A post-translational modification cascade employing HDAC9-PIASy-RNF4 axis regulates chondrocyte hypertrophy by modulating Nkx3.2 protein stability

Highlights

• Identification of HDAC9 as a critical protein de-acetylase regulated by chondrocyte hypertrophy during skeletal development

• Molecular characterization of a post-translational modification cascade employing HDAC9/HDRP-PIASy-RNF4 axis

• Elucidation of a novel pathway for Nkx3.2 suppression triggered by HDAC9 upon the onset of chondrocyte hypertrophy

Abstract

While Nkx3.2/Bapx1 promotes chondrogenic differentiation and plays a role in maintaining chondrocyte viability and suppressing chondrocyte hypertrophy, the regulatory mechanisms of Nkx3.2 remain poorly understood. Here we show that p300- and HDAC9-induced Nkx3.2 acetylation and de-acetylation, respectively, play critical roles in controlling Nkx3.2 protein stability. In addition, we also found that HDAC9-dependent de-acetylation of Nkx3.2 triggers PIASy-mediated sumoylation and subsequent RNF4-mediated SUMO-targeted ubiquitination. Furthermore, we demonstrate that Nkx3.2 regulation by HDAC9 can be linked to the management of chondrocyte survival and hypertrophic maturation during cartilage development. Finally, our results together reveal a novel mechanism of protein stability control involving complex interplay between acetylation, de-acetylation, sumoylation, and ubiquitination, and suggest that this post-translational modification of Nkx3.2 employing HDAC9-PIASy-RNF4 axis plays a crucial role in controlling chondrocyte viability and hypertrophic maturation during skeletal development in vertebrates.

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.