N-Glycosylation Is Required for Matriptase-2 Autoactivation and Ectodomain Shedding

Matriptase-2 is a hepatic membrane serine protease that regulates iron homeostasis. Defects in matriptase-2 cause iron deficiency anemia. In cells, matriptase-2 is synthesized as a zymogen. To date, how matriptase-2 expression and activation are regulated remains poorly understood. Here we expressed human matriptase-2 in HEK293 and hepatic BEL-7402, SMMC-7721, and QGY-7703 cells. By labeling cell surface proteins and Western analysis, we examined matriptase-2 cell surface expression, zymogen activation, and ectodomain shedding. Our results show that matriptase-2 was activated on the cell surface but not intracellularly. Activated matriptase-2 underwent ectodomain shedding, producing soluble fragments in the conditioned medium. By testing inactive mutants, R576A and S762A, we found that matriptase-2 activation and shedding were mediated by its own catalytic activity and that the one-chain form of matriptase-2 had little activity in ectodomain shedding. We made additional matriptase-2 mutants, N136Q, N184Q, N216Q, N338Q, N433Q, N453Q, and N518Q, in which each of the predicted N-glycosylation sites was mutated. All of these mutants were expressed on the cell surface. However, mutants N216Q, N453Q, and N518Q, but not the other mutants, had impaired zymogen activation and ectodomain shedding. Our results indicate that N-glycans at specific sites are critical for matriptase-2 activation. Together, these data provide new insights into the cell surface expression, zymogen activation, and ectodomain shedding of matriptase-2.     

Precise Regulation of Porcupine Activity Is Required for Physiological Wnt Signaling

Gradients of diverse Wnt proteins regulate development, renewal, and differentiation. Porcupine (PORCN) is a membrane-bound O-acyltransferase that is required for post-translational modification of all Wnts to enable their transport, secretion, and activity. Mutations in PORCN are associated with focal dermal hypoplasia (FDH), whereas gene deletion causes embryonic lethality in mice. To study the protein in more detail, zinc finger nucleases were used to edit the PORCN genomic locus, establishing two HT1080 fibrosarcoma clones null for PORCN activity that facilitate the study of PORCN structure and function. We establish that PORCN is a key non-redundant node for the regulation of global Wnt signaling because PORCN null cells are completely incapable of autocrine Wnt signaling. The strength of Wnt signaling is exquisitely sensitive to PORCN expression, with a dynamic range of at least 3 orders of magnitude, suggesting that PORCN activity is a key modulator of all Wnt ligand activity. Consistent with this, we find that multiple FDH-associated mutants have only subtle alterations in enzyme activity yet are associated with a severe FDH phenotype. These studies support an essential regulatory role of PORCN in shaping Wnt signaling gradients.     

Nubp1 Is Required for Lung Branching Morphogenesis and Distal Progenitor Cell Survival in Mice

The lung is a complex system in biology and medicine alike. Whereas there is a good understanding of the anatomy and histology of the embryonic and adult lung, less is known about the molecular details and the cellular pathways that ultimately orchestrate lung formation and affect its health. From a forward genetic approach to identify novel genes involved in lung formation, we identified a mutated Nubp1 gene, which leads to syndactyly, eye cataract and lung hypoplasia. In the lung, Nubp1 is expressed in progenitor cells of the distal epithelium. Nubp1(m1Nisw) mutants show increased apoptosis accompanied by a loss of the distal progenitor markers Sftpc, Sox9 and Foxp2. In addition, Nubp1 mutation disrupts localization of the polarity protein Par3 and the mitosis relevant protein Numb. Using knock-down studies in lung epithelial cells, we also demonstrate a function of Nubp1 in regulating centrosome dynamics and microtubule organization. Together, Nubp1 represents an essential protein for lung progenitor survival by coordinating vital cellular processes including cell polarity and centrosomal dynamics.     

Osmotic Regulation Is Required for Cancer Cell Survival under Solid Stress

For a solid tumor to grow, it must be able to support the compressive stress that is generated as it presses against the surrounding tissue. Although the literature suggests a role for the cytoskeleton in counteracting these stresses, there has been no systematic evaluation of which filaments are responsible or to what degree. Here, using a three-dimensional spheroid model, we show that cytoskeletal filaments do not actively support compressive loads in breast, ovarian, and prostate cancer. However, modulation of tonicity can induce alterations in spheroid size. We find that under compression, tumor cells actively efflux sodium to decrease their intracellular tonicity, and that this is reversible by blockade of sodium channel NHE1. Moreover, although polymerized actin does not actively support the compressive load, it is required for sodium efflux. Compression-induced cell death is increased by both sodium blockade and actin depolymerization, whereas increased actin polymerization offers protective effects and increases sodium efflux. Taken together, these results demonstrate that cancer cells modulate their tonicity to survive under compressive solid stress.     

