Differential dependence of regulatory volume decrease behavior in rabbit corneal epithelial cells on MAPK superfamily activation

Abstract

We characterized the dependence of hypotonicity-induced regulatory volume decrease (RVD) responses on mitogen-activated protein kinase (MAPK) pathway signaling in SV40-immortalized rabbit corneal epithelial cells (RCEC). Following calcein-AM loading, RVD was monitored using a microplate fluorescence reader. Western blot analysis determined MAPK activation. After 30 min, the RVD response restored the relative cell volume to nearly isotonic values, whereas it was inhibited when cells were bathed either in a Cl − -free solution or with the Cl − -channel inhibitors: 5-nitro-2-(3-phenylpropylamino) benzoic acid or niflumic acid. Similar declines occurred with either a high-K + (20 mM) supplemented solution or the K + channel inhibitor 4-aminopyridine. Activation of extracellular signal-regulated kinase (ERK), p38, and stress-activated protein kinase/c-Jun Nterminal kinase (SAPK/JNK) was time and tonicity-dependent. Stimulation of ERK and SAPK/JNK was maximized earlier than that of p38. Activation of ERK and SAPK/JNK was insensitive to Cl − and K + channel inhibitors, whereas inhibition with either PD98059 or SP600125, respectively, blocked RVD. However, inhibition of p38 with SB203580 had no effect on RVD. Suppression of RVD instead blocked p38 activation. Differences in the dependence of RVD activation on Erk1/2 and p38 signaling were validated in dominant negative (d/n) -Erk1 and d/n-p38 cells. Volumesensitive Cl − and K + channel activation contributes, in concert, to RVD in RCEC. Therefore, swelling-induced ERK and SAPK/JNK stimulation precedes Cl − and K + channel activation, whereas p38 activation occurs as a consequence of RVD.

Physiology of Cell Volume Regulation in Vertebrates

“…Indeed, there is evidence to suggest the involvement of ERK, p38 MAPK, and JNK in control of RVI (285,558,803,854) and RVD (759,1049) in at least some cell types. MAPK activation may also contribute to the volume-dependent reorganization of the actin cytoskeleton (sect.…”

“…Moreover, there are several examples of swelling-induced stimulation of JNK and p38 MAPK activity (e.g., Refs. 624,713,759,993,1049), raising the question of to which extent changes in MAPK activity are part of the volume response per se, as opposed to being general stress responses activated by cell volume perturbations. Finally, a recent study demonstrated that also ERK5 is activated by hyperosmotic stress in fibroblasts and appears to promote cell survival under these conditions by downregulating Fas ligand expression (1067).…”

Dependence of regulatory volume decrease on transient receptor potential vanilloid 4 (TRPV4) expression in human corneal epithelial cells

“…Such restoration is in part dependent on activation of Na-K-2Cl 1 co-transporter (NKCC1)-induced osmolyte influx coupled to Na-K pump stimulation [11]. On the other hand, during exposure to a hypotonic challenge, CEC initially swell, and activate a RV decrease (RVD) response to restore their isotonic volume [12]. In human CEC (HCEC), RVD is dependent on increases in K + and Cl − efflux through volume-sensitive ion channels [9,12] and K-Cl cotransporter 1 (KCC1) [13].…”

“…Their association in HCEC is requisite for NKCC1 activation and RVI induction [15]. On the other hand, activation of MAPKs ERK and JNK is required for RVD whereas p38 stimulation occurs subsequent to RVD in rabbit CEC (RCEC) [12]. Ca 2+ signaling is critical to induce RVD during exposure to a hypotonic challenge.…”

“…On the other hand, during exposure to a hypotonic challenge, CEC initially swell, and activate a RV decrease (RVD) response to restore their isotonic volume [12]. In human CEC (HCEC), RVD is dependent on increases in K + and Cl − efflux through volume-sensitive ion channels [9,12] and K-Cl cotransporter 1 (KCC1) [13]. Each of these regulatory volume responses is essential for maintaining corneal transparency, since they restore CEC isotonic volume and prevent compromise of their barrier function [14].…”