A post-translational modification cascade employing HDAC9-PIASy-RNF4 axis regulates chondrocyte hypertrophy by modulating Nkx3.2 protein stability
Introduction
Nkx3.2 (also known as Bapx1, the vertebrate homologue of the Drosophila protein bagpipe), is a homeobox-containing transcription factor shown to promote chondrogenic cell fate commitment of mesenchymal cells [1], [2], [3], [4], [5], [6]. Interestingly, Nkx3.2 expression is maintained in early stage immature and proliferative chondrocytes, whereas its expression is suppressed upon the onset of chondrocyte hypertrophy during terminal stages of chondrogenesis [7], [8], [9]. Consistent with this expression pattern, forced expression of Nkx3.2 can inhibit chondrocyte maturation [10], [11]. Moreover, Nkx3.2 plays a key role in maintaining chondrocyte viability by permitting constitutive ligand-independent activation of p65-RelA [12], [13]. Although the pathways controlled by Nkx3.2 have been well established, regulatory mechanisms that modify the expression and function of Nkx3.2 remain poorly understood.
Post-translational modifications (PTMs) including glycosylation, phosphorylation, acetylation, ubiquitination, sumoylation, neddylation, nitrosylation, methylation, lipidation and proteolysis are known to be important mechanisms in eukaryotic cells to regulate protein function and biological processes. Protein acetylation was originally documented in histone proteins [14], [15], though further studies have demonstrated that non-histone proteins are also regulated in an acetylation-dependent manner [16], [17]. For example, acetylation of the p53 C-terminal regulatory domain modulates the activation processes of its target genes [18], [19] and Smad7 acetylation has been shown to control its stability [20], [21]. Histone de-acetylases (HDACs) are a class of enzymes originally identified based on their ability to remove acetyl groups of histone lysine residues [22], [23]. HDACs are important regulators of proliferation, differentiation, cell survival, and cell death. Four HDAC classes have been identified in vertebrates [24], [25]. Class I HDACs (HDAC1, 2, 3, and 8) are expressed in the nucleus, class II HDACs (HDAC4, 5, 6, 7, 9, and 10) shuttle between the nucleus and the cytoplasm, Class III HDACs (e.g. sirtuins) are characterized by their unique requirement for NAD + for enzymatic activity, and Class IV HDACs (e.g. HDAC11) remain poorly understood [26], [27].
SUMO is a small ubiquitin-related modifier and, similar to ubiquitination, SUMO modification (i.e., sumoylation) occurs through a multi-enzymatic pathway. This pathway includes the E1 SUMO-activating enzyme, the E2 SUMO-conjugating enzyme, and the E3 SUMO ligase [28], [29]. In vertebrates, different SUMO isoforms (i.e., SUMO-1, SUMO-2, SUMO-3, and SUMO-4) have been identified, and various PIAS genes including PIAS1, PIAS3, PIASxα, PIASxβ, and PIASy have been characterized as major SUMO ligases [23], [30], [31], [32]. Over the past few years, sumoylation has been shown to play a critical role in a broad range of biological processes, including nuclear transport, transcriptional regulation, and cell division [30], [33], [34]. Furthermore, sumoylation has been shown to function together with ubiquitination in various proteasomal degradation pathways [35].
Here we have demonstrated that p300-mediated acetylation of Nkx3.2 at lysines 260/262 increases stability of the protein, while HDAC9/HDRP-induced de-acetylation of Nkx3.2 promotes PIASy-mediated sumoylation, which in turn triggers SUMO-targeted ubiquitination by RNF4. Together, these findings have elucidated the molecular mechanisms by which acetylation/de-acetylation regulates Nkx3.2 protein stability. We further demonstrated, both in vitro and in vivo, that HDAC9-dependent Nkx3.2 regulation plays a role in controlling chondrocyte viability and hypertrophic maturation.
