Heparanase cleavage of perlecan heparan sulfate modulates FGF10 activity during ex vivo submandibular gland branching morphogenesis

Heparan sulfate proteoglycans are essential for biological processes regulated by fibroblast growth factors (FGFs). Heparan sulfate (HS) regulates the activity of FGFs by acting as a coreceptor at the cell surface, enhancing FGF-FGFR affinity, and being a storage reservoir for FGFs in the extracellular matrix (ECM). Here we demonstrate a critical role for heparanase during mouse submandibular gland (SMG) branching morphogenesis. Heparanase, an endoglycosidase, colocalized with perlecan in the basement membrane and in epithelial clefts of SMGs. Inhibition of heparanase activity in organ culture decreased branching morphogenesis, and this inhibition was rescued specifically by FGF10 and not by other FGFs. By contrast, exogenous heparanase increased SMG branching and MAPK signaling and, surprisingly, when isolated epithelia were cultured in a three-dimensional ECM with FGF10, it increased the number of lateral branches and end buds. In a solid-phase binding assay, an FGF10-FGFR2b complex was released from the ECM by heparanase. In addition, surface plasmon resonance (SPR) analysis showed that FGF10 and the FGF10-FGFR2b complex bound to purified perlecan HS and could be released by heparanase. We used the FGF10-FGFR2b complex as a probe for HS in SMGs, and it colocalized with perlecan in the basement membrane and partly colocalized with syndecan 1 in the epithelium, and binding was reduced by treatment with heparanase. In summary, our results show heparanase releases FGF10 from perlecan HS in the basement membrane, increasing MAPK signaling, epithelial clefting, and lateral branch formation, which results in increased branching morphogenesis.     

Immortalized Hertwig's epithelial root sheath cell line works as model for epithelial–mesenchymal interaction during tooth root formation

Hertwig's epithelial root sheath (HERS) is critical for epithelial–mesenchymal interaction (EMI) during tooth root formation. However, the exact roles of HERS in odontogenic differentiation by EMI have not been well characterized, because primary HERS cells are difficult to obtain. Immortalized cell lines constitute crucial scientific tools, while there are few HERS cell lines available. Our previous study has successfully established immortalized HERS cell lines. Here, we confirmed the phenotype of our HERS-H1 by verifying its characteristics and functions in odontogenic differentiation through EMI. The HERS-H1-conditioned medium (CM-H1) effectively enhanced odontogenic differentiation of dental papilla cells (DPCs) in vitro. Furthermore, Smad4 and p-Smad1/5/8 were significantly activated in DPCs treated with CM-H1, and this activation was attenuated by noggin. In vivo, our implanted recombinants of HERS-H1 and DPCs exhibited mineralized tissue formation and expression of Smad4, p-Smad1/5/8, and odontogenic differentiation markers. Our results indicated that HERS-H1 promoted DPCs odontoblastic differentiation via bone morphogenetic protein/Smad signaling. HERS-H1 exhibits relevant key molecular characteristics and constitutes a new biological model for basic research on HERS and the dental EMI during root development and regeneration.     

Fate of HERS during tooth root development

Tooth root development begins after the completion of crown formation in mammals. Previous studies have shown that Hertwig's epithelial root sheath (HERS) plays an important role in root development, but the fate of HERS has remained unknown. In order to investigate the morphological fate and analyze the dynamic movement of HERS cells in vivo, we generated K14-Cre;R26R mice. HERS cells are detectable on the surface of the root throughout root formation and do not disappear. Most of the HERS cells are attached to the surface of the cementum, and others separate to become the epithelial rest of Malassez. HERS cells secrete extracellular matrix components onto the surface of the dentin before dental follicle cells penetrate the HERS network to contact dentin. HERS cells also participate in the cementum development and may differentiate into cementocytes. During root development, the HERS is not interrupted, and instead the HERS cells continue to communicate with each other through the network structure. Furthermore, HERS cells interact with cranial neural crest derived mesenchyme to guide root development. Taken together, the network of HERS cells is crucial for tooth root development.     

