AKT and MET signalling mediates antiapoptotic radioresistance in head neck cancer cell lines.

OBJECTIVES: Induction of apoptosis is a major mechanism of radiosensitivity in different types of cancer. In contrast, EGFR/PI3K/AKT signalling and recently the presence of so-called cancer stem cells are discussed as reasons for radioresistance. MATERIALS AND METHODS: The study investigates mechanisms of apoptosis, key oncogenes of the PI3K/AKT pathway and the presence of cancer cells with stem cell properties during irradiation in two cell lines (PCI-9A, and PCI-15) of head and neck squamous cell carcinoma. WST-1-tests, qRT-PCR, western blots and FACS analysis were performed for analysis. RESULTS: The two cell lines presented different degrees of cell death upon irradiation. The radiosensitive cell line PCI-9A showed increased apoptosis after irradiation measured by expressed cleaved caspases 3 and 7 while the radioresistant cell line PCI-15 upregulated antiapoptotic Survivin and BCL2A1 mRNA. Besides, increased PI3K/AKT- and ERK1/2-signalling was associated with radioresistance accompanied by loss of PTEN function through phosphorylation on S380. Blockade of pAKT increased radiation-induced cell death, and moreover, led to an upregulation of pMET in the radioresistant cell line. The percentage of ALDH-positive tumour cells was markedly decreased after irradiation in the radiosensitive cell line. CONCLUSIONS: Functional apoptosis is mandatory for sensitivity to irradiation in head neck cancer cells. Upregulation of the AKT-pathway seems to be one reason for poor radioresponse. Activated MET may also predict radioresistance, possibly through ERK1/2 signalling. Moreover MET may indicate the presence of cancer stem cells facilitating radioresistance as shown by increased ALDH expression.

Evaluation of implant-materials as cell carriers for dental stem cells under in vitro conditions.

BACKGROUND: Dental stem cells in combination with implant materials may become an alternative to autologous bone transplants. For tissue engineering different types of soft and rigid implant materials are available, but little is known about the viability and the osteogenic differentiation of dental stem cells on these different types of materials. According to previous studies we proposed that rigid bone substitute materials are superior to soft materials for dental tissue engineering. METHODS: We evaluated the proliferation, the induction of apoptosis and the osteogenic differentiation of dental stem/progenitor cells on a synthetic bone-like material and on an allograft product. The soft materials silicone and polyacrylamide (PA) were used for comparison. Precursor cells from the dental follicle (DFCs) and progenitor cells from the dental apical papilla of retained third molar tooth (dNC-PCs) were applied as dental stem cells in our study. RESULTS: Both dental cell types attached and grew on rigid bone substitute materials, but they did not grow on soft materials. Moreover, rigid bone substitute materials only sustained the osteogenic differentiation of dental stem cells, although the allograft product induced apoptosis in both dental cell types. Remarkably, PA, silicone and the synthetic bone substitute material did not induce the apoptosis in dental cells. CONCLUSIONS: Our work supports the hypothesis that bone substitute materials are suitable for dental stem cell tissue engineering. Furthermore, we also suggest that the induction of apoptosis by bone substitute materials may not impair the proliferation and the differentiation of dental stem cells.

Collagen I induces the expression of alkaline phosphatase and osteopontin via independent activations of FAK and ERK signalling pathways.

OBJECTIVE: Dental follicle cells (DFCs) are the genuine precursors of alveolar osteoblasts. Previous studies suggested that collagen I supports the osteogenic differentiation of DFCs. This study investigated the effect of collagen I on the osteogenic differentiation of human DFCs. MATERIALS AND METHODS: We modified the cell culture surface with collagen I and evaluated the osteogenic differentiation of DFCs by the gene expression of alkaline phosphatase (ALP) and osteopontin (OPN) and by the assessment of the ALP-activity and Alizarin red staining. FAK and ERK signalling pathways regulation were investigated by Western blot analyses. Cell culture media were supplemented with specific inhibitors of FAK (PF573228) or ERK signalling pathways (PD98059). RESULTS: During the osteogenic differentiation collagen I induced the ALP activity and the expression of the late osteogenic differentiation markers OPN, but it did not stimulate mineralization. The FAK/ERK signalling pathway was activated on collagen I and after the induction of osteogenic differentiation. The inhibition of FAK repressed also the activation of ERK signalling in DFCs and the expression of osteogenic markers ALP and OPN on standard cell culture dishes. After cultivation on collagen I, however, the inhibition of ERK was slightly reverted in DFCs. Here, the expression of OPN was restored, while the expression of ALP was still repressed. Interestingly, the expression of OPN was repressed after the inhibition of ERK signalling. CONCLUSION: Collagen I induced independently the expression of the osteogenic differentiation markers ALP and OPN via the FAK and ERK signalling pathways, respectively.

