Hyperosmotic stress strongly potentiates serum response factor (SRF)‐dependent transcriptional activity in ehrlich lettré ascites cells through a mechanism involving p38 mitogen‐activated protein kinase

Abstract

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 Ehrlich Lettré Ascites (ELA) fibroblasts, the MEK1-ERK1/2 pathway is transiently inhibited while p38 MAPK is activated, in turn impacting on cell survival (Pedersen et al., 2007, Cell Physiol Biochem 20: 735-750). Here, we show that downstream of these kinases, two transcription factors with major roles in control of cell proliferation and death, serum response factor (SRF) and cAMP response element-binding protein (CREB) are differentially regulated in ELA cells. SRF Ser(103) phosphorylation and SRF-dependent transcriptional activity were strongly augmented 5-30 min and 24 h, respectively, after hyperosmotic stress (50% increase in extracellular ionic strength), in a p38 MAPK-dependent manner. In contrast, CREB Ser(133) was transiently dephosphorylated upon osmotic shrinkage. The ERK1/2 effector ribosomal S kinase (RSK) and the ERK1/2- and p38 MAPK effector mitogen- stress-activated protein kinase 1 (MSK1) both phosphorylate CREB at Ser(133) . RSK and MSK1 were dephosphorylated within 5 min of shrinkage. MSK1 phosphorylation recovered within 30 min in a p38-MAPK-dependent manner. CREB was transiently dephosphorylated after shrinkage in a manner exacerbated by p38 MAPK inhibition or MSK1 knockdown, but unaffected by inhibition of RSK. In conclusion, in ELA cells, hyperosmotic stress activates SRF in a p38 MAPK-dependent manner and transiently inactivates CREB, likely due to MSK1 inactivation. We suggest that these events contribute to shrinkage-induced changes in gene transcription and death/survival balance.

Hyperosmotic stress regulates the distribution and stability of myocardin-related transcription factor, a key modulator of the cytoskeleton

“…This observation is consistent with the fact that hyperosmolarity is a weak and transient stimulus for ERK in many cells, while in others it actually inhibits this kinase (Ref. 24; see also Ref. 29).…”

“…Specifically, LCM-triggered MRTF translocation is suppressed by p38 inhibition, indicating that p38 is needed for upstream signaling steps induced by contact disruption (61). More importantly, SRF activity itself can be enhanced by p38, as indicated by the observation that p38 inhibition significantly reduced the hypertonicity-provoked activation of the SRF reporter in ELA cells (24). The likely mechanism underlying this finding is that p38 activates MAPKAP kinase-2, which directly phosphorylates and activates SRF (28,58).…”

“…Cells were harvested and lysed, and the obtained lysates were assayed for caspase-3 activity, using a fluorigenic substrate as described in MATERIALS pathways and the consequent cytoskeletal changes themselves are not only responsible for the acute structural adaptation, but they also mobilize transcription factors that can impact the expression of cytoskeletal genes. Our recent studies have shown that SRF is phosphorylated and activated upon hyperosmotic stimulation in ELA cells (24). However, SRF is a dual-function transcription factor that can drive both proliferation/survival-promoting early genes and cytoskeleton/muscle differentiation-specific genes (41).…”

“…Briefly, cells were plated onto 12-well plates and at ϳ60% confluence were transfected with 0.5 g/well reporter plasmid pGL3-3DA-Luc, which contains the firefly luciferase gene under the control of a CArG box triplet (64), or pSMA-Luc, which harbors the proximal 765-base pair segment of the rat SMA promoter in the pGL3 vector (40). Normalization was carried out by cotransfection of the enhancer-less Renilla luciferase control plasmid pHRG-B (Promega) (0.125 g/well), which is optimal for this purpose, as it is not responsive to hypertonicity, as verified in our previous studies (24). At 16 -20 h posttransfection, cells were serum deprived for 3 h, and treatments were initiated by adding fresh, serum-free medium supplemented with NaCl, in the presence or absence of inhibitors, as indicated.…”

PDGFRα signaling in the primary cilium regulates NHE1-dependent fibroblast migration via coordinated differential activity of MEK1/2-ERK1/2-p90RSK and AKT signaling pathways

“…Previous studies by us and others have shown that p90 RSK localizes to the nucleus, nuclear membrane region, and cytoplasm (e.g. Anjum and Blenis, 2008;Gorbatenko et al, 2011), but this is the first study to report its localization at the primary cilium. Confirming the specificity of binding, pre-incubation of the anti-p-p90 ) labeling both at the ciliary base region and in the nucleus (Fig.…”