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Previous Article | Next Article 
Molecular and Cellular Biology, June 2007, p. 4306-4316, Vol. 27, No. 12
0270-7306/07/$08.00+0 doi:10.1128/MCB.00041-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
11ß1 Integrin-Dependent Regulation of Periodontal Ligament Function in the Erupting Mouse Incisor
Wouter Beertsen,3
Paola Zigrino,4
Attila Aszodi,5
Nicolai Miosge,6
Erik Forsberg,7 and
Donald Gullberg1,2*
Department of Biomedicine,1 Department of Oral Sciences-Periodontics, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway,8 Department of Medical Biochemistry and Microbiology, Uppsala University, BMC, Box 582, Uppsala SE-751 23, Sweden,2 Academic Center for Dentistry, Amsterdam,The Netherlands,3 Department of Dermatology, University of Cologne, Cologne, Germany,4 Max Planck Institute for Biochemistry, Martinsried, Germany,5 Department of Prosthodontics, Georg-August-Universität, Göttingen, Germany,6 Amersham Biosciences AB, Uppsala, Sweden7
Received 9 January 2007/ Returned for modification 2 February 2007/ Accepted 28 March 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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11ß1 is a key receptor for fibrillar collagens. To study the potential function of 11 in vivo, we generated a null allele of the 11 gene. Integrin 11–/– mice are viable and fertile but display dwarfism with increased mortality, most probably due to severely defective incisors. Mutant incisors are characterized by disorganized periodontal ligaments, whereas molar ligaments appear normal. The primary defect in the incisor ligament leads to halted tooth eruption. 11ß1-defective embryonic fibroblasts displayed severe defects in vitro, characterized by (i) greatly reduced cell adhesion and spreading on collagen I, (ii) reduced ability to retract collagen lattices, and (iii) reduced cell proliferation. Analysis of matrix metalloproteinase in vitro and in vivo revealed disturbed MMP13 and MMP14 synthesis in 11–/– cells. We show that 11ß1 is the major receptor for collagen I on mouse embryonic fibroblasts and suggest that 11ß1 integrin is specifically required on periodontal ligament fibroblasts for cell migration and collagen reorganization to help generate the forces needed for axial tooth movement. Our data show a unique role for 11ß1 integrin during tooth eruption. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunits and 8 ß subunits. Out of the 24 integrin heterodimers, 1ß1, 2ß1, 10ß1, and 11ß1 act as primary receptors for native collagens. The 3ß1 integrin, initially described as a collagen receptor, was later shown to act as a receptor for laminin-5 and does not display any measurable affinity for collagen I (21). The observed role of vß3 integrin in collagen lattice remodeling in vitro (13, 33) and in vivo (33) may be related to indirect binding to RGD ligands which are locally deposited in the collagen matrix.
Collagen-binding integrins bind native collagens via their I domain, recognizing a GFOGER motif (30, 52) or similar sequences with varying specificities and affinities depending on the collagen type and fibrillar status (27). In addition to mediating cell adhesion, cell spreading, and cell migration (22), collagen-binding integrins regulate collagen turnover (32) and take part in assembling (31, 49) and reorganizing three-dimensional collagen matrices (23).
Fibroblasts express characteristic collagen receptor repertoires in a tissue-specific manner, partly reflecting their different embryonic origins. We have previously demonstrated that 11ß1 is the only detectable collagen-binding integrin in the incisor periodontal ligament (PDL) fibroblasts of mice (38). The PDL in the mouse incisor is composed of a relatively dense connective tissue on the side facing the tooth and a loose connective tissue region rich in blood vessels on the side facing the bone. The odontogenic ectomesenchyme is the source of the cells populating these two domains in the PDL, which are sometimes referred to as tooth-associated fibroblasts and bone-associated fibroblasts, respectively. Both cell populations are characterized by the expression of Runx2 (50), periostin (29, 43), and collagen XII (41), whereas markers distinguishing between these two cell populations in incisors and molars are scarce.
Studies of tooth eruption have indicated that it is a complex multifactorial process, initially dependent on osteoclast activity to generate an eruptive path through the bone (50). In the continuously erupting rodent incisor, the PDL has been shown to play a central role during the subsequent supraosseous phase of tooth eruption (37), although the exact role of the ligament in this process is controversial.
