines a unique class of antibody derivatives

Selenocysteine is cotranslationally inserted into proteins by recoding the stop codon UGA from termination to selenocysteine insertion. The nucleophilic selenol group of selenocysteine endows this rare amino acid with unique chemical reactivity that allows regiospecific covalent conjugation in the presence of the other natural amino acids. Using a mammalian expression system, we generated an IgG1-derived Fc fragment with a C-terminal selenocysteine in yields comparable to conventional monoclonal antibodies and conjugated it to an electrophilic derivative of a peptidomimetic that binds with high affinity and specificity to integrin alpha(4)beta(1). Through this conjugation, both the biological and chemical components are endowed with pharmacological advantages. We demonstrate that whereas the Fc protein increases the circulatory half-life from minutes to days and mediates transcytosis through binding to the neonatal Fc receptor, the peptidomimetic introduces cross-species binding to cell surface integrin alpha(4)beta(1) and blocks its interaction with vascular cell adhesion molecule-1. Compared with conventional monoclonal antibodies, our technology benefits economically from combining a generic biological component with a variable chemical component.     

Structural and biochemical studies identify tobacco SABP2 as a methyl salicylate esterase and implicate it in plant innate immunity

Salicylic acid (SA) is a critical signal for the activation of plant defense responses against pathogen infections. We recently identified SA-binding protein 2 (SABP2) from tobacco as a protein that displays high affinity for SA and plays a crucial role in the activation of systemic acquired resistance to plant pathogens. Here we report the crystal structures of SABP2, alone and in complex with SA at up to 2.1-A resolution. The structures confirm that SABP2 is a member of the alpha/beta hydrolase superfamily of enzymes, with Ser-81, His-238, and Asp-210 as the catalytic triad. SA is bound in the active site and is completely shielded from the solvent, consistent with the high affinity of this compound for SABP2. Our biochemical studies reveal that SABP2 has strong esterase activity with methyl salicylate as the substrate, and that SA is a potent product inhibitor of this catalysis. Modeling of SABP2 with MeSA in the active site is consistent with all these biochemical observations. Our results suggest that SABP2 may be required to convert MeSA to SA as part of the signal transduction pathways that activate systemic acquired resistance and perhaps local defense responses as well.     

Posttranslational processing of mouse and human BMP-15: potential implication in the determination of ovulation quota

There has been significant attention to the growing recognition that oocytes have a critical capacity to organize and govern surrounding somatic cells. Bone morphogenetic protein 15 (BMP-15) is an oocyte-secreted factor that has raised particular interest due to its established role in determining ovulation quota and female fertility in mammals. As a first step in determining whether there are species-specific differences in the BMP-15 system that may play causal roles in the differences in ovulation quota observed in different mammalian species, we here compare the molecular characteristics of BMP-15 of polyovulatory mice with that of monoovulatory humans. We found that, although human BMP-15 mature protein is readily produced, there are defects in the production of mouse BMP-15 mature protein in an in vitro system of transfected cells. The generation of chimeric constructs consisting of different combinations of mouse and human BMP-15 proregions, cleavage sites, and mature regions indicates that the defects in the production of mouse BMP-15 mature protein depend on the presence of the mouse BMP-15 proregion. The mouse proregion also caused a significant reduction in the production of human BMP-15 mature protein. The coexpression with a convertase cleavage enzyme, furin, results in complete processing of all these chimeras; however, no mouse mature protein is detected in either secreted or cell-confined forms except when associated with the human proregion. Based on the biological role of BMP-15, defects in the production of mouse BMP-15 mature protein could correlate with the high ovulation quota and litter size observed in mice.     

Alterations of mast cells and TGF-β1 on the silymarin treatment for CCl4-induced hepatic fibrosis

AIM: Silymarin is a potent antioxidant, antiinflammatory and anti-fibrogenic agent in the liver, which is mediated by alteration of hepatic Kupffer cell function, lipid peroxidation, and collagen production, Especially, in hepatic fibrogenesis, mast cells are expressed in chronic inflammatory conditions, and promote fibroblast growth and stimulate production of the extracellular matrix by hepatic stellate cells. METHODS: We examined the inhibitory mechanism of silymarin on CCl4-induced hepatic cirrhosis in rats. At 4, 8, and 12 wk, liver tissues were examined histopathologically for fibrotic changes produced by silymarin treatment. RESULTS: In the silymarin with CCl4-treated group, increase of hepatic stellate cells and TGF-β1 production were lower than in the CCl4-treated group at early stages. Additionally, at the late fibrogenic stage, expressions of TGF-β1 were weaker and especially not expressed in hepatocytes located in peripheral areas. Moreover, the number of mast cell in portal areas gradually increased and was dependent on the fibrogenic stage, but those of CCl4+silymarin-treated group decreased significantly. CONCLUSION: Anti-fibrotic and antiinflammatory effects of silymarin were associated with activation of hepatic stellate cells through the expression of TGF-β1 and stabilization of mast cells, These results suggest that silymarin prevent hepatic fibrosis through suppression of inflammation and hypoxia in the hepatic fibrogenesis.     

RBBP9: A tumor-associated serine hydrolase activity required for pancreatic neoplasia

Pancreatic cancer is one of the most lethal malignancies. To discover functionally relevant modulators of pancreatic neoplasia, we performed activity-based proteomic profiling on primary human ductal adenocarcinomas. Here, we identify retinoblastoma-binding protein 9 (RBBP9) as a tumor-associated serine hydrolase that displays elevated activity in pancreatic carcinomas. Whereas RBBP9 is expressed in normal and malignant tissues at similar levels, its elevated activity in tumor cells promotes anchorage-independent growth in vitro as well as pancreatic carcinogenesis in vivo. At the molecular level, RBBP9 activity overcomes TGF-β-mediated antiproliferative signaling by reducing Smad2/3 phosphorylation, a previously unknown role for a serine hydrolase in cancer biology. Conversely, loss of endogenous RBBP9 or expression of mutationally inactive RBBP9 leads to elevated Smad2/3 phosphorylation, implicating this serine hydrolase as an essential suppressor of TGF-β signaling. Finally, RBBP9-mediated suppression of TGF-β signaling is required for E-cadherin expression as loss of the serine hydrolase activity leads to a reduction in E-cadherin levels and a concomitant decrease in the integrity of tumor cell–cell junctions. These data not only define a previously uncharacterized serine hydrolase activity associated with epithelial neoplasia, but also demonstrate the potential benefit of functional proteomics in the identification of new therapeutic targets.     

Growth/differentiation factor 3 signals through ALK7 and regulates accumulation of adipose tissue and diet-induced obesity

Growth/differentiation factor 3 (GDF3) is highly expressed in adipose tissue, and previous overexpression experiments in mice have suggested that it may act as an adipogenic factor under conditions of high lipid load. GDF3 has been shown to signal via the activin receptor ALK4 during embryogenesis, but functional receptors in adipose tissue are unknown. In this study, we show that Gdf3(-/-) mutant mice accumulate less adipose tissue than WT animals and show partial resistance to high-fat diet-induced obesity despite similar food intake. We also demonstrate that GDF3 can signal via the ALK4-homolog ALK7 and the coreceptor Cripto, both of which are expressed in adipose tissue. In agreement with a role for ALK7 in GDF3 signaling in vivo, mutant mice lacking ALK7 also showed reduced fat accumulation and partial resistance to diet-induced obesity. We propose that GDF3 regulates adipose-tissue homeostasis and energy balance under nutrient overload in part by signaling through the ALK7 receptor.