The SUMO-specific isopeptidase SENP3 regulates MLL1/MLL2 methyltransferase complexes and controls osteogenic differentiation.

The ubiquitin-like SUMO system regulates gene expression, but the molecular insights into this process are incomplete. We show that the SUMO-specific isopeptidase SENP3 controls H3K4 methylation by regulating histone-modifying SET1/MLL complexes. SET1/MLL complexes are composed of a histone methyltransferase and the regulatory components WDR5, RbBP5, Ash2L, and DPY-30. MLL1/MLL2 complexes contain menin as additional component and are particularly important for the activation of HOX genes. We demonstrate that SENP3 is associated with MLL1/MLL2 complexes and catalyzes deSUMOylation of RbBP5. This is required for activation of a subset of HOX genes, including the developmental regulator DLX3. In the absence of SENP3, the association of menin and Ash2L with the DLX3 gene is impaired, leading to decreased H3K4 methylation and reduced recruitment of active RNA polymerase II. Importantly, the SENP3-DLX3 pathway dictates osteogenic differentiation of human stem cells, thus delineating the importance of balanced SUMOylation for epigenetic control of gene expression programs.

Laminin regulates the osteogenic differentiation of dental follicle cells via integrin-α2/-ß1 and the activation of the FAK/ERK signaling pathway.

Dental follicle cells (DFCs) are ideal for studies concerning the differentiation of dental precursor cells into alveolar osteoblasts and cementoblasts. Previous investigations have suggested that the extracellular matrix (ECM) protein laminin and the ECM receptor integrin-α2/-ß1 play regulatory roles during the osteogenic differentiation of DFCs. Our present data indicate that laminin impairs alkaline phosphatase (ALP) activity following osteogenic induction while inducing integrin-α2/-ß1 expression, osteogenic differentiation marker elevation, and DFC biomineralization. Integrin-α2/-ß1 facilitates the laminin-dependent expression of osteogenic differentiation markers and the laminin-dependent inhibition of ALP activity. Moreover, these laminin-dependent effects on the osteogenic differentiation of DFCs can be reversed by the inhibition of the FAK/ERK signaling pathway. Thus, laminin regulates the inhibition of early osteogenic differentiation markers and the induction of late osteogenic differentiation markers via integrin-α2/-ß1 and the activation of the FAK/ERK signaling pathway.

NOTCH1 signaling regulates the BMP2/DLX-3 directed osteogenic differentiation of dental follicle cells.

Dental follicle cells (DFCs) are dental stem/progenitor cells and the genuine precursors of alveolar osteoblasts and dental cementoblasts. A previous study showed that the transcription factor DLX3 (distal less homeobox 3) supports the osteogenic differentiation in DFCs via a positive feedback loop with the bone morghogenetic protein (BMP) 2. Until today, however, the control of this BMP2/DLX3 pathway by additional signaling pathways remains elusive. Previous studies also suggested that the NOTCH signaling pathway plays a role in the osteogenic differentiation of DFCs. In this study we showed that DLX3 overexpression and the initiation of the osteogenic differentiation by BMP2 or dexamethasone induced the NOTCH signaling pathway in DFCs. However, the induction of NOTCH-signaling impaired not only the osteogenic differentiation (ALP activity and mineralized nodules) but also the expression of the transcription factor DLX3 and the activation of the BMP-signaling pathway. So, NOTCH signaling plays a regulatory role for the osteogenic differentiation of DFCs. In conclusion, results of our study suggest that the NOTCH-signaling pathway, which is activated during the osteogenic differentiation of DFCs, regulates the BMP2/DLX3 directed differentiation of DFCs via a negative feed-back loop.

Transcription factors for dental stem cell differentiation.

Dental stem cells are excellent for oral and craniofacial tissue engineering. A profound knowledge about molecular processes in dental stem cells is necessary to create treatment approaches in oral medicine. Transcription factors regulate gene expression and provide decisive information for cellular functions. In recent years, the authors have investigated transcriptomes in dental stem cells before and after osteogenic differentiation. The present paper reports on the potential role of selected transcription factors, including ZBTB16, TP53, and SP1, in dental stem cell differentiation. This review discusses putative molecular processes in dental stem cells and summarizes the current knowledge.

Rigid matrix supports osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED).