Two central functions of the PDL, independent of its role in tooth eruption, are to anchor the tooth in its socket and to act as a shock absorber during mastication. For this purpose an elaborate network of collagen fibrils exists. Collagen turnover in the PDL is high, most likely reflecting the constant need for remodeling in order to resist the forces of mastication. Collagen synthesis occurs throughout the mouse incisor PDL, but remodeling has been reported to occur mainly in a middle region called the transitional zone (5). Elaborate control mechanisms exist to maintain a constant width of the PDL and to prevent bone and cementum from encroaching into the PDL space. The transcription factor Msx2 has recently been suggested as a molecular defense mechanism that prevents mineralization in the PDL (51), but the nature of the specific molecular regulators on the surface of the PDL fibroblasts that "sense" the matrix status and control collagen turnover has remained elusive.
We describe here the identification of 11ß1 integrin as a central molecule on the surface of PDL fibroblasts. Mice deficient in the 11 integrin chain are dwarfed, show signs of malnutrition, and display increased mortality due to defective incisors. Data from our in vivo and in vitro models suggest that the tooth phenotype is due to a primary defect in incisor PDL and support a model in which 11ß1 is needed during the migration/reorganization of fibroblasts throughout the ligament in order to help generate the forces needed for tooth eruption.
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MATERIALS AND METHODS |
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11 integrin-deficient mice.
Four bacterial artificial chromosome (BAC) clones were identified from a spotted 129/SvJ genomic library (BAC Mouse Release II; Genome Systems) using a 0.95-kb probe from the 5' end of human 11 cDNA (nucleotides –37 to 917) (48). BAC clones were obtained from Genome Systems. After cleavage with SacI, a 12-kb fragment reactive with the 0.95-kb probe was identified, subcloned into the pBluescript SK(+) cloning vector, and sequenced (MWG Biotech). The cell culture, homologous recombination, and microinjection of embryonic stem (ES) cell clones into C57BL/6J blastocysts were carried out as described previously (17). Two mouse strains originating from clones 95 and 215 were obtained by mating the chimeras with C57BL/6J mice. Homozygous mice were obtained by intercrossing of heterozygous mice. The genotyping of the mice was determined by Southern blotting or genomic PCR (38). In order to remove the PGK neo cassette, Itga11–/– mice were bred with a mouse expressing Cre recombinase in the tyrosine hydroxylase locus (34).
Western blotting and immunoprecipitations.
Western blotting for detection of the integrin 11 protein and immunoprecipitations were performed as described elsewhere (38). Antibodies used for cell cycle analysis were rabbit anti-mouse p16, mouse anti-human p53 (both Santa Cruz Biotechnology, Inc.), mouse anti-human Rb (BD PharMingen), and rabbit anti-human phospho-Rb (Ser795; Cell Signaling).
RT-PCR. Total RNA isolation from mouse embryonic fibroblasts (MEFs) and PDL tissue as well as cDNA synthesis was is described elsewhere (38). The sequences of the primers amplifying collagen-binding integrins have been described previously (38). Sequences of additional primers used are listed in Table 1. Total RNA from MEFs cultured in gels was harvested by solubilizing the gels directly in RNAzol, and the RNA was extracted according to the manufacturer's instructions (Wak-Chemie Medical GmbH). After a 15-min DNase treatment (Boehringer) and phenol-chloroform extraction, reverse transcription-PCR (RT-PCR) was performed by following the manufacturer's instructions (Gene Amp RNA PCR kit; Perkin Elmer). The cDNA was used to amplify specific transcripts by PCR by following the manufacturer's instructions (REDTaq ReadyMix PCR Mix; Sigma). Amplification of murine S26 was used for normalization.
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11 knockout (ko) and wild-type (wt) MEFs were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum (FCS; PAA Laboratories) and antibiotics. In order to obtain immortalized MEFs, primary fibroblast cultures from wt and Itga11 ko embryos were infected with recombinant retrovirus-transducing simian virus 40 (SV40) large T at 37°C for 2 h (a gift from S. Johansson, Uppsala University) (26). After infection, cells were cultured in DMEM plus 10% FCS for approximately 14 days. Noninfected cells died during this period, and growing noncloned cells were expanded and used as bulk-immortalized cells.