Section snippets
Expression plasmids and molecular cloning
Expression inserts for △N and KR mutants were generated by two-step PCR mutagenesis of WT and cloned into pCS vectors. Expression plasmid of p300-HA was a generous gift from Dr. Suk-Chul Bae (Chungbuk National University, Cheongju, Korea). SUMO and ARD1 constructs were kindly provided from Dr. Jong-Bok Yoon (Yonsei University, Seoul, Korea) and Dr. Jong-Wan Park (Seoul National University College of Medicine, Seoul, Korea), respectively. Human PCAF, HDAC1, and PIAS (1, y, xα, xβ) cDNAs were
Protein stability of Nkx3.2 can be controlled by its acetylation status
To investigate whether Nkx3.2 regulation can be mediated by acetylation/de-acetylation, we first employed Trichostatin A (TSA), a well-known HDAC inhibitor [36]. Interestingly, pharmacological inhibition of HDACs significantly increased endogenous Nkx3.2 protein levels in human primary chondrocytes such as HFC and HAC (Fig. 1A). Additionally, treatment of these cells with TSA dramatically increased Nkx3.2 acetylation (Fig. 1B). Further, Nkx3.2 protein levels in murine ATDC5 chondrocytes were
Discussion
Here we describe a novel mechanism behind Nkx3.2 protein stability control. While we and others have shown diverse functions of Nkx3.2 during cartilage development, it remains poorly understood how Nkx3.2 can be regulated. Although we previously demonstrated that Nkx3.2 protein stability can be regulated by the Ihh-Wnt5a axis [10] and also by phosphatidylinositol-3-kinase signaling [53], both of which engage downstream effectors that modulate Nkx3.2 ubiquitination via phosphorylation.
Disclosure statement
The authors declare no conflict of interest.
Acknowledgements
This work was supported by the grants (NRF-2012M3A9A9055078; 2015K000180) funded by the Korean Ministry of Science, ICT and Future Planning.
References (71)
- et al.
The chick transcriptional repressor Nkx3.2 acts downstream of Shh to promote BMP-dependent axial chondrogenesis
Dev. Cell
(2001) - et al.
Hypertrophic differentiation during chondrogenic differentiation of progenitor cells is stimulated by BMP-2 but suppressed by BMP-7
Osteoarthr. Cartil.
(2013) - et al.
Characterization of Nkx3.2 DNA binding specificity and its requirement for somitic chondrogenesis
J. Biol. Chem.
(2003) - et al.
Acetylation and deacetylation of non-histone proteins
Gene
(2005) - et al.
Acetylation of non-histone proteins modulates cellular signalling at multiple levels
Int. J. Biochem. Cell Biol.
(2009) - et al.
Control of Smad7 stability by competition between acetylation and ubiquitination
Mol. Cell
(2002) - et al.
The balance between acetylation and deacetylation controls Smad7 stability
J. Biol. Chem.
(2005) - et al.
Genome-wide mapping of HATs and HDACs reveals distinct functions in active and inactive genes
Cell
(2009) - et al.
The role of histone deacetylases (HDACs) in human cancer
Mol. Oncol.
(2007) - et al.
Cloning and functional characterization of HDAC11, a novel member of the human histone deacetylase family
J. Biol. Chem.
(2002)
SP-RING for SUMO: new functions bloom for a ubiquitin-like protein
Cell
Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A
J. Biol. Chem.
The histone deacetylase 9 gene encodes multiple protein isoforms
J. Biol. Chem.
The Sumo-targeted ubiquitin ligase RNF4 regulates the localization and function of the HTLV-1 oncoprotein Tax
Blood
SUMO-targeted ubiquitin ligases
Biochim. Biophys. Acta
Acetylation stabilizes ATP-citrate lyase to promote lipid biosynthesis and tumor growth
Mol. Cell
Transforming growth factor-beta stimulates p300-dependent RUNX3 acetylation, which inhibits ubiquitination-mediated degradation
J. Biol. Chem.
Regulation and destabilization of HIF-1alpha by ARD1-mediated acetylation
Cell
Histone deacetylase 4 controls chondrocyte hypertrophy during skeletogenesis
Cell
Histone deacetylase 3 suppression increases PH domain and leucine-rich repeat phosphatase (Phlpp)1 expression in chondrocytes to suppress Akt signaling and matrix secretion
J. Biol. Chem.
Histone deacetylase 7 (Hdac7) suppresses chondrocyte proliferation and beta-catenin activity during endochondral ossification
J. Biol. Chem.
Histone deacetylase1 promotes TGF-beta1-mediated early chondrogenesis through down-regulating canonical Wnt signaling
Biochem. Biophys. Res. Commun.
Class II histone deacetylases act as signal-responsive repressors of cardiac hypertrophy
Cell
Targeted disruption of the homeobox transcription factor bapx1 results in lethal skeletal dysplasia with asplenia and gastroduodenal malformation
Genes Cells
Transcriptional control of chondrocyte fate and differentiation
Birth Defects Res. C Embryo Today
The mouse bagpipe gene controls development of axial skeleton, skull, and spleen
Proc. Natl. Acad. Sci. U. S. A.
The murine Bapx1 homeobox gene plays a critical role in embryonic development of the axial skeleton and spleen
Development
Shh establishes an Nkx3.2/Sox9 autoregulatory loop that is maintained by BMP signals to induce somitic chondrogenesis
Genes Dev.
Smad-dependent recruitment of a histone deacetylase/Sin3A complex modulates the bone morphogenetic protein-dependent transcriptional repressor activity of Nkx3.2
Mol. Cell. Biol.
Indian Hedgehog signalling triggers Nkx3.2 protein degradation during chondrocyte maturation
Biochem. J.
Nkx3.2/Bapx1 acts as a negative regulator of chondrocyte maturation
Development
Constitutive RelA activation mediated by Nkx3.2 controls chondrocyte viability
Nat. Cell Biol.
Exogenous signal-independent nuclear IkappaB kinase activation triggered by Nkx3.2 enables constitutive nuclear degradation of IkappaB-alpha in chondrocytes
Mol. Cell. Biol.
Histone acetyltransferases
Annu. Rev. Biochem.
Histone acetylation: a switch between repressive and permissive chromatin. Second in review series on chromatin dynamics
EMBO Rep.
Cited by (13)
RNF4-mediated SUMO-targeted ubiquitination relieves PARIS/ZNF746-mediated transcriptional repression
2020, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Interaction between the SUMO moiety of SUMOylated PARIS and PIASy through its SIM2 may block the SUMO-RNF4 SIM interaction that is essential for ubiquitination, and consequently stabilize the PARIS SUMOylation status. In contrast to our observation, it has been reported that PIASy increases RNF4-mediated ubiquitination of Nkx3.2 [36]. The ubiquitination inhibition activity of PIAS proteins may show different substrate specificities in addition to their SUMOylation promoting activity.
Nkx3.2 induces oxygen concentration-independent and lysosome-dependent degradation of HIF-1α to modulate hypoxic responses in chondrocytes
2017, Cellular SignallingCitation Excerpt :Nkx3.2 (also known as Bapx1) encodes the NK-2 class homeodomain transcription factor that is initially expressed in chondrocyte progenitor cells and promotes chondrogenic cell fate [26,35–39]. Once chondrogenic differentiation is induced, Nkx3.2 expression is maintained in proliferating chondrocytes but downregulated in hypertrophic chondrocytes [40–42]. Consistent with these results, Nkx3.2 promotes chondrogenic differentiation.
Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration
2023, Cellular and Molecular Biology LettersEpigenetic Regulation in Knee Osteoarthritis
2022, Frontiers in GeneticsBioinformatics analysis of miRNA and mRNA expression profiles to reveal the key miRNAs and genes in osteoarthritis
2021, Journal of Orthopaedic Surgery and Research