Toll-like receptor 4 signaling plays a role in triggering periodontal infection

Toll-like receptors (TLRs) are a group of sensors on the surface of antigen-presenting cells, such as dendritic cells and macrophages, which recognize microbial pathogens and induce innate and adaptive immune responses. Periodontitis is an inflammatory disease characterized by the destruction of tooth-supporting structures. In order to address whether TLR4 signaling plays a role in periodontitis, we studied the gene expression change in human periodontal ligament cells (HPDLCs) in response to TLR4 ligand, lipopolysaccharide treatment by microarray analysis. Expression of TLR4 was detected in HPDLCs. Lipopolysaccharide treatment increased the expression of 12 genes (more than twofold), including TLR4, TLR5, TLR7, Pellino 1, colony stimulating factor 2 (CSF2) and IL-6. In addition, the expression of 15 genes (less than equal to twofold) was decreased, including Fos, LY64 and LY86. In addition, real-time PCR was used to confirm the change of gene expression of TLR4, IL-6 and Fos. We also showed that the upregulation of IL-6 by lipopolysaccharide treatment was TLR4-dependent. This pattern of gene expression indicates that pathogens may trigger TLR4 signaling and cause periodontitis. Manipulating TLR4 signaling may potentially become one of the recognized therapies for periodontitis.     

The wnt receptor ryk plays a role in Mammalian planar cell polarity signaling

Wnts are essential for a wide range of developmental processes, including cell growth, division, and differentiation. Some of these processes signal via the planar cell polarity (PCP) pathway, which is a β-catenin-independent Wnt signaling pathway. Previous studies have shown that Ryk, a member of the receptor tyrosine kinase family, can bind to Wnts. Ryk is required for normal axon guidance and neuronal differentiation during development. Here, we demonstrate that mammalian Ryk interacts with the Wnt/PCP pathway. In vitro analysis showed that the Wnt inhibitory factor domain of Ryk was necessary for Wnt binding. Detailed analysis of two vertebrate model organisms showed Ryk phenotypes consistent with PCP signaling. In zebrafish, gene knockdown using morpholinos revealed a genetic interaction between Ryk and Wnt11 during the PCP pathway-regulated process of embryo convergent extension. Ryk-deficient mouse embryos displayed disrupted polarity of stereociliary hair cells in the cochlea, a characteristic of disturbed PCP signaling. This PCP defect was also observed in mouse embryos that were double heterozygotes for Ryk and Looptail (containing a mutation in the core Wnt/PCP pathway gene Vangl2) but not in either of the single heterozygotes, suggesting a genetic interaction between Ryk and Vangl2. Co-immunoprecipitation studies demonstrated that RYK and VANGL2 proteins form a complex, whereas RYK also activated RhoA, a downstream effector of PCP signaling. Overall, our data suggest an important role for Ryk in Wnt/planar cell polarity signaling during vertebrate development via the Vangl2 signaling pathway, as demonstrated in the mouse cochlea.     

Phosphoinositide signaling plays a key role in cytokinesis

To perform the vital functions of motility and division, cells must undergo dramatic shifts in cell polarity. Recent evidence suggests that polarized distributions of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, which are clearly important for regulating cell morphology during migration, also play an important role during the final event in cell division, which is cytokinesis. Thus, there is a critical interplay between the membrane phosphoinositides and the cytoskeletal cortex that regulates the complex series of cell shape changes that accompany these two processes.     

An essential role for FGF receptor signaling in lens development

Since the days of Hans Spemann, the ocular lens has served as one of the most important developmental systems for elucidating fundamental processes of induction and differentiation. More recently, studies in the lens have contributed significantly to our understanding of cell cycle regulation and apoptosis. Over 20 years of accumulated evidence using several different vertebrate species has suggested that fibroblast growth factors (FGFs) and/or fibroblast growth factor receptors (FGFRs) play a key role in lens development. FGFR signaling has been implicated in lens induction, lens cell proliferation and survival, lens fiber differentiation and lens regeneration. Here we will review and discuss historical and recent evidence suggesting that (FGFR) signaling plays a vital and universal role in multiple aspects of lens development.     

