Conclusions

If TGF-b indeed functions as coupling factor in peripheral tissues, targeted manipulation of its signaling pathway will enable to answer many open questions in the field of chronobiology. Are peripheral circadian oscillators coupled? Is the mechanism conserved? What is the functional relevance of this coupling? How does peripheral coupling contribute to or protect from circadian misalignment?

Based on the results presented here we strongly believe that coupling among peripheral circadian oscillators exists and that it is mediated by paracrine communication of single cell oscillators within tissue networks. We have accumulated evidence that TGF-b functions as paracrine coupling factor in peripheral oscillator networks (Figure 4-3). We propose a mechanism by which cAMP response element driven, immediate early expression of Per2 (or PER2 in human models) elicits temporally gated phase responses and phase-synchronization of neighboring oscillators (Figure 4-3). Subsequently, increased phase coherence may result in frequency-locking and amplitude resonance of synchronized oscillators. In agreement with theoretical models, this initial synchronization will increase the coupling strength of the network and recruit more and more oscillators into the synchronized pack [212].

Once a critical coupling threshold is reached the network transitions from the incoherent to the coupled state [172], [212]. For peripheral circadian oscillators intercellular coupling appears to be weak, at least in vitro, likely resulting in partial network synchronization. However, we suspect that peripheral coupling in vivo may be strengthened by more complex tissue microenvironments and formation of 3-dimensional cell-extracellular matrix interactions.

Figure 4-3: Model of TGF-b coupling among peripheral circadian oscillators

TGF-b transcription may be regulated rhythmically by BMAL1/CLOCK activity [335]. It gets secreted from peripheral circadian oscillators as inactive form and is distributed and stored in the extracellular matrix until its release is triggered by ECM components, including proteases and integrins. ECM components themselves may be produced and/or regulated rhythmically. Thus, active TGF-b may signal rhythmically via its TGF-b type I/type II receptor complexes resulting in intracellular activation and assembly of SMAD proteins, which translocate to the nucleus where they associate with additional transcriptional factors and/or transcriptional regulators. Binding of SMAD/CREB/CBP/p300 complex to SBE and CRE sites in the Per2 promoter may result in the immediate early expression of Per2 and respective phase responses of the receiving oscillator. Additionally, TGF-b signaling may result in feedback regulation of its own signaling pathway and extracellular availability further modulating intercellular TGF-b coupling among peripheral circadian oscillators. (TGF-b=Transforming Growth Factor beta, ECM=extracellular matrix, CREB=cAMP response element binding protein, CBP=CREB binding protein, p300=p300 histone acetyltransferase, CRE=cAMP response element, SBE=SMAD binding element, E-box=enhancer box, SMAD2/3 = R-SMADs, SMAD4 = Co-SMAD).

Known mechanisms of TGF-b secretion, ECM disposition, distribution, and activation support the idea that peripheral coupling is achieved by global mean field coupling and collective synchronization rather than by local effects. Latent TGF-b has been reported to have a half-life of > 100 minutes, while its active form is stable for 2-3 minutes [452].

Half-lives suggest that latent TGF-b may be distributed over a wide range of cells by simple diffusion before its activating results in the rapid induction of downstream signaling pathways. In agreement with Gonze et al. (2005) rapid diffusion relative to the 24 hour circadian cycle enables global synchronization of oscillators by rhythmic paracrine factors [213]. While TGF-b has been described to be under rhythmic control of BMAL1/CLOCK driven E-box transcription [334], [335], we are not aware of studies investigating rhythms in TGF-b secretion and/or activity. Nevertheless, functional secretory pathway has been demonstrated to play an important role for rhythmic secretion of ECM components, as well as for regulation of circadian rhythmicity [293], [297]. Thus, we suspect that rhythms in TGF-b signaling may be introduced by rhythmic production and secretion of latent TGF-b or rhythmic release of active of TGF-b (including rhythms in ECM components that promote the release). Rhythmic response to TGF-b, e.g. rhythmic expression of the receptor or SMADs [337], [338], as well as delayed feedback regulations of TGF-b signaling [331], [453]–[455] may contribute to coupling among peripheral oscillators (Figure 4-3). Computational models predict that rhythmic receptor expression can enhance amplitudes and modulate entrainment range of oscillator networks [233]. Additionally, duration and dose (molecules per cell) of TGF-b signals have been described to dictate dynamics of SMAD activity , receptor recycling, cellular responses and target gene expression [453], [454], [456]. Thus, all these regulatory layers may contribute to the fine-tuning of TGF-b dependent intercellular coupling in peripheral oscillator networks.

Alternatively, constant paracrine signals may promote spontaneous and rapid (phase-) synchronization, e.g. by sudden reduction of noisiness in the network or strong resetting signals. Whether or not this could explain bidirectionality of coupling as observed for co-culture experiments is unclear. We suggest that future studies of TGF-b dependent peripheral coupling may TGF-be supported TGF-by computational models to answer the following questions: Is peripheral coupling dependent on rhythmic coupling factors?

Is synchronization achieved by global or local coupling? Do mutual feedback regulations in response to paracrine signals play a role or is unidirectional information transfer from one oscillator to the other is sufficient?

Lastly, we propose that intercellular coupling between peripheral circadian oscillators is of functional relevance in vivo. In the SCN, coupling maintains tissue rhythmicity

a major determinant of clock precision. Compared to central coupling, we and others ([61]–[63]) have demonstrated that peripheral coupling, at least in vitro, is weak.

Moreover, SCN derived and rhythmic external signals have been shown to be indispensable for high-amplitude peripheral oscillations and the maintenance of normal phase relationships between tissue clocks [35], [85], [457]. However, due to its interconnection with oscillator robustness, entrainment, and response to Zeitgeber signals, coupling in peripheral tissue clocks likely plays a role for the temporal coordination of rhythmic organ functions. We suggest that, rather than governing precision and rigidity of tissue oscillations, peripheral coupling is required for fine-tuning responses of peripheral tissues to incoming Zeitgeber signals as it modulates robustness and plasticity of tissue clocks. The mammalian circadian system is exposed to a multitude of internal and external perturbations on a daily basis, e.g. mealtimes, physical activity, temperature changes, hormone levels, and humoral (metabolic) signals. Thus, with increasing evidence for an association between severe health consequences and chronic circadian disruption and/or misalignment, a better understanding of peripheral coupling and its relation to circadian tissue physiology may help to uncover sources and potential treatment options of “circadian diseases”.

Thus, to close with Jürgen Aschoffs' words: “the self-sustained circadian oscillator has to be taken into account. Its main properties seem to be the same in human beings as in all other organisms. We have to study them before we can discuss practical problems successfully. As always in science, a better understanding of the basic phenomena will be the first step toward a proper application in practice.” [1]