Abstract
The survival of renal medullary interstitial cells (RMICs) requires their adaptation to rapid shifts in ambient tonicity normally occurring in the renal medulla. Previous studies determined that cyclooxygenase-2 (COX 2) activation is critical for this adaptation. The present studies find that these adaptive mechanisms are dampened by the simultaneous activation of an apoptotic pathway linked to a glycogen synthase kinase 3β (GSK 3β). Inhibition of GSK 3 by LiCl or specific small molecule GSK inhibitors increased RMIC survival following hypertonic stress, and transduction of RMICs with a constitutively active GSK 3β (AdGSK 3βA9) significantly increased apoptosis, consistent with a proapoptotic role of GSK 3β. Following GSK 3β inhibition, increased survival was accompanied by increased COX 2 expression and COX 2 reporter activity. In contrast, GSK 3β overexpression reduced COX 2 reporter activity. Importantly, enhanced RMIC survival produced by GSK 3β inhibition was completely dependent on COX 2 because it was abolished by a COX 2-specific inhibitor, SC58236. The signaling pathway by which GSK 3β suppresses COX 2 expression was then explored. GSK 3β inhibition increased both NFκB and β-catenin activity associated with decreased IκB and increased β-catenin levels. The increase in COX 2 following GSK 3β inhibition was entirely blocked by NFκB inhibition using mutant IκB adenovirus. However, adenoviral overexpression of β-catenin did not increase COX 2 levels. These findings suggest that GSK 3β negatively regulates COX 2 expression and that GSK 3β inhibitors protect RMICs from hypertonic stress via induction of NFκB-COX 2-dependent pathway.
Highlights
Signaling cascades that either lead to cell death or promote cell survival
Recent studies demonstrate that renal medullary interstitial cells (RMICs)1 depend on robust COX 2 activity to adapt to hypertonic stress, both in vitro and in vivo [2, 8, 9]
Hypertonic Stress Increases GSK 3 Activity in Cultured RMICs—Measurement of GSK 3 enzymatic activity using primed glycogen synthase as a substrate showed that hypertonic stress significantly increased GSK 3 activity in a timedependent manner to 150% of that present in non-stressed cells within 10 min (150 Ϯ 13 versus 100 Ϯ 9; p Ͻ0.005, n ϭ 4) and 171% by 30 min (171 Ϯ 9.5 versus 100 Ϯ 9; p Ͻ0.005, n ϭ 4) (Fig. 1A)
Summary
Signaling cascades that either lead to cell death or promote cell survival. The balance between these two pathways determines cell fate. Recent studies demonstrate that renal medullary interstitial cells (RMICs) depend on robust COX 2 activity to adapt to hypertonic stress, both in vitro and in vivo [2, 8, 9]. A link between GSK 3 activity and neuronal cell survival following hypertonic stress has been suggested, though the downstream mechanisms of these effects are uncharacterized [18]. Some data suggest that COX 2 is a downstream target of Wnt/GSK 3/-catenin signaling, promoting proliferation and tumorigenesis [19], and previous studies have shown that COX 2 is critical for renal interstitial cell survival. The aim of the present study was to examine whether GSK 3 modulates renal medullary interstitial cell survival through a COX 2-dependent mechanism
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