Age and oxidative stress regulate Nrf2 homeostasis in human articular chondrocytes.

OBJECTIVE: The purpose of this study was to investigate the effect of age and oxidative stress on regulation of nuclear factor erythroid-2-related factor 2 (Nrf2) in young, old, and osteoarthritic (OA) human articular chondrocytes. DESIGN: Levels of Nrf2 in primary human chondrocytes isolated from young, old, and OA donors were measured by immunoblotting, qPCR, and immunohistochemistry. Effects on levels of Nrf2, antioxidant proteins regulated by Nrf2, as well as p65, and the anabolic response to insulin-like growth factor-1 (IGF-1) were evaluated after induction of oxidative stress with menadione, Nrf2 knockdown with siRNA, and/or Nrf2 activation with RTA-408. RESULTS: Nrf2 protein levels were significantly lower in older adult chondrocytes (∼0.59 fold; p = 0.034) and OA chondrocytes (∼0.50 fold; p = 0.016) compared to younger cells. Menadione significantly increased Nrf2 protein levels in young chondrocytes by just under four-fold without changes in old chondrocytes. Nrf2 knockdown and activation differentially regulated levels of anti-oxidant proteins including sulfiredoxin and NAD(P)H quinone dehydrogenase 1. Nrf2 activation with RTA-408 also decreased basal p65 phosphorylation, increased aggrecan and type II collagen gene expression, and increased production of proteoglycans in OA chondrocytes treated with IGF-1. CONCLUSIONS: Targeted therapeutic strategies aimed at maintaining Nrf2 activity could be useful in maintaining chondrocyte homeostasis through maintenance of intracellular antioxidant function and redox balance.

GIRK3 deletion facilitates kappa opioid signaling in chondrocytes, delays vascularization and promotes bone lengthening in mice.

Long bones are formed and repaired through the process of endochondral ossification. Activation of G protein-coupled receptor (GPCR) signaling pathways is crucial for skeletal development and long bone growth. G protein-gated inwardly-rectifying K+ (GIRK) channel genes are key functional components and effectors of GPCR signaling pathways in excitable cells of the heart and brain, but their roles in non-excitable cells that directly contribute to endochondral bone formation have not been studied. In this study, we analyzed skeletal phenotypes of Girk2-/-, Girk3-/- and Girk2/3-/- mice. Bones from 12-week-old Girk2-/- mice were normal in length, but femurs and tibiae from Girk3-/- and Girk2/3-/- mice were longer than age-matched controls at 12-weeks-old. Epiphyseal chondrocytes from 5-day-old Girk3-/- mice expressed higher levels of genes involved in collagen chain trimerization and collagen fibril assembly, lower levels of genes encoding VEGF receptors, and produced larger micromasses than wildtype chondrocytes in vitro. Girk3-/- chondrocytes were also more responsive to the kappa opioid receptor (KOR) ligand dynorphin, as evidenced by greater pCREB expression, greater cAMP and GAG production, and upregulation of Col2a1 and Sox9 transcripts. Imaging studies showed that Kdr (Vegfr2) and endomucin expression was dramatically reduced in bones from young Girk3-/- mice, supporting a role for delayed vasculogenesis and extended postnatal endochondral bone growth. Together these data indicate that GIRK3 controls several processes involved in bone lengthening.

Pleckstrin homology (PH) domain and Leucine Rich Repeat Phosphatase 1 (Phlpp1) Suppresses Parathyroid Hormone Receptor 1 (Pth1r) Expression and Signaling During Bone Growth.

