Ascorbic acid induces collagenase-1 in human periodontal ligament cells but not in mc3t3-e1 osteoblast-like cells: potentia.

Ascorbic Acid Induces Collagenase-1 in Human Periodontal Ligament Cells but Not in
MC3T3-E1 Osteoblast-Like Cells: Potential Association Between Collagenase
Expression and Changes in Alkaline Phosphatase Phenotype
MOMOTOSHI SHIGA,1 YVONNE L KAPILA,2 QIN ZHANG,1 TAKAYUKI HAYAMI,1 and SUNILKAPILA1,2 1Department of Growth and Development, University of California San Francisco, San Francisco, California,USA.
2Department of Stomatology, University of California San Francisco, San Francisco, California, USA.
ABSTRACTAscorbic acid (AA) enhances osteoblastic differentiation by increasing collagen accumulation, which in turn,results in increased alkaline phosphatase (AP) expression in some osteogenic cells. However, in other cells,including human periodontal ligament (PDL) cells, additional osteoinductive agents are required for thisresponse. To understand the potential basis for the maintenance of the AP phenotype of PDL cells exposed toAA, we examined the modulation of the tissue-degrading matrix metalloproteinases (MMPs) and their inhibitorsby AA in short-term cell cultures. Early passage PDL cells in serum-free medium were exposed to AA for 5days. The samples were analyzed for MMPs and their inhibitors, the tissue inhibitors of metalloproteinases(TIMPs), AP, collagen I(1), and osteocalcin. We found that AA dose-dependently increased the expression ofcollagenase-1, and minimally TIMP-1, but not stromelysin-1 or TIMP-2. Additionally, AA caused substantialincreases in levels of type I collagen. AA was unable to increase AP activity or osteocalcin messenger RNA inPDL cells. However, the cells retained the ability to show a significantly greater AP expression in high- versuslow-density cultures, and increased osteocalcin as well as AP levels when cultured in the presence ofdexamethasone. Moreover, in cells exposed to dexamethasone, increases in AP and osteocalcin wereaccompanied by a repression of collagenase-1 expression. In contrast to PDL cells, AA did not inducecollagenase but produced a significant increase in AP expression in MC3T3-E1 cells. These findings provide thefirst evidence that AA, by modulating both collagen and collagenase-1 expression in PDL cells, most likelycontributes to a net matrix remodeling response in these cells. Furthermore, the relationship between changes incollagenase expression and alterations in AP activity in PDL and MC3T3-E1 cells suggests a potential role forcollagenase in modulating the AP phenotype of cells with osteoblastic potential. (J Bone Miner Res 2003;18:67-77) DISCUSSION ACKNOWLEDGMENTS REFERENCESKey words: human periodontal ligament cells; collagenase-1; stromelysin-1; tissue inhibitors ofmetalloproteinases; alkaline phosphatase INTRODUCTIONASCORBIC ACID (AA) induces the synthesis of type I collagen in various connective tissue cells, includingosteoblasts and chondrocytes. It enhances the accumulation of the extracellular matrices that in turn influence thedifferentiation of these cells. AA increases collagen matrix formation mainly by stimulating the prolinehydroxylation pool, increasing the secretion and processing of type I procollagen components, stabilizingmessenger RNA (mRNA), and slightly increasing procollagen synthesis and gene expression.((1-3)) In mostosteogenic cells, the accumulation of an appropriate collagenous extracellular matrix in response to AA is aprerequisite for the osteoblastic differentiation of these cells. Thus, addition of AA to cultured mouse calvaria-derived MC3T3-E1 cells((2,4-6)) and primary rat calvarial osteoblast-like cells((7)) produces a temporalsequence of gene expression, with initial deposition of type I collagen matrix followed by the induction of genesspecific to an osteoblastic phenotype, such as the bone/liver/kidney isozyme of alkaline phosphatase (AP) andosteocalcin.((2,4,8-10)) More evidence that the AA-mediated increase in collagen expression is required for thedifferentiation of MC3T3-E1 cells is provided by studies showing that inhibition of collagen synthesis blocks theinduction of AP and osteocalcin.((2,4)) In vivo studies showing defective bone regeneration in AA-deficient ratsprovide further evidence of a relationship between AA, collagen synthesis, and bone repair.((11)) The fact that a stable collagenous matrix is necessary for osteoblastic differentiation is further highlighted bystudies showing that the presence of exogenous bacterial collagenase((2)) or endogenous collagenase induced byinterleukin (IL)-1((12)) inhibits osteoblastic differentiation. These findings suggest that the increaseddegradation of the collagen matrix may affect the accumulation of the appropriate matrices, which in turn limitsthe ability of the compromised matrix to induce osteoblastic differentiation. Interstitial collagenase, one of the very few enzymes that degrades type I collagen, belongs to the matrix metalloproteinase (MMP) family ofenzymes. MMPs constitute the major proteolytic enzyme group degrading extracellular matrix components.
