Functional role of Rho-kinase in ameloblast differentiation.

During tooth development, inner enamel epithelial (IEE) cells differentiate into enamel-secreting ameloblasts, a polarized and elongated cellular population. The molecular underpinnings of this morphogenesis and cytodifferentiation, however, are not well understood. Here, we show that Rho-associated coiled-coil-containing protein kinase (ROCK) regulates ameloblast differentiation and enamel formation. In mouse incisor organ cultures, inhibition of ROCK, hindered IEE cell elongation and disrupted polarization of differentiated ameloblasts. Expression of enamel matrix proteins, such as amelogenin and ameloblastin, and formation of the terminal band structure of actin and E-cadherin were also perturbed. Cultures of dental epithelial cells revealed that ROCK regulates cell morphology and cell adhesion through localization of actin bundles, E-cadherin, and ß-catenin to cell membranes. Moreover, inhibition of ROCK promoted cell proliferation. Small interfering RNA specific for ROCK1 and ROCK2 demonstrated that the ROCK isoforms performed complementary functions in the regulation of actin organization and E-cadherin-mediated cell-cell adhesion. Thus, our results have uncovered a novel role for ROCK in amelogenesis.

Activity of matrix metalloproteinases in bovine versus human dentine.

Metalloproteinases (MMPs) have been implicated with metabolism of collagen in physiological and pathological processes in human dentine. As bovine teeth have been used as a substitute for human teeth in laboratory analysis, this study evaluated the activity of MMP-2 and -9 in bovine versus human dentine. Bovine and human dentine fragments, from crowns and roots, were powderized. Protein extraction was performed by two protocols: a neutral extraction with guanidine-HCl/EDTA (pH 7.4) and an acidic extraction with citric acid (pH 2.3). Gelatinolytic activities of extracts were revealed by zymography. MMP-2 and -9 were detected in crown and root dentine from bovine and human teeth. Total activities of MMP-2 were 11.4 ± 2.2, 14.6 ± 2.0, 9.7 ± 1.2 and 12.4 ± 0.9 ng/ml for bovine root, human root, bovine crown and human crown dentine, respectively. Corresponding activities for MMP-9 were 14.9 ± 2.0, 15.3 ± 1.3, 15.4 ± 1.3 and 15.5 ± 1.3 ng/ml, respectively. Bovine dentine was found to be a reliable substrate for studies involving the activity of MMP-2 and -9.

Expression pattern of transglutaminases in the early differentiation stage of erupting rat incisor.

Several studies demonstrated that transglutaminases play a key role in extracellular matrix stabilization needed for cell differentiation. We evaluated transglutaminase expression and activity in the pre-secretory stage of differentiation of the continuously erupting rat incisor. We observed that transglutaminase-mediated incorporation of monodansylcadaverine into protein substrates was specifically located in the apical loop, and along the basement membrane joining mesenchyme and inner dental epithelium in the odontogenic organ. Enzyme activity was associated with mRNAs for transglutaminase 1 and 2. Notably, labelling cells for these isoenzymes were observed in both mesenchymal and epithelial compartments, but not in the basement membrane, in the ameloblast facing pulp anterior region, where ameloblast and odontoblast differentiation begins. These findings demonstrate that transglutaminase 1 and transglutaminase 2 are expressed at a major extent in the pre-secretory stage of regenerating rat incisor, where they probably play complementary roles in cell signalling between mesenchyme and epithelium and extracellular matrix.

Transglutaminase crosslinking of SIBLING proteins in teeth.

Transglutaminase 2 (TG2), a protein-crosslinking enzyme, participates in extracellular matrix maturation and cell adhesion in cartilage and bone. We hypothesized that TG2 has similar roles in teeth. A TG activity assay and immunoblotting of rat tooth extracts showed TG activity and the presence of high-molecular-weight forms of the SIBLING (Small Integrin-Binding LIgand N-linked Glycoprotein) proteins: dentin matrix protein 1 (DMP1), dentin phosphoprotein (DPP), and bone sialoprotein (BSP). DMP1 and BSP, each containing both glutamine and lysine residues critical for crosslink formation, readily formed polymers in vitro when incubated with TG2. The ability of glutamine-lacking DPP to form polymers in vitro and in vivo demonstrates that it could act as a lysine donor for crosslinking, potentially having protein crosslinking partner(s) in teeth. Consistent with a role in cell adhesion, the TG2 isoform was co-localized by immunohistochemistry with its substrates at cell-matrix adhesion sites, including along odontoblast tubules (DMP1 and DPP), in the pericellular matrix of cementocytes (DMP1), and in predentin (BSP).

