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. 2011 Apr;90(4):489–494. doi: 10.1177/0022034510390043

Fluoride Does Not Inhibit Enamel Protease Activity

CE Tye 1, JV Antone 1, JD Bartlett 1,*
PMCID: PMC3077535  NIHMSID: NIHMS258202  PMID: 21118795

Abstract

Fluorosed enamel can be porous, mottled, discolored, hypomineralized, and protein-rich if the enamel matrix is not completely removed. Proteolytic processing by matrix metalloproteinase-20 (MMP20) and kallikrein-4 (KLK4) is critical for enamel formation, and homozygous mutation of either protease results in hypomineralized, protein-rich enamel. Herein, we demonstrate that the lysosomal proteinase cathepsin K is expressed in the enamel organ in a developmentally defined manner that suggests a role for cathepsin K in degrading re-absorbed enamel matrix proteins. We therefore asked if fluoride directly inhibits the activity of MMP20, KLK4, dipeptidyl peptidase I (DPPI) (an in vitro activator of KLK4), or cathepsin K. Enzyme kinetics were studied with quenched fluorescent peptides with purified enzyme in the presence of 0–10 mM NaF, and data were fit to Michaelis-Menten curves. Increasing concentrations of known inhibitors showed decreases in enzyme activity. However, concentrations of up to 10 mM NaF had no effect on KLK4, MMP20, DPPI, or cathepsin K activity. Our results show that fluoride does not directly inhibit enamel proteolytic activity.

Keywords: enamel, fluoride, proteases/proteinases

Introduction

Concentrations of 1.6–1.8 ppm fluoride in drinking water are the threshold for fluorosis risk (Whitford, 1996). Enamel fluorosis is characterized by mottled, discolored, porous enamel that contains increased protein content and decreased overall mineral content if the enamel matrix is not removed completely (Wright et al., 1996; Robinson et al., 2004). High fluoride levels in drinking water are endemic throughout the world, and these areas in India were recently mapped (Viswanathan et al., 2009).

Dental enamel is formed in stages. During the secretory stage, the ameloblast cells secrete proteins that form the enamel matrix, and they also secrete matrix metalloproteinase-20 (MMP20), which cleaves this matrix. The ameloblasts then go through a short transition stage and on to the maturation stage of development, where they secrete the serine protease kallikrein-4 (KLK4) (Hu et al., 2000, 2002). KLK4 further degrades the enamel proteins, which are ultimately removed, resulting in virtually protein-free mature enamel. Mature enamel is composed of about 97% mineral and 3% organic content (Robinson et al., 1988). Proteolytic processing of the enamel matrix by both MMP20 and KLK4 is critical for proper enamel formation, and homozygous mutation of either protease causes amelogenesis imperfecta, characterized by enamel that is hypomineralized and protein-rich (Hart et al., 2004; Kim et al., 2005; Ozdemir et al., 2005; Wright et al., 2006; Papagerakis et al., 2008; Lee et al., 2010).

Cathepsin K is a cysteine protease that is well-characterized for its expression in osteoclasts and role in bone resorption. Expression of cathepsin K is not restricted to osteoclasts. High expression is also seen in the ovary, small intestine, and colon and at lower levels in the heart, skeletal muscle, placenta, lung, prostate, testes, spleen, thymus, kidney, pancreas, and liver (Bromme and Okamoto, 1995). The expression of cathepsin K in developing teeth has not been well-documented, but we recently identified cathepsin K in the maturation-stage enamel organ during a lysosomal protease screen (Tye et al., 2009a). Here we demonstrate that cathepsin K expression increases during the maturation stage of enamel development, which is consistent with its playing a role in the degradation of enamel proteins within the lysosome.

Why the enamel matrix is not properly removed from fluorosed enamel is poorly understood. Several hypotheses exist, including reduced secretion of degradative enzymes, reduced protein absorption, and delayed and/or reduced matrix degradation (Bronckers et al., 2009). Several studies have characterized protease activity in fluorosed enamel, but with ambiguous results (DenBesten and Heffernan, 1989; Gerlach et al., 2000; Zhang et al., 2006, 2007). Most studies have focused on the secretory-stage enzyme MMP20. However, epidemiological studies have shown that the most critical time for susceptibility to fluorosis in permanent human maxillary incisors is between 15 and 30 mos of age (Evans and Darvell, 1995). This corresponds to the early maturation stage. Fluoride exposure prior to this time presents less risk of fluorosis than continued exposure for up to 36 mos beyond this critical time (Evans and Stamm, 1991). Thus, the maturation stage of enamel development is the stage most susceptible to fluoride exposure. So, we focused on the maturation stage for our proteinase kinetic activity assays.

