Phorbol ester stimulation of RasGