Oxidative metabolism is impaired by phosphate deficiency during fracture healing and is mechanistically related to BMP induced chondrocyte differentiation.

Prior studies of acute phosphate restriction during the endochondral phase of fracture healing showed delayed chondrocyte differentiation was mechanistically linked to decreased bone morphogenetic protein signaling. In the present study, transcriptomic analysis of fracture callus gene expression in three strains of mice was used to identify differentially expressed (FDR = q ≤ 0.05) genes in response to phosphate (Pi) restriction. Ontology and pathway analysis of these genes showed that independent of genetic background, a Pi-deficient diet downregulated (p = 3.16 × 10-23) genes associated with mitochondrial oxidative phosphorylation pathways as well as multiple other pathways of intermediate metabolism. Temporal clustering was used to identify co-regulation of these specific pathways. This analysis showed that specific Ox/Phos, tricarboxylic acid cycle, pyruvate dehydrogenase. Arginine, proline metabolism genes, and prolyl 4-hydroxylase were all co-regulated in response to dietary Pi restriction. The murine C3H10T½ mesenchymal stem cell line was used to assess the functional relationships between BMP2-induced chondrogenic differentiation, oxidative metabolism and extracellular matrix formation. BMP2-induced chondrogenic differentiation of C3H10T½ was carried out in culture media in the absence or presence of ascorbic acid, the necessary co-factor for proly hydroxylation, and in media with normal and 25 % phosphate levels. BMP2 treatment led to decreased proliferation, increased protein accumulation and increased collagen and aggrecan gene expression. Across all conditions, BMP2 increased total oxidative activity and ATP synthesis. Under all conditions, the presence of ascorbate further increased total protein accumulation, proly-hydroxylation and aggrecan gene expression, oxidative capacity and ATP production. Lower phosphate levels only diminished aggrecan gene expression with no other effects of metabolic activity being observed. These data suggest that dietary phosphate restriction controls endochondral growth in vivo indirectly through BMP signaling, which upregulates oxidative activity that is linked to overall protein production and collagen hydroxylation.

Suppression of human T cell activation by derivatives of glycerol monolaurate.

Glycerol monolaurate (GML), a naturally occurring monoglyceride, is widely used commercially for its antimicrobial properties. Interestingly, several studies have shown that GML not only has antimicrobial properties but is also an anti-inflammatory agent. GML inhibits peripheral blood mononuclear cell proliferation and inhibits T cell receptor (TCR)-induced signaling events. In this study, we perform an extensive structure activity relationship analysis to investigate the structural components of GML necessary for its suppression of human T cell activation. Human T cells were treated with analogs of GML, differing in acyl chain length, head group, linkage of acyl chain, and number of laurate groups. Treated cells were then tested for changes in membrane dynamics, LAT clustering, calcium signaling, and cytokine production. We found that an acyl chain with 12-14 carbons, a polar head group, an ester linkage, and a single laurate group at any position are all necessary for GML to inhibit protein clustering, calcium signaling, and cytokine production. Removing the glycerol head group or replacing the ester linkage with a nitrogen prevented derivative-mediated inhibition of protein cluster formation and calcium signaling, while still inhibiting TCR-induced cytokine production. These findings expand our current understanding of the mechanisms of action of GML and the of GML needed to function as a novel immunosuppressant.