Stem cell fate can be induced by the grade of stiffness of the extracellular matrix, depending on the developed tissue or complex tissues. For example, a rigid extracellular matrix induces the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs), while a softer surface induces the osteogenic differentiation in dental follicle cells (DFCs). To determine whether differentiation of ectomesenchymal dental precursor cells is supported by similar grades of extracellular matrices (ECMs) stiffness, we examined the influence of the surface stiffness on the proliferation and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED). Cell proliferation of SHED was significantly decreased on cell culture surfaces with a muscle-like stiffness. A dexamethasone-based differentiation medium induced the osteogenic differentiation of SHED on substrates of varying mechanical stiffness. Here, the hardest surface improved the induction of osteogenic differentiation in comparison to that with the softest stiffness. In conclusion, our study showed that the osteogenic differentiation of ectomesenchymal dental precursor cells SHED and DFCs are not supported by similar grades of ECM stiffness.

Transcription factors TP53 and SP1 and the osteogenic differentiation of dental stem cells.

Dental follicle is a loose connective tissue that surrounds the developing tooth. Dental follicle cells (DFCs) have a promising potential for tissue engineering applications including periodontal and bone regeneration. However, little is known about the molecular mechanisms underlying osteogenic differentiation. In a previous study we detected that more than 35% of genes that are regulated during osteogenic differentiation of DFCs have promoter binding sites for the transcription factors TP53 and SP1. However, the role of these transcription factors in dental stem cells is still unknown. We hypothesize that both factors influence the processes of cell proliferation and differentiation in dental stem cells. Therefore, we transiently transfected DFCs and dental pulp stem cells (SHED; Stem cells from human exfoliated decidiuous teeth) with expression vectors for these transcription factors. After overexpression of SP1 and TP53, SP1 influenced cell proliferation and TP53 osteogenic differentiation in both dental cell types. The effects on cell proliferation and differentiation were less pronounced after siRNA mediated silencing of TP53 and SP1. This indicates that the effects we observed after TP53 and SP1 overexpression are indirect and subject of complex regulation. Interestingly, upregulated biological processes in DFCs after TP53-overexpression resemble the downregulated biological processes in SHED after SP1-overexpression. Here, regulated processes are involved in cell motility, wound healing and programmed cell death. In conclusion, our study demonstrates that SP1 and TP53 influence cell proliferation and differentiation and similar biological processes in both SHED and DFCs.

The transcription factor DLX3 regulates the osteogenic differentiation of human dental follicle precursor cells.

The transcription factor DLX3 plays a decisive role in bone development of vertebrates. In neural-crest derived stem cells from the dental follicle (DFCs), DLX3 is differentially expressed during osteogenic differentiation, while other osteogenic transcription factors such as DLX5 or RUNX2 are not highly induced. DLX3 has therefore a decisive role in the differentiation of DFCs, but its actual biological effects and regulation are unknown. This study investigated the DLX3-regulated processes in DFCs. After DLX3 overexpression, DFCs acquired a spindle-like cell shape with reorganized actin filaments. Here, marker genes for cell morphology, proliferation, apoptosis, and osteogenic differentiation were significantly regulated as shown in a microarray analysis. Further experiments showed that DFCs viability is directly influenced by the expression of DLX3, for example, the amount of apoptotic cells was increased after DLX3 silencing. This transcription factor stimulates the osteogenic differentiation of DFCs and regulates the BMP/SMAD1-pathway. Interestingly, BMP2 did highly induce DLX3 and reverse the inhibitory effect of DLX3 silencing in osteogenic differentiation. However, after DLX3 overexpression in DFCs, a BMP2 supplementation did not improve the expression of DLX3 and the osteogenic differentiation. In conclusion, DLX3 influences cell viability and regulates osteogenic differentiation of DFCs via a BMP2-dependent pathway and a feedback control.

Soft matrix supports osteogenic differentiation of human dental follicle cells.

The differentiation of stem cells can be directed by the grade of stiffness of the developed tissue cells. For example a rigid extracellular matrix supports the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs). However, less is known about the relation of extracellular matrix stiffness and cell differentiation of ectomesenchymal dental precursor cells. Our study examined for the first time the influence of the surface stiffness on the proliferation and osteogenic differentiation of human dental follicle cells (DFCs). Cell proliferation of DFCs was only slightly decreased on cell culture surfaces with a bone-like stiffness. The osteogenic differentiation in DFCs could only be initiated with a dexamethasone based differentiation medium after using varying stiffness. Here, the softest surface improved the induction of osteogenic differentiation in comparison to that with the highest stiffness. In conclusion, different to bone marrow derived MSCs, soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs.

ß-tricalcium-phosphate stimulates the differentiation of dental follicle cells.

The use of dental progenitor cells is a straightforward strategy for regenerative dentistry. For example a cell based therapy with dental follicle cells (DFCs) could be a novel therapeutic strategy for the regeneration of oral tissues in the future. For the regeneration of large bone defects for example dental progenitor cells have to be combined with bone substitutes as scaffolds. This study therefore investigated cell attachment (scanning electron microscopy), cell vitality/proliferation (WST-1 assay) and cell differentiation (under in vitro conditions) of human DFCs on synthetic ß-tricalcium phosphate (TCP). DFCs showed considerable cell attachment and proliferation on TCP. Moreover, TCP stimulates osteogenic differentiation in comparison to DFCs with a standard protocol. Here, for example, the osteoblast marker bone sialoprotein (BSP) was highly expressed on TCP, but almost absent in differentiated DFCs without TCP. In conclusion, our study shows that TCP is an excellent scaffold for DFCs for oral tissue regeneration.

