Sanjiv Neupane

Developmental regulations of Perp in mice molar morphogenesis

Teraspanin transmembrane protein, Perp (P53 apoptosis effector related to PMP22), which is found in the plasma membrane as a component of the desmosome, is reported to be involved in the morphogenesis of the epithelium and the enamel formation of the incisor. However, its expression pattern and signaling regulation during molar development have not been elucidated in detail. We have examined the precise expression patterns of Perp in developing lower molars and employed the knock-down of Perp by antisense oligodeoxynucleotide treatment during in vitro organ cultivation at embryonic day 13 to define the precise developmental function of Perp. Perp was expressed mainly in the dental lamina and stellate reticulum regions at the bud and cap stages. After Perp knock-down, the tooth germ showed disruption of the dental lamina and stellate reticulum with altered apoptosis and proliferation. The changed expression levels of related signaling molecules from the enamel knot and desmosome were evaluated by real-time quantitative polymerase chain reaction. A renal capsule transplantation method was employed to examine the effects of Perp knock-down on molar crown development. Ultrastructural observations revealed that enamel was deposited more densely in an irregular pattern in the cusp region, and that dentin was hypo-mineralized after Perp knock-down at the cap stage. Thus, Perp might play important roles in the formation and integration of stellate reticulum, dental lamina structure and enamel formation through signaling interactions with the enamel knot and desmosome-related signaling molecules at the cap stage of lower molar development.

Mesenchymal signaling in dorsoventral differentiation of palatal epithelium

After palatal fusion, the dorsal and ventral epithelia of the palatal shelf differentiate into the nasal and oral mucosa, respectively. The tissue-specific differentiation of palatal epithelia along the dorsal–ventral axis is regulated by the signaling molecules expressed in the underlying mesenchyme. Thus, as in many other epithelial organs, differentiation relies on epithelial–mesenchymal interactions. To screen for region-specific mesenchymal signaling molecules that determine the fate of the palatal epithelia, we employed a laser microdissection (LMD) method. LMD allowed us to collect region-specific mesenchymal tissues at E13, prior to palatal fusion and the development of distinct dorsal and ventral epithelial morphology. Genome-wide screening was performed on the tissues collected using LMD to identify candidate mesenchymal signaling molecules. The microarray results were validated using real-time quantitative (qPCR) and in situ hybridization methods. The developmental role and interactions of the candidate genes were evaluated in in vitro-cultivated E13 palates using an anti-sense oligodeoxynucleotide (AS-ODN)-based loss-of-function approach. Apparent changes in the expression patterns of Runt-related transcription factor 2 (Runx2) and LIM homeobox 8 (Lhx8) were observed after knocking down each gene. Knock-down of Runx2 and Lhx8 also altered the immunolocalization pattern of cytokeratin18 (CK18), an established marker for nasal epithelium. These results were confirmed using Runx2 heterozygote mice. The mesenchymal signaling molecules Runx2 and Lhx8, which possess region-specific expression patterns along the dorsoventral axis, functionally interact to regulate the cellular and molecular characteristics of dorsal and ventral epithelia, suggesting that mesenchymal signaling molecules determine the dorsoventral fate of epithelial structures in the developing palate.

Immunolocalization patterns of cytokeratins during salivary acinar cell development in mice

Embryonic development of the mouse salivary glands begins with epithelial thickening and continues with sequential changes from the pre-bud to terminal bud stages. After birth, morphogenesis proceeds, and the glands develop into a highly branched epithelial structure that terminates with saliva-producing acinar cells at the adult stage. Acinar cells derived from the epithelium are differentiated into serous, mucous, and seromucous types. During differentiation, cytokeratins, intermediate filaments found in most epithelial cells, play vital roles. Although the localization patterns and developmental roles of cytokeratins in different epithelial organs, including the mammary glands, circumvallate papilla, and sweat glands, have been well studied, their stage-specific localization and morphogenetic roles during salivary gland development have yet to be elucidated. Therefore, the aim of this study was to determine the stage and acinar cell type-specific localization pattern of cytokeratins 4, 5, 7, 8, 13, 14, 18, and 19 in the major salivary glands (submandibular, sublingual, and parotid glands) of the mouse at the E15.5, PN0, PN10, and adult stages. In addition, cell physiology, including cell proliferation, was examined during development via immunostaining for Ki67 to understand the cellular mechanisms that govern acinar cell differentiation during salivary gland morphogenesis. The distinct localization patterns of cytokeratins in conjunction with cell physiology will reveal the roles of epithelial cells in salivary gland formation during the differentiation of serous, mucous or seromucous salivary glands.

Bortezomib prevents ovariectomy-induced osteoporosis in mice by inhibiting osteoclast differentiation

Bone homeostasis is achieved through coordinated activities of bone-forming osteoblasts and bone-resorbing osteoclasts. When the balance is skewed in favor of osteoclasts due to hormonal or inflammatory issues, pathologic bone loss occurs leading to conditions such as osteoporosis, rheumatoid arthritis, and periodontitis. Bortezomib is the first in-class of proteasome inhibitors used as an anti-myeloma agent. In the present study, we show that bortezomib directly inhibited the receptor activator of nuclear factor κB ligand (RANKL)—dependent osteoclast differentiation of mouse bone marrow macrophages. Bortezomib significantly reduced the induction of osteoclast marker genes and proteins including nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1). The intraperitoneal injection of bortezomib reduced ovariectomy-induced osteoclastogenesis and protected the mice from bone loss. These data propose novel use of bortezomib as a potential anti-resorptive agent.

Involvement of PI3K and PKA pathways in mouse tongue epithelial differentiation.

In mice, tongue epithelial differentiation is mainly regulated by the interactions among various signalling molecules including Fgf signalling pathways. However, the subsequent signalling modulations for epithelial maturation, initiated by Fgf signalling, remain to be elucidated. Therefore, we employed an in vitro tongue organ cultivation system along with the applications of various pharmacological inhibitors against the intracellular signalling molecules of Fgf signalling pathways, including H89, LY294002, PD98059, and U0126. Following treatments with LY294002 and H89, inhibitors for PI3K and PKA, respectively, the decreased thickness of the tongue epithelium was observed along with the alteration in cell proliferative and apoptotic patterns. Meanwhile, cultivated tongues treated with MEK inhibitor U0126 or PD98059 showed significantly decreased cell proliferation in the tongue epithelium and the mesenchyme. Based on these results, we suggest that the tongue epithelium is differentiated into multiple epithelial cell layers via the PI3K and PKA pathways in tissue-specific manner during the epithelial-mesenchymal interactions.

Gene profiling involved in fate determination of salivary gland type in mouse embryogenesis

Salivary gland (SG) development involves dynamic epithelial-mesenchymal interactions resulting in the formation of highly branched epithelial structures that produce and secrete saliva. The SG epithelium differentiates into saliva-producing terminal buds, i.e., acini, and transporting ducts. Most studies on the salivary gland have focused on branching morphogenesis; however, acinar cell differentiation underlying the determination of serous or mucous salivary glands is unclear. The objective of this study was to identify the mesenchymal signaling molecules involved in the epithelial differentiation of the salivary gland type as serous or mucous. Salivary glands undergoing stage-specific development, including the parotid gland (PG) and the sublingual gland (SLG) at embryonic day 14.5 (E14.5) were dissected. The glands were treated with dispase II to separate the epithelium and the mesenchyme. RNA from mesenchyme was processed for microarray analysis. Thereafter, microarray data were analyzed to identify putative candidate molecules involved in salivary gland differentiation and confirmed via quantitative reverse transcription polymerase chain reaction. The microarray analysis revealed the expression of 31,873 genes in the PG and SLG mesenchyme. Of the expressed genes 21,026 genes were found to be equally expressed (Fold change 1.000) in both PG and SLG mesenchyme. The numbers of genes expressed over onefold in the PG and SLG mesenchyme were found to be 5247 and 5600 respectively. On limiting the fold-change cut off value over 1.5 folds, only 214 and 137 genes were expressed over 1.5 folds in the PG and the SLG mesenchyme respectively. Our findings suggest that differential expression patterns of the mesenchymal signaling molecules are involved in fate determination of the salivary acinar cell types during mouse embryogenesis. In the near future, functional evaluation of the candidate genes will be performed using gain- and loss-of-function mutation studies during in vitro organ cultivation.

Developmental roles of Meox2 in palatal mucosa differentiation

During palatogenesis, the anterior palate is covered with ortho or parakeratinized epithelium while the posterior palate is covered with non-keratinized epithelium. To elucidate the developmental mechanisms underlying these region-specific differentiation patterns of palatal epithelium along the antero-posterior axis, we employed the tissue recombination assay during in vitro organ cultivation of the developing palate at E16 for 2 days. The recombination assay results revealed that epithelial differentiation with specific localization patterns of Cytokeratin10 and Ki67 are modulated by mesenchymal tissues. Based on these results, we examined the underlying signaling regulations that modulate epithelial differentiation, using laser microdissection and genome wide screening. Our screening data suggested Meox2 (Mesenchyme homeobox 2) to be a key regulator that controls epithelial differentiation in the mesenchymal tissue. To examine the developmental function of Meox2, we employed in vitro organ cultivation along with the knockdown and overexpression of Meox2 using siRNA andMeox2 overexpression vector, respectively, at E14.5 for 2 and 4 days. After 2-day cultivation, we examined the altered expression patterns of related signaling molecules such as Shh and Bmp, using in situ hybridization and RT-qPCR. After 4-day cultivation, we examined the altered histogenesis and localization patterns of Cytokeratin10 and Ki67. Based on the restricted and specific expression of candidate mesenchymal genes and the results of the recombination assay, we conclude that posteriorly expressed Meox2 is involved in the determination of non-keratinized epithelial differentiation through complex signaling regulations in mice palatogenesis.

Grhl3 modulates epithelial structure formation of the circumvallate papilla during mouse development

Department of Biochemistry, School of Dentistry, Kyungpook National University, Daegu, South Korea Department of Dental Hygiene, Gachon University College of Health Science, Incheon, South Korea Department of Oral and Maxillofacial Radiology, School of Dentistry, Kyungpook National University, Daegu, South Korea School of Life Science and Biotechnology, Kyungpook National University, Daegu, South Korea Institute for Hard Tissue and Bio-tooth Regeneration, Kyungpook National University, Daegu, South Korea

Immunolocalization patterns of cytokeratins during mouse salivary gland morphogenesis

Department of Biochemistry, School of Dentistry, Kyungpook National University, Daegu, South Korea Department of Dental Hygiene, Gachon University College of Health Science, Incheon, South Korea Department of Oral and Maxillofacial Radiology, School of Dentistry, Kyungpook National University, Daegu, South Korea School of Life Science and Biotechnology, Kyungpook National University, Daegu, South Korea Institute for Hard Tissue and Bio-tooth Regeneration, Kyungpook National University, Daegu, South Korea

Bortezomib Facilitates Reparative Dentin Formation after Pulp Access Cavity Preparation in Mouse Molar

Introduction The aim of this study was to evaluate in vitro and ex vivo roles of bortezomib, a proteasome inhibitor that binds to the active site of the 26S proteasome, in tertiary dentin formation. Methods We established pulpal access cavity preparation that was treated with or without bortezomib before direct pulp capping with a calcium hydroxide‐based material. We also analyzed bone morphogenetic protein (Bmp)‐ and Wnt‐related signaling molecules using quantitative real‐time polymerase chain reaction. Results In the short‐term observation period, the bortezomib‐treated pulp specimens showed the period‐altered immunolocalization patterns of nestin, CD31, and myeloperoxidase, whereas the control specimens did not. The bortezomib‐treated group showed a complete dentin bridge with very few irregular tubules after 42 days. The micro‐computed tomographic images showed more apparent dentin bridge structures in the treated specimens than were in the controls. Quantitative real‐time polymerase chain reaction analysis showed up‐regulated Bmp and Wnt. Conclusions These findings revealed that treatment with 1 &mgr;mol/L bortezomib induced reparative dentin formation that facilitated the maintenance of the integrity of the remaining pulpal tissue via early vascularization and regulation of Bmp and Wnt signaling. HIGHLIGHTSBortezomib facilitates reparative dentin formation through modulated vascularization and inflammatory response.Molecular mechanisms underlying bortezomib treatment would be related with Bmp and Wnt signaling.Pulpal access cavity preparation using a mice model system permits us to evaluate a range of drugs for their ability of reparative dentin formation.