BMP4 signaling regulates formation of Hertwig’s epithelial root sheath during tooth root development

Although Hertwig’s epithelial root sheath (HERS) performs an important function in the formation of the tooth root, the developmental mechanisms that control HERS growth and differentiation remain to be thoroughly elucidated. Bone morphogenetic protein 4 (BMP4), which is secreted by mesenchymal cells, acts on the dental epithelium as a regulator of cell differentiation during crown formation. In an effort to determine whether BMP4 specifically regulates the development of HERS in the dental epithelium, we assessed the localizations of BMP4, BMP receptor-IB (BMPR-IB), and BMPR-II during molar root formation in the mouse. HERS cells were shown to express BMPR-IB and BMPR-II. BMP4-positive cells were detected densely in the dental papillae around HERS, thereby suggesting that BMP4 participated in HERS formation. Beads soaked in BMP4, NOGGIN, or phosphate-buffered saline (PBS) were implanted into the pulp cavity under culture conditions, and the length of HERS was evaluated with regard to the proliferating cells. After 12 h, both groups exhibited a similar HERS developmental pattern, with the length and shape of HERS bearing a close resemblance to one another. However, after 48 h, the observed HERS elongation was significantly shorter in the BMP4-treated group. In addition, proliferative cell nuclear antigens were detectable only in the NOGGIN- and PBS-treated groups. These findings demonstrate that mesenchymally expressed BMP4 regulates HERS development by preventing elongation and maintaining cell proliferation. BMP4 may, therefore, prove useful as a root-formation regulatory agent in a variety of tissue-engineering applications. Akihiro Hosoya and Ji-Youn Kim contributed equally to this work. This work was supported by the Seoul R&BD Program (NT070139).     

Regulatory mechanisms of Hertwig׳s epithelial root sheath formation and anomaly correlated with root length

Teeth are composed of two domains, the enamel-covered crown and cementum-covered root. The mechanism for determining the transition from crown to root is important for understanding root anomaly diseases. Hertwig?s epithelial root sheath (HERS) is derived from the dental epithelium and is known to drive the growth of root dentin and periodontal tissue. Some clinical cases of hypoplastic tooth root are caused by the cessation of HERS development. Understanding the mechanisms of HERS development will contribute to the study of the disease and dental regenerative medicine. However, the developmental biology of tooth root formation has not been fully studied, particularly regarding HERS formation. Here, we describe the mechanisms of HERS formation on the basis of analysis of cell dynamics using imaging and summarize how the growth factor and its receptor regulate cell behavior of the dental epithelium.     

Establishment of Hertwig's epithelial root sheath cell line from cells involved in epithelial-mesenchymal transition

The epithelial–mesenchymal transition (EMT) is an important event in the developmental process of various organs. In periodontal development during root formation of a tooth, this EMT has been a subject of controversy. Hertwig’s epithelial root sheath (HERS), consisting of two epithelial layers, plays a role of inducing odontogenesis during root development and thereafter becomes fragmented. Some researchers have maintained that in the process of this fragmentation, some HERS cells change from epithelial to mesenchymal cells. Here, we established a HERS cell line (HERS01a) and examined its gene and protein expression. Immunohistochemical staining and real-time PCR analysis showed that HERS01a cells expressed vimentin and N-cadherin as mesenchymal markers as well as cytokeratin14, E-cadherin, and p63 as epithelial stem cell markers. In the presence of TGF-β, HERS01a cells also expressed many more mesenchymal markers, as well as snail1 and 2 as EMT markers. Taken together, our data show that HERS01a displayed unique features associated with EMT in the root formation process, and will thus be useful for analyzing the biological characteristics of HERS and the molecular mechanism underlying the EMT.     

Immunohistochemical localization of calbindin D28k during root formation of rat molar teeth

