Dataset Information

Pleiotropic Roles of NOTCH1 Signaling in the Loss of Maturational Arrest of Human Osteoarthritic Chondrocytes.

ABSTRACT: Notch signaling has been identified as a critical regulator of cartilage development and homeostasis. Its pivotal role was established by both several joint specific Notch signaling loss of function mouse models and transient or sustained overexpression. NOTCH1 is the most abundantly expressed NOTCH receptors in normal cartilage and its expression increases in osteoarthritis (OA), when chondrocytes exit from their healthy "maturation arrested state" and resume their natural route of proliferation, hypertrophy, and terminal differentiation. The latter are hallmarks of OA that are easily evaluated in vitro in 2-D or 3-D culture models. The aim of our study was to investigate the effect of NOTCH1 knockdown on proliferation (cell count and Picogreen mediated DNA quantification), cell cycle (flow cytometry), hypertrophy (gene and protein expression of key markers such as RUNX2 and MMP-13), and terminal differentiation (viability measured in 3-D cultures by luminescence assay) of human OA chondrocytes. NOTCH1 silencing of OA chondrocytes yielded a healthier phenotype in both 2-D (reduced proliferation) and 3-D with evidence of decreased hypertrophy (reduced expression of RUNX2 and MMP-13) and terminal differentiation (increased viability). This demonstrates that NOTCH1 is a convenient therapeutic target to attenuate OA progression.

SUBMITTER: Minguzzi M

PROVIDER: S-EPMC8585104 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Pleiotropic Roles of NOTCH1 Signaling in the Loss of Maturational Arrest of Human Osteoarthritic Chondrocytes.

Minguzzi Manuela M Panichi Veronica V D'Adamo Stefania S Cetrullo Silvia S Cattini Luca L Flamigni Flavio F Mariani Erminia E Borzì Rosa Maria RM

International journal of molecular sciences 20211105 21

Notch signaling has been identified as a critical regulator of cartilage development and homeostasis. Its pivotal role was established by both several joint specific Notch signaling loss of function mouse models and transient or sustained overexpression. NOTCH1 is the most abundantly expressed NOTCH receptors in normal cartilage and its expression increases in osteoarthritis (OA), when chondrocytes exit from their healthy "maturation arrested state" and resume their natural route of proliferatio ...[more]

PMID: 34769441

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Project description:BackgroundCellular 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.MethodsChondrocytes, 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.ResultsCompared 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.ConclusionsCBX4 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.

Project description:BackgroundOsteoarthritis (OA) is a multifactorial disease characterized by destruction of the articular cartilage due to environmental, mechanical and genetic components. The genetics of OA is complex and is not completely understood. Recent works have demonstrated the importance of microRNAs (miRNAs) in cartilage function. MiRNAs are a class of small noncoding RNAs that regulate gene expression and are involved in different cellular process: apoptosis, proliferation, development, glucose and lipid metabolism. The aim of this study was to identify and characterize the expression profile of miRNAs in normal and OA chondrocytes and to determine their role in the OA.MethodsChondrocytes were moved to aggregate culture and evaluated using histological and qPCR techniques. miRNAs were isolated and analyzed using the Agilent Human miRNA Microarray.ResultsOf the 723 miRNAs analyzed, 7 miRNAs showed a statistically significant differential expression. Amongst these 7 human miRNAs, 1 was up-regulated in OA chondrocytes (hsa-miR-483-5p) and 6 were up-regulated in normal chondrocytes (hsa-miR-149*, hsa-miR-582-3p, hsa-miR-1227, hsa-miR-634, hsa-miR-576-5p and hsa-miR-641). These profiling results were validated by the detection of some selected miRNAs by qPCR. In silico analyses predicted that key molecular pathways potentially altered by the miRNAs differentially expressed in normal and OA chondrocytes include TGF-beta, Wnt, Erb and mTOR signalling; all of them implicated in the development, maintenance and destruction of articular cartilage.ConclusionsWe have identified 7 miRNAs differentially expressed in OA and normal chondrocytes. Our potential miRNA target predictions and the signalling cascades altered by the differentially expressed miRNAs supports the potential involvement of the detected miRNAs in OA pathology. Due to the importance of miRNA in mediating the translation of target mRNA into protein, the identification of these miRNAs differentially expressed in normal and OA chondrocyte micropellets could have important diagnostic and therapeutic potential. Further studies are needed to know the function of these miRNAs, including the search of their target mRNA genes, which could lead to the development of novel therapeutic strategies for the OA treatment.

Dataset Information

Pleiotropic Roles of NOTCH1 Signaling in the Loss of Maturational Arrest of Human Osteoarthritic Chondrocytes.

Publications

Pleiotropic Roles of NOTCH1 Signaling in the Loss of Maturational Arrest of Human Osteoarthritic Chondrocytes.

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