Currently this family of proteinases comprises more than 20 members, and is divided into five distinct groups:collagenases, stromelysins, gelatinases, membrane-type MMPs, and others.((13)) Although some variations existamong the individual members, the MMPs generally share several similar characteristics. Chief among these aretheir extracellular matrix substrate specificity, zinc-dependent activity, extracellular inhibition by tissueinhibitors of metalloproteinases (TIMPs), secretion as inactive proenzymes or zymogens, and sequencesimilarities.((14)) Although the ability of AA to induce osteoblastic differentiation in several osteogenic cells is wellestablished,((2,4,6,15)) some osteoprogenitor cells require the presence of other osteoinductive agents forincreased AP expression and mineralized nodule formation. Among the latter group of cells are humanperiodontal ligament (PDL) cells((16-18)) that in culture are a heterogeneous population composed offibroblastic and mineralized tissue-forming cells derived from fibrous and cellular connective tissues attachingteeth to bone.((19)) While a large percentage of PDL cells in mixed cell cultures are likely to be determinedfibroblasts,((20,21)) a substantial proportion of the cells show an osteogenic response to appropriatestimulation.((12,17,22-28)) The mediators of osteogenic responses in these cells have been well characterizedand include dexamethasone,((17,22,23)) dexamethasone and -glycerophosphate,((12,24,25)) -estradiol,((26))1,25 dihydroxyvitamin D3,((24,27)) and retinoic acid.((28)) These mediators enhance the expression of AP, typeI collagen, osteocalcin, osteopontin, and bone sialoprotein, and increase the formation of mineralized nodules inPDL cells retrieved from humans and rats.((12,16-18,22-28)) However, no study has demonstrated that AAalone enhances an osteoblastic phenotype in PDL cells. Thus, it seems that, unlike MC3T3-E1 cells that undergodifferentiation on exposure to AA alone, mixed PDL cell cultures require osteogenic stimuli in addition to AAfor induction of an osteoblastic phenotype.((12)) The reason for this difference in the responsiveness to AA ofcells with osteogenic potential remains unknown, but could potentially result from an increased degradation ofcollagen, particularly if AA itself causes a concomitant induction of collagenase in these cells. Thus, although itis presently accepted that AA produces a specific matrix anabolic response by inducing collagen expression, it isplausible that in PDL cells, this response is linked to a generalized increase in matrix remodeling activityresulting from AA's concomitant modulation of MMPs. AA's induction of collagenase in turn could limit itsability to enhance osteoblastic differentiation of PDL cellsa mechanism previously proposed to be responsiblefor inhibition of osteoblastic differentiation of these cells when exposed to appropriate exogenous agents in thepresence of IL-1.((12)) Our primary goal in these studies was to characterize the effects of AA on the expression of specific MMPsand TIMPs in PDL cells. We also evaluated whether the AP phenotype of PDL cells remains consistent in thepresence of AA, and if so, whether the cells retain the ability to show increased AP phenotype under appropriateculture conditions. To assess the potential relationship between collagenase expression and changes in APphenotype in PDL cells, we determined whether dexamethasone's modulation of an osteoblastic phenotype inthese cells is accompanied by downregulation of collagenase. We also studied AA's modulation of collagenaseand AP expression in the osteoblast-like MC3T3-E1 cells that are known to undergo osteoblastic differentiationin the presence of AA. Because these studies were limited to short-term cultures, when modulation of anosteoblastic phenotype manifests primarily as changes in expression of type I collagen and AP,((2)) our studiesfocused on determining changes in the levels of these early markers of osteoblastic differentiation. Our findingsshow for the first time that AA induces collagenase-1 expression in PDL cells but not in MC3T3-E1 cells, andthat the maintenance or decrease in collagenase expression is accompanied by increases in AP phenotype of PDLcells.
MATERIALS AND METHODS
PDL and MC3T3-E1 cell cultures
Human PDL cells, obtained from patients undergoing therapeutic third molar extractions or extraction of
premolars for orthodontic reasons, were retrieved as described previously.((29)) Briefly, extracted teeth were
washed twice with phosphate-buffered saline (PBS; 5 penicillin and streptomycin, and 1 fungizone). PDL tissue
attached to the mid-third of the root was removed with a surgical scalpel. The PDL tissue was minced and placed
in 35-mm tissue culture dishes. The explants were then covered with sterilized glass coverslips and kept in -
minimum essential medium (MEM) with 10% fetal bovine serum (FBS) at 37°C in 5% CO2 in humidified air
until cells grew out of the explants and reached confluency. Cells were then trypsinized and cells from passages
one to five were used in subsequent experiments.