Mechanical signaling through the discoidin domain receptor 1 plays a central role in tissue fibrosis

ABSTRACT

The preservation of tissue and organ architecture and function depends on tightly regulated interactions of cells with the extracellular matrix (ECM). These interactions are maintained in a dynamic equilibrium that balances intracellular, myosin-generated tension with extracellular resistance conferred by the mechanical properties of the extracellular matrix. Disturbances of this equilibrium can lead to the development of fibrotic lesions that are associated with a wide repertoire of high prevalence diseases including obstructive cardiovascular diseases, muscular dystrophy and cancer. Mechanotransduction is the process by which mechanical cues are converted into biochemical signals. At the core of mechanotransduction are sensory systems, which are frequently located at sites of cell-ECM and cell-cell contacts. As integrins (cell-ECM junctions) and cadherins (cell-cell contacts) have been extensively studied, we focus here on the properties of the discoidin domain receptor 1 (DDR1), a tyrosine kinase that mediates cell adhesion to collagen. DDR1 expression is positively associated with fibrotic lesions of heart, kidney, liver, lung and perivascular tissues. As the most common end-point of all fibrotic disorders is dysregulated collagen remodeling, we consider here the mechanical signaling functions of DDR1 in processing of fibrillar collagen that lead to tissue fibrosis.     

CrkL is recruited through its SH2 domain to the erythropoietin receptor and plays a role in Lyn-mediated receptor signaling.

The erythropoietin (Epo) receptor transduces its signals by activating physically associated tyrosine kinases, mainly Jak2 and Lyn, and thereby inducing tyrosine phosphorylation of various substrates including the Epo receptor (EpoR) itself. We previously demonstrated that, in Epo-stimulated cells, an adapter protein, CrkL, becomes tyrosine-phosphorylated, physically associates with Shc, SHP-2, and Cbl, and plays a role in activation of the Ras/Erk signaling pathway. Here, we demonstrate that Epo induces binding of CrkL to the tyrosine-phosphorylated EpoR and SHIP1 in 32D/EpoR-Wt cells overexpressing CrkL. In vitro binding studies showed that the CrkL SH2 domain directly mediates the EpoR binding, which was specifically inhibited by a synthetic phosphopeptide corresponding to the amino acid sequences at Tyr(460) in the cytoplasmic domain of EpoR. The CrkL SH2 domain was also required for tyrosine phosphorylation of CrkL in Epo-stimulated cells. Overexpression of Lyn induced constitutive phosphorylation of CrkL and activation of Erk, whereas that of a Lyn mutant lacking the tyrosine kinase domain attenuated the Epo-induced phosphorylation of CrkL and activation of Erk. Furthermore, Lyn, but not Jak2, phosphorylated CrkL on tyrosine in in vitro kinase assays. Together, the present study suggests that, upon Epo stimulation, CrkL is recruited to the EpoR through interaction between the CrkL SH2 domain and phosphorylated Tyr(460) in the EpoR cytoplasmic domain and undergoes tyrosine phosphorylation by receptor-associated Lyn to activate the downstream signaling pathway leading to the activation of Erk and Elk-1.     

Helix 8 plays a crucial role in bradykinin B(2) receptor trafficking and signaling

Upon activation the human bradykinin B(2) receptor (B(2)R) acts as guanine nucleotide exchange factor for the G proteins G(q/11) and G(i). Thereafter, it gets phosphorylated by G protein-coupled receptor kinases (GRKs) and recruits β-arrestins, which block further G protein activation and promote B(2)R internalization via clathrin-coated pits. As for most G protein-coupled receptors of family A, an intracellular helix 8 after transmembrane domain 7 is also predicted for the B(2)R. We show here that disruption of helix 8 in the B(2)R by either C-terminal truncation or just by mutation of a central amino acid (Lys-315) to a helix-breaking proline resulted in strong reduction of surface expression. Interestingly, this malfunction could be overcome by the addition of the membrane-permeable B(2)R antagonist JSM10292, suggesting that helix 8 has a general role for conformational stabilization that can be accounted for by an appropriate antagonist. Intriguingly, an intact helix 8, but not the C terminus with its phosphorylation sites, was indispensable for receptor sequestration and for interaction of the B(2)R with GRK2/3 and β-arrestin2 as shown by co-immunoprecipitation. Recruitment of β-arrestin1, however, required the presence of the C terminus. Taken together, our results demonstrate that helix 8 of the B(2)R plays a crucial role not only in efficient trafficking to the plasma membrane or the activation of G proteins but also for the interaction of the B(2)R with GRK2/3 and β-arrestins. Additional data obtained with chimera of B(2)R with other G protein-coupled receptors of family A suggest that helix 8 might have similar functions in other GPCRs as well.     

