Circadian time series proteomics reveals daily dynamics in cartilage physiology

M Dudek,C Angelucci,D Pathiranage,P Wang,V Mallikarjun,C Lawless,J Swift,K.E Kadler,R.P Boot-Handford,J.A Hoyland,S.R Lamande,J.F Bateman,Q.-J Meng

doi:10.1016/j.joca.2021.02.008

Abstract

SummaryObjectiveCartilage in joints such as the hip and knee experiences repeated phases of heavy loading and low load recovery during the 24-h day/night cycle. Our previous work has shown 24 h rhythmic changes in gene expression at transcript level between night and day in wild type mouse cartilage which is lost in a circadian clock knock-out mouse model. However, it remains unknown to what extent circadian rhythms also regulate protein level gene expression in this matrix rich tissue.MethodsWe investigated daily changes of protein abundance in mouse femoral head articular cartilage by performing a 48-h time-series LC-MS/MS analysis.ResultsOut of the 1,177 proteins we identified across all time points, 145 proteins showed rhythmic changes in their abundance within the femoral head cartilage. Among these were molecules that have been implicated in key cartilage functions, including CTGF, MATN1, PAI-1 and PLOD1 & 2. Pathway analysis revealed that protein synthesis, cytoskeleton and glucose metabolism exhibited time-of-day dependent functions. Analysis of published cartilage proteomics datasets revealed that a significant portion of rhythmic proteins were dysregulated in osteoarthritis and/or ageing.ConclusionsOur circadian proteomics study reveals that articular cartilage is a much more dynamic tissue than previously thought, with chondrocytes driving circadian rhythms not only in gene transcription but also in protein abundance. Our results clearly call for the consideration of circadian timing mechanisms not only in cartilage biology, but also in the pathogenesis, treatment strategies and biomarker detection in osteoarthritis.

Highlights

The night and day cycle governs the circadian (24 hourly) rhythms of rest/activity, physiology and metabolism of most animals on this planet, including humans
Using Guanidine HCL buffer protein extraction and nanoliquid chromatography coupled tandem mass spectrometry, we were able to identify 1,177 proteins by a minimum of two unique peptides (Supplementary Table 1). 70% of identified proteins overlapped with datasets from two single time point studies using similar cartilage protein extraction methods [Supplementary Fig. 1(A)]7,8
To reveal the functional relevance of the rhythmic proteins we identified in cartilage in a disease setting, we compared our rhythmic proteins with published ageing or osteoarthritis-regulated cartilage proteomic studies

Summary

Introduction

The night and day cycle governs the circadian (24 hourly) rhythms of rest/activity, physiology and metabolism of most animals on this planet, including humans. A central circadian clock in the anterior hypothalamus of the brain co-. Ordinates the rhythmic behaviour (such as the sleep/wake cycle), and synchronizes various local circadian oscillators that are found in most peripheral organs. The circadian oscillator is based on a transcriptional/translational negative feedback loop composed of interlocked transcriptional activators (BMAL1 and CLOCK) and repressors (CRYs and PERs). In addition to driving the expression of core clock genes, these clock factors control rhythmic expression of hundreds of genes in a tissue specific manner[1]. Time series transcriptome studies in mouse and primate have revealed that over 80% of protein-coding genes exhibit 24-h rhythms of mRNA expression in at least one tissue[2].

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