Herein we performed a kinetic analysis of fluoride’s effect on enamel protease activity. The goal was to determine if fluoride exposure directly affects enzyme activity. We have examined the effect of fluoride on KLK4, two potential activators of KLK4, MMP20 and dipeptidyl peptidase I (DPPI), and on the lysosomal protease cathepsin K.

Materials & Methods

Six-month-old pig mandibles were purchased from a local slaughterhouse, and we followed approved animal use protocols.

KLK4 Activity Assay

We activated recombinant human KLK4 (rhKLK4; R&D Systems, Minneapolis, MN, USA) [EC 3.4.21] by incubating 0.2 µg/µL of inactive pro-KLK4 with 0.002 µg/µL thermolysin (Sigma, St. Louis, MO, USA) in 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5, at 37°C for 2 hrs, following the manufacturer’s protocol. Activation was stopped by the addition of 1,10 phenanthroline (10 mM; Sigma). To determine the effect of fluoride on rhKLK4 activity, we incubated 50 ng of activated KLK4 with 1–100 µM Boc-Val-Pro-Arg-AMC (R&D Systems) in 200 µL of 50 mM Tris, pH 9.0, with 0–10 mM NaF. We measured hydrolysis of the substrate by monitoring fluorescence every 1 min for 45 min with 370 nm excitation and 460 nm emission at room temperature. As a reference standard, 28.5–7125 nM of 7-amino-4-methylcoumarin (AMC; EMD Chemicals, Gibbstown, NJ, USA) was assayed. 4- (2-Aminoethyl)benzenesulfonylfluoride (AEBSF; 0.2–3 mM; EMD) was run separately as a positive control inhibitor.

MMP20 Activity Assay

To determine if fluoride inhibits MMP20 [EC 3.4.24.] activity, we incubated 10 ng recombinant human MMP20 catalytic domain (rhMMP20; Enzo Life Sciences, Plymouth Meeting, PA, USA) with increasing concentrations of Mca-Pro-Leu-Gly-Leu-Dap (Dnp)-Ala-Arg-NH2 (0.3125–3.125 nM) in 200 µL of 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.05% Brij35, 5% DMSO, pH 7.4, with 0–10 mM NaF in a 96-well plate. We measured hydrolysis of the substrate by monitoring fluorescence every 4 min for 1 hr at 37°C with 320 nm excitation and 405 nm emission. Mca-PL-OH (2.25–225 nM; EMD) was run as a reference compound, and GM6001 (1–5 pM; Biomol, Enzo Life Sciences) was included in each run as a positive control inhibitor.

DPPI Activity Assay

We determined the effect of fluoride on DPPI [EC 3.4.14.1] activity by measuring the rate of increase in fluorescence produced by enzymatic hydrolysis of H-Gly-Arg-AMC (Bachem, Torrance, CA, USA). The 200-µL reaction mixture contained 10 ng of recombinant active mouse DPPI (rmDPPI; R&D Systems), 0.625–125 nM of H-Gly-Arg-AMC, and 0–10 mM NaF in 50 mM MES, 50 mM NaCl, 5 mM DTT, pH 5.5. We measured substrate cleavage by monitoring fluorescence every 1 min for 30 min at 30°C with excitation and emission wavelengths of 370 and 460 nm, respectively. As a reference standard, 28.5–2850 nM of AMC was included with each measurement, and E64 (15–50 nM; Enzo Life Sciences) was run as a positive control inhibitor.

