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. 2023 Oct 12;25(1):197.
doi: 10.1186/s13075-023-03183-8.

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

Yu-Hsiu Chen  1   2   3 , Xin Zhang  1   4 , David Attarian  4 , Virginia Byers Kraus  5   6   7   8
Affiliations

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

Yu-Hsiu Chen et al. Arthritis Res Ther. .

Abstract

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 β-galactosidase [SA-β-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-1β, 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-1β, 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-1β 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.

Keywords: CBX4; Cellular senescence; DPP4; Osteoarthritis; Senomorphic.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Effects of CBX4 on primary human chondrocytes. CBX4 was overexpressed in human OA chondrocytes using CBX4 WT lentiviral particles. A CBX4 gene expression (n = 6) and protein expression (representative Western blot) in the CBX4 WT and control lentiviral construct (Ctrl) transduced OA chondrocytes. Upon longer exposure of the Western blot, CBX4 protein expression in control transduced primary chondrocytes was comparable to levels of expression we observe in senescent WI-38 (passage 55) cells (data not shown). CBX4: 75 kDa, β-actin: 42 kDa. B SA-β-gal activity, protein expression of p16INK4A and DPP4, and EdU proliferation were detected using flow cytometry. C Senescence-associated secretory phenotype (SASP) secretion was measured in the culture supernatants of CBX4 WT and vector only control (Ctrl)-transduced OA chondrocytes. Ratio-paired t-tests were performed to compare control and CBX4 WT presented as a dot line graph; P values are listed in the figure
Fig. 2
Fig. 2
Mapping effects of CBX4 (wild-type and mutants) on primary human chondrocytes. Expression of senescence markers and cell proliferation of primary human chondrocytes transduced by CBX4 wild-type (WT) or mutants. A CBX4 gene expression (n = 6) and B protein expression by Western blot (representative blot with quantification of n = 5 biological replicates in dot plot) in cells transduced by the empty vector Control (Ctrl), CBX4 WT, or its mutants. The expected molecular weights of CBX4 WT, CBX4 CDM, CBX4 ΔSIMs, CBX4 ΔC-box, and CBX4 CDMΔSIM were 75 kDa, 75 kDa, 74 kDa, 72 kDa, and 74 kDa, respectively. C Protein-level expression of senescence markers DPP4, SA-β-gal activity, and p16.INK4A and cell proliferation marker EdU quantified by flow cytometry and plotted as a dot bar graph. Repeated measures with Dunnett’s post hoc test were performed to compare the effects of CBX4 WT and mutants. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001
Fig. 3
Fig. 3
Effects of CBX4 wild-type and mutants on SASP secretion by primary human OA chondrocytes. Comparison of SASP secretion, including IL-1β, IL-6, IL-8, TNF-α, MMP-1, MMP-3, and MMP-9, by primary human OA chondrocytes transduced with CBX4 wild-type (WT) or mutants. Repeated measures with Dunnett’s post hoc test were performed to compare WT and mutant CBX4. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001
Fig. 4
Fig. 4
Senescence-related gene expression profiling of CBX4 wild-type and mutants in primary human OA chondrocytes. The heatmap depicts the relative gene expression by primary human OA chondrocytes transduced with CBX4 wild-type (WT) or four CBX4 mutants of a select group of genes quantified by qPCR array. Gene expression is shown as a log2 ratio of expression level by CBX4 mutant-transduced cells relative to the CBX4 WT-transduced condition. The genes in the heatmap are organized vertically based on their functional properties: green text labels (top y-axis) are genes associated with cell proliferation; black text labels (middle y-axis) are genes related to DNA damage repair and apoptosis; red text labels (bottom y-axis) are genes related to SASPs (senescence-associated secretory phenotypes) and cellular senescence. The most deleterious impact on CBX4 senomorphic capability was caused by the combination of CDM and SIM mutations (far right column). #P ≤ 0.1 *P ≤ 0.05, **P ≤ 0.01
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