Molekulare Untersuchung des Einflusses der Proteinkinase C auf die osteogene Differenzierung von dentalen Follikelzellen

Dentale Follikelzellen (DFCs, engl. Dental follicle cells) sind die endogenen Stamm-/Vorläuferzellen des Zahnhalteapparats und können in alveoläre Osteoblasten, Zementoblasten und Fibroblasten des parodontalen Ligaments differenzieren. DFCs besitzen großes Potential für regenerative Therapien von (dentalem) Knochengewebe, wobei zunächst eine genaue Kenntnis der molekularen Mechanismen während der osteogenen Differenzierung, welche sich in vitro durch BMP2 (engl. Bone morphogenetic protein 2) oder Dexamethason induzieren lässt, erforderlich ist. Nachdem Vorarbeiten zeigten, dass klassische Isoformen der Proteinkinase C (PKC) während der Osteogenese von DFCs herunterreguliert werden und diese hemmen, sollten in der vorliegenden Arbeit die zugrundeliegenden Mechanismen evaluiert werden. Hierfür wurde die Regulierung potentieller Zielproteine und Signalwege nach osteogener Induktion und Hemmung klassischer PKCs untersucht sowie deren Einfluss auf osteogene Marker evaluiert. Dabei wurde festgestellt, dass klassische PKCs die Kinase Akt regulieren und stromabwärts durch Phosphorylierung von GSK3β (Glykogensynthase-Kinase 3β) die Aktivität des Proteins β-Catenin beeinflussen, welches eine wichtige Rolle für die Induktion der Osteogenese spielt. Während Akt zwar die Expression von aktivem β-Catenin unterstützte, führte eine Überaktivierung der Kinase jedoch zur Inhibition der Differenzierung und des Signalwegs stromabwärts von BMP2. Darüber hinaus konnte eine Regulierung des NF-κB (engl. Nuclear factor kappa B)-Signalwegs durch klassische PKCs und Akt nachgewiesen werden, wobei NF-κB die Proliferation und osteogene Differenzierung von DFCs unterdrückte. Dahingegen konnte keine direkte Beteiligung von klassischen PKCs oder Akt an der BMP2-induzierten Aktivierung der Proteinkinase A (PKA), welche die Osteogenese unterstützt, gefunden werden. Stattdessen zeigten die Versuche, dass die PKA-Aktivierung von der intrazellulären Expression des Proteins PTHrP (engl. Parathyroid hormone-related protein) abhängt, und dass PKA den Signalweg stromabwärts von BMP2 durch eine negative Rückkopplung hemmt. Des Weiteren konnte eine Induktion des mitochondrialen Energiemetabolismus und des oxidativen Stresses während der osteogenen Differenzierung und nach Inhibition klassischer PKCs festgestellt werden, während eine Hemmung des mitochondrialen Stoffwechsels die Mineralisierung unterdrückte. Eine Evaluierung von Proteinen, welche oxidativen Stress neutralisieren können, zeigte eine Stimulierung der Catalase-Expression und eine Herunterregulierung der Expression von GPX1 (Glutathionperoxidase 1) während der Differenzierung und nach Inhibition klassischer PKCs. Überdies führte eine Hemmung der Catalase zu einer verringerten Mineralisierung. Stromaufwärts von PKC wurde PTHrP für die Aktivität der Kinase benötigt, während das Protein WNT5A (engl. Wnt family member 5A) diese unterdrückte. Die Ergebnisse legten dar, dass klassische Isoformen von PKC die osteogene Differenzierung von DFCs über verschiedene Mechanismen hemmen, wobei insbesondere biologische Prozesse reguliert werden, die in späteren Differenzierungsphasen wichtig sind

Classical isoforms of protein kinase C (PKC) and Akt regulate the osteogenic differentiation of human dental follicle cells via both β-catenin and NF-κB

