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Amelogenin isoforms, including full-length amelogenin (AMEL) and leucine-rich amelogenin peptide (LRAP), are major components of the enamel matrix, and are considered as signaling molecules in epithelial-mesenchymal interactions regulating tooth development and periodontal regeneration. Nevertheless, the molecular mechanisms involved are still poorly understood. The aim of the present study was to identify novel binding partners for amelogenin isoforms in the cementoblast (OCCM-30), using an affinity purification assay (GST pull-down) followed by mass spectrometry and immunoblotting. Protein-protein interaction analysis for AMEL and LRAP evidenced the plasminogen activation system (PAS) as a potential player regulating OCCM-30 response to amelogenin isoforms. For functional assays, PAS was either activated (plasmin) or inhibited (ε-aminocaproic acid [aminocaproic]) in OCCM-30 cells and the cell morphology, mineral nodule formation, and gene expression were assessed. PAS inhibition (EACA 100 mM) dramatically decreased mineral nodule formation and expression of OCCM-30 differentiation markers, including osteocalcin (Bglap), bone sialoprotein (Ibsp), osteopontin (Spp1), tissue-nonspecific alkaline phosphatase (Alpl) and collagen type I (Col1a1), and had no effect on runt-related transcription factor 2 (Runx2) and Osterix (Osx) mRNA levels. PAS activation (plasmin 5 µg/µl) significantly increased Col1a1 and decreased Bglap mRNA levels (p < .05). Together, our findings shed new light on the potential role of plasminogen signaling pathway in the control of the amelogenin isoform-mediated response in cementoblasts and provide new insights into the development of targeted therapies., (© 2019 Wiley Periodicals, Inc.)

Background: Periodontal ligament (PDL) has been reported to be a source of mesenchymal stem cells (MSCs).New vascular networks from undifferentiated cells are essential for repair/regeneration of specialized tissues, including PDL. The current study aims to determine potential of CD105(+)-enriched cell subsets of periodontal ligament cells (PDLSCs) to differentiate into endothelial cell (EC)-like cells and to give insights into the mechanism involved., Methods: CD105(+)-enriched PDLSCs were induced to EC differentiation by endothelial growth medium 2 (EGM-2) for 3, 7, 14, and 21 days, with mRNA/protein levels and functional activity assessed by: 1) real-time polymerase chain reaction; 2) Western blotting; 3) fluorescence-activated cell sorting; 4) immunohistochemistry; 5) immunofluorescence; 6) matrigel; and 7) small interfering RNA assays., Results: Data analyses demonstrated that EGM-2 treated PDLSCs presented increased expression of EC markers, including: 1) CD105; 2) kinase domain-containing receptor; and 3) Ulex europaeus agglutinin 1, and were able to form cord/tube-like structures. Gene and protein expression analysis showed that neuropilin 2 (NRP2), a key factor for vascular development, was significantly downregulated during EC differentiation. NRP2 was constitutively expressed in mouse PDL tissues by immunohistochemistry analysis, and NRP2 knockdown in CD105(+)-enriched PDLSCs resulted in increased cord/tube-like structures in a matrigel assay., Conclusion: These findings demonstrated the potential of CD105(+)-enriched PDLSCs to support angiogenesis, and NRP2 as a pivotal factor regulating this process.

The double-stranded RNA-dependent protein kinase (PKR) plays a critical role in various biological responses including antiviral defense, cell differentiation, apoptosis, and tumorigenesis. In this study, we investigated whether PKR could affect the post-translational modifications of STAT1 protein and whether these modifications regulate osteoblast differentiation. We demonstrated that PKR was necessary for the ubiquitination of STAT1 protein. The expressions of bone-related genes such as type I collagen, integrin binding sialoprotein, osteopontin, and osterix were suppressed in osteoblasts lacking PKR activity. In contrast, the expressions of interleukin-6 and matrix metalloproteinases 8 and 13 increased in PKR-mutated osteoblasts. The expression and degradation of STAT1 protein were regulated by PKR in a SLIM-dependent pathway. Inhibition of SLIM by RNA interference resulted in the decreased activity of Runx2 in osteoblasts. Stimulation of interleukin-6 expression and suppression of alkaline phosphatase activity were regulated through by SLIM-dependent pathway. However, expressions of bone-related genes and MMPs were regulated by SLIM-independent pathway. Our present results suggest that the aberrant accumulation of STAT1 protein induced by loss of PKR regulate osteoblast differentiation through both SLIM/STAT1-dependent and -independent pathways.