Literature context: D: RRID:AB_490941 Twist1 Santa Cruz sc-81417; RRI
Cancer cachexia is characterized by extreme skeletal muscle loss that results in high morbidity and mortality. The incidence of cachexia varies among tumor types, being lowest in sarcomas, whereas 90% of pancreatic ductal adenocarcinoma (PDAC) patients experience severe weight loss. How these tumors trigger muscle depletion is still unfolding. Serendipitously, we found that overexpression of Twist1 in mouse muscle progenitor cells, either constitutively during development or inducibly in adult animals, caused severe muscle atrophy with features reminiscent of cachexia. Using several genetic mouse models of PDAC, we detected a marked increase in Twist1 expression in muscle undergoing cachexia. In cancer patients, elevated levels of Twist1 are associated with greater degrees of muscle wasting. Finally, both genetic and pharmacological inactivation of Twist1 in muscle progenitor cells afforded substantial protection against cancer-mediated cachexia, which translated into meaningful survival benefits, implicating Twist1 as a possible target for attenuating muscle cachexia in cancer patients.
Literature context: Signaling Cat# 3108; RRID:AB_490941 rabbit anti-SMAD2/3 Cell Signal
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 β (TGF-β) 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.
Literature context: Signaling Technology Cat#3108; RRID:AB_490941 Rabbit monoclonal anti-Smad2 Ce
Endothelial-to-mesenchymal transition (EndoMT) is a cellular process often initiated by the transforming growth factor β (TGF-β) family of ligands. Although required for normal heart valve development, deregulated EndoMT is linked to a wide range of pathological conditions. Here, we demonstrate that endothelial fatty acid oxidation (FAO) is a critical in vitro and in vivo regulator of EndoMT. We further show that this FAO-dependent metabolic regulation of EndoMT occurs through alterations in intracellular acetyl-CoA levels. Disruption of FAO via conditional deletion of endothelial carnitine palmitoyltransferase II (Cpt2E-KO) augments the magnitude of embryonic EndoMT, resulting in thickening of cardiac valves. Consistent with the known pathological effects of EndoMT, adult Cpt2E-KO mice demonstrate increased permeability in multiple vascular beds. Taken together, these results demonstrate that endothelial FAO is required to maintain endothelial cell fate and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions.
Literature context: nology Cat#3108; RRID:AB_490941 anti-pSmad3 Cell Signaling Tech
Upon stimulation of cells with transforming growth factor β (TGF-β), Smad proteins form trimeric complexes and activate a broad spectrum of target genes. It remains unresolved which of the possible Smad complexes are formed in cellular contexts and how these contribute to gene expression. By combining quantitative mass spectrometry with a computational selection strategy, we predict and provide experimental evidence for the three most relevant Smad complexes in the mouse hepatoma cell line Hepa1-6. Utilizing dynamic pathway modeling, we specify the contribution of each Smad complex to the expression of representative Smad target genes, and show that these contributions are conserved in human hepatoma cell lines and primary hepatocytes. We predict, based on gene expression data of patient samples, increased amounts of Smad2/3/4 proteins and Smad2 phosphorylation as hallmarks of hepatocellular carcinoma and experimentally verify this prediction. Our findings demonstrate that modeling approaches can disentangle the complexity of transcription factor complex formation and its impact on gene expression.
Literature context: Signaling Technology Cat#3108; RRID:AB_490941 Rabbit monoclonal anti-TAK1 Cel
Breast tumor recurrence and metastasis represent the main causes of cancer-related death in women, and treatments are still lacking. Here, we define the lipogenic enzyme acetyl-CoA carboxylase (ACC) 1 as a key player in breast cancer metastasis. ACC1 phosphorylation was increased in invading cells both in murine and human breast cancer, serving as a point of convergence for leptin and transforming growth factor (TGF) β signaling. ACC1 phosphorylation was mediated by TGFβ-activated kinase (TAK) 1, and ACC1 inhibition was indispensable for the elevation of cellular acetyl-CoA, the subsequent increase in Smad2 transcription factor acetylation and activation, and ultimately epithelial-mesenchymal transition and metastasis induction. ACC1 deficiency worsened tumor recurrence upon primary tumor resection in mice, and ACC1 phosphorylation levels correlated with metastatic potential in breast and lung cancer patients. Given the demonstrated effectiveness of anti-leptin receptor antibody treatment in halting ACC1-dependent tumor invasiveness, our work defines a "metabolocentric" approach in metastatic breast cancer therapy.
