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Keywords:

  • ameloblast;
  • tight junction;
  • claudin;
  • PAR3;
  • occludin

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Tight junctions (TJs) create a paracellular permeability barrier to restrict the passage of ions, small solutes, and water. Ameloblasts are enamel-forming cells that sequentially differentiate into preameloblasts, secretory, transition, and ruffle-ended and smooth-ended maturation ameloblasts (RAs and SAs). TJs are located at the proximal and distal ends of ameloblasts. TJs at the distal ends of secretory ameloblasts and RAs are well-developed zonula occludens, but other TJs are moderately developed but incomplete zonula occludens (ZO) or less-developed macula occludens. We herein examined the immunofluorescence localization of TJ proteins, 10 claudin isoforms, occludin, ZO-1, and PAR3, a cell polarity-related protein, in ameloblasts of rat upper incisors. ZO-1 and claudin-1 were detected at both ends of all ameloblasts except for the distal ends of SAs. Claudin-4 and occludin were detected at both ends of transition and maturation ameloblasts except for the distal ends of SAs. PAR3 was detected at the proximal TJs of all ameloblasts and faintly at the distal TJs of early RAs. These results indicate that functional zonula occludens formed at the distal ends of the secretory ameloblasts and RAs consisted of different TJ proteins. Therefore, the distal TJs of secretory ameloblasts and RAs may differentially regulate the paracellular permeability to create a microenvironment suitable for enamel deposition and enamel maturation, respectively. In addition, PAR3 may be principally involved in the formation and maintenance of the proximal, but not distal, TJs. Anat Rec, 291:577–585, 2008. © 2008 Wiley-Liss, Inc.

Tight junctions (TJs) function as a barrier to regulate the paracellular transport of ions, small solutes, and water (Gumbiner,1993). In addition, TJs are a multiprotein complex consisting of integral membrane proteins and cytoplasmic plaque proteins and play a role in the regulation of cell polarity, proliferation, and differentiation (Shin et al.,2006). Claudins, occludin, tricellulin, and junctional adhesion molecules (JAMs) are integral membrane proteins at the TJs. Claudins form a multigene family (Morita et al.,1999) composed of at least 24 members in mice. More than two claudin isoforms are usually expressed in a single cell, and the combination and mixing ratios of claudins determine the TJ properties including paracellular permeability. Zonula occludens (ZO)-1, ZO-2, and ZO-3 are three of the well-characterized proteins among many cytoplasmic plaque proteins at TJs. They link occludin and claudins to the actin cytoskeleton to stabilize the TJ structure or to promote the clustering of claudins/occludin to polymerize into TJ strands (Furuse et al.,1994; Haskins et al.,1998; Itoh et al.,1999a,b,1997) 1.

Table 1. Summary for the expression pattern of ZO-1, claudin-1, claudin-4, occludin, and PAR3 in the ameloblasts during differentiationa
 PreameloblastsSecretory ameloblastsTransition ameloblastsMaturation ameloblasts
EarlyLateEarly RAMid SAMid RALate SALate RA
  • a

    prox., proximal ends of the lateral membrane of ameloblasts; dist., distal ends of the lateral membrane of ameloblasts; ZO, zonula occludens; PAR, partitioning defective. Intensity of immunostaining: −, negative; ±, weak (occasionally expressed); +, weak (always expressed); ++, moderate; +++, strong; ++++, very strong.

ZO-1
 prox.++++++++++++++++++++++++
 dist.+++++++++++++±+++±+++
Claudin-1
 prox.± ˜ ++++±± ˜ +++
 dist.±+++++++++++±+++
Claudin-4
 prox.± ˜ ++± ˜ ++++
 dist.± ˜ +++±++±+
Occludin
 prox.+++++++
 dist.+++++±++++++++
PAR3
 prox.± ˜ ++ ˜ +++++++++++++++++
 dist.± ˜ +

The establishment of epithelial cell polarity requires atypical protein kinase C (aPKC) activity (Suzuki et al.,2001). The Crumbs complex and the partitioning defective (PAR) complex have been shown to play critical roles in the development of cell polarity (Shin et al.,2006). The PAR3/PAR6/aPKC complex localizes at TJs (Ohno,2001; Macara,2004). PAR6 links PAR3/PAR6/aPKC complex to the active form of Cdc42, a Rho GTPase, and these interactions are important for the formation or maintenance of TJs in MDCK epithelial cells (Joberty et al.,2000).