In order to restore the function of integrin 11, 10 µg of full-length human 11 cDNA was transfected into SV40-immortalized ko cells as described previously (47), and highly expressed clones were isolated. Two independently obtained clones were used in further experiments.
Histology and immunohistochemistry. For histological analysis, the lower jaws or knee joints were fixed overnight in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), pH 7.4, and decalcified in 10% EDTA-PBS for 3 to 4 weeks. After the jaw and joints were embedded in paraffin, 6- to 8-µm sections were cut and stained with hematoxylin and eosin (H&E), toluidine blue, or sirius red. Images were visualized under a Leica microscope equipped with a ProgRess LCD camera.
For immunohistochemistry, specimens were collected in polyvinylpyrrolidone-EDTA solution without fixation and subjected to decalcification for 1 to 2 weeks (28). Following decalcification, the specimens were snap-frozen in liquid nitrogen and sectioned on a Leica cryostat. The primary antibodies were hamster anti-1 integrin (Ha31/8; BD Pharmingen), rat anti-2 integrin (Emfret Analytics), rabbit anti-10 integrin (10), rabbit anti-11 integrin (38), rat anti-PECAM (CD31; BD PharMingen), rabbit anti-collagen I, rabbit anti-collagen III (both obtained from J. Risteli, University of Oulu, Finland), rabbit anti-collagen XII (obtained from M. Koch, University of Cologne, Germany), rat anti-F4/80 antigen (Serotec), rat anti-tenascin-C (46), and mouse anti-human alpha-smooth muscle actin (clone 1A4; Sigma). The secondary antibodies were Cy2, Cy3, or biotin-conjugated goat anti-rat, goat anti-rabbit, and goat anti-mouse immunoglobulin G (Jackson ImmunoResearch Laboratories). An ABC kit (Vector Laboratories) and DAB substrate kit (Zymed Laboratories) were used to detect biotinylated secondary antibodies. Images were visualized under a Zeiss Axioscope microscope equipped with optics for observing fluorescence and were captured using a digital AxioCam MRm camera.
Detection of proliferation and apoptosis. The proliferation index in vivo was analyzed using bromodeoxyuridine (BrdU) incorporation as described elsewhere (1). At least three animals of each genotype were used per time point. Three sections from each animal were analyzed. The proliferation index in vitro was analyzed using an immunofluorescence assay for the detection of BrdU incorporated according to the manufacturer's instructions (5'-bromo-2'-deoxy-uridine labeling and detection kit I; Roche Diagnostics). The coverslips were mounted in Vectashield medium with 4',6'-diamidino-2-phenylindole (DAPI; Vector Laboratories). The total number of nuclei (DAPI) and number of BrdU-positive nuclei were counted. Five fields of 150 to 200 cells were counted for each cell type.
A terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay using sections of mouse PDL of different ages (2 months, 6 months, and 1 year) was performed to detect apoptotic nuclei. The sections were processed using a TdT in situ apoptosis detection kit (R&D Systems) according to manufacturer's instructions. Three animals of each genotype were analyzed. Nuclei were counted in whole PDL at the level of the molar block, and the apoptotic index was calculated as the ratio of TdT-positive nuclei to the total number of nuclei.
Micro-CT. Three-dimensional, high-resolution reconstructions of the upper and lower incisors were obtained using a microcomputed tomography (micro-CT) system. The tissues were fixed in buffer containing 4% PFA, 1% glutaraldehyde, and 0.1 mM sodium cacodylate (pH 7.4) and processed as described elsewhere (36).
Electron microscopy.
The mice were anesthetized with ether and sacrificed by cervical dislocation, and the lower jaw bones were removed. The tissues were fixed in 4% PFA and 0.5% glutaraldehyde for 15 min, dehydrated in a graded series of ethanol up to 70%, and embedded in the acrylic resin LR-Gold (London Resin Company). Semithin (1 µm) and ultrathin (0.8 µm) sections were cut according to procedures previously described in detail (19). For orientation purposes, semithin sections were stained with toluidine blue. For electron microscopy, the sections were rinsed with water and stained with uranyl acetate (10 min) and lead citrate (8 min). Sections were examined with an LEO 906E electron microscope. Three wild-type mice and three 11 knockout mice were investigated.
