Structure and Innervation of the Inner Ear Sensory Organs in an Otophysine Fish,the Upside–down Catfish (Synodontis nigriventrisDavid)

All the sensory epithelia of the inner ear in the upside–down catfish (Synodontis nigriventrisDavid) were examined by light microscopy. The morphology of the membranous labyrinth and the orientation of the hair cells is similar to what has been found in other otophysine fishes. The sensory cells are of variable size both inter– and intraepithelially; particularly the macula sacculi is equipped with heterogeneous receptors. Regional differences in the hair cell density are presented for all the otolith organs plus the papilla neglecta. Nerve stainings reveal regional differentiation. The central areas are innervated by stout and stubbly nerve endings intermingled with a few thin nerve fibres while the peripheral parts are reached exclusively by thin axons. In the anterior region of the macula sacculi are found unique cup–shaped axon terminations which surround the basal parts of a single or a few sensory cells. The number and diameter range of the myelinated nerve fibres as well as the hair cell/axon ratio are presented. Electron microscopy demonstrates the presence of unmyelinated axons in all inner ear nerve ramuli.     

Proteolytic Processing Regulates Placental Growth Factor Activities

Placental growth factor (PlGF) is a critical mediator of blood vessel formation, yet mechanisms of its action and regulation are incompletely understood. Here we demonstrate that proteolytic processing regulates the biological activity of PlGF. Specifically, we show that plasmin processing of PlGF-2 yields a protease-resistant core fragment comprising the vascular endothelial growth factor receptor-1 binding site but lacking the carboxyl-terminal domain encoding the heparin-binding domain and an 8-amino acid peptide encoded by exon 7. We have identified plasmin cleavage sites, generated a truncated PlGF118 isoform mimicking plasmin-processed PlGF, and explored its biological function in comparison with that of PlGF-1 and -2. The angiogenic responses induced by the diverse PlGF forms were distinct. Whereas PlGF-2 increased endothelial cell chemotaxis, vascular sprouting, and granulation tissue formation upon skin injury, these activities were abrogated following plasmin digestion. Investigation of PlGF/Neuropilin-1 binding and function suggests a critical role for heparin-binding domain/Neuropilin-1 interaction and its regulation by plasmin processing. Collectively, here we provide new mechanistic insights into the regulation of PlGF-2/Neuropilin-1-mediated tissue vascularization and growth.     

外力作为信号诱导基因的选择性剪接与力生长因子表达

许多种类的细胞都响应力信号,人们将这些细胞称为力效应细胞(mechanocyte).应力可引起细胞在基因水平或表达水平的调控,其中胰岛素样生长因子Ⅰ(insulin-like growth factor-Ⅰ,IGFⅠ)是力学敏感因子.对骨骼肌的长期拉伸实验发现,IGF-Ⅰ不仅表达量受到拉伸刺激的调控,而且存在多种变异体形式,其中一种对力刺激敏感,只在拉伸作用下产生,命名为力生长因子(mechano growth factor,MGF).进一步研究发现,MGF能激活卫星细胞、促进成肌细胞增殖,在治疗肌损失、预防心肌损伤和修复神经损伤等方面有重要的作用.机械拉伸也可以使成骨细胞表达MGF,研究表明,对成骨细胞施加应变为15%的周期性拉伸刺激,细胞的IGFⅠ表达量增加,同时表达MGF剪接变异体.对MGF的深入研究可望在疾病治疗和组织工程修复领域取得广泛的应用.     

The F-BAR Protein PACSIN2 Regulates Epidermal Growth Factor Receptor Internalization

Signaling via growth factor receptors, including the epidermal growth factor (EGF) receptor, is key to various cellular processes, such as proliferation, cell survival, and cell migration. In a variety of human diseases such as cancer, aberrant expression and activation of growth factor receptors can lead to disturbed signaling. Intracellular trafficking is crucial for proper signaling of growth factor receptors. As a result, the level of cell surface expression of growth factor receptors is an important determinant for the outcome of downstream signaling. BAR domain-containing proteins represent an important family of proteins that regulate membrane dynamics. In this study, we identify a novel role for the F-BAR protein PACSIN2 in the regulation of EGF receptor signaling. We show that internalized EGF as well as the (activated) EGF receptor translocated to PACSIN2-positive endosomes. Furthermore, loss of PACSIN2 increased plasma membrane expression of the EGF receptor in resting cells and increased EGF-induced phosphorylation of the EGF receptor. As a consequence, EGF-induced activation of Erk and Akt as well as cell proliferation were enhanced in PACSIN2-depleted cells. In conclusion, this study identifies a novel role for the F-BAR-domain protein PACSIN2 in regulating EGF receptor surface levels and EGF-induced downstream signaling.     

