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Circadian control of the secretory pathway maintains collagen homeostasis

DOI:10.1038/s41556-019-0441-z
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Joan Chang at The University of Manchester
Joan Chang
Richa Garva at The University of Manchester
Richa Garva
Adam Pickard at The University of Manchester
Adam Pickard
Ching-Yan Chloé Yeung at Institute of Sports Medicine Copenhagen
Ching-Yan Chloé Yeung
  • Institute of Sports Medicine Copenhagen
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Abstract and Figures

Collagen is the most abundant secreted protein in vertebrates and persists throughout life without renewal. The permanency of collagen networks contrasts with both the continued synthesis of collagen throughout adulthood and the conventional transcriptional/translational homeostatic mechanisms that replace damaged proteins with new copies. Here, we show circadian clock regulation of endoplasmic reticulum-to-plasma membrane procollagen transport by the sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis and degradation affects a pool of newly synthesized collagen, while maintaining the persistent collagen network. Disabling the circadian clock causes abnormal collagen fibrils and collagen accumulation, which are reduced in vitro by the NR1D1 and CRY1/2 agonists SR9009 and KL001, respectively. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis wherein a sacrificial pool of collagen maintains tissue function. Here, Chang et al. show that the circadian clock regulates secretion resulting in nocturnal procollagen synthesis and daytime collagen fibril assembly in mice to maintain the homeostasis of the collagen network.
Electron microscopy and biomechanics a, Collagen fibril diameter distributions measured from transverse TEM images of tail tendons sampled through a circadian period. Mice were housed in 12-hour dark/12-hour light cycles. ZT, zeitgeber time (hours into light). Fibrils (n = 1400) were measured for each panel. Black lines show 3-Gaussian fit curves. b, Typical scanning transmission electron microscopy image of fibrils from mechanically disrupted tendon. A set of 50 similar STEM images were acquired for each of 30 tendon samples. Bar, 500 nm. c, Representative mass-per-unit-length distribution measured from scanning transmission electron microscopy images of mechanically disrupted whole tendons. Time point shown is ZT12. The data shown is for n- = 997 fibrils from a single Achilles tendon. All mass-per-unit-length distributions from 30 tendon samples showed a characteristic prominent D1 peak. Percentage of D1 fibrils at all time points over 24 h is shown in Fig. 1e. d, Elastic modulus of Achilles tendon is not time-of-day dependent. Bars show mean ± s.e.m., n = 4 biological replicates, unpaired t-test, p = 0.46. e, Representative hysteresis curves from cyclic loading show the energy loss is greater in tendons taken at ZT15 compared with ZT3, n = 4 biological replicates, n = 5 hysteresis cycles for each displacement rate for each tendon sample. f, Time constants derived from stress–relaxation from an initial 1 N load of Achilles tendon sampled at ZT3 compared to ZT15 (T1, T2 and T3). Bars show mean ± s.e.m., n = 4 biological replicates; the p-values were 0.114, 0.026 and 0.75 for the time constants T1, T2 and T3, respectively. g, Comparison of energy loss values of Achilles tendons at ZT3 versus ZT15 for a 5-fold range of strain rates showing a consistent ~ 40% greater energy loss at ZT15 compared with ZT3. Bars show mean ± s.e.m., n = 4 biological replicates, p-values using the unpaired t-test are 0.27, 0.26, 0.21, 0.004, 0.01 for displacement rates of 1, 2, 3, 4 and 5 mm/min, respectively. See also Statistical Source Extended Data Fig. 1. Source data
Electron microscopy and biomechanics a, Collagen fibril diameter distributions measured from transverse TEM images of tail tendons sampled through a circadian period. Mice were housed in 12-hour dark/12-hour light cycles. ZT, zeitgeber time (hours into light). Fibrils (n = 1400) were measured for each panel. Black lines show 3-Gaussian fit curves. b, Typical scanning transmission electron microscopy image of fibrils from mechanically disrupted tendon. A set of 50 similar STEM images were acquired for each of 30 tendon samples. Bar, 500 nm. c, Representative mass-per-unit-length distribution measured from scanning transmission electron microscopy images of mechanically disrupted whole tendons. Time point shown is ZT12. The data shown is for n- = 997 fibrils from a single Achilles tendon. All mass-per-unit-length distributions from 30 tendon samples showed a characteristic prominent D1 peak. Percentage of D1 fibrils at all time points over 24 h is shown in Fig. 1e. d, Elastic modulus of Achilles tendon is not time-of-day dependent. Bars show mean ± s.e.m., n = 4 biological replicates, unpaired t-test, p = 0.46. e, Representative hysteresis curves from cyclic loading show the energy loss is greater in tendons taken at ZT15 compared with ZT3, n = 4 biological replicates, n = 5 hysteresis cycles for each displacement rate for each tendon sample. f, Time constants derived from stress–relaxation from an initial 1 N load of Achilles tendon sampled at ZT3 compared to ZT15 (T1, T2 and T3). Bars show mean ± s.e.m., n = 4 biological replicates; the p-values were 0.114, 0.026 and 0.75 for the time constants T1, T2 and T3, respectively. g, Comparison of energy loss values of Achilles tendons at ZT3 versus ZT15 for a 5-fold range of strain rates showing a consistent ~ 40% greater energy loss at ZT15 compared with ZT3. Bars show mean ± s.e.m., n = 4 biological replicates, p-values using the unpaired t-test are 0.27, 0.26, 0.21, 0.004, 0.01 for displacement rates of 1, 2, 3, 4 and 5 mm/min, respectively. See also Statistical Source Extended Data Fig. 1. Source data