Eruption measurement. Mice were anesthetized with an intraperitoneal injection of avertin (0.20 mg/ml, 100 µl per 10 g of body weight), and a horizontal mark was made at the gum level on one of the lower incisors using a dental drill (Silfradent) equipped with a diamond saw. The distance between the mark and the gum was measured 1 week later. The measurements were repeated once a week for 5 weeks, making new marks when necessary. The eruption rate was estimated as the average value of the five measurements for each animal. At least five 3-week-old and 6-month-old mice of each genotype were used.
Cell attachment and cell spreading. Forty-eight-well plates (Nunc Roskilde) were coated with different concentrations of collagen type I or type IV overnight at 4°C and blocked with 2% bovine serum albumin in PBS for 1 h at 37°C, and cell attachment experiments were performed as previously described (47).
To analyze the number of focal contacts formed, cells were left to attach on collagen type I for 1 h and fixed with 2% PFA. Fixed and washed cells were incubated with mouse anti-human vinculin antibodies (clone hVIN-1; Sigma) and fluorescein isothiocyanate-labeled phalloidin (Sigma) and mounted for microscopy. Images were visualized under a Zeiss Axioscope microscope. For cell spreading, the cells were allowed to spread for 45 min, washed with PBS, and stained with 0.1% crystal violet for 30 min. The number of spread cells and total number of attached cells were counted in six fields of each cell type at x20 magnification.
Collagen gel contraction.
The collagen gel contraction assay has been described previously (23). DMEM with or without blocking antibodies (anti-1 Ha31/8, anti-2 Ha 1/29, or both; BD Pharmingen) was ejected into the wells to detach the gels. Free-floating gels were further incubated at 37°C, and gel diameters were measured under a microscope at the time points indicated.
Flow cytometry.
For fluorescence-activated cell sorter analysis, 75-cm2 flasks were coated with collagen type I as described above. Confluent 11 ko and wt MEFs in passage 5 were serum starved for 12 h, washed in PBS, and trypsinized. Following trypsinization, 5 x 105 cells were seeded into collagen-coated flasks in DMEM containing 10% FCS. After 2 h, medium was replaced with DMEM containing 2% FCS. Cells were collected after 48 h and resuspended in PBS. MEFs were permeabilized with cold ethanol overnight at 4°C and stained with propidium iodide (500 µg/ml; Sigma) according to a previously described method (45). Cell cycle data were collected using a FACSCalibur flow cytometer (BD Bioscience) and analyzed with CellQuest Pro software (BD Bioscience).
Analysis of matrix metalloproteinases. MEFs were used at passages 3 and 4. Three-dimensional collagen gels were performed as described earlier (35). Control cultures of MEFs were performed on uncoated (monolayers) tissue culture plates. Twenty-four hours postseeding, cultures were washed three times with PBS and medium was replaced with serum-free DMEM for 24 h.
Serum-free supernatants from monolayers and collagen gel cultures were centrifuged (5 min, 272 x g) to eliminate debris and suspend cells, and then they were analyzed. Equal amounts of supernatants, normalized to the cell number (volume/2,000 cells/well), were used for gelatin zymography. Collagen gels or monolayers were further processed for RNA preparation.
For gelatin zymography, supernatants were separated on nonreducing sodium dodecyl sulfate (SDS) polyacrylamide gels (10%) containing 1 mg/ml bovine gelatin (Sigma). After electrophoresis the gels were washed in 2.5% Triton X-100 for 30 min to remove SDS and then incubated in enzyme substrate buffer (50 mM Tris-HCl, pH 8, 5 mM CaCl2) overnight at 37°C. Gels were stained with Coomassie blue R250 and destained in water.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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11-deficient mouse strains.