Rac Regulates Vascular Endothelial Growth Factor Stimulated Motility

During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood.

Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF.

These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent. VEGF stimulated chemotaxis, is critically dependent on Rac activation. Osteopontin was a potent matrix activator of motility, and perhaps one explanation for the absence of a VEGF plus osteopontin effect is that osteopontin stimulated motility was inhibitory to the Rac pathway.     

Rac Regulates Vascular Endothelial Growth Factor Stimulated Motility

During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood.

Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF.

These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent. VEGF stimulated chemotaxis, is critically dependent on Rac activation. Osteopontin was a potent matrix activator of motility, and perhaps one explanation for the absence of a VEGF plus osteopontin effect is that osteopontin stimulated motility was inhibitory to the Rac pathway.     

The Distribution of Nerve Growth Factor in the Male Sex Organs of Mammals

Abstract: The Nerve Growth Factor (NGF) content of male sex organs of the mouse, rat, guinea pig, hamster, rabbit, human, and bull has been investigated using both a biological assay and a two-site radioimmunoassay. The prostate glands of the rabbit and bull have been found to contain moderate levels of NGF, these being lower than the concentrations found in the guinea pig prostate and mouse submaxillary glands. The sex organs investigated of the mouse, rat, hamster, and human contained no detectable NGF activity. Genital organs, other than the prostate glands, of the guinea pig and rabbit were also devoid of NGF. The NGFs from the rabbit and bull are immunologically related to those found in the submaxillary glands of the mouse and the prostate glands of the guinea pig, but immunodiffusion and radioimmunoassay experiments show that there are also clear differences between the NGFs. The use of a two-site radioimmunoassay, based on purified antibodies against mouse submaxillary gland NGF, for the determination of NGF levels in species other than the mouse, is described. It is essential during such applications to compensate for the fact that the NGFs from different species are sufficiently distinct that only part of the antibody population (raised against mouse NGF) is capable of recognizing NGF from species other than the mouse. The results of radioimmunoassay and biological assay determinations are in reasonable agreement, if corrections for this feature are made.     

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.Open in a separate windowClick here to view.^{(51M, flv)}     

Disulfide Bond Formation Between Nerve Growth Factor and the Nerve Growth Factor Receptor from Embryonic Sensory Neurons

Recent studies with sympathetic neurons using radiolabeled nerve growth factor have indicated that a high-molecular-weight covalent complex is formed. This complex is between the nerve growth factor and the high-affinity (type I) receptor and occurs through the formation of a disulfide bond. Studies presented in the present article demonstrate a similar complex is formed on chicken embryonic sensory neurons. The formation of this complex is inhibited by the addition of unlabeled nerve growth factor, metabolic energy inhibitors (dinitrophenol and NaF), and of sulfhydryl reagents. On the other hand, formation of this complex is not inhibited by temperature, or by the addition of insulin or epidermal growth factor. The receptor involved in the covalent complex formation is the high-affinity (type I) receptor. The molecular weight of this complex is approximately 232,000 daltons. Evidence indicates that this covalent complex may be required for the biological activity of the nerve growth factor.     

The DNA Replication Licensing Factor Miniature Chromosome Maintenance 7 Is Essential for RNA Splicing of Epidermal Growth Factor Receptor,c-Met,and Platelet-derived Growth Factor Receptor

Miniature chromosome maintenance 7 (MCM7) is an essential component of DNA replication licensing complex. Recent studies indicate that MCM7 is amplified and overexpressed in a variety of human malignancies. In this report, we show that MCM7 binds SF3B3. The binding motif is located in the N terminus (amino acids 221–248) of MCM7. Knockdown of MCM7 or SF3B3 significantly increased unspliced RNA of epidermal growth factor receptor, platelet-derived growth factor receptor, and c-Met. A dramatic drop of reporter gene expression of the oxytocin exon 1-intron-exon 2-EGFP construct was also identified in SF3B3 and MCM7 knockdown PC3 and DU145 cells. The MCM7 or SF3B3 depleted cell extract failed to splice reporter RNA in in vitro RNA splicing analyses. Knockdown of SF3B3 and MCM7 leads to an increase of cell death of both PC3 and DU145 cells. Such cell death induction is partially rescued by expressing spliced c-Met. To our knowledge, this is the first report suggesting that MCM7 is a critical RNA splicing factor, thus giving significant new insight into the oncogenic activity of this protein.     

Nerve Growth Factor Regulates Neurolymphatic Remodeling during Corneal Inflammation and Resolution

The cellular and physiologic mechanisms that regulate the resolution of inflammation remain poorly defined despite their widespread importance in improving inflammatory disease outcomes. We studied the resolution of two cardinal signs of inflammation–pain and swelling–by investigating molecular mechanisms that regulate neural and lymphatic vessel remodeling during the resolution of corneal inflammation. A mouse model of corneal inflammation and wound recovery was developed to study this process in vivo. Administration of nerve growth factor (NGF) increased pain sensation and inhibited neural remodeling and lymphatic vessel regression processes during wound recovery. A complementary in vivo approach, the corneal micropocket assay, revealed that NGF-laden pellets stimulated lymphangiogenesis and increased protein levels of VEGF-C. Adult human dermal lymphatic endothelial cells did not express canonical NGF receptors TrkA and p75^NTR or activate downstream MAPK- or Akt-pathway effectors in the presence of NGF, although NGF treatment increased their migratory and tubulogenesis capacities in vitro. Blockade of the VEGF-R2/R3 signaling pathway ablated NGF-mediated lymphangiogenesis in vivo. These findings suggest a hierarchical relationship with NGF functioning upstream of the VEGF family members, particularly VEGF-C, to stimulate lymphangiogenesis. Taken together, these studies show that NGF stimulates lymphangiogenesis and that NGF may act as a pathogenic factor that negatively regulates the normal neural and lymphatic vascular remodeling events that accompany wound recovery.     

Structure and Innervation of Inner Ear Sensory Epithelia in the European Eel (Anguilla anguilla L.)

Hair cell orientations of all inner ear sensory epithelia in glass eel, yellow eel and silver eel are presented. The patterns of hair cell orientation do not change with age. All sensory epithelia increase in area during growth of the eel. Examination of the hair cell population in macula utriculi show constant hair cell densities and increased hair cell population during development. Further, regional differences in hair cell densities and hair cell types are observed. The hair cells/axons ratio increases 3-fold from glass eel to silver eel stadium. Nerve stainings in silver eel reveal complex innervation patterns with large stubby fibres confined to restricted regions. Histograms of nerve fiber diameters show marked differences from glass eel to silver eel. Growth of sensory epithelia is discussed.     

Adipose Vascular Endothelial Growth Factor Regulates Metabolic Homeostasis through Angiogenesis

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Highlights? Two-way modulations of adipose VEGF were generated with aP2-Cre transgene ? Adipose VEGF KO reduces vasculature, increases hypoxia and inflammation in fat ? Adipose VEGF KO accelerates the development of metabolic disease in high-fat diet ? Induced adipose VEGF has opposite effect on fat and restores metabolic homeostasis     

HCN Channels Are Not Required for Mechanotransduction in Sensory Hair Cells of the Mouse Inner Ear

The molecular composition of the hair cell transduction channel has not been identified. Here we explore the novel hypothesis that hair cell transduction channels include HCN subunits. The HCN family of ion channels includes four members, HCN1-4. They were orginally identified as the molecular correlates of the hyperpolarization-activated, cyclic nucleotide gated ion channels that carry currents known as I_f, I_Q or I_h. However, based on recent evidence it has been suggested that HCN subunits may also be components of the elusive hair cell transduction channel. To investigate this hypothesis we examined expression of mRNA that encodes HCN1-4 in sensory epithelia of the mouse inner ear, immunolocalization of HCN subunits 1, 2 and 4, uptake of the transduction channel permeable dye, FM1-43 and electrophysiological measurement of mechanotransduction current. Dye uptake and transduction current were assayed in cochlear and vestibular hair cells of wildtype mice exposed to HCN channel blockers or a dominant-negative form of HCN2 that contained a pore mutation and in mutant mice that lacked HCN1, HCN2 or both. We found robust expression of HCNs 1, 2 and 4 but little evidence that localized HCN subunits in hair bundles, the site of mechanotransduction. Although high concentrations of the HCN antagonist, ZD7288, blocked 50–70% of the transduction current, we found no reduction of transduction current in either cochlear or vestibular hair cells of HCN1- or HCN2- deficient mice relative to wild-type mice. Furthermore, mice that lacked both HCN1 and HCN2 also had normal transduction currents. Lastly, we found that mice exposed to the dominant-negative mutant form of HCN2 had normal transduction currents as well. Taken together, the evidence suggests that HCN subunits are not required for mechanotransduction in hair cells of the mouse inner ear.