The present study was undertaken to examine the localization of calbindin D28k (CB)-like immunoreactivity (-LI) during the root formation of the rat molar. In the adult rat, CB-LI was detected in some of the cells of the epithelial rest of Malassez at the bifurcational region and in certain cells between the root dentin and cementum at the apical region. These cells had indented nuclei and many tonofilaments, and cementocytes lacked CB-LI. Moreover, CB-LI was observed in the periodontal fibroblasts in the alveolar half of the apical region. During root formation, the cells in the Hertwig's epithelial root sheath (HERS) lacked CB-LI, but most fragmented cells along the root surface began to express CB-LI when HERS was disrupted. Preodontoblasts and odontoblasts at the apical portion of the root also showed CB-LI. After the formation of cellular cementum, the CB-immunoreactive (-IR) cells were entrapped between the root dentin and cementum in the apical portion of the root. The number of CB-IR cells at the root surface decreased gradually, while that between the root dentin and cementum increased. The fibroblasts in the periodontal ligament began to express CB-LI after commencement of the occlusion, and the number and the staining intensity of CB-IR fibroblasts increased gradually with the passage of time. The present results suggest that CB may play an important role in the survival of the epithelial cells, in the cellular responses of periodontal fibroblasts against mechanical forces caused by the occlusion, and in the initial mineralization by the odontoblasts through the regulation of intracellular Ca(2+) concentration.     

Perlecan from human epithelial cells is a hybrid heparan/chondroitin/keratan sulfate proteoglycan

Perlecan is a multidomain proteoglycan, usually substituted with heparan sulphate (HS), and sometimes substituted with both HS and chondroitin sulphate (CS). In this paper, we describe perlecan purified from HEK-293 cells substituted with HS, CS and keratan sulphate (KS). KS substitution was confirmed by immunoreactivity with antibody 5D4, sensitivity to keratanase treatment, and fluorophore-assisted carbohydrate electrophoresis. HEK-293 perlecan failed to promote FGF-dependent cell growth in an in vitro assay. This study is the first to report perlecan containing KS, and makes perlecan one of only a very few proteoglycans substituted with three distinct types of glycosaminoglycan chains.     

Heparanases and tumor metastasis

The successful penetration of endothelial basement membranes is an important process in the formation of hematogenous tumor metastases. Heparan sulfate (HS) proteoglycan is a major constituent of endothelial basement membranes, and we have found that HS-degradative activities of metastatic B16 melanoma sublines correlate with their lung-colonizing potentials. The melanoma HS-degrading enzyme is a unique endo-beta-D-glucuronidase (heparanase) that cleaves HS at specific intrachain sites and is detectable in a variety of cultured human malignant melanomas. The treatment of B16 melanoma cells with heparanase inhibitors that have few other biological activities, such as N-acetylated N-desulfated heparin, results in significant reductions in the numbers of experimental lung metastases in syngeneic mice, indicating that heparanase plays an important role in melanoma metastasis. HS-degrading endoglycosidases are not tumor-specific and have been found in several normal tissues and cells. There are at least three types of endo-beta-D-glucuronidases based on their substrate specificities. Melanoma heparanase, an Mr approximately 96,000 enzyme with specificity for beta-D-glucuronosyl-N-acetylglucosaminyl linkages in HS, is different from platelet and mastocytoma endoglucuronidases. Elevated levels of heparanase have been detected in sera from metastatic tumor-bearing animals and malignant melanoma patients, and a correlation exists between serum heparanase activity and extent of metastases. The results suggest that heparanase is potentially a useful marker for tumor metastasis.     

Establishment of Hertwig’s Epithelial Root Sheath/Epithelial Rests of Malassez Cell Line from Human Periodontium

Human Hertwig’s epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare population in periodontium and the primary isolation of them is considered to be difficult. To overcome these problems, we immortalized primary HERS/ERM cells isolated from human periodontium using SV40 large T antigen (SV40 LT) and performed a characterization of the immortalized cell line. Primary HERS/ERM cells could not be maintained for more than 6 passages; however, immortalized HERS/ERM cells were maintained for more than 20 passages. There were no differences in the morphological and immunophenotypic characteristics of HERS/ERM cells and immortalized HERS/ERM cells. The expression of epithelial stem cell and embryonic stem cell markers was maintained in immortalized HERS/ERM cells. Moreover, immortalized HERS/ERM cells could acquire mesenchymal phenotypes through the epithelial-mesenchymal transition via TGF-β1. In conclusion, we established an immortalized human HERS/ERM cell line with SV40 LT and expect this cell line to contribute to the understanding of the functional roles of HERS/ERM cells and the tissue engineering of teeth.     