In contrast to many previous studies, all our experiments were performed in serum-free conditions with a
previously defined supplement((29)) to eliminate the complex effects of serum on the responses of the cells. For
the first set of studies to determine the modulation of MMPs by AA, two different cell isolates from two subjects
were plated at 3.0 104 cells/cm2 in six-well plates in MEM with 10% FBS. After 24-48 h, the cells were washed
with PBS, and the medium was replaced with serum-free medium (MEM plus 0.2% lactalbumin hydrolysate
[LAH]). Cells were rinsed again after 6 h, and fresh serum-free medium, with or without 50 µg/ml AA, was
added. The medium was replaced with fresh medium without or with AA every 24 h for 5 days. A 5-day time
period was selected because previous studies((2)) show that AA causes initial deposition of type I collagen,which continues to increase for up to 3 days, followed by the induction of AP, which is first detected at aboutday 3 in osteogenic cells.((2)) After 5 days of culture, the cell-conditioned medium was collected and stored at -70°C until further analysis. Cells were washed in PBS, lysed in distilled water, scraped, and the lysate wasassayed for AP and total protein. For AA dose-response experiments, PDL cells were cultured at 3.0 104cells/cm2 as described above and exposed to 0, 5, 10, 25, 50, and 100 µg/ml of AA for 5 days. All otherprocedures were performed as described above. Three wells were used for each cell isolate, and subsequentassays were performed in triplicate.
For studies to determine changes in collagen I(1) and collagenase-1 mRNA expression, two PDL cell isolateswere cultured and exposed to AA for 5 days as described above. The cells were washed twice in cold Ca2+- andMg2+-free PBS, and total RNA was extracted.
To determine whether the PDL cells retain the ability to show an increased AP phenotype, we compared theAP expression in high-density versus low-density cultures; the former conditions having previously been shownto enhance the expression of osteoblastic markers.((30,31)) In these experiments, PDL cells from fourindependent cell isolates (including one from the previous experiment) from different subjects were seeded inMEM containing 10% FBS at an initial density of 6.0 104 cells/cm2 (high density) or at 1.5 104 cells/cm2 (lowdensity). After 24 h the cells were washed with PBS, and subsequent procedures were performed as describedabove. For all experiments, three wells were used for each cell isolate, and assays for total protein, MMPs, andAP were performed in triplicate.
For experiments testing the ability of dexamethasone to modulate markers of osteoblastic phenotype in PDLcells, the cells were plated at 3 104 cells/cm2 in medium containing 10% FBS. After 24 h the cells were washedand serum-free medium was added. Cells were rinsed again after 6 h, and fresh serum-free medium, without orwith 50 µg/ml AA, or with 50 µg/ml AA plus 10 nM of dexamethasone was added. The medium was replacedwith fresh medium without or with AA or AA and dexamethasone every 24 h for 5 days. Subsequent procedureswere as described above for the first experiment. Additionally, total RNA was extracted as described below forosteocalcin RT-PCR. For all experiments, three wells were used for one cell isolate, and assays for total protein,collagenase-1, and AP were performed in triplicate.
To determine that the PDL cells used in our experiments indeed have the ability to form mineralized nodules,long-term experiments were performed in the presence of serum-containing medium. Briefly, cells were platedin -MEM with 10% FBS alone or with 50 µg/ml of AA, or AA plus 10 nM dexamethasone or AA plusdexamethasone plus10 mM -glycerophosphate. The latter two mediators served as positive controls forosteoblastic differentiation and mineralized nodule formation as demonstrated previously.((12,16,25)) Themedium was changed every 3 days and the experiments terminated at day 27 of culture. Mineralized noduleswere visualized by von Kossa stain.((12)) The cells were fixed in 10% formaldehyde, washed, incubated in 5%silver nitrate for 5 minutes under sunlight, washed, and incubated for 2 minutes in 5% sodium thiosulfate. Themineralized nodules per well were counted and imaged.
Because the response of MC3T3-E1 cells to AA, including the induction of type I collagen, AP, and otherosteoblastic markers, has been well characterized,((2,4,15)) we used these cells as positive controls to show AA'sability to enhance an osteoblastic phenotype. MC3T3-E1 cells were plated at a density of 3.0 104 cells/cm2 inMEM containing 10% FBS for 2 days in the presence or absence of 50 µg/ml AA. The cells were washed twicewith PBS (Ca2-and Mg2+-free) and the medium changed to MEM supplemented with 0.1% bovine serumalbumin (BSA; Fisher Chemical, Fair Lawn, NJ, USA), with or without AA. Six hours later, the cells were againwashed twice with PBS (Ca2+- and Mg2+-free) and fresh MEM (0.1% BSA), with or without AA, was added.