The first sign of TJ formation was observed in preameloblasts abutting the unmineralized dentine at the distal ends of the lateral membrane, and later, TJ strands formed an anastomosing meshwork at the proximal and distal ends of the preameloblasts. However, these TJs did not form a complete seal around the cell body circumference (Sasaki et al.,1982,1990). When the newly secreted enamel matrix reached 7–8 μm in thickness, the distal TJ in the secretory ameloblasts exhibited a functional zonular TJ (zonula occludens). The proximal TJ formed by two secretory ameloblasts was moderately developed but there was an open space at the corner formed by three secretory ameloblasts (Sasaki et al.,1982,1990). In transition ameloblasts, the proximal and distal TJs did not completely occlude the extracellular space (Sasaki et al.,1990). In RAs, the distal TJ is a zonula occludens but the proximal TJ is a macula occludens (Sasaki et al.,1983a,1990). In SAs, the proximal TJ is a structurally incomplete zonula occludens but the distal TJ is a macula occludens (Sasaki et al.,1983b,1990).

Intercellular junctions, tight, adherens, and gap junctions, not only organize tissue architecture but also are involved in physiological function, morphogenesis, and cell differentiation. The distributions of integral membrane proteins forming intercellular junctions such as occludin, claudins, cadherins, and connexins in ameloblasts have been reported previously (Inai et al.,1997; Obara et al.,1998; João and Arana-Chavez,2004; Bello et al.,2007; Ohazama and Sharpe,2007). Early and late differentiating rat ameloblasts at the bell stage expressed claudin-1 at the proximal and distal TJs and also expressed occludin except for the distal TJs of late differentiating ameloblasts (João and Arana-Chavez,2004). In developing human teeth, strong immunoreactivities for claudin-1 and -7 were detected in the enamel organ, ameloblasts, and enamel matrix, but moderate staining for claudin-4 was detected in the outer enamel epithelium (Bello et al.,2007). An in situ hybridization analysis of claudin-1 to -11 showed that only claudin-2 was expressed in mouse ameloblasts at the early bell stage (Ohazama and Sharpe,2007). We herein examined the expression of TJ proteins, including 10 claudins, occludin, ZO-1, and PAR3 in preameloblasts, secretory, transition, and maturation ameloblasts, because no researchers have reported the localization of TJ proteins in maturation ameloblasts.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Antibodies

Primary antibodies and their working dilutions are as follows: rabbit anti–claudin-1 polyclonal antibody (pAb; 1:50), mouse anti–claudin-2 monoclonal antibody (mAb; 1:100), anti–claudin-3 pAb (1:100), anti–claudin-4 mAb (1:100), anti–claudin-5 mAb (1:200), anti–claudin-6 pAb (1:100), anti–claudin-7 pAb (1:100), anti–claudin-8 pAb (1:100), anti–claudin-12 pAb (1:50), anti–claudin-15 pAb (1:50), anti-occludin mAb (1:200), anti-occludin pAb (1:200), anti–ZO-1 pAb (1:400), and anti–ZO-1 mAb (1:1,000). Anti–claudin-6 pAb and anti–claudin-7 pAb were purchased from IBL (Takasaki, Japan), and the remaining antibodies were purchased from Zymed (South San Francisco, CA). Anti-mouse Ig and anti-rabbit Ig conjugated with either Alexa 488 or Alexa 594 (Molecular Probes, Eugene, OR) were used as secondary antibodies at a 1:400 dilution.

Specimens

Three 21-day-old Wistar rats were purchased from Kyudo (Tosu, Japan). They were anesthetized intraperitoneally with Nembutal (30 mg/kg body weight) before perfusion with fixative. They were perfused systemically from the left ventricle with 1% paraformaldehyde in phosphate buffered saline (PBS, pH 7.4) under anesthesia. The upper incisors were dissected and then were immersed in the same fixative on ice for 4 hr. After washing in PBS, the upper incisors were decalcified in 10% ethylenediaminetetraacetic acid, pH 7.4, for 4 days at 4°C, washed with PBS, and rapidly frozen in liquid nitrogen-cooled OCT compound. Cryosections (5 μm) were cut and mounted on glass slides.

Immunohistochemistry

The cryosections were washed in PBS, incubated in 0.2% Triton X-100 in PBS for 15 min, washed again in PBS, and then incubated with 1% bovine serum albumin in PBS for 30 min at room temperature to block nonspecific binding. They were incubated with primary antibodies for 1 hr in a moist chamber. After rinsing in PBS 5 times for 3 min each, they were incubated with secondary antibodies for 30 min in the dark. They were washed 5 times in PBS for 3 min each and mounted in Vectashield mounting medium (Vecta Laboratories, Burlingame, CA). The specimens were then examined with a Zeiss LSM 510 confocal microscope (Oberkochen, Germany).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The upper incisors contained preameloblasts, secretory, transition, and maturation ameloblasts. We examined the localization of 10 claudin isoforms and only claudin-1 and -4 were detected in the ameloblasts. Claudin-5 was expressed in vascular endothelial cells but not in ameloblasts.