Cytochemical localization of Ca2+-Mg2+ adenosine triphosphatase in rat incisor ameloblasts during enamel secretion and maturation.

A modified Wachstein-Meisel medium containing lead or cerium as capturing ions was used to localize Ca2+-Mg2+ adenosine triphosphatase (ATPase; EC 3.6.1.3) in rat incisor ameloblasts during enamel formation. Sections representing different developmental stages were processed for electron microscopic cytochemistry. Distribution and intensity of the observed reaction product, which was almost exclusively associated with cell membranes, varied according to the stage of enamel formation. During the secretory stage, intense reaction product was evident along the entire plasma membrane of ameloblasts and papillary cells. The early transitional ameloblasts showed reaction product on their proximal and lateral cell membranes, but not distally. In late transitional (pre-absorptive) ameloblasts, distal cell membranes exhibited intense reaction product. During enamel maturation, smooth-ended ameloblasts showed reaction product proximally and laterally, but not distally. Ruffle-ended maturative ameloblasts exhibited intense reaction product along their lateral and distal membranes. The intensity of the latter was decreased but not eliminated by levamisole. In the transition from smooth-ended to ruffle-ended cells, the reaction product became evident distally, concomitant with the appearance of cell membrane invaginations. These data are consistent with a possible role for Ca2+-Mg2+ ATPase in controlling calcium availability at the enamel mineralization front.

Immunolocalization of enamelysin (matrix metalloproteinase-20) in the forming rat incisor.

In the rat model, we used the continuously growing incisor to study the expression pattern of matrix metalloproteinase-20 (MMP-20) during the formation of mineralized dental tissues. Casein zymography analysis of extracts of the forming part of the incisor revealed lysis bands corresponding to both the latent form at 57 kD and the active 46- and 41-kD forms, whereas omission of proteinase inhibitors during protein extraction resulted in a single band at 21 kD. A higher molecular weight form of 78 kD was also stained with MMP-20 and TIMP-2 antibodies in Western blotting, and was therefore believed to correspond to an MMP-20/TIMP-2 complex. Immunohistochemical and immunogold electron microscopic results demonstrated strong MMP-20 staining in the forming outer enamel, which diminished near the dentino-enamel junction, but dentin and predentin were unstained. A strong concentration of MMP-20 was seen in the stratum intermedium (SI), particularly at the earlier stages of enamel development. Our results confirm the presence of MMP-20 protein in ameloblasts and odontoblasts of rat incisor and show it to be localized in the same sites of the forming enamel as amelogenin. Their expression is transient in odontoblasts but persists in ameloblasts, and in both cases the expression of amelogenin preceded that of MMP-20 suggesting a developmentally controlled regulation.

A comparison of ATP-degrading enzyme activities in rat incisor odontoblasts.

In active odontoblasts from the rat incisor, used as a model system for biologic calcification, two distinguishable enzyme activities capable of degrading adenosine monophosphate (ATP) exist. Once can be inhibited ny 1-tetramisole, (+/-)-2,3,5,6,-tetrahydro-6-phenylimidazo (2.1B) THIAZOLE HYDROCHLORIDE (Levamisol) and (+/-)-6(m-bromophenyl)-5.6-dehydroimidazo (2.1-b) thiazole oxalate (R823) and is probably identical with nonspecific alkaline phosphatase (EC 3.1.3.1). The activity of the other enzyme, named Ca2+-ATPase, is dependent on the presence of Ca2+ or Mg2+ and is activated by these ions. The pH optimum of Ca2+-ATPase is 9.8. The Ca2+-ATPase is unaffected by Levamisole, R 8231, ouabain, ruthenium red, Na+ and K+ ions. Maximal activity was found against ATP, whereas adenosine diphosphate, guanosine triphosphate, inosine triphosphate and adensoine monophosphate were hydrolysed at lower rate. It may be speculated that the Ca2+-ATPase is concerned with the transmembranous transport of Ca2+ ions to the mineralization front.

Ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase) activity to the intermediate saccules of the Golgi apparatus in rat incisor ameloblasts.

Cytochemical evidence for the existence of a Golgi-associated phosphatase activity that hydrolyzes nicotinamide adenine dinucleotide phosphate (NADP) at acid pH in rat incisor ameloblasts was obtained by incubating sections from glutaraldehyde-fixed teeth in a medium containing NADP as substrate and lead ions as capture agent. Following incubation for 1 hr at 37 degrees C and pH 5.0, the Golgi saccules situated between those at the cis (immature) and trans (mature) faces of the ameloblast Golgi apparatus were marked by reaction product with the heaviest deposit in the middle saccule. Reaction product was otherwise seen in trace amounts only over some elements of the GERL system as well as a few lysosomal dense bodies and immature secretory granules. Control experiments established that the selective staining of intermediate Golgi saccules at pH 5.0 could only be duplicated by using substrates that resembled the complete NADP molecule, and not just the portion containing the adenosine 2'-monophosphate group. As well, no deposits of reaction product were seen within the Golgi saccules of ameloblasts incubated at pH 5.0 with nictoinamide adenine dinucleotide (NAD) as the substrate or that were incubated at pH 7.2 or pH 9.0 with NADP as the substrate. It was concluded that a specific, acid-NADPase activity is present in the intermediate Golgi saccules of secretory ameloblasts. Preliminary observations on other cells suggest that the localization of NADPase activity to Golgi saccules may constitute a general phenomenon.

Ultrastructural localization of coenzyme A phosphatase (CoA-Pase) activity to the GERL system in secretory ameloblasts of the rat incisor.

Enzymatic hydrolysis of the monoester phosphate group from coenzyme A (CoA) was studied in rat incisor ameloblasts by incubating specimens from glutaraldehyde-fixed teeth in a cytochemical medium prepared with acetyl-CoA as substrate and lead ions as capture agent for phosphate. Ameloblasts incubated for 1 hr at 37 degrees C and at pH 5.0 in this medium showed reaction product localized almost exclusively along the trans (mature) aspect of the Golgi apparatus within a network of small granules and interconnecting tubular channels that comprise the GERL system in this cell. Reaction product was otherwise seen in trace amounts only within some Golgi saccules, a few lysosomal dense bodies and, in rare instances, within an occasional focal area of the endoplasmic reticulum. No selective staining of the GERL system was seen in control ameloblasts incubated at either pH 7.2 or pH 9.0 with acetyl-CoA as substrate, or incubated at pH 5.0 with dephospho-CoA as substrate. Control experiments at pH 5.0 also revealed that reaction product selectively stained the GERL system in ameloblasts when other molecules resembling CoA were used as substrate (e.g., crotonyl-CoA, 3'-NADP+), but not when adenosine 3'-monophosphate (3'-AMP) was used as substrate. That is, ameloblasts incubated at pH 5.0 with 3'-AMP showed heavy deposits of reaction product at many sites throughout the cell, including most lysosomal dense bodies, the Golgi saccules, the GERL system, most secretory granules, the nucleus, and extensively throughout the endoplasmic reticulum. These findings suggest that the GERL system of ameloblasts contains a CoA-specific phosphatase activity that may function to convert CoA to dephospho-CoA at acid pH. Biochemical studies included with this investigation further indicate that CoA-Pase activity saturates at exceptionally low concentrations of substrate (KM = 30 microM CoA) compared to other acid-dependent phosphatases.

Tubular lysosomes in ruffle-ended ameloblasts associated with enamel maturation in rat incisor.

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were localized to investigate the lysosomal system, particularly tubular lysosomes, in ruffle-ended ameloblasts associated with maturation of enamel in rat incisor. Demineralized specimens were incubated for TMPase and for CMPase in a modified medium where cerium was used as the capture ion. Ruffle-ended ameloblasts showed distal invaginations and membrane-bound bodies filled with fine granular material, some of which displayed CMPase reaction product. Elongated tubular configurations 80-140 nm wide were distributed throughout the cytoplasm and were reactive with both TMPase and CMPase, thus characterizing these structures as lysosomes. They often contained fine granular material morphologically similar to that present in multivesicular bodies. During late enamel maturation, fewer tubular lysosomes were observed when compared to early maturation. These cytochemical results demonstrate the presence of tubular lysosomes in ruffle-ended ameloblasts, and it is suggested that they are elements of the endosomal system in these cells. These findings are also consistent with a resorptive function for ruffle-ended ameloblasts during enamel maturation.

Inhibition studies of alkaline phosphatase in hard tissue-forming cells.