The medium was replaced with fresh medium without or with AA every 24 h. BSA was used as a serum-freesupplement in these experiments instead of 0.2% LAH because MC3T3-E1 cells were not viable over extendedperiods of time in medium with LAH, and because BSA-containing medium is compatible with viability andnormal function of MC3T3-E1 cells. Because a different medium supplement was used with MC3T3-E1 cellsthan used in the previous experiments with PDL cells, we performed concurrent experiments with PDL cellsusing the 0.1% BSA media supplement. The experiments were terminated at day 5 of culture. The cell-conditioned medium was collected and stored at -70°C for future protein and proteinase assays. The cells werewashed three times with PBS, lysed in deionized water, and stored at -70°C for future assays for AP. Allexperiments were performed in triplicate wells and repeated three times.
Total protein
Total protein in cell lysates and cell-conditioned medium was assayed for standardization of assays using the
Bradford microassay (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions.
Substrate zymograms and reverse zymograms
Gelatin substrate zymography was used to detect and characterize proteinase activity in PDL and MC3T3-E1
cell-conditioned medium. The conditioned medium, standardized by total protein, was mixed with 4 sample
buffer and electrophoresed in 10% SDS-PAGE gels containing 2 mg/ml of gelatin as described previously.((32))
After, SDS was removed by washing the gels in 2.5% Triton X-100. The gels were placed in incubation buffer at
37°C for 24 h and stained with 0.5% Coomassie blue and destained until proteinase bands were clearly visible.
Proteinase bands were further characterized by incubating zymograms in incubation buffer containing 0.3 mM1,10-phenanthroline (Sigma, St. Louis, MO, USA), a zinc chelator, and metalloproteinase inhibitor.
Reverse zymograms were used to detect proteinase inhibitors in PDL cell-conditioned medium by using 18%SDS-PAGE as described above, except that after being washed with Triton X-100, the gels were incubated for15 minutes with 4-aminophenylmercuric acetate-activated rabbit skin-conditioned medium. The gels were thenincubated in incubation buffer for 18 h, stained, and destained as described above.
Images of the substrate zymograms and reverse zymograms were video-digitized by a CCD camera andanalyzed with image software (Image Version 1.42; National Institutes of Health, Bethesda, MD, USA). Westandardized the gel imaging by capturing the zymograms at the same focal length and exposure and quantifiedthe intensity and area of proteolytic and inhibitor activities by video densitometry.
Western blots
Western blots were used to identify collagenase-1, stromelysin-1, TIMP-1, and TIMP-2, and to validate the
findings of the substrate and reverse zymograms. After electrophoretic resolution of the conditioned medium
using 10-18% SDS-PAGE gels, the proteins were transferred to nitrocellulose membranes. Nonspecific binding
was blocked with 3% dry low-fat milk in tris-buffered saline (TBS) for 1 h, and membranes were washed twice
with TBS and incubated for 1 h either with rabbit anti-human collagenase-1 (MMP-1) antibody (Chemicon
International, Temecula, CA, USA), mouse anti-human stromelysin-1 (MMP-3) antibody (Oncogene Science,
Cambridge, MA, USA), or rabbit anti-human TIMP-1 or TIMP-2 antibodies (Triple Point Biologics, Forest
Grove, OR, USA). After further washes with TBS or TBS with 0.1% Tween, the membranes were incubated
with a 1:1000 dilution of peroxidase-conjugated goat anti-mouse (Jackson Immuno Research Laboratories, Inc.,
West Grove, PA, USA) or goat anti-rabbit (Pierce, Rockford, IL, USA) secondary antibody in TBS for 1 h. The
membranes were washed again and the bands were visualized by incubating the blot in 20 µg/ml of NBT/BCIP
solution (Bio-Rad).
AP activity assay
AP activity was assayed in the cell lysates by enzymatic conversion of p-nitrophenylphosphate substrate to p-
nitrophenol. The amount of p-nitrophenol produced was measured spectrophotometrically at a wavelength of
410 nm and quantified against a standard curve in nanomolar per microgram of protein per minute.
Determination of collagenase-1, type I collagen, and osteocalcin mRNA levels
Total RNA was extracted for Northern blots by lysing, scraping, and homogenizing cells in 1 ml of lysis buffer
(Ultraspec RNA Isolation System; BIOTECX, Houston, TX, USA). The RNA was precipitated and extracted by
chloroform and isopropanol using standard procedures.((33)) The yield and purity of RNA was determined by
ultraviolet (UV) spectroscopy.
