Objective: Long-term osmotic stress results in altered gene transcription, however, with the exception of the TonE/TonEBP system, the underlying mechanisms are poorly understood. We previously showed that upon osmotic shrinkage of interphase fibroblasts, the MEK1-ERK1/2 pathway is transiently inhibited while p38 MAPK and JNK are activated, all in turn impacting on cell survival [1]. Here, we identify novel effectors up- and downstream of these MAPKs in shrinkage- induced transcriptional regulation. Methods: Immunofluorescence, Western blotting, subcellular fractionation, and luciferase reporter assays were used to dissect signaling events after osmotic shrinkage (50% increase in extracellular ionic strength) of interphase fibroblasts. Results: Within 0-30 min after shrinkage, the ERK1/2 effector, p90 Ribosmal S kinase (p90RSK) and the ERK1/2- and p38 MAPK effector Mitogen-and-stress activated protein kinase 1 (Msk1) were transiently inactivated and exported from the nucleus. Upon p38 MAPK inhibition, basal p90RSK activity was augmented while Msk1 inactivation was exacerbated. Serum response factor (SRF) and cAMP response element binding protein (CREB) are major p90RSK and Msk1 effectors and regulators of cell proliferation and death. SRF S¹⁰³ and CREB S¹³³ phosphorylation, which is associated with increased DNA binding, were oppositely regulated by osmotic shrinkage: SRF was phosphorylated in a p90RSK-independent, p38 MAPK-dependent manner, while CREB was dephosphorylated. Finally, SRF-dependent transcriptional activity, monitored as the activity of a triple CArG box promoter, was greatly increased 24 h after osmotic shrinkage, in a p38 MAPK dependent manner. Conclusion: Signaling via p90RSK, Msk1, CREB, and SRF is rapidly altered in response to brief periods of osmotic stress. These pathways may play important roles in hyperosmotic stress- induced changes in gene transcription and death/survival balance. [1] Pedersen SF et al. (2007) Cell Physiol Biochem, 20(6):735-750