Cell proliferation and death of Hertwig's epithelial root sheath in the rat

Hertwig's epithelial root sheath (HERS) degenerates immediately after root dentin is formed. However, odontogenic tumors or cysts may originate from residual cells, although little is known about how HERS proliferates and disappears. This study investigated whether cell death is provoked in the tissues surrounding the root during eruption of the rat upper molar. We employed the TdT-mediated-dUTP nick end labeling (TUNEL) method and transmission electron microscopy (TEM) to observe the morphological features of cell death. We examined the activity of cell proliferation immunohistochemically using proliferative cell nuclear antigen (PCNA) and the continuity of HERS using polyclonal keratin antibody (PK). Cell death resembling apoptosis and apoptotic bodies phagocytosed by neighboring mesenchymal cells were detected in only a few cells by both TUNEL and TEM. We also found cells with electron-lucent cytoplasm which contained dilated or ruptured mitochondria and remarkably dilated rough endoplasmic reticulum (rER) which lay sparsely along the root. These cells seemed to be dead HERS cells based on their ultrastructural features, location, and stage. PCNA-positive cells were found in the apical end of the HERS cells, fibroblasts of the periodontal ligament, and odontoblasts. PK reacted with HERS; however, PK-positive cells partially disappeared after the 15th postnatal day when the root dentin had formed slightly. These results may indicate that HERS cells migrate into the periodontal ligament or die immediately after root dentin is formed and that various types of cell death such as apoptosis and cytoplasmic type occur in the tissues surrounding the root during tooth development.     

Localization of STRO-1, BMP-2/-3/-7, BMP receptors and phosphorylated Smad-1 during the formation of mouse periodontium

Bone morphogenetic proteins (BMPs) and BMP receptors (BMPRs) are known to regulate the development of calcified tissues by directing mesenchymal precursor cells differentiation. However, their role in the formation of tooth-supporting tissues remains unclear. We investigated the distribution pattern of STRO-1, a marker of mesenchymal progenitor cells and several members of the BMP pathway during the development of mouse molar periodontium, from the post-natal days 6 to 23 (D6 to D23). STRO-1 was mainly localized in the dental follicle (DF) at D6 and 13 then in the periodontal ligament (PDL) at D23. BMP-2 and -7 were detected in Hertwig's epithelial root sheath (HERS) and in DF, then later in differentiated periodontal cells. BMP-3 was detected after D13 of the periodontal development. BMPRs-Ib, -II, the activin receptor-1 (ActR-1) and the phosphorylated Smad1 were detected in DF and HERS at D6 and later more diffusely in the periodontium. BMPR-Ia detection was restricted to alveolar bone. These findings were in agreement with others data obtained with mouse immortalized DF cells. These results suggest that STRO-1 positive DF cells may be target of BMPs secreted by HERS. BMP-3 might be involved in the arrest of this process by inhibiting the signaling provided by cementogenic and osteogenic BMPs.     

Ameloblastin in Hertwig’s Epithelial Root Sheath Regulates Tooth Root Formation and Development

Tooth root formation begins after the completion of crown morphogenesis. At the end edge of the tooth crown, inner and outer enamel epithelia form Hertwig’s epithelial root sheath (HERS). HERS extends along with dental follicular tissue for root formation. Ameloblastin (AMBN) is an enamel matrix protein secreted by ameloblasts and HERS derived cells. A number of enamel proteins are eliminated in root formation, except for AMBN. AMBN may be related to tooth root formation; however, its role in this process remains unclear. In this study, we found AMBN in the basal portion of HERS of lower first molar in mice, but not at the tip. We designed and synthesized small interfering RNA (siRNA) targeting AMBN based on the mouse sequence. When AMBN siRNA was injected into a prospective mandibular first molar of postnatal day 10 mice, the root became shorter 10 days later. Furthermore, HERS in these mice revealed a multilayered appearance and 5-bromo-2′-deoxyuridine (BrdU) positive cells increased in the outer layers. In vitro experiments, when cells were compared with and without transiently expressing AMBN mRNA, expression of growth suppressor genes such as p21^Cip1 and p27^Kip1 was enhanced without AMBN and BrdU incorporation increased. Thus, AMBN may regulate differentiation state of HERS derived cells. Moreover, our results suggest that the expression of AMBN in HERS functions as a trigger for normal root formation.     

Morphogenetic roles of perlecan in the tooth enamel organ: an analysis of overexpression using transgenic mice

Perlecan, a heparan sulfate proteoglycan, is enriched in the intercellular space of the enamel organ. To understand the role of perlecan in tooth morphogenesis, we used a keratin 5 promoter to generate transgenic (Tg) mice that over-express perlecan in epithelial cells, and examined their tooth germs at tissue and cellular levels. Immunohistochemistry showed that perlecan was more strongly expressed in the enamel organ cells of Tg mice than in wild-type mice. Histopathology showed wider intercellular spaces in the stellate reticulum of the Tg molars and loss of cellular polarity in the enamel organ, especially in its cervical region. Hertwig's epithelial root sheath (HERS) cells in Tg mice were irregularly aligned due to excessive deposits of perlecan along the inner, as well as on the outer sides of the HERS. Tg molars had dull-ended crowns and outward-curved tooth roots and their enamel was poorly crystallized, resulting in pronounced attrition of molar cusp areas. In Tg mice, expression of integrin β1 mRNA was remarkably higher at E18, while expression of bFGF, TGF-β1, DSPP and Shh was more elevated at P1. The overexpression of perlecan in the enamel organ resulted in irregular morphology of teeth, suggesting that the expression of perlecan regulates growth factor signaling in a stage-dependent manner during each step of the interaction between ameloblast-lineage cells and mesenchymal cells.     

Not all perlecans are created equal: interactions with fibroblast growth factor (FGF) 2 and FGF receptors

Human basement membrane heparan sulfate proteoglycan (HSPG) perlecan binds and activates fibroblast growth factor (FGF)-2 through its heparan sulfate (HS) chains. Here we show that perlecans immunopurified from three cellular sources possess different HS structures and subsequently different FGF-2 binding and activating capabilities. Perlecan isolated from human umbilical arterial endothelial cells (HUAEC) and a continuous endothelial cell line (C11 STH) bound similar amounts of FGF-2 either alone or complexed with FGFRalpha1-IIIc or FGFR3alpha-IIIc. Both perlecans stimulated the growth of BaF3 cell lines expressing FGFR1b/c; however, only HUAEC perlecan stimulated those cells expressing FGFR3c, suggesting that the source of perlecan confers FGF and FGFR binding specificity. Despite these differences in FGF-2 activation, the level of 2-O- and 6-O-sulfation was similar for both perlecans. Interestingly, perlecan isolated from a colon carcinoma cell line that was capable of binding FGF-2 was incapable of activating any BaF3 cell line unless the HS was removed from the protein core. The HS chains also exhibited greater bioactivity after digestion with heparinase III. Collectively, these data clearly demonstrate that the bioactivity of HS decorating a single PG is dependent on its cell source and that subtle changes in structure including secondary interactions have a profound effect on biological activity.     

Directing the differentiation of human dental follicle cells into cementoblasts and/or osteoblasts by a combination of HERS and pulp cells

It is known that the dental follicle (DF) consists of progenitor cells that give rise to the cementum, periodontal ligament, and alveolar bone; but little information is available about the regulation of DF cell differentiation into either cementogenic or osteogenic cell lineages for the regeneration of diseased periodontal tissue. Here, we investigated the roles of DF, Hertwig’s epithelial root sheath (HERS), and pulp cells in the cementum and during alveolar bone formation. We cultured these cells; transplanted them alone or in combination into immunocompromised mice; and observed their effects at 6 and 12 weeks. Histological and immunohistochemical results revealed that DF cells formed cementum-like tissues with immunoreactivity to cementum-derived attached protein, bone sialoprotein, type I collagen, and alkaline phosphatase. In addition, HERS cells played a role in the induction and maturation of cementum-like tissues formed by DF cells. In contrast, implants of DF cells in the presence of pulp cells led to the formation of bone-like tissues. Interestingly, in the presence of both HERS and pulp cells, DF cells formed both cementum-like and bone-like tissues. We demonstrated that while HERS cells are able to induce DF cell differentiation into cementoblasts and promote cementum formation, pulp cells could direct DF cell differentiation into osteoblasts and enhance alveolar bone formation. These results suggest that the combined use of DF, HERS, and pulp cells could direct DF cell differentiation into cementoblasts and/or osteoblasts in vivo, thus providing a novel strategy for the successful repair and regeneration of diseased periodontal tissue.