Background
All species face environmental stresses, including temperature fluctuations, mechanical forces, and variations in osmotic conditions. At the cellular level, the plasma membrane (PM) is the first line of defence, rapidly adapting and responding to these stresses. Osmotic stress, for instance, causes cell swelling (hypoosmotic stress) or cell shrinking (hyperosmotic stress), which requires the cell to adjust its cell volume.
Changes that occur at the cell membrane activate multiple signaling cascades that trigger gene expression changes, ultimately leading to physiological responses. Hypoosmotic stress, for instance, occurs when the concentration of solutes outside the cell is lower than inside, leading to water influx and cell swelling. This process activates an array of genes downstream, with even mild hypoosmotic stress inducing significant shifts in transcriptional activity over the course of 12–36 h.
Interestingly, genes associated with circadian rhythms also change expression under osmotic stress. In their recent study, “Hypoosmotic stress shifts transcription of circadian genes,” published in the February 4, 2025 Issue of Biophysical Journal, Qifti et al. explore this link by exposing cultured smooth muscle cells to mild hypoosmotic stress. This study reveals an intriguing link between cell stress and genes that regulate circadian rhythms, shedding light on how cells may synchronize their molecular clocks in response to environmental changes.
Downstream effects of hypoosmotic stress: what happens after cell swelling?
To explore the effects of osmotic stress on gene expression, Qifti et al. first subjected rat smooth muscle cells (WKO-3M22) to mild hypoosmotic stress (150 mOsm) at specific time points—0 min, 5 min, 12 h, and 24 h. At each time point, cells were harvested, and RNA was extracted for sequencing and analysis.
Although 5-min exposure to osmotic stress induced no significant genetic changes, the 12- and 24-h time points triggered differential expression of >100 genes. Notably, the authors found that among the overexpressed genes, many were associated with the circadian clock, including ciart (circadian associated repressor of transcription). Indeed, hypoosmotic stress increased both the transcript and protein levels of Ciart, which follows the roughly 24-h circadian cycle.
Hypoosmotic stress and the cell cycle
To further investigate the effect of osmotic stress, Qifti et al. looked at its effect on cell cycle progression. Specifically, they assessed how osmotic stress affects cellular localization of Bmal1 (brain and muscle Arnt1-like-1), a core mammalian circadian clock transcription factor. Bmal1 dimerizes with Clock; together, they initiate the transcription of circadian genes.
The authors synchronized smooth muscle cells to the G1 phase and compared cells under normal (isoosmotic) conditions to those subjected to hypoosmotic stress. Interestingly, although Bmal1 nuclear localization was significantly reduced in cells exposed to hypoosmotic stress after 12 h of stress, the two conditions showed no significant differences after 24 h. This indicates that cells may adapt to osmotic stress over time and be able to resume their cell cycle. Interestingly, this effect was found to be cell type specific, highlighting the possible complexity of cellular responses to osmotic stress.
Caveolae: linking osmotic stress and circadian genes
Perhaps the most intriguing finding of this study is the role that caveolae play in the response to osmotic stress. Caveolae are protein-rich PM invaginations, formed by the assembly of Cavin1-3 and Caveolin1-2 proteins. These structures help cells adapt to mechanical and osmotic stresses by accommodating increases in cell volume, preventing PM damage or rupture. Interestingly, their role is twofold in this context: not only do they help buffer membrane tension changes, but they also promote release of Cavin-1 from caveolae. Once released, cavin-1 translocates to the nucleus, where it promotes gene transcription.
The authors hypothesized that cavin-1 may serve as the molecular link between osmotic stress and circadian rhythm genes. Indeed, when cavin-1 is downregulated, osmotic stress fails to regulate circadian gene expression, including Ciart levels, which showed significantly lower levels compared to controls and failed to respond to hypoosmotic stress. When looking at Bmal1, Qifti et al. found Bmal1 localization to be independent of osmotic stress with cavin-1 downregulation at both 12 and 24 h of osmotic stress. These findings strongly indicate a role for cavin-1 in regulating circadian rhythm genes under osmotic stress.
Conclusions
The current study by Qifti et al. provides interesting evidence linking hypoosmotic stress to changes in circadian rhythm gene transcription, with caveolae playing a key role in this process. As illustrated in their simple model (Figure 1), their findings suggest that osmotic stress induces cavin-1 dissociation from caveolae in the PM, allowing it to translocate to the nucleus and promote transcription of circadian genes. These genes appear to include Ciart and Bmal1. Under normal conditions (Figure 1, left), Bmal1 dimerizes with Clock and enters the nucleus to promote the transcription of circadian proteins. Instead, under hypoosmotic stress (Figure 1, right), Bmal1 no longer enters the nucleus. Notably, this response is abolished when cavin-1 is knocked down, indicating a key role of caveolae and cavin-1 in this adaptive mechanism.
Altogether, Qifti et al. highlight a notable link between cell stress and circadian rhythms. These findings open new avenues for understanding how cellular stress might influence not only cell growth and function but also our daily biological cycles.
