Circadian control of the secretory pathway maintains collagen homeostasis

Chang, Joan; Garva, Richa; Pickard, Adam; Yeung, Ching-Yan Chloé; Mallikarjun, Venkatesh; Swift, Joe; Holmes, David; Calverley, Ben; Lu, Yinhui; Adamson, Antony; Raymond-Hayling, Helena; Jensen, Oliver E.; Shearer, Tom; Meng, Qing‐Jun; Kadler, Karl E.

doi:10.1038/s41556-019-0441-z

Cited by 151 publications

(182 citation statements)

References 68 publications

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“…4B). The time of peak levels of intracellular luminescence was 12.2 hours post synchronisation (estimated to be circadian time CT0), which aligns well with observations of peak PC-I levels in tendon in vivo 6 . These findings provided direct evidence that the circadian clock influences the synthesis of NLuc-PC-I in nluc::Col1a2 cells.…”

Section: Resultssupporting

confidence: 86%

“…Therefore, cells concentrate procollagen molecules in preparation for collagen fibril formation. Our ability to measure the number of PC-I molecules in individual cells enabled a time-series study of procollagen synthesis, in which we showed that the synthesis of PC-I was rhythmic with a ~24-hour period, and thereby confirmed previous proteomic data that the synthesis of PC-I is under circadian clock control 6 .…”

Section: Discussionsupporting

confidence: 80%

“…3A). It has recently been shown that PC-I levels in tendon fluctuate rhythmically during 24 hours under the control of the circadian clock 6 . Therefore, to explore the possibility that the variation in NLuc levels observed in individual cells could reflect differences in PC-I levels in cells at different stages of the circadian cycle, we synchronised nluc-Col1a2 cells every 4 hours (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Dynamic protein quantitation (DyProQ) of procollagen-I by CRISPR-Cas9 NanoLuciferase tagging

Calverley

Kadler

Pickard

2020

Preprint

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The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery but remains a major technical challenge. Here, we describe dynamic protein quantitation (DyProQ) which is effective across microscopy and multiwell platforms. Using collagen as a test protein, CRISPR-Cas9-mediated introduction of nluc (encoding NanoLuciferase, NLuc) into the Col1a2 locus enabled simplification and miniaturisation of procollagen-I (PC-I) quantitation. We robustly assessed extracellular, intracellular, and subcellular PC-I levels, by correlating to known concentrations of recombinant NLuc in the presence of substrate. Loss of collagen causes tissue degeneration whereas excess collagen results in fibrosis (often with poor-outcome) and is evident in aggressive cancers; however, treatment options are extremely limited. Using collagen-DyProQ, we screened a library of 1,971 FDA-approved compounds and identified 10 candidates for repurposing in the treatment of fibrotic and 7 for degenerative diseases.

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Section: Resultssupporting

confidence: 86%

Section: Discussionsupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

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Dynamic protein quantitation (DyProQ) of procollagen-I by CRISPR-Cas9 NanoLuciferase tagging

Calverley

Kadler

Pickard

2020

Preprint

Self Cite

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“…In most cases, it is evident that a healthy biological response to loading modifies the course of damage accumulation and prevents the development of connective tissue injuries due to normal physical activity. Studies directly investigating cellular collagen production in tendons have revealed increased collagen production and incorporation within hours or days of exercise or direct mechanical loading (26,27) and have demonstrated circadian regulation of collagen synthesis, cellular export, and degradation as a possible mechanism for maintaining tissue homeostasis (28). The exact process by which tendon damage is identified and new collagen is incorporated into noninjured loaded tendons or tendons healing after injury is unclear, but we know that the body has solved this engineering design problem.…”

Section: Discussionmentioning

confidence: 99%

Accumulation of collagen molecular unfolding is the mechanism of cyclic fatigue damage and failure in collagenous tissues

et al. 2020

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Overuse injuries to dense collagenous tissues are common, but their etiology is poorly understood. The predominant hypothesis that micro-damage accumulation exceeds the rate of biological repair is missing a mechanistic explanation. Here, we used collagen hybridizing peptides to measure collagen molecular damage during tendon cyclic fatigue loading and computational simulations to identify potential explanations for our findings. Our results revealed that triple-helical collagen denaturation accumulates with increasing cycles of fatigue loading, and damage is correlated with creep strain independent of the cyclic strain rate. Finite-element simulations demonstrated that biphasic fluid flow is a possible fascicle-level mechanism to explain the rate dependence of the number of cycles and time to failure. Molecular dynamics simulations demonstrated that triple-helical unfolding is rate dependent, revealing rate-dependent mechanisms at multiple length scales in the tissue. The accumulation of collagen molecular denaturation during cyclic loading provides a long-sought “micro-damage” mechanism for the development of overuse injuries.

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“…In summary, Egr1 appears to be required for the correct expression of matrix genes during tendon formation, homeostasis, and ageing in vitro and in vivo, but also during tendon healing. A recent discovery is that tendons are peripheral circadian clock tissues, reviewed in [105], in which collagen synthesis and homeostasis is under a circadian control [106]. Interestingly, the circadian clock is disturbed in Egr1 −/− mice, associated with impaired locomotor activity and body temperature [107], suggesting that Egr1 could be involved in the circadian synthesis of tendon-associated collagens.…”

Section: Egr1 Function In Tendon Healingmentioning

confidence: 99%

EGR1 Transcription Factor is a Multifaceted Regulator of Matrix Production in Tendons and Other Connective Tissues

Havis

Duprez

2020

IJMS

366

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Although the transcription factor EGR1 is known as NGF1-A, TIS8, Krox24, zif/268, and ZENK, it still has many fewer names than biological functions. A broad range of signals induce Egr1 gene expression via numerous regulatory elements identified in the Egr1 promoter. EGR1 is also the target of multiple post-translational modifications, which modulate EGR1 transcriptional activity. Despite the myriad regulators of Egr1 transcription and translation, and the numerous biological functions identified for EGR1, the literature reveals a recurring theme of EGR1 transcriptional activity in connective tissues, regulating genes related to the extracellular matrix. Egr1 is expressed in different connective tissues, such as tendon (a dense connective tissue), cartilage and bone (supportive connective tissues), and adipose tissue (a loose connective tissue). Egr1 is involved in the development, homeostasis, and healing processes of these tissues, mainly via the regulation of extracellular matrix. In addition, Egr1 is often involved in the abnormal production of extracellular matrix in fibrotic conditions, and Egr1 deletion is seen as a target for therapeutic strategies to fight fibrotic conditions. This generic EGR1 function in matrix regulation has little-explored implications but is potentially important for tendon repair.

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Circadian control of the secretory pathway maintains collagen homeostasis

Cited by 151 publications

References 68 publications

Dynamic protein quantitation (DyProQ) of procollagen-I by CRISPR-Cas9 NanoLuciferase tagging

Dynamic protein quantitation (DyProQ) of procollagen-I by CRISPR-Cas9 NanoLuciferase tagging

Accumulation of collagen molecular unfolding is the mechanism of cyclic fatigue damage and failure in collagenous tissues

EGR1 Transcription Factor is a Multifaceted Regulator of Matrix Production in Tendons and Other Connective Tissues

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