ZO-1

ZO-1 was first detected at the proximal ends of very early preameloblasts and eventually at the distal ends (Fig. 1). Intense signals for ZO-1 were consistently observed at the proximal and distal ends of preameloblasts, secretory, transition, and maturation ameloblasts (Figs. 1–5) although not at the distal ends of the SAs (Figs. 2,5).

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Figure 1. Immunofluorescence localization of claudin-1, zonula occludens (ZO) -1, and occludin in the preameloblasts and secretory ameloblasts. Cryosections were doubly stained with anti–claudin-1 and anti–ZO-1 antibodies or anti-occludin and anti–ZO-1 antibodies. Green signals for claudin-1 are first detected at the proximal ends of early preameloblasts and then at the distal ends of preameloblasts (long arrow). Red signals for ZO-1 are first detected at the proximal ends of early preameloblasts and then at the distal ends of preameloblasts (short arrow). Signals for claudin-1 are weak at the proximal and distal ends of preameloblasts and at the proximal ends of secretory ameloblasts, but strong at the distal ends of secretory ameloblasts. Dot-like staining for claudin-1 is also detected in the cytoplasm of secretory ameloblasts. The signals for ZO-1 are moderate to strong at the proximal ends of preameloblasts, weak to moderate at the distal ends of preameloblasts, and strong at the both ends of secretory ameloblasts. The signals for occludin are detected in the stellate reticulum and stratum intermedium, but not in secretory ameloblasts. An asterisk indicates the artificial gap between late preameloblasts and predentin/dentin located between the arrowheads. An oblique arrow indicates Tomes' processes of secretory ameloblasts. cldn-1, claudin-1; pAm, preameloblast; Am, ameloblast; SI, stratum intermedium; SR, stellate reticulum; Od, odontoblast. Scale bars = 20 μm.

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Figure 2. Immunofluorescence localization of claudin-1, claudin-4, occludin, and zonula occludens (ZO) -1 in transition and maturation ameloblasts. Cryosections were doubly stained with anti–claudin-1 and anti–ZO-1 antibodies, anti–claudin-4 and anti–ZO-1 antibodies, or anti-occludin and anti–ZO-1 antibodies. In transition ameloblasts, weak signals for claudin-1, strong signals for ZO-1, negative to weak signals for claudin-4, and weak signals for occludin are detected at both ends. In maturation ameloblasts, intense red signals for ZO-1 are detected at both ends of the RAs and at the proximal ends of the SAs, but are negative to weak at the distal ends of the SAs. At the proximal ends of the RAs, negative to weak signals for claudin-1, weak signals for claudin-4, and weak to moderate signals for occludin are detected. At the distal ends of the RAs, strong signals for claudin-1, moderate to weak signals for claudin-4, and very strong signals for occludin are detected. At the proximal ends of SAs, negative to weak signals for claudin-1 and claudin-4, and weak signals for occludin are detected. At the distal ends of the SAs, negligible signals for claudin-1, claudin-4, and occludin are detected. Dot-like staining for claudin-1 is also detected in the cytoplasm of ameloblasts. The signals for claudin-4 detected in the papillary layer may be nonspecific staining, especially in the transition ameloblasts and early RAs. RA, ruffle-ended ameloblast; SA, smooth-ended ameloblast; PL, papillary layer. Scale bar = 20 μm.

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Figure 3. Immunofluorescence localization of partitioning defective (PAR) 3 and zonula occludens (ZO) -1 in preameloblasts. Cryosections were doubly stained with anti-PAR3 and anti–ZO-1 antibodies. Very weak signals for PAR3 are first detected at the proximal ends of early preameloblasts and gradually increase toward late preameloblasts. However, PAR3 is not detected at the distal ends of preameloblasts. Red signals for ZO-1 are first detected at the proximal ends of early preameloblasts, after a while at the distal ends (arrow), and later at both ends. An asterisk indicates the artificial gap between late preameloblasts and predentin/dentin. pAm, preameloblast. Scale bar = 20μm.