To introduce a null mutation in the mouse integrin 11 gene (Itga11), we replaced parts of exon 3 and intron 3 with an internal ribosome entry site, lacZ, and a PGK neo cassette by gene targeting (Fig. 1A and B). The targeting construct was introduced into R1 ES cells by electroporation, and resistant colonies were selected with G418. Southern blotting was used to identify four homologously targeted clones out of 325 that were screened (data not shown). The clones 95 and 215 were selected for further work. Southern blotting of these was also used to verify that integration had occurred only once (Fig. 1C). These clones, with the correctly targeted alleles, were injected into blastocysts of C57BL/6J mice. Chimeric males were mated with C57BL/6J mice, and the offspring were screened with Southern blots of tail DNA for germ line transmission of the targeted allele. Both recombinant ES clones transmitted the mutant allele to the germ line. Intercrossing of heterozygous F1 mice gave rise to live homozygous offspring (Fig. 1D and E) with the expected Mendelian ratios and with no obvious phenotypic defects at birth. The homozygous males and females were fertile.
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11 transcripts in the knockout mice, RNA was isolated from E14.5 wild-type (+/+) and null (–/–) embryonic fibroblasts and RT-PCR analysis was performed (Fig. 1F). No PCR product was obtained from the –/– cells using primers located in exons 2 and 5, whereas RT-PCR using primers downstream of the deletion amplified a small amount of 11 RNA from the null cells, most likely representing out-of-frame RNA transcripts resulting from readthrough. Immunohistochemistry and Western blotting of tissues from the wt and –/– mice showed an absence of any specific 11 immunoreactivity in the latter (Fig. 1G and H).
Characteristics of the skeletal system of 11 integrin-deficient mice.
When the tails were cut for genotyping at 3 weeks of age, it was noted that each litter contained mice that were smaller than their littermates (Fig. 2A). Both the female and male homozygous mice revealed a 20 to 30% reduction in weight on average that persisted into adulthood (Fig. 2B). X-rays showed that the overall size of the skeleton was smaller, compatible with proportional dwarfism (data not shown). To rule out any effect of the neo cassette, the cassette which is flanked by loxP sites in the targeting construct was Cre deleted by crossing with Cre deleter mice. The same phenotype was observed in the mice in which the neo cassette had been removed (data not shown), confirming that the weight difference is due to the inactivation of 11 integrin alleles. The neo-deleted mice were used in all further characterizations.
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11 is heavily expressed in the perichondrium (Fig. 1G) (38, 47), we initially focused our analysis on the growing skeletal system. However, at the light microscopic level we could not observe any structural defects of the forming cartilage or bone at 1 to 8 weeks (data not shown), nor could we detect any significant differences in chondrocyte proliferation rate between the normal and mutant mice (data not shown).
Tooth phenotype in the absence of 11.
The 11-deficient mice showed increased mortality starting from around 1 year of age, and 10 such mice died between 12 and 19 months, whereas only one control mouse died (data not shown). Necropsy of the 11 null mice revealed severe malnutrition, which led our attention to the digestive system, including the teeth.
We have previously established that 11 is coexpressed with 2 in villus cluster fibroblasts (38), raising the question of whether defects in these cells could affect the villus structure, with consequences for nutrient absorption. The analyses performed so far have revealed no such defects, however (S. N. Popova, D. Gullberg, and P. Simon-Assman, unpublished data).
In the older mice a phenotype in the incisors was clearly visible, prompting closer analysis of the forming teeth. The smaller size and tooth phenotype were present in both strains of independently generated Itga11-deleted mice. All future work was performed on the neo-deleted strain derived from ES clone 95. In the mutant mice there was a clear delay in the time of incisor eruption (Fig. 3A), and an altered tooth shape was observed after this delayed eruption (Fig. 3B and C). In the older mice the incisal part of the upper incisors was frequently missing (Fig. 3C), whereas the intraalveolar part proved to be still present upon removal of the soft tissue (Fig. 3D). Monitoring of tooth eruption showed that it was reduced at 3 to 6 weeks and had essentially stopped at 6 to 7 months (Fig. 3E). Micro-CT of 2-month-old incisors showed increased thickness of the dentin layers in the apical region and, as a consequence, pulp closure (Fig. 3F). The enamel layer proved to be mineralized until well into the apical area (data not shown), and the incisors of the older animals showed buckling of their apices (Fig. 3G and H).
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In vitro phenotype of 11-deficient mouse embryonic fibroblasts.
MEFs isolated from wt and ko embryos were tested for adhesion to collagen I and collagen IV using fibronectin as a control. Attachment to collagen I was reduced to less than 20% of that in the control cells in the absence of 11 (Fig. 4A), whereas cell attachment to collagen IV was only marginally affected (Fig. 4B). Cell attachment to fibronectin was not affected by the absence of integrin 11 (Fig. 4C). The proportion of cells that had spread on collagen I was reduced by 50% in the integrin 11–/– fibroblasts but was normal in the case of spreading to fibronectin (Fig. 4D). 11–/– MEFs formed fewer vinculin-positive focal contacts on collagen I (Fig. 4E) and were found to be less efficient in reorganizing a collagen lattice (Fig. 4F). Whereas wt cells incubated with a mixture of 1 and 2 integrin-blocking antibodies efficiently reduced the collagen lattice to 20% of its original size, gels populated with 11–/– MEFs and incubated with antibodies to 1 and 2 reduced the lattices only to 60% of their original size. To confirm that the changed collagen gel reorganizing properties were 11 dependent, we retransfected SV40-immortalized 11–/– cells with human 11 cDNA and were able to efficiently correct the collagen gel contraction defect (Fig. 4G). We also compared levels of MMP expression (MMP13 and MMP14) and activity (MMP2 and MMP9) for cells cultured on monolayers in floating and attached collagen gels. Although the absolute levels of MMP RNA varied with cell isolates, a reproducible drop in MMP13 and MMP14 RNA levels was observed in the 11–/– MEFs (Fig. 5) cultured in floating collagen gels, whereas MMP2 and MMP9 gelatinase activity in MEFs was unaffected by 11 integrin expression.
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11–/– cells stopped growing at earlier passages than wt cells. In accordance with this, cells that were homozygous for the 11 deletion showed reduced proliferation and BrdU incorporation on collagen I (Fig. 6A). Fluorescence-activated cell sorter analysis revealed blockage of cells at the G1/S transition (Fig. 6B), but we failed to detect changed levels of p53, pRb, or p16 in MEFs deficient in 11 (data not shown).
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11 RNA and immunohistochemical analysis of 11 protein in periodontal ligaments.
To gain an understanding of the differential phenotype in the incisors, we followed the expression of 11 RNA and protein during E14 to E17 and postnatally (Fig. 7A to D). 11 was expressed in the dental follicular mesenchyme that forms the PDL and in the preodontoblasts of the developing molars and incisors. Immunohistochemical staining of the embryonic/postnatal follicular mesenchyme revealed no differences in the levels of expression of integrin 11 or the repertoires of collagen-binding integrins between the incisor and molar PDLs (data not shown). 1 and 2 were present in capillaries, whereas 10 was lacking completely in the PDL. This expression pattern was largely retained in the adult incisor PDL (Fig. 7E to I). One noticeable difference, however, was that the levels of 11 in the molar PDL tissue were low (Fig. 7I) compared to those of the adult incisors (Fig. 7H). We failed to detect any compensatory regulation of collagen-binding integrins in the incisor or molar PDL in the absence of 11 (Fig. 7J to M). This was also confirmed by semiquantitative PCR (Fig. 7N).
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Since disturbed blood vessel formation during wound healing has been reported in other animal models lacking collagen-binding integrins, we also analyzed CD31-positive cells in the 11–/– PDL but found no appreciable differences relative to the control mice (Fig. 7S and T). Staining for macrophages failed to reveal any substantial amounts of these cells in either the control or mutant PDL (data not shown). Due to the denaturing conditions in the decalcification protocols, we could not assay MMP activity by in situ methods.
PCR analysis indicated unchanged RNA levels of the PDL marker periostin and of the matrix metalloproteinase MMP2. Interestingly, an increase in the mRNA levels of MMP9 and a decrease in MMP14 mRNA levels were consistently observed in the integrin 11-deficient incisor PDL tissue (Fig. 7U).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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11 integrin chain is most closely related to the 10 integrin chain, and both subunits display restricted expression patterns, being confined embryonically to different interstitial connective tissues. Comparison with the expression patterns of other collagen-binding integrins suggests that the expression of 11 partially overlaps with 2 and is complementary to that of 10 in many locations (38).
