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Journal of Dental Research
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Clinical

The Effect of LRAP on Enamel Organ Epithelial Cell Differentiation

T.Q. Le*, Y. Zhang, W. Li and P.K. DenBesten

Department of Orofacial Sciences, University of California at San Francisco, 513 Parnassus Avenue, P.O. Box #0422; San Francisco, CA 94143–0422, USA

Correspondence: * corresponding author, Thuan.Le{at}ucsf.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leucine-rich amelogenin peptide (LRAP) is an alternatively spliced amelogenin found in the developing enamel organ. LRAP functions to regulate the development of mesenchymal-derived cells; however, its effect on cells of the enamel organ remains unclear. The hypothesis tested in this study is that LRAP also regulates human enamel organ epithelial cells. Recombinant human LRAP (rH58) was synthesized in E. coli, purified, and exogenously added to cultures of human primary enamel epithelial cells, which were analyzed for changes in cell proliferation and differentiation. rH58 had no effect on cell proliferation, but altered enamel epithelial cell morphology, resulting in larger, more rounded cells. Immunofluorescence showed that rH58 treatment increased amelogenin synthesis, but down-regulated Notch1 expression in enamel epithelial cells. LAMP-1, a membrane receptor for LRAP in mesenchymal cells, was identified and was up-regulated in the presence of rH58. These results suggest that rH58 promotes differentiation of human enamel organ epithelial cells.

Key Words: leucine-rich amelogenin peptide (LRAP) • rH58 • alternative splicing • cell differentiation • enamel organ epithelial cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leucine-rich amelogenin peptide (LRAP, [A-4] or rH58) was first isolated and characterized as one of the principal components of lower-molecular-weight amelogenins (~5–6 kDa) in the secretory enamel matrix proteins (Fincham et al., 1981). The peptide was generated by alternative splicing of the amelogenin pre-mRNA (Gibson et al., 1991). LRAP is identical to the full-length amelogenin at its amino and carboxyl termini, but it lacks a majority of the exon-6-coded segment found in the central region of the full-length protein. LRAP or [A-4] has been shown to promote chondrogenesis and osteogenesis (Veis et al., 2000; Tompkins and Veis, 2002; Veis, 2003; Tompkins et al., 2005), and to stimulate dental pulp cell proliferation in vitro (Ye et al., 2006), suggesting a role in mesenchymal cell signaling. The precise function of LRAP in enamel formation is still unknown.

Cell-surface receptors of LRAP have been recently identified and characterized in mesenchymal-derived mouse fetal myoblasts, as lysosome-associated membrane protein 1 (LAMP-1) (Tompkins et al., 2006). LAMP-1 is also associated with differentiation of mouse epithelial-derived mammary cells (Cella et al., 1996). In addition, up-regulated LAMP-1 expression in human keratinocytes is strongly correlated with cell differentiation (Sarafian et al., 2006).

Notch1 signaling has been shown to promote proliferation of human epithelial-derived cells (Zagouras et al., 1995; Gray et al., 1999). Interestingly, Harada and co-workers have shown that Notch1 maintains dental epithelial stem cells in the cervical loop of the continuously growing mouse incisor (Harada et al., 1999; Harada and Ohshima, 2004). However, these authors suggest that Jagged1-induced Notch1 activation, which was detected in the stratum intermedium of rat incisors, was also responsible for differentiation of an immortalized dental epithelial progenitor cell line (HAT-7) into stratum intermedium (Harada et al., 2006).

In this study, we investigated the role of LRAP in enamel organ epithelial cell proliferation and differentiation. We used LAMP-1 and Notch1 to track changes in the differentiation of enamel organ epithelial cells, following exposure of these cells to recombinant human LRAP (rH58). This study provides new insights into the effect of rH58 on the synthesis of proteins that control the process of dental epithelial cell differentiation, regulating tooth enamel formation.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Expression, Purification, and Characterization of Recombinant Human LRAP (rH58)
Expression and purification of rH58 was conducted according to methods previously published (Le et al., 2006) (see APPENDIX). The purified rH58 was characterized by SDS-PAGE, Western blot, and mass spectrometry (see APPENDIX Fig., A and B).

Isolation of Epithelial Cells from Human Enamel Organs
Enamel organ epithelial cells (including ameloblast-lineage cells) were isolated from human fetal cadaver tissue as previously described (DenBesten et al., 2005; Yan et al., 2006; Zhang et al., 2006) (see APPENDIX).

Effect of rH58 on Enamel Organ Epithelial Cell Proliferation
Enamel organ epithelial cells were grown at 37°C in 5% CO2 in KGM-2 media (Cambrex, Walkersville, MD, USA) to a density of 2.5 x 103 cells/well in clear-bottomed black 96-well plates. At 60% confluence, cells were synchronized in keratinocyte basal medium 2 (KBM-2) (Cambrex) for 24 hrs, and then rH58 was added to triplicate cultures at various final concentrations (0, 30, 90, 180 nM). Cells were maintained in this medium for another 24 hrs, and cell proliferation was measured by means of an ELISA BrdU-labeling kit (Roche, Indianapolis, IN, USA), according to the manufacturer’s instructions. Statistical analyses between groups were performed by ANOVA with Tukey’s post-test analysis, with GraphPad Prism version 4.0a software (GraphPad Software, San Diego, CA, USA).

Localization of LAMP-1 in Human Enamel Organ Epithelium
Tooth organs obtained from 21-week-old human fetal tissue were embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Hatfield, PA, USA) within 3 hrs of collection, frozen in a mixture of dry ice/2-methylbutane, and cryosectioned for immunocytochemistry. Tissue collection was conducted according to guidelines and approval of the University of California-San Francisco committee on human research.

Frozen tissue sections were fixed with a mixture of 5% acetic acid and 95% methanol for 30 min at –20°C. Non-specific binding sites were blocked by the incubation of samples with 10% horse serum for 1 hr, followed by incubation for 1 hr at room temperature with rabbit anti-human LAMP-1 (1/500 dilution) (Abcam, Cambridge, MA, USA), or anti-human Notch1 separately as a primary antibody (1/100 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). In addition, human enamel organ tissue sections were stained with primary antibodies of rabbit anti-human cytokeratin 14 (1/200 dilution) (Chemicon, Temecula, CA, USA) or pre-immune rabbit IgG (1/200 dilution) (Sigma, St. Louis, MO, USA) as a positive or negative control, respectively. After being washed for the removal of unbound antibodies, the samples were incubated for another 1 hr in the dark with fluorescence-conjugated anti-rabbit IgG-FITC (1/500 dilution) (Sigma). The slides were then washed with PBS, and cell nuclei were counterstained with 1 µg/mL bis-benzimide or Hoechst dye (Molecular Probes, Eugene, OR, USA) for 5 min. After the unbound dyes were removed with PBS, these slides were mounted with SlowFade anti-fading agent (Molecular Probes), observed under a Nikon Eclipse E800 fluorescent microscope, and photographed with SimplePCI Version 5.3.1 software (Compix, Cranberry Township, PA, USA).

RT- PCR for LAMP-1 Expression in Enamel Organ Epithelial Cells
Human fetal primary enamel organ epithelial cells were plated in 100-mm Primaria culture dishes (Becton Dickinson Labware, Franklin Lakes, NJ, USA). At 60% confluence, total RNA was isolated from the cultures with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The purified total RNA (3 µg) was used for reverse-transcription (RT) with an oligo-dT primer, according to the SuperScript II RT kit (Invitrogen, Carlsbad, CA, USA), followed by PCR in 50-µL reaction tubes: 30 µ L H2O, 5 µL 10X PCR buffer, 1.5 µL of 50 mM MgCl2, 1 µL of 10 mM dNTP, 0.5 µL Taq polymerase, 2 µL DNA template, 5 µL sense primer, and 5 µL anti-sense primer. A pair of primers (sense 5'-CCTCATCGTCCTCATCGCCTA-3' and anti-sense 5'-CTCAGAGACAGCGGCATTCCA-3') was used for amplification of a segment of LAMP-1 cDNA, resulting in a 513-bp PCR product. Human enamel organ cDNA library (2 µL) was used as a positive control, while a 2-µL quantity of dH2O was used as a negative control. PCR reactions were incubated in an Eppendorf Mastercycler cycler (Eppendorf, New York, NY, USA). The cycles for PCR were 1 cycle at 94°C for 3 min, 34 cycles of 94°C for 30 sec, 65°C for 30 sec, and 72°C for 2 min. The final extension time was 7 min at 72°C. After amplification, the reactions were analyzed by 1% agarose gel electrophoresis. PCR products were subjected to DNA sequencing to reconfirm the identity of the amplified products (data not shown).

Characterization of Enamel Organ Epithelial Cells Cultured with rH58
Enamel organ epithelial cells were grown on glass chamber slides (Lab-Tek, Naperville, IL, USA) in KGM-2 media (Cambrex). At 60% confluence, cells were synchronized in KBM-2 media (Cambrex) for 16 hrs, followed by the addition of rH58 to a final concentration of 180 nM. This concentration of rH58 resulted in easily detectable changes in cell phenotype after 24-hour incubation, and thus, was used for subsequent experiments. Cells without rH58 treatment were used as controls. The protein-treated and non-treated control cells were incubated for another 24 hrs at 37°C. Cells were immunofluorescent-stained with rabbit anti-human antibodies against LAMP-1 (1/500 dilution) (Abcam), Notch1 (1/100 dilution) (Santa Cruz Biotechnology), or amelogenin (1/1000 dilution). Human amelogenin antibody was purified from serum of a rabbit immunized with recombinant human full-length amelogenin by Protein A affinity chromatography. In the negative control, enamel epithelial cells were stained by pre-immune rabbit IgG at 1/1000 dilution.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
No Effect of rH58 on Enamel Organ Epithelial Cell Proliferation
Exposure of enamel organ epithelial cells to different concentrations of rH58 had no significant effect on cell proliferation, as measured by BrdU incorporation into DNA contents of rH58-treated and untreated control cells (p > 0.05, N = 3) (Fig. 1Go).


Figure 1
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  		Figure 1. The effect of rH58 on enamel organ epithelial cell proliferation. Exogenous rH58 at various concentrations added to enamel organ epithelial cell cultures had no significant effect on cell proliferation, as measured by an ELISA BrdU-labeling cell proliferation assay (p > 0.05, N = 3). The average RFU ± SD values obtained for cell treatments of rH58 at 0 nM, 30 nM, 90 nM, and 180 nM were 162.8 ± 9.4, 194.3 ± 18.1, 214.4 ± 61.4, and 216.6 ± 12.8, respectively.

 
LAMP-1 Expression in Human Tooth Stratum Intermedium
Immunofluorescent staining of tooth enamel organ showed positive reactivity for LAMP-1 localized in the stratum intermedium and stellate reticulum (Fig. 2AGo). However, differentiated ameloblasts did not show any detectable LAMP-1 staining (Fig. 2AGo). Additional immuno-fluorescent staining of the enamel organ sections showed that Notch1 was weakly positive in the stellate reticulum, while it was hardly detectable in the stratum intermedium and differentiated ameloblasts (Fig. 2BGo). In the positive control, strong positive immunostaining for cytokeratin-14 was observed in the stratum intermedium and stellate reticulum, with weaker positive staining detected in the ameloblasts (Fig. 2CGo). In the negative control, the human tooth enamel organ epithelium showed no staining with the pre-immune IgG (Fig. 2DGo).


Figure 2
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  		Figure 2. Detection of LAMP-1 in the stratum intermedium of human enamel organ. (A) Immunofluorescent staining of LAMP-1 showed a strong positive staining for LAMP-1 in the stratum intermedium (SI) and stellate reticulum (SR) of a frozen section of a human enamel organ, while immunostaining for LAMP-1 in ameloblasts (Am) was not detectable. (B) Immunostaining of the human enamel organ showed that Notch1 was weakly positive in the SR, while it was hardly detectable in the SI and differentiated Am. (C) The positive control showed strong immunostaining for cytokeratin-14 in the SI and SR, but weaker staining in the Am. (D) The negative control stained with the pre-immune IgG was immunofluorescent-negative. Cell nuclei were counterstained with Hoechst dye (blue).

 
Expression and Immunostaining of LAMP-1 in Human Enamel Epithelial Cell Cultures
LAMP-1 mRNA expression was detected in enamel organ epithelial cells by reverse-transcription PCR, similar to the positive (+) control with the tooth enamel organ epithelium cDNA library (Fig. 3AGo). No amplified PCR product was detected in the negative (–) control. Immunofluorescent analysis of enamel organ epithelial cells in vitro showed a weak positive staining for LAMP-1 (Fig. 3BGo). In the negative control, these epithelial cells, incubated with pre-immune IgG, were immunonegative (Fig. 3CGo).


Figure 3
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  		Figure 3. Expression and immunostaining of LAMP-1 in human enamel organ epithelial cell cultures. (A) Reverse-transcription (RT)-PCR product showed positive LAMP-1 mRNA expression in human enamel organ epithelial (EOE) cells, indicated by a 513-bp band in 1% gel. Human enamel organ cDNA library was used as a positive (+) control, while 2 µL of sterile dH2O was used as a negative (–) control. (B) Immunofluorescence of enamel epithelial cells in vitro showed a weak positive staining for LAMP-1. (C) Pre-immune IgG staining of enamel epithelial cells showed immunonegativity for LAMP-1.

 
rH58 Treatment Promoted Enamel Epithelial Cell Differentiation by Up-regulating LAMP-1 and Amelogenin, while Down-regulating Notch1
Treatment of enamel organ epithelial cells with rH58 resulted in a change in cell morphology. Cells became more rounded and larger (Fig. 4AGo), as compared with the smaller, cobblestone-like shapes of the untreated control cells (Fig. 4BGo).


Figure 4
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  		Figure 4. Characterization of rH58-treated enamel organ epithelial cells. Phase-contrast images showed that, after treatment with rH58 (A), enamel organ epithelial cells became rounded and larger, while the untreated control cells (B) retained their small, cobblestone-like shapes. Immunofluorescent results indicated strong positive staining for LAMP-1 (C) and amelogenin (E), but decreased immunostaining for Notch1 (G) in rH58-treated epithelial cells. The untreated control cells were stained weakly for LAMP-1 (D) and were immuno-negative for amelogenin (F), but were immunopositive for Notch1 (H). Cell nuclei were counterstained with Hoechst dye (blue). Bars = 50 µM.

 
Immunofluorescent staining showed a strong increase of LAMP-1 reactivity after the cells were incubated with rH58 (Fig. 4CGo), while the untreated cells showed weak positive staining for LAMP-1 (Fig. 4DGo) Amelogenin immunostaining was also up-regulated with rH58 treatment (Fig. 4EGo); however, the control untreated cells were not immunoreactive for amelogenin (Fig. 4FGo). In contrast, rH58 treatment resulted in immunonegative staining for Notch1 (Fig. 4GGo), as compared with positive immunoreactivity for Notch1 detected in the untreated control cells (Fig. 4HGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we showed positive expression of LAMP-1 at both mRNA and protein levels in human enamel organ epithelial cells in vitro. In vivo, the most intense LAMP-1 staining was localized in the stratum intermedium of the developing human enamel organ. This finding is in agreement with the results from a study by Tompkins and co-workers, who used "Far Western" immunohistochemistry with biotin-labeled mouse LRAP or [A-4] as a primary ligand to bind receptor LAMP-1 in embryonic day 18 (E18) mouse incisors, suggesting that LAMP-1 immunostaining was most prominent in the stratum intermedium (Tompkins et al., 2006). In addition, weaker immunopositive staining for LAMP-1 in mouse polarized ameloblasts was gradually reduced as ameloblasts and odontoblasts became separated by mineralizing dentin, suggesting that LAMP-1 was down-regulated in more mature, differentiated mouse ameloblasts (Tompkins et al., 2006). We did not detect LAMP-1 immunofluorescent staining in ameloblasts of human enamel organ sections, reconfirming down-regulation of LAMP-1 in mature ameloblasts. However, differences in LAMP-1 localization in our study and that of Tompkins and co-workers (Tompkins et al., 2006) may be related to different stages of ameloblast differentiation, or to species differences (mouse vs. human) in these model systems.

LAMP-1 has been shown to be a receptor of LRAP or [A-4] in mesenchymal-derived mouse fetal myoblasts (Tompkins et al., 2006). In this study, we found that recombinant human LRAP (rH58) up-regulated LAMP-1 to promote differentiation of enamel epithelial cells into the stratum intermedium. These rH58-treated cells appeared to be more rounded and larger, in comparison with the smaller, cobblestone-shaped, untreated control cells. Similar to our results, up-regulation of LAMP-1 expression in epithelial-derived mammary cells and keratinocytes was associated with cell differentiation (Cella et al., 1996; Sarafian et al., 2006). Thus, LAMP-1, which is up-regulated as epithelial cells differentiate, may have a role in mediating the differentiation of epithelial cells in the developing human enamel organ.

LRAP secreted by the stratum intermedium cells could interact with the LAMP-1 receptor in an autocrine manner to maintain stratum intermedium differentiation from the undifferentiated early-forming dental epithelium. Clearly, further studies are needed to understand the roles of LRAP and LAMP-1 in the differentiation of ameloblasts, in the stratum intermedium, and in enamel organ development.

Notch1 functions in the maintenance of undifferentiated epithelial stem cells (Harada et al., 1999). A strong expression of Notch1 at both mRNA and protein levels has been reported in dental epithelial stem cells, located in the cervical loop of the mouse incisor, which includes stellate reticulum (Harada et al., 1999; Harada and Ohshima, 2004). These studies suggest that Notch1 may be down-regulated as dental epithelial stem cells differentiate. Immunofluorescent staining of the 21-week-old human enamel organ in our study showed positive reactivity for Notch1 in the stellate reticulum, but not in the stratum intermedium. Moreover, in vitro immunostaining showed that enamel epithelial cells express Notch1, suggesting that these are young and undifferentiated epithelial cells. However, rH58 treatment resulted in down-regulation of Notch1 signal, supporting the evidence that rH58 promotes differentiation of enamel organ epithelial cells into stratum-intermedium-like cells.

Harada et al. suggested that Notch1 activation promotes dental epithelial cell differentiation into the stratum intermedium lineage (Harada et al., 2006). However, our results did not indicate a role for Notch1 signaling in the differentiation of the stratum intermedium. The discrepancy between these two studies is not clear, but may be related to different species (mouse vs. human) or tooth type (incisor vs. molar).

The enamel epithelial cells were mostly immunonegative for amelogenins, again supporting their early lineage. However, after rH58 treatment, LAMP-1 and amelogenin were up-regulated in subpopulations of these cells. Expression patterns and tissue distribution of amelogenins have shown that amelogenins are present in the stratum intermedium (Papagerakis et al., 2005; Iacob and Veis, 2006). In view of these results, we suggest that enamel organ epithelial cells can differentiate to stratum intermedium in the presence of rH58.

In conclusion, this study showed that rH58 up-regulates LAMP-1 receptors, and promotes tooth organ epithelial cells to differentiate into stratum intermedium in vitro. These results suggest that, rather than a structural role in enamel formation, rH58 signals early enamel epithelial cell specification and differentiation.


   ACKNOWLEDGMENTS
 
This research was supported by research grants T32-DE07306–09, P01-DE009859, and R01-DE015821 from the National Institute of Dental and Craniofacial Research, Bethesda, MD 20892, USA.


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received for publication November 21, 2006. Revision received June 8, 2007. Accepted for publication July 10, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Journal of Dental Research, Vol. 86, No. 11, 1095-1099 (2007)
DOI: 10.1177/154405910708601114
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