The effects of the alkaline phosphatase inhibitors levamisole and R 8231 on p-nitro-phenylphosphatase, inorganic pyrophosphatase and adenosine triphosphatase (ATPase) activities in dentingenically active odontoblasts were studied. The p-nitrophenylphosphatase and inorganic pyrophosphatase activities were inhibited, while 40% of the ATP-splitting enzyme activity remained under the assay condition used. This finding, togeather with earlier studies, indicates that at least two different phosphatase are active at alkaline pH in hard tissue-forming cells; on nonspecific alkaline phosphatase and one specific ATPase. The ATPase activity is uninfluenced by ouabain and ruthenium red and is activated by Ca-2+ ions.

Cytochemical localization of calcium and Ca2+,Mg2(+)-adenosine triphosphatase in colchicine-altered rat incisor ameloblasts.

Colchicine is known to affect secretory, transport, and degradative functions of ameloblasts. The effects of colchicine on membrane-associated calcium and Ca2+,Mg2(+)-ATPase in secretory and maturation ameloblasts were investigated cytochemically. The pyroantimonate (PPA) method was used for localizing calcium and a modified Wachstein-Meisel medium was used to localize Ca2+,Mg2(+)-ATPase. Sections representing secretory and early maturation stages were examined by transmission electron microscopy. Morphological changes induced by colchicine included dislocated organelles and other well-established reactions to such anti-microtubule drugs. Calcium pyroantimonate (Ca-PA) deposits in most ameloblast types were markedly reduced, with the greater reduction occurring in those cells more severely altered morphologically. However, the cell membranes of both control and experimental smooth-ended maturation ameloblasts were essentially devoid of Ca-PA. The normal distribution and intensity of Ca2+,Mg2(+)-ATPase was not affected by colchicine. Because the observed reduction of membrane-associated calcium is apparently not mediated by Ca2+,Mg2(+)-ATPase in this case, other aspects of the calcium regulating system of ameloblasts are apparently targeted by colchicine.

Correlated alkaline phosphatase histochemistry and quantitative backscattered electron imaging in the study of rat incisor ameloblasts and enamel mineralization.

The different functional conditions of pre-ameloblasts, secretory ameloblasts, and maturation ameloblasts in 9 day rat incisors were recognized using high resolution light microscopic alkaline phosphatase histochemistry: Digital backscattered electron imaging was performed using the block surfaces from which thin sections were taken for histochemical study. It was possible to correlate exact locations in histochemical sections with positions in the block face at all stages of enamel mineralization from early secretion through late maturation. The first steep increase in the rate of mineralization of completed enamel matrix occurs after the first transition from smooth ended ameloblasts to ruffle ended ameloblasts. In the 9 day rat incisors used for this purpose, there are only two smooth to ruffle cyclical transitions, and the width of successional smooth ended bands of ameloblasts in the maturation cycling process is always narrow. Nevertheless, there seems to be a good correlation between mineralization increase and the acquisition of the high alkaline phosphatase activity in the deeply enfolded distal cytoplasm of the ruffle-ended maturation stage ameloblasts.

Carbonic anhydrase isozyme II immunoreactivity in the mechanoreceptive Ruffini endings of the periodontal ligament in rat incisor.

The present study describes the distribution of carbonic anhydrase isozyme II (CA II) in the lingual periodontal ligament of the rat incisor. Some thick nerve fibers in the nerve bundle displayed CA II-like immunoreactivity (LI) as well as non-neuronal elements such as osteoclasts. At the alveolar half of the lingual periodontal ligament of the incisor, thick CA II-like immunoreactive (-IR) nerve fibers showed a tree-like raminification, but thin and beaded CA II-IR nerve fibers were rare. Under the electron microscope, CA II-LI were diffusely localized in the axoplasm of the axon terminals surrounded by Schwann sheaths which were immunonegative for CA II. The cell bodies of the terminal Schwann cells associated with the periodontal Ruffini endings did not exhibit CA II-LI. The present immunohistochemical evidence indicates that CA II may participate in the regulation of the intra-neuronal ion in the periodontal Ruffini endings which are thought to be in a state of high neuronal activity.

Immunocytochemical detection of superoxide dismutases (SODs) in the periodontal Ruffini endings of the rat incisor.

The expression of immunoreactivities for superoxide dismutases (SODs), Mn-SOD and Cu/Zn-SOD, was immunohistochemically investigated in the lingual periodontal ligament and toe pads of adult rats. Immunocytochemistry for SODs revealed that the axon terminals of both the periodontal Ruffini endings and cutaneous Meissner's corpuscles showed mitochondrial Mn-SOD immunoreactivity, but not cytosolic Cu/Zn-SOD immunoreactivity, indicating Mn-SOD is a useful marker for identifying the mechanoreceptors. It is likely that Mn-SOD in the axon terminals of mechanoreceptors exerts protective action against nerve injury and neuronal death under severe conditions, serving to scavenge free radicals from the axon terminals.

Prolyl-hydroxylase activity in odontoblasts from bovine incisor and premolar teeth.

Odontoblasts of incisor and premolar teeth showed high prolyl-hydroxylase (EC 1.14.11.2; proline, 2-oxoglutarate dioxygenase) activity, but the activities in other regions of these dental pulps were low. These results suggest that these odontoblasts are concerned with the biosynthesis and processing of dentine collagen.

Purification and kinetic properties of phosphofructokinase from dental pulps of rat incisors.

Phosphofructokinase (EC 2.7.1.11) was partially purified 19-fold from dental pulps of rat incisors, by ammonium-sulphate fractionation and phenyl-Sepharose chromatography with a recovery of about 95 per cent. With a 0.05 M tris-HCl buffer, the pH optimum of the enzyme was determined to be 8.4. At this pH, substrate inhibition of the enzyme by either fructose-6-phosphate (F6P) or ATP was not observed, and the relationship between reaction velocity and each substrate concentration was well explained by the Michaelis-Menten equation. The Km values were determined to be 5 X 10(-5) and 1.8 X 10(-4) M for ATP and F6P, respectively. The enzyme, however, showed different catalytic properties at pH 7.6, i.e. the kinetic behaviour was sigmoidal with respect to F6P and it was inhibited by high concentrations of ATP. The Hill coefficient for F6P was determined graphically to be 1.4. At pH 7.6, the enzyme activity was inhibited by citrate and phosphoenolpyruvate (PEP), neither of which showed any inhibitory effect on the enzyme at pH 8.4.

Influence of bivalent cations, phosphate and complexing substances on inorganic pyrophosphate in the microsomal fraction of isolated rat odontoblasts.

The influence of three diphosphonates, ethane-1-hydroxy 1.1. diphosphonate, methane-diphosphonate and dichloromethane diphosphonate, on inorganic pyrophosphatase (PPiase, E.C. 3.6.1.1.) was investigated. The inhibition by the diphosphonates appeared to be due to their complexation with a bivalent cation, probably Zn2+, which acts as a cofactor for the enzyme. The influence of calcium and inorganic phosphate (Pi) on the PPiase activity was studied in the presence and absence of several complexing substances, including the diphosphonates. Ca2+ alone inhibited the enzyme whereas, in the presence of EDTA, a Ca2+ stimulation of the PPiase was observed which was substrate-dependent and had an optimal activity at a Ca2+:PP4-i ratio of 1:1. There was a small inhibition of the PPiase activity by Pi which was not influenced by the substrate used.

EMMPRIN/CD147 deficiency disturbs ameloblast-odontoblast cross-talk and delays enamel mineralization.

Tooth development is regulated by a series of reciprocal inductive signaling between the dental epithelium and mesenchyme, which culminates with the formation of dentin and enamel. EMMPRIN/CD147 is an Extracellular Matrix MetalloPRoteinase (MMP) INducer that mediates epithelial-mesenchymal interactions in cancer and other pathological processes and is expressed in developing teeth. Here we used EMMPRIN knockout (KO) mice to determine the functional role of EMMPRIN on dental tissue formation. We report a delay in enamel deposition and formation that is clearly distinguishable in the growing incisor and associated with a significant reduction of MMP-3 and MMP-20 expression in tooth germs of KO mice. Insufficient basement membrane degradation is evidenced by a persistent laminin immunostaining, resulting in a delay of both odontoblast and ameloblast differentiation. Consequently, enamel volume and thickness are decreased in adult mutant teeth but enamel maturation and tooth morphology are normal, as shown by micro-computed tomographic (micro-CT), nanoindentation, and scanning electron microscope analyses. In addition, the dentino-enamel junction appears as a rough calcified layer of approximately 10±5µm thick (mean±SD) in both molars and growing incisors of KO adult mice. These results indicate that EMMPRIN is involved in the epithelial-mesenchymal cross-talk during tooth development by regulating the expression of MMPs. The mild tooth phenotype observed in EMMPRIN KO mice suggests that the direct effect of EMMPRIN may be limited to a short time window, comprised between basement membrane degradation allowing direct cell contact and calcified matrix deposition.