Dlk1-Mediated Temporal Regulation of Notch Signaling Is Required for Differentiation of Alveolar Type II to Type I Cells during Repair

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Dhrs3 Protein Attenuates Retinoic Acid Signaling and Is Required for Early Embryonic Patterning

All-trans-retinoic acid (atRA) is an important morphogen involved in many developmental processes, including neural differentiation, body axis formation, and organogenesis. During early embryonic development, atRA is synthesized from all-trans-retinal (atRAL) in an irreversible reaction mainly catalyzed by retinal dehydrogenase 2 (aldh1a2), whereas atRAL is converted from all-trans-retinol via reversible oxidation by retinol dehydrogenases, members of the short-chain dehydrogenase/reductase family. atRA is degraded by cytochrome P450, family 26 (cyp26). We have previously identified a short-chain dehydrogenase/reductase 3 (dhrs3), which showed differential expression patterns in Xenopus embryos. We show here that the expression of dhrs3 was induced by atRA treatment and overexpression of Xenopus nodal related 1 (xnr1) in animal cap assay. Overexpression of dhrs3 enhanced the phenotype of excessive cyp26a1. In embryos overexpressing aldh1a2 or retinol dehydrogenase 10 (rdh10) in the presence of their respective substrates, Dhrs3 counteracted the action of Aldh1a2 or Rdh10, indicating that retinoic acid signaling is attenuated. Knockdown of Dhrs3 by antisense morpholino oligonucleotides resulted in a phenotype of shortened anteroposterior axis, reduced head structure, and perturbed somitogenesis, which were also found in embryos treated with an excess of atRA. Examination of the expression of brachyury, not, goosecoid, and papc indicated that convergent extension movement was defective in Dhrs3 morphants. Taken together, these studies suggest that dhrs3 participates in atRA metabolism by reducing atRAL levels and is required for proper anteroposterior axis formation, neuroectoderm patterning, and somitogenesis.     

Toll-Like Receptor 3 Signaling on Macrophages Is Required for Survival Following Coxsackievirus B4 Infection

Toll-like receptor 3 (TLR3) has been proposed to play a central role in the early recognition of viruses by sensing double stranded RNA, a common intermediate of viral replication. However, several reports have demonstrated that TLR3 signaling is either dispensable or even harmful following infection with certain viruses. Here, we asked whether TLR3 plays a role in the response to coxsackievirus B4 (CB4), a prevalent human pathogen that has been associated with pancreatitis, myocarditis and diabetes. We demonstrate that TLR3 signaling on macrophages is critical to establish protective immunity to CB4. TLR3 deficient mice produced reduced pro-inflammatory mediators and are unable to control viral replication at the early stages of infection resulting in severe cardiac damage. Intriguingly, the absence of TLR3 did not affect the activation of several key innate and adaptive cellular effectors. This suggests that in the absence of TLR3 signaling on macrophages, viral replication outpaces the developing adaptive immune response. We further demonstrate that the MyD88-dependent signaling pathways are not only unable to compensate for the loss of TLR3, they are also dispensable in the response to this RNA virus. Our results demonstrate that TLR3 is not simply part of a redundant system of viral recognition, but rather TLR3 plays an essential role in recognizing the molecular signatures associated with specific viruses including CB4.     

BAF200 Is Required for Heart Morphogenesis and Coronary Artery Development

ATP-dependent SWI/SNF chromatin remodeling complexes utilize ATP hydrolysis to non-covalently change nucleosome-DNA interactions and are essential in stem cell development, organogenesis, and tumorigenesis. Biochemical studies show that SWI/SNF in mammalian cells can be divided into two subcomplexes BAF and PBAF based on the subunit composition. ARID2 or BAF200 has been defined as an intrinsic subunit of PBAF complex. However, the function of BAF200 in vivo is not clear. To dissect the possible role of BAF200 in regulating embryogenesis and organ development, we generated BAF200 mutant mice and found they were embryonic lethal. BAF200 mutant embryos exhibited multiple cardiac defects including thin myocardium, ventricular septum defect, common atrioventricular valve, and double outlet right ventricle around E14.5. Moreover, we also detected reduced intramyocardial coronary arteries in BAF200 mutants, suggesting that BAF200 is required for proper migration and differentiation of subepicardial venous cells into arterial endothelial cells. Our work revealed that PBAF complex plays a critical role in heart morphogenesis and coronary artery angiogenesis.     

