A novel role for hSMG-1 in stress granule formation

hSMG-1 is a member of the phosphoinositide 3 kinase-like kinase (PIKK) family with established roles in nonsense-mediated decay (NMD) of mRNA containing premature termination codons and in genotoxic stress responses to DNA damage. We report here a novel role for hSMG-1 in cytoplasmic stress granule (SG) formation. Exposure of cells to stress causing agents led to the localization of hSMG-1 to SG, identified by colocalization with TIA-1, G3BP1, and eIF4G. hSMG-1 small interfering RNA and the PIKK inhibitor wortmannin prevented formation of a subset of SG, while specific inhibitors of ATM, DNA-PK(cs), or mTOR had no effect. Exposure of cells to H(2)O(2) and sodium arsenite induced (S/T)Q phosphorylation of proteins. While Upf2 and Upf1, an essential substrate for hSMG-1 in NMD, were present in SG, NMD-specific Upf1 phosphorylation was not detected in SG, indicating hSMG-1's role in SG is separate from classical NMD. Thus, SG formation appears more complex than originally envisaged and hSMG-1 plays a central role in this process.     

A novel role for 14-kDa phosphohistidine phosphatase in lamellipodia formation

Cell migration involves dynamic regulation of the actin cytoskeleton, which exhibits rapid actin polymerization at the leading edge of migrating cells. This process relies on regulated recruitment of actin nucleators and actin-binding proteins to the leading edge to polymerize new actin filaments. Many of these proteins have been identified, including the actin-related protein (Arp) 2/3 complex, which has emerged as the core player in the initiation of actin polymerization. However, the functional coordination of these proteins is unclear. Previously, we have demonstrated that the 14-kDa phosphohistidine phosphatase (PHP14) is involved in cell migration regulation and affects actin cytoskeleton reorganization. Here, we show that PHP14 may regulate actin remodeling directly and play an important role in dynamic regulation of the actin cytoskeleton. We observed a colocalization of PHP14 with Arp3 and F-actin at the leading edge of migrating cells. Moreover, PHP14 was recruited to the actin remodeling sites in parallel with Arp3 during lamellipodia formation. Furthermore, PHP14 knockdown impaired Arp3 localization at the leading edge of lamellipodia, as well as lamellipodia formation. Most importantly, we found that PHP14 was a novel F-actin-binding protein, displaying an Arp2/3-dependent localization to the leading edge. Collectively, our results indicated a crucial role for PHP14 in the dynamic regulation of the actin cytoskeleton and cell migration.     

The divergent expression of periostin mRNA in the periodontal ligament during experimental tooth movement

A novel 90-kDa protein named periostin, which is preferentially expressed in the periosteum and the periodontal ligament (PDL), may play a role in bone metabolism and remodeling. However, the precise role of periostin in the PDL remains unclear. Therefore, we examined the expression of periostin mRNA during experimental tooth movement. Experimental tooth movement was achieved in 7-week-old male Sprague-Dawley rats. In control specimens without tooth movement, the expression of periostin mRNA was uniformly observed in the PDL surrounding the mesial and distal roots of the upper molars and was weak in the PDL of the root furcation area. The periostin mRNA-expressing cells were mainly fibroblastic cells in the PDL and osteoblastic cells on the alveolar bone surfaces. The divergent expression of periostin mRNA in the PDL began to be observed at 3 h and continued up to 96 h after tooth movement. The maximum changes, which showed stronger staining in the pressure sites than in the tension sites, were observed at 24 h. The expression of periostin mRNA in the PDL 168 h after tooth movement exhibited a similar distribution to that of the control specimens. These results suggest that periostin is one of the local contributing factors in bone and periodontal tissue remodeling following mechanical stress during experimental tooth movement.     

A novel regulatory role of TRAPPC9 in L-plastin-mediated osteoclast actin ring formation

Trafficking protein particle complex 9 (TRAPPC9) is a major subunit of the TRAPPII complex. TRAPPC9 has been reported to bind nuclear factor κB kinase subunit β (IKKβ) and NF-kB-inducing kinase (NIK) where it plays a role in the canonical and noncanonical of nuclear factor-κB (NF-kB) signaling pathways, receptively. The role of TRAPPC9 in protein trafficking and cytoskeleton organization in osteoclast (OC) has not been studied yet. In this study, we examined the mRNA expression of TRAPPC9 during OC differentiation. Next, we examined the colocalization of TRAPPC9 with cathepsin-K, known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking pathway within OC. To identify TRAPPC9 protein partners important for OC-mediated cytoskeleton re-organization, we conducted immunoprecipitation of TRAPPC9 in mature OCs followed by mass spectrometry analysis. Our data showed that TRAPPC9 binds various protein partners. One protein with high recovery rate is L-plastin (LPL). LPL localizes at the podosomes and reported to play a crucial role in actin aggregation thereby actin ring formation and OC function. Although the role of LPL in OC-mediated bone resorption has not fully reported in detail. Here, first, we confirmed the binding of LPL to TRAPPC9 and, then, we investigated the potential regulatory role of TRAPPC9 in LPL-mediated OC cytoskeleton reorganization. We assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is colocalized with LPL at the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression on LPL recruitment to the actin ring using a viral system. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared with control cultures. In addition, we observed that TRAPPC9 overexpression reorganizes actin clusters/aggregates and regulates vinculin recruitment into the OC periphery to initiate podosome formation.     

A novel role for cytochrome c: Efficient catalysis of S-nitrosothiol formation

Although S-nitrosothiols are regarded as important elements of many NO-dependent signal transduction pathways, the physiological mechanism of their formation remains elusive. Here, we demonstrate a novel mechanism by which cytochrome c may represent an efficient catalyst of S-nitrosation in vivo. In this mechanism, initial binding of glutathione to ferric cytochrome c is followed by reaction of NO with this complex, yielding ferrous cytochrome c and S-nitrosoglutathione (GSNO). We show that when submitochondrial particles or cell lysates are exposed to NO in the presence of cytochrome c, there is a robust formation of protein S-nitrosothiols. In the case of submitochondrial particles protein S-nitrosation is paralleled by an inhibition of mitochondrial complex I. These observations raise the possibility that cytochrome c is a mediator of S-nitrosation in biological systems, particularly during hypoxia, and that release of cytochrome c into the cytosol during apoptosis potentially releases a GSNO synthase activity that could modulate apoptotic signaling.     

Identification of a novel putative signaling center,the tertiary enamel knot in the postnatal mouse molar tooth

The final shape of the molar tooth crown is thought to be regulated by the transient epithelial signaling centers in the cusp tips, the secondary enamel knots (SEKs), which are believed to disappear after initiation of the cusp growth. We investigated the developmental fate of the signaling center using the recently characterized Slit1 enamel knot marker as a lineage tracer during morphogenesis of the first molar and crown calcification in the mouse. In situ hybridization analysis showed that after Fgf4 downregulation in the SEK, Slit1 expression persisted in the deep compartment of the knot. After the histological disappearance of the SEK, Slit1 expression was evident in a novel epithelial cell cluster, which we call the tertiary enamel knot (TEK) next to the enamel-free area (EFA)-epithelium at the cusp tips. In embryonic tooth, Slit1 was also observed in the stratum intermedium (SI) and stellate reticulum cells between the parallel SEKs correlating to the area where the inner enamel epithelium cells do not proliferate. After birth, the expression of Slit1 persisted in the SI cells of the transverse connecting lophs of the parallel cusps above the EFA-cells. These results demonstrate the presence of a novel putative signaling center, the TEK, in the calcifying tooth. Moreover, our results suggest that Slit1 signaling may be involved in the regulation of molar tooth shape by regulating epithelial cell proliferation and formation of EFA of the crown.     