For collagenase-1 and type I collagen Northern blots, the RNA was standardized by total RNA concentrations
and loaded on a 1% agarose-formaldehyde gel. After electrophoresis, the RNA was transferred to nylon
membranes through passive transfer and crosslinked by ultraviolet transillumination. Plasmids pUC and SP64
containing complementary DNA (cDNA) inserts for human collagen I(1)((34)) or collagenase-1((35)) were
amplified in E. coli, the DNA was isolated, and the cDNA insert was retrieved by restriction digests and
separation on 1% low-melting agarose gel.((33)) This cDNA template was used to synthesize a
[32P]deoxycytidine triphosphate-labeled probe using a random priming kit (Amersham, Arlington Heights, IL,
USA) according to the manufacturer's instructions. The probe was purified and hybridized to the nylon
membrane for 1 h at 65°C. After washes with 2 SSC buffer containing 0.1% SDS, the signal was detected by
autoradiography on Kodak X-OMAT film (Eastman Kodak Co., Rochester, NY, USA). The message was
quantitated by video-densitometry using glyceraldehyde-3-phosphate-dehydrogenase (G3PDH) as an internal
standard to normalize the densitometric data for total RNA loaded. The fold-induction of collagenase-1 and
collagen I(1) mRNA by AA relative to baseline levels was determined.
For osteocalcin reverse transcriptase-polymerase chain reaction (RT-PCR), 0.1 µg total RNA was used in the
initial reaction mix. The human osteocalcin primer sequences used were as described previously,((36)) yielding a
297-bp product. Reverse transcription and amplification reactions with MuLV reverse transcriptase and
AmpliTaq DNA polymerase, respectively, were performed according to the manufacturer's instructions
(GeneAmp, RNA PCR kit; Applied Biosystems, Foster City, CA, USA). All amplifications were performed for
40 cycles with the annealing temperature set at 55°C. For each primer pair, parallel reactions with water to
replace template were run to control for template contamination.
Statistical analysis
The effects of ascorbic acid and plating density on the expression of AP, collagenase-1, and mineralized nodule
formation was determined by a two-way analysis of variance (ANOVA), and the intergroup differences were
determined by Scheffé multiple comparisons test with the level of significance set at p < 0.05. The statistical
significance of the fold-induction for collagenase-1, stromelysin-1, and TIMPs mediated by AA was analyzed by
paired t-test.
RESULTSAA dose-dependently and specifically induces collagenase-1 but not stromelysin-1 and minimally modulatesTIMPs in PDL cells Using two cell isolates from two subjects, we first determined by substrate zymography, Western blot, andNorthern blots whether AA mediated any changes in the expression of collagenase. PDL cells in the absence orpresence of AA expressed three gelatin-degrading activities at 72, 53/58, and 43/48 kDa (Fig. 1A). All theseproteinases were inhibited by 1,10-phenanthroline (Fig. 1A, lane 5), a metalloproteinase inhibitor, and thereforeare likely to be the MMPs 72-kDa gelatinase (MMP-2), and inactive (proMMP-1) and active collagenase (MMP-1), respectively. The 53/58-kDa gelatinolytic proteinase was identified as procollagenase-1 by Western blot (Fig.
1B). The 43/48-kDa proteinase, which is likely active collagenase, was observed in the gelatin zymogram but notin the Western blot because of the relatively greater sensitivity of the former assay. AA produced substantialincreases in levels of procollagenase and active collagenase in both cell isolates. The increases in expression ofcollagenase-1 protein were paralleled by changes in mRNA levels for this enzyme (Figs. 1C and 1D). AA'smodulation of collagenase expression was further determined with dose-response experiments. Cells exposed toincreasing concentrations of AA demonstrated a dose-dependent increase in expression of gelatin-degradingproteinase at 53/58 kDa (procollagenase) that was paralleled by increases in its activated 43/48-kDa enzyme(collagenase; Fig. 1E).
Because the expression of stromelysin is often coordinately regulated with that of collagenase,((37)) we alsodetermined whether AA induced stromelysin-1 in parallel with collagenase-1. Western blots showed that incontrast to AA's upregulation of collagenase-1, it produced minimal changes in the expression of stromelysin-1(data not shown). These findings were further confirmed by dose-response experiments that demonstratedminimal changes in expression of stromelysin-1 with increasing concentrations of AA (data not shown). Thesefindings suggest that in PDL cells, AA's regulation of collagenase-1 is uncoupled from that of stromelysin-1.
To determine whether the induction of collagenase-1 is accompanied by changes in TIMPs, which couldpotentially negate any increase in matrix-degradative activity, we evaluated the effects of AA on expression ofTIMPs. PDL cells produced basal levels of two proteinase inhibitors of approximately 30 and 20 kDa (Fig. 2A,lanes 1 and 3), corresponding to TIMP-1 and 2, respectively. Exposure of the cells to AA produced a slightincrease in expression of the 30-kDa inhibitor, whereas the levels of the 20-kDa inhibitor decreased slightly (Fig.