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Figure 4. Immunofluorescence localization of partitioning defective (PAR) 3 and zonula occludens (ZO) -1 in the secretory and transition ameloblasts. Cryosections were doubly stained with anti-PAR3 and anti–ZO-1 antibodies. Strong signals for PAR3 are detected at the proximal ends of early secretory, mid secretory, and transition ameloblasts, but not at the distal ends. Strong signals for ZO-1 are detected at both ends. Green signals for PAR3 are not perfectly colocalized with red signals for ZO-1 and PAR3 is localized slightly apical to ZO-1. sAm, secretory ameloblast; Am, ameloblast; SI, stratum intermedium. Upper short scale bar = 20 μm. Lower long scale bar = 10 μm.

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Figure 5. Immunofluorescence localization of partitioning defective (PAR) 3 and zonula occludens (ZO) -1 in maturation ameloblasts. Cryosections were doubly stained with anti-PAR3 and anti–ZO-1 antibodies. Green signals for PAR3 are basically detected at the proximal ends of maturation ameloblasts, including the RAs and SAs, and then they gradually decrease toward the late RAs. Very weak signals for PAR3 are also detected at the distal ends of the early RAs (arrowheads). Red signals for ZO-1 are detected at both ends in the early, mid, and late RAs, and only at the proximal ends of the mid SAs. RA, ruffle-ended ameloblast; SA, smooth-ended ameloblast; PL, papillary layer. Scale bar = 20 μm.

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Claudin-1

Claudin-1 was first detected at the proximal ends of early preameloblasts and eventually at the distal ends (Fig. 1). Staining for claudin-1 gradually increased toward the late preameloblasts (Fig. 1). In the secretory ameloblasts, intense staining for claudin-1 was detected at the distal ends and weak staining was detected at the proximal ends (Fig. 1). Weak staining for claudin-1 was detected at both ends of transition ameloblasts (Fig. 2). In RAs, negative to weak staining for claudin-1 was observed at the proximal ends and intense staining was detected at the distal ends (Fig. 2). In the SAs, negative to weak staining for claudin-1 was observed at the proximal ends and no staining was detected at the distal ends (Fig. 2).

Claudin-4

Claudin-4 was not detected in the preameloblasts and secretory ameloblasts (data not shown). Claudin-4 was first detected at both ends of the late transition ameloblasts (Fig. 2). Moderate staining for claudin-4 was observed at the distal ends of early and mid RAs. Weak staining was detected at the proximal ends of the RAs and SAs and at the distal ends of late RAs (Fig. 2). Staining for claudin-4 was not detected at the distal ends of the SAs (Fig. 2).

Occludin

Occludin was not detected in either the preameloblasts or secretory ameloblasts, but it was detected in the stratum intermedium, stellate reticulum, and papillary layer (Figs. 1, 2). Occludin was first detected at the proximal and distal ends of the transition ameloblasts (Fig. 2). Intense or weak staining for occludin was observed at the distal or proximal ends of RAs, respectively (Fig. 2). Occludin was negative at the distal ends of SAs but weak signals for occludin were detected at the proximal ends of SAs (Fig. 2).

PAR3

PAR3 was first detected at the proximal ends of early preameloblasts and gradually increased toward the late preameloblasts (Fig. 3). Intense staining for PAR3 was observed at the proximal ends of secretory and transition ameloblasts (Fig. 4). PAR3 was localized slightly apical to ZO-1 and not perfectly colocalized with ZO-1 (Fig. 4). Intense staining for PAR3 was detected at the proximal ends of early RAs, and thereafter, it gradually decreased with the development of the ameloblasts (Fig. 5). PAR3 was not detected at the distal ends of ameloblasts except for early RAs (Figs. 3–5).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The distribution and signal intensity of claudin-1 and -4, TJ-forming integral membrane proteins, in this study closely coincided with the development of TJs in the ameloblasts as previously examined by freeze-fracture electron microscopy. The distal TJs in the secretory ameloblasts and RAs are a well-developed zonula occludens which encircles each cell body, the proximal TJs in secretory ameloblasts and SAs are a moderately developed but incomplete zonular type, and other proximal and distal TJs in ameloblasts are basically a less-developed macula occludens (Sasaki et al.,1982,1983a,b,1990). Intense immunoreactivity for ZO-1 was constantly detected at both ends of ameloblasts, although development of the proximal TJs was generally insufficient. This phenomenon is probably due to the fact that ZO-1 can associate with not only TJ proteins such as occludin and claudins (Furuse et al.,1994; Itoh et al.,1999a) but also with adherens junction proteins such as cadherin/catenin complex (Itoh et al.,1997) or with gap junction proteins such as connexin (Cx) 43 (Giepmans and Moolenaar,1998; Toyofuku et al.,1998). Indeed, Cx43 and ZO-1 always coexisted in differentiating ameloblasts, thus suggesting the existence of a close relationship between the two proteins (João and Arana-Chavez,2003).

