A novel library screen identifies immunosuppressors that promote osteoblast differentiation.

Bone homeostasis can be compromised by an increase in osteoclast-mediated resorption and/or a decrease in osteoblast-mediated bone deposition. While many efforts have focused on treating osteoclast resorption, there has been less emphasis on identifying strategies for promoting osteoblast function. Herein, we describe a high-throughput screening assay to select for small molecules that augment bone morphogenetic protein-2 (BMP-2)-mediated osteoblast lineage commitment. After an initial screen of 5405 compounds; consisting of FDA-approved drugs, known bioactives, and compounds with novel chemical makeup, we identified 45 small molecules that promoted osteoblast commitment. Of the 45 candidates, there was a broad array of classes that included nine retinoid analogs/derivatives and four immunosuppressants, notably rapamycin and FK-506, which were chosen for further study. Treatment of osteoblast precursor cells with rapamycin or FK-506, either alone, or synergistically with BMP-2, increased levels of phospho-Smad 1/5/8 protein and transcription of Runx-2, Osx and Smad-7, consistent with a role in promoting osteoblast differentiation. Only FK-506 was able to enhance osteocalcin transcripts and Alizarin Red staining, both late markers for differentiation. When osteoblast differentiation was suppressed with exogenous TGF-ß1 treatment, rapamycin (but not FK-506) was able to rescue expression of differentiation markers, indicating distinct but overlapping activity of these compounds. Collectively, these data add to an understanding of pathways engaged in osteoblastogenesis, support a role for non-redundant immunosuppressant signaling, and provide a novel approach for the discovery of potentially therapeutic compounds that affect bone remodeling.

The RNA editor gene ADAR1 is induced in myoblasts by inflammatory ligands and buffers stress response.

Muscle atrophy remains a significant concern in multiple inflammatory conditions, including injury, sepsis, cachexia, and HIV-associated wasting. Herein, we show that inflammatory stressors, including TNF-alpha, IFN-gamma, or lipopolysaccharide, potently induced the novel expression of the RNA editor ADAR1, an observation not previously described in muscle cells. We also observed that cytokine stimulation suppressed muscle-associated microRNAs, an observation also not previously demonstrated. To map potential effects of ADAR1 induction in the muscle program, we conducted knockdown and overexpression studies in the mouse C2C12 muscle precursor cell (MPC) line and in primary human MPCs. We show that knockdown of stress-induced ADAR1 increased inflammation-mediated declines in the muscle differentiation markers Myogenin and myosin heavy chain, and knockdown reduced levels of active phosphorylated Akt (phospho-Akt), but had no effect on microRNA transcript levels, suggesting a role for ADAR1 in buffering inflammatory stress effects on myogenic transcription and protein synthesis pathways. In addition, overexpression of recombinant ADAR1 suppressed active phosphorylated double-stranded RNA (dsRNA)-dependent protein kinase (phospho-PKR), consistent with a role for ADAR1 in limiting inflammation-driven catabolic atrophy pathways. Collectively, these data identify a novel regulatory role for ADAR1 activation under inflammatory stress to both promote muscle protein synthesis pathways and limit atrophy pathways.

RNA editing genes associated with extreme old age in humans and with lifespan in C. elegans.

BACKGROUND: The strong familiality of living to extreme ages suggests that human longevity is genetically regulated. The majority of genes found thus far to be associated with longevity primarily function in lipoprotein metabolism and insulin/IGF-1 signaling. There are likely many more genetic modifiers of human longevity that remain to be discovered. METHODOLOGY/PRINCIPAL FINDINGS: Here, we first show that 18 single nucleotide polymorphisms (SNPs) in the RNA editing genes ADARB1 and ADARB2 are associated with extreme old age in a U.S. based study of centenarians, the New England Centenarian Study. We describe replications of these findings in three independently conducted centenarian studies with different genetic backgrounds (Italian, Ashkenazi Jewish and Japanese) that collectively support an association of ADARB1 and ADARB2 with longevity. Some SNPs in ADARB2 replicate consistently in the four populations and suggest a strong effect that is independent of the different genetic backgrounds and environments. To evaluate the functional association of these genes with lifespan, we demonstrate that inactivation of their orthologues adr-1 and adr-2 in C. elegans reduces median survival by 50%. We further demonstrate that inactivation of the argonaute gene, rde-1, a critical regulator of RNA interference, completely restores lifespan to normal levels in the context of adr-1 and adr-2 loss of function. CONCLUSIONS/SIGNIFICANCE: Our results suggest that RNA editors may be an important regulator of aging in humans and that, when evaluated in C. elegans, this pathway may interact with the RNA interference machinery to regulate lifespan.