2A, lanes 2 and 4). Western blots identified these proteinase inhibitors as TIMP-1 (Fig. 2B) and TIMP-2,respectively (Fig. 2C), and also confirmed the slight modulation of these inhibitors by AA. Using a biggersample size of four cell isolates from four subjects cultured at low density, we found the AA-mediated changesin TIMP-1 and TIMP-2 expression were not statistically significant (p > 0.05; Fig. 2D).
AA increases type I collagen mRNA but not AP expression in PDL cells
We next determined whether AA has the ability to modulate the expression of type I collagen and AP in PDL
cells. Northern blot analysis revealed that AA produced a 3- and 7.5-fold increase in type I(1) collagen mRNA in
two cell isolates (Figs. 3A and 3B). In contrast, neither of the two isolates showed any increases in AP
expression when exposed to AA (Fig. 3C). These findings suggest that AA's induction of type I collagen is not
related to its modulation of AP in short-term PDL cell cultures.
PDL cells retain the ability to show increased AP expression and mineralized nodule formation underappropriate conditions To determine whether the PDL cells do indeed retain the ability to show an increased AP phenotype underappropriate conditions, we next assessed the AP levels in these cells when grown in low-density and high-density cultures; the latter conditions were previously shown to mediate osteoblastic differentiation.((30,31)) Wealso evaluated whether the manipulation of AP status impacted on modulation of collagenase-1 expression byAA.
These experiments were performed on one cell isolate used in the previous experiments and three additionalcell isolates from four subjects. The four cell isolates demonstrated substantial heterogeneity in their basal levelsof AP (data not shown), as reported previously.((22,38)) All four cell isolates demonstrated significantly greater(p < 0.05) AP expression when plated at high versus low density (Fig. 4A). However, as observed in ourprevious experiments, AP expression remained relatively unchanged on exposure of both low- and high-densitycell cultures to AA. On the average, high-density cultures had approximately 1.5-fold greater levels of AP thanlow-density cultures both in the absence or presence of AA. These findings reflect significant effects of platingdensity (p = 0.006), but not of AA (p = 0.8), on fold-increase in AP expression in PDL cells.
Irrespective of the plating density and AP phenotype, exposure of all four cell isolates to AA produced asignificant (ANOVA p < 0.001) 2- and 3-fold increase in collagenase-1 in low- and high-density cultures,respectively (Figs. 4B and 4C). The lack of modulation of stromelysin expression by AA in both low-(p = 0.19)and high-density (p = 0.48) cultures (data not shown) confirmed our previous observations. Similarly, TIMP-1 was also not significantly modulated by AA in either low-density (p = 0.12) or high-density (p = 0.70) cultures(data not shown).
To determine that the PDL cells used in the short-term serum-free experiments have the ability to generatemineralized nodules, we performed long-term experiments in serum-containing medium. We found that cellscultured under control conditions or in the presence of AA alone showed minimal numbers and very smallmineralized nodules (Figs. 5A, 5B, and 5E). Addition of dexamethasone and AA caused a statisticallyinsignificant increase (Scheffé p > 0.05) in mineralized nodules (Figs. 5C and 5E) relative to control and AA-treated cells. Mineralized nodule formation was significantly enhanced (ANOVA p < 0.0001) in cells incubatedin the presence of AA, dexamethasone, and -glycerophosphate relative to control (Scheffé p < 0.0003), AA-treated (p < 0.0004), and AA plus dexamethasone-treated (p < 0.01) cells (Figs. 5D and 5E).
Dexamethasone's repression of collagenase-1 is accompanied by an increase in AP and osteocalcin expression inPDL cells Further evidence that PDL cells have the ability for increased expression of osteoblastic markers withappropriate stimulation was provided by studies in which the cells were cultured in the presence ofdexamethasone and AA, which have previously been shown to enhance the osteogenic responses in thesecells.((12,24,25)) These experiments demonstrated that cells exposed to dexamethasone and AA hadsignificantly increased AP activity (p < 0.05) and substantially greater osteocalcin mRNA expression thencontrol cells or those cultured in the presence of AA alone (Figs. 6A and 6B, respectively). We also determinedwhether the induction of AP activity and osteocalcin mRNA expression by dexamethasone was accompanied bychanges in collagenase-1 expression in PDL cells. As observed previously, AA increased the expression ofcollagenase-1 relative to control baseline levels (Fig. 6C, lanes 1 and 2). However, the expression ofcollagenase-1 was completely repressed in cells exposed to dexamethasone and AA. These findings show thatdexamethasone's inhibition of constitutively expressed and AA-induced collagenase-1 is accompanied by aninduction of AP and osteocalcin in PDL cells.
