Multiple mitogen-activated protein kinases are regulated by hyperosmolality in mouse IMCD cells

Abstract

Inner medullary collecting duct (IMCD) cells adapt to a hypertonic environment by synthesizing transporters that allow for accumulation of organic osmolytes. To examine for activation of additional mitogen-activated protein (MAP) kinases, extracts of IMCD-3 cells subjected to a hypertonic medium (600 mosmol/kgH2O) for 15 min were fractionated by Mono Q fast-performance liquid chromatography and assayed with the epidermal growth factor receptor [EGFR-(662-681)] peptide as substrate. Three peaks of activity were identified. Western blotting revealed that these peaks coincided with Jun NH2-terminal kinase (JNK), extracellular signal-regulated protein kinases, ERK1 and ERK2, and p38 MAP kinase. To assess the functional significance of ERK2 activation in IMCD-3 cells, the effect of PD-098059, an inhibitor of the upstream regulatory protein kinase MAP/ERK kinase (MEK) was assessed. PD-098059 inhibited ERK activation by hypertonicity. Yet, the stimulation of inositol uptake, a marker of adaptation, after 16 h was unaltered. Direct measurements of JNK activity [phosphorylation of GST-cJun-(1-79)] revealed a marked (20- to 40-fold) increase in activity as medium osmolality was increased from 300 to 900 mosmol/kgH2O with either NaCl or mannitol. Urea induced a more modest increase in activity. The response is prompt and detected as early as 2 min after exposure, reaching a maximum activation at 10-15 min. Downregulation of cellular protein kinase C (PKC) by chronic exposure to phorbol esters only minimally attenuated the JNK response to hyperosmolality, indicating a lack of involvement of PKC. We conclude that, in IMCD-3 cells, inhibition of ERK activation by hyperosmolality does not prevent osmoregulatory increase in inositol transport. This is not consistent with a role for ERKs in the response. The roles for JNK and p38 have not been ruled out, and these pathways may represent the initiating event in the subsequent transcription of organic osmolyte transporter genes and adaptation to extracellular hypertonicity.

Acute Hypertonicity Alters Aquaporin-2 Trafficking and Induces a MAPK-dependent Accumulation at the Plasma Membrane of Renal Epithelial Cells

“…MAPK signaling has been shown to modulate intracellular trafficking of a number of receptors and transporters (48 -53). Similar to observations of the present study, previous reports using inner medullary collecting duct cells indicate that urea induces activation of ERK1/2 (54, 55) but not p38 (55) and only induces weak activation of JNK as compared with the effect produced by NaCl and mannitol (43). Since urea did not induce accumulation of AQP2 at the cell surface, increased ERK1/2 activity alone by hypertonicity cannot account for increased AQP2 plasma membrane expression.…”

“…Similar to results of the present study, p38, ERK1/2, and JNK MAPK activation by hypertonicity has been demonstrated in inner medullary collecting duct cells and in cells of the medullary thick ascending limb of Henle's loop (43)(44)(45). A role of p38, ERK and JNK in the transcriptional regulation of various aquaporins by hypertonicity has been shown in renal and lung epithelial cells and in astrocytes (45)(46)(47).…”

Hypertonicity-Induced Expression of Monocyte Chemoattractant Protein-1 through a Novel Cis-Acting Element and MAPK Signaling Pathways

“…Several lines of evidence have demonstrated that MAPKs, such as p38MAPK (21,22,24), ERK (21)(22)(23)(24) and JNK (21,22,24), are activated by hypertonic stress associated with NaCl. Thus, to further characterize the cell signaling pathways stimulated by hypertosmolality, involvement of MAPKs in the regulation of MCP1 expression was analyzed.…”

“…In kidney tubular cells, such as Madin-Darby canine kidney cells (21), mouse inner medullary collecting duct cells (20,22,23), and rabbit kidney papillary epithelial (PAP-HT25) cells (24), p38MAPK, ERK, and JNK have been found to be involved in hypertonicity-mediated cell signaling cascades. The present study also demonstrated that hypertonic NaCl treatment results in the activation of p38MAPK, ERK, and JNK in NRK52E cells (Fig.…”

“…In addition to the BGT1 and AR genes, the TonE/ORE has been identified in the proximal region of the 59-flanking region of stress protein genes, such as Hsp70 (19) and Osp94 (20), which protect cells from the deleterious effects of hyperosmolity. Recent studies on intracellular signaling pathways responsive to hypertonicity have suggested that MAPKs pathways play a role in the cellular mechanisms underling the regulation of gene expression in response to hypertonicity (21)(22)(23)(24).…”