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Nuclear factor I-C (NFIC) regulates dentin sialophosphoprotein (DSPP) and E-cadherin via control of Kruppel-like factor 4 (KLF4) during dentinogenesis.

ABSTRACT: Odontoblasts are a type of terminally differentiated matrix-secreting cells. A number of molecular mechanisms are involved in the differentiation of odontoblasts. Several studies demonstrated that Krüppel-like factor 4 (KLF4) promotes odontoblast differentiation via control of dentin sialophosphoprotein (DSPP). Because nuclear factor I-C (NFIC) is also known to control DSPP, we investigated the relationship between NFIC and KLF4 during odontoblast differentiation. Klf4 mRNA expression was significantly decreased in Nfic(-/-) pulp cells compared with wild type cells. In immunohistochemistry assays, dentin matrix protein 1 (Dmp1), and DSP protein expression was barely observed in Nfic(-/-) odontoblasts and dentin matrix. Nfic bound directly to the Klf4 promoter and stimulated Klf4 transcriptional activity, thereby regulating Dmp1 and DSPP expression during odontoblast differentiation. Nfic or Klf4 overexpression promoted mineralized nodule formation in MDPC-23 cells. In addition, Nfic overexpression also decreased Slug luciferase activity but augmented E-cadherin promoter activity via up-regulation of Klf4 in odontoblasts. Our study reveals important signaling pathways during dentinogenesis: the Nfic-Klf4-Dmp1-Dspp and the Nfic-Klf4-E-cadherin pathways in odontoblasts. Our results indicate the important role of NFIC in regulating KLF4 during dentinogenesis.

SUBMITTER: Lee HK

PROVIDER: S-EPMC4192478 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Nuclear factor I-C (NFIC) regulates dentin sialophosphoprotein (DSPP) and E-cadherin via control of Krüppel-like factor 4 (KLF4) during dentinogenesis.

Lee Hye-Kyung HK Lee Dong-Seol DS Park Su-Jin SJ Cho Kwang-Hee KH Bae Hyun-Sook HS Park Joo-Cheol JC

The Journal of biological chemistry 20140819 41

Odontoblasts are a type of terminally differentiated matrix-secreting cells. A number of molecular mechanisms are involved in the differentiation of odontoblasts. Several studies demonstrated that Krüppel-like factor 4 (KLF4) promotes odontoblast differentiation via control of dentin sialophosphoprotein (DSPP). Because nuclear factor I-C (NFIC) is also known to control DSPP, we investigated the relationship between NFIC and KLF4 during odontoblast differentiation. Klf4 mRNA expression was signif ...[more]

PMID: 25138274

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Project description:BackgroundWe determined recently that dentin sialophosphoprotein (DSPP), a member of the SIBLING (Small integrin-binding ligand N-linked glycoproteins) family of phosphoglycoproteins, is highly upregulated in human oral squamous cell carcinomas (OSCCs) where upregulation is associated with tumor aggressiveness. To investigate the effects of DSPP-silencing on the tumorigenic profiles of the oral cancer cell line, OSC2, short-hairpin RNA (shRNA) interference was employed to silence DSPP in OSC2 cells.Methodology/principal findingsMultiple regions of DSPP transcript were targeted for shRNA interference using hDSP-shRNA lentiviral particles designed to silence DSPP gene expression. Control shRNA plasmid encoding a scrambled sequence incapable of degrading any known cellular mRNA was used for negative control. Following puromycin selection of stable lines of DSSP-silenced OSC2 cells, phenotypic hallmarks of oral carcinogenesis were assayed by western blot and RT-PCR analyses, MTT (cell-viability), colony-formation, modified Boyden-Chamber (migration and invasion), and flow cytometry (cell-cycle and apoptosis) analyses. DSPP-silenced OSC2 cells showed altered cell morphology, reduced viability, decreased colony-formation ability, decreased migration and invasion, G0/G1 cell-cycle arrest, and increased tumor cell sensitivity to cisplatin-induced apoptosis. Furthermore, MMP-2, MMP-3, MMP-9, VEGF, Ki-67, p53, and EGFR were down-regulated. There was a direct correlation between the degree of DSPP-silencing and MMP suppression, as indicated by least squares regression: MMP-2 {(y?=?0.850x, p<0.001) (y?=?1.156x, p<0.001)}, MMP-3 {(y?=?0.994x, p<0.001) (y?=?1.324x, p?=?0.004)}, and MMP-9 {(y?=?1.248x, p?=?0.005, y?=?0.809, p?=?0.013)}.Conclusions/significanceDSPP-silencing in OSC2 cell decreased salient hallmarks of oral tumorigenesis and provides the first functional evidence of a potential key role for DSPP in oral cancer biology. The down-regulation of MMP-2, MMP-3, MMP-9, p53 and VEGF in DSPP-silenced OSC2 cells provides a significant functional/molecular framework for deciphering the mechanisms of DSPP activities in oral cancer biology.