MC3T3-E1 cells in short-term cultures show no change in collagenase levels but increased AP expression onexposure to AA Finally, we sought further evidence of a potential link between the induction of collagenase by AA and themodulation of the AP phenotype, by evaluating whether MC3T3-E1 cells which undergo osteoblasticdifferentiation in the presence of AA alone, show any changes in collagenase expression on exposure to AA. Incontrast to our findings with PDL cells, AA did not cause any modulation of the constitutively expressed 53/58-kDa gelatinolytic proteinase (procollagenase) in short-term MC3T3-E1 cell cultures (Fig. 7A). Additionally, asopposed to the lack of an AP response to AA in PDL cells, MC3T3-E1 cells showed significant (p < 0.001)increases in AP expression on exposure to AA (Fig. 7B). Although it is helpful to compare changes in APexpression between PDL cells and MC3T3-E1 cells, it should be noted that MC3T3-E1 cells have substantiallyhigher basal levels of AP expression than PDL cells. Therefore, PDL cells should not be equated directly withclonal osteoblastic cells such as the MC3T3-E1 cells.
DISCUSSIONOur studies show that besides having the ability to increase the expression of type I collagen, AA has acollagenase-1-inductive effect in PDL cells. Among the MMP family, and that of its inhibitors, the TIMPs, thisinduction was specific for collagenase-1, but was not seen for stromelysin-1, 72-kDa gelatinase, or TIMP-2. AAcaused a small but statistically insignificant increase in TIMP-1. Our studies also show that although PDL cellsretain their ability for increased AP expression under high-density culture conditions, and in the presence ofdexamethasone, AA alone did not increase the expression of AP in these cells. We also noted thatdexamethasone's induction of AP and osteocalcin was accompanied by a repression in collagenase-1 expressionin PDL cells. In contrast to PDL cells, AA did not induce collagenase expression in short-term MC3T3-E1 cellcultures, but was capable of increasing their AP phenotype.
While there is some discussion on the predominant phenotype and origin of various cell types in theperiodontal ligament,((20,21)) early passage PDL cell populations as used in our studies usually contain cellsthat show an osteogenic response to appropriate stimulation.((17,22,25,28)) The percentage of cell cultures thatshow some sort of osteoblastic response range from 40% of cloned PDL cells((22)) to 100% of mixed PDL cellpopulations.((17,25,28)) As with osteoblastic cells, mixed PDL cell populations respond todexamethasone,((12,17,22-25)) -estradiol,((26)) and 1,25 dihydroxyvitamin D3((24,27)) by the induction of AP,type I collagen, osteocalcin, osteopontin, and bone sialoprotein, and formation of mineralized nodules.
Additionally, human PDL cells exposed to dexamethasone show a dose-dependent cAMP response to PTH,indicating the presence of osteoblast-like PDL cell populations in vitro.((39)) Based on these studies and ourfindings, it is very likely that the responses to dexamethasone and differences in AP phenotype between high- and low-density cultures of PDL cells used in our studies indicate the presence of cells with osteoblasticcharacteristics within our cultures.
Several studies in which collagen synthesis is perturbed((4,8)) or its degradation enhanced((2,12)) indicatethat, in cells with osteogenic potential, the deposition of type I collagen is a prerequisite for increased APexpression, followed by induction of osteocalcin((4,8)) and subsequent mineralized nodule formation in long-term cultures.((8,12)) In contrast to these previous long-term studies, the limitation on cell viability imposed byserum-free culture conditions used in our investigation necessitates short-term studies such that subsequentmarkers of osteoblastic differentiation including mineralized nodule formation cannot be readily monitored.
Because in our short-term studies the PDL cells may not be receptive to the downstream effects of AA, thefindings may not necessarily mimic in vivo responses or those observed in long-term in vitro studies. However,the findings of studies using serum-free medium enables one to obtain insights into likely mechanisms of cellularresponses to specific mediators in the absence of the numerous agents present in serum. Nevertheless, ourcurrent findings and those of others((25,26,40)) on long-term serum-containing cultures of human PDL cellshave demonstrated that these cells have the ability to form mineralized nodules in the presence ofdexamethasone and -glycerophosphate. As such, our study suggests a potential relationship between collagenaseexpression and changes in AP and osteocalcin expression that may be difficult to discern in the presence ofserum.