A novel role for Niemann-Pick disease type 2C protein in papillae formation

Background

Despite the presence of papillary structures and papillary tumors in humans, the mechanism of papillae formation is unknown. We describe herein a novel role for Niemann-Pick disease type 2C (NPC2) protein, a cholesterol binding protein in the lysosome, in papillae formation.

Methodology/Principal Finding

We examined NPC2 protein expression in surgical samples of papillary tissues by immunohistochemical stain, and all papillary tissues expressed NPC2 protein in the epithelium. To examine our hypothesis of NPC2 protein-mediated papillae formation, we carried out xenograft experiments using wild H460 cells (large cell lung carcinoma cell line) that constitutively expressed abundant NPC2 protein and NPC2 protein-depleted H460 cells by NPC2 shRNA. The xenografts of wild H460 cells and empty shRNA vector cells showed distinct papillae formation, whereas NPC2 protein-depleted H460 cells displayed markedly reduced or no papillae. Since all papillary tissues have open spaces we examined whether NPC2 protein might also contribute to the creation of open spaces. The TUNEL assay in the xenografts of wild and empty shRNA vector H460 cells showed massive cell death, and NPC2 protein-depleted cells displayed minimal cell death. Measurement of caspase 3/7 activities in cultured H460 cells supported NPC2 protein-mediated apoptotic cell death. The presence of excess NPC2 protein, however, did not always produce papillae as seen in the xenografts of CHO cells that were stably transfected with NPC2.

Conclusions/Significance

The NPC2 protein of certain cells forms papillae coupled with apoptosis that creates open space. This protein may have future applications to modulate papillae formation and papillary growth in tumor tissues.     

A potential role for tetranectin in mineralization during osteogenesis

Tetranectin is a protein shared by the blood and the extracellular matrix. Tetranectin is composed of four identical, noncovalently bound polypeptides each with a molecular mass of approximately 21 kD. There is some evidence that tetranectin may be involved in fibrinolysis and proteolysis during tissue remodeling, but its precise biological function is not known. Tetranectin is enriched in the cartilage of the shark, but the gene expression pattern in the mammalian skeletal system has not been determined. In the present study we have examined the expression pattern and putative function of tetranectin during osteogenesis. In the newborn mouse, strong tetranectin immunoreactivity was found in the newly formed woven bone around the cartilage anlage in the future bone marrow and along the periosteum forming the cortex. No tetranectin immunoreactivity was found in the proliferating and hypertrophic cartilage or in the surrounding skeletal muscle. Using an in vitro mineralizing system, we examined osteoblastic cells at different times during their growth and differentiation. Tetranectin mRNA appeared in the cultured osteoblastic cells in parallel with mineralization, in a pattern similar to that of bone sialoprotein, which is regarded as one of the late bone differentiation markers. To explore the putative biological role of tetranectin in osteogenesis we established stably transfected cell lines (PC12-tet) overexpressing recombinant tetranectin as evidenced by Northern and Western blot analysis and immunoprecipitation. Both control PC12 cells and PC12-tet cells injected into nude mice produced tumors containing bone material, as evidenced by von Kossa staining for calcium and immunostaining with bone sialoprotein and alkaline phosphatase antiserum. Nude mice tumors established from PC12-tet cells contained approximately fivefold more bone material than those produced by the untransfected PC12 cell line or by the PC12 cells transfected with the expression vector with no insert (Mann Whitney rank sum test, p < 0.01), supporting the notion that tetranectin may play an important direct and/or indirect role during osteogenesis. In conclusion, we have established a potential role for tetranectin as a bone matrix protein expressed in time and space coincident with mineralization in vivo and in vitro.     

A model of postnatal formation of alveoli in rat lung.

