Lysosomal pH Plays a Key Role in Regulation of mTOR Activity in Osteoclasts.

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in the regulation of cell growth. It has been shown to play an important role in osteoclast differentiation, particularly at the earlier stages of osteoclastogenesis. mTOR activation and function, as part of mTORC1 complex, is dependent on lysosomal localization and the vacuolar H(+) -ATPase (V-ATPase) activity; however, the precise mechanism is still not well understood. Using primary mouse osteoclasts that are known to have higher lysosomal pH due to R740S mutation in the V-ATPase a3 subunit, we investigated the role of lysosomal pH in mTORC1 signaling. Our results demonstrated that +/R740S cells had increased basal mTOR protein levels and mTORC1 activity compared to +/+ osteoclasts, while mTOR gene expression was decreased. Treatment with lysosomal inhibitors chloroquine and ammonium chloride, compounds known to raise lysosomal pH, significantly increased mTOR protein levels in +/+ cells, confirming the importance of lysosomal pH in mTOR signaling. These results also suggested that mTOR could be degraded in the lysosome. To test this hypothesis, we cultured osteoclasts with chloroquine or proteasomal inhibitor MG132. Both chloroquine and MG132 increased mTOR and p-mTOR protein levels in +/+ osteoclasts, suggesting that mTOR undergoes both lysosomal and proteasomal degradation. Treatment with cycloheximide, an inhibitor of new protein synthesis, confirmed that mTOR is constitutively expressed and degraded. These results show that, in osteoclasts, the lysosome plays a key role not only in mTOR activation but also in its deactivation through protein degradation, representing a novel molecular mechanism of mTOR regulation.

The influence of implant design on the kinetics of osseointegration and bone anchorage homeostasis.

Titanium implants have shown considerable success in terms of achieving quick and long-lasting stability in bone through the process of osseointegration. Further work aims to improve implant success rates by modifying implant design on the nano-, micro-, and macro- scales with the goal of achieving higher levels of bone anchorage more quickly. However, the most frequently used methods of analysis do not investigate bone anchorage as a whole but as a series of discrete points, potentially missing relevant insight which could inform the effects of topography on these 3 scale ranges. Herein we utilize an asymptotic curve fitting method to obtain a biologically relevant description of reverse torque data and compare the anchorage of 12 different implant groups. Implant surface topography had a significant effect on the rate and degree of anchorage achieved during the initial bone formation period of osseointegration but was not found to influence the relative change in anchorage during bony remodeling. Threaded implants significantly decreased the time required to reach peak anchorage compared to non-threaded implants and implants with micro-topographically complex surfaces required greater torque to be removed than implants without such features. Nanotopography increased overall anchorage and decreased the time required to reach peak anchorage but to a lesser degree than microtopography or macrogeometry respectively. The curve fitting method utilized in the present study allows for a more integrated analysis of bone anchorage and permits investigation of osseointegration with respect to time, which may lead to a more targeted approach to implant design.

Mitochondrial function is a critical determinant of IL-1-induced ERK activation.

Interleukin-1 (IL-1) is a potent, proinflammatory cytokine, but local environmental factors in inflamed sites or in sepsis may affect cell metabolism and energetics, including the amplitude and duration of IL-1-induced signals, thereby leading to loss of tissue homeostasis. Currently, the mechanisms by which disruption of cell energetics affects inflammatory signaling are incompletely understood. Here, we examined the impact of cell energetics and mitochondrial function on the regulation of IL-1-induced Ca2+ signals and ERK activation in human gingival fibroblasts, cells that are important targets for IL-1-induced destruction of extracellular matrix in inflamed connective tissues. In untreated cells, IL-1 induced a prolonged increase of free intracellular calcium, which was required for ERK activation. Inhibition of cellular energetics by selective depolarization of mitochondria blocked Ca2+ uptake and almost completely abolished IL-1-induced cytosolic Ca2+ signals and ERK activation. IL-1 caused rapid Ca2+ release from the endoplasmic reticulum (ER), concomitant with mitochondrial Ca2+ uptake from ER and non-ER stores. Disruption of mitochondrial energetics abrogated IL-1 induced Ca2+ release from the ER but left other vital cellular functions intact. The negative effect of mitochondrial depolarization on ER release was bypassed by BAPTA/AM, indicating that mitochondrial Ca2+ buffering is the key mechanism in regulating ER release. Thus, in gingival fibroblasts, mitochondrial Ca2+ uptake is essential not only for shaping the kinetics and duration, but also the generation of, IL-1-induced Ca2+ signals. Consequently, mitochondria regulate key downstream effectors of IL-1, including MAP kinases.

