SUMMARY
Nutritional supplementation with probiotics can prevent pathologic bone
loss. Here we examined the impact of supplementation with Lactobacillus
rhamnosus GG (LGG) on bone homeostasis in eugonadic young mice.
Micro-computed tomography revealed that LGG increased trabecular bone volume in
mice, which was due to increased bone formation. Butyrate produced in the gut
following LGG ingestion, or butyrate fed directly to germ-free mice, induced the
expansion of intestinal and bone marrow (BM) regulatory T (Treg) cells.
Interaction of BM CD8+ T cells with Treg cells resulted in increased
secretion of Wnt10b, a bone anabolic Wnt ligand. Mechanistically, Treg cells
promoted the assembly of a NFAT1-SMAD3 transcription complex in CD8+
cells, which drove expression of
Wnt10b−/−. Reducing Treg cell
numbers, or reconstitution of TCRβ−/− mice with
CD8+ T cells from
Wnt10b−/− mice, prevented
butyrate-induced bone formation and bone mass acquisition. Thus, butyrate
concentrations regulate bone anabolism via Treg cell-mediated regulation of
CD8+ T cell Wnt10b production.
Cyclin-dependent kinase 9 (CDK9) is a well-characterized subunit of the positive transcription elongation factor b complex in which it regulates transcription elongation in cooperation with cyclin T. However, CDK9 also forms a complex with cyclin K, the function of which is less clear. Using a synthetic lethal RNA interference screen in human cells, we identified CDK9 as a component of the replication stress response. Loss of CDK9 activity causes an increase in spontaneous levels of DNA damage signalling in replicating cells and a decreased ability to recover from a transient replication arrest. This activity is restricted to CDK9-cyclin K complexes and is independent of CDK9-cyclin T complex. CDK9 accumulates on chromatin in response to replication stress and limits the amount of single-stranded DNA in cells under stress. Furthermore, we show that CDK9 and cyclin K interact with ataxia telangiectasia and Rad3-related protein and other checkpoint signalling proteins. These results reveal an unexpectedly direct role for CDK9-cyclin K in checkpoint pathways that maintain genome integrity in response to replication stress.
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