In rats, and many other species, most lung alveoli are formed after birth. Septation of the large air saccules existing at birth has been considered as the main mechanism for alveoli formation. However, other undefined means of alveolarization have also been postulated to account for the large increase in gas-exchange surface area that takes place in the lung as the rat grows larger. Moreover, recent results show that the majority of alveoli in rat lung are formed by means other than septation of saccules existing at birth, but these mechanisms have not been identified up to the present. In this study, a mathematical model of alveolarization in rat lung is presented. The model is based on three postulates: (a) new saccules continue to be formed up to adulthood according to certain rules; (b) all these saccules subsequently septate generating a certain number of alveoli; (c) once formed, the saccules (and alveoli) do not change in volume, but newly-formed saccules are larger than the preceding ones according to a given law. The model accurately predicts the experimentally-known values at different ages of total alveolar volume, alveolar number, volume of the average alveolus, gas-exchange surface area, and alveolar volume distribution for normal rats and for rats in which septation is inhibited by treatment with dexamethasone or hypoxia during the early postnatal weeks of life.     

A dual role for Ikk alpha in tooth development

IKK alpha is a component of the I kappa B kinase (IKK) complex that plays a key role in the activation of NF-kappa B. In Ikk alpha mutant mice and mice expressing a transdominant negative mutant of I kappa B alpha (cI kappa B alpha Delta N), molars have abnormal cusps, indicating that Ikk alpha is involved in cusp formation through the NF-kappa B pathway. However, Ikk alpha mutant incisors also have an earlier phenotype where epithelium evaginates outward into the developing oral cavity rather than invaginating into the underlying mesenchyme. A similar evagination of epithelium was also observed in whisker development, suggesting that Ikk alpha contributes to the direction of epithelial growth during the early stages of development in many ectodermal appendages. Since cI kappa B alpha Delta N mice have normal incisor epithelial invagination, Ikk alpha's role appears to be NF-kappa B independent. Changes in Notch1, Notch2, Wnt7b, and Shh expression found in incisor epithelium of Ikk alpha mutants suggest that this NF-kappa B-independent function is mediated by Notch/Wnt/Shh signaling pathways.     

Mechanisms of leaf tooth formation in Arabidopsis

Serration found along leaf margins shows species‐specific characters. Whereas compound leaf development is well studied, the process of serration formation is largely unknown. To understand mechanisms of serration development, we investigated distinctive features of cells that could give rise to tooth protrusion in the simple‐leaf plant Arabidopsis. After the emergence of a tooth, marginal cells, except for cells at the sinuses and tips, started to elongate rapidly. Localized cell division seemed to keep cells at the sinus smaller, rather than halt cell elongation. As leaves matured, the marginal cell number between teeth became similar in any given tooth. These results suggest that teeth are formed by repetition of an unknown mechanism that spatially monitors cell number and regulates cell division. We then examined the role of CUP‐SHAPED COTYLEDON 2 (CUC2) in serration development. cuc2‐3 forms fewer hydathodes and auxin maxima, visualized by DR5rev::GFP, at the leaf margin, suggesting that CUC2 patterns serration through the regulation of auxin. In contrast to a previous interpretation, comparison of leaf outlines revealed that CUC2 promotes outgrowth of teeth rather than suppression of growth at the sinuses. We found that mutants with increased CUC2 expression form ectopic tissues and mis‐express SHOOT MERISTEMLESS (STM) at the sinus between the enhanced teeth. Similar but infrequent STM expression was found in the wild type, indicating STM involvement in the serration of simple leaves. Our study provides insights into the morphological and molecular mechanisms for leaf development and tooth formation, and highlights similarities between serration and compound leaf development.     

Unicuspid and bicuspid tooth crown formation in squamates

The molecular and developmental factors that regulate tooth morphogenesis in nonmammalian species, such as snakes and lizards, have received relatively little attention compared to mammals. Here we describe the development of unicuspid and bicuspid teeth in squamate species. The simple, cone-shaped tooth crown of the bearded dragon and ball python is established at cap stage and fixed in shape by the differentiation of cells and the secretion of dental matrices. Enamel production, as demonstrated by amelogenin expression, occurs relatively earlier in squamate teeth than in mouse molars. We suggest that the early differentiation in squamate unicuspid teeth at cap stage correlates with a more rudimentary tooth crown shape. The leopard gecko can form a bicuspid tooth crown despite the early onset of differentiation. Cusp formation in the gecko does not occur by the folding of the inner enamel epithelium, as in the mouse molar, but by the differential secretion of enamel. Ameloblasts forming the enamel epithelial bulge, a central swelling of cells in the inner enamel epithelium, secrete amelogenin at cap stage, but cease to do so by bell stage. Meanwhile, other ameloblasts in the inner enamel epithelium continue to secrete enamel, forming cusp tips on either side of the bulge. Bulge cells specifically express the gene Bmp2, which we suggest serves as a pro-differentiation signal for cells of the gecko enamel organ. In this regard, the enamel epithelial bulge of the gecko may be more functionally analogous to the secondary enamel knot of mammals than the primary enamel knot.     

A novel role for VEGF in endocardial cushion formation and its potential contribution to congenital heart defects

Normal cardiovascular development is exquisitely dependent on the correct dosage of the angiogenic growth factor and vascular morphogen vascular endothelial growth factor (VEGF). However, cardiac expression of VEGF is also robustly augmented during hypoxic insults, potentially mediating the well-established teratogenic effects of hypoxia on heart development. We report that during normal heart morphogenesis VEGF is specifically upregulated in the atrioventricular (AV) field of the heart tube soon after the onset of endocardial cushion formation (i.e. the endocardium-derived structures that build the heart septa and valves). To model hypoxia-dependent induction of VEGF in vivo, we conditionally induced VEGF expression in the myocardium using a tetracycline-regulated transgenic system. Premature induction of myocardial VEGF in E9.5 embryos to levels comparable with those induced by hypoxia prevented formation of endocardial cushions. When added to explanted embryonic AV tissue, VEGF fully inhibited endocardial-to-mesenchymal transformation. Transformation was also abrogated in AV explants subjected to experimental hypoxia but fully restored in the presence of an inhibitory soluble VEGF receptor 1 chimeric protein. Together, these results suggest a novel developmental role for VEGF as a negative regulator of endocardial-to-mesenchymal transformation that underlies the formation of endocardial cushions. Moreover, ischemia-induced VEGF may be the molecular link between hypoxia and congenital defects in heart septation.     

A novel model for cyanobacteria bloom formation: the critical role of anoxia and ferrous iron

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Akt signaling and its role in postnatal neovascularization

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Functional role of periostin in development and wound repair: implications for connective tissue disease

Integrity of the extracellular matrix (ECM) is essential for maintaining the normal structure and function of connective tissues. ECM is secreted locally by cells and organized into a complex meshwork providing physical support to cells, tissues, and organs. Initially thought to act only as a scaffold, the ECM is now known to provide a myriad of signals to cells regulating all aspects of their phenotype from morphology to differentiation. Matricellular proteins are a class of ECM related molecules defined through their ability to modulate cell-matrix interactions. Matricellular proteins are expressed at high levels during development, but typically only appear in postnatal tissue in wound repair or disease, where their levels increase substantially. Members of the CCN family, tenascin-C, osteopontin, secreted protein acidic rich in cysteine (SPARC), bone sialoprotein, thrombospondins, and galectins have all been classed as matricellular proteins. Periostin, a 90 kDa secreted homophilic cell adhesion protein, was recently added to matricellular class of proteins based on its expression pattern and function during development as well as in wound repair. Periostin is expressed in connective tissues including the periodontal ligament, tendons, skin and bone, and is also prominent in neoplastic tissues, cardiovascular disease, as well as in connective tissue wound repair. This review will focus on the functional role of periostin in tissue physiology. Fundamentally, it appears that periostin influences cell behaviour as well as collagen fibrillogenesis, and therefore exerts control over the structural and functional properties of connective tissues in both health and disease. Periostin is a novel matricellular protein with close homology to Drosophila fasciclin 1. In this review, the functional role of periostin is discussed in the context of connective tissue physiology, in development, disease, and wound repair.