Careful analyses of the phenotypes of mice deficient in individual collagen-binding integrins have revealed relatively mild phenotypes in unchallenged mutant mice (8, 11, 18, 25). The unchallenged 11-deficient mice described in this report have an incisor phenotype causing malnutrition and increased mortality.
The relatively mild phenotypes of the individual collagen receptor mutant mice suggest a fine-tuning role for these receptors in cell-collagen adhesive events under normal physiological conditions. Alternatively, these phenotypes may reflect overlapping roles for individual integrin receptors. Circumstantial evidence does indeed suggest that integrins have redundant functions in certain tissues (2, 20, 44). Future studies aimed at inactivating multiple collagen-binding integrins should resolve whether they have overlapping functions. The use of 1 and 2 integrin-deficient mice in disease models has revealed important functions for these integrins in angiogenesis (39), fibrosis (14), inflammation (15), and mast cell activation (16). Little is known about the behavior of mice that are deficient in 10 and 11 integrin chains in disease models.
11 mice fed on a regular laboratory chow are dwarfed, display increased mortality, and suffer from malnutrition. Despite the abundant expression of 11 in perichondrial cells, 11 expression did not correlate with any cartilage growth defects. Analysis of other tissues in which 11 is normally expressed, such as the cornea, intervertebral disc, intestine, and skin, did not reveal any obvious phenotypes in the corresponding unchallenged 11–/– tissues (data not shown). Instead, the malnutrition and smaller size observed in 11-deficient mice appear to correlate with the tooth phenotype. Upon supplementing the diet with soft food, the mice no longer suffered from malnutrition and had a normal life span (data not shown). Interestingly, the mutant mice fed on soft food were still smaller, indicating that growth defects must exist in addition to the tooth phenotype. The smaller size of the mice at the time of weaning also supports the notion of a tooth-independent growth defect. Unlike the situation observed recently in periostin-deficient mice (43), the major tooth phenotype in the 11–/– mice persisted during supplementation of the diet with soft food (data not shown).
The phenotype observed in the 11-deficent mice is highly selective for the incisors, which, unlike the molars, erupt continuously in rodents. Where 11 appears to be expressed in the follicle mesenchyme of both molars and incisors, in the adult dentition it is mainly expressed in the incisors, which most likely explains the restriction of the tooth phenotype to the incisors. Since molar eruption occurs in part by a different mechanism, it is unclear what role integrins and collagen receptors might play in it. Our analysis of the collagen-binding integrin expression patterns suggests that receptors other than collagen-binding integrins may mediate the attachment of molar PDL cells to the collagen matrix.
A number of morphogenic differences exist between molars and incisors, including the types of transcription factors expressed during embryonic development, the growth factors mediating paracrine cellular communication, and the types of MMPs needed at different stages in the tooth eruption process. Epidermal growth factor affects only incisor eruption, whereas CSF-1 preferentially affects the osteoclast-dependent stage of molar eruption (12). FGF-10 supports the stem cell compartment required for growth of the incisors, and incisors, but not molars, are missing in its absence (24). Finally, a lack of MT1-MMP (MMP14) has been reported to affect root development and eruption of molars (3, 7). The list of molecular mechanisms that differ between molars and incisors now also includes a matrix receptor, integrin 11ß1.
The PDL has been shown to play a central role during rodent incisor eruption, but the exact nature of this role has been controversial. According to one school, periodontal ligament fibroblasts migrate occlusally through the PDL space and create the tractional force that pulls the tooth towards the surface of the oral mucosa (6), while another school maintains that this eruptive force is provided by the hydrostatic tissue pressure within the vascular tissue of the periodontal ligament (9). Many existing mutations described for the tooth affect the neural crest-derived cells and disturb tooth morphogenesis as a result, but relatively few of these selectively affect tooth eruption.