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Time-series transcriptomics a,b, Expression of core clock genes (Npas2, Per1, Per2, Per3, Arntl, Cry1, and Cry2) mRNA. Transcripts were detected by microarray over a 48-h period. If present, multiple probe sets are shown, n = 2 mice. Metacycle analyses BHQ (Benjamini–Hochberg q-⁶³) values indicated on top right-hand corner. ArrayExpress Accession number E-MTAB-7743 and ref. ¹⁷. c, Expression of fibrillar collagen genes in wild-type mouse tail tendon tissue detected by different probe sets in a time series microarray study were not rhythmic, n = 2 mice. Grey shadow indicates subjective night phase. ArrayExpress Accession number E-MTAB-7743 and ref. ¹⁷. See also Statistical Source Extended Data Fig. 2. Source data
Time-series transcriptomics a,b, Expression of core clock genes (Npas2, Per1, Per2, Per3, Arntl, Cry1, and Cry2) mRNA. Transcripts were detected by microarray over a 48-h period. If present, multiple probe sets are shown, n = 2 mice. Metacycle analyses BHQ (Benjamini–Hochberg q-⁶³) values indicated on top right-hand corner. ArrayExpress Accession number E-MTAB-7743 and ref. ¹⁷. c, Expression of fibrillar collagen genes in wild-type mouse tail tendon tissue detected by different probe sets in a time series microarray study were not rhythmic, n = 2 mice. Grey shadow indicates subjective night phase. ArrayExpress Accession number E-MTAB-7743 and ref. ¹⁷. See also Statistical Source Extended Data Fig. 2. Source data
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Rhythmic ribosome docking a, Images of electron microscopy of transverse sections across mouse tendons fibroblasts in vivo showing the endoplasmic reticulum (ER) at different time points, n = 2 biological repeats. Bars 500 nm. b, Quantification of ribosome docking onto ER show differences at ZT2 and ZT15.5 in tendon fibroblasts in vivo, n = 2 biological repeats with 37 (ZT2) and 20 ZT15.5 regions scored across the two samples. Samples were scored by two independent researchers, all samples were blinded (p = 0.00000000034, two-tailed unpaired t-test). The mean and SD for each sample is shown. c, The transcription factor binding site prediction tool Contra v3 http://bioit2.irc.ugent.be/contra/v3 was used to search for binding sites in the promoter and upstream sequences of the genes shown. The default (pragmatic) positional weight matrices (PWMs) score matching stringency in a 500 base promoter region was used. E-box: Ebox, CLOCK/BMAL, Myc, Myc/Max. Cry1/Cry2 binding sites are E-boxes/CLOCK/BMAL and ROR/REV-ERB. D-box: E4BP, NFIL3, ATF2, CEBP. GRE: GRE, NR3C1. ROR: RORA, RORB, RORC. See also Statistical Source Extended Data Fig. 3. Source data
Rhythmic ribosome docking a, Images of electron microscopy of transverse sections across mouse tendons fibroblasts in vivo showing the endoplasmic reticulum (ER) at different time points, n = 2 biological repeats. Bars 500 nm. b, Quantification of ribosome docking onto ER show differences at ZT2 and ZT15.5 in tendon fibroblasts in vivo, n = 2 biological repeats with 37 (ZT2) and 20 ZT15.5 regions scored across the two samples. Samples were scored by two independent researchers, all samples were blinded (p = 0.00000000034, two-tailed unpaired t-test). The mean and SD for each sample is shown. c, The transcription factor binding site prediction tool Contra v3 http://bioit2.irc.ugent.be/contra/v3 was used to search for binding sites in the promoter and upstream sequences of the genes shown. The default (pragmatic) positional weight matrices (PWMs) score matching stringency in a 500 base promoter region was used. E-box: Ebox, CLOCK/BMAL, Myc, Myc/Max. Cry1/Cry2 binding sites are E-boxes/CLOCK/BMAL and ROR/REV-ERB. D-box: E4BP, NFIL3, ATF2, CEBP. GRE: GRE, NR3C1. ROR: RORA, RORB, RORC. See also Statistical Source Extended Data Fig. 3. Source data
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Rhythmic collagen fibril assembly a, MEFs were synchronized and collagen-I deposition was assessed by indirect immunofluorescence every 4 h during 60 h. Representative images from n = 3 for each time point are shown with similar results. Culture of synchronized cells was cultured in the presence of 1 mM DMOG as a control for no collagen secretion. Bars, 10 µm. b, Metacycle analyses values are shown (n = 3 independent experiments; mean ± SD). See also Statistical Source Extended Data Fig. 4. Source data
Rhythmic collagen fibril assembly a, MEFs were synchronized and collagen-I deposition was assessed by indirect immunofluorescence every 4 h during 60 h. Representative images from n = 3 for each time point are shown with similar results. Culture of synchronized cells was cultured in the presence of 1 mM DMOG as a control for no collagen secretion. Bars, 10 µm. b, Metacycle analyses values are shown (n = 3 independent experiments; mean ± SD). See also Statistical Source Extended Data Fig. 4. Source data
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Collagen-I intracellular localization a, MEFs were synchronized and collagen-I co-localization with GM130 was assessed by indirect immunofluorescence every 4 h during 24 h (up to 15 h post synchronization shown). Arrows point to the Golgi. Bars 10 µm. n = 4 biological repeats. b, Enlarged frames. c, Quantification of the co-localization of collagen-I and GM130 after circadian clock synchronization. d, Quantification of the co-localization of HSP47 and GM130 after circadian clock synchronization. N = 4 independent experiments, mean ± s.e.m. See also Statistical Source Extended Data Fig. 5. Source data
+12
Collagen-I intracellular localization a, MEFs were synchronized and collagen-I co-localization with GM130 was assessed by indirect immunofluorescence every 4 h during 24 h (up to 15 h post synchronization shown). Arrows point to the Golgi. Bars 10 µm. n = 4 biological repeats. b, Enlarged frames. c, Quantification of the co-localization of collagen-I and GM130 after circadian clock synchronization. d, Quantification of the co-localization of HSP47 and GM130 after circadian clock synchronization. N = 4 independent experiments, mean ± s.e.m. See also Statistical Source Extended Data Fig. 5. Source data
