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HUGO (FNDC3A): a New Gene Overexpressed in Human Odontoblasts
F. Carrouel1,2,3,4,5,6,
M.-L. Couble1,2,3,4,5,6,
C. Vanbelle1,7,
M.-J. Staquet1,2,3,4,5,6,
H. Magloire1,2,3,4,5,6 and
F. Bleicher1,2,3,4,5,6,*
1 Université de Lyon, Villeurbanne, F-69000, France;
2 Université Lyon 1, Faculté d’Odontologie, Lyon, F-69008, France;
3 CNRS, UMR 5242, IGFL, Lyon, F-69007, France;
4 INRA, UMR 1288, Lyon, F-69007, France;
5 ENSL, Lyon, F-69007, France;
6 INSERM, ERI 16, Lyon, F-69008, France; and
7 Université Lyon 1, CeCIL, IFR62, Lyon, F-69008, France
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Figure 1. Characterization of HUGO gene and protein sequences. (A) Northern blot analysis of HUGO expression reveals the presence of two transcripts in odontoblast-like cells. (B) Schematic representation of exon-intron structures of HUGO1 and HUGO2 isoforms. HUGO1 is composed of 26 exons and 25 introns, and HUGO2 consists of 24 exons and 23 introns. The difference between HUGO1 and HUGO2 structures is observed at the 5' end. Exon 4 of HUGO1 corresponds to Exon 2 of HUGO2. (C) Amino acid sequence of HUGO. The entire sequence corresponds to HUGO1, whereas the sequence of HUGO2 is indicated in bold characters. Type III-fibronectin domains are boxed in grey. The underlined sequence corresponds to the proline-rich region. The framed sequence indicates the transmembrane domain. The two peptides used for rabbit immunization are double-framed.
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Figure 2. HUGO gene expression pattern. Real-time PCR analysis of HUGO, HUGO1, and HUGO2 in precursor pulp cells, odontoblast-like cells, and COS-1 cells (A) and in different tissues (B). Expression of each target gene was normalized to the cyclophilin A housekeeping gene, with the use of RelQuant software (Roche). Results were expressed as fold-change values relative to precursor pulp cells. A significant increase of gene expression for HUGO (11-fold), HUGO1 (3.5-fold), and HUGO2 (nine-fold) can be observed in odontoblast-like cells compared with precursor pulp cells. In COS-1 cells, the HUGO2 transcript is expressed, whereas HUGO1 is not detected. The transcripts of HUGO, HUGO1, and HUGO2 can be detected mainly in the trachea and, to a lesser extent, in the brain, liver, kidney, and lung, but not in the heart (B). (C,D,E,F) In situ hybridization on a human dental pulp section. (C) Methylene blue counter-stained section shows the odontoblast cell bodies organized as a layer (double arrows) at the periphery of the pulp, and pulp cells (arrows) scattered in the underlying tissue. (D) With the antisense probe, the HUGO transcript is detected in the odontoblast layer. (E) The HUGO transcript is also expressed in nerve fibers. (F) Negative control with the sense probe. No significant signal is detected in the pulp and the odontoblast layer. DP, dental pulp; Od, odontoblasts; PPC, precursor pulp cells; NF, nerve fibers. Bars represent 50 µm.
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Figure 3. Characterization of HUGO expression. (A) Western blot analysis of HUGO expression in precursor pulpal cells (1,5), odontoblast-like cells (2,6), COS-1 (3,7), and COS-1 overexpressing V5-6xHis-tagged HUGO protein (4,8). Antibodies (anti-HUGO and anti-V5) were used at 0.3 µg/mL and 0.06 µg/mL, respectively. Anti-V5 antibody (1–4) recognizes a 130-kDa protein only in COS-1 overexpressing V5-6xHis-tagged HUGO (4). Anti-HUGO antibody (5–8) recognizes HUGO1 and HUGO2 isoforms in precursor pulp cells (5) and odontoblast-like cells (6). Only the HUGO2 isoform is detected in COS-1 (7). The additional band of 130 kDa, recognized in COS-1 overexpressing V5-6xHis-tagged HUGO (8), is identical to that identified by the anti-V5 antibody (4). Controls were performed with anti-rabbit IgG (9), anti- mouse IgG2a (10), or the omission of primary antibody (11). (B) Flow cytometry analysis of HUGO expression. Cells were obtained following trypsin/EDTA treatment of cultures and directly labeled with anti-HUGO polyclonal antibody (filled histograms). HUGO was detected only in permeabilized cells, indicating a cytoplasmic localization. Controls with normal rabbit IgG are shown as empty histograms. Results are representative of three independent experiments. PPC: precursor pulpal cells. (C,D,E,F) Immunostaining of dental pulp and odontoblast-like cells. A positive labelling can be seen in odontoblasts with the anti-HUGO antibody (C), compared with the control section (D). In the pulp core, nerve bundles are also labeled (E). In odontoblast-like cells, HUGO is clearly identified around the nucleus (F). Bars represent 50 µm. (G,H) Electron micrograph of odontoblast-like cells showing indirect immunoperoxidase HUGO staining. Some Golgi vesicles were strongly labeled as dots of peroxidase deposits, particularly in close association with the vesicle membranes (arrows in H). In contrast, the rough endoplasmic reticulum was devoid of peroxidase deposits. N: nucleus. We created negative controls by omitting the primary antibody, by incubating cells with normal rabbit or mouse IgG and with pre-absorbed antibody (data not shown). Bars represent 1 µm. P, dental pulp; Od, odontoblasts; NF, nerve bundles; GV, Golgi vesicle; ER, endoplasmic reticulum; N, nucleus.
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Figure 4. Co-localization of HUGO with Golgi vesicles. (A,B,C,D) Double staining shows a co-localization of the Golgi zone and HUGO within odontoblast-like cells (arrow). Note that only some specific vesicles (yellow) co-expressed both HUGO and the Golgi marker. The inset shows the same co-localization in the z axis. (D) Higher magnification of the Golgi region in (C) (arrow). (E,F,G,H) Co-localization of the Golgi zone and HUGO in odontoblasts in vivo. Yellow patches were strongly detected in some Golgi vesicles (arrow), well-identified in (H). The inset shows the double staining according to the z axis. (I,J,K) Double staining with β2 tubulin and HUGO reveals no co-localization between the two molecules, as demonstrated in (K) (inset). These confocal images were extracted from Z-series of optical sections. Negative controls were performed as described in Fig. 3 .
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Journal of Dental Research, Vol. 87, No. 2,
131-136 (2008)
DOI: 10.1177/154405910808700209
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