The bone morphogenetic protein (BMP) signaling pathways have important roles in embryonic development and cellular homeostasis, with aberrant BMP signaling resulting in a broad spectrum of human ...disease. We report that BMPs unexpectedly signal through the canonical transforming growth factor β (TGF‐β)‐responsive Smad2 and Smad3. BMP‐induced Smad2/3 signaling occurs preferentially in embryonic cells and transformed cells. BMPs signal to Smad2/3 by stimulating complex formation between the BMP‐binding TGF‐β superfamily receptors, activin receptor‐like kinase (ALK)3/6, and the Smad2/3 phosphorylating receptors ALK5/7. BMP signaling through Smad2 mediates, in part, dorsoventral axis patterning in zebrafish embryos, whereas BMP signaling through Smad3 facilitates cancer cell invasion. Consistent with increased BMP‐mediated Smad2/3 signaling during cancer progression, Smad1/5 and Smad 2/3 signaling converge in human cancer specimens. Thus, the signaling mechanisms used by BMPs and TGF‐β superfamily receptors are broader than previously appreciated.—Holtzhausen, A., Golzio, C., How, T., Lee, Y.‐H., Schiemann, W. P., Katsanis, N., Blobe, G. C. Novel bone morphogenetic protein signaling through Smad2 and Smad3 to regulate cancer progression and development. FASEB J. 28, 1248–1267 (2014). www.fasebj.org
Bone morphogenetic proteins (BMPs) have an emerging role in human cancers. Here we demonstrate that the BMP-signaling pathway is intact and functional in human pancreatic cancer cells, with several ...BMP signaling components and transcriptional targets upregulated in human pancreatic cancer specimens compared with normal pancreatic tissue. Functionally, multiple BMP family members, including BMP-2, BMP-4 and BMP-7, induce an epithelial to mesenchymal transition (EMT) in the human pancreatic cancer cell line Panc-1, as demonstrated by morphological alterations and loss of E-cadherin expression. BMP-mediated EMT results in an increase in invasiveness of Panc-1 cells, in part through increased expression and activity of matrix metalloproteinase (MMP)-2, a known mediator of pancreatic cancer cell invasiveness. Accompanying EMT, BMP reduces expression of the transforming growth factor (TGF)-β superfamily receptor, transforming growth factor-β type III receptor (TβRIII), for which we have previously demonstrated loss of expression during pancreatic cancer progression. Maintaining TβRIII expression inhibits BMP-mediated invasion and suppresses Smad1 activation. Further, Smad1 is required for BMP-induced invasiveness and partially responsible for BMP-mediated increases in MMP-2 activity. These data suggest that BMP signaling, through Smad1 induction and upregulation of MMP-2, is an important mediator of pancreatic cancer invasiveness and a potential therapeutic target for treating this deadly disease.
Naïve T cell activation requires antigen presentation combined with costimulation through CD28, both of which optimally occur in secondary lymphoid tissues such as lymph nodes and the spleen. ...Belatacept impairs CD28 costimulation by binding its ligands, CD80 and CD86, and in doing so, impairs de novo alloimmune responses. However, in most patients belatacept is ineffective in preventing allograft rejection when used as a monotherapy, and adjuvant therapy is required for control of costimulation‐blockade resistant rejection (CoBRR). In rodent models, impaired access to secondary lymphoid tissues has been demonstrated to reduce alloimmune responses to vascularized allografts. Here we show that surgical maneuvers, lymphatic ligation, and splenectomy, designed to anatomically limit access to secondary lymphoid tissues, control CoBRR and facilitate belatacept monotherapy in a nonhuman primate model of kidney transplantation without adjuvant immunotherapy. We further demonstrate that animals sustained on belatacept monotherapy progressively develop an increasingly naïve T and B cell repertoire, an effect that is accelerated by splenectomy and lost at the time of belatacept withdrawal and rejection. These pilot data inform the role of secondary lymphoid tissues on the development of CoBRR and the use of costimulation molecule–focused therapies.
Introducing two surgical maneuvers—lymphatic ligation and splenectomy, designed to anatomically limit access to secondary lymphoid tissues—successfully controls costimulation blockade–resistant rejection and facilitates belatacept monotherapy in a nonhuman primate model of kidney transplantation without adjuvant immunotherapy.
