Histone Deacetylase regulated the chondrocyte transcriptome by modifying the chromatin landscape and controlling non-histone protein activity. Carpio et al. found that HDAC3 promotes bone growth by restricting the secretion of inflammatory factors from chondrocytes and they publish their article on Science Signalling (Sci. Signal. 09 Aug 2016: Vol. 9, Issue 440, pp. ra79 DOI: 10.1126/scisignal.aaf3273). Here we share part of this wonderful article, and Creative Biomart Provides kinds of recombinant proteins for the research applications.
[table caption=”Related Products” width=”800″ colwidth=”150|280|180″ colalign=”left|left|center|center|center”]
Cat. #,Product name,Source(Host),Species,Conjugate
HDAC2-3565H,Recombinant Human HDAC2,insect cells,Human,His
HDAC3-94H,Recombinant Human HDAC3 Protein,Insect cell,Human,GST
HDAC5-96H,Recombinant Human HDAC5 Protein,Insect cell,Human,His
HDAC11-586C,Recombinant Cynomolgus HDAC11 Protein,Mammalian Cells,Cynomolgus,His
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Introduction
Histone deacetylases (HDACs) affect various cellular processes but are best known as transcriptional corepressors that epigenetically control gene transcription by removing acetyl groups from lysine side chains of histone tails. The removal of these post-translational modifications from histones prevents the recruitment of readers, such as bromodomain- or YEATS domain–containing proteins, thereby promoting chromatin compaction and repression of RNA polymerase II–dependent gene expression. Humans and mice have just 18 HDACs, which are divided into four classes on the basis of their structure and function. Class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8) predominately localize to the nucleus, although HDAC3 has also been detected at plasma membranes. Class I HDACs are ubiquitously expressed and have high enzymatic activity toward histone substrates and thus serve as the enzymatic subunits of multiprotein repressive complexes. Class II HDACs (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10) vary from class I HDACs in that they shuttle between the nucleus and cytoplasm and have more temporal and spatial gene expression patterns. Class II HDACs have low intrinsic enzymatic activity and therefore often recruit class I HDACs for most of their enzymatic activity. Class III HDACs [sirtuins (SIRTs)] are substantially different from the other HDACs in that they require nicotinamide adenine dinucleotide (NAD+) instead of zinc (Zn2+) for their catalytic activity and thus are inhibited by different small molecules. Class IV consists of only HDAC11, which shares characteristics of both class I and class II HDACs.
HDACs can deacetylate proteins other than histones, including transcription factors [nuclear factor κB (NF-κB), RUNX2, p53, and signal transducer and activator of transcription 3 (STAT3)], to posttranslationally influence their stability and activity. HDACs are particularly important during development when gene expression programs change quickly as cell fate and function are determined. Small molecules with inhibitory activity for class I and II HDACs have been used to treat many cancers and mood disorders, and are in clinical trials for the treatment of neurological disorders and arthritis. However, many of these inhibitors, such as suberanilohydroxamic acid (SAHA), which is also known as vorinostat, are nonspecific and target multiple HDACs. As a result, off-target effects are common, particularly in the context of skeletal development and repair. For example, in utero exposure to HDAC inhibitors causes birth defects, and long-term exposure increases fracture risk in children and adults and reduces bone density in mice. Therefore, it is important to define the biological roles of individual HDACs in the skeleton.
HDAC3, HDAC4, HDAC5, and HDAC7 are crucial for endochondral ossification. HDAC3 is highly expressed by osteoblasts and chondrocytes and acts as a corepressor for RUNX2, ZFP521, class II HDACs (HDAC4, HDAC5, and HDAC7), and other transcriptional regulators. Germline deletion of Hdac3 causes embryonic lethality during midgestation [embryonic day 9.5 (E9.5)] several days before skeletogenesis begins. Tissue-specific ablation of Hdac3in osteoblasts [using osteocalcin (OCN)–Cre], in osteoprogenitors [using osterix (OSX1)–Cre], and in neural crest cells (using WNT1-Cre or PAX3-Cre) has demonstrated its importance in long bone and craniofacial bone development; however, the specific role of HDAC3 in chondrocyte maturation remains to be investigated.