Cathepsin K Activity Assay

Recombinant human procathepsin K (pro-rhCatK; EMD Chemicals) was activated by incubation in 100 mM sodium acetate, 10 mM DTT, 5 mM EDTA, pH 3.9, at room temperature for 40 min. Methyl-methanthiosulfonate was added to a final concentration of 1 mM to reduce autoproteolysis, and aliquots were stored at −80°C. To determine if fluoride had any effect on cathepsin K (EC 3.4.22.38) activity, we incubated 10 ng of activated rhCatK with 0.3125–12.5 nM of Z-Leu-Arg-AMC (Enzo Life Sciences) in 200 µL of 50 mM 2-(N-morpholino)ethanesulfonic acid, 5 mM DTT, 2.5 mM EDTA, pH 5.5, with 0–10 mM NaF in a 96-well plate. We measured substrate cleavage by monitoring fluorescence every 1 min for 60 min at 30°C with 370 nm excitation and 460 nm emission. As a reference standard, 2.25–112.5 nM of AMC was included with each measurement, and E64 (0.025–0.1 nM) was run as a positive control inhibitor.

Northern Blot

Approximately 30 µg of total RNA extracted from 6-month-old porcine pulp and enamel organs was characterized by Northern blot analysis as described previously (Caron et al., 1998). The pulp and enamel organ were at the secretory (third molar), early maturation (second molar), and maturation (third premolar) stages of development (Robinson et al., 1987). A 384-bp cathepsin K cDNA generated by RT-PCR (5′ primer, 5′-GGAGGGCCAACTCAAG AAGAAA-3′; 3′ primer, 5′-GTGGTTGAGATTATCGCTATT-3′) was radiolabeled for use as a probe. The same filter was Stripped and re-probed for β-actin.

Data Analysis

For accurate velocity readings, only the linear initial reactions were used. At each substrate concentration, we used the amount of substrate cleaved over the set time period to calculate the initial velocity. All activity assays were performed on duplicate samples. We fit these data to the Michaelis-Menten equation by linear regression analysis with the Prism 5 software suite (GraphPad Software, La Jolla, CA, USA) to determine Vmax.

Results

KLK4 Kinetics

KlK4 activity was assessed by incubation of activated recombinant human KLK4 (rhKLK4) with a quenched fluorescent peptide substrate in the presence of increasing concentrations of NaF (Fig. 1A) or with the irreversible serine proteinase inhibitor AEBSF (Fig. 1B). Dose-dependent inhibition occurred with AEBSF, but not with NaF. To determine if the rate of hydrolysis was inhibited, we generated substrate-velocity plots with increasing amounts of substrate. Incubation of rhKLK4 with increasing concentrations of AEBSF resulted in decreased Vmax, and Michaelis-Menten plots demonstrated AEBSF to be a non-competitive inhibitor (Fig. 1D). Michaelis-Menten plots with increasing concentrations of NaF were overlapping and did not demonstrate inhibition of rhKLK4 (Fig. 1C).

Figure 1.

Figure 1.

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Effect of fluoride on rhKLK4 activity. The substrate Boc-Val-Pro-Arg-AMC was incubated in assay buffer at a final concentration of 50 µM with 50 ng of rhKLK4 and increasing concentrations of NaF (A) or AEBSF (B). NaF concentrations were 0 (□), 1 µM (○), 10 µM (Δ), 100 µM (◊), 1 mM (X), and 10 mM (*). AEBSF concentrations were 0 (□), 0.2 mM (○), 0.5 mM (Δ), 1 mM (◊), 2 mM (X), and 3 mM (*). We calculated reaction rates by increasing the substrate concentration from 1 to 100 µM. We used data from the first 10 min to calculate V (nM per min). Michaelis-Menten plots of 50 ng of rhKLK4 incubated with increasing concentrations of NaF at 10 min (C) and the irreversible serine protease inhibitor AEBSF (D) were generated. Values represent the mean ± standard deviation, with 2 replicates per concentration.

MMP20 Kinetics

To determine the effect of fluoride on MMP20 activity, we monitored the rate of hydrolysis of a quenched fluorescent peptide. rhMMP20 incubated with increasing concentrations of the inhibitor GM6001 exhibited the expected dose-dependent inhibition (Fig. 2B), whereas incubation of rhMMP20 with increasing concentrations of sodium fluoride (Fig. 2A) did not result in a decrease in substrate cleavage. Incubation of rhMMP20 with increasing concentrations of GM6001 resulted in decreased Vmax, and Michaelis-Menten plots demonstrated GM6001 to be a non-competitive inhibitor (Fig. 2D), whereas NaF did not inhibit rhMMP20 (Fig. 2C).