Background Human dental follicle cells (DFCs) are the precursor cells of the periodontium with a high potential for regenerative therapies of (alveolar) bone. However, the molecular mechanisms of osteogenic differentiation are inadequately understood. Classical isoforms of protein kinase C (PKC) are reported to inhibit osteogenesis of stem/precursor cells. This study evaluated the role of classical PKCs and potential downstream targets on the osteogenic differentiation of DFCs. Methods DFCs were osteogenic differentiated with dexamethasone or bone morphogenetic protein 2 (BMP2). Expression of PKC and potential upstream/downstream regulators was manipulated using activators, inhibitors, and small interfering ribonucleic acid (siRNA). Expression of proteins was examined by Western blot analysis, while the activation levels of enzymes and transcription factors were examined by their phosphorylation states or by specific activation assays. Expression levels of osteogenic markers were examined by RT-qPCR (reverse transcription-quantitative polymerase chain reaction) analysis. Activity of alkaline phosphatase (ALP) and accumulation of calcium nodules by Alizarin Red staining were measured as indicators of mineralization. Results Classical PKCs like PKCα inhibit the osteogenic differentiation of DFCs, but do not interfere with the induction of differentiation. Inhibition of classical PKCs by Gö6976 enhanced activity of Akt after osteogenic induction. Akt was also regulated during differentiation and especially disturbed BMP2-induced mineralization. The PKC/Akt axis was further shown to regulate the canonical Wnt signaling pathway and eventually nuclear expression of active β-catenin during dexamethasone-induced osteogenesis. Moreover, the nuclear factor “kappa-light-chain-enhancer” of activated B cells (NF-κB) pathway is regulated during osteogenic differentiation of DFCs and via the PKC/Akt axis and disturbs the mineralization. Upstream, parathyroid hormone-related protein (PTHrP) sustained the activity of PKC, while Wnt5a inhibited it. Conclusions Our results demonstrate that classical PKCs like PKCα and Akt regulate the osteogenic differentiation of DFCs partly via both β-catenin and NF-κB

High endogenous expression of parathyroid hormone-related protein (PTHrP) supports osteogenic differentiation in human dental follicle cells

Dental follicle cells (DFCs) are progenitor cells for mineralizing cells such as alveolar osteoblasts, but little is known about the mechanisms of the differentiation. Interestingly, different cell lines sometimes have different potentials to differentiate into mineralizing cells. In this study, we compared two different DFC lines, with one cell line (DFC_B) showing a high alkaline phosphatase (ALP) activity in long-term cultures with standard medium and a reliable mineralizing potential. However, the other cell line DFC_A shows low ALP activity in standard medium and almost no mineralization. Known osteogenic markers such as RUNX2 were similarly expressed in both cell lines. However, the proosteogenic signaling pathway of the bone morphogenetic protein (BMP) is induced in DFC_B, and the parathyroid hormone-related protein (PTHrP), which is involved in tooth root development, was also expressed more strongly. Previous studies have shown that the secreted PTHrP negatively regulate the transition from pre-osteoblastic progenitors to osteoblasts, but we showed that an inhibition of PTHrP gene expression reduced the ALP activity and the BMP-signaling pathway. In addition, endogenously expressed PTHrP is located in the cell nucleus. In contrast, supplementation of PTHrP or an inhibitor for the PTHrP receptor did not affect the ALP activity of DFC_B. In conclusion, our data suggest that a high endogenous expression of PTHrP in DFCs supports the induction of osteogenic differentiation via an intracrine mode

AMP-activated protein kinase and the down-stream activated process of autophagy regulate the osteogenic differentiation of human dental follicle cells

Objective: Dental follicle cells (DFCs) are progenitors of alveolar osteoblasts. AMP-activated protein kinase (AMPK) and the down-stream activated autophagy process play a key role in cellular energy and metabolic homeostasis and are involved in many biological processes including differentiation. Previous studies showed ambiguous results about the role of AMPK and autophagy in osteogenic differentiation of various osteogenic progenitors, but the role of AMPK and autophagy in DFCs is unknown. This study examined the role of AMPK and autophagy in the osteogenic differentiation of DFCs. Materials and methods: We evaluated the expression of AMPK isoforms and autophagy markers during osteogenic differentiation via Western Blot analyses and the impact of AMPK / autophagy activators and inhibitors and siRNAs on osteogenic differentiation via ALP activity assay, Alizarin Red staining and Real-Time Reverse-Transcription PCR. Results: We have shown that expression of AMPK and autophagy markers are regulated during osteogenic differentiation and that activation of AMPK inhibits the ALP activity and other osteogenic markers after induction of osteogenic differentiation, while inhibition of AMPK and autophagy increased the expression of some osteogenic markers. In long-term cultures with osteogenic differentiation medium, however, both the activation and the inhibition of AMPK significantly inhibited biomineralization of DFCs. In contrast, activation or inhibition of autophagy barely affected early differentiation markers, while autophagy inhibition enhanced biomineralization and autophagy activation diminished mineralization capability of DFCs. Conclusions: AMPK regulates the osteogenic differentiation in earlier stages while indirectly affecting biomineralization at least partly via autophagy. The osteogenic differentiation of DFCs is sensitive to changes in AMPK and autophagic activity