Dental implant surface treatments may modulate cytokine secretion in Porphyromonas gingivalis-stimulated human gingival fibroblasts: a comparative study.

Peri-implantitis is an inflammation that affects dental implants and can lead to implant loss. The aim of this study was to analyze the in vitro effect of different implant surface treatments on cytokine production by human gingival fibroblasts (HGFs) stimulated or not with Porphyromonas gingivalis lipopolysaccharide (PgLPS). Six different titanium implants were tested: turned, sandblasted, anodized, acid-etched, TiO2-blasted/acid-etched, and grit-blasted/acid-etched. HGFs were seeded with each implant in a 6-well plate and assayed before LPS treatment (-LPS) or after 36 h of LPS (+LPS) treatment. Protein concentrations were measured using a Pierce bicinchoninic acid (BCA) assay and cytokine secretions were analyzed using a multiplex cytokine array. Scanning electron microscopy was performed for sterile implants and after cell attachment. Protein levels were consistent across all implants indicating that cell growth was uniform (p > 0.05). Sandblasted and turned surfaces significantly increased the secretion of interleukin (IL)-6, -8, -10, MCP-1 and VEGF (p < 0.05) when compared with the other surfaces. PgLPS stimulus increased cytokine secretion in all tested surfaces. In conclusion, different implant surfaces had various effects on HGFs' cytokine secretion. The findings may provide insights into the progression of peri-implantitis.

Modulation of human neutrophil functions in vitro by Treponema denticola major outer sheath protein.

In this study of human polymorphonuclear leukocytes (PMNs), pretreatment with Treponema denticola major outer sheath protein (Msp) inhibited formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis, phagocytosis of immunoglobulin G-coated microspheres, fMLP-stimulated calcium transients, and actin assembly. Msp neither altered oxidative responses to phorbol myristate or fMLP nor induced apoptosis. Msp selectively impairs chemotaxis and phagocytosis by impacting the PMN cytoskeleton.

The major outer sheath protein of Treponema denticola inhibits the binding step of collagen phagocytosis in fibroblasts.

Bacterial infections contribfute to the chronicity of connective tissue lesions in part by perturbing extracellular matrix remodelling processes. We examined a novel mechanism by which the major outer sheath protein (Msp) of the spirochaete Treponema denticola disrupts matrix remodelling mediated by intracellular digestion of collagen. The initial collagen-binding step of phagocytosis was examined in human gingival fibroblasts and Rat-2 fibroblasts. Cells were pretreated with Msp or vehicle, and binding of collagen-coated beads was measured by flow cytometry. Exposure to Msp induced a dose- and time-dependent decrease in cells that bound collagen beads; the inhibition of binding was reversed by absorption with anti-Msp antibodies. Msp-treated fibroblasts remained viable but underwent actin reorganization, including the assembly of a dense meshwork of subcortical actin filaments. Shear force assays showed that Msp abrogated collagen-binding interactions in the minimal affinity range required for stable adhesion. Fluorescence microscopy and immunoblotting showed equivalent amounts of beta1 integrin associated with collagen beads bound to Msp- and vehicle-treated cells. Photobleaching experiments found a similar percentage mobile fraction of beta1 integrins recovered in bleached areas of the plasma membrane. In contrast, Msp-induced inhibition of collagen binding was reversed by beta1 integrin affinity-activating antibodies and by latrunculin B, which prevented subcortical actin assembly. We conclude that native Msp of T. denticola inhibits the binding step of collagen phagocytosis in fibroblasts by inducing subcortical actin filament assembly and restricting affinity modulation of beta1 integrins. We suggest that, like Msp, bacterial toxins that target the cytoskeleton may also perturb the signalling networks required for cellular engagement of matrix ligands.