Evidence that a stable collagenous matrix is important for the progression of osteoblastic differentiation,particularly during the preosteoblastic phase of development, is provided by studies showing that inhibition ofcollagen expression((4,8)) or its increased degradation((2,12)) leads to a decrease or delay in the expression ofmarkers of osteoblastic differentiation. Thus, when MC3T3-E1 cells are exposed to AA, they show increasedexpression of AP that is partially inhibited when the cells are grown in the presence of bacterial collagenase.((2))A further indication that collagen remodeling affects the differentiation of MC3T3-E1 cells is provided bystudies((6)) in which cells stably transfected with a plasmid expressing high-turnover type I collagen chainsproduced abnormal collagen fibrils, resulting in a delayed and attenuated increase in AP. Finally, when PDLcells are cultured in the presence of medium containing dexamethasone and AA, which supports osteoblasticdifferentiation, and exposed to IL-1, they undergo a significant increase in collagenase mRNA expression that isassociated with an inability to form mineralized nodules.((12)) Although the cells exposed to IL-1 do not formmineralized nodules, they retain the ability to synthesize collagen, suggesting that IL-1 inhibits mineralizednodule formation through increased collagen-degradative activity. These studies, together with our findings,provide evidence on the important role of collagenase in the regulation of osteoblastic differentiation. However,our findings are unique in showing that AA, which is traditionally thought to induce collagen leading toosteoblastic differentiation, may not produce this response in PDL cells because it concurrently increases theexpression of collagenase. These findings suggest that, despite AA's induction of collagen in PDL cells, itsconcomitant increase in collagenase expression may lead to enhanced collagen turnover that in turn counteractsAA's expected stimulation of osteoblastic differentiation of these cells.
Because the PDL is comprised of a heterogenous pool of cells with differing physiologic functions, it is likelythat the various cellular phenotypes show varied responses to AA. Thus, for example, as observed withestablished osteoblastic cells,((2,4,8-10)) bone-forming PDL cells lining the lamina dura may undergo furtherdifferentiation in the presence of AA. In contrast, specific pools of PDL cells that maintain the fibrouscomposition of the PDL may respond to AA by increasing both collagen and collagenase-1 expression, therebymaintaining a high state of matrix turnover necessary for an actively remodeling tissue like the PDL. AA'sincreased turnover of collagen may also limit the signals mediated by the matrix that are necessary to increasecellular AP and subsequent osteoblastic differentiation of this pool of PDL cells, further aiding in themaintenance of fibrous tissues and preventing ankylosis. This postulate on the role of AA in the matrixremodeling processes in vivo remains to be tested.
In contrast to MC3T3-E1 cells, which undergo osteoblastic differentiation when exposed to AAalone,((2,4,6,15)) human PDL cells require dexamethasone for the upregulation of AP expression as shown inour and previous studies.((12,16-18)) AA alone is not sufficient for modulating the AP phenotype of PDL cells,but dexamethasone when used on its own stimulates AP expression in these cells.((17)) Although themechanisms by which dexamethasone increases AP expression in PDL cells remain poorly understood, it isplausible that it modulates osteoblastic differentiation by downregulating collagenase expression in PDL cells,thereby counteracting the induction of collagenase by AA. Dexamethasone's repression of both constitutivelyexpressed collagenase or collagenase induced by interleukins or other stimulatory agents in fibroblastic cells iswell documented.((41-43)) Our studies show that dexamethasone has the ability to repress both constitutive andAA-induced collagenase-1 expression in PDL cells, and this is accompanied by an increase in AP phenotype andosteocalcin mRNA levels. Because the net accretion of collagen is dependent on its synthesis and degradation, itis plausible that the AA-enhanced secretion of collagen and the dexamethasone-mediated decrease in collagenaseexpression together potentiate the accumulation of an appropriate collagenous matrix by PDL cells, therebyproviding an environment conducive to their differentiation.
Because the mean concentration of AA in gingival crevicular fluid from healthy volunteers is 207 ± 81.8 µMor 36.46 ± 14.40 µg/ml,((44)) our findings on the response of PDL cells to AA at concentrations of 50 µg/mlmay have some physiological relevance. Based on our findings on the effects of AA in the induction of collagenase, it is plausible that AA at physiologic concentrations may participate in the normal turnover of thePDL or could possibly prevent osteoblastic differentiation of PDL cells by enhancing collagen degradationthrough induction of collagenase. Further in vivo studies are recommended to determine the contribution of AAin oral fluids including gingival crevicular fluid to the physiology of the healthy PDL.
ACKNOWLEDGMENTSThis study was funded in part by the American Association of Orthodontists Foundation to SK. We thank MsEvangeline Leash for editorial assistance.
The authors have no conflict of interest.
REFERENCES: 1-44Address reprint requests to:Sunil Kapila, DDS, MS, PhDDepartment of Growth and DevelopmentUniversity of California San Francisco521 Parnassus AvenueSan Francisco, CA 94143-0640, USA Received in original form April 15, 2001; in revised form July 1, 2002; accepted July 15, 2002.
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