The differentiation and gene expression profile of human dental follicle cells.

Human dental follicle cells (DFCs) are progenitor cells. Recent studies supposed that osteogenic differentiation of DFCs is controlled by growth factors such as BMP2 and IGF2, but their influence on the differentiation of DFCs has not been investigated in detail. We examined DFCs after the induction of osteogenic differentiation with BMP2, IGF2 and a standard osteogenic differentiation medium (ODM) with dexamethasone. The alkaline phosphatase (ALP) activity and the calcium accumulation demonstrated osteogenic differentiation after all treatments, but with the most effective differentiation by ODM. Interestingly, markers of the process of osteoblast differentiation were much higher up-regulated in BMP2- or IGF2-treated cells than in ODM-treated cells. To evaluate the reason of these differences, we compared genome-wide expression profiles at an early stage of differentiation. Chondroblast markers in BMP2-differentiated cells and general markers for cell differentiation/proliferation in IGF2-treated cells were significantly regulated. However, ODM-treated DFCs expressed late markers of osteogenic-differentiated DFCs such as the transcription factor ZBTB16 that is not expressed in BMP2- or IGF2-differentiated cells. Importantly, although the BMP-antagonist noggin (NOG) diminishes the phosphorylation of SMAD1 in DFCs, it did not inhibit osteogenic differentiation by ODM and the expression of ZBTB16. In conclusion, this study demonstrates that osteogenic differentiation of DFCs can be stimulated with all tested inducers but also independently of BMP signaling. To evaluate this mechanism, the transcription factor ZBTB16 is a target for further investigations.

Gene expression profiles of dental follicle cells before and after osteogenic differentiation in vitro.

Recently, osteogenic precursor cells were isolated from human dental follicles, which differentiate into cementoblast- or osteoblast-like cells under in vitro conditions after the induction with dexamethasone or insulin. However, mechanisms for osteogenic differentiation are not understood in detail. In a previous study, real-time RT-PCR results demonstrated molecular mechanisms in dental follicle cells (DFCs) during osteogenic differentiation that are different from those in bone-marrow-derived mesenchymal stem cells. We analysed gene expression profiles in DFCs before and after osteogenic differentiation with the Affymetrix GeneChip(R) Human Gene 1.0 ST Array. Transcripts of 98 genes were up-regulated after differentiation. These genes could be clustered into subcategories such as cell differentiation, cell morphogenesis, and skeletal development. Osteoblast-specific transcription factors like osterix and runx2 were constitutively expressed in differentiated DFCs. In contrast, the transcription factor ZBTB16, which promotes the osteoblastic differentiation of mesenchymal stem cells as an up-stream regulator of runx2, was differentially expressed after differentiation. Transcription factors NR4A3, KLF9 and TSC22D3, involved in the regulation of cellular development, were up-regulated as well. In conclusion, we present the first transcriptome of human DFCs before and after osteogenic differentiation. This study sheds new light on the complex mechanism of osteogenic differentiation in DFCs.

Crystallization and preliminary characterization of a novel haem-binding protein of Streptomyces reticuli.

Streptomyces reticuli is a soil-growing Gram-positive bacteria that has been shown to secrete a novel haem-binding protein known as HbpS. Sequence analysis reveals that homologues of HbpS are found in a wide variety of bacteria, including different Actinobacteria and the Gram-negative Vibrio cholera and Klebsiella pneumoniae. The in vivo production of HbpS is greatly increased when S. reticuli is cultured in the presence of the natural antibiotic haemin (Fe3+ oxidized form of haem). Mutational analysis demonstrated that HbpS significantly increases the resistance of S. reticuli to toxic concentrations of haemin. Previous data show that the presence of the newly identified two-component sensor system SenS-SenR also considerably enhances the resistance of S. reticuli to haemin and the redox-cycling compound plumbagin, suggesting a role in the sensing of redox changes. Specific interaction between HbpS and SenS-SenR, which regulates the expression of the catalase-peroxidase CpeB, as well as HbpS, has been demonstrated in vitro. HbpS has been recombinantly overexpressed, purified and crystallized in space group P2(1)3, with a cell edge of 152.5 A. Diffraction data were recorded to a maximal resolution of 2.25 A and phases were obtained using the SAD method from crystals briefly soaked in high concentrations of sodium bromide.