Protein kinase A is activated during bone morphogenetic protein 2-induced osteogenic differentiation of dental follicle stem cells via endogenous parathyroid hormone-related protein

Objective: The aim of this study was to investigate the mechanisms of how protein kinase A (PKA) is activated during bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation in dental follicle stem cells. Design: Human dental follicle stem cells were cultured and treated with a BMP2-containing osteogenic differentiation medium or differentiation medium without BMP2. Specific siRNAs and substances/proteins were used to modulate pathways. PKA activity and activity of alkaline phosphatase were determined. Expression of targets was measured by Western Blots and reverse transcription-quantitative polymerase chain reaction, while protein interactions were investigated by immunoprecipitation. Immunofluorescence staining was used for subcellular target localization. Results: PKA activity is stimulated after osteogenic induction by BMP2. Differentiation medium without BMP2 strongly induces BMP2 gene expression, which correlates with downstream target expression. Elevation of cAMP levels does not affect alkaline phosphatase activity and PKA does not directly interact with Smad 4. However, PKA activation requires expression of parathyroid hormone-related protein (PTHrP), which is stimulated after BMP2-induced differentiation. Furthermore, neither supplementation with PTHrP nor with the receptor antagonist parathyroid hormone (7-34) affects PKA activity. Thus, endogenous PTHrP expression is required for PKA activation and immunofluorescence staining shows that PTHrP is mainly located in the nucleus of dental follicle stem cells. Beyond, knockdown of PKA stimulates the BMP2 signaling pathway and down-stream expression of PTHrP. Conclusions: BMP2-induced osteogenic differentiation activates PKA in dental follicle stem cells via endogenous expression of PTHrP. Additionally, PKA inhibits BMP2 signaling and expression of PTHrP in a negative feedback loop

Analysis of the phosphoproteome in human dental follicle cells during osteogenic differentiation

Dental follicle cells (DFCs) are osteogenic progenitor cells and are well suited for molecular studies of differentiation of alveolar osteoblasts. A recent study examined the metabolism in DFCs during osteogenic differentiation and showed that energy metabolism is increased after 14 days of differentiation (mid phase). However, previous studies have examined proteomes at early (2 h, 24 h) or very late (28 days) stages of differentiation, but not during the phase of increased metabolic activity. In this study, we examined the phosphoproteome at the mid phase (14 days) of osteogenic differentiation. Analysis of DFC phosphoproteomes showed that during this phase of osteogenic differentiation, proteins that are part of signal transduction are significantly regulated. Proteins involved in the regulation of the cytoskeleton and apoptosis were also increased in expression. As osteogenic differentiation induced oxidative stress and apoptosis in DFCs, the oxidative stress defense protein, catalase, was also upregulated during osteogenic differentiation, which supports the biomineralization of DFCs. In summary, this study revealed that during the middle phase (14 days) of osteogenic differentiation, processes in DFCs related to the control of cell organization, apoptosis, and oxidative stress are regulated

p53 inhibits the osteogenic differentiation but does not induce senescence in human dental follicle cells

Replicative senescence causes a reduced osteogenic differentiation potential of senescent dental follicle cells (DFCs). The transcription factor p53 is often involved in the induction of cellular senescence, but little is known about its role in DFCs. This study examined for the first time the role of p53 compared to its pro-proliferative antagonist E2F-1 in terms of osteogenic differentiation potential and induction of senescence. Protein expression of E2F-1 decreased during cell aging, while p53 was expressed constitutively. Gene silencing of E2F1 (E2F-1) inhibited the proliferation rate of DFCs and increased the induction of cellular senescence. The induction of cellular senescence is regulated independently of the gene expression of TP53 (p53), since its gene expression depends on the expression of E2F1. Moreover, gene silencing of TP53 induced E2F1 gene expression and increased cell proliferation, but did not affect the rate of induction of cellular senescence. TP53 knockdown further induced the alkaline phosphatase and mineralization in DFCs. However, the simultaneous silencing of TP53 and E2F1 did not inhibit the inductive effect of TP53 knockdown on osteogenic differentiation, indicating that this effect is independent of E2F-1. In summary, our results suggest that p53 inhibits osteogenic differentiation and cell proliferation in senescent DFCs, but is not significantly involved in senescence induction

Energy Metabolism and Lipidome Are Highly Regulated during Osteogenic Differentiation of Dental Follicle Cells

Dental follicle cells (DFCs) are stem/progenitor cells of the periodontium and give rise to alveolar osteoblasts. However, understanding of the molecular mechanisms of osteogenic differentiation, which is required for cell-based therapies, is delimited. This study is aimed at analyzing the energy metabolism during the osteogenic differentiation of DFCs. Human DFCs were cultured, and osteogenic differentiation was induced by either dexamethasone or bone morphogenetic protein 2 (BMP2). Previous microarray data were reanalyzed to examine pathways that are regulated after osteogenic induction. Expression and activity of metabolic markers were evaluated by western blot analysis and specific assays, relative amount of mitochondrial DNA was measured by real-time quantitative polymerase chain reaction, the oxidative state of cells was determined by a glutathione assay, and the lipidome of cells was analyzed via mass spectrometry (MS). Moreover, osteogenic markers were analyzed after the inhibition of fatty acid synthesis by 5-(tetradecyloxy)-2-furoic acid or C75. Pathway enrichment analysis of microarray data revealed that carbon metabolism was amongst the top regulated pathways after osteogenic induction in DFCs. Further analysis showed that enzymes involved in glycolysis, citric acid cycle, mitochondrial activity, and lipid metabolism are differentially expressed during differentiation, with most markers upregulated and more markedly after induction with dexamethasone compared to BMP2. Moreover, the cellular state was more oxidized, and mitochondrial DNA was distinctly upregulated during the second half of differentiation. Besides, MS of the lipidome revealed higher lipid concentrations after osteogenic induction, with a preference for species with lower numbers of C-atoms and double bonds, which indicates a de novo synthesis of lipids. Concordantly, inhibition of fatty acid synthesis impeded the osteogenic differentiation of DFCs. This study demonstrates that energy metabolism is highly regulated during osteogenic differentiation of DFCs including changes in the lipidome suggesting enhanced de novo synthesis of lipids, which are required for the differentiation process

2019 updated consensus statement on the diagnosis and treatment of pediatric pulmonary hypertension : The European Pediatric Pulmonary Vascular Disease Network (EPPVDN), endorsed by AEPC, ESPR and ISHLT

The European Pediatric Pulmonary Vascular Disease Network is a registered, non-profit organization that strives to define and develop effective, innovative diagnostic methods and treatment options in all forms of pediatric pulmonary hypertensive vascular disease, including pulmonary hypertension (PH) associated with bronchopulmonary dysplasia, PH associated with congenital heart disease (CHD), persistent PH of the newborn, and related cardiac dysfunction. The executive writing group members conducted searches of the PubMed/MEDLINE bibliographic database (1990–2018) and held face-to-face and web-based meetings. Ten section task forces voted on the updated recommendations, based on the 2016 executive summary. Clinical trials, meta-analyses, guidelines, and other articles that include pediatric data were searched using the term “pulmonary hypertension” and other keywords. Class of recommendation (COR) and level of evidence (LOE) were assigned based on European Society of Cardiology/American Heart Association definitions and on pediatric data only, or on adult studies that included >10% children or studies that enrolled adults with CHD. New definitions by the World Symposium on Pulmonary Hypertension 2018 were included. We generated 10 tables with graded recommendations (COR/LOE). The topics include diagnosis/monitoring, genetics/biomarkers, cardiac catheterization, echocardiography, cardiac magnetic resonance/chest computed tomography, associated forms of PH, intensive care unit/lung transplantation, and treatment of pediatric PH. For the first time, a set of specific recommendations on the management of PH in middle- and low-income regions was developed. Taken together, these executive, up-to-date guidelines provide a specific, comprehensive, detailed but practical framework for the optimal clinical care of children and young adults with PH