Expression of UNCL during development of periodontal tissue and response of periodontal ligament fibroblasts to mechanical stress in vivo and in vitro
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- Kim, H., Choi, Y.S., Jeong, M. et al. Cell Tissue Res (2007) 327: 25. doi:10.1007/s00441-006-0304-3
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Mutations in two genes, uncoordinated (unc) and uncoordinated-like (uncl), lead to a failure of mechanotransduction in Drosophila. UNCL, the human homolog of unc and uncl, is preferentially expressed in periodontal ligament (PDL) fibroblasts compared with gingival fibroblasts. However, the precise role of UNCL in the PDL remains unclear. The aim of the present study has been to examine whether mechanical stimuli modulate the expression of UNCL in the human PDL in vivo and in vitro and to examine the roles of UNCL in the development, regeneration, and repair of the PDL. We have investigated the expression pattern of UNCL during the development of periodontal tissue and the response of PDL fibroblasts to mechanical stress in vivo and in vitro. The expression of UNCL mRNA and protein increases with PDL fibroblast differentiation from the confluent to multilayer stage but slightly decreases on mineralized nodule formation. UNCL has also been localized in ameloblasts and adjacent cells, differentiating cementoblasts, and osteoblasts of the developing tooth. Strong distinct UNCL expression has further been observed in the differentiating cementoblasts of the tooth periodontium at the site of tension after orthodontic tooth movement. Application of cyclic mechanical stress on PDL fibroblasts increases the expression of UNCL mRNA. These results indicate that UNCL plays important roles in the development, differentiation, and maintenance of periodontal tissues and also suggest a potential role of UNCL in the mechanotransduction of PDL fibroblasts.
KeywordsUNCLPeriodontal ligamentFibroblastMechanical stressCementoblastDifferentiationHumanRat (Sprague-Dawley)
Periodontal ligament (PDL) is the connective tissue located between the tooth root and alveolar bone. It functions in several capacities, including tooth eruption, anchorage of the tooth in the jaw, and physiological mobility during mastication, and is a source of osteogenic cells (Lekic et al. 1996). PDL fibroblasts, in particular, can differentiate in order to contribute to the regeneration and repair of the PDL and to the remodeling of the surrounding hard tissue, cementum, and alveolar bone (Lekic and McCulloch 1996). Furthermore, PDL fibroblasts can undergo differentiation in response to a variety of extracellular stimuli (Arceo et al. 1991; Basdra and Komposch 1997; Carnes et al. 1997). These cells are subject to continuous mechanical strain under physiological conditions, i.e., masticatory forces, and during orthodontic and orthopedic treatment (Wilde et al. 2003). Mechanical stimulation has been demonstrated to activate signal transduction pathways affecting the form and function of PDL, cementum, and alveolar bone (Kawarizadeh et al. 2005). However, little is known about these pathways; this reflects a deficiency in our understanding of mechanotransduction mechanisms.
In a previous study, we have isolated UNCL (uncoordinated-like), the human homolog of the Caenorhabditis elegans gene unc-50 (Genebank accession no. AF077038, DKFZp564G0222, PDLs22), by using subtractive hybridization between cultured PDL fibroblasts and gingival fibroblasts (Park et al. 2001). UNCL is preferentially expressed in PDL fibroblasts compared with gingival fibroblasts. Although UNCL has been reported as an inner nuclear RNA-binding protein in neuronal nicotinic acetylcholine receptor (nAChR) subunits (Fitzgerald et al. 2000; Wanamaker et al. 2003), its function in periodontal tissues has not as yet been described. Interestingly, mutations in two genes, viz., uncoordinated (unc) and uncoordinated-like (uncl), have been found to eliminate mechanotransduction and show uncoordination in Drosophila (Kernan et al. 1994). The aim of the present study has been to examine whether mechanical stimuli modulate the expression of UNCL in the human PDL in vivo and in vitro and to examine the roles of UNCL in the development, regeneration, and repair of PDL. We have investigated the distribution patterns of UNCL during the development of periodontal tissues in vivo and during the differentiation of PDL fibroblasts in vitro. Furthermore, we have examined the relationship between mechanical stress and UNCL in the PDL by the application of orthodontic tooth movement in vivo and mechanical stretching of PDL fibroblasts in vitro.
Materials and methods
Cell culture and Northern blot
PDL fibroblasts were obtained from an explant culture of healthy human PDL tissue. These biopsies were performed under the guidelines of the Chosun University for Animal and Human Subjects Research (Gwang-Ju, Korea). The PDL tissue attached to the middle third of the root were minced, plated, and cultured in Dulbecco’s modified Eagle medium (Gibco BRL) supplemented with 10% heat-inactivated fetal bovine serum and antibiotics at 37°C in humidified air containing 5% CO2. When 80%–90% confluence was obtained, the cells were collected by digestion with 0.15% trypsin and 0.5 mM EDTA in phosphate-buffered saline (PBS) and plated at a density of 7×105 cells/60-mm dish. To induce PDL cell differentiation and mineralized nodule formation, confluent cells were treated with 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate (GP), and 5 μM dexamethasone (Dex) for up to 2 weeks.
Total RNAs were isolated from cultured PDL fibroblasts at 0, 4, 7, and 14 days by using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The blot containing total RNA (20 μg) from cultured PDL fibroblasts was probed with the 32P-labeled cDNA fragment of human UNCL, secreted protein acidic and rich in cysteine (SPARC), alkaline phosphatase (ALP), bone sialoprotein (BSP), and human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) as previously described (Chien et al. 1999). For re-hybridization, membranes were stripped in a boiled 0.1% SDS solution. The radioactive band was quantified by densitometry (Pharmacia Ultrascan XL, Pharmacia LKB) and normalized to the actual amount of the GAPDH mRNA loaded. The differences in mRNA expression level were presented as the fold increase of each RNA species and were expressed as the mean (±SEM).
Antibody preparation and Western blot
The peptide, ISNKYLVKRQSRD, was selected from the human UNCL mRNA sequence (Fitzgerald et al. 2000) and was chemically synthesized by a solid-phase procedure with the aid of an automated peptide synthesizer (Model 430, Applied Biosystems). To immunize the rabbits, the synthetic peptide was conjugated to a carrier protein, viz., keyhole limpet hemocyanin, through sulfhydryl groups by the MBS coupling method. After immunization, the polyclonal antiserum was affinity-purified at Peptron (Seoul, Korea) by using CNBr-sepharose 4B (Amersham Pharmacia). The polyclonal antibody to UNCL recognized rat and human UNCL.
After protein transfer from 15% SDS-polyacrylamide gels, nitrocellulose membranes were blocked with 5% (m/V) non-fat milk in 0.05% Tween 20/PBS (PBS-T). The membranes were washed (3× for 10 min) with PBS-T, further incubated with polyclonal anti-UNCL antibody (1:500) in 5% non-fat milk-PBS-T for 1 h, treated with anti-rabbit IgG peroxidase conjugate (Sigma; 1:1,000) in 5% non-fat milk-PBS-T, and washed as above. Bands were revealed with ECL reagent (Amersham Pharmacia). The change in UNCL protein expression was quantified by densitometry as described above.
Preparation of developing tissue and immunohistochemistry
Embryonic (E16, E18) and postnatal (PN2, PN7, PN14, PN21, PN28, PN41) rat tissues were fixed in 4% paraformaldehyde, decalcified in a 10% EDTA (ethylenediaminetetraacetic acid, pH 7.4) solution at 4°C for 4 weeks, embedded in paraffin, and cut into identical mesio-distal sections. Tissue sections were pre-incubated with 1% bovine serum albumin in PBS for 30 min, incubated for 1 h with anti-UNCL (1:150) antibody and then for 1 h at room temperature with secondary antibody, and reacted with avidine-biotin-peroxidase complex (Vector Laboratories) in PBS for 1 h. After color development with 0.05% diaminobenzidine tetrahydrochloride, they were counterstained with hematoxylin. Rabbit pre-immune serum was used as a negative control.
Orthodontic tooth movement and immunohistochemistry
Eight 7-week-old male Sprague-Dawley rats were used. A titanium-nickel closed-coil spring was applied to the occlusal surface of the maxillary first molar (M1) with a hook and the upper incisors with a ligature wire. The coil spring exerted a mesially directed force of 40 g for 1 h per day. At 3 days after orthodontic tooth movement, the tissues were prepared and immunostained with anti-UNCL (1:150) and anti-osteocalcin (Santa Cruz; 1:200) antibodies as described above.
Application of mechanical tensile force to PDL fibroblasts and reverse transcription/polymerase chain reaction
PDL fibroblasts were seeded at 5×104/35-mm Flexercell plate dish (Flexcell Internatonal). Mechanical tensile force was applied to the PDL fibroblasts by using a Flexcell strain unit (FX-2000; Flexcell Internatonal), which was capable of controlling the magnitude and the frequency of cell deformation. The PDL fibroblasts were subjected to 9% of maximum strain for 5 s, followed by a 5-s relaxation (6 cycle/min) for 6 days (Matsuda et al. 1998).
Total RNAs were isolated from the PDL fibroblasts by using TRIzol reagent (Invitrogen). First-strand cDNA synthesis was carried out with oligo (dT) primers (Invitrogen) according to the manufacturer’s instructions. This cDNA was then used as a substrate for polymerase chain reaction (PCR) amplification over 30 cycles. We used the following PCR primers pairs: 5′-ATGAGAGCCCTCAGACTCCTC-3′ and 5′-CGGGCCGTAGAAAGCGCCGATA-3′ (55°C) for osteocalcin; 5′-ACGTGGGAATCGCAGGAT-3′ and 5′-CCTTCC CAACACCAGACAGT-3′ (53°C) for UNCL; 5′-CCATGG AGA AGGCTGGG-3′ and 5′-CAA AGT TGTCATGGATGACC-3′ (55°C) for GAPDH. GAPDH was used for standardization in all reverse transcription/PCR (RT-PCR) experiments. Controls were performed in the absence of reverse transcriptase. The change in UNCL and osteocalcin mRNAs expression was quantified by densitometry as described above.
Expression of UNCL mRNA and protein during PDL fibroblast differentiation
Expression of UNCL protein during tooth development
Expression of UNCL protein in periodontal tissues after orthodontic tooth movement
Expression of UNCL mRNAs after application of mechanical tensile force on PDL fibroblasts
Mechanical stress plays an important role in the homeostasis of human tissues during normal development (Ziros et al. 2002; Schild and Trueb 2004) and under specific conditions, such as tooth movement and mastication (Doi et al. 2003; Myokai et al. 2003; Takahashi et al. 2003; Komatsu et al. 2004). During tooth movement and mastication, PDL fibroblasts must therefore be able to sense mechanical stimuli and to respond to these changes by alterations in the metabolism of biomolecules and by adapting the surrounding extracellular matrix (Howard et al. 1998; Ozaki et al. 2005). The differential expression of genes in PDL fibroblasts under physiological stress, such as that caused by an occlusal force, is thought to be orchestrated not only for the remodeling of PDL itself, but also for the repair and regeneration of periodontal tissues. However, little is known about the genes expressed in PDL fibroblasts under mechanical stress; this reflects a deficiency in our understanding of mechanotransduction mechanisms.
The genes unc and uncl have previously been identified during the intensive mutagenesis and mapping of genes based on the X-chromosome of Drosophila (Perrimon et al. 1989) and are related to unc-50 of C. elegans. In addition, the alignment of predicted protein sequences from C. elegans, rat, and human have revealed a striking degree of conservation between these three proteins (Fitzgerald et al. 2000). Mutation in two genes, viz., unc and uncl, produces uncoordination, touch-insensitive larva, adult behavioral defects, and failure to elicit a scratch reflex of Drosophila (Kernan et al. 1994). This study suggests that UNCL, the human homolog of unc and uncl, plays an important role in the mechanotransduction of mammalian tissue.
PDL fibroblasts differentiate and form mineralized nodules when cultured in the presence of Dex (5 μM), GP (10 mM), and ascorbic acid (50 μg/ml) for up to 2 weeks (Cho et al. 1992). In the present study, the expression of UNCL mRNA and protein has been shown to increase with PDL fibroblast differentiation from the confluent to multilayer stage but slightly to decrease with mineralized nodule formation. This suggests that UNCL plays important roles in PDL fibroblast proliferation and differentiation, although it may also be related to mineralized nodule formation of the cells.
The development and regeneration of periodontal tissues presumably require similar cellular activities. Clues for defining the role of specific factors in the regeneration of periodontal tissues have come from developmental studies directed at examining the expression of genes/proteins during the development of the periodontium (Foster and Somerman 2005). In the present study, UNCL protein appears to be strongly expressed in ameloblasts (internal dental epithelium) and adjacent cells at the crown stage and in the Malassez epithelial cell rest of the PDL at the erupted and functional stages of the tooth (data not shown). At the root formation and PDL alignment stage, UNCL has been localized in differentiating pre-cementoblasts and pre-osteoblasts along the developing root surface and alveolar bone. These results suggest a potential role of UNCL in tooth root development including cementoblast differentiation. Although mechanical stimulation has not been demonstrated to play a role in tooth root development, mechanical signal transduction clearly influences PDL, cementum, and alveolar bone remodeling (Bolcato-Bellemin et al. 2000; Yamashiro et al. 2001). After the completion of crown formation, the internal and external dental epithelium (devoid of stellate reticulum and stratum intermedium) fuse below the level of crown cervical enamel to produce a bilayered epithelial sheath termed Hertwig’s epithelial root sheath. The current perception is that disintegration of this sheath into epithelial fragments allows ectomesenchymal cells from the dental follicle proper to come in contact with the dentin surface where they differentiate into cementoblasts (Spouge 1980; MacNeil and Thomas 1993). However, accumulating evidence indicates that epithelial cells from Hertwig’s epithelial root sheath may undergo epithelial-mesenchymal transformation into cementoblasts during development (Zeichner-David et al. 2003).
Although an intermittent orthodontic force, viz., the 1:23-h force protocol (Konoo et al. 2001; Hayashi et al. 2004), is ineffective at stimulating typical mesial tooth movement, we have found that the force induces cementoblast differentiation at the site of tension. Osteocalcin is a marker protein of osteoblastic/cementoblastic differentiation in the PDL because cementoblasts and osteoblasts may be considered as phenotypically similar, and any differences noted between these cells may be related to the local environment (Shimono et al. 2003; Bosshardt 2005). PDL fibroblasts have been shown to possess osteogenic/cementogenic potential in vitro and to undergo osteoblastic/cementoblastic differentiation in response to mechanical stimuli in vivo (Basdra and Komposch 1997). In the present study, UNCL and osteocalcin protein have been found to be up-regulated more in the tension side than in the compression side after mechanical stress during experimental tooth movement. This suggests that UNCL system participates in the mechanotransduction of PDL during orthodontic tooth movement and, thereby, in the regulation of cementoblastic/osteoblastic differentiation of the PDL cells.
Of note, PDL is subjected to the intermittent loading of bite forces. We have applied intermittent mechanical force to produce 9% of elongation of the periphery of the wall in the PDL fibroblasts in vitro. In the present study, UNCL and osteocalcin mRNAs have been shown to be strongly expressed after mechanical stretching of the cells. Thus, a mechanical stimulus up-regulates UNCL in the PDL fibroblasts. However, our results do not directly support the function of the UNCL in the mechanotransduction pathway. A yeast two-hybrid assay is currently being undertaken to detect protein-protein interactions in vivo. In addition, we are examining the way that the intracellular signals generated by mechanical stress affect the expression of UNCL.
We thank K.-Y. Lee and M.-H. Lee for their excellent technical assistance, and Dr. M.-I. Cho of SUNY at Buffalo for valuable suggestions and critical reading of the manuscript.