Here, we examined the role of HDAC3 in hyaline cartilage development by genetically deleting it in vitro and in vivo. Prenatal deletion of Hdac3 in chondrocytes caused embryonic lethality, but postnatal deletion using an inducible conditional knockout (CKO) system produced animals with several skeletal abnormalities, including delayed secondary ossification center (SOC) formation, delayed maturation of epiphyseal plate cartilage, and increased osteoclastogenesis. Transcriptomic and chromatin analyses of HDAC3-deficient immature murine chondrocytes (IMCs) revealed increased expression of cytokine and matrix-degrading genes and reduced expression of genes related to extracellular matrix composition and deposition, bone development, and ossification. The altered secretome of HDAC3-deficient chondrocytes had autocrine and paracrine effects on bone formation and matrix remodeling, suggesting an important role of HDAC3 in controlling the coupling of chondrocyte maturation and bone modeling. Inhibition of cytokine-controlled pathways [namely, Janus kinase (JAK)–STAT or NF-κB] or of bromodomain extraterminal (BET) proteins that recognize acetylated proteins rescued many of the defects observed in Hdac3-depleted chondrocytes. Furthermore, inhibition of JAK signaling suppressed osteoclast recruitment in Hdac3-CKO mice. Together, these results demonstrate that HDAC3 plays an important role in endochondral bone formation by maintaining temporal and spatial regulation of gene expression and downstream signaling factors that are crucial in chondrocyte maturation, proper long bone growth, and ossification. Furthermore, our study provides genetic evidence that HDAC3 normally attenuates cytokine expression during skeletal development to control the JAK-STAT and NF-κB pathways, and thereby supports cartilage extracellular matrix formation and remodeling.
Discussion
This study elucidates the essential functions for autologous HDAC3 in proper growth plate chondrocyte maturation and long bone development. Mice in which Hdac3 is deleted in chondrocytes (Hdac3-CKOCol2ERT) also exhibit a number of cartilage-extrinsic phenotypes including delayed angiogenesis, accelerated bone resorption, and severely reduced bone mineral density. These results demonstrate that HDAC3 controls cell-cell communication between chondrocytes and cell types required for the integrated development of cartilage, bone, and blood vessels during skeletal development. In particular, we observed that HDAC3 loss in chondrocytes deregulates the cell-autonomous expression of many cytokines and MMPs that appear to be required for skeletal matrix modeling during endochondral bone formation.
Cytokines like IL-6 are classically thought of as proinflammatory or anti-inflammatory factors based on their potent biological effects as systemic factors. Yet, our results indicate that these ligands also have key roles as mediators of cell communication during normal bone development. Although cartilage lacks major blood vessels, chondrocytes communicate with each other and other cell types in neighboring bone and periosteal tissues including osteoclast and chondroclast precursors and endothelial cells through several mechanisms. Factors produced by growth plate chondrocytes can diffuse through a permissive region in the midplane of the growth plate near the prehypertrophic zone to the periosteum at a rate inversely proportional to their size. MMPs that digest matrix components and fluid flow could further facilitate the movement and release of molecules that may be stored in the matrix. Our results indicate that HDAC3 specifically controls the expression of MMPs as well as important cytokines in growth plate chondrocytes, and this may have systemic effects. Together, we propose that HDAC3-mediated suppression of cytokines and matrix remodeling genes in chondrocytes may facilitate coordination of cartilage development and bone formation during normal endochondral ossification, as well as perhaps in cartilaginous callous formation during bone fracture repair.