Essential role of glycosaminoglycans in Fgf signaling during mouse gastrulation

In vitro studies have suggested that proteoglycans facilitate signaling by mammalian growth factors, but genetic evidence supporting this role has been lacking. Here, we characterize the ENU-induced mutation lazy mesoderm (lzme), which disrupts the single mouse gene encoding UDP-glucose dehydrogenase (Ugdh), an enzyme required for the synthesis of the glycosaminoglycan (GAG) side chains of proteoglycans. lzme mutants arrest during gastrulation with defects in migration of mesoderm and endoderm, a phenotype similar to that of mutants in the fibroblast growth factor (Fgf) pathway. Analysis of the expression of molecular markers indicates that Fgf signaling is blocked in lzme mutant embryos. In contrast, signaling by the growth factors Nodal and Wnt3, which are also essential during mouse gastrulation, appears to be normal in lzme embryos. The results demonstrate that proteoglycans are required during mouse gastrulation specifically to promote Fgf signaling.     

Notch signaling plays a key role in cardiac cell differentiation

Results from lineage tracing studies indicate that precursor cells in the ventricles give rise to both cardiac muscle and conduction cells. Cardiac conduction cells are specialized cells responsible for orchestrating the rhythmic contractions of the heart. Here, we show that Notch signaling plays an important role in the differentiation of cardiac muscle and conduction cell lineages in the ventricles. Notch1 expression coincides with a conduction marker, HNK-1, at early stages. Misexpression of constitutively active Notch1 (NIC) in early heart tubes in chick exhibited multiple effects on cardiac cell differentiation. Cells expressing NIC had a significant decrease in expression of cardiac muscle markers, but an increase in expression of conduction cell markers, HNK-1, and SNAP-25. However, the expression of the conduction marker connexin 40 was inhibited. Loss-of-function study, using a dominant-negative form of Suppressor-of-Hairless, further supports that Notch1 signaling is important for the differentiation of these cardiac cell types. Functional studies show that the expression of constitutively active Notch1 resulted in abnormalities in ventricular conduction pathway patterns.     

Phospholipase D1 plays a key role in TNF-alpha signaling

The primary characteristic features of any inflammatory or infectious lesions are immune cell infiltration, cellular proliferation, and the generation of proinflammatory mediators. TNF-alpha is a potent proinflammatory and immuno-regulatory cytokine. Decades of research have been focused on the physiological/pathophysiological events triggered by TNF-alpha. However, the signaling network initiated by TNF-alpha in human leukocytes is still poorly understood. In this study, we report that TNF-alpha activates phospholipase D1 (PLD1), in a dose-dependent manner, and PLD1 is required for the activation of sphingosine kinase and cytosolic calcium signals. PLD1 is also required for NFkappaB and ERK1/2 activation in human monocytic cells. Using antisense oligonucleotides to reduce specifically the expression of PLD isozymes showed PLD1, but not PLD2, to be coupled to TNF-alpha signaling and that PLD1 is required to mediate receptor activation of sphingosine kinase and calcium transients. In addition, the coupling of TNF-alpha to activation of the phosphorylation of ERK1/2 and the activation of NFkappaB were inhibited by pretreating cells with antisense to PLD1, but not to PLD2; thus, demonstrating a specific requirement for PLD1. Furthermore, use of antisense oligonucleotides to reduce expression of PLD1 or PLD2 demonstrated that PLD1 is required for TNF-alpha-induced production of several important cytokines, such as IL-1beta, IL-5, IL-6, and IL-13, in human monocytes. These studies demonstrate the critical role of PLD1 in the intracellular signaling cascades initiated by TNF-alpha and its functional role for coordinating the signals to inflammatory responses.     

Retinoic acid signaling plays a restrictive role in zebrafish primitive myelopoiesis

Retinoic acid (RA) is known to regulate definitive myelopoiesis but its role in vertebrate primitive myelopoiesis remains unclear. Here we report that zebrafish primitive myelopoiesis is restricted by RA in a dose dependent manner mainly before 11 hpf (hours post fertilization) when anterior hemangioblasts are initiated to form. RA treatment significantly reduces expressions of anterior hemangioblast markers scl, lmo2, gata2 and etsrp in the rostral end of ALPM (anterior lateral plate mesoderm) of the embryos. The result indicates that RA restricts primitive myelopoiesis by suppressing formation of anterior hemangioblasts. Analyses of ALPM formation suggest that the defective primitive myelopoiesis resulting from RA treatment before late gastrulation may be secondary to global loss of cells for ALPM fate whereas the developmental defect resulting from RA treatment during 10-11 hpf should be due to ALPM patterning shift. Overexpressions of scl and lmo2 partially rescue the block of primitive myelopoiesis in the embryos treated with 250 nM RA during 10-11 hpf, suggesting RA acts upstream of scl to control primitive myelopoiesis. However, the RA treatment blocks the increased primitive myelopoiesis caused by overexpressing gata4/6 whereas the abolished primitive myelopoiesis in gata4/5/6 depleted embryos is well rescued by 4-diethylamino-benzaldehyde, a retinal dehydrogenase inhibitor, or partially rescued by knocking down aldh1a2, the major retinal dehydrogenase gene that is responsible for RA synthesis during early development. Consistently, overexpressing gata4/6 inhibits aldh1a2 expression whereas depleting gata4/5/6 increases aldh1a2 expression. The results reveal that RA signaling acts downstream of gata4/5/6 to control primitive myelopoiesis. But, 4-diethylamino-benzaldehyde fails to rescue the defective primitive myelopoiesis in either cloche embryos or lycat morphants. Taken together, our results demonstrate that RA signaling restricts zebrafish primitive myelopoiesis through acting downstream of gata4/5/6, upstream of, or parallel to, cloche, and upstream of scl.     

Hedgehog signaling plays a cell-autonomous role in maximizing cardiac developmental potential

Elucidation of the complete roster of signals required for myocardial specification is crucial to the future of cardiac regenerative medicine. Prior studies have implicated the Hedgehog (Hh) signaling pathway in the regulation of multiple aspects of heart development. However, our understanding of the contribution of Hh signaling to the initial specification of myocardial progenitor cells remains incomplete. Here, we show that Hh signaling promotes cardiomyocyte formation in zebrafish. Reduced Hh signaling creates a cardiomyocyte deficit, and increased Hh signaling creates a surplus. Through fate-mapping, we find that Hh signaling is required at early stages to ensure specification of the proper number of myocardial progenitors. Genetic inducible fate mapping in mouse indicates that myocardial progenitors respond directly to Hh signals, and transplantation experiments in zebrafish demonstrate that Hh signaling acts cell autonomously to promote the contribution of cells to the myocardium. Thus, Hh signaling plays an essential early role in defining the optimal number of cardiomyocytes, making it an attractive target for manipulation of multipotent progenitor cells.     