Project description:Dentin Sialoprotein (DSP) and phosphophoryn (PP) are two most dominant non-collagenous proteins in dentin, which are the cleavage products of the DSPP (dentin sialophosphoprotein) precursor protein. The absence of the DSPP gene in DSPP knock-out (KO) mice results in characteristics that are consistent with dentinogenesis imperfecta type III in humans. Symptoms include thin dentin, bigger pulp chamber with frequent pulp exposure as well as abnormal epithelial-mesenchymal interactions, and the appearance of chondrocyte-like cells in dental pulp. To better understand how DSPP influences tooth development and dentin formation, we used a bacterial artificial chromosome transgene construct (BAC-DSPP) that contained the complete DSPP gene and promoter to generate BAC-DSPP transgenic mice directly in a mouse DSPP KO background. Two BAC-DSPP transgenic mouse strains were generated and characterized. DSPP mRNA expression in BAC-DSPP Strain A incisors was similar to that from wild-type (wt) mice. DSPP mRNA expression in BAC-DSPP Strain B animals was only 10% that of wt mice. PP protein content in Strain A incisors was 25% of that found in wt mice, which was sufficient to completely rescue the DSPP KO defect in mineral density, since microCT dentin mineral density analysis in 21-day postnatal animal molars showed essentially identical mineral density in both strain A and wt mice. Strain B mouse incisors, with 5% PP expression, only partially rescued the DSPP KO defect in mineral density, as microCT scans of 21-day postnatal animal molars indicated a reduced dentin mineral density compared to wt mice, though the mineral density was still increased over that of DSPP KO. Furthermore, our findings showed that DSPP dosage in Strain A was sufficient to rescue the DSPP KO defect in terms of epithelial-mesenchymal interactions, odontoblast lineage maintenance, along with normal dentin thickness and normal mineral density while DSPP gene dosage in Strain B only partially rescued the aforementioned DSPP KO defect.

Project description:Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein that is highly expressed in odontoblasts, but only transiently expressed in presecretory ameloblasts during tooth development. We previously generated a knockin mouse model expressing a mouse equivalent (DSPP, p.P19L) of human mutant DSPP (p.P17L; referred to as "DsppP19L/+ "), and reported that DsppP19L/+ and DsppP19L/P19L mice manifested a dentin phenotype resembling human dentinogenesis imperfecta (DGI). In this study, we analyzed pathogenic effects of mutant P19L-DSPP on enamel development in DsppP19L/+ and DsppP19L/P19L mice. Micro-Computed Tomography (μCT) analyses of 7-week-old mouse mandibular incisors showed that DsppP19L/P19L mice had significantly decreased enamel volume and/or enamel density at different stages of amelogenesis examined. Acid-etched scanning electron microscopy (SEM) analyses of mouse incisors demonstrated that, at the mid-late maturation stage of amelogenesis, the enamel of wild-type mice already had apparent decussating pattern of enamel rods, whereas only minute particulates were found in DsppP19L/+ mice, and no discernible structures in DsppP19L/P19L mouse enamel. However, by the time that incisor enamel was about to erupt into oral cavity, distinct decussating enamel rods were evident in DsppP19L/+ mice, but only poorly-defined enamel rods were revealed in DsppP19L/P19L mice. Moreover, μCT analyses of the mandibular first molars showed that DsppP19L/+ and DsppP19L/P19L mice had a significant reduction in enamel volume and enamel density at the ages of 2, 3, and 24weeks after birth. Backscattered and acid-etched SEM analyses revealed that while 3-week-old DsppP19L/+ mice had similar pattern of enamel rods in the mandibular first molars as age-matched wild-type mice, no distinct enamel rods were observed in DsppP19L/P19L mice. Yet neither DsppP19L/+ nor DsppP19L/P19L mice showed well-defined enamel rods in the mandibular first molars by the age of 24weeks, as judged by backscattered and acid-etched SEM. In situ hybridization showed that DSPP mRNA level was markedly reduced in the presecretory ameloblasts, but immunohistochemistry revealed that DSP/DSPP immunostaining signals were much stronger within the presecretory ameloblasts in Dspp mutant mice than in wild-type mice. These results suggest that mutant P19L-DSPP protein caused developmental enamel defects in mice, which may be associated with intracellular retention of mutant DSPP in the presecretory ameloblasts.

Dataset Information

Nuclear factor I-C (NFIC) regulates dentin sialophosphoprotein (DSPP) and E-cadherin via control of Kruppel-like factor 4 (KLF4) during dentinogenesis.

Publications

Nuclear factor I-C (NFIC) regulates dentin sialophosphoprotein (DSPP) and E-cadherin via control of Krüppel-like factor 4 (KLF4) during dentinogenesis.

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