Immune Cell Induced Migration of Osteoprogenitor Cells Is Mediated by TGF-β Dependent Upregulation of NOX4 and Activation of Focal Adhesion Kinase

Abstract

The cytokines secreted by immune cells have a large impact on the tissue, surrounding a fracture, e.g., by attraction of osteoprogenitor cells. However, the underlying mechanisms are not yet fully understood. Thus, this study aims at investigating molecular mechanisms of the immune cell-mediated migration of immature primary human osteoblasts (phOBs), with transforming growth factor beta (TGF-β), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) and focal adhesion kinase (FAK) as possible regulators. Monocyte- and macrophage (THP-1 cells ± phorbol 12-myristate 13-acetate (PMA) treatment)-conditioned media, other than the granulocyte-conditioned medium (HL-60 cells + dimethyl sulfoxide (DMSO) treatment), induce migration of phOBs. Monocyte- and macrophage (THP-1 cells)-conditioned media activate Smad3-dependent TGF-β signaling in the phOBs. Stimulation with TGF-β promotes migration of phOBs. Furthermore, TGF-β treatment strongly induces NOX4 expression on both mRNA and protein levels. The associated reactive oxygen species (ROS) accumulation results in phosphorylation (Y397) of FAK. Blocking TGF-β signaling, NOX4 activity and FAK signaling effectively inhibits the migration of phOBs towards TGF-β. In summary, our data suggest that monocytic- and macrophage-like cells induce migration of phOBs in a TGF-β-dependent manner, with TGF-β-dependent induction of NOX4, associated production of ROS and resulting activation of FAK as key mediators.

Feasibility of Cell Lines for In Vitro Co-Cultures Models for Bone Metabolism

“…Recently, there is growing evidence that osteogenic and osteoclastic cells are not only influenced by the surrounding matrix [116,117] and mechanical stimuli [220], but also strongly by immune cells [221,222], adipose cells [223][224][225], nerve cells [226,227], or endothelial cells [228] (for overview see Figure 6). However, considering the cell-cell-and cell-matrix-interactions, as well as the 3D conformation and mechanical stimuli and including these or other cells within the model systems would rapidly increase the complexity of the system, such that analytical limits are quickly reached [229].…”

“…Primary human osteoblasts from spongy bone tissue (ethical vote: 539/2016BO2) were isolated by collagenase digestion as described before [222,230,231]. Cells were expanded in DMEM medium supplemented with 5% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μM L-ascorbate-2-phosphate, 50 μM β-glycerol phosphate.…”

“…SCP-1 cells were cultured in MEMα Medium supplemented with 5% FCS. The osteogenic cell lines MG-63, Cal-72, and SaOS-2 (obtained from the DSMZ) were all expanded in RPMI 1640 medium, supplemented with 5% FCS [222]. The myeloid cell lines THP-1 and HL-60 (DSMZ) were used as osteoclastic precursor cells.…”

“…The myeloid cell lines THP-1 and HL-60 (DSMZ) were used as osteoclastic precursor cells. Cells were expanded as suspension culture in RPMI 1640 Medium, supplemented with 5% FCS [222].…”

Material-Dependent Formation and Degradation of Bone Matrix—Comparison of Two Cryogels

“…For maturation of SaOS-2 cells, the culture medium was replaced by osteogenic medium (RPMI 1640, 2% FCS, 200 μM L-ascorbic acid 2-phosphate, 5 mM β-glycerol phosphate, 25 mM HEPES, 1.5 mM CaCl 2 , and 5 μM cholecalciferol) [ 29 ]. 3D-cultures were maintained at 37 °C (5% CO 2 , humidified atmosphere).…”