Figure 2.

Figure 2.

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Effect of fluoride on rhMMP20 activity. The substrate Mca-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2 was incubated in assay buffer at a final concentration of 2.5 nM with 10 ng of rhMMP20 and increasing concentrations of NaF (A) or GM6001 (B). NaF concentrations were 0 (□), 1 µM (○), 10 µM (Δ), 100 µM (◊), 1 mM (X), and 10 mM (*). GM6001 concentrations were 0 (□), 1 pM (○), 2.5 pM (Δ), and 5 pM (◊). We calculated reaction rates by increasing the substrate concentration from 0.3125 to 4.375 nM. We used data from the entire 60 min to calculate V (nM per min). Michaelis-Menten plots of 10 ng of rhMMP20 incubated with increasing concentrations of NaF (C) and with increasing concentrations of the irreversible metalloproteinase inhibitor GM6001 (D) were generated. Six separate experiments were combined, and values represent the mean ± standard deviation.

DPPI Kinetics

DPPI is slowly inactivated by E64 (Fig. 3B), which is a non-competitive, irreversible inhibitor of cysteine proteases (Barrett et al., 1982). DPPI was very active against the quenched peptide, and hydrolysis was not linear over the 30-minute time scale; therefore, the rate of reaction was calculated for the first 6 min. Increasing concentrations of E64 resulted in a decreased Vmax (Fig. 3D). Sodium fluoride, however, had no effect on rmDPPI activity (Figs. 3A, 3C).

Figure 3.

Figure 3.

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Effect of fluoride on rmDPPI activity. The substrate H-Gly-Arg-AMC was incubated in assay buffer at a final concentration of 6.25 nM with 10 ng of rmDPPI and increasing concentrations of NaF (A) or the irreversible inhibitor E64 (B). We calculated reaction rates by increasing the substrate concentration from 0.625 to 125 nM. We used data from the first 6 min to calculate V (nM per min). Michaelis-Menten plots of 10 ng of rmDPPI incubated with increasing concentrations of NaF (C) and E64 (D) were generated. NaF concentrations were 0 (□), 1 µM (○), 10 µM (Δ), 100 µM (◊), 1 mM (X), and 10 mM (*). E64 concentrations were 0 (□), 5 nM (○), 10 nM (Δ), 15 nM (◊), 25 nM (X), and 50 nM (*). Seven separate experiments were combined, and values represent the mean ± standard deviation.

Cathepsin K Expression and Kinetics

Northern blot analysis of stage-specific porcine enamel organs demonstrated a strong increase in cathepsin K expression during the maturation stage of enamel development (Fig. 4A). This is when the ameloblasts are actively re-absorbing enamel matrix proteins from the hardening enamel. Therefore, cathepsin K activity may be important for enamel maturation, and its inhibition might result in enamel defects and/or increased enamel protein content. We asked if cathepsin K activity was inhibited in the presence of NaF.

Figure 4.

Figure 4.

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Stage-specific cathepsin K expression in enamel organs and effect of fluoride on rhCathepsin K activity. Porcine teeth at specific stages of development were assessed for cathepsin K transcript levels by Northern blot analysis. (A) Note that in the enamel organ (EO), cathepsin K expression was low during the secretory stage (S), increased during the early maturation stage (EM), and peaked during the maturation stage (M) of enamel development. The respective normalized densitometry values were 1.0, 5.7, and 13.6. This enamel organ stage-specific expression did not occur in the pulp organ (PO), where the respective densitometry values for S, EM, and M were 5.5, 5.0, and 5.4. For the cathepsin K activity analysis, the substrate Z-Leu-Arg-AMC was incubated in assay buffer at a final concentration of 3.125 nM with 10 ng of rhCatK and increasing concentrations of NaF (B) or E64 (C). We calculated reaction rates by increasing the substrate concentration from 0.3125 to 31.25 nM. We used data from the first 10 min to calculate Vo (nM per min). Michaelis-Menten plots of 10 ng of rhCatK incubated with increasing concentrations of NaF (D) and the irreversible metalloproteinase inhibitor E64 (E) were generated. NaF concentrations were 0 (□), 1 µM (○), 10 µM (Δ), 100 µM (◊), 1 mM (X), and 10 mM (*). E64 concentrations were 0 (□), 0.025 nM (○), 0.05 nM (Δ), 0.10 nM (◊), and 0.15 nm (X). Seven separate experiments with 2 replicates were combined, and values represent the mean ± standard deviation.