The transforming growth factor-beta (TGF-beta) superfamily members, TGF-beta, activin, and inhibin, all have prominent roles in regulating normal ovarian function. Betaglycan, or the type III ...TGF-beta receptor, is a coreceptor that regulates TGF-beta, activin, and inhibin signaling. Here, we show that betaglycan expression is frequently decreased or lost in epithelial derived ovarian cancer at both the mRNA and protein level, with the degree of loss correlating with tumor grade. Treatment of ovarian cancer cell lines with the methyltransferase inhibitor 5-aza-2-deoxycytidine and the histone deacetylase inhibitor trichostatin A resulted in significant synergistic induction of betaglycan message levels and increased betaglycan protein expression, indicating that epigenetic silencing may play a role in the loss of betaglycan expression observed in ovarian cancer. Although restoring betaglycan expression in Ovca429 ovarian cancer cells is not sufficient to restore TGF-beta-mediated inhibition of proliferation, betaglycan significantly inhibits ovarian cancer cell motility and invasiveness. Furthermore, betaglycan specifically enhances the antimigratory effects of inhibin and the ability of inhibin to repress matrix metalloproteinase levels in these cells. These results show, for the first time, epigenetic regulation of betaglycan expression in ovarian cancer, and a novel role for betaglycan in regulating ovarian cancer motility and invasiveness.
Background
Membrane cofactor protein CD46 attenuates the complement cascade by facilitating cleavage of C3b and C4b. In solid organ xenotransplantation, organs expressing CD46 have been shown to ...resist hyperacute rejection. However, the incremental value of human CD46 expression for islet xenotransplantation remains poorly defined.
Methods
This study attempted to delineate the role of CD46 in early neonatal porcine islet engraftment by comparing Gal‐knocked out (GKO) and hCD46‐transgenic (GKO/CD46) islets in a dual transplant model. Seven rhesus macaques underwent dual transplant and were sacrificed at 1 hour (n = 4) or 24 hours (n = 3). Both hemilivers were recovered and fixed for immunohistochemistry (CD46, insulin, neutrophil elastase, platelet, IgM, IgG, C3d, C4d, CD68, Caspase 3). Quantitative immunohistochemical analysis was performed using the Aperio Imagescope.
Results
Within 1 hour of intraportal infusion of xenografts, no differences were observed between the two types of islets in terms of platelet, antibody, or complement deposition. Cellular infiltration and islet apoptotic activity were also similar at 1 hour. At 24 hours, GKO/CD46 islets demonstrated significantly less platelet deposition (P = 0.01) and neutrophil infiltration (P = 0.01) compared to GKO islets. In contrast, C3d (P = 0.38) and C4d (P = 0.45) deposition was equal between the two genotypes.
Conclusions
Our findings suggest that expression of hCD46 on NPIs potentially provides a measurable incremental survival advantage in vivo by reducing early thrombo‐inflammatory events associated with instant blood‐mediated inflammatory reaction (IBMIR) following intraportal islet infusion.
β-Arrestins bind to activated seven transmembrane-spanning (7TMS) receptors (G protein-coupled receptors) after the receptors are phosphorylated by G protein-coupled receptor kinases (GRKs), thereby ...regulating their signaling and internalization. Here, we demonstrate an unexpected and analogous role of β-arrestin 2 (βarr2) for the single transmembrane-spanning type III transforming growth factor-β (TGF-β) receptor (TβRIII, also referred to as beta-glycan). Binding of βarr2 to TβRIII was also triggered by phosphorylation of the receptor on its cytoplasmic domain (likely at threonine 841). However, such phosphorylation was mediated by the type II TGF-β receptor (TβRII), which is itself a kinase, rather than by a GRK. Association with βarr2 led to internalization of both receptors and down-regulation of TGF-β signaling. Thus, the regulatory actions of β-arrestins are broader than previously appreciated, extending to the TGF-β receptor family as well.
Transforming growth factor β (TGF-β) signals through two distinct pathways to regulate endothelial cell proliferation, migration, and angiogenesis, the ALK-1/Smad 1/5/8 and ALK-5/Smad2/3 pathways. ...Endoglin is a co-receptor predominantly expressed in endothelial cells that participates in TGFβ-mediated signaling with ALK-1 and ALK-5 and regulates critical aspects of cellular and biological responses. The embryonic lethal phenotype of knock-out mice because of defects in angiogenesis and disease-causing mutations resulting in human vascular diseases both support essential roles for endoglin, ALK-1, and ALK-5 in the vasculature. However, the mechanism by which endoglin mediates TGF-β signaling through ALK-1 and ALK-5 has remained elusive. Here we describe a novel interaction between endoglin and GIPC, a scaffolding protein known to regulate cell surface receptor expression and trafficking. Co-immunoprecipitation and immunofluorescence confocal studies both demonstrate a specific interaction between endoglin and GIPC in endothelial cells, mediated by a class I PDZ binding motif in the cytoplasmic domain of endoglin. Subcellular distribution studies demonstrate that endoglin recruits GIPC to the plasma membrane and co-localizes with GIPC in a TGFβ-independent manner, with GIPC-promoting cell surface retention of endoglin. Endoglin specifically enhanced TGF-β1-induced phosphorylation of Smad 1/5/8, increased a Smad 1/5/8 responsive promoter, and inhibited endothelial cell migration in a manner dependent on the ability of endoglin to interact with GIPC. These studies define a novel mechanism for the regulation of endoglin signaling and function in endothelial cells and demonstrate a new role for GIPC in TGF-β signaling.
