FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family
2000; Springer Nature; Volume: 19; Issue: 15 Linguagem: Inglês
10.1093/emboj/19.15.4046
ISSN1460-2075
AutoresIlona N. Holcomb, Rhona C. Kabakoff, Betty Chan, Thad W. Baker, Austin Gurney, William J. Henzel, Chris Nelson, Henry B. Lowman, Barbara D. Wright, Nicholas J. Skelton, Gretchen Frantz, Daniel B. Tumas, Franklin Peale, David L. Shelton, Caroline C. Hébert,
Tópico(s)Epigenetics and DNA Methylation
ResumoArticle1 August 2000free access FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family Ilona N. Holcomb Ilona N. Holcomb Department of Pathology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Rhona C. Kabakoff Rhona C. Kabakoff Department of Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Betty Chan Betty Chan Department of Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Thad W. Baker Thad W. Baker Department of Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Austin Gurney Austin Gurney Department of Molecular Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author William Henzel William Henzel Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Chris Nelson Chris Nelson Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Henry B. Lowman Henry B. Lowman Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Barbara D. Wright Barbara D. Wright Department of Pathology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Nicholas J. Skelton Nicholas J. Skelton Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Gretchen D. Frantz Gretchen D. Frantz Department of Pathology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Daniel B. Tumas Daniel B. Tumas Department of Pathology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Franklin V. Peale, Jr Corresponding Author Franklin V. Peale, Jr Department of Pathology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author David L. Shelton David L. Shelton Department of Neuroscience, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Caroline C. Hébert Caroline C. Hébert Department of Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Ilona N. Holcomb Ilona N. Holcomb Department of Pathology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Rhona C. Kabakoff Rhona C. Kabakoff Department of Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Betty Chan Betty Chan Department of Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Thad W. Baker Thad W. Baker Department of Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Austin Gurney Austin Gurney Department of Molecular Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author William Henzel William Henzel Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Chris Nelson Chris Nelson Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Henry B. Lowman Henry B. Lowman Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Barbara D. Wright Barbara D. Wright Department of Pathology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Nicholas J. Skelton Nicholas J. Skelton Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Gretchen D. Frantz Gretchen D. Frantz Department of Pathology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Daniel B. Tumas Daniel B. Tumas Department of Pathology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Franklin V. Peale, Jr Corresponding Author Franklin V. Peale, Jr Department of Pathology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author David L. Shelton David L. Shelton Department of Neuroscience, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Caroline C. Hébert Caroline C. Hébert Department of Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA Search for more papers by this author Author Information Ilona N. Holcomb1, Rhona C. Kabakoff2, Betty Chan2, Thad W. Baker2, Austin Gurney3, William Henzel4, Chris Nelson4, Henry B. Lowman5, Barbara D. Wright1, Nicholas J. Skelton5, Gretchen D. Frantz1, Daniel B. Tumas1, Franklin V. Peale, 1, David L. Shelton6 and Caroline C. Hébert2 1Department of Pathology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA 2Department of Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA 3Department of Molecular Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA 4Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA 5Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA 6Department of Neuroscience, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080 USA *Corresponding author. Genentech, Inc., Mailstop 72B, 1 DNA Way, South San Francisco, CA 94080-4996, USA, E-mail: [email protected] The EMBO Journal (2000)19:4046-4055https://doi.org/10.1093/emboj/19.15.4046 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Bronchoalveolar lavage fluid from mice with experimentally induced allergic pulmonary inflammation contains a novel 9.4 kDa cysteine-rich secreted protein, FIZZ1 (found in inflammatory zone). Murine (m) FIZZ1 is the founding member of a new gene family including two other murine genes expressed, respectively, in intestinal crypt epithelium and white adipose tissue, and two related human genes. In control mice, FIZZ1 mRNA and protein expression occur at low levels in a subset of bronchial epithelial cells and in non-neuronal cells adjacent to neurovascular bundles in the peribronchial stroma, and in the wall of the large and small bowel. During allergic pulmonary inflammation, mFIZZ1 expression markedly increases in hypertrophic, hyperplastic bronchial epithelium and appears in type II alveolar pneumocytes. In vitro, recombinant mFIZZ1 inhibits the nerve growth factor (NGF)-mediated survival of rat embryonic day 14 dorsal root ganglion (DRG) neurons and NGF-induced CGRP gene expression in adult rat DRG neurons. In vivo, FIZZ1 may modulate the function of neurons innervating the bronchial tree, thereby altering the local tissue response to allergic pulmonary inflammation. Introduction Asthma, defined clinically by abnormal, reversible bronchoconstriction in response to a variety of non-specific stimuli, affects 7–10% of children and 5% of adults living in the United States. Approximately two-thirds of cases are caused by allergic sensitivity to environmental antigens, but clinically identical disease can be caused by respiratory infections, inhaled chemical irritants, exercise, emotional stress or cold. Despite the diversity of precipitating factors, the pathological changes in all cases are relatively similar. Interactions between mast cells, eosinophils, lymphocytes and bronchial tissue result in persistent morphological and physiological changes in all layers of the airways. Typically, there is hypertrophy and hyperplasia of the bronchial epithelium, thickening of the basal lamina, hypertrophy of the bronchial submucosal glands with increased mucus production and hypertrophy of bronchial smooth muscle (McFadden and Gilbert, 1992). Most importantly, peribronchial smooth muscle, controlled by sympathetic, parasympathetic and non-adrenergic, non-cholinergic (NANC) inputs, shows increased excitability resulting in inappropriate but reversible constriction of the airways (airway hyperresponsiveness; AHR), the hallmark clinical presentation of asthma (Barnes, 1996). By correlating findings in human asthma with those in experimental animal models, a variety of small molecule and protein mediators, including histamine, bradykinin, prostaglandins, leukotrienes and cytokines have been implicated in promoting allergic inflammation and bronchoconstriction. These molecules may be released from inflammatory cells, nerve terminals or respiratory epithelial cells, and may promote AHR in at least four ways: (i) by amplifying the inflammatory process; (ii) by damaging the respiratory epithelium, thereby increasing irritant access to sensory nerve endings (Davies et al., 1993); (iii) by lowering the threshold for vagal afferent sensory fiber stimulation (Spina et al., 1998); or (iv) by increasing responsiveness of bronchial smooth muscle cells to efferent stimulation (Braun et al., 1998). Nerve growth factor (NGF), well known for its role as a sympathetic and sensory nerve growth factor, has been shown to have additional activities both within and outside the nervous system. Of particular relevance to the pathophysiology of asthma, NGF induces acute changes in the excitability of peptidergic sensory neurons (Shu and Mendell, 1999), longer term changes in levels of bioactive peptides such as substance P and calcitonin gene-related peptide (CGRP; Lindsay and Harmar, 1989; Lindsay et al., 1989), and changes in the neuronal expression of receptors for bradykinin (Petersen et al., 1998). NGF is among the mediators released from mast cells during allergic inflammation, and NGF stimulation of mast cells results in degranulation, potentially amplifying the inflammatory reaction. Elevated serum NGF has been reported in human patients with asthma (Bonini et al., 1996). NGF is elevated in serum and bronchoalveolar lavage fluid (BALF) from mice with experimentally induced asthma, and this elevation is correlated with increased bronchial smooth muscle hyperresponsiveness (Braun et al., 1998). In order to identify additional molecules associated with allergic inflammation and AHR, we screened for novel secreted proteins expressed in murine ovalbumin (OVA)-induced asthma. This model reproduces the perivascular and peribronchial inflammation, bronchial epithelial hypertrophy and increased mucus production described in human asthma (Blyth et al., 1996), making it appropriate for identification of key cellular and biochemical components of the disease state. Here we describe a new gene family, one member of which encodes a protein whose expression in vivo and function in vitro implicate the molecule as a possible mediator of neuronal function and airway hyperreactivity. Results Identification of the FIZZ gene family SDS–acrylamide gel analysis of BALF collected from mice with OVA-induced pulmonary inflammation revealed a band, co-migrating with an 8.3 kDa marker protein, that was not present in control BALF (Figure 1). The apparent abundance of protein in this band did not correlate with BALF serum albumin concentration as assessed by ELISA (data not shown), indicating that the protein was not simply a component of plasma that had leaked into the alveolar airspace. Concentrations of this protein in the BALF, estimated by comparison with the marker protein, were as high as 0.5–0.75 μM (∼5 μg/ml). Microsequencing of the isolated protein allowed the subsequent isolation of a 536 bp cDNA from normal mouse lung. Named FIZZ1 (found in inflammatory zone), the sequence encodes 111 amino acids with an N-terminal signal peptide (amino acids 1–23) and a C-terminal cysteine-rich domain (Figure 2A). The predicted molecular weight and pI of the secreted form of the protein are 9431 Da and 4.83, respectively. Figure 1.SDS–acrylamide gel analysis of BALF. Equal volumes (10 μl) of BALF from control mice and BALF obtained from mice with OVA-induced allergic pulmonary inflammation were analyzed under reducing conditions by SDS–PAGE on a Tricine-buffered 16% acrylamide gel. BALF from mice with allergic pulmonary inflammation (lane 2) contains a unique band, co-migrating with an 8.3 kDa molecular weight marker (IL-8, 50 ng, lane 3), which is not present in BALF from control mice (lane 1). Download figure Download PowerPoint Figure 2.Sequences of the FIZZ protein family. (A) The amino acid sequences of murine and human FIZZ proteins. The consensus sequence (Cons.) indicates the position of the conserved residues. Underlined residues represent predicted signal peptide sequences. The corresponding nucleotide sequences are entered in the DDBJ/EMBL/GenBank databases under the following accession Nos: mFIZZ1, AF205951; mFIZZ2, EST AA245405; mFIZZ3, EST W42069; hFIZZ1, EST AA524300; hFIZZ3, AF205952. (B) Amino acid identity (upper right) and homology (lower left) for the five members of the FIZZ gene family (based on PAM250 matrix). Download figure Download PowerPoint Nucleotide homology searches of the DDBJ/EMBL/GenBank database identified two additional mouse genes and two human genes with homology to murine (m) FIZZ1 (Figure 2A). The relative amino acid homology of various FIZZ family members to each other is illustrated in Figure 2B. All five genes encode proteins with 105–114 amino acids containing signal peptide sequences 10–23 amino acids long, with 10 cysteine residues in the C-terminus having identical spacing [1CX112CX83CX4CX35CX106CX7CX8CX99C10C]. Three of the 10 C-terminal cysteines are embedded within two highly conserved motifs, (A/G)5CGSW(D/E)(I/V) and DW(A/T) XAR9C10C. With the exception of mFIZZ1, all family members have an additional cysteine in the N-terminal domain of the processed form of the protein. The FIZZ proteins lack significant homology to any proteins outside the family, as determined by BLASTP (Altschul et al., 1990, 1997), and HMMER 2.1.1 Pfam software (Bateman et al., 1999). The nearest Pfam match was to cysteine knot proteins, although the degree of homology was very low (E-values = 22, 11 and 41 for mFIZZ-1, -2 and -3, respectively). Secondary structure prediction indicates that the processed form of the mFIZZ1 protein may contain a helix at the N-terminus (residues Ile6 to Ala17). Examination of the mFIZZ1 amino acid sequence with the threading package ProCyon (Flöckner et al., 1997) indicated a poor quality of the match between mFIZZ1 and cysteine knot proteins including NGF, neurotrophin 3 (NT3), brain-derived neurotrophin factor (BDNF), transforming growth factor-β, glial cell line-derived neurotrophic factor (GDNF) and platelet-derived growth factor, consistent with the large E-value given by the Pfam homology search. In all cases, secondary structure elements or locations of cysteine residues were not compatible with the mFIZZ1 sequence. However, the epidermal growth factor (EGF) fold gave high scores in both the ‘pair/surface’- and ‘sequence’-based potentials. Tissue expression of FIZZ genes Northern blot analysis of adult mouse tissues using an oligonucleotide probe complementary to mFIZZ1 shows expression of a single 750–800 base mRNA primarily in the lung, with ∼10-fold lower levels detectable in heart and skeletal muscle (Figure 3). Adult mouse multi-tissue northern blots hybridized with mFIZZ3-specific probes revealed expression at low levels in many organs (data not shown). Recent DDBJ/EMBL/GenBank expressed sequence tag (EST) entries with homology to the various FIZZ genes are as follows: mFIZZ1, AA945994 (rat lung) and AA712003 (mouse mammary gland); mFIZZ2, AA711012 (mouse colon); mFIZZ3, AI746650 (mouse kidney) and AA796118 (mammary gland); human (h) FIZZ1, AI732383 (colon cancer); hFIZZ3, AA311223 (Jurkat T cells). Figure 3.mFIZZ1 northern blot. A Clontech adult mouse multiple tissue northern blot was probed with an oligonucleotide corresponding to nucleotides 176–225 of the full-length mFIZZ1 sequence, and exposed for 16 h or 5 days as shown. The same blot, stripped and reprobed with an oligonucleotide for β-actin, and exposed for 7 days, is shown at the bottom of the figure. Download figure Download PowerPoint Expression analysis by in situ hybridization and immunohistochemistry To characterize the cell type-specific expression of mFIZZ1 in detail, we examined normal and inflamed murine lungs by in situ hybridization and immunohistochemistry. In control adult lung, mFIZZ1 mRNA was expressed at low levels in the large airways in small discrete clusters of epithelial cells (Figure 4A, C and E), and in scattered isolated cells in the peribronchiolar stroma (Figure 4G and I). Consistent with the BALF analysis, expression of mFIZZ1 mRNA in lungs with OVA-induced allergic inflammation was markedly increased, with widespread uniform expression in the bronchial mucosal epithelial cells (Figure 4B, D and F). Additionally, in inflamed but not control lungs, mFIZZ1 message was present throughout the lung in scattered cells associated with the alveolar wall, consistent with type II pneumocytes; significantly, no signal was seen in alveolar macrophages (Figure 4H and J). Figure 4.In situ hybridization of mFIZZ 1 in adult mouse lung. A 33P-labeled mFIZZ1 riboprobe detected patchy expression in bronchial epithelium of control (OVA-challenged, non-immunized mouse) lung after a 4-week exposure (A, C and E). In inflamed lung, a 2-week exposure with the same probe detected diffuse strong expression in bronchial epithelium (B, D and F) and type II pneumocytes (arrows, H and J), while alveolar macrophages (arrowheads, H and J) were negative. Murine FIZZ1 expression was also present in discrete cells in neurovascular bundles in peribronchial interstitium (arrowheads, C, G and I). Dark-field images: A–D, G and H. Corresponding bright-field images: E, F, I and J. Scale bars represent 500 μm (A and B), 50 μm (C–F) or 25 μm (G–J). ar, artery; br, bronchiole; alv, alveolar space. Download figure Download PowerPoint Polyclonal rabbit antiserum to mFIZZ1 was generated against a peptide derived from the N-terminal helical domain, a portion of the protein sharing little homology with other members of the FIZZ family. Western blots (Figure 5) of inflamed lung BALF, resolved under reducing conditions and probed with mFIZZ1 antiserum, detected a single band consistent with mFIZZ1 (lane 2) while pre-immune serum did not detect any specific proteins (lane 1). Whole-lung homogenates from animals with allergic inflammation revealed a similar band under reducing conditions (lane 3). However, under non- reducing conditions, two additional bands were detected, one migrating only slightly more slowly than mFIZZ1, another migrating with an apparent size of 25–30 kDa (lane 4). Figure 5.Western blot analysis of BALF and lung homogenates from mice with allergic pulmonary inflammation. Lane 1, BALF sample resolved under reducing conditions and analyzed with pre-immune serum; lane 2, BALF sample resolved under reducing conditions and analyzed with a rabbit anti-FIZZ1 peptide antiserum. Whole-lung homogenate (Hom.) resolved under reducing (lane 3) or non-reducing conditions (lane 4) and analyzed with the same rabbit antiserum. The migration of molecular size markers (kDa) is indicated on the left. Download figure Download PowerPoint Immunohistochemistry using the same polyclonal antiserum confirmed the in situ hybridization expression results. Limited patchy FIZZ1 protein expression was observed in control bronchial mucosa (Figure 6A and C), whereas inflamed mucosa showed uniform high protein levels (Figure 6B). In alveoli of the inflamed lung, but not in the control lung, scattered plump alveolar epithelial cells, consistent with type II pneumocytes, were strongly positive for FIZZ1 protein (Figure 6D). Alveolar macrophages and many tissue components in the inflamed lungs were weakly positive by immunohistochemistry. Enhanced expression of FIZZ1 in inflamed lung tissue was not restricted to the ovalbumin model: similar changes were seen in a second allergic inflammation model that utilized dust mite extract as allergen and which induced similar bronchial epithelial pathology (data not shown). Figure 6.Immunohistochemical detection of mFIZZ1 protein in inflamed and control lung. (A and C) Murine FIZZ1 expression in control (OVA-challenged, non-immunized mouse) lung is limited to small patches of bronchial epithelial cells. (B and D) In the inflamed lungs of immunized, OVA-challenged mice, mFIZZ1 protein expression in bronchial epithelium is both more diffuse and more intense. In addition, expression is seen in alveolar epithelial cells with granular cytoplasm, consistent with type II pneumocytes (arrows, D). Alveolar macrophages (arrrowheads, D) and stromal cellular components stain reproducibly, but weakly with anti-FIZZ antibody, despite being negative for FIZZ mRNA by in situ hybridization (Figure 4H and J). Scale bars represent 100 μm (A and B), 10 μm (C) or 25 μm (D). Download figure Download PowerPoint We examined other tissues for mFIZZ1 mRNA and protein expression. FIZZ1 transcripts and protein were seen in discrete cells adjacent to neurovascular bundles, primarily in the submucosa of the large intestine (Figure 7A–C), and throughout the small bowel wall (not shown). In the colon, mFIZZ1-expressing cells were most often located adjacent to cells expressing neuronal and Schwann cell markers including protein gene product 9.5 (PGP9.5), neuron-specific enolase (NSE) and S-100 (Figure 7D). Occasional mFIZZ1-positive cells were seen in the muscularis propria of the large bowel, generally associated with neurovascular bundles, but no mFIZZ1-positive cells were specifically associated with Auerbach's plexus. Similar cells in the peribronchial stroma (Figures 4G, I and 7E) were adjacent to PGP9.5-positive neurons (Figure 7F). Scattered mFIZZ1-positive cells were noted in mammary gland subcutaneous tissue, in the heart and mediastinum (not shown). There was no epidermal expression of mFIZZ1. Figure 7.Expression of mFIZZ1 mRNA in colonic submucosal and peribronchial neurovascular bundles. (A and B) In situ hybridization of mFIZZ1 in colon shows strong expression in discrete cells (arrows) in the submucosa after a 4-week exposure. (C–F) Immunohistochemistry of FIZZ1 (C and E) and PGP9.5 (D and F) in serial sections of colonic submucosal (C and D) and peribronchial (E and F) tissue. mFIZZ1-positive cells (arrows) are adjacent to neuronal cell bodies and fibers (arrowheads). Similar results were obtained with antibodies to S-100 and NSE (not shown). Scale bars represent 50 μm (A and B) or 25 μm (C–F). mu, mucosa; sm, submucosa; mp, muscularis propria; ve, venule; ar, arteriole; ag, autonomic ganglion; br, bronchiole; pa, pulmonary artery. Download figure Download PowerPoint Strong expression of mFIZZ2 mRNA was found in the adult colon in the mucosal crypt epithelial cells; expression was highest in the lower one-half to one-third of the crypt and diminished in the more superficial epithelium (Figure 8A and B). Murine FIZZ2 expression was segmental: in situ hybridization using histological sections of ‘jelly-rolled’ bowel showed positive signal in contiguous regions ∼2 cm long alternating with segments of approximately the same length having no detectable expression (Figure 8C and D). In the small bowel, mFIZZ2 was also expressed more highly in the crypts than in the villi (not shown). All other tissues examined were negative. Figure 8.Expression of mFIZZ2 and mFIZZ3 mRNA. (A–D) In situ hybridization of mFIZZ2 to a section of colon shows strong signal in crypt epithelium after a 2-week exposure. In (C and D), the ‘jelly roll’ orientation of the colon has artificially brought two segments of colon back-to-back; one segment (arrowhead) is intensely positive for FIZZ2 expression, while the other segment (arrow), several centimeters from the first, is negative. (E and F) In situ hybridization of mFIZZ3 to small bowel and mesentery (4-week exposure). Expression is limited to adipose tissue (arrow) in mesentery. (G and H) In situ hybridization of mFIZZ3 to peritracheal tissue (4-week exposure); expression is limited to adipose tissue (arrow) adjacent to thyroid gland (Th) and trachea (Tr). Dark-field images: A, C, E and G. Corresponding bright-field images: B, D, F and H. Scale bars represent 35 μm (A, B and E–H) or 100 μm (C and D). mu, mucosa; mp, muscularis propria. Download figure Download PowerPoint Murine FIZZ3 mRNA was expressed exclusively in white adipose tissue in a variety of organs (Figure 8E–H). All other tissues, including brown adipose tissue, were negative. The tissue-specific expression patterns of mFIZZ-1, -2 and -3 are summarized in Table I. Table 1. mFIZZ expression summary Gene Tissue Expression pattern mFIZZ1 normal lung scattered bronchial epithelial cells; peribronchial stromal cells adjacent to nerves inflamed lung most bronchial epithelial cells; type II pneumocytes small, large intestine submucosal cells adjacent to nerves mammary gland scattered cells in subcutaneous tissue mFIZZ2 small, large intestine crypt epithelium mFIZZ3 white adipose tissue diffuse expression Functional studies Allergic pulmonary inflammation is associated with infiltration of inflammatory cells into the peribronchial tissue, transmigration of inflammatory cells into the airways, increased bronchial secretions and enhanced airway smooth muscle reactivity. Because the expression of mFIZZ1 was significantly upregulated in allergic inflammation in vivo, we asked whether the molecule could modify related biological responses in vitro. Murine FIZZ1 had no detectable inhibitory activity in assays to measure human neutrophil elastase, trypsin, chymotrypsin or cathepsin G activity (data not shown). Because mFIZZ1 is expressed by cells intimately associated with submucosal bronchial nerves, we asked whether there was a relationship between mFIZZ1 and NGF activity. We first examined the ability of mFIZZ1 to affect the NGF-induced survival of rat embryonic day (E) 14 dorsal root ganglion (DRG) neurons, the majority of which require NGF for survival in vitro and in vivo at this age. In agreement with previous results (Levi-Montalcini and Angeletti, 1963), addition of NGF to cultures resulted in increased neuronal survival after 3 days. Recombinant mFIZZ1-his, added to cultures at a concentration equivalent to that found in BALF (4 μg/ml), significantly inhibited the NGF-induced survival (Figure 9A) without inducing toxicity in non-neuronal cells (not shown). Figure 9.FIZZ1 inhibits NGF-mediated neuronal survival and gene expression. (A) Surviving rat E14 DRG neurons were counted after 3 days in culture. Recombinant mFIZZ1 at 4 μg/ml inhibited neuronal survival without inducing non-neuronal cell toxicity. (B) NGF induces higher levels of CGRP immunoreactivity in neurons, and mFIZZ1 inhibits the NGF-induced increase in CGRP content. Open bars, no mFIZZ1; hatched bars, 0.4 μg/ml (∼40 nM) mFIZZ1; solid bars, 4 μg/ml (∼0.4 μM) mFIZZ1. mFIZZ1 at 4 μg/ml inhibits the increase in CGRP induced by 1 ng/ml NGF (p <0.002) or 10 ng/ml NGF (p <0.01). Download figure Download PowerPoint To rule out the possibility that mFIZZ1-his is selectively toxic to neurons, we took advantage of the fact that adult DRG neurons no longer require NGF for their survival, but do still respond to NGF by induction of CGRP (Lindsay and Harmar, 1989; Horton et al., 1998). In agreement with previous work, addition of NGF to cultures of adult DRG neurons did not change the number of surviving neurons as measured by the number of NeuN-positive cells, nor did it change the number of CGRP-positive cells (not shown). However, NGF dramatically increased the CGRP content of adult DRG neurons (Figure 9B). Addition of mFIZZ1-his to these cultures had no effect on neuron survival, nor on the number of CGRP-positive neurons (not shown), ruling out non-specific toxic effects. However, mFIZZ1-his did inhibit, in a dose-dependent fashion, the NGF-mediated increase in neuronal CGRP content (Figure 9B). To explore the mechanism of this inhibition, we asked if mFIZZ1 inhibited the binding of NGF to its signal transducing receptor, trkA. We examined the effect of mFIZZ1 on NGF binding utilizing a cell-free system of the cloned trkA receptor bound to ELISA plates. This system accurately replicates the affinity and selectivity of the various trk receptors for their natural ligands (Shelton et al., 1995). Inclusion of mFIZZ1 in the binding experiments resulted in no change in NGF binding at any concentration tested (D.L.Shelton, data not shown). In order to test for direct interactions between mFIZZ1 and either trkA or NGF, we coupled mFIZZ1, NGF, trkA–IgG or an irrelevant IgG to a surface plasmon resonance (SPR) biosensor chip. Soluble mFIZZ1 showed significant but weak association with immobilized mFIZZ1 (Figure 10A). Soluble mFIZ
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