Scavenger receptor BI plays a role in facilitating chylomicron metabolism

The function of scavenger receptor class B type I (SR-BI) in mediating the selective uptake of high density lipoprotein (HDL) cholesterol esters is well established. However, the potential role of SR-BI in chylomicron and chylomicron remnant metabolism is largely unknown. In the present investigation, we report that the cell association of 160 nm-sized triglyceride-rich chylomicron-like emulsion particles to freshly isolated hepatocytes from SR-BI-deficient mice is greatly reduced (>70%), as compared with wild-type littermate mice. Competition experiments show that the association of emulsion particles with isolated hepatocytes is efficiently competed for (>70%) by the well established SR-BI ligands, HDL and oxidized low density lipoprotein (LDL), whereas LDL is ineffective. Upon injection into SR-BI-deficient mice the hepatic association of emulsion particles is markedly decreased ( approximately 80%) as compared with wild-type mice. The relevance of these findings for in vivo chylomicron (remnant) metabolism was further evaluated by studying the effect of SR-BI deficiency on the intragastric fat load-induced postprandial triglyceride response. The postprandial triglyceride response is 2-fold higher in SR-BI-deficient mice as compared with wild-type littermates (area-under-the-curve 39.6 +/- 1.2 versus 21.1 +/- 3.6; p < 0.005), with a 4-fold increased accumulation of chylomicron (remnant)-associated triglycerides in plasma at 6 h after intragastric fat load. We conclude that SR-BI is important in facilitating chylomicron (remnant) metabolism and might function as an initial recognition site for chylomicron remnants whereby the subsequent internalization can be exerted by additional receptor systems like the LDL receptor and LDL receptor-related protein.     

A direct role for Fgf but not Wnt in otic placode induction

Induction of the otic placode, which gives rise to all tissues comprising the inner ear, is a fundamental aspect of vertebrate development. A number of studies indicate that fibroblast growth factor (Fgf), especially Fgf3, is necessary and sufficient for otic induction. However, an alternative model proposes that Fgf must cooperate with Wnt8 to induce otic differentiation. Using a genetic approach in zebrafish, we tested the roles of Fgf3, Fgf8 and Wnt8. We demonstrate that localized misexpression of either Fgf3 or Fgf8 is sufficient to induce ectopic otic placodes and vesicles, even in embryos lacking Wnt8. Wnt8 is expressed in the hindbrain around the time of otic induction, but loss of Wnt8 merely delays expression of preotic markers and otic vesicles form eventually. The delay in otic induction correlates closely with delayed expression of fgf3 and fgf8 in the hindbrain. Localized misexpression of Wnt8 is insufficient to induce ectopic otic tissue. By contrast, global misexpression of Wnt8 causes development of supernumerary placodes/vesicles, but this reflects posteriorization of the neural plate and consequent expansion of the hindbrain expression domains of Fgf3 and Fgf8. Embryos that misexpress Wnt8 globally but are depleted for Fgf3 and Fgf8 produce no otic tissue. Finally, cells in the preotic ectoderm express Fgf (but not Wnt) reporter genes. Thus, preotic cells respond directly to Fgf but not Wnt8. We propose that Wnt8 serves to regulate timely expression of Fgf3 and Fgf8 in the hindbrain, and that Fgf from the hindbrain then acts directly on preplacodal cells to induce otic differentiation.     

Angiopoietin 2 signaling plays a critical role in neural crest cell migration

Background

Collective neural crest cell migration is critical to the form and function of the vertebrate face and neck, distributing bone, cartilage, and nerve cells into peripheral targets that are intimately linked with head vasculature. The vasculature and neural crest structures are ultimately linked, but when and how these patterns develop in the early embryo are not well understood.

Results

Using in vivo imaging and sophisticated cell behavior analyses, we show that quail cranial neural crest and endothelial cells share common migratory paths, sort out in a dynamic multistep process, and display multiple types of motion. To better understand the underlying molecular signals, we examined the role of angiopoietin 2 (Ang2), which we found expressed in migrating cranial neural crest cells. Overexpression of Ang2 causes neural crest cells to be more exploratory as displayed by invasion of off-target locations, the widening of migratory streams into prohibitive zones, and differences in cell motility type. The enhanced exploratory phenotype correlates with increased phosphorylated focal adhesion kinase activity in migrating neural crest cells. In contrast, loss of Ang2 function reduces neural crest cell exploration. In both gain and loss of function of Ang2, we found disruptions to the timing and interplay between cranial neural crest and endothelial cells.

Conclusions

Together, these data demonstrate a role for Ang2 in maintaining collective cranial neural crest cell migration and suggest interdependence with endothelial cell migration during vertebrate head patterning.

     

Follicle-stimulating hormone plays a role in the induction of ovarian follicular cysts in hypophysectomized rats.

Unabated stimulation by low doses of LH-like activity produces ovarian follicular cysts in both progesterone-synchronized immature rats and pregnant rats. Serum FSH is maintained in both of these models at values similar to those observed on diestrus. To determine whether unabated stimulation by basal serum FSH affects the ability of LH-like activity to induce cystic ovaries, immature hypophysectomized (HYPOXD) rats were given either no hormone (control); 2 micrograms ovine FSH (oFSH) once daily for 14 days beginning on Day 27; 0.5 IU hCG twice daily for 13 days beginning on Day 28 of age; or both oFSH and hCG (FSH + hCG) beginning on Day 27 and Day 28, respectively. By the end of the in vivo treatments (Day 40 of age), the largest follicles in the ovaries of control and hCG-treated HYPOXD rats were at the preantral stage of development, whereas the largest follicles present in ovaries from FSH-treated animals were atretic and at the small antral stage of development. In contrast, ovaries from rats treated with FSH + hCG displayed large follicular cysts by Day 37 of age. Of the serum steroids analyzed, only estradiol and androstenedione concentrations for animals treated with FSH + hCG were consistently elevated above values observed for control HYPOXD rats. Serum testosterone and dihydrotestosterone values were similar for hCG-treated and control HYPOXD rats throughout the in vivo treatments. In contrast, these steroids were elevated between Days 3 and 5 of FSH treatment (+/- hCG treatment). Serum progesterone and estrone values for all in vivo gonadotropin treatment groups were similar to those of controls. Serum insulin concentrations were not affected by any in vivo treatment. Incubates of follicles/cysts from FSH + hCG-treated HYPOXD rats contained more progesterone, androstenedione, and estradiol than incubates of follicles from any other in vivo treatment group. Follicles from all in vivo treatment groups responded to 8-bromo cAMP (cAMP) with increased in vitro progesterone accumulation. However, only follicles from FSH-treated and FSH + hCG-treated rats responded to cAMP with increased androstenedione and estradiol accumulation in vitro. Inclusion of 400 ng of either androstenedione or testosterone in the incubation medium enhanced progesterone accumulation in follicular incubates from control, hCG-treated, and FSH-treated HYPOXD rats, but did not enhance progesterone accumulation in follicular incubates from FSH + hCG-treated animals. Both androstenedione and estradiol production increased markedly under these conditions for follicles from all in vivo treatment groups.(ABSTRACT TRUNCATED AT 400 WORDS)     

Caveolin-1-mediated negative signaling plays a critical role in the induction of regulatory dendritic cells by DNA and protein coimmunization

Induction of Ag-specific regulatory T cells (iTregs) by vaccination is a promising strategy for treating autoimmune diseases. We previously demonstrated that DNA and protein covaccination converted naive T cells to Ag-specific iTregs by inducing CD11c(+)CD40(low)IL-10(+) regulatory dendritic cells (DCregs). However, it is unclear how coimmunization induces the DCregs. In this paper, we report that the event is initiated by coentry of sequence-matched DNA and protein immunogens into the same DC via caveolae-mediated endocytosis, which leads to inhibition of phosphorylation of caveolin-1 (Cav-1), the main component of caveolae, and upregulation of Tollip. This triggers downstream signaling that upregulates suppressor of cytokine signaling 1 and downregulates NF-κB and STAT-1α. Silencing either Cav-1 or Tollip blocks the negative signaling, leading to upregulated expression of CD40, downregulated production of IL-10, and loss of iTreg-inducing function. We further show that DCregs can be induced in culture from primary DCs and JAWS II DC lines by feeding them sequence-matched DNA and protein immunogens. The in vitro-generated DCregs are effective in ameliorating autoimmune and inflammatory diseases in several mouse models. Our study thus suggests that DNA and protein coimmunization induces DCregs through Cav-1- and Tollip-mediated negative signaling. It also describes a novel method for generating therapeutic DCregs in vitro.