The transforming growth factor-β (TGF-β) superfamily has essential roles in lung development, regulating cell proliferation, branching morphogenesis, differentiation and apoptosis. Although most lung ...cancers become resistant to the tumor suppressor effects of TGF-β, and loss or mutation of one of the components of the TGF-β signaling pathway, including TβRII, Smad2 and Smad4 have been reported, mutations are not common in non-small cell lung cancer (NSCLC). Here we demonstrate that the TGF-β superfamily co-receptor, the type III TGF-β receptor (TβRIII or betaglycan) is lost in the majority of NSCLC specimens at the mRNA and protein levels, with loss correlating with increased tumor grade and disease progression. Loss of heterozygosity at the TGFBR3 genomic locus occurs in 38.5% of NSCLC specimens and correlates with decreased TβRIII expression, suggesting loss of heterozygosity as one mechanism for TβRIII loss. In the H460 cell model of NSCLC, restoring TβRIII expression decreased colony formation in soft agar. In the A549 cell model of NSCLC, restoring TβRIII expression significantly decreased cellular migration and invasion through Matrigel, in the presence and absence of TGF-β1, and decreased tumorigenicity in vivo. In a reciprocal manner, shRNA-mediated silencing of endogenous TβRIII expression enhanced invasion through Matrigel. Mechanistically, TβRIII functions, at least in part, through undergoing ectodomain shedding, generating soluble TβRIII, which is able to inhibit cellular invasiveness. Taken together, these results support TβRIII as a novel tumor suppressor gene that is commonly lost in NSCLC resulting in a functional increase in cellular migration, invasion and anchorage-independent growth of lung cancer cells.
In the present study, we tested the hypothesis that lipopolysaccharide (LPS)-induced expression of nitric oxide synthase (iNOS) by splenocytes is modulated through the activation of endogenous ...opioids in the central nervous system. The initial studies determined the parameters of LPS-induced expression of iNOS by splenocytes. Rats were injected with LPS at doses of 0, 1, 10, 100, and 1000 microg/kg, and measures of both iNOS mRNA and protein showed a dose-dependent increase in expression. In a time course study, rats received 100 microg/kg LPS and were killed at 0, 2, 4, 8, and 16 h postinjection. Both iNOS mRNA and protein expression was detectable at the 2-h time point, with peak expression occurring at 8 h. To evaluate the involvement of endogenous opioids, the opioid receptor antagonist naltrexone was administered at 0, 0.1, 1, or 10 mg/kg s.c. in combination with LPS (100 microg/kg), with a second injection of naltrexone at the same dose 4 h after the injection of LPS. Naltrexone induced a pronounced dose-dependent reduction in iNOS mRNA and protein expression by splenocytes. The modulation of iNOS expression occurs via central opioid receptors as intracerebroventricular administration but not peripheral administration of N-methylnaltrexone, the quaternary form of naltrexone that does not readily cross the blood-brain barrier, reduced the expression of iNOS. For all of the manipulations, nitrite/nitrate levels in the plasma showed effects similar to those for iNOS mRNA and protein. Collectively, these findings indicate that central opioid receptors are involved in the in vivo regulation of splenic nitric oxide production.
Recent studies implicate a role for cell mechanics in cancer progression. The epithelial-to-mesenchymal transition (EMT) regulates the detachment of cancer cells from the epithelium and facilitates ...their invasion into stromal tissue. Although classic EMT hallmarks include loss of cell-cell adhesions, morphology changes, and increased invasion capacity, little is known about the associated mechanical changes. Previously, force application on integrins has been shown to initiate cytoskeletal rearrangements that result in increased cell stiffness and a stiffening response. Here we demonstrate that transforming growth factor β (TGF-β)-induced EMT results in decreased stiffness and loss of the normal stiffening response to force applied on integrins. We find that suppression of the RhoA guanine nucleotide exchange factors (GEFs) LARG and GEF-H1 through TGF-β/ALK5-enhanced proteasomal degradation mediates these changes in cell mechanics and affects EMT-associated invasion. Taken together, our results reveal a functional connection between attenuated stiffness and stiffening response and the increased invasion capacity acquired after TGF-β-induced EMT.
PDF