Transcriptome data and clinical findings support this interpretation that originates from skeletal phenotyping of our Hdac3 conditional null mice. Our RNA-seq data show that normal primary mouse chondrocytes expressed a number of cytokines and chemokines, but their abundances were significantly increased when HDAC3 was inactivated. The physiological production of these soluble factors in growth plate and articular chondrocytes suggests that these ligands have a normal role during mouse skeletal formation and maintenance when produced at appropriate levels. Chronic inflammation has direct and indirect effects on chondrocytes and interrupts endochondral ossification during development, resulting in many clinical manifestations, including permanently shortened limbs. Because HDAC3-mediated control of cytokines appears to be an essential physiological mechanism for skeletogenesis, as well as normal cartilage formation and maintenance, these clinical findings can perhaps be reevaluated as a potential perturbation of normal cytokine-mediated signaling pathways.
Our data show that deletion of Hdac3 derepressed proinflammatory and matrix degrading signaling pathways by hyperacetylating histones and NF-κB in chondrocytes. Robust NF-κB activation and expression of cytokines are also observed when Hdac3 was deleted in T regulatory lymphocytes. Among the many genes that were highly induced and hyperacetylated, Il-6 andMmp13 were validated in vivo. The increased production of IL-6 and MMP13 had autocrine effects on HDAC3-depleted chondrocyte cultures. IL-6 activated the JAK-STAT signaling pathway in the HDAC3-depleted micromasses, and a JAK inhibitor partially reduced Il-6 and Mmp13 expression but did not increase Col2a1 expression or decrease Saa3 expression. These reductions in Il-6 andMmp13 are consistent with the ability of JAK inhibitors to suppress inflammatory cytokine production in models of aging and frailty. In comparison, blockade of NF-κB signaling suppressed Mmp13 and Saa3 expression but also did not affect Col2a1 expression. Additionally, preventing BET proteins from recognizing the acetylated marks significantly suppressed Il-6, Mmp13, and Saa3 expression. In summary, our study provides a molecular mechanistic explanation for the physiological deregulation of cytokine signaling in chondrocytes, because deacetylation of histones and NF-κB promotes gene induction as predicted and Hdac3 deletion affects signaling pathways in chondrocytes that control the expression of JAK-STAT– and NF-κB–dependent cytokines and MMPs.
This work is of biomedical importance because HDAC inhibitors are increasingly recognized and clinically tested as potential therapies for many conditions and diseases including cancer and neurological disorders due to their epigenetic reprogramming capabilities. These conditions include several forms of arthritis. In animal models of osteoarthritis, which typically occurs after the skeleton is mature, HDAC inhibitors slowed disease progression. Moreover, in models of rheumatoid arthritis and periodontitis, HDAC inhibitors blocked bone resorption and osteoclast activation. These favorable outcomes may be due to the ability of HDAC inhibitors to neutralize leukocyte populations that are major sources of inflammatory cytokines and chemokines. However, evidence that HDAC inhibitors have detrimental effects on the developing skeleton also exists. This study demonstrates that endochondral ossification was unfavorably affected in a number of ways by HDAC3 depletion in growth plate chondrocytes. Additional studies will need to be performed on osteoclasts and articular chondrocytes in adult mice to fully comprehend the role of HDAC3 in joint health and disease.
In summary, HDAC3 plays a crucial role in regulating the chondrocyte transcriptome by modifying the chromatin landscape and controlling non-histone protein activity, both of which allow for necessary temporal and spatial control of gene expression during the highly orchestrated process of endochondral bone formation. Loss of HDAC3 in culture models and in vivo delays ossification, alters terminal chondrocyte hypertrophy, and disrupts the coupling of chondrocyte maturation to ossification, by decreasing angiogenesis and increasing osteoclast activity. Furthermore, our study provides both physiological and molecular mechanistic evidence that these skeletal phenotypes are linked to HDAC3 control of histone acetylation, as well as the JAK-STAT and NF-κB signaling in response to the production of cytokines, chemokines, and MMPs in chondrocytes. The developmental effects of perturbing HDAC3-controlled pathways have lasting consequences by compromising bone quality, architecture, and overall skeletal health.