… 
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Articles
https://doi.org/10.1038/s41556-019-0441-z
1Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science
Centre, Manchester, UK. 2School of Mathematics, Faculty of Science and Engineering, University of Manchester, Manchester, UK. 3Present address:
Institute of Sports Medicine Copenhagen, Bispebjerg Hospital, Copenhagen, Denmark. 4These authors contributed equally: Joan Chang, Richa Garva,
Adam Pickard, Ching-Yan Chloé Yeung. *e-mail: qing-jun.meng@manchester.ac.uk; karl.kadler@manchester.ac.uk
One-third of the eukaryote proteome enters the secretory
pathway1, including the collagens that assemble into centi-
metre-long fibrils in the extracellular matrix (ECM)2. These
fibrils account for one-third of the mass of vertebrates3 and are the
sites of attachment for a wide range of macromolecules, including
integrins, making them essential for metazoan development3. A
remarkable feature of collagen fibrils is that they are formed dur-
ing embryogenesis4 and remain without turnover for the life of the
animal5–8. This has led to the idea that collagen fibrils are static and
unchanging. However, the difficulty with zero turnover is that it does
not explain the absence of fatigue failure, which would be expected
in the face of life-long cyclic loading. In contrast to the evidence
of zero replacement, fibroblasts synthesize collagen in response to
mechanical loading9, and microdialysis of human Achilles tendon
shows elevated levels of the C-propeptides of procollagen-I (PC-I;
the precursor of collagen-I) after moderate exercise10.
These opposing observations led to the alternative hypothesis
presented in this study, in which zero turnover and continued syn-
thesis can coexist. We hypothesized that a pool of ‘persistent’ col-
lagen coexists with a pool of ‘sacrificial’ collagen, in which the latter
is synthesized and removed on a daily basis under the control of the
circadian clock. Support for this alternative hypothesis comes from
observations of a circadian oscillation in the serum concentrations
of the C-propeptides of PC-I11 and of collagen degradation products
in bone12. However, despite physiological and clinical observations,
direct mechanistic support for these observations was lacking.
Although the suprachiasmatic nucleus of the hypothalamus is the
master circadian pacemaker, almost all tissues have self-sustaining
circadian pacemakers that synchronize rhythmic tissue-specific
gene expression in anticipation of environmental cycles of light and
dark13. Disruption of the circadian clock leads to musculoskeletal
abnormalities—for example, chondrocyte-specific disruptions of
the circadian clock result in progressive degeneration of articular
cartilage14 and fibrosis in the intervertebral disc15, and mice with
a global knockout of Bmal1 (ref. 16) or the ClockΔ19 mutation17
develop thickened and calcified tendons with associated immobi-
lization. These observations are indicative of circadian control of
ECM homeostasis.
Here, we performed time-series electron microscopy, tran-
scriptomics and proteomics over day–night cycles, which showed
that the synthesis and transport of PC-I by the protein secretory
pathway in fibroblasts is regulated by the circadian clock. We
show that SEC61, TANGO1, PDE4D and VPS33B regulate col-
lagen secretion, are 24-h rhythmic, and are located at the entry
and exit points of the endoplasmic reticulum (ER), Golgi and
post-Golgi compartments, respectively. CTSK is a collagen-
degrading proteinase, which is rhythmic in-phase with colla-
gen degradation to maintain collagen homeostasis. The result is
nocturnal PC-I synthesis and a daily wave of collagen-I with no
net change in the total collagen content of the tissue. Crucially,
we discovered that arrhythmic ClockΔ19 and Scleraxis–Cre-
dependent Bmal1-deletion mutant mice accumulate collagen and
have a disorganized and structurally abnormal collagen matrix
that is mechanically abnormal. Finally, we show that ClockΔ19
fibroblasts in vitro amass collagen fibres compared with con-
trol cells and treatment of ClockΔ19 fibroblasts with the NR1D1
agonist SR9009 (ref. 18) or the cryptochrome (CRY1/2) agonist
KL001 (ref. 19) reduces the number of collagen fibres. Wild-type
fibroblasts treated with KL001 lose their circadian rhythm and
generate more collagen fibres. Together, these results provide
insights into the importance of the circadian clock in maintaining
collagen homeostasis.
Circadian control of the secretory pathway
maintains collagen homeostasis
Joan Chang1,4, Richa Garva1,4, Adam Pickard1,4, Ching-Yan Chloé Yeung1,3,4, Venkatesh Mallikarjun 1,
Joe Swift 1, David F. Holmes1, Ben Calverley1,2, Yinhui Lu1, Antony Adamson 1,
Helena Raymond-Hayling1,2, Oliver Jensen 2, Tom Shearer2, Qing Jun Meng 1* and Karl E. Kadler 1*
Collagen is the most abundant secreted protein in vertebrates and persists throughout life without renewal. The permanency
of collagen networks contrasts with both the continued synthesis of collagen throughout adulthood and the conventional tran-
scriptional/translational homeostatic mechanisms that replace damaged proteins with new copies. Here, we show circadian
clock regulation of endoplasmic reticulum-to-plasma membrane procollagen transport by the sequential rhythmic expression
of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly
in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis
and degradation affects a pool of newly synthesized collagen, while maintaining the persistent collagen network. Disabling
the circadian clock causes abnormal collagen fibrils and collagen accumulation, which are reduced invitro by the NR1D1 and
CRY1/2 agonists SR9009 and KL001, respectively. In conclusion, our study has identified a circadian clock mechanism of
protein homeostasis wherein a sacrificial pool of collagen maintains tissue function.
