Egyptian Dental Journal (2006) 52, 1399-1407

Immunohistochemical Detection of Cytokeratin 14 in

Developing Enamel Organ of Rat Molars

Hisham I. Osman*+ and Azza S. Kora**

* Assistant professor of oral biology, faculty of dentistry, Alexandria University ** Lecturer of oral biology, faculty of dentistry, Alexandria University

Abstract Cytokeratins (CKs) are cytoskeletal intermediate filament proteins characteristic of odontogenic epithelia. The CK expression patterns of odontogenic epithelia are still poorly described. Most studies have searched for pools of CKs instead of individual polypeptides. The objective of this study was to clarify the immunohistochemical expression of individual CK polypeptides 14 in the enamel organ of rat molar at different developmental stages using monoclonal antibody LL002- Cks14.The results showed that all cells of the enamel organ were positive for CK14 and its configuration showed differences related to the stage-specific state of differentiation. A strong label for CK 14 was present at the inner dental epithelium at early bell stage, preameloblasts and amelobalsts It is conclude that the monoclonal antibodies to CK 14 are well documented and should serve as useful tools for tracing the development and differentiation of ameloblasts. The developing enamel organ may be a suitable model for investigating the relationship between keratin expression and cell function. +Corresponding author. Tel.: +996502105253 E-mail address: hiosman2005@yahoo.com Introduction Dental epithelial cells in tooth buds differentiate into various cell types during development of tooth germs, inner enamel epithelium, stratum intermedium, stellate reticulum, and outer enamel epithelium. The inner enamel epithelial cells subsequently

differentiate into ameloblasts, which then form enamel. The

mechanisms responsible for this

cellular differentiation are not completely understood (1). The cytoskeleton of the most cells is structured by scaffolding composed of actin microfilaments, microtubules, and intermediate

filaments. Intermediate filaments, so named because their 10-nanometer diameter is intermediate between that of microfilaments (6 nanometers) and microtubules (23 nanometers), assemble into an

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anastomosed network within the cytoplasm. They provide flexible

intracellular scaffolding whose function is to structure cytoplasm

and to resist stresses externally applied to the cell. Mutations that weaken this structural framework increase the risk of cell rupture and cause a variety of human disorders

(2). The intermediate filaments are a large multigene family of fibrous protein that includes cytokeratins, which are characteristic of epithelia and vimentin which is typical of cells of mesenchymal origin. The dental lamina and enamel organ are formed by epithelial cells which are characterized by the presence of specific sets of cytokeratins (CK) (3). Cytokeratins are a subfamily of intermediate filament proteins and are characterized by remarkable biochemical diversity, represented in epithelial tissues by at least 20 different polypeptides. They range in molecular weight between 40 kDa and 68 kDa and isoelectric pH (4.9 – 7.8). The individual cytokeratin polypeptides are designated 1 to 20. Cytokeratin 1 has the highest molecular weight and the highest isoelectric pH, while cytokeratin 19 has the lowest molecular weight and a low isoelectric pH. The cytokeratins are divided into the type I and type II subgroups, the type II family members

comprising the basic to neutral cytokeratins 1-8, while the type I group comprises the acidic cytokeratins 9-20.. Thus, CK polypeptides are markers for tissue-specific and stage-specific states of differentiation. Knowledge of CK expression patterns in dental epithelia is useful not only for cell typing and monitoring the progression of epithelial cell differentiation, but also for understanding odontogenic tumor histogenesis and contributing to tumor classification (4). The pattern of CK expression within a particular epithelium varies with several parameters: cell type, developmental stage (5), state of differentiation (6), anatomical location (7), and degree of complexity (8).

Recent progress in understanding the biology of keratins, together with the development of monoclonal antibodies to individual keratin proteins, provides the foundation for studying keratin expression in normal and pathological oral epithelia. The expression of CKs is closely linked to epithelium differentiation, particularly during embryogenesis. They are useful as epithelial marker proteins because of their high abundance, stability and antigenicity. The pattern of CK expression within a particular

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epithelium varies with its anatomical location, developmental stage and state of differentiation. Epithelia can therefore be characterized by the specific pattern of polypeptides they express (9).

Different CKs have been shown to characterize epithelial type: In keratinized stratified epithelia, such as the epidermis, CKs 1, 2, 5, 10, 11, and 14 are expressed, whereas in nonkeratinized stratified epithelia, such as the oesophageal epithelium, CKs 4, 5, 13, 14 and 19 are found. The relatively low molecular-weight CKs (CKs 7, 8, 18, 19 and 20) have been identified in simple-type epithelia, including glandular (9), odontogenic (10,11) and intestinal tissues (12). Site-related differences have also been reported. CKs 2, 6, and 16 characterize the keratinized stratified squamous epithelium of the hard palate. CK 13 is characteristic of the foreskin, whereas CK 15 marks the skin of the sole (13, 14).

Cytokeratin 14, a member of the family of acidic type I cytokeratins, consists of a conserved rod domain with four _-

helical regions separated by short non-helical linker sequences and flanked by non-helical globular sequences commonly referred to as head (N-terminal) and tail (C-terminal) domains. Self-assembly of CK14 leads to formation of intermediate filaments of 10 nm diameter (15). Only limited information is available on the cytokeratins of the enamel organ epithelium. Immunohistochemical studies on keratin expression in enamel organs during development have yielded rather variable and, in some cases, contradictory results (16, 17). Most studies have searched for pools of CKs instead of individual polypeptides. There is, thus, a lack in the characterization of individual CK expression in dental epithelia. The aim of this work was to analyze the distribution of individual CK polypeptides 14 in odontogenic epithelia. Knowing that the tooth development has been divided into three distinct phases; initiation, morphogenesis and cell differentiation.

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Material and methods Ten normal, healthy, female pregnant rats were used in this study .Developing mandibles were carefully dissected from the offsprings including lower first molar regions buds from embryonic day 10 to postnatal day 7 so that to cover the period of embryonic and postnatal growth of first molar development and were fixed in 4% paraformaldehyde in phosphate-buffered saline. Tissues from postnatal rats were decalcified in 10% EDTA for about 3 weeks. Paraffin stained sections H&E (5 um) were prepared for routine histological examination. The tooth germs were classified into bud stage, cap stage, bell stage and initial dental hard tissues formation. Immunohistochemistry An indirect streptavidin-biotin immunohistochemical technique (18) was used to detect the monoclonal mouse anti-human cytokeratin 14 (clone LL002)*. This monoclonal antibody reacts with the acidic intermediate filament protein (50kD) identified as cytokeratin 14 (CK14). Counterstaining was performed using Meyer's haematoxylin for one minute. For the negative controls the primary antibody was

omitted and goat antiserum antibody was substituted Staining Assessment Cells were considered immunochemically positive for anti cytokeratin 14 when distinct, reddish-brown, intracytoplasmic staining was identified. The staining intensity in cytokeratin cases was categorized as follows: - = Negative, no staining reaction + = slightly positive, densely stained reaction ++ = strongly positive, faintly stained cytoplasmic reaction The collected data were tabulated for evaluation of staining of cytokeratin at the different developmental stage of tooth formation * DAKO, Copenhagen, Denmark

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Results Positive reaction for monoclonal anti CK 14 in the stained sections among the epithelial cells of the developing enamel organ displayed a widely distributed temporospatial pattern. Variations in staining intensity were verified among cells of different locations during development of rat enamel organ shown in table 1. A closer analysis of the CK14 expression in the various stages of tooth development revealed the following: Initiation of tooth development and bud stage At the early tooth formation, no immunoreactivity CK14 could be detected at the epithelial thickenings or in nearby tissues (fig.1). As histodifferentiation proceeds, at early bud stage of developing molar enamel organ, positive reactivity could only be seen in the stellate reticulum (fig.2). None of the other cells of the enamel organ were reacting positively at this stage of development. No immunoreactivity was detected in the ectomesenchymal tissues around the dental epithelium. At bud stage, the streak of staining cells extended to the epithelial bud (fig.3).

Early bell stage During bell stage, cuspal morphogenesis continues and odontoblasts as well as ameloblasts undergo terminal differentiation. As at earlier stages, the distribution of immunohistochemical staining of CK 14 was temporally and spatially regulated. At this stage, Ck14 was located in the epithelial cells of the dental lamina and outer enamel epithelium. The inner enamel epithelium revealed strong positively for CK 14. (fig.4). Remnants of the dental lamina showed an intense positive reaction The stellate reticulum exhibited a weak immunoreactions for cytokeratin ( fig 5) Late bell stage With terminal differentiation of the amelobalsts from the inner enamel epithelium, more extensive staining of the enamel organ was observed. There was intense staining of the stratum intermedium (stronger than the stellate reticulum) and weak or no staining reaction of cytokeratin in the inner enamel epithelium (fig.6). Cervical loop Immunoreactivity for CK14 was observed among the progenitor cells of the cervical loop especially

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within the outer and inner enamel epithelium (fig.7) Amelogenesis in developing molars With the beginning of the process of enamel formation, the preameloblasts, ameloblast cell

layer and stratum intermedium exhibited a stronger positive reaction of CK14 in comparison to the rest of the cell layer (fig.8, 9). After complete enamel formation, the reduced enamel epithelium showed a relatively fainted ck14 staining (fig.10).

Fig.1 Molar tooth germ at bud stage stained with CK 14. No immunoreactivity of CK14 is detected either in all cell layers of the enamel

organ or in the surrounding tissues X 200

Fig.2 Bud stage of tooth germ. Staining reaction extend from the oral surface epithelium to the central part of the budding dental epithelium (stellate reticulum) indicating a strongly positive reaction for CK14. The outer

epithelial layer shows relatively weak staining X 400

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Fig.3 Further growth of the enamel organ. The enamel knot and the inner enamel epithelium showing marked

immunoreactivity of CK14 X 400.

Fig.4 Early bell stage of molar tooth germ. All epithelial layers exhibited positive staining reaction. Note the strongly

positive stain CK 14 of the inner enamel epithelium X 200.

Fig.5 Bell stage of tooth development ,weak Ck 14 reaction of the stellate

reticulum

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Fig. 6 Late bell stage of molar tooth germ showing terminal differentiation of the ameloblasts with absence of staining reaction of the inner enamel epithelium. The stratum intermedium displays high expression of the CK14

Fig.7 Positive immunoreactivity of CK14 at the cervical loop of the developing enamel organ

Fig. 8 Positive reactivity of the ameloblast cell layer and stratum intermedium. Note the relatively staining intensity of the stellate reticulum

and outer enamel epithelium. X 200

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Fig. 9 Strong positive CK 14 staining of the ameloblasts X 400

Fig. 10 Uniform positive CK 14 of the reduced enamel epithelium. Remnants of dental lamina show weak immunoreactivity. Note the presence

of positive cytokeratin staining in the covering epithelium indicating the presence of cytokeratin X 100

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Table 1: Expression pattern of CK14 among the developmental different stage of rat molar enamel organ

Enamel Organ Early bell

stage Late bell stage Developing Tooth Bud

stage Cap

stage

IEE

St. R

OEE

IEE

St.I

St. R

OEE

Pre -amelobl

asts

Amelobalsts

Cervical

loop

REE

CK 14

+ ++ ++ - + - ++ + + ++ ++ ++ +

- = Negative, + = slightly positive ++ = strongly positive IEE; Inner enamel epithelium, OEE; Outer enamel epithelium, St. R; stellate reticulum, St I; Stratum intermedium, REE; Reduced enamel epithelium

Discussion Cells of the enamel organ express keratin proteins typically associated with stratified epithelium. However, immunostaining patterns using specific antikeratin antibodies change with development and tooth eruption. These results demonstrate a relationship between keratin expression and morphologic changes as ameloblasts differentiate to squamous cells of the reduced enamel epithelium. Changes in keratin expression have been previously documented in the epidermis during fetal development and in normal differentiation of epidermal cells

(19). In the present study, it may be possible to relate changes in keratin staining with the dramatic changes in the enamel organ ,ameloblast differentiation and formation of reduced enamel epithelium. CK is a filament that plays a central role in epithelial tissue and, like the polypeptides of intermediate filaments in general, shows a high degree of tissue specificity. The pattern of CK distribution in the dental epithelium during tooth formation is very complex (20). The differential reactivity of the various cell-layers of the enamel

organ during development observed in this study indicates a temporo-spatial programme of keratin expression during odontogenesis. In this work, one type of cytokeratin was used instated of using several types of cytokeratins and this provides a more clear results about the temporarily changes that occur in the individual cell layers of the enamel organ during development This was in agreement with Leoset et al.(17) and they added that the use of monoclonal antibodies has allowed detection of more changes in the reactivity of the stellate reticulum and stratum intermedium than those seen with polyclonal keratin antibodies. Cytokeratin 14 (molecular weight 50 DK), an acidic (type I) cytokeratin protein, is one of the cytokeratin pair (50/58 KD) that distinguish stratified epithelial types from simple epithelial types. It is a homogenously expressed in all cells of the keratinizing squamous epithelium and is confined to the basal and parabasal cells in the nonkeratinizing squamous epithelium of the normal adult urinary tract (21) Cytokeratin 14 is an anchorage protein capable of preserving the mechanical integrity of epithelial cells (22). In the present work, the immunohistochemical study

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showed that the epithelial cells of enamel organ and dental lamina expressed CKs 14 with slight changes in their pattern during the differentiation phase of odontogenesis. The most striking finding was the presence of strong label for CK 14 in the inner enamel epithelium at early bell stage, which was changed into minimal amount indicated by weak label at the late bell stage when the ameloblasts were fully differentiated. Domingues et al. (23), were in agreement with the previous result and stated that at the early bell stage, the inner enamel epithelium and the underlying mesenchymal cells should be well connected to interact for developing morphogenetic functions. Thus, the presence of CK 14 at this stage might have a role in preserving and supporting the epithelial- mesenchymal interactions. On the other hand, concurrently with dentin formation, the ameloblast to be functional must be loosely connected to the mesenchyme to migrate and gain space for depositing the enamel matrix. For achieving this, it might be speculated that the ameloblast needs to lose CK 14 to fully disengage its anchorage. Crivelin et al. (24) supported the previous statement and added that CK14 as the main intermediate filament of odontogenic epithelium, which

was observed in the dental lamina, in the reduced enamel epithelium and in almost all cells of the enamel organ. Its absence in regions of advanced amelogenesis may suggest loss of CK14 as a consequence of the cellular secretory activity. In the present study, during enamel formation, the ameloblasts exhibited pronounced accumulation of CK14. Similar dynamics has earlier been observed with E-cadherin, a cell adhesion molecule that is accumulated during the terminal differentiation of ameloblasts (25). This terminal differentiation of ameloblasts requires the integrity of the cytoskeleton. Actin has been shown to redistribute and accumulate at the apical poles of ameloblasts. E-cadherin has been suggested to be involved in local accumulation of proteins during polarization of epithelial cells (26).Other molecules have also been involved in this process, such as BMPs 4, 5 and 7 (27). It may be concluded that CK 14 could be regarded as a marker of ameloblast differentiation. In another study done by Tabata et al.,(9) they were in accordance with previous conclusion and stated that CK14 and amelogenin as ameloblast-differentiation markers, while amelogenin is the postnatal differentiation marker of

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ameloblasts, the presence of CK14 in ameloblasts 48–96 h earlier than the expression of amelogenin indicates that CK14 is a stage-specific differentiation marker for ameloblasts. The expression pattern of CKs 14 in early bell stage cells was mostly similar to those of the late bell stage, except for the staining of inner enamel epithelium. This result was similar to those of previous reports (28,16), except for the labelling of some individual CK polypeptides, such as CK 16 and CK 17, the presence of which was behind the scope of this research. During differentiation of the inner enamel epithelium to ameloblasts, a change of the cell polarity occurs. This previous process is confirmed in this study by the accumulation of strong positive staining of CK14 at both ends of the ameloblasts. The ameloblast secretes enamel matrix during the enamel apposition phase, and then undergoes morphologic and functional changes into a short, relatively cuboidal cell, the reduced ameloblast. These cells form part of the reduced enamel epithelium (REE), a stratified integument which protects enamel from the adjacent connective tissue. During tooth eruption, the REE fuses with oral epithelium to

provide the primary epithelial attachment to enamel. At time of tooth eruption, the reduced ameloblast differentiates into a typical squamous junctional epithelial cell. Thus, the enamel organ is an unusual stratified epithelium which undergoes many functional changes during development, including functions typically associated with simple or glandular epithelia.(29-31) In this study, a positively strong immune clarified the formation of the REE by using staining the monoclonal antibody cytokeratin as a marker Several problems can limit the interpretation of antikeratin immunohistochemical staining data. (32) We used a highly sensitive staining method (streptavidin-biotin), however the extensive hard tissue decalcification required in this study produced weak keratin staining reaction. Conclusion This result demonstrates a relationship between keratin expression and morphologic changes during different developmental stages of rat enamel organ and ameloblasts differentiate to squamous cells of the reduced enamel epithelium. Thus, the developing enamel organ may be a suitable model for investigating the relationship between keratin expression and cell function.

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الملخص العربى

فى برعم14آشف هستوآيميائى مناعى عن وجود سيتوآراتين

الضروس عند الفئران

ة ا الطلائي زة للخلاي ة المتوسطة الطول الممي إن السيتوآيراتين يعتبر من الألياف البروتينية الهيكلي

ا البروتي .. السنية ذه الخلاي ائى له ة المتوسطة الطول ،من وحتى الآن يعتبر وصف الترآيب البن ني

رآزت معظم الأبحاث العلمية حول السيتوآيراتين على التعامل معه آمجموعة وقد ت ..الأشياء القليلة

والغرض من .آبيرة متكاملة من الأنواع المختلفة بدلا من البحث حول نوع معين من انواعه العديدة

م سيتوآيراين رق روتين ال ع الب ة توزي و الكشف عن طريق ذا البحث ه ة 14ه وار المختلف ى الأط ف

د ضروس عن رعم ال اعى لب ستوآيميائى المن ران باستخدام الكشف الهي د أسفر البحث عن . الفئ وق

سيتوآيراتين بغة ال ة ص ة 14إيجابي ت درج ضروس ،وان اختلف رعم ال ة لب ا المكون ع الخلااي لجمي

سنة وين ال اء تك ا اثن ر به ى تم ة الت ة التطوري اختلاف المرحل ضا .استجابتها ب ر البحث أي د أظه وق

رعم الضروس فى وجود صبغة السيتوآيرا داخلى لب ى ال تين بدرجة ايجابية عالية فى النسيج الطلائ

ونستخلص من هذا البحث إمكانية اعتبار . مرحلة الجرس الأولى وايضا خلال تكوين ميناء الأسنان

.برعم الضروس آنموذج للبحث حول علاقة ظهور الكيراتين ووظيفة الخلية

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