Until now there has been a lack of good genetic models to study tooth eruption. We believe that the integrin 11-deficient mouse now offers one such model. In vitro analysis of 11–/– cells is a powerful tool for corroborating the in vivo 11–/– PDL phenotype. The lack of the 11 integrin chain in fibroblasts was seen to reduce cell attachment to collagen, reduce cell spreading on collagen I, and reduce cell-mediated collagen reorganization in a three-dimensional collagen I matrix in vitro. The major reduction in cell attachment and cell spreading on collagen I of 11–/– cells agrees well with the observations that 1–/– (18) or 2–/– (53) fibroblasts display only slightly reduced interactions with collagen I and shows that 11ß1 is the major receptor for collagen I on MEFs. It will be important to determine the relative importance of 11ß1 for cell adhesion to fibrillar collagens on other types of fibroblasts. We have previously demonstrated that inactivation of 11ß1 in MEFs expressing 1ß1, 2ß1, and 11ß1 increases cell migration on collagen I (38), presumably due to a lower strength of binding to collagen, offering less of a restraint on migration but also leading to less tractional force generation. In the absence of 11ß1, PDL cells will bind PDL collagen with lower affinity. We predict that this changed interaction with collagen fibrils disturbs the tractional forces being developed, in turn contributing to the disturbed tissue homeostasis.
The mutant incisor periodontal ligament lacking in 11 is characterized by a distinctly increased thickness, the presence of a wider acellular cementum, and an increased number of rests of Mallasez. The increased width of the cementum layer is most likely related to decreased eruption rate (4). Maintaining the correct width of the PDL is an essential function of the periodontal fibroblast, and this regulation is obviously lost in the absence of periodontal ligament fibroblast 11ß1. The present data thus identify 11ß1 in periodontal ligament fibroblasts as a central regulator of PDL width. As judged by sirius red staining, collagen accumulates in the mutant PDL. Integrins are known to regulate both collagen and MMP synthesis (40, 42), and we attempted to determine whether increased synthesis was the mechanism behind this collagen accumulation, but quantification by biochemical methods was not possible due to the small amount of tissue. The numbers of PDL fibroblasts that can be isolated from the mouse incisor PDL are too low to allow analysis of collagen in primary cells. Immortalization of this cell population from wt and ko animals could possibly allow such an analysis in the future. Quantitative PCR analysis of RNA from PDL tissue and MEFs cultured under different conditions failed to detect any changes in the RNA levels of collagen I and III (data not shown). However, increased collagen accumulation could be due to a number of mechanisms, including increased RNA stability and posttranslational mechanisms. In the case of the MMPs, we would predict that MMP activity is reduced in the knockout mouse. However, since the phenotype is manifested only postnatally, when ossification has occurred, sectioning of the PDL tissue involves a lengthy decalcification scheme, preventing reliable in situ zymography to detect changes in MMP activity. Interestingly, analysis of MMPs from MEFs cultured inside a collagen gel revealed reduced induction of MMP13 and MMP14 RNA in the 11–/– cells. Similar analysis of PDL tissue showed increased levels of MMP9 RNA and reduced levels of MMP14 RNA in the mutant PDL tissue. In summary, the exact mechanisms whereby 11 deficiency leads to a change in collagen turnover remain to be clarified, but they appear to involve changes in MMP levels, leading to altered matrix turnover in the absence of integrin 11.
In summary, our data support a role for 11ß1 in tooth eruption. We predict that 11ß1 is needed on the surface of incisor fibroblasts to generate the tractional force needed for tooth eruption. Consistent with this model, the reduced capacity of PDL fibroblasts to adhere to and remodel the collagen matrix in vitro fits well with the phenotype of reduced tooth eruption observed in vivo.
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ACKNOWLEDGMENTS |
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This project was supported by grants to D. Gullberg from Helse Vest, the Research Council of Norway (172330 V40), and the Swedish Research Council (VR NTK 2002-4309), Cartela AB (Lund, Sweden), and to Paola Zigrino (Deutsche Forschungsgemeinschaft through the SFB 589). Svetlana Popova is supported by a postdoc scholarship from the Research Council of Norway.
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FOOTNOTES |
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Present address: Department of Microbiology, University of Gothenburg, Gothenburg, Sweden.
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