NATURE CELL BIOLOGY | VOL 22 | JANUARY 2020 | 74–86 | www.nature.com/naturecellbiology
74
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... Fibrils exhibit a regular, wavy, longitudinal morphology called crimp, thought to undulate in either a two-dimensional (2D) planar or helical fashion [15][16][17] over an approximately 100 μm period for adult mouse tendon [18] . Their diameter remains constant along most of their length, with some exceptions [19] , but there is a distribution of diameters in any given tendon cross-section. Recent work has shown that the diameter distribution can be described well by a trimodal Gaussian distribution [19] . ...
... Their diameter remains constant along most of their length, with some exceptions [19] , but there is a distribution of diameters in any given tendon cross-section. Recent work has shown that the diameter distribution can be described well by a trimodal Gaussian distribution [19] . ...
... An increase of 50% in fibril diameter has previously been found to account for a five-fold increase in ultimate tensile strength (the maximum stress before failure) and Young's modulus in human fibroblast tendon constructs [20] . Collagen fibrils in tendon are structurally continuous through large longitudinal distances ( ∼ 10 −2 m) [21] , and can be as small as 50 nm in diameter, and as large as 500 nm [19] , giving them an aspect ratio of 10 5 -10 6 . ...
A fibre tracking algorithm for volumetric microstructural data - application to tendons
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Tendons are crucial connective tissues made almost entirely of bundles of long, near-parallel collagen fibrils, and are vitally important to skeletal stability and mechanical function. Tendon structure is typically quantified in 2D, whereas, in this work, we have used serial block face-scanning electron microscopy to image tendons in 3D. We present a custom fibre tracking algorithm (FTA), with which we have characterised the 3D microstructure of tendon. Currently available tools for fibre tracing were unsuitable for tracking large numbers of fibrils and handling imaging artefacts associated with EM. We have tracked fibrils through a representative tendon volume and measured their relative length, diameter, orientation, chirality, tortuosity and volume fraction, which are just some of the measurements it is possible to make with the FTA depending on the research question. This algorithm has been developed in a general way and can be applied to a range of biological research questions relating to tendon structure-function relationships, on topics such as ageing, disease, development and injury. The FTA is also applicable to other fibrous biological materials, as well as engineered materials and textiles; it is written using Python and is freely available to download. Statement of significance We have created an algorithm for tracking fibres in 3D image stacks and applied it to tendon tissue. Previous studies have examined tendon structure in 2D, whereas we have imaged tendons in 3D using volumetric electron microscopy. Currently available fibre tracing tools could not track the large numbers of fibres or tolerate the artefacts present in biological imaging data. Using our algorithm, we have reconstructed and characterised the geometrical properties of the collagen fibrils (length, width, alignment, area, location). This algorithm could help to answer questions in biology which relate tissue microstructure to function in areas such as ageing, disease, development and injury. It could also be used to study engineered materials and textiles and is available freely to download.
... Recently, it was found that fibrillar collagen molecules are in fact undergoing a remarkable remodelling that is controlled by the circadian clock [103]. In the Achilles tendon, while collagen The table lists the half-lives of fibrillar matrix components reported in literature, the origin of the samples (organism and tissue), the age or body weight of source animals, and the method used to infer the half-lives. ...
... However, these results seem to disagree with the previous reports that collagen does not turnover daily [97,[99][100][101]. To solve this problem, it was proposed that there exist a large amount of 'persistent' collagen and a small pool of 'sacrificial' collagen, and the latter is undergoing the circadian turnover [103]. It remains to be elucidated exactly how much of collagen molecules are 'persistent' and 'sacrificial' in vivo. ...
... Disruption of the circadian clock leads to a number of abnormalities in connective tissues, e. g. the fibrosis, calcification, and degeneration of cartilages and tendons [104][105][106][107]. Failure in the circadian turnover of fibrillar matrices may underlie these diverse pathologies [103]. It is unknown why fibrillar matrices should undergo a circadian turnover. ...
Dynamic movement and turnover of extracellular matrices during tissue development and maintenance
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Extracellular matrices (ECMs) are essential for the architecture and function of animal tissues. ECMs have been thought to be highly stable structures; however, too much stability of ECMs would hamper tissue remodelling required for organ development and maintenance. Regarding this conundrum, this article reviews multiple lines of evidence that ECMs are in fact rapidly moving and replacing components in diverse organisms including hydra, worms, flies, and vertebrates. Also discussed are how cells behave on/in such dynamic ECMs, how ECM dynamics contributes to embryogenesis and adult tissue homoeostasis, and what molecular mechanisms exist behind the dynamics. In addition, it is highlighted how cutting-edge technologies such as genome engineering, live imaging, and mathematical modelling have contributed to reveal the previously invisible dynamics of ECMs. The idea that ECMs are unchanging is to be changed, and ECM dynamics is emerging as a hitherto unrecognized critical factor for tissue development and maintenance.
... We have a poor understanding of how biomechanical overload timing and severity from sports training influences the in vivo proteolytic activity that may drive ACL microtrauma beyond the threshold for natural repair homeostasis [19,113]. High frequency, intensity, or total volume sport training may create situations where youth and adolescent athletes are at greater risk for compromising any of a number of developmental processes through chronic overtraining and hormonal dysregulation [37,58,61,68]. ...
... The nature and acuity of this healing response can prompt either an anabolic, homeostatic, or catabolic state, in which ECM production and structural properties are respectively either increased, maintained, or reduced. Increased ECM collagen production and incorporation occurs within hours of loading [53,86] with circadian regulation of collagen synthesis, cellular export, and collagen degradation attempting to maintain or restore tissue homeostasis [19]. Excessive sports training intensity, frequency, or total volume, however, may upset this balance. ...
