Synergistic roles of CBX4 chromo and SIM domains in regulating senescence of primary human osteoarthritic chondrocytes.

BACKGROUND: Cellular senescence is a critical factor contributing to osteoarthritis (OA). Overexpression of chromobox homolog 4 (CBX4) in a mouse system was demonstrated to alleviate post-traumatic osteoarthritis (PTOA) by reducing cellular senescence. Additionally, replicative cellular senescence of WI-38 fibroblasts can be attenuated by CBX4. However, the mechanisms underlying this senomorphic function of CBX4 are not fully understood. In this study, we aimed to investigate the role of CBX4 in cellular senescence in human primary osteoarthritic chondrocytes and to identify the functional domains of CBX4 necessary for its function in modulating senescence. METHODS: Chondrocytes, isolated from 6 individuals undergoing total knee replacement for OA, were transduced with wild-type CBX4, mutant CBX4, and control lentiviral constructs. Senescence-related phenotypic outcomes included the following: multiple flow cytometry-measured markers (p16INK4A, senescence-associated ß-galactosidase [SA-ß-gal] activity and dipeptidyl peptidase-4 [DPP4], and proliferation marker EdU), multiplex ELISA-measured markers in chondrocyte culture media (senescence-associated secretory phenotypes [SASPs], including IL-1ß, IL-6, IL-8, TNF-α, MMP-1, MMP-3, and MMP-9), and PCR array-evaluated senescence-related genes. RESULTS: Compared with control, CBX4 overexpression in OA chondrocytes decreased DPP4 expression and SASP secretion and increased chondrocyte proliferation confirming CBX4 senomorphic effects on primary human chondrocytes. Point mutations of the chromodomain domain (CDM, involved in chromatin modification) alone were sufficient to partially block the senomorphic activity of CBX4 (p16INK4A and DPP4 increased, and EdU decreased) but had minimal effect on SASP secretion. Although having no effect on p16INK4A, DPP4, and EdU, deletion of two small-ubiquitin-like-modifier-interaction motifs (CBX4 ΔSIMs) led to increased SASP secretion (IL-1ß, TNF-α, IL-8). The combination CBX4 CDMΔSIMs altered all these measures adversely and to a greater degree than the single domain mutants. Deletion of the C-terminal (CBX4 ΔC-box) involved with transcriptional silencing of polycomb group proteins increased IL-1ß slightly but significantly but altered none of the other senescence outcome measures. CONCLUSIONS: CBX4 has a senomorphic effect on human osteoarthritic chondrocytes. CDM is critical for CBX4-mediated regulation of senescence. The SIMs are supportive but not indispensable for CBX4 senomorphic function while the C-box is dispensable.

Expression profile of polycomb group proteins in odontogenic keratocyst and ameloblastoma.

Polycomb group (PcG) proteins are repressive chromatin modifiers required for proliferation and development. PcG proteins form two large repressive complexes, namely, Polycomb Repressive Complex 1 and 2. These proteins have been shown to drive tumorigenesis by repressing cell-type specific sets of target genes. Using immunohistochemistry, we investigated the expression patterns of five human PcG proteins, including Bmi-1, Ring1b, Mel-18, Ezh2, and Suz12, in various cellular components of odontogenic keratocysts (OKCs), ameloblastomas and, pericoronal follicles (PFs). In OKCs, expression of PcG proteins were found in the majority of cases while the expression pattern was relatively different for each PcG proteins. All PcG proteins were strongly expressed in the basal cells while some proteins showed variable expression in the parabasal and luminal cell layer of OKCs. In ameloblastomas, almost all PcG proteins showed a similar expression pattern of moderate to strong staining in the peripheral ameloblast-like cells and metaplastic squamous cells. Some of the central stellate reticulum-like cells also showed positive reaction to most PcG proteins. In PFs, most PcG proteins were intensely expressed in odontogenic epithelium lining the follicles, except Mel-18 and Suz12. The present study provides the initial evidence regarding epigenetic involvement by PcG proteins in these odontogenic lesions. Although these proteins are known to be in the same repressive group proteins, differential expression patterns of these proteins in OKCs and ameloblastomas indicates that these proteins may play different roles in pathogenesis of these odontogenic lesions.