Because rhCatK-mediated hydrolysis of the quenched fluorescent peptide was not linear over the 60-minute time scale (Figs. 4B, 4C), the rate of hydrolysis was calculated for the first 10 min. We used E64 as a control to demonstrate the inhibition of rhCatK. Increasing concentrations of E64 resulted in a decreased Vmax (Fig. 4E). Addition of up to 10 mM NaF, however, had no discernible effect on the rate of hydrolysis by rhCatK (Fig. 4D).

Discussion

In this study, we directly measured the effect of fluoride on protease activity using substrates and conditions optimized for enzyme activity. We examined the effect of fluoride on the activity of the maturation-stage protease KLK4. Although the in vivo activator of KLK4 is undefined, KLK4 can be activated in vitro by either MMP20 or DPPI (Ryu et al., 2002; Tye et al., 2009b). If the activity of either of these proteases is reduced by the presence of fluoride, this may result in decreased KLK4 activation. Therefore, we investigated the effect of fluoride on the activity of each of these activator enzymes. We also tested cathepsin K activity in the presence of fluoride, because its expression pattern suggests that it may degrade re-absorbed enamel matrix proteins.

Although a full kinetic analysis was not previously completed, the effect of fluoride on MMP20 activity was studied. It was reported that 100 µM fluoride decreased the ability of MMP20 to cleave the tyrosine-rich amelogenin peptide (DenBesten et al., 2002). MMP20 was also shown to be inhibited at pH 6.0 with as little as 2 µM fluoride (DenBesten et al., 2002). Conversely, another group used pooled secretory- and maturation-stage enamel matrix from rat incisors and examined the effects of increasing concentrations of fluoride on these pooled samples. A colorimetric assay and zymography demonstrated that there was no significant decrease in total protease activity with up to 10 mM fluoride (Gerlach et al., 2000). Because of these discrepancies, we believed that a full kinetic analysis of proteases that are, or may be, important in enamel development was warranted.

DPPI may be an important KLK4 activation enzyme (Tye et al., 2009b), and salivary DPPI (cathepsin C) was previously suggested to be inhibited by fluoride (Dabrowska et al., 2005). In this study, individuals were given various aminofluoride treatments commonly used in oral hygiene, which resulted in a decrease in DPPI activity. Treatment with these fluoride solutions also increased the pH of saliva from 7.0 to 7.1 -7.35. The pH optimum for peptide hydrolysis for DPPI is 5.0–6.0 (Turk et al., 2004). Therefore, it is not clear if the resulting decrease in activity was caused by the change in pH or if enzyme activity was directly affected by fluoride. Here, we directly examined the effect of NaF on DPPI activity using a fluorescent peptide under optimum conditions for the enzyme. Our findings demonstrate that DPPI activity is not inhibited by fluoride at concentrations up to 10 mM.

The increased protein content in fluorosed enamel may be due to other factors affecting the enamel proteases. It has been demonstrated that treatment of an ameloblast-like cell line with fluoride inhibited protein secretion (Sharma et al., 2008). Fluoride-treated rats were also previously demonstrated to have decreased protease activity per unit of sample protein (DenBesten et al., 2002). We have shown that KLK4 transcripts were decreased in fluoride-treated rats, and this was attributed to an ameloblast cell stress response (Sharma et al., 2010). Alternatively, it was postulated that fluoride may induce a molecular or structural change in the enamel proteins, which makes them less susceptible to proteolysis (Crenshaw and Bawden, 1981; Drinkard et al., 1983).

In any case, analysis of the data presented here definitively demonstrates that, in conditions that have been optimized for each protease’s activity, fluoride does not directly inhibit KLK4, MMP20, DPPI, or cathepsin K. If proteolytic activity is reduced within fluorosed dental enamel, then it must result from a process other than direct inhibition of individual proteases thought to be important in enamel formation.

Acknowledgments

This investigation was supported by NIDCR grants DE016276, DE016276-04S1, and DE018106 to JDB.

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