ACL microtrauma: healing through nutrition, modified sports training, and increased recovery time
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Purpose Sports injuries among youth and adolescent athletes are a growing concern, particularly at the knee. Based on our current understanding of microtrauma and anterior cruciate ligament (ACL) healing characteristics, this clinical commentary describes a comprehensive plan to better manage ACL microtrauma and mitigate the likelihood of progression to a non-contact macrotraumatic ACL rupture. Methods Medical literature related to non-contact ACL injuries among youth and adolescent athletes, collagen and ACL extracellular matrix metabolism, ACL microtrauma and sudden failure, and concerns related to current sports training were reviewed and synthesized into a comprehensive intervention plan. Results With consideration for biopsychosocial model health factors, proper nutrition and modified sports training with increased recovery time, a comprehensive primary ACL injury prevention plan is described for the purpose of better managing ACL microtrauma, thereby reducing the incidence of non-contact macrotraumatic ACL rupture among youth and adolescent athletes. Conclusion Preventing non-contact ACL injuries may require greater consideration for reducing accumulated ACL microtrauma. Proper nutrition including glycine-rich collagen peptides, or gelatin-vitamin C supplementation in combination with healthy sleep, and adjusted sports training periodization with increased recovery time may improve ACL extracellular matrix collagen deposition homeostasis, decreasing sudden non-contact ACL rupture incidence likelihood in youth and adolescent athletes. Successful implementation will require compliance from athletes, parents, coaches, the sports medicine healthcare team, and event organizers. Studies are needed to confirm the efficacy of these concepts. Level of evidence V
... 72,73 Fibroblasts in culture show a circadian expression pattern of clock components 74 and seem to function in a rhythmic manner to maintain collagen homeostasis in surrounding tissues. 75 Lung fibroblasts also showed robust circadian rhythms that are accentuated in fibrotic areas. 76 However, the circadian biology of cardiac fibroblast function remains to be elucidated. ...
Circulation Research The Circadian Biology of Heart Failure
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Driven by autonomous molecular clocks that are synchronized by a master pacemaker in the suprachiasmatic nucleus, cardiac physiology fluctuates in diurnal rhythms that can be partly or entirely circadian. Cardiac contractility, metabolism, and electrophysiology, all have diurnal rhythms, as does the neurohumoral control of cardiac and kidney function. In this review, we discuss the evidence that circadian biology regulates cardiac function, how molecular clocks may relate to the pathogenesis of heart failure, and how chronotherapeutics might be applied in heart failure. Disrupting molecular clocks can lead to heart failure in animal models, and the myocardial response to injury seems to be conditioned by the time of day. Human studies are consistent with these findings, and they implicate the clock and circadian rhythms in the pathogenesis of heart failure. Certain circadian rhythms are maintained in patients with heart failure, a factor that can guide optimal timing of therapy. Pharmacologic and nonpharmacologic manipulation of circadian rhythms and molecular clocks show promise in the prevention and treatment of heart failure.
... Multi-tissues time-serie proteomic data come from: Mauvoisin et al. [17] for the liver, Noya et al. [45] for the forebrain, Chang et al. [46] for the tendon, and Dudek et al. [47] for the cartilage. Finally, single-cell data of thousand of cells in organs for which we had time-series datasets, i.e. liver, lung, kidney, muscle, aorta, and heart, were downloaded from figshare using R objects from FACS single-cell datasets [29]. ...
Rhythmicity is linked to expression cost at the protein level but to expression precision at the mRNA level
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Many genes have nycthemeral rhythms of expression, i.e . a 24-hours periodic variation, at either mRNA or protein level or both, and most rhythmic genes are tissue-specific. Here, we investigate and discuss the evolutionary origins of rhythms in gene expression. Our results suggest that rhythmicity of protein expression could have been favored by selection to minimize costs. Trends are consistent in bacteria, plants and animals, and are also supported by tissue-specific patterns in mouse. Unlike for protein level, cost cannot explain rhythm at the RNA level. We suggest that instead it allows to periodically reduce expression noise. Noise control had the strongest support in mouse, with limited evidence in other species. We have also found that genes under stronger purifying selection are rhythmically expressed at the mRNA level, and we propose that this is because they are noise sensitive genes. Finally, the adaptive role of rhythmic expression is supported by rhythmic genes being highly expressed yet tissue-specific. This provides a good evolutionary explanation for the observation that nycthemeral rhythms are often tissue-specific.
... Up to 20% of a tissue's proteome is regulated by the circadian clock [6,7]. This co-ordinates the temporal compartmentalisation of vital cellular processes to anticipate the differing demands of the day or night, generating time-of-day specific cellular physiology [8][9][10]. For example, the O'Neill lab has demonstrated that the circadian clock regulates actin dependent processes such as cell migration and adhesion, which ultimately creates remarkable time-of-day differences in wound healing efficiency [9]. ...
Oscillations of the circadian clock protein, BMAL-1, align to daily cycles of mechanical stimuli: a novel means to integrate biological time within predictive in vitro model systems
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Purpose In vivo, the circadian clock drives 24-h rhythms in human physiology. Isolated cells in vitro retain a functional clockwork but lack necessary timing cues resulting in the rapid loss of tissue-level circadian rhythms. This study tests the hypothesis that repeated daily mechanical stimulation acts as a timing cue for the circadian clockwork. The delineation and integration of circadian timing cues into predictive in vitro model systems, including organ-on-a-chip (OOAC) devices, represent a novel concept that introduces a key component of in vivo physiology into predictive in vitro model systems. Methods Quiescent bovine chondrocytes were entrained for 3 days by daily 12-h bouts of cyclic biaxial tensile strain (10%, 0.33 Hz, Flexcell) before sampling during free-running conditions. The core clock protein, BMAL-1, was quantified from normalised Western Blot signal intensity and the temporal oscillations characterised by Cosinor linear fit with 24-h period. Results Following entrainment, the cell-autonomous oscillations of the molecular clock protein, BMAL-1, exhibited circadian (24 h) periodicity ( p < 0.001) which aligned to the diurnal mechanical stimuli. A 6-h phase shift in the mechanical entrainment protocol resulted in an equivalent shift of the circadian clockwork. Thus, repeated daily mechanical stimuli synchronised circadian rhythmicity of chondrocytes in vitro. Conclusion This work demonstrates that daily mechanical stimulation can act as a timing cue that is sufficient to entrain the peripheral circadian clock in vitro. This discovery may be exploited to induce and sustain circadian physiology within into predictive in vitro model systems, including OOAC systems. Integration of the circadian clock within these systems will enhance their potential to accurately recapitulate human diurnal physiology and hence augment their predictive value as drug testing platforms and as realistic models of human (patho)physiology.
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Article
Brain-derived neurotrophic factor (BDNF)/tyrosine kinase receptor B (TrkB) pathway is a therapeutic target in cardiac diseases. A BDNF mimetic, 7,8-dihydroxyflavone (7,8-DHF), is emerging as a protective agent in cardiomyocytes; however, its potential role in cardiac fibroblasts (CFs) and fibrosis remains unknown. Thus, we aimed to explore the effects of 7,8-DHF on cardiac fibrosis and the possible mechanisms. Myocardial ischemia (MI) and transforming growth factor-β1 (TGF-β1) were used to establish models of cardiac fibrosis. Hematoxylin & eosin and Masson's trichrome stains were used for histological analysis and determination of collagen content in mouse myocardium. Cell viability kit, EdU (5-ethynyl-2′-deoxyuridine) assay and immunofluorescent stain were employed to examine the effects of 7,8-DHF on the proliferation and collagen production of CFs. The levels of collagen I, α-smooth muscle actin (α-SMA), TGF-β1, Smad2/3, and Akt as well as circadian rhythm-related signals including brain and muscle Arnt-like protein 1 (Bmal1), period 2 (Per2), and cryptochrome 2 (Cry2) were analyzed. Treatment with 7,8-DHF markedly alleviated cardiac fibrosis in MI mice. It inhibited the activity of CFs accompanied by decreasing number of EdU-positive cells and downregulation of collagen I, α-SMA, TGF-β1, and phosphorylation of Smad2/3. 7,8-DHF significantly restored the dysregulation of Bmal1, Per2, and Cry2, but inhibited the overactive Akt. Further, inhibition of Bmal1 by SR9009 effectively attenuated CFs proliferation and collagen production of CFs. In summary, these findings indicate that 7,8-DHF attenuates cardiac fibrosis and regulates circadian rhythmic signals, at least partly, by inhibiting Bmal1/Akt pathway, which may provide new insights into therapeutic cardiac remodeling.
Circadian lncRNA ADIRF-AS1 binds PBAF and regulates renal clear cell tumorigenesis
Article
Full-text available
  • Oct 2022
We identify ADIRF-AS1 circadian long non-coding RNA (lncRNA). Deletion of ADIRF-AS1 in U2OS cells alters rhythmicity of clock-controlled genes and expression of extracellular matrix genes. ADIRF-AS1 interacts with all components of the PBAF (PBRM1/BRG1) complex in U2OS cells. Because PBRM1 is a tumor suppressor mutated in over 40% of clear cell renal carcinoma (ccRCC) cases, we evaluate ADIRF-AS1 in ccRCC cells. Reducing ADIRF-AS1 expression in ccRCC cells decreases expression of some PBAF-suppressed genes. Expression of these genes is partially rescued by PBRM1 loss, consistent with ADIRF-AS1 acting in part to modulate PBAF. ADIRF-AS1 expression correlates with survival in human ccRCC, particularly in PBRM1 wild-type, but not mutant, tumors. Loss of ADIRF-AS1 eliminates in vivo tumorigenesis, partially rescued by concurrent loss of PBRM1 only when co-injected with Matrigel, suggesting a PBRM1-independent function of ADIRF-AS1. Our findings suggest that ADIRF-AS1 functions partly through PBAF to regulate specific genes as a BMAL1-CLOCK-regulated, oncogenic lncRNA.
Biglycan Regulation Of Regional Tendon Development Via The Pericellular Matrix
Article
  • Jan 2022
Tendons are a unique orthopaedic tissue that rely on a highly ordered tissue matrix for proper function. Tendon disease degrades this matrix order, causing deviations in resident cell behavior that result in decreased tissue function. Treatments for tendon disease remain ineffective due to a knowledge gap in the factors most vital for maintaining tendon health. Many matrix molecules help regulate tendon growth and maintenance, including biglycan and collagen VI. These molecules are attributed to the pericellular matrix, a critical matrix structure that preserves cellular health across multiple contexts. The role of the PCM, and how interactions between biglycan and collagen VI govern tendon health, however, remain unknown. This dissertation defined the coordinate roles of biglycan and collagen VI and determined that while both molecules are key for tendon health, collagen VI is a more robust regulator, and that biglycan and collagen VI do not play additive roles in tendon. This work sought to further refine biglycan’s regulatory mechanism in tendon by leveraging a unique model system of distinct tendon matrix environments—“wrap-around” tendons. In addition to the characteristic, aligned tendon matrix, wrap-around tendons contain a matrix that more closely mimics fibrocartilage. This work analyzed the effect of biglycan knockout across these distinct tissue contexts to determine the molecular mechanism by which biglycan regulates tendon function. In doing so, we mapped the postnatal development of regional tendon properties for the first time in mice. Results from this work demonstrate that while biglycan may regulated tendon function through the PCM, this mechanism is likely independent of collagen VI interactions. Instead, biglycan may regulate tendon properties by directly organizing the collagen matrix. Overall, this work provides unique insight into the role of biglycan across distinct tendon matrix environments and lays the foundation for future work that may identify the factors most essential for preserving tendon health. Such knowledge is critical for the prevention and treatment of tendon disease.
Food plant extracts for sleep-related skin health: Mechanisms and prospects
Article
  • Aug 2022
Chronic sleep deficiency, a public health epidemic, triggers an elevated risk for physical and mental disorders and may cause unpleasant sensations or experiences in daily life. Importantly, sleep-associated skin diseases attract public attention because most people have anxiety about their facial appearance in modern society. Amounts of health food, especially derived from edible and efficient plant extracts, have been developed for skincare via improvement of sleep quality. Mechanisms of good sleep and healthy skin have been studied, however, the relationship between sleep-promoting herbs and skincare is less elucidated. In this review, we summarize the main signaling pathways of neurotransmitter-mediated skin beauty and list dozens of functional plant extracts for sleep assistance. We conclude several plant candidates for oral cosmetics application through sleep-related neurotransmitters regulation. This review provides pieces of evidence for the application of oral cosmetic food derived from plant extracts in sleep-related beauty.
SR9009 has REV-ERB-independent effects on cell proliferation and metabolism
Article
Full-text available
  • May 2019
The nuclear receptors REV-ERBα and -β link circadian rhythms and metabolism. Like other nuclear receptors, REV-ERB activity can be regulated by ligands, including naturally occurring heme. A putative ligand, SR9009, has been reported to elicit a range of beneficial effects in healthy as well as diseased animal models and cell systems. However, the direct involvement of REV-ERBs in these effects of SR9009 has not been thoroughly assessed, as experiments were not performed in the complete absence of both proteins. Here, we report the generation of a mouse model for conditional genetic deletion of REV-ERBα and -β. We show that SR9009 can decrease cell viability, rewire cellular metabolism, and alter gene transcription in hepatocytes and embryonic stem cells lacking both REV-ERBα and -β. Thus, the effects of SR9009 cannot be used solely as surrogate for REV-ERB activity.
TANGO1 builds a machine for collagen export by recruiting and spatially organizing COPII, tethers and membranes
Article
Full-text available
Collagen export from the endoplasmic reticulum (ER) requires TANGO1, COPII coats, and retrograde fusion of ERGIC membranes. How do these components come together to produce a transport carrier commensurate with the bulky cargo collagen? TANGO1 is known to form a ring that corrals COPII coats and we show here how this ring or fence is assembled. Our data reveal that a TANGO1 ring is organized by its radial interaction with COPII, and lateral interactions with cTAGE5, TANGO1-short or itself. Of particular interest is the finding that TANGO1 recruits ERGIC membranes for collagen export via the NRZ (NBAS/RINT1/ZW10) tether complex. Therefore, TANGO1 couples retrograde membrane flow to anterograde cargo transport. Without the NRZ complex, the TANGO1 ring does not assemble, suggesting its role in nucleating or stabilising of this process. Thus, coordinated capture of COPII coats, cTAGE5, TANGO1-short, and tethers by TANGO1 assembles a collagen export machine at the ER.
Pharmacological activation of REV-ERBs is lethal in cancer and oncogene-induced senescence
Article
Full-text available
The circadian clock imposes daily rhythms in cell proliferation, metabolism, inflammation and DNA damage response. Perturbations of these processes are hallmarks of cancer and chronic circadian rhythm disruption predisposes individuals to tumour development. This raises the hypothesis that pharmacological modulation of the circadian machinery may be an effective therapeutic strategy for combating cancer. REV-ERBs, the nuclear hormone receptors REV-ERBα (also known as NR1D1) and REV-ERBβ (also known as NR1D2), are essential components of the circadian clock. Here we show that two agonists of REV-ERBs-SR9009 and SR9011-are specifically lethal to cancer cells and oncogene-induced senescent cells, including melanocytic naevi, and have no effect on the viability of normal cells or tissues. The anticancer activity of SR9009 and SR9011 affects a number of oncogenic drivers (such as HRAS, BRAF, PIK3CA and others) and persists in the absence of p53 and under hypoxic conditions. The regulation of autophagy and de novo lipogenesis by SR9009 and SR9011 has a critical role in evoking an apoptotic response in malignant cells. Notably, the selective anticancer properties of these REV-ERB agonists impair glioblastoma growth in vivo and improve survival without causing overt toxicity in mice. These results indicate that pharmacological modulation of circadian regulators is an effective antitumour strategy, identifying a class of anticancer agents with a wide therapeutic window. We propose that REV-ERB agonists are inhibitors of autophagy and de novo lipogenesis, with selective activity towards malignant and benign neoplasms.
An Optimized Shotgun Strategy for the Rapid Generation of Comprehensive Human Proteomes
Article
Full-text available
  • Jun 2017
This study investigates the challenge of comprehensively cataloging the complete human proteome from a single-cell type using mass spectrometry (MS)-based shotgun proteomics. We modify a classical two-dimensional high-resolution reversed-phase peptide fractionation scheme and optimize a protocol that provides sufficient peak capacity to saturate the sequencing speed of modern MS instruments. This strategy enables the deepest proteome of a human single-cell type to date, with the HeLa proteome sequenced to a depth of ∼584,000 unique peptide sequences and ∼14,200 protein isoforms (∼12,200 protein-coding genes). This depth is comparable with next-generation RNA sequencing and enables the identification of post-translational modifications, including ∼7,000 N-acetylation sites and ∼10,000 phosphorylation sites, without the need for enrichment. We further demonstrate the general applicability and clinical potential of this proteomics strategy by comprehensively quantifying global proteome expression in several different human cancer cell lines and patient tissue samples.
ConTra v3: A tool to identify transcription factor binding sites across species, update 2017
Article
Full-text available
Transcription factors are important gene regulators with distinctive roles in development, cell signaling and cell cycling, and they have been associated with many diseases. The ConTra v3 web server allows easy visualization and exploration of predicted transcription factor binding sites (TFBSs) in any genomic region surrounding coding or non-coding genes. In this updated version, with a completely re-implemented user interface using latest web technologies, users can choose from nine reference organisms ranging from human to yeast. ConTra v3 can analyze promoter regions, 5΄-UTRs, 3΄-UTRs and introns or any other genomic region of interest. Thousands of position weight matrices are available to choose from for detecting specific binding sites. Besides this visualization option, additional new exploration functionality is added to the tool that will automatically detect TFBSs having at the same time the highest regulatory potential, the highest conservation scores of the genomic regions covered by the predicted TFBSs and strongest co-localizations with genomic regions exhibiting regulatory activity. The ConTra v3 web server is freely available at http://bioit2.irc.ugent.be/contra/v3.
The intervertebral disc contains intrinsic circadian clocks that are regulated by age and cytokines and linked to degeneration
Article
Full-text available
  • Aug 2016
Objectives The circadian clocks are internal timing mechanisms that drive ∼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration. Methods Clock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containing Per2::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock gene Bmal1. Results Here we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1β but not tumour necrosis factor α. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout of Bmal1 in their disc cells demonstrated age-related degeneration of IVDs. Conclusions We have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain.
Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing
Article
  • Jan 1995
The common approach to the multiplicity problem calls for controlling the familywise error rate (FWER). This approach, though, has faults, and we point out a few. A different approach to problems of multiple significance testing is presented. It calls for controlling the expected proportion of falsely rejected hypotheses — the false discovery rate. This error rate is equivalent to the FWER when all hypotheses are true but is smaller otherwise. Therefore, in problems where the control of the false discovery rate rather than that of the FWER is desired, there is potential for a gain in power. A simple sequential Bonferronitype procedure is proved to control the false discovery rate for independent test statistics, and a simulation study shows that the gain in power is substantial. The use of the new procedure and the appropriateness of the criterion are illustrated with examples.
Circadian actin dynamics drive rhythmic fibroblast mobilization during wound healing
Article
  • Nov 2017
Fibroblasts are primary cellular protagonists of wound healing. They also exhibit circadian timekeeping, which imparts an approximately 24-hour rhythm to their biological function. We interrogated the functional consequences of the cell-autonomous clockwork in fibroblasts using a proteome-wide screen for rhythmically expressed proteins. We observed temporal coordination of actin regulators that drives cell-intrinsic rhythms in actin dynamics. In consequence, the cellular clock modulates the efficiency of actin-dependent processes such as cell migration and adhesion, which ultimately affect the efficacy of wound healing. Accordingly, skin wounds incurred during a mouse’s active phase exhibited increased fibroblast invasion in vivo and ex vivo, as well as in cultured fibroblasts and keratinocytes. Our experimental results correlate with the observation that the time of injury significantly affects healing after burns in humans, with daytime wounds healing ~60% faster than nighttime wounds. We suggest that circadian regulation of the cytoskeleton influences wound-healing efficacy from the cellular to the organismal scale.
A subcellular map of the human proteome
Article
  • May 2017
Mapping the proteome Proteins function in the context of their environment, so an understanding of cellular processes requires a knowledge of protein localization. Thul et al. used immunofluorescence microscopy to map 12,003 human proteins at a single-cell level into 30 cellular compartments and substructures (see the Perspective by Horwitz and Johnson). They validated their results by mass spectroscopy and used them to model and refine protein-protein interaction networks. The cellular proteome is highly spatiotemporally regulated. Many proteins localize to multiple compartments, and many show cell-to-cell variation in their expression patterns. Presented as an interactive database called the Cell Atlas, this work provides an important resource for ongoing efforts to understand human biology. Science , this issue p. eaal3321 ; see also p. 806
Evidence that interfibrillar load transfer in tendon is supported by small diameter fibrils and not extrafibrillar tissue components: INTERFIBRILLAR LOAD TRANSFER IN TENDON
Article
Collagen fibrils in tendon are believed to be discontinuous and transfer tensile loads through shear forces generated during interfibrillar sliding. However, the structures that transmit these interfibrillar forces are unknown. Various extrafibrillar tissue components (e.g., glycosaminoglycans, collagens XII and XIV) have been suggested to transmit interfibrillar loads by bridging collagen fibrils. Alternatively, collagen fibrils may interact directly through physical fusions and interfibrillar branching. The objective of this study was to test whether extrafibrillar proteins are necessary to transmit load between collagen fibrils or if interfibrillar load transfer is accomplished directly by the fibrils themselves. Trypsin digestions were used to remove a broad spectrum of extrafibrillar proteins and measure their contribution to the multiscale mechanics of rat tail tendon fascicles. Additionally, images obtained from serial block-face scanning electron microscopy were used to determine the three-dimensional fibrillar organization in tendon fascicles and identify any potential interfibrillar interactions. While trypsin successfully removed several extrafibrillar tissue components, there was no change in the macroscale fascicle mechanics or fibril:tissue strain ratio. Furthermore, the imaging data suggested that a network of smaller diameter fibrils (<150 nm) wind around and fuse with their neighboring larger diameter fibrils. These findings demonstrate that interfibrillar load transfer is not supported by extrafibrillar tissue components and support the hypothesis that collagen fibrils are capable of transmitting loads themselves. Conclusively determining how fibrils bear load within tendon is critical for identifying the mechanisms that impair tissue function with degeneration and for restoring tissue properties via cell-mediated regeneration or engineered tissue replacements. This article is protected by copyright. All rights reserved.