EGF-induced changes in Erk1/2 and p38 phosphorylation status are dependent on PP-mediated crosstalk. This control modulates the magnitude of growth factor-induced increases in corneal epithelial cell migration.
We examined the ability of SV40-immortalized human and rabbit corneal epithelial cells (HCEC and RCEC, respectively) to adapt to chronic hypertonic stress. Under isotonic conditions, in the presence of 50 microm bumetanide, proliferation measured as (3)H-thymidine incorporation declined in RCEC and HCEC by 8 and 35%, respectively. After 48 hr exposure to 375 mOsm medium, RCEC proliferation fell by 19% whereas in HCEC it declined by 45%. Light scattering behavior demonstrated that both cell lines mediate nearly complete regulatory volume increase (RVI) responses to an acute hypertonic (375 mOsm) challenge, which in part depend on bumetanide-sensitive Na-K-2Cl cotransporter (NKCC) activity. Following exposing RCEC for 48 hr to 375 mOsm medium, their RVI response to an acute hypertonic challenge was inhibited by 17%. However, in HCEC this response declined by 68%. During exposure to 375 mOsm medium for up to 24 hr, only RCEC upregulated NKCC gene and protein expression as well as bumetanide-sensitive (86)Rb influx. These increases are consistent with the smaller declines in RVI and proliferation capacity occurring during this period in RCEC than in HCEC. Therefore, adaptation by RCEC to chronic hypertonic stress is dependent on stimulation of NKCC gene and protein expression and functional activity. On the other hand, under isotonic conditions, HCEC RVI and proliferation are more dependent on NKCC activity than they are in RCEC.
In hypertonicity-stressed (i.e., 600 mOsm) SV40-immortalized rabbit and human corneal epithelial cell layers (RCEC and HCEC, respectively), we characterized the relationship between time-dependent changes in translayer resistance, relative cell volume and modulation of MAPK superfamily activities. Sulforhodamine B permeability initially increased by 1.4- and 2-fold in RCEC and HCEC, respectively. Subsequently, recovery to its isotonic level only occurred in RCEC. Light scattering revealed that in RCEC 1) regulatory volume increase (RVI) extent was 20% greater; 2) RVI half-time was 2.5-fold shorter. However, inhibition of Na-K-2Cl cotransporter and Na/K-ATPase activity suppressed the RVI response more in HCEC. MAPK activity changes were as follows: 1) p38 was wave-like and faster as well as larger in RCEC than in HCEC (90- and 18-fold, respectively); 2) increases in SAPK/JNK activity were negligible in comparison to those of p38; 3) Erk1/2 activity declined to 30-40% of their basal values. SB203580, a specific p38 inhibitor, dose dependently suppressed the RVI responses in both cell lines. However, neither U0126, which inhibits MEK, the kinase upstream of Erk, nor SP600125, inhibitor of SAPK/JNK, had any effect on this response. Taken together, sufficient activation of the p38 limb of the MAPK superfamily during a hypertonic challenge is essential for maintaining epithelial cell volume and translayer resistance. On the other hand, Erk1/2 activity restoration seems to be dependent on cell volume recovery.
The capacity of the corneal epithelium to adapt to hypertonic challenge is dependent on the ability of the cells to upregulate the expression and activity of cell membrane-associated Na-K-2Cl cotransporter1 (NKCC1). Yet, the signaling pathways that control this response during hypertonic stress are still unclear. We studied stress-induced changes in proliferation and survival capacity of SV40-immortalized human (HCEC) and rabbit (RCEC) corneal epithelial cells as a function of (i) the magnitude of the hypertonic challenge, (ii) differential changes in activation of mitogen-activated protein kinase (MAPK), and (iii) the extent of p38MAPK interaction with NKCC1. Cells were incubated in hypertonic (up to 600 mOsm) media for varying time periods up to 24 h. Phosphorylated forms of p44/42, p38, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) MAPK were immunoprecipitated from cell lysates, and the amount of each activated NKCC1-associated MAPK was evaluated by Western blot/ECL assay. DNA integrity was assessed by electrophoresis in a 2% agarose gel. Cell survival and proliferation were evaluated based on three criteria: protein content, cell count, and the MTT assay. Exposure to media of 325-350 mOsm increased proliferation of HCEC up to 75%, whereas this response was limited to <16% in RCEC. At higher osmolarities, cell proliferation decreased in both species. SAPK/JNK activity increased 150-fold in HCEC and <10-fold in RCEC, while DNA fragmentation occurred only in HCEC. Compared to HCEC, the better RCEC survival rate was associated with higher p38MAPK activity and near complete restoration of p44/42MAPK activity after the first 30 min. In both cell lines, the amount of phospho-NKCC1 that coimmunoprecipitated with phospho-p38MAPK was proportional to the magnitudes of their respective activation levels. However, no such associations occurred between amounts of phosphorylated p44/42MAPK or SAPK/JNK and phospho-NKCC1. Under isotonic conditions, with bumetanide-induced inhibition of RCEC and HCEC NKCC1 activities, p44/42MAPK activity declined by 40 and 60%, respectively. Such declines led to proportional decreases in cell proliferation. Survival of hypertonicity-stressed corneal epithelial cells depends both on p38MAPK activation capacity and the ability of p38MAPK to stimulate NKCC1 activity through protein-protein interaction. The level of NKCC1 activation affects the extent of cell volume recovery and, in turn, epithelial survival capacity.
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