MEKK3-mediated signaling to p38 kinase and TonE in hypertonically stressed kidney cells

Mitogen-activated protein kinase (MAPK) cascades contain a trio of kinases, MAPK kinase kinase (MKKK) → MAPK kinase (MKK) → MAPK, that mediate a variety of cellular responses to different signals including hypertonicity. The signaling response to hypertonicity is conserved across evolution from yeast to mammals in that it involves activation of p38/SAPK. However, very little is known about which upstream protein kinases mediate activation of p38 by hypertonicity in mammals. The MKKKs, MEKK3 and MEKK4, are upstream regulators of p38 in many cells. To investigate these signaling proteins as potential activators of p38 in the hypertonicity response, we generated stably transfected MDCK cells that express activated versions of MEKK3 or MEKK4, utilized RNA interference to deplete MEKK3, and employed pharmacological inhibition of p38 kinase. MEKK3-transfected cells demonstrated increased betaine transporter (BGT1) mRNA levels and upregulated tonicity enhancer (TonE)-driven luciferase activity under isotonic (basal) and hypertonic conditions compared with empty vector-transfected controls; small-interference RNA-mediated depletion of MEKK3 downregulated the activity of p38 kinase and decreased the expression of BGT1 mRNA. p38 Kinase inhibition abolished the effects of MEKK3 activation on BGT1 induction. In contrast, the response to hypertonicity in MEKK4-kA-transfected cells was similar to that observed in empty vector-transfected controls. Our data are consistent with the existence of an input from MEKK3 →→ p38 kinase →→ TonE.

many organisms, including bacteria, yeast, plants, and animals, adapt to sustained hypertonic stress by the preferential accumulation of compatible organic osmolytes (56). In water-deprived mammals, for example, the extracellular osmolality of the kidney medulla may exceed 4,000 mosmol/kgH2O (38). Roughly one-half of the prevailing medullary interstitial solutes consist of urea, whereas the other half is composed of NaCl (15). Urea easily equilibrates across biological membranes and does not cause water shift between the intracellular and extracellular compartments. However, NaCl remains confined to the extracellular space, owing to the action of the Na-K-ATPase. An increase in the extracellular concentration of NaCl contributes to dehydration of the intracellular milieu (hypertonic stress), and restoration of intracellular volume in hypertonically stressed kidney cells requires the induction of a group of genes that lead to the accumulation of organic osmolytes intracellulary [BGT1 for betaine transporter (54), SMIT for inositol transporter (55), taurine transporter (49), and the aldose reductase enzyme (AR), which catalyze the reduction of d-glucose to the organic solute sorbitol (3)]. The transcriptional “machinery” that drives the expression of these genes [SMIT, BGT1, taurine transporter, and AR] under hypertonic conditions is similar (13, 19, 36, 44) and involves interaction between the cis-element [tonicity enhancer (TonE) (36), also known as osmotic response element (ORE) (13); referred to herein as TonE] and transcription factor TonE binding protein [TonEBP; (37), also known also as ORE binding protein (OREBP) (24) as well as NFAT5 (34); referred to herein as TonEBP]. Activation of TonE-mediated gene expression by hypertonicity is not unique to kidney cells [Madin-Darby canine kidney (MDCK) (36); rabbit kidney papillary epithelial cells (PAP-HT25) (25); mouse inner medullar collecting duct cells (mIMCD) (48)], as it has been shown to occur in neurons (35), human liver-derived HepG2 (41), Chang liver, Cos-7, and HeLa cells (24). Deletion of the TonEBP gene in mice blocks the expression of TonE-mediated gene expression in the kidney medulla almost completely, as evidenced by the diminished expression of the BGT1, SMIT, and AR genes. Remarkably, mice lacking TonEBP show atrophy of the renal medulla, which contains smaller cells and displays increased apoptosis (33).

While transcriptional control of hypertonicity-induced genes in mammalian cells is reasonably well characterized, the signaling pathways leading to TonE-mediated gene expression need further delineation. In yeast, the adaptation to osmotic stress is dependent on the p38 MAPK homolog high-osmolarity glycerol 1 (HOG1) (7). Similarly, the induction of TonE-mediated gene expression in mammalian cells is p38 kinase dependent (41, 46) but requires cooperative action of Fyn, the catalytic subunit of PKA and the DNA damage-inducible kinase ATM (reviewed in Ref. 47). While ERK and JNK are induced by hypertonicity, the significance of their activation is not clear, as JNK and ERK do not appear to have an effect on TonE-mediated gene expression (reviewed in Ref. 47).

In the current experiments, we sought to determine upstream signaling molecules in the p38 kinase cascade that “drive” the expression of hypertonicity-induced genes (represented by the betaine transporter BGT1) and affect TonE-mediated gene expression in kidney cells. The activity of p38 kinase is dependent on MAPK kinases (MKKs) and their activators, the MAPK kinase kinases (MKKKs; see review in Ref. 52). MEK kinase 1 (MEKK1; 1 of the MKKKs) is linked to JNK activation, whereas MEKK2 is linked to JNK and ERK activation (reviewed in Ref. 27). On the other hand, MEKK3 may activate ERK, JNK, and p38, whereas MEKK4 may activate JNK and p38 kinase (27). Hence, we hypothesized that MEKK3 and/or MEKK4 are likely mediators of p38 kinase activation in kidney cells under hypertonic conditions. Our data are consistent with the existence of MEKK3 → → → p38 kinase input to drive TonE-mediated gene expression.