Histone deacetylase 3 supports endochondral bone formation by controlling cytokine signaling and matrix remodeling

Abstract

Histone deacetylase (HDAC) inhibitors are efficacious epigenetic-based therapies for some cancers and neurological disorders; however, each of these drugs inhibits multiple HDACs and has detrimental effects on the skeleton. To better understand how HDAC inhibitors affect endochondral bone formation, we conditionally deleted one of their targets, Hdac3, pre- and postnatally in type II collagen α1 (Col2α1)–expressing chondrocytes. Embryonic deletion was lethal, but postnatal deletion of Hdac3 delayed secondary ossification center formation, altered maturation of growth plate chondrocytes, and increased osteoclast activity in the primary spongiosa. HDAC3-deficient chondrocytes exhibited increased expression of cytokine and matrix-degrading genes (Il-6, Mmp3, Mmp13, and Saa3) and a reduced abundance of genes related to extracellular matrix production, bone development, and ossification (Acan, Col2a1, Ihh, and Col10a1). Histone acetylation increased at and near genes that had increased expression. The acetylation and activation of nuclear factor κB (NF-κB) were also increased in HDAC3-deficient chondrocytes. Increased cytokine signaling promoted autocrine activation of Janus kinase (JAK)–signal transducer and activator of transcription (STAT) and NF-κB pathways to suppress chondrocyte maturation, as well as paracrine activation of osteoclasts and bone resorption. Blockade of interleukin-6 (IL-6)–JAK–STAT signaling, NF-κB signaling, and bromodomain extraterminal proteins, which recognize acetylated lysines and promote transcriptional elongation, significantly reduced Il-6 and Mmp13 expression in HDAC3-deficient chondrocytes and secondary activation in osteoclasts. The JAK inhibitor ruxolitinib also reduced osteoclast activity in Hdac3 conditional knockout mice. Thus, HDAC3 controls the temporal and spatial expression of tissue-remodeling genes and inflammatory responses in chondrocytes to ensure proper endochondral ossification during development.

Histone deacetylase 3 suppresses Erk phosphorylation and matrix metalloproteinase (Mmp)-13 activity in chondrocytes

“…We previously demonstrated Hdac3 is expressed during skeletal development, as early as E14.5 and continues to be robustly expressed by growth plate chondrocytes into adulthood (34, 52, 53). Tissue-specific deletion of Hdac3 in chondrocytes with tamoxifen-inducible type II collagen alpha 1 Cre (Col2a1ERT-Cre) caused dramatic delays in endochondral ossification (34, 53).…”

“…Tissue-specific deletion of Hdac3 in chondrocytes with tamoxifen-inducible type II collagen alpha 1 Cre (Col2a1ERT-Cre) caused dramatic delays in endochondral ossification (34, 53). Genome-wide transcriptional profiling of Hdac3-depleted chondrocytes showed that markers of chondrocyte maturation, extracellular matrix, and bone development were suppressed with Hdac3-deficiency, while cartilage catabolic genes, like matrix metalloproteinases (e.g., Mmp13) were highly induced (53). To understand these Hdac3-dependent changes in matrix degrading enzymes, immature murine chondrocytes (IMCs) were isolated from 1-week-old Hdac3 fl/fl mice, plated in micromasses, and transduced with Ad-GFP or Ad-Cre to generate control and Hdac3-deficient IMCs, respectively.…”

“…Hdac3 fl/fl mice were crossed to tamoxifen-inducible type II collagen alpha 1Cre (Col2a1ERT-Cre) mice as previously reported (53, 55). To generate Hdac3-CKO Col2ERT mice, 5-day-old pups were administered a single intraperitoneal injection of tamoxifen (1 mg/kg, Sigma Aldrich, St. Louis, MO) or corn oil (53, 55).…”

“…To generate Hdac3-CKO Col2ERT mice, 5-day-old pups were administered a single intraperitoneal injection of tamoxifen (1 mg/kg, Sigma Aldrich, St. Louis, MO) or corn oil (53, 55). All animals were housed in an accredited facility under a 12-hour light/dark cycle and provided water and food (PicoLab® RodentDiet20, LabDiet) ad libitum .…”

“…While germline deletion of Hdac3 is embryonic lethal (49) conditional knockout mouse models have demonstrated the importance of Hdac3 in maintaining proper bone mass during aging, as well as long bone development through actions in osteoblasts and chondrocytes (50–52). We recently showed that conditional deletion of Hdac3 in chondrocytes (with inducible type II collagen alpha 1, (Col2ERT)-Cre) alters cytokine signaling and matrix metalloproteinase expression suggesting an important role in regulating extracellular matrix remodeling (53). …”