Endochondral ossification is tightly controlled by a coordinated network of signaling cascades including parathyroid hormone (PTH). Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 (Phlpp1) affects endochondral ossification by suppressing chondrocyte proliferation in the growth plate, longitudinal bone growth, and bone mineralization. As such, Phlpp1-/- mice have shorter long bones, thicker growth plates, and proportionally larger growth plate proliferative zones. The goal of this study was to determine how Phlpp1 deficiency affects PTH signaling during bone growth. Transcriptomic analysis revealed greater PTH receptor 1 (Pth1r) expression and enrichment of histone 3 lysine 27 acetylation (H3K27ac) at the Pth1r promoter in Phlpp1-deficient chondrocytes. PTH (1-34) enhanced and PTH (7-34) attenuated cell proliferation, cAMP signaling, cAMP response element-binding protein (CREB) phosphorylation, and cell metabolic activity in Phlpp1-inhibited chondrocytes. To understand the role of Pth1r action in the endochondral phenotypes of Phlpp1-deficient mice, Phlpp1-/- mice were injected with Pth1r ligand PTH (7-34) daily for the first 4 weeks of life. PTH (7-34) reversed the abnormal growth plate and long-bone growth phenotypes of Phlpp1-/- mice but did not rescue deficits in bone mineral density or trabecular number. These results show that elevated Pth1r expression and signaling contributes to increased proliferation in Phlpp1-/- chondrocytes and shorter bones in Phlpp1-deficient mice. Our data reveal a novel molecular relationship between Phlpp1 and Pth1r in chondrocytes during growth plate development and longitudinal bone growth. © 2021 American Society for Bone and Mineral Research (ASBMR).

Histone Mutations and Bone Cancers.

Primary bone tumors are rare cancers that cause significant morbidity and mortality. The recent identification of recurrent mutations in histone genes H3F3A and H3F3B within specific bone cancers, namely, chondroblastomas and giant cell tumors of bone (GCTB), has provided insights into the cellular and molecular origins of these neoplasms and enhanced understanding of how histone variants control chromatin function. Somatic mutations in H3F3A and H3F3B produce oncohistones, H3.3G34W and H3.3K36M, in more than nine of ten GCTB and chondroblastomas, respectively. Incorporation of the mutant histones into nucleosomes inhibits histone methyltransferases NSD2 and SETD2 to alter the chromatin landscape and change gene expression patterns that control cell proliferation, survival, and differentiation, as well as DNA repair and chromosome stability. The discovery of these histone mutations has facilitated more accurate diagnoses of these diseases and stratification of malignant tumors from benign tumors so that appropriate care can be delivered. The broad-scale epigenomic and transcriptomic changes that arise from incorporation of mutant histones into chromatin provide opportunities to develop new and disease-specific therapies. In this chapter, we review how mutant histones inhibit SETD2 and NSD2 function in bone tumors and discuss how this information could lead to better treatments for these cancers.

Histone Deacetylase 3 Deletion in Mesenchymal Progenitor Cells Hinders Long Bone Development.

Long bone formation is a complex process that requires precise transcriptional control of gene expression programs in mesenchymal progenitor cells. Histone deacetylases (Hdacs) coordinate chromatin structure and gene expression by enzymatically removing acetyl groups from histones and other proteins. Hdac inhibitors are used clinically to manage mood disorders, cancers, and other conditions but are teratogenic to the developing skeleton and increase fracture risk in adults. In this study, the functions of Hdac3, one of the enzymes blocked by current Hdac inhibitor therapies, in skeletal mesenchymal progenitor cells were determined. Homozygous deletion of Hdac3 in Prrx1-expressing cells prevented limb lengthening, altered pathways associated with endochondral and intramembranous bone development, caused perinatal lethality, and slowed chondrocyte and osteoblast differentiation in vitro. Transcriptomic analysis revealed that Hdac3 regulates vastly different pathways in mesenchymal cells expressing the Prxx1-Cre driver than those expressing the Col2-CreERT driver. Notably, Fgf21 was elevated in Hdac3-CKOPrrx1 limbs as well as in chondrogenic cells exposed to Hdac3 inhibitors. Elevated expression of Mmp3 and Mmp10 transcripts was also observed. In conclusion, Hdac3 regulates distinct pathways in mesenchymal cell populations and is required for mesenchymal progenitor cell differentiation and long bone development. © 2017 American Society for Bone and Mineral Research.