Circadian Rhythmic Localization of tPA and PAI-1 in the SCN 2.2 Cell Culture May Provide Evidence for Determining the Mechanism of Gating Photic Phase Shifts

Mammalian circadian rhythms are controlled by a central pacemaker located in the suprachiasmatic nucleus (SCN) of the brain. The SCN exhibits endogenous rhythms in neuronal activity and entrains to external stimuli, particularly light. Interestingly, phase shifts in response to light only occur at night and the mechanisms gating phase shifting are not well characterized. Our lab demonstrated that the extracellular protease, tissue-type plasminogen activator (tPA) and its inhibitor, plasminogen activator inhibitor (PAI-1), help gate phase shifting. Total tPA and PAI-1 expression are rhythmic in mouse SCN. These proteins mediate different functions depending on their exact subcellular localization. Therefore, knowing where they are located within the SCN will clarify their actions with respect to SCN clock phase regulation. The immortalized rat SCN2.2 cell culture exhibits rhythms in protein expression in vitro that mirror those found in vivo and can be separated into cellular, extracellular matrix, and media fractions. Here, we investigate tPA and PAI-1 expression using western blotting in the cellular fraction of the SCN2.2 line over a 36-hr timecourse. Preliminary results suggest a rhythm of PAI-1 levels inside the cell with peak expression in the early subjective night. Future studies are aimed toward elucidating the subcellular localization and temporal expression patterns of these proteins in the SCN.