TGF- b signaling pathway promotes intercellular coupling

Described findings suggested that peripheral circadian oscillators weakly couple with each other to establish (partially) synchronized networks rhythms. Moreover, TGF-b signaling pathway appeared to be involved in the maintenance of such synchronized network rhythms. In agreement with the Kuramoto model, progressive phase synchronization can result in transitions from incoherent to coherent (coupled) network states. Thus, we suggest that TGF-b may act as peripheral coupling factor mediating phase synchronization among single cell oscillators by the temporally gated induction of CRE enhancer element and immediate early expression of PER2/Per2.

Previous co-culture experiments of low-density, low-amplitude, highly damped U-2 OS reporter cells with increasing numbers of non-reporter cells showed that co-culture results in amplitude expansion and decreased damping of the reporter cell population in a density dependent manner (Figure 3-4). We assumed that amplitude increases, and damping decreases are due to intercellular coupling leading to amplitude resonance and decreased desynchronization of the co-cultured populations. To test whether of TGF-b signaling mediates these (coupling) effects, co-cultures were performed upon pharmacological inhibition of TGF-b receptor.

While solvent controls displayed density dependent increases in amplitudes of low-density U-2 OS Bmal1:Luc reporter cells (as seen in Figure 3-4), TGF-b receptor inhibition abolished density dependent amplitude expansion (Figure 3-13 A,B). This suggests that disruption of TGF-b signaling results in a reduction of intercellular coupling and thus attenuation of amplitude resonance effects among coupled (frequency-locked) oscillators. However, oppositely to expectations, TGF-b receptor inhibition resulted in larger absolute amplitudes, especially for low co-culture numbers (Figure 3-13 A,B). We suspect that amplitude increases are an artifact of dexamethasone synchronization prior to recording. Since TGF-b receptor inhibitor was

added to the co-cultures already during seeding (one day prior to recording), it appears likely that cells were already desynchronized when dexamethasone was applied.

Consistent with theoretical predictions, reduced network synchrony enhances the susceptibility to Zeitgeber pulses [57], thereby leading to stronger dexamethasone responses when intercellular coupling is disturbed. This assumption is further supported by increased damping of Bmal1:Luc rhythms upon TGF-b receptor inhibition (Figure 3-13 A,C), suggesting that, following initial (dexamethasone) synchronization, disruption of TGF-b signaling results in even faster desynchronization of single cell oscillators within the co-cultured ensemble. Alternatively, absolute amplitude increases could be a consequence of stronger intercellular coupling upon perturbation of TGF-b signaling. This however is not consistent with previous observations and appears unlikely.

If TGF-b signaling promotes intercellular coupling and synchronized rhythmicity, it should render oscillator networks more robust against perturbation by Zeitgeber stimuli. Thus, as described above, oscillator ensembles are expected to respond to Zeitgeber pulses with larger phase shifts upon perturbation of TGF-b signaling. To test this U-2 OS Bmal1:Luc circadian reporter cells were subjected to a 20°C temperature pulse following pharmacological inhibition of TGF-b receptor. Indeed, compared to control, perturbation of TGF-b signaling resulted in much larger phase shifts of circadian oscillations following an 8 hour 20°C temperature pulse (Figure 3-13 D,E).

This suggests that TGF-b signaling promotes intercellular coupling and thus robust networks rhythmicity. Interestingly however, reducing culture density alone did not increase susceptibility to the applied temperature pulse and strong phase responses were only observed upon additional TGF-b receptor inhibition (Figure 6-7 A,B). This result was unexpected because, consistent with previous findings, sparse oscillator networks were assumed to be less coupled and therefore more susceptible to perturbation by a Zeitgeber pulse. We suspect that, relative to residual intercellular coupling among sparsely cultured U-2 OS cells, the applied temperature pulse was only a weak Zeitgeber. Therefore, additional disruption of intercellular coupling by TGF-b receptor inhibition would be required to elicit temperature responses in both, dense and sparse peripheral oscillator networks. Additionally, timing of the temperature pulse may not have been optimal. Based on the conditioned medium PRC, the trough of PER2 expression (inferred from nearly anti-phasic Bmal1:Luc

expression) was chosen as timepoint of the temperature pulse. Nevertheless, since temperature dependent phase resetting depends on other input routes than CM, e.g.

via heat shock proteins binding to HSE sites, PRCs may differ. Thus, density effects on intercellular coupling and on responses to Zeitgeber stimuli may become more apparent at timepoints when temperature induced shifts are maximal.

Figure 3-13: Pharmacological perturbation of TGF-b signaling attenuates intercellular coupling TGF-b receptor inhibitor (LY2109761) was used to assess whether perturbation of TGF-b signaling attenuates intercellular coupling, as characterized by a lack of amplitude resonance, increased damping

0.0 0.5 1.0 1.5 2.0 2.5 3.0

cells/dish) U-2 OS Bmal1:Luc reporter cells with increasing numbers of non-reporter cells were seeded into 35-mm dishes. TGF-b receptor inhibitor was applied during seeding and bioluminescence imaging.

Co-cultures were synchronized, and luciferase activity continuously monitored. (A) Detrended time series of a representative co-culture experiment with or without TGF-b receptor inhibitor. (B,C) Quantification of amplitudes (B) and damping (E) of Bmal1:Luc oscillations under co-culture conditions and upon treatment with TGF-b receptor inhibitor or solvent control (n=3 repeat experiment with 2 technical replicates, individual values and connecting line displayed, linear regression test and Unpaired one-tailed student’s t-test against respective solvent groups: *p<0.05, **p<0.01). (D,E) U-2 OS cells harboring a Bmal1:Luc reporter gene were seeded at high density (3.0 x10⁵ cells/dish) into 35-mm dishes with or without TGF-b receptor inhibitor. An 8 hour, 20°C temperature pulse was applied at the inferred trough of PER2 expression (nearly anti-phasic to Bmal1:Luc peak). (D) Detrended time series of a representative temperature pulse experiment with or without TGF-b receptor inhibitor. (E) Quantification of temperature induced phase shifts of Bmal1:Luc oscillations upon treatment with TGF-b receptor inhiTGF-bitor or solvent control (n=4 repeat experiment with 2 technical replicates each, mean ± SD, individual values displayed, Unpaired one-tailed student’s t-test against solvent group: **p<0.01).

Overall, these findings suggest that paracrine TGF-b signaling promotes robustness of circadian rhythmicity against perturbation by external Zeitgeber stimuli. Thereby, further supporting the role of TGF-b as potential intercellular coupling factor in peripheral oscillator networks.