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RESEARCH REPORT |
Louisiana State University Health Science Center, Department of Cell Biology and Anatomy, Center of Excellence in Oral and Craniofacial Biology, School of Dentistry, 1100 Florida Avenue, New Orleans, LA 70119, USA; tlalli{at}lsuhsc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: neuropilin • plexin • osteogenesis • cementogenesis • differentiation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The regulation of neural crest cell migration is not fully understood (Bronner-Fraser, 1994, 2000); however, it is partly regulated by the expression of cell-cell and cell-extracellular matrix adhesion molecules (e.g., CAMs, cadherins, and integrins). The regulation of these adhesive molecules involves localized cell-surface signals utilizing members of the ephrin and semaphorin families of molecules. Ephrin B1/Eph tyrosine kinase receptor interaction is involved with regulating the metameric patterning of neural crest cells in the thoracic region of chick embryos (Krull et al., 1997). Semaphorin 3A/neuropilin receptor interactions are also involved in this process, as well as in the guidance of neural-crest-derived sensory axons (Eickholt et al., 1999; Brown et al., 2001; Feiner et al., 2001).
Semaphorin (Collapsin) Structure and Function
Semaphorins are a family of cell-surface and secreted glycoproteins that influence axon guidance (Kolodkin et al., 1993; Luo et al., 1995). These molecules exist either as cell-surface transmembrane proteins, or as secreted proteins that associate with the cell surface. Many semaphorins appear to act as negative cues in axon guidance, and may be responsible for regulating the pathfinding of new growth cones, and possibly target selection (Kolodkin et al., 1993; Luo et al., 1995; Puschel et al., 1995). Semaphorins have been associated with several functions, including the regulation of axon guidance, regulation of blood vessel patterning, and the regulation of leukocyte movement. Mice deficient in Sema3A expression display alterations in sensory neuron guidance, as well as skeletal and cardiac malformations (Behar et al., 1996; Taniguchi et al., 1997). In addition, Sema4D is involved in B-cell aggregation within the immune system (Hall et al., 1996), and monocytes express Sema7A (Holmes et al., 2002). Analysis of these data indicates that these molecules play a key role not only in proper neuronal guidance and target selection, but also in non-neural cell guidance and organogenesis.
Neuropilin and Plexin (Semaphorin Receptors)
Neuropilins and plexins act as cell-surface receptors for semaphorins. Neuropilin-1 has been identified as having significant binding affinity for Sema3A (He and Tessier-Lavigne, 1997; Kolodkin et al., 1997; Nakamura et al., 1998; Feiner et al., 2001). A related protein, neuropilin-2, acts as a receptor for Sema3F (Chen et al., 1997; Giger et al., 1998). In addition, both neuropilin-1 and -2 were also demonstrated to bind other Class III semaphorins, Sema3C and Sema3E (Chen et al., 1997, 1998). Similarly, hippocampal neurons expressing neuropulin-1 are sensitive to Sema3A, while others expressing neuropilin-2 are responsive to Sema3F (Chedotal et al., 1998). Analysis of these data, taken together, indicates that neuropilins act as receptors for the Class III soluble/membrane-associated semaphorins.
Here we have chosen to investigate the role of semaphorins in regulating the formation of the laminated periodontal attachment apparatus. As a first step, we present evidence for the expression of most of the vertebrate semaphorins, neuropilins, and plexins in several adult periodontal mesenchymal cell types (osteoblasts, PDL, and gingival fibroblasts) as well as in dermal fibroblasts.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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MEM containing 10% fetal calf serum (FCS), 200 units/mL of penicillin, and 200 µg/mL streptomycin (GIBCO, Grand Island, NY, USA). PDL and gingival fibroblasts between the 5th and 12th passages were used. Four independent isolates of gingival and PDL fibroblasts were compared in this study. Cell lines of human dermal fibroblasts (ATCC CRL-1502, CRL-1474, CRL-1489, and CRL-1497) and pre-osteoblasts (ATCC-CRL-11372) were obtained from the American Type Culture Collection. The dermal fibroblasts were isolated from the skin of both male and female neonates, children, adults, and embryos. The pre-osteoblasts are a line of fetal osteoblasts (obtained from a spontaneous miscarriage) that have been transfected with a temperature-sensitive expression vector (pUCCSVtsA58) and with the neomycin resistance expression vector pSV2-neo (Harris et al., 1995). These cells were grown at 33°C to prevent their temperature-sensitive auto-differentiation. Pre-osteoblasts were maintained in DMEM/F12 (without phenol red) media containing 10% fetal calf serum (FCS), 200 units/mL of penicillin, and 200 µg/mL streptomycin (GIBCO, Grand Island, NY, USA). All of these cell types have been more fully characterized (Lallier et al., 2004).
RNA Isolation and Reverse Transcriptase Polymerase Chain-reaction (RT-PCR)
RNA was extracted from gingival, dermal, and periodontal ligament fibroblasts with the use of guanidine thiocyanate, and gene expression was determined by RT-PCR (Palaiologou et al., 2001). We chose the number of cycles by creating a standard curve using serial dilutions of DNA template known to include the genes in question, and determined those PCR conditions that reveal two-fold differences in gene expression over 3 orders of magnitude. From 25 to 35 cycles of PCR was determined to be within the linear range of detection for all of the genes examined. The primers for these studies were derived from the published DNA sequences for human semaphorins, neuropilins, and plexins (Table
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Cell-sorting
Cell segregation was measured in vitro by means of Cell Tracker dyes (Molecular Probes, Eugene, OR, USA). Cells were grown to near-confluence, and labeled with 1 of 2 fluorescent dyes, by incubation in a PBS solution containing from 1 to 10 µM dye and 0.1% DMSO for 30 min at 37°C. The 2 dyes used were CellTracker Orange (5- and 6- [4-chloromethylbenzoylaminotetramenthylrhodamine] mixed isomers) and CellTracker Green CMFDA (5-chloromethylfluorescein diacetate). Cells were rinsed extensively with PBS, trypsinized to free them from their substrates, and triturated into a single cell suspension. Cells from different sources were labeled with different dyes and mixed at a ratio of 1:1. The mixtures of cells were then plated into multiple 60-mm tissue culture dishes and allowed to adhere for 1–2 hrs, before being fixed with 4% paraformaldehyde for 30 min, rinsed in PBS, and photographed under a Nikon Eclipse E600 fluorescence microscope (Tokyo, Japan). Matching dishes of cells were incubated for various times from 12–72 hrs before fixation and observation. For each experiment, 10 random, non-overlapping fields of view were photographed. The degree of cell segregation was determined based on the ratio of cell-cell contacts made by each cell within a field of view to each of its adjacent neighbors and was represented as the percentage of cells in contact with cells of similar type (similar color).
ELISA Assays
Semaphorin and plexin antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used for the detection of Sema3A (N-15), Plexin-A1 (S-16), and Olexin-C1 (N-17) protein expression. These antibodies were raised in rabbits and immunopurified to human peptides. Non-immune rabbit antiserum was used as a control, and antibody specificity was determined by peptide competition. Briefly, extracts of cell proteins (100 µg/mL in 50 mM PBS with 100 mM NaCl and 1 mM phenylmethylsulfonate) were plated into 48-well plates and allowed to adhere for 24 hrs at 4°C. Non-specific protein binding was inhibited with the use of 1 mg/mL BSA. A 10-µg/mL quantity of semaphorin- or plexin-specific antibody was incubated with each sample for 2 hrs at 4°C and probed with alkaline-phosphatase-conjugated secondary antibodies and visualized with thes use of 1 mg/mL p-nitrophenol phosphate (PNPP) in 0.1 M diethanolamine (pH 8.3) with 5 mM levamisole (incubated at 25°C for 30 min with gentle agitation). The enzymatic color reaction was stopped by the addition of 500 µL of 0.75 N NaOH, and the mixture was assayed for 405-nm absorbance in a microplate reader (FL600, BioTek, Winooski, VT, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). PDL and gingival fibroblasts displayed little ability to separate in culture (Fig. 1A). In contrast, pre-osteoblasts displayed a greater tendency to separate from both PDL and gingival fibroblasts (Fig. 1B). This separation increased over time. In addition, gingival fibroblasts and pre-osteoblasts segregated more readily than did PDL fibroblasts and pre-osteoblasts (Fig. 1C). Thus, this in vitro model system for cell-sorting reflects the ability of pre-osteoblasts to segregate from adjacent mesenchymal cells in vivo.
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). Analysis of these data demonstrated that dermal, gingival, and PDL fibroblasts express many of these molecules in common (semaphorins 3A, 3B, 3C, 3D, 3E, 3F, 4A, 4B, 4C, 4D, 4E, 5B, 6B, and 6C; neuropilins 1, 2, and EDSN; and Plexin-B1 and Plexin-C1). These fibroblast populations varied from one another only in the expression of Sema3D, Sema5A, Sema7A, and Plexin-A1 (Fig. 2B). In contrast, pre-osteoblasts differed from all fibroblasts in their expression of Sema3B, Sema4C, Sema6B, Sema6C, Plexin-B1, and Plexin-C1 (Fig. 2C). In addition, pre-osteoblasts differ from at least one fibroblast population in Sema3D, Sema7A, and Plexin-A1. Sema3A and Plexin-A1 and Plexin-C1 protein expression mirrored their RNA expression (Fig. 3). In addition, intact PDL expressed the same semaphorin and plexin proteins as did PDL fibroblasts in culture, although at reduced levels. Therefore, pre-osteoblasts displayed greater differences in the expression of these cell-surface signaling molecules to fibroblasts than was seen between fibroblast populations. (Fig. 2D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Thus, members of these three families of molecules are expressed in a highly complex pattern in oral mesenchymal tissues and represent potential regulators of tissue movements during periodontal development and regeneration.
Similarities between Gingival and PDL Fibroblasts
Analysis of our current data suggests a high degree of similarity between fibroblast populations. This is in stark contrast to previous data suggesting a greater degree of difference between these populations (Lallier et al., 2004). In that study, PDL and gingival fibroblasts were more similar to each other than was either to dermal fibroblasts. In the current study, these same 3 populations varied only in the expression of 3 or 4 semaphorins and plexins (Fig. 2D). This may reflect an inherent difference between the transcripts being examined in each study. In the previous study, these fibroblast populations differed in their expression of transcripts involved with osteogenesis, indicating a potential difference in the developmental potential of these cell populations. In contrast, this study focused upon transcripts that may be involved in aiding cells in sorting themselves from their neighbors. Analysis of our in vitro cell-sorting data supports the finding that these cells do not segregate, and is supported by their similarity in semaphorin, neuropilin, and plexin expression. Analysis of these data, taken together, supports the supposition that these cells are functionally similar with regard to sorting, yet distinct in their osteogenic potential.
Similarities between Pre-osteoblast and Fibroblast Populations
While fibroblasts display great similarity to one another, these 3 cell populations display greater differences to pre-osteoblasts. Pre-osteoblasts express Sema6B to a greater degree than do all 3 fibroblast populations. In contrast, pre-osteoblasts also express Sema3B, Sema4C, Sema5B, Sema6C, Plexin-B1, and Plexin-C1 at significantly lower levels than do any of the 3 fibroblast populations. Pre-osteoblasts differed from dermal and gingival fibroblasts in their expression of 9 transcripts, and from PDL fibroblasts in the expression of 7 transcripts. This may reflect the ability of pre-osteoblasts to segregate from adjacent mesenchyme in vivo. This ability to segregate is mimicked by our in vitro assay system.
PDL fibroblasts display a greater ability to interact with pre-osteoblasts in vivo. This is reflected by the intermingling of collagen fibers (Sharpey’s fibers) between ligaments and calcified tissue (bone or cementum). In our experiments, this is reflected by a smaller number of differentially expressed semaphorins and plexins between pre-osteoblasts and PDL fibroblasts (7) and gingival fibroblasts (9). This is mimicked in our in vitro assay system by the greater segregation seen between gingival fibroblasts and pre-osteoblasts. Thus, analysis of these data supports the potential role of semaphorin signaling in the segregation of pre-osteoblasts from the mesenchymal cell types of the developing and regenerative periodontal attachment apparatus. Further studies will be required to determine how these molecules function in this process. In addition, the current study points out the extensive and complex pattern of expression of these families of molecules in a few distinct cell types. This could have broader implications, indicating a greater need to evaluate families of molecules in their entireties rather than focusing on the role of individual members of these families in isolation.
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ACKNOWLEDGMENTS |
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Received May 15, 2003; Last revision June 6, 2004; Accepted June 29, 2004
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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