Carbonic anhydrase III protects osteocytes from oxidative stress.

Osteocytes are master orchestrators of bone remodeling; they control osteoblast and osteoclast activities both directly via cell-to-cell communication and indirectly via secreted factors, and they are the main postnatal source of sclerostin and RANKL (receptor activator of NF-kB ligand), two regulators of osteoblast and osteoclast function. Despite progress in understanding osteocyte biology and function, much remains to be elucidated. Recently developed osteocytic cell lines-together with new genome editing tools-has allowed a closer look at the biology and molecular makeup of these cells. By using single-cell cloning, we identified genes that are associated with high Sost/sclerostin expression and analyzed their regulation and function. Unbiased transcriptome analysis of high- vs. low-Sost/sclerostin-expressing cells identified known and novel genes. Dmp1 (dentin matrix protein 1), Dkk1 (Dickkopf WNT signaling pathway inhibitor 1), and Phex were among the most up-regulated known genes, whereas Srpx2, Cd200, and carbonic anhydrase III (CAIII) were identified as novel markers of differentiated osteocytes. Aspn, Enpp2, Robo2, Nov, and Serpina3g were among the transcripts that were most significantly suppressed in high-Sost cells. Considering that CAII was recently identified as being regulated by Sost/sclerostin and capable of controlling mineral homeostasis, we focused our attention on CAIII. Here, we report that CAIII is highly expressed in osteocytes, is regulated by parathyroid hormone both in vitro and in vivo, and protects osteocytes from oxidative stress.-Shi, C., Uda, Y., Dedic, C., Azab, E., Sun, N., Hussein, A. I., Petty, C. A., Fulzele, K., Mitterberger-Vogt, M. C., Zwerschke, W., Pereira, R., Wang, K., Divieti Pajevic, P. Carbonic anhydrase III protects osteocytes from oxidative stress.

Chemically induced reprogramming to reverse cellular aging.

A hallmark of eukaryotic aging is a loss of epigenetic information, a process that can be reversed. We have previously shown that the ectopic induction of the Yamanaka factors OCT4, SOX2, and KLF4 (OSK) in mammals can restore youthful DNA methylation patterns, transcript profiles, and tissue function, without erasing cellular identity, a process that requires active DNA demethylation. To screen for molecules that reverse cellular aging and rejuvenate human cells without altering the genome, we developed high-throughput cell-based assays that distinguish young from old and senescent cells, including transcription-based aging clocks and a real-time nucleocytoplasmic compartmentalization (NCC) assay. We identify six chemical cocktails, which, in less than a week and without compromising cellular identity, restore a youthful genome-wide transcript profile and reverse transcriptomic age. Thus, rejuvenation by age reversal can be achieved, not only by genetic, but also chemical means.

Parathyroid hormone signaling in mature osteoblasts/osteocytes protects mice from age-related bone loss.

Aging is accompanied by osteopenia, characterized by reduced bone formation and increased bone resorption. Osteocytes, the terminally differentiated osteoblasts, are regulators of bone homeostasis, and parathyroid hormone (PTH) receptor (PPR) signaling in mature osteoblasts/osteocytes is essential for PTH-driven anabolic and catabolic skeletal responses. However, the role of PPR signaling in those cells during aging has not been investigated. The aim of this study was to analyze the role of PTH signaling in mature osteoblasts/osteocytes during aging. Mice lacking PPR in osteocyte (Dmp1-PPRKO) display an age-dependent osteopenia characterized by a significant decrease in osteoblast activity and increase in osteoclast number and activity. At the molecular level, the absence of PPR signaling in mature osteoblasts/osteocytes is associated with an increase in serum sclerostin and a significant increase in osteocytes expressing 4-hydroxy-2-nonenals, a marker of oxidative stress. In Dmp1-PPRKO mice there was an age-dependent increase in p16Ink4a/Cdkn2a expression, whereas it was unchanged in controls. In vitro studies demonstrated that PTH protects osteocytes from oxidative stress-induced cell death. In summary, we reported that PPR signaling in osteocytes is important for protecting the skeleton from age-induced bone loss by restraining osteoclast's activity and protecting osteocytes from oxidative stresses.

Transcription factor NF-κB in a basal metazoan, the sponge, has conserved and unique sequences, activities, and regulation.

Herein, we characterize transcription factor NF-κB from the demosponge Amphimedon queenslandica (Aq). Aq-NF-κB is most similar to NF-κB p100/p105 among vertebrate proteins, with an N-terminal DNA-binding domain, a C-terminal Ankyrin (ANK) repeat domain, and a DNA binding-site profile akin to human NF-κB proteins. Like mammalian NF-κB p100, C-terminal truncation allows nuclear translocation of Aq-NF-κB and increases its transcriptional activation activity. Expression of IκB kinases (IKKs) induces proteasome-dependent C-terminal processing of Aq-NF-κB in human cells, and processing requires C-terminal serines in Aq-NF-κB. Unlike NF-κB p100, C-terminal sequences of Aq-NF-κB do not inhibit its DNA-binding activity. Tissue of a black encrusting demosponge contains NF-κB site DNA-binding activity, as well as nuclear and processed NF-κB. Treatment of sponge tissue with LPS increases both DNA-binding activity and processing of NF-κB. A. queenslandica transcriptomes contain homologs to upstream NF-κB pathway components. This is first functional characterization of NF-κB in sponge, the most basal multicellular animal.