Na⁺/K⁺/Cl⁻ cotransporter activates mitogen‐activated protein kinase in fibroblasts and lymphocytes

Abstract

In a previous work, we have shown that overexpression of the Na(+)/K(+)/Cl(-) cotransporter (NKCC1) induces cell proliferation and transformation. We investigate in the present study the role of the NKCC1 in the mitogenic signal transduction. We show that overexpression of the cotransporter gene (NKCC1) in stablely transfected cells (Balb/c-NKCC1), resulted in enhanced phosphorylation of the extracellular regulated kinase (ERK) to produce double phosphorylated ERK (DP-ERK). Furthermore, the level of DP-ERK was reduced by 50-80% following the addition of bumetanide, a specific inhibitor of the Na(+)/K(+)/Cl(-) cotransporter, in quiescent as well as in proliferating cultures of the Balb/c-NKCC1 clone. In order to explore further the role of the Na(+)/K(+)/Cl(-) cotransporter in mitogenic signal transduction, we measured the effect of the two specific inhibitors of the cotransporter; bumetanide and furosemide, on DP-ERK level in immortalized non-transformed cells. In Balb/c 3T3 fibroblasts stimulated with FGF, bumetanide, and furosemide inhibited 50-60% of the ERK 1/2 phosphorylation. The inhibitor concentration needed for maximal inhibition of ERK 1/2 phosphorylation was similar to the concentration needed to block the K(+) influx mediated by the Na(+)/K(+)/Cl(-) cotransporter in these cells. To analyze whether the Na(+)/K(+)/Cl(-) cotransporter has a role in the mitogenic signal of normal cells, we measured the effect of bumetanide on ERK phosphorylation in human peripheral blood lymphocytes. The phosphorylation of ERK 1/2 in resting human lymphocytes, as well as in lymphocytes stimulated with phytohemagglutinin (PHA) was inhibited by bumetanide. The effect of bumetanide on ERK 2 phosphorylation was much lower than that of ERK 1 phosphorylation. The finding that the Na(+)/K(+)/Cl(-) cotransporter controls the ERK/MAPK (mitogen-activated protein kinase) signal transduction pathway, support our hypothesis that Na(+) and K(+) influxes mediated by this transporter plays a central role in the control of normal cell proliferation. Exploring the cellular ionic currents and levels, mediated by the Na(+)/K(+)/Cl(-) cotransporter, should lead to a better comprehension of cell proliferation and transformation machinery.

Acquisition of Cisplatin-resistance in Malignant Mesothelioma Cells Abrogates Na^+,K^+,2Cl^-;-cotransport Activity and Cisplatin-induced Early Membrane Blebbing

“…Because NKCC1 activity can have a role in membrane blebbing [11][12][13][14], the lack of effect of bumetanide on K + fluxes in P31res1.2 cells led us to investigate the morphological response of P31res1.2 cells to cisplatin, and compare this to the response of P31 cells to cisplatin in absence and presence of bumetanide.…”

“…Cell membrane blebbing can commence within minutes of start of insult [8][9][10]. There are several reports that indicate a role for the secretory Na + ,K + ,2Cl --cotransporter (NKCC1) in membrane blebbing via interactions with actin, the p38 mitogen activated protein kinases (p38) and extra-cellular regulated kinase 1/2 (ERK1/2) [11][12][13][14].…”

Ion Transporter NKCC1, Modulator of Neurogenesis in Murine Olfactory Neurons

“…In summary, the lack of NKCC1 leads to a reduced number of mature neurons due to an inhibition of cell proliferation. NKCC1 is a wellknown cell cycle regulator in cultured cell lines (20,21). In addition, previous studies suggested a role of NKCC1 in regeneration after nerve injuries and postnatal neurogenesis in the brain (23,25).…”

“…NKCC1 regulates the resting phase and early interphase (G1) of the cell cycle in mouse fibroblasts. Upon NKCC1 overexpression, proliferation is induced, and mouse fibroblasts undergo phenotypic transformation (20,21). Conversely, treatment of the PC12D rat pheochromocytoma cell line with nerve growth factor induces NKCC1 up-regulation, demonstrating the relationship between neurite outgrowth and NKCC1 expression (22).…”

Dependence of Corneal Epithelial Cell Proliferation on Modulation of Interactions Between ERK1/2 and NKCC1

“…Nevertheless, in fibroblasts there is some uncertainty regarding the cause and effect relationship between ERK1/2 and NKCC1 activation since it was reported that growth factor-induced NKCC1 activation precedes ERK1/2 phosphorylation [26]. However, ERK1/2 and PKC activation induced NKCC1 phosphorylation in human tracheal epithelial cells [21].…”