It has been proposed that each claudin isoform may form pores with a unique charge selectivity determined by charged amino acids in the first extracellular loop of each claudin in the TJ (Colegio et al.,2002; Van Itallie et al.,2003), thus suggesting that the overall physiological properties of the paracellular space may be determined by the combination and mixing ratios of claudin isoforms expressed in the cells. In occludin-deficient mice, the morphology of TJs and barrier function of intestinal epithelium were normal, but histological abnormalities were found in several tissue specimens (Saitou et al.,2000). In contrast, the second extracellular domain peptide of occludin reduced the amount of occludin localized at the TJ and reversibly disrupted the transepithelial permeability barrier (Wong and Gumbiner,1997). Therefore, the function of occludin is still obscure, but occludin may nevertheless modulate the TJ permeability in epithelial cells. Speculating from these results, the paracellular permeability of TJs at the distal ends of RAs may be different from that of secretory ameloblasts because of the difference in combinations of TJ proteins regulating paracellular permeability. This speculation is supported by the findings of a previous report that describe the distal TJ in RAs to be closed to calcium, whereas the proximal and distal TJs in secretory ameloblasts were not (Kawamoto and Shimizu,1997).

Calcium ions are provided from blood vessels to mineralizing enamel across the layer of ameloblasts through the paracellular pathway and/or transcellular pathway. 45Ca autoradiography of rat lower incisors with accurate control of exposure time showed that the proximal TJ in SAs and the distal TJ in RAs were closed to calcium, whereas the distal TJ in SAs, the proximal TJs in RAs, and the proximal and distal TJs in secretory ameloblasts were not (Kawamoto and Shimizu,1997). High radioactivity was detected in RAs and the time for calcium to diffuse across RAs was much longer than that across SAs and secretory ameloblasts. From these results, the authors concluded that calcium moves to mineralizing enamel through the paracellular pathway in SAs and secretory ameloblasts but through the transcellular pathway in RAs. Because claudin-4 formed pores in TJs reducing cation entry (Van Itallie et al.,2001,2003; Colegio et al.,2002), the distal TJ in RAs constituted by claudin-4 may restrict the calcium movement between the extracellular fluid and enamel matrix fluid and maintain concentration of calcium ions transported through cytoplasm of RAs. Although the overexpression of claudin-1 increased transepithelial electrical resistance in MDCK cells (Inai et al.,1999), the charge selectivity of the pores formed by claudin-1 has not yet been determined. Therefore, it is premature to discuss the function of claudin-1 in ameloblasts.

The PAR3/PAR6/aPKC complex localizes at TJs (Macara,2004; Ohno,2001) and it also plays a role in the formation or maintenance of TJs (Joberty et al.,2000). The number of TJ strands was higher in the distal rather than proximal renal tubules, but there was no significant difference in the signal intensity for PAR3 between the proximal and distal renal tubules (Hirose et al.,2002). These results suggest that the expression level of PAR3 may not be related to the development of TJ. Therefore, it is not surprising that PAR3 was consistently localized at the proximal ends of ameloblasts where less-developed TJs were usually observed. Double immunogold labeling of the distal renal tubules for PAR3 and ZO-1 showed that PAR3 was detected in the cytoplasm located at the apical edge of the TJ, but ZO-1 distributed alongside the TJ slightly basal to PAR3 (Hirose et al.,2002). This result is consistent with the present result; PAR3 was localized slightly apical to ZO-1 in the proximal TJ of secretory ameloblasts (Fig. 4). The present result raises a new question as to why TJs are formed at the distal ends of ameloblasts without PAR3.

In summary, the proximal and distal TJs in ameloblasts differentially expressed TJ proteins including ZO-1, claudin-1, claudin-4, and occludin during ameloblast development. In particular, the distal TJ of secretory ameloblasts consisted of claudin-1, while that of the RAs consisted of claudin-1, claudin-4, and occludin. Therefore, the distal TJs in secretory ameloblasts and RAs may differently regulate the paracellular permeability against the enamel surface to perform their functions appropriately; namely, enamel formation and enamel maturation, respectively. In addition, the consistent localization of PAR3 at the proximal TJ during ameloblast development suggests that the proximal, but not distal, TJ in the ameloblasts may be formed or maintained by the PAR3/PAR6/aPKC complex.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED