Original Article KLK4 Is Synchronically Expressed to CTSC in Ameloblasts During Amelogenesis Molars

Int J Clin Exp Pathol 2017;10(5):5751-5757 www.ijcep.com /ISSN:1936-2625/IJCEP0047909

Original Article KLK4 is synchronically expressed to CTSC in ameloblasts during amelogenesis molars

Lijie Wang1,2*, Xiaohua Xie1*, Yunduan Sun3, Jingyu Wang1, Yan Wang1, Xiumei Wang1

1Department of Stomatology, The 2nd Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China; 2Department of Stomatology, General Hospital of Daqing Oil Field, Daqing, Heilongjiang Prov- ince, China; 3The 1st Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China. *Equal contributors. Received January 2, 2017; Accepted January 27, 2017; Epub May 1, 2017; Published May 15, 2017

Abstract: The aim of this study was to provide the evidence that the CTSC product, DPPI is the activator of KLK4 during amelogenesis by examining the expression pattern of KLK4 and CTSC in developing teeth. The 1st mandibular molars from E16.5 embryos and postnatal mice, and incisor from P5 mandibles were selected for the immunohistochemistry with antibodies against KLK4 and DPPI. The expression of KLK4 and CTSC were initiated from postnatal day 1 (P1) expressed in molar ameloblasts. From P5 on, KLK4 was distributed not only in ameloblasts, but also in the degenerating satellite reticulum of molar enamel organ. However, DPPI distribution is always restricted to the molar ameloblasts. In the P5 incisor, the distribution of KLK4 and DPPI were spatiotemporally consistent in the ameloblasts. The coincidence of KLK4 and DPPI distribution in ameloblasts strongly suggested that DPPI activated KLK4. The distribution of KLK4 in satellite reticulum suggested that KLK4 most likely plays a proteolytic role in degeneration of enamel organ.

Keywords: Amelogenesis, DPPI, KLK4, CTSC, papillon-lefèvre syndrome

Introduction leukotoxin from anaerobic pathogens [6]. These findings explained how and why the mutations Papillon-Lefèvre syndrome (PLS) is a rare of CTSC or the loss of DPPI lead to hyperkerato- autosomal recessive disorder characterized by sis, prepubertal periodontitis and premature palmoplantar hyperkeratosis, prepubertal peri- tooth loss in PLS. However, not all of the mani- odontitis and premature loss of both deciduous festations in PLS patients or CTSC deficient and permanent teeth. The mutations of Cathe- mice was verified to result from this mecha- psin C gene (CTSC) are attributed to PLS [1]. A nism. For example, micro-hardness testing con- cysteine lysosomal protease belonging to the firmed that the enamel ofCTSC knock-out mice papaya superfamily is encoded by CTSC, name- is significantly softened compared with the nor- ly dipeptidyl-peptidase I (DPPI) which hydrolyz- mal littermates [7]. Whether the serine prote- es proteins or peptides in lysosomes by its ase activity of DPPI contributes to the soft endopeptidase and carboxypeptidase activities enamel of CTSC deficient mice is an attracting [2, 3]. In PLS patients, dysfunction of DPPI concern in PLS study. decreases protein degradation resulting in hyperkeratosis [4]. Meanwhile, DDPI also plays a Ameloblasts produce matrix metalloprotein- key role in activating chymotrypin-like serine ase-20 (MMP20) for matrix processing during proteases by removing one or more N-terminal the secretory stage of amelogenesis, while se- dipeptides from zymogen [5]. PLS patients ex- cret the serine proteinase, Kallikrein-4 (KLK4) hibited reduced activities of polymorphonucle- during the transition and mature stages of ame- ar leukocyte (PMN)-derived serine proteinases, logenesis [8]. The absence or insufficiency of such as elastase, cathepsin G and proteinase MMP20 and KLK4 were demonstrated to cause 3, which in turn decreased the production of amelogenesis imperfect in both human and antimicrobial peptide and the neutralization of mouse [9-12]. Since expressed in late stages KLK4 and CTSC in ameloblasts

and responsible for the ultimate removal of matrix proteins from enamel rods, the activity of KLK4 is regarded more critical for enamel maturation and hardness [10, 12]. KLK4 is produced in a pre- pro-enzyme form in endoplasm reticulum, secreted into extracellular space as a pro-enzyme after the removal of signal peptide, and transformed into active serine proteinase by cutting N-terminal propeptide off [13-15]. However, the extracellular activator of KLK4 is still unknown. Although MMP20 could activate KLK4 in vitro, their short overlap during amelogenesis and the activation of KLK4 in MMP20 knock-out mice revealed that the in vivo activation of KLK4 is performed by enzymes other than MMP20 [16].

As a serine proteinase activating extracellular zymogens, DPPI was found to be secreted into extracellular space [17]. Since the reduced hardness of enamel was detected in both CTSC and KLK4 deficiency mice [7], DPPI was pro- posed as the activator of KLK4 during amelogenesis. Examining the expression pattern of CTSC and KLK4 during amelogenesis is the first step to verify this hypoth- esis. Only when the expression of KLK4 is spatiotemporally consistent with that of CTSC, the possi- bility of DPPI activating KLK4 can be established.

Materials and methods

Animals and tissue preparations

All the experiments were approv- ed by animal ethics committee of Harbin medical university and co- nducted in accordance with the Figure 1. KLK4 expression in mouse molar germs. The immunohisto- requirements of ethics commit- chemistry results of KLK4 expression for P1 (A1, A2), P5 (B1, B2), P74 tee. C57/BL6 male and female (C1, C2), P9 (D1, D2), P11 (E1, E2) and P14 (F1, F2), respectively. (A1- mice was mating and the morning F1) are images at the magnitude of 200 folds; (A2-F2) are the magnified images at 400 folds of the corresponding boxes in (A1-F1). (Am: amelo- vaginal plug appeared was record- blasts; Od: odontoblast; SR: satellite reticulum; the scale bar in (A1-F1) ed as embryonic day 0.5 (E0.5) to stands for 200 µm and in (A2-F2) for 20 µm). get E16.5 embryos. The pregnant

5752 Int J Clin Exp Pathol 2017;10(5):5751-5757 KLK4 and CTSC in ameloblasts

mice were sacrificed by cervical dislocation and the embryonic mandibles were fixed in 4% polyformaldehyde for 24 h at 4°C. For postnatal tissue preparation, the mandibles were separated from the mice after 10% chloral hydra- te intraperitoneal anesthesia and fixed in 4% polyformaldehyde for 24 h at 4°C. Then, the postnatal mandibles were put into 8% EDTA for decalcification. Both the embryonic and postnatal mandibles were dehydrated by gradient et- hanol and embedded in paraffin after xylene treatment. All sam- ples were sectioned into 4 um slices, adhered to polylysine am- monia acid treated slides, dried at 37°C and finally treated at 70°C oven overnight.

Antibodies

For immunohistochemical examination, rabbit polyclonal antibody against KLK4 (Biorbyt, CA, USA) and rat polyclonal antibody against DPPI were applied (Santa Cruz, CA, USA). Secondary antibodies against rabbit and rat conju- gated with horseradish peroxidase were purchased from ZSGB- BIO Inc, Beijing, China.

Immunohistochemistry

Paraffin sections were deparaf- finized in xylene and rehydrated with gradient alcohol. Endogenous peroxidase was inactivated in 3% hydrogen peroxide at room temperature for 10 minutes. Then, the sections were rinsed with distilled water and placed in phosphate buffer saline (PBS) for 5 minutes. Sections citrate sustained-release -2 Figure 2. CTSC expression and DPPI distribution in mouse molar germs. liquid (PH6.0, 10 M) were heated The immunohistochemistry results of DPPI distribution in P1 (A1, A2), by microwave for antigen retrieval P5 (B1, B2), P7 (C1, C2), P9 (D1, D2), P11 (E1, E2) and P14 (F1, F2), (high heat for 2 minutes, and then respectively. (A1-F1) are images at the magnitude of 200 folds; (A2-F2) low heat for 5 minutes for KLK4; are the magnified images at 400 folds of the corresponding boxes in (A1-F1). (Am: ameloblasts; Od: odontoblasts; SR: satellite reticulum; the high heat for 5 minutes for DPPI). scale bar in (A1-F1) stands for 200 um and in (A2-F2) for 20 um). After cooled down to room tem-

5753 Int J Clin Exp Pathol 2017;10(5):5751-5757 KLK4 and CTSC in ameloblasts

Figure 3. KLK4 expression and DPPI distribution in mouse P5 incisor.The KLK4 immunohistochemistry results in P5 incisor (A). KLK4 distribution in mature (A1), secretory (A2) and pre-secretory stage (A3), respectively. The DPPI immunohistochemistry results in P5 incisor (B). DPPI distribution in mature (B1), secretory (B2) and pre-secretory stage (B3), respectively. (A, B) are images at the magnitude of 200 folds; (A1-A3 and B1-B3) are the magnified images at 400 folds of the corresponding boxes in (A and B). (Am: ameloblasts; Od: odontoblast; *: mesenchyme adjacent to cervical loop; the scale bar in (A and B) stands for 200 um and in A1-A3 and B1-B3 for 10 um). perature and rinsed with PBS, the sections 4°C. Then, the sections were rinsed with PBS were blocked with bovine serum for 20 min and and incubated with secondary antibody for 20 subsequently incubated with KLK4 antibody min at room temperature before develop the (1:100) or DPPI antibody (1:50) overnight at color with freshly prepared DAB.

5754 Int J Clin Exp Pathol 2017;10(5):5751-5757 KLK4 and CTSC in ameloblasts

Results Distributions of KLK4 and DPPI in postnatal incisor Distribution of KLK4 in developing molar germs The P5 incisors which contain ameloblasts from progenitor to mature status were applied The immunohistochemistry examinations sh- to the examination of the KLK4 and CTSC own that at the E16.5 day, neither the pre-ame- expression. In the para-sagittal section of the loblasts nor pre-odontoblasts of the mouse P5 incisor, the KLK4 expression was initiated molar germs exhibited the KLK4 expression the pre-secretory ameloblasts which started (data not shown). In the 1st molar at P1, the the polarization to transform into high colum- KLK4 expression was first detected in the ame- nar morphology (Figure 3A and 3A3). Intrigu- loblasts and odontoblasts at secretory stage, ingly, although the epithelial cells between the but absent from the satellite reticulum of cervical loop and the polarizing domain were enamel organ (Figure A1 and A2). At the P5 devoid of KLK4 expression, the mesenchyme molar enamel organ, the KLK4 distribution was adjacent to cervical loop exhibited a strong detected not only in the ameloblasts at mature staining of KLK4 signal (Figure 3A). In contrast, stage, but also even stronger in the satellite both the polarized ameloblasts and odonto- reticulum (Figure B1 and B2). However, the blasts showed the continuous expression of KLK4 distribution in the P5 odontoblasts was KLK4 (Figure 3A2 and 3A1). On the other hand, concentrated at the cusps region, instead of the DPPI distribution exhibited the consistent the entire layer of odontoblasts (Figure C1 and pattern with that of KLK4 in the P5 incisor, C2). In the P7 molar germs, the KLK4 signals in except was absent from the mesenchyme adja- odontoblasts almost diminished, but insisted in cent to cervical loop (Figure 3B and 3B1-B3). the ameloblasts and the degenerating satel- Noticeably, both KLK4 and CSTC displayed lite reticulum (Figure D1 and D2). From P9 on, the more robust expression in ameloblasts the intensity of KLK4 became weaker and than in odontoblasts (Figure 3A, 3A1-A3 and weaker in both the ameloblasts andsatellite 3B, 3B1-B3). Figure E1 E2 reticulum ( and ). To the P14, the Discussion KLK4 signal was almost undetectable in ameloblasts, though still obvious in degeneratedsat- As the highest mineralized tissue, 95% volume ellite reticulum (Figure F1 and F2). of the mature enamel is hydroxyl apatite, while the matrix proteins only account for the other Expression of CTSC and distribution of DPPI in 5% volume [18]. The persistence of too much developing molar germs enamel matrix proteins in the maturing enamel impairs the formation of the enamel rods of Similar to the expression of KLK4, the immuno- hydroxyapatite crystal [19]. Previous studies histochemistry with antibody against DPPI gave demonstrate that MMP20 and KLK4, especi- rise to no signal in the E16.5 molar germs (data ally KLK4, are the essential proteinases for not shown). Even at the P1 when DPPI was enamel maturation by processing and remov- intensively detected in the alveolar bone sur- ing enamel matrix proteins [20]. The deficiency rounding molar germs, there was only slight sig- or insufficient production of MMP20 and/or nals in the ameloblasts and odontoblasts KLK4 could result in Amelogenesis Imperfecta (Figure 2A1 and 2A2). However, from P5 to P7, [9-12]. Since persistently and solely expressed the DPPI intensity decreased dramatically in in the transition and mature stages of amelo- the alveolar bone and almost diminished in genesis and exhibits a higher efficiency in odontoblasts, while its intensity increased in degrading amelogenin, KLK4 is regarded more the ameloblasts (Figure 2B1-C2). Such increase important than MMP20 in the proteolysis of was reversed at P9 and decreased gradually enamel matrix proteins [21]. KLK4 expression until P14. Meanwhile, the surrounding alveolar was initiated at P1 and secreted as an inac- bone, odontoblasts and degenerating satellite tive zymogen into the extracellular space [22]. reticulum were almost devoid of DPPI distribu- How the pro-KLK4 is transformed into the tion (Figure 2D1-F2). active form remains unknown. MMP20 was

5755 Int J Clin Exp Pathol 2017;10(5):5751-5757 KLK4 and CTSC in ameloblasts once thought as the activator of KLK4, howev- Acknowledgements er, there were emerging evidences denying this notion. First, MMP20 and KLK4 expression This study is funded by: the Sciences and overlap few chronically [20, 22]; second, KLK4 technology program of Education Bureau in is normally activated in MMP20 knock-out mice Heilongjiang Province (12541499: The relation- and also able to inactivate MMP20 in vitro [16, ship between AEG-1 expression and prognosis 23]; finally, the severity of amelogenesis impe- in oral squamous cell cancer), Funds on leading fecta in MMP20 null mouse is milder than KLK4 candidates for future leaders in Heilongjiang deficient mouse [12, 24]. Therefore, KLK4 is Province, and Natural Science Foundation of activated by proteinases other than MMP20. Heilongjiang Province of China (Grant H2014- 18) and the grant from Natural Science Our study showed that the lysosomal protein- Foundation of China (NSFC NO. 81600848). ase, DPPI encoded by CTSC exhibits the expression pattern in ameloblasts identical to KLK4. Disclosure of conflict of interest Additionally, KLK4 expression reached the None. climax at P5 to P7, which was consistent with the DPPI expression. Previous study demon- Address correspondence to: Dr. Xiumei Wang, De- strated that the reduced enamel hardness was partment of Stomatology, The 2nd Affiliated Hospital observed in KLK4 deficient mouse as well of Harbin Medical University, 246 Xuefu Road, as CTSC deficient mouse [7]. These findings Harbin 150086, Heilongjiang, China. Tel: (+86) 451- strongly suggested that DPPI could serve as 86605544; E-mail: [email protected] the activator of KLK4. However, there is few reports on the reduced enamel hardens in the References PLS patients, which may result from the early loss of teeth. So it still needs further investiga- [1] Toomes C, James J, Wood AJ, Wu CL, McCor- tion to address if the hardness of PLS teeth is mick D, Lench N, Hewitt C, Moynihan L, Rob- reduced. erts E, Woods CG, Markham A, Wong M, Wid- mer R, Ghaffar KA, Pemberton M, Hussein IR, The most interesting discovery in our study is Temtamy SA, Davies R, Read AP, Sloan P, Dixon that the distribution of KLK4 is much broader MJ, Thakker NS. Loss-of-function mutations in than that of DPPI confined in ameloblasts. the cathepsin C gene result in periodontal dis- Considering the previous study showing that ease and palmoplantar keratosis. Nat Genet KLK4 was only transcribed in ameoloblasts 1999; 23: 421-424. [22], the extended spread of KLK4 to the [2] McGrath ME. The lysosomal cysteine proteas- degenerating enamel organ suggested that es. Annu Rev Biophys Biomol Struct 1999; 28: KLK4 was supposed to be activated by DPPI 181-204. around ameloblasts and then, transported by [3] Santilman V, Jadot M, Mainferme F. Impor- other binging proteins to satellite reticulum. tance of the propeptide in the biosynthetic The broader distribution of KLK4 also suggest- maturation of rat cathepsin C. Eur J Cell Biol ed that KLK4 was most likely play a proteolytic 2002; 81: 654-663. [4] Meade JL, de Wyntr EA, Brett P, Sharif SM, role in the degeneration of enamel organ. Woods CG, Markham AF, Cook GP. A family with The other intriguingly findings in our study is the papillon-lefèvre syndrome reveals a require- detection of KLK4-expressing domains sur- ment for cathepsin C in granzyme B activation rounding the cervical loops of incisor. As shown and NK cell cytolytic activity. Blood 2006; 107: in previous studies, the mesenchyme adjacent 3665-3668. [5] Tran TV, Ellis KA, Kam CM, Hudig D, Power JC. to cervical loop of incisor acts as the stem cell Dipeptidyl peptidase I: importance of progran- pool of odontoblasts [25]. Our discovery implies zyme activation sequences, other dipeptide that KLK4 contributes to the maintenance or sequences,and the N-terminal amino group of renewal of the mesenchymal stem cell pool, synthetic substrates for enzyme activity. Arch and may fulfill the role through its protelytic Biochem Biophys 2002; 403: 160-170. function. On the other hand, DPPI was not [6] de Haar SF, Hiemstra PS, van Steenbergen MT, detected in the same mesenchymal region, Everts V, Beertsen W. Role of polymorphonu- suggesting that DPPI is not the sole enzyme clear leukocyte-derived serine proteinases in capable of activating KLK4. defense against actinobacillus actinomycete-

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