Circadian control of the secretory pathway maintains collagen homeostasis

Abstract

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.

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Fig. 1: Circadian regulation of the tendon collagen ECM.
Fig. 2: Rhythmic expression of genes regulating collagen trafficking and fibre assembly.
Fig. 3: Circadian clock-regulated PC-I trafficking and collagen fibre assembly in culture.
Fig. 4: Golgi and post-Golgi transport of PC-I is rhythmic.
Fig. 5: Mathematical prediction of PC-I and collagen-I secretion.
Fig. 6: Time-series proteomics analysis of proteins in mouse tendons.
Fig. 7: Dysregulated collagen fibre homeostasis in the absence of a circadian rhythm.
Fig. 8: Circadian clock-deficient tendons have abnormal collagen homeostasis.

Data availability

The mouse tail tendon time-series microarray data17 have been deposited to the ArrayExpress repository with the dataset identifier (ArrayExpress accession number E-MTAB-7743). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [1] with the dataset identifier PXD013450. All other data supporting the study are available from the corresponding authors on reasonable request. Source data for Figs. 13, 68 and Extended Data Figs. 19 are available online.

Code availability

The code for the secretory pathway model was written in Mathematica (version 11.3) and is available from GitHub at https://github.com/bcalvr/Col1_005.

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Acknowledgements

The research was funded by Wellcome (grant nos. 110126/Z/15/Z and 203128/Z/16/Z to K.E.K.), the MRC (grant no. MR/P010709/1 to Q.J.M.) and Arthritis Research UK (grant no. 20875 to Q.J.M.). V.M. was supported by a studentship from the Sir Richard Stapley Educational Trust. J.S. was funded by a Biotechnology and Biological Sciences Research Council (BBSRC) David Phillips Fellowship (grant no. BB/L024551/1). B.C. is supported by a Wellcome four-year PhD studentship (grant no. 210062/Z/17/Z). H.R.-H. is supported by a University of Manchester four-year PhD studentship. The authors would like to thank R. Schweitzer (Shriners Hospital for Children) for the ScxCre mice; A. Hallworth, R. Hodgkiss and M. Jackson and the University of Manchester Biological Support Facility for assistance with animal welfare and husbandry; and M. Dudek and N. Yang (University of Manchester) for assistance with bioluminescence imaging. The proteomics was performed at the Biological Mass Spectrometry Facility in the Faculty of Biology, Medicine and Health (University of Manchester) with the assistance of S. Warwood and R. O’Cualain, and electron microscopy was performed in the Electron Microscopy Facility, Faculty of Biology, Medicine and Health (University of Manchester).

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J.C., A.P., C.-Y.C.Y. and R.G. designed and performed experiments, interpreted data and drafted the manuscript. D.F.H., Y.L., B.C. and H.R.-H. designed and performed experiments and interpreted data. V.M., J.S. and A.A. contributed materials and analysis tools. O.J. and T.S. supervised the mathematical aspects of the research. Q.J.M. and K.E.K. conceived the project and supervised the experiments. K.E.K. wrote the manuscript.

Corresponding authors

Correspondence to Qing Jun Meng or Karl E. Kadler.

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Extended data

Extended Data Fig. 1 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

Extended Data Fig. 2 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

Extended Data Fig. 3 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

Extended Data Fig. 4 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

Extended Data Fig. 5 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

Extended Data Fig. 6 Secretory pathway protein knockdown.

a, Relative expression of Sec61a1 and Sec61a2 mRNA was reduced in MEFs after CRISPR–Cas9-mediated deletion of both Sec61a1 and Sec61a2 compared to control wild-type MEFs (representative results from n = 3 biological repeats; mean and SD errors are shown *p = 0.0159, p = 0.0143 for Sec61a1 levels; ***p = 0.0007, p = 0.0002 for Sec61a2 levels, two-tailed unpaired t-test). b, Immunofluorescence analysis of collagen-I entry into the ER (detected via the marker calnexin) showed little co-localization of collagen-I and calnexin in Sec61a1 and Sec61a2 KO MEFs compared to wild-type MEFs. c, Relative expression of Mia3 mRNA was reduced in MEFs treated with siRNA targeting Mia3 compared to cells treated with scrambled control siRNA (representative results each showing similar degree of knockdown, n = 3 independent experiments, mean and SD errors are shown; ***p = 0.0004 for KD#2 and #9, two-tailed unpaired t-test). d, Relative expression of Pde4d mRNA was reduced in MEFs treated with siRNA targeting Pde4d compared to cells treated with scrambled control siRNA (n = 3 independent experiments; mean and SD errors shown, ***p = 0.0001, two-tailed unpaired t-test). e, Western blot analysis of MEFs treated with siRNAs targeting Pde4d showed depletion of PDE4D protein (Pde4d KD). Levels of GAPDH protein served as a loading control. f, Relative expression of Vps33b mRNA was significantly reduced in MEFs treated with CRISPR/Cas9n targeting Vps33b compared to control. Controls were wild-type MEFs that underwent electroporation with Cas9 protein but without gRNA (n = 3 biologically independent experiments; mean and SD errors are shown, **p = 0.021, two-tailed t-test). g, Relative expression of Vps33b mRNA in iTTFs after CRISPR/Cas9-mediated deletion of Vps33b (n = 3 biologically independent experiments; mean and SD errors shown, **p = 0.024, two-tailed t-test). h, Western blot analysis of VPS33b protein from Vps33b KO immortalized tail tendon fibroblasts (iTTFs). Vinculin protein served as a loading control. i, TEM of mouse tail tendons prepared in PBS, n = 2. Bar, 1 µm. j, TEM of mouse tail tendons after disruption in SL-DOC and collection by centrifugation, n = 2. Bar, 1 µm. k, Diameter distribution of collagen fibrils in unextracted tendon (untreated) and after extraction by SL-DOC (pellet). l, Hydroxyproline content of dissected tail tendons from mice incubated with SL-DOC. Data are expressed in percentage of total collagen extracted from tendons demonstrating that SL-DOC extracts ~ 18% of total collagen in the tendon (n = 2 animals). See also Statistical Source Extended Data Fig. 6 and Unprocessed Blots Extended Fig. 6. Source data

Extended Data Fig. 7 Rhythmic procollagen synthesis.

a, The 141 rhythmic proteins were separated into CT1-12 (day) and CT13-24 (night). The numbers of proteins in each group are shown. These two lists were analysed using ENRICHR, the significance of the enrichment of proteins within an ontology is shown on the right and is computed using Fisher’s exact test63. b, Gene ontology terms based on molecular function for each group are shown. c,d, Achilles and tail tendon tissues were harvested at the day phase (ZT3, day) or night phase (ZT15, night) and labelled with 14C-proline for 1 h in the presence of exogenous L-ascorbate. Higher incorporation of 14C was observed in the night phase in both Achilles (n = 6 animals; *p = 0.0423 two-tailed unpaired t-test) and tail tendons (tendons isolated from n = 3 animals; *p = 0.0395 two-tailed unpaired t-test). The central point indicates the mean values for each time point. e, Phosphoimager analysis of 1 h 14C-proline labelling of mouse Achilles tendon. Proteins were extracted into neutral buffer containing NP40 buffer extraction. Higher incorporation of 14C is observed in the night phase. f, Western blot analysis of the samples shown in e. Collagen-I was observed with an anti-collagen-I antibody. e and f are representative blots from two independent experiments consisting of all 8 time points. See also Statistical Source Data Extended Fig. 7 and Unprocessed Blots Extended Fig. 7. Source data

Extended Data Fig. 8 Rhythmic signal peptide-containing proteins.

a, Peptides in the time-series proteomics analysis (tail tendons from n = 4 independent animals) that contained a signal peptide were identified by overlapping the dataset with the signal peptide database41. The abundance of all signal peptide containing proteins was plotted as a function of circadian time (black). The abundance of the subset of rhythmic proteins (blue) exhibits two peaks at CT7–11 and CT27–31. b, A heat map of the twenty rhythmic signal peptide-containing proteins. See also Statistical Source Extended Data Fig. 8. Source data

