Redox Regulation of PPARγ in Polarized Macrophages

The peroxisome proliferator-activated receptor (PPARγ) is a central mediator of cellular lipid metabolism and immune cell responses during inflammation. This is facilitated by its role as a transcription factor as well as a DNA-independent protein interaction partner. We addressed how the cellular redox milieu in the cytosol and the nucleus of lipopolysaccharide (LPS)/interferon-γ- (IFNγ-) and interleukin-4- (IL4-) polarized macrophages (MΦ) initiates posttranslational modifications of PPARγ, that in turn alter its protein function. Using the redox-sensitive GFP2 (roGFP2), we validated oxidizing and reducing conditions following classical and alternative activation of MΦ, while the redox status of PPARγ was determined via mass spectrometry. Cysteine residues located in the zinc finger regions (amino acid fragments AA 90-115, AA 116-130, and AA 160-167) of PPARγ were highly oxidized, accompanied by phosphorylation of serine 82 in response to LPS/IFNγ, whereas IL4-stimulation provoked minor serine 82 phosphorylation and less cysteine oxidation, favoring a reductive milieu. Mutating these cysteines to alanine to mimic a redox modification decreased PPARγ-dependent reporter gene transactivation supporting a functional shift of PPARγ associated with the MΦ phenotype. These data suggest distinct mechanisms for regulating PPARγ function based on the redox state of MΦ

Hypochlorhydria-induced calcium malabsorption does not affect fracture healing but increases post-traumatic bone loss in the intact skeleton

Efficient calcium absorption is essential for skeletal health. Patients with impaired gastric acidification display low bone mass and increased fracture risk because calcium absorption is dependent on gastric pH. We investigated fracture healing and post‐traumatic bone turnover in mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells. Cckbr−/− mice display hypochlorhydria, calcium malabsorption, and osteopenia. Cckbr−/− and wildtype (WT) mice received a femur osteotomy and were fed either a standard or calcium‐enriched diet. Healed and intact bones were assessed by biomechanical testing, histomorphometry, micro‐computed tomography, and quantitative backscattering. Parathyroid hormone (PTH) serum levels were determined by enzyme‐linked immunosorbent assay. Fracture healing was unaffected in Cckbr−/− mice. However, Cckbr−/− mice displayed increased calcium mobilization from the intact skeleton during bone healing, confirmed by significantly elevated PTH levels and osteoclast numbers compared to WT mice. Calcium supplementation significantly reduced secondary hyperparathyroidism and bone resorption in the intact skeleton in both genotypes, but more efficiently in WT mice. Furthermore, calcium administration improved bone healing in WT mice, indicated by significantly increased mechanical properties and bone mineral density of the fracture callus, whereas it had no significant effect in Cckbr−/− mice. Therefore, under conditions of hypochlorhydria‐induced calcium malabsorption, calcium, which is essential for callus mineralization, appears to be increasingly mobilized from the intact skeleton in favor of fracture healing. Calcium supplementation during fracture healing prevented systemic calcium mobilization, thereby maintaining bone mass and improving fracture healing in healthy individuals whereas the effect was limited by gastric hypochlorhydria. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1914–1921, 2016

Inhibition of Midkine Augments Osteoporotic Fracture Healing.

The heparin-binding growth and differentiation factor midkine (Mdk) is proposed to negatively regulate osteoblast activity and bone formation in the adult skeleton. As Mdk-deficient mice were protected from ovariectomy (OVX)-induced bone loss, this factor may also play a role in the pathogenesis of postmenopausal osteoporosis. We have previously demonstrated that Mdk negatively influences bone regeneration during fracture healing. Here, we investigated whether the inhibition of Mdk using an Mdk-antibody (Mdk-Ab) improves compromised bone healing in osteoporotic OVX-mice. Using a standardized femur osteotomy model, we demonstrated that Mdk serum levels were significantly enhanced after fracture in both non-OVX and OVX-mice, however, the increase was considerably greater in osteoporotic mice. Systemic treatment with the Mdk-Ab significantly improved bone healing in osteoporotic mice by increasing bone formation in the fracture callus. On the molecular level, we demonstrated that the OVX-induced reduction of the osteoanabolic beta-catenin signaling in the bony callus was abolished by Mdk-Ab treatment. Furthermore, the injection of the Mdk-Ab increased trabecular bone mass in the skeleton of the osteoporotic mice. These results implicate that antagonizing Mdk may be useful for the therapy of osteoporosis and osteoporotic fracture-healing complications

Midkine (Mdk) serum levels during fracture healing in 3-month-old sham-operated and ovariectomized (OVX) mice in pg/ml.

<p>Midkine (Mdk) serum levels during fracture healing in 3-month-old sham-operated and ovariectomized (OVX) mice in pg/ml.</p

Midkine-antibody (Mdk-Ab) treatment accelerated osteoporotic fracture healing.

<p>Biomechanical, micro-computed tomography (μCT) and histomorphometric analysis of the fractured femurs at day 23. Biomechanical testing: A) relative flexural rigidity of the fractured femur in comparison with intact femur determined by biomechanical testing. Parameters determined by μCT analysis (volume of interest 2): B) bone volume to tissue volume ratio and C) tissue volume (n = 6–7 per group). Parameters determined by histomorphometric analysis of the whole fracture callus at day 10 (white bars) and day 23 (grey bars): D) bone area to tissue area ratio, E) cartilage area to tissue area ratio and F) fibrous tissue area to tissue area ratio. *Significantly different from sham+vehicle or OVX+vehicle group (p<0.05) by Kruskal-Wallis test. (n = 5–7 per group.) G) Representative images of sections from undecalcified femurs at day 23, stained using Giemsa; scale bar: 250 μm.</p

Midkine-antibody (Mdk-Ab) treatment increased the bone content in the intact femur of ovariectomized (OVX) mice after short-time treatment.

<p>Micro-computed tomography (μCT) analysis of the cortical bone at the midshaft of the intact femur volume of interest 2 (VOI 2): A) cortical tissue mineral density (TMD) and B) cortical thickness. μCT analysis of the distal part of the intact femur (VOI 3): C) trabecular TMD, D) bone volume to tissue volume ratio, E) trabecular thickness and F) trabecular number. *Significantly different from sham+vehicle or OVX+vehicle group (p<0.05) by Kruskal-Wallis test. (n = 6–7 per group.) G) Three-dimensional reconstructions of the trabecular region of the distal intact femur (VOI 3), representative images are shown.</p

Midkine-antibody (Mdk-Ab) treatment increased the bone content in vertebral bodies of ovariectomized (OVX) mice after short-time treatment.

<p>Micro-computed tomography (μCT) analysis of the vertebral bodies (volume of interest 4): A) two-dimensional images of the second caudal vertebral body, representative images are shown. B) Trabecular tissue mineral density, C) trabecular bone volume to tissue volume ratio, D) trabecular thickness and E) trabecular number. *Significantly different from sham+vehicle or OVX+vehicle group (p<0.05) by Kruskal-Wallis test. (n = 6−7 per group).</p

Cellular parameters in the fracture callus and the intact femur at day 23.

<p>Cellular parameters in the fracture callus and the intact femur at day 23.</p