Literature context: y; 138D4, RRID:AB_490941), anti-LEF
Type XVII collagen (COL17) is a transmembrane protein located at the epidermal basement membrane zone. COL17 deficiency results in premature hair aging phenotypes and in junctional epidermolysis bullosa. Here, we show that COL17 plays a central role in regulating interfollicular epidermis (IFE) proliferation. Loss of COL17 leads to transient IFE hypertrophy in neonatal mice owing to aberrant Wnt signaling. The replenishment of COL17 in the neonatal epidermis of COL17-null mice reverses the proliferative IFE phenotype and the altered Wnt signaling. Physical aging abolishes membranous COL17 in IFE basal cells because of inactive atypical protein kinase C signaling and also induces epidermal hyperproliferation. The overexpression of human COL17 in aged mouse epidermis suppresses IFE hypertrophy. These findings demonstrate that COL17 governs IFE proliferation of neonatal and aged skin in distinct ways. Our study indicates that COL17 could be an important target of anti-aging strategies in the skin.
Remodeling of maternal spiral arteries during pregnancy requires a subpopulation of extravillous cytotrophoblasts (EVTs) to differentiate into endovascular EVTs. Activin A, which is abundantly expressed at the maternal-fetal interface, has been shown to promote trophoblast invasion, but its role in endovascular differentiation remains unknown. Vascular endothelial growth factor-A (VEGF-A) is well recognized as a key regulator in trophoblast endovascular differentiation. Whether and how activin A might regulate VEGF-A production in human trophoblasts and its relationship to endovascular differentiation have yet to be determined. In the present study, we found that activin A increased VEGF-A production in primary and immortalized (HTR8/SVneo) human EVT cells. In addition, activin A enhanced HTR8/SVneo endothelial-like tube formation, and these effects were attenuated by pretreatment with small interfering RNA targeting VEGF-A or the VEGF receptor 1/2 inhibitor SU4312. Pretreatment with the activin/TGF-β type 1 receptor (ALK4/5/7) inhibitor SB431542 abolished the stimulatory effects of activin A on phosphorylated mothers against decapentaplegic (SMAD)-2/3 phosphorylation, VEGF-A production, and endothelial-like tube formation. Moreover, small interfering RNA-mediated down-regulation of SMAD2, SMAD3, or common SMAD4 abolished the effects of activin A on VEGF-A production and endothelial-like tube formation. In conclusion, activin A may promote human trophoblast cell endothelial-like tube formation by up-regulating VEGF-A production in an SMAD2/3-SMAD4-dependent manner. These findings provide insight into the cellular and molecular events regulated by activin A during human implantation.
TGFβ superfamily ligands, receptors, and second messengers, including activins A and B, have been identified in pancreatic islets and proposed to have important roles regulating development, proliferation, and function. We previously demonstrated that Fstl3 (an antagonist of activin activity) null mice have larger islets with β-cell hyperplasia and improved glucose tolerance and insulin sensitivity in the absence of altered β-cell proliferation. This suggested the hypothesis that increased activin signaling influences β-cell expansion by destabilizing the α-cell phenotype and promoting transdifferentiation to β-cells. We tested the first part of this hypothesis by treating α- and β-cell lines and sorted mouse islet cells with activin and related ligands. Treatment of the αTC1-6 α cell line with activins A or B suppressed critical α-cell gene expression, including Arx, glucagon, and MafB while also enhancing β-cell gene expression. In INS-1E β-cells, activin A treatment induced a significant increase in Pax4 (a fate determining β-cell gene) and insulin expression. In sorted primary islet cells, α-cell gene expression was again suppressed by activin treatment in α-cells, whereas Pax4 was enhanced in β-cells. Activin treatment in both cell lines and primary cells resulted in phosphorylated mothers against decapentaplegic-2 phosphorylation. Finally, treatment of αTC1-6 cells with activins A or B significantly inhibited proliferation. These results support the hypothesis that activin signaling destabilized the α-cell phenotype while promoting a β-cell fate. Moreover, these results support a model in which the β-cell expansion observed in Fstl3 null mice may be due, at least in part, to enhanced α- to β-cell transdifferentiation.
Liver receptor homolog-1 (LRH-1) is an orphan nuclear receptor that belongs to the NR5A subgroup of nuclear receptors. LRH-1 induces key genes to regulate metabolic process, ovarian function, cancer cell proliferation, and steroidogenesis. In the breast, LRH-1 modulates and synergizes with endogenous estrogen signaling to promote breast cancer cell proliferation. We used small interfering RNA knockdown strategies to deplete LRH-1 in breast cancer cells and followed with microarray analysis to identify LRH-1-dependent mechanisms. We identified key genes involved in TGF-β signaling to be highly responsive to LRH-1 knockdown. This relationship was validated in 2 breast cancer cell lines overexpressing LRH-1 in vitro and in a novel transgenic mouse with targeted LRH-1 overexpression in mammary epithelial cells. Notably, TGF-β signaling was activated in LRH-1-overexpressing breast cancer cells and mouse mammary glands. Further analyses of mammary gross morphology revealed a significant reduction in mammary lateral budding after LRH-1 overexpression. These findings suggest that the altered mammary morphogenesis in LRH-1 transgenic animals is mediated via enhanced TGF-β expression. The regulation of TGF-β isoforms and SMAD2/3-mediated downstream signaling by LRH-1 also implicates a potential contribution of LRH-1 in breast cancer. Collectively, these data demonstrate that LRH-1 regulates TGF-β expression and downstream signaling in mouse mammary glands.
The immortal C2C12 cell line originates from dystrophic mouse thigh muscle and has been used to study the endocrine control of muscle cell growth, development, and function, including those actions regulated by myostatin. Previous studies suggest that high concentrations of recombinant myostatin generated in bacteria inhibit C2C12 proliferation and differentiation. Recombinant myostatin generated in eukaryotic systems similarly inhibits the proliferation of primary myosatellite cells, but consequently initiates, rather than inhibits, their differentiation and is bioactive at far lower concentrations. Our studies indicate that 2 different sources of recombinant myostatin made in eukaryotes stimulate, not inhibit, C2C12 proliferation. This effect occurred at different cell densities and serum concentrations and in the presence of IGF-I, a potent myoblast mitogen. This stimulatory effect was comparable to that obtained with TGFβ1, a related factor that also inhibits primary myosatellite cell proliferation. Attenuating the myostatin/activin (ie, Acvr2b) and TGFβ1 receptor signaling pathways with the Alk4/5 and Alk5 inhibitors, SB431542 and SB505142, respectively, similarly attenuated proliferation induced by serum, myostatin or TGFβ1 and in a dose-dependent manner. In serum-free medium, both myostatin and TGFβ1 stimulated Smad2 phosphorylation, but not that of Smad3, and a Smad3 inhibitor (SIS3) only inhibited proliferation in cells cultured in high serum. Thus, myostatin and TGFβ1 stimulate C2C12 proliferation primarily via Smad2. These results together question the physiological relevance of the C2C12 model and previous studies using recombinant myostatin generated in bacteria. They also support the alternative use of primary myosatellite cells and recombinant myostatin generated in eukaryotes.
In an effort to expand human islets and enhance allogeneic islet transplant for the treatment of type 1 diabetes, identifying signaling pathways that stimulate human β-cell proliferation is paramount. TGF-β superfamily members, in particular activin-A, are likely involved in islet development and may contribute to β-cell proliferation. Nodal, another TGF-β member, is present in both embryonic and adult rodent islets. Nodal, along with its coreceptor, Cripto, are pro-proliferative factors in certain cell types. Although Nodal stimulates apoptosis of rat insulinoma cells (INS-1), Nodal and Cripto signaling have not been studied in the context of human islets. The current study investigated the effects of Nodal and Cripto on human β-cell proliferation, differentiation, and viability. In the human pancreas and isolated human islets, we observed Nodal mRNA and protein expression, with protein expression observed in β and α-cells. Cripto expression was absent from human islets. Furthermore, in cultured human islets, exogenous Nodal stimulated modest β-cell proliferation and inhibited α-cell proliferation with no effect on cellular viability, apoptosis, or differentiation. Nodal stimulated the phosphorylation of mothers against decapentaplegic (SMAD)-2, with no effect on AKT or MAPK signaling, suggesting phosphorylated SMAD signaling was involved in β-cell proliferation. Cripto had no effect on human islet cell proliferation, differentiation, or viability. In conclusion, Nodal stimulates human β-cell proliferation while maintaining cellular viability. Nodal signaling warrants further exploration to better understand and enhance human β-cell proliferative capacity.