Inhibition of caspase-8 cascade restrains the osteoclastogenic fate of bone marrow cells.

Osteoclasts are multinucleated cells of hematopoietic origin, with a pivotal role in bone development and remodeling. Failure in osteoclast differentiation and activation leads to various bone disorders; thus, attention has focused on a search of molecules involved in osteoclast regulatory pathways. Caspase-8 appears to be an interesting candidate for further exploration, due to its potential function in bone development and homeostasis. Mouse bone marrow cells were differentiated into osteoclasts by RANKL stimulation. Increased activation of caspase-8 and its downstream executioner caspases (caspase-3 and caspase-6) was found during osteoclastogenesis. Subsequent inhibition of caspase-8, caspase-3, or caspase-6, respectively, during osteoclast differentiation showed distinct changes in the formation of TRAP-positive multinucleated cells and reduced expression of osteoclast markers including Acp5, Ctsk, Dcstamp, and Mmp9. Analysis of bone matrix resorption confirmed significantly reduced osteoclast function after caspase inhibition. The results clearly showed the role of caspases in the proper development of osteoclasts and contributed new knowledge about non-apoptotic function of caspases.

Markers of dental pulp stem cells in in vivo developmental context.

Teeth and their associated tissues contain several populations of mesenchymal stem cells, one of which is represented by dental pulp stem cells (DPSCs). These cells have mainly been characterised in vitro and numerous positive and negati ve markers for these cells have been suggested. To investigate the presence and localization of these molecules during development, forming dental pulp was examined using the mouse first mandibular molar as a model. The stages corresponding to postnatal (P) days 0, 7, 14, and 21 were investigated. The expression was monitored using customised PCR Arrays. Additionally, in situ localization of the key trio of markers (Cd73, Cd90, Cd105 coded by genes Nt5e, Thy1, Eng) was performed at prenatal and postnatal stages using immunohistochemistry. The expression panel of 24 genes assigned as in vitro markers of DPSCs or mesenchymal stem cells (MSCs) revealed their developmental dynamics during formation of dental pulp mesenchyme. Among the positive markers, Vcam1, Fgf2, Nes were identified as increasing and Cd44, Cd59b, Mcam, Alcam as decreasing between perinatal vs. postnatal stages towards adulthood. Within the panel of negative DPSC markers, Cd14, Itgb2, Ptprc displayed increased and Cd24a decreased levels at later stages of pulp formation. Within the key trio of markers, Nt5e did not show any significant expression difference within the investigated period. Thy1 displayed a strong decrease between P0 and P7 while Eng increased between these stages. In situ localization of Cd73, Cd90 and Cd105 showed them overlap in differentiated odontoblasts and in the sub-odontoblastic layer that is speculated to host odontoblast progenitors. The highly prevalent expression of particularly Cd73 and Cd90 opens the question of potential multiple functions of these molecules. The results from this study add to the in vitro based knowledge by showing dynamics in the expression of DPSC/MSC markers during dental pulp formation in an in vivo context and thus with respect to the natural environment important for commitment of stem cells.

Both Hypoxia-Inducible Factor 1 and MAPK Signaling Pathway Attenuate PI3K/AKT via Suppression of Reactive Oxygen Species in Human Pluripotent Stem Cells.

Mild hypoxia (5% O2) as well as FGFR1-induced activation of phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) and MAPK signaling pathways markedly support pluripotency in human pluripotent stem cells (hPSCs). This study demonstrates that the pluripotency-promoting PI3K/AKT signaling pathway is surprisingly attenuated in mild hypoxia compared to the 21% O2 environment. Hypoxia is known to be associated with lower levels of reactive oxygen species (ROS), which are recognized as intracellular second messengers capable of upregulating the PI3K/AKT signaling pathway. Our data denote that ROS downregulation results in pluripotency upregulation and PI3K/AKT attenuation in a hypoxia-inducible factor 1 (HIF-1)-dependent manner in hPSCs. Using specific MAPK inhibitors, we show that the MAPK pathway also downregulates ROS and therefore attenuates the PI3K/AKT signaling-this represents a novel interaction between these signaling pathways. This inhibition of ROS initiated by MEK1/2-ERK1/2 may serve as a negative feedback loop from the MAPK pathway toward FGFR1 and PI3K/AKT activation. We further describe the molecular mechanism resulting in PI3K/AKT upregulation in hPSCs-ROS inhibit the PI3K's primary antagonist PTEN and upregulate FGFR1 phosphorylation. These novel regulatory circuits utilizing ROS as second messengers may contribute to the development of enhanced cultivation and differentiation protocols for hPSCs. Since the PI3K/AKT pathway often undergoes an oncogenic transformation, our data could also provide new insights into the regulation of cancer stem cell signaling.