MT1-MMP Is Required for Myeloid Cell Fusion via Regulation of Rac1 Signaling

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Highlights? Myeloid cells fuse during osteoclast and giant cell formation ? MT1-MMP is required for this fusion in vitro and in vivo ? MT1-MMP regulates the GTPase Rac1 through p130Cas binding ? MT1-MMP proteolytic activity is not required     

The ERK-1/2 Signaling Pathway Is Involved in the Stimulation of Branching Morphogenesis of Fetal Mouse Submandibular Glands by EGF

We have previously reported that epidermal growth factor (EGF) stimulates branching morphogenesis of the fetal mouse submandibular gland (SMG) (M. Kashimata and E. W. Gresik, 1997, Dev. Dyn. 208, 149–161) and that the EGF receptor (EGFR) is localized principally, if not exclusively, on the epithelial components of the fetal SMG (E. W. Gresik, M. Kashimata, Y. Kadoya, R. Mathews, N. Minami, and S. Yamashina, 1997, J. Histochem. Cytochem. 45, 1651–1657). The EGFR is a receptor tyrosine kinase, and after binding of its ligand, it triggers several intracellular signaling cascades, among them the one activating the mitogen-activated protein kinases (MAPK) ERK-1/2. Here we investigated whether EGF utilizes the ERK-1/2 signaling cascade to stimulate branching morphogenesis in the fetal mouse SMG. SMG rudiments were collected as matched pairs at E14, E16, and E18 (E0 = day of vaginal plug); placed into wells of defined medium (BGJb); and exposed to EGF for 5 or 30 min or to medium alone (controls). By Western blotting we found that EGF induced the appearance of multiple bands of phosphotyrosine-containing proteins, including bands at 170 kDa and 44 kDa/42 kDa, presumably corresponding to the phosphorylated forms of EGFR and ERK-1/2, respectively. Other blots showed the specific appearance of the phosphorylated EGFR and of phospho-ERK-1/2 in response to EGF. Immunohistochemical staining for phosphotyrosine increased at the plasma membrane after EGF stimulation for 5 or 30 min. Diffuse cytoplasmic staining for MEK-1/2 (the MAPK kinase that activates ERK-1/2) increased near the cell membrane after EGF stimulation. Phospho-ERK-1/2 was localized in the nuclei of a few epithelial cells after EGF for 5 min, but in the nuclei of many cells after EGF for 30 min. PD98059, an inhibitor of phosphorylation and activation of MEK-1/2, by itself inhibited branching morphogenesis and, furthermore, decreased the stimulatory effect of EGF on branching. Western blots confirmed that this inhibitor blocked phosphorylation of ERK-1/2 in fetal SMGs exposed to EGF. These results show that components of the ERK-1/2 signaling cascade are present in epithelial cells of the fetal SMG, that they are activated by EGF, and that inhibition of this cascade perturbs branching morphogenesis. However, EGF did not cause phosphorylation of two other MAPKs, SAPK/JNK or p38MAPK, in fetal SMGs. These results imply that the ERK-1/2 signaling is responsible, at least in part, for the stimulatory effect of EGF on branching morphogenesis of the fetal mouse SMG.     

LGR4 Is Required for the Cell Survival of the Peripheral Mesenchyme at the Embryonic Stages of Nephrogenesis

In mice, homozygous Lgr4 inactivation results in hypoplastic kidneys. To understand better the role of LGR4 in kidney development, we performed an analysis of kidneys in Lgr4 ^-/- embryos. We stained Lgr4 ^-/- kidneys with anti-WT1 and anti-Cleaved Caspase3 antibodies at E16.5, and observed that the structures of the cap mesenchyme were disrupted and that apoptosis increased. In addition, the expression of PAX2, an anti-apoptotic factor in kidney development, was also significantly decreased at E16.5. We found that the LGR4 defect caused an increase in apoptosis in the peripheral mesenchyme during kidney development.