Extended Data Fig. 9 SR9009 affects collagen fibril assembly.

a, Immunofluorescence analysis showed more collagen-I fibres assembled by the fibroblasts of ClockΔ19 mice. N = 3 biological repeats. Bars 10 µm. b, Kill curve of SR9009 showing that concentration of 10 µM (indicated with arrow) is well tolerated by WT and Δ19 fibroblasts. c,d, Luminometry recordings of circadian rhythms of dexamethasone-synchronized fibroblasts isolated from PER2::Luciferase (WT) and ClockΔ19::PER2::Luciferase (Δ19) mice. SR2.5, 2.5 µM SR9009. SR10, 10 µM SR9009. Arrow indicates addition of SR9009. e,f, Immunofluorescence analysis of wild-type and ClockΔ19 fibroblasts either treated with 10 µM SR9009 or mock-treated with dimethylsulfoxide (Ctrl). Blue, DAPI. Red, anti-collagen-I antibody. Number of DAPI-stained nuclei (that is number of cells) and number of fibres were blind scored from randomly-selected regions from n = 3 biological repeats by 3 observers. Number of collagen fibres per cell is shown for control and SR9009-treated fibroblasts. Fold change from qPCR analysis of gene expression is shown for control and SR9009-treated fibroblasts. *p = 0.0127, WT Ctrl vs WT + SR9009; ***p = 0.0005 Δ19Ctrl vs Δ19SR9009. Q-PCR analysis for SR9009-treated WT cells: *p = 0.0476, Sec61a2; *p = 0.0072, Mia3; **p = 0.0042, Serpinh1; *p = 0.0226, Pde4d. Q-PCR analysis for SR9009-treated Δ19 cells: **p = 0.0072, Vps33b; two-tailed paired t-test, n = 4 biological replicates. Bars show s.e.m. See also Statistical Source Data Extended Fig. 9. Source data

Supplementary information

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Reporting Summary

Supplementary Video 1

Step-through movie of SBF-SEM of mouse Achilles tendon at 09:00. Bar, 7 µm. Z-depth, 486 µm, images recorded at 9.1 µm intervals to reduce file size.

Supplementary Video 2

Step through movie displaying individual colourised fibrils in small region of mouse tail tendon, showing merging of D1 fibrils onto larger D2 and D3 fibrils. Data gathered from SBF-SEM stack of images. Area shown is 4 × 4 µm, each frame is a longitudinal step of 100 nm through the tendon.

Supplementary Video 3

Step-through movie of SBF-SEM of mouse Achilles tendon at 09:00. Bar, 500 nm. D3 fibril highlighted with a blue line. Filled circles indicate D1 fibrils.

Supplementary Video 4

Step-through movie of SBF-SEM of mouse Achilles tendon showing diameter variability along the length of fibrils and tortuosity of D1 fibrils. Bar, 700 nm. Z-depth, 12.5 µm.

Supplementary Video 5

Step-through animation showing the relative proportion of PC-I in the ER, ER exit sites, Golgi, post-Golgi, plasma membrane and ECM starting at CT0.

Supplementary Video 6

Real-time bioluminescence microscopy of dissected Achilles tendon from wild-type PER2::Luciferase mice during 5 d.

Supplementary Video 7

Real-time bioluminescence microscopy of dissected Achilles tendon from Scx–Cre::Bmal1 lox/lox PER2::Luciferase mice during 5 d. The brightness for the Scx–Cre::Bmal1 lox/lox tendon recording was increased to enable visualization.

Supplementary Table 1

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Chang, J., Garva, R., Pickard, A. et al. Circadian control of the secretory pathway maintains collagen homeostasis. Nat Cell Biol 22, 74–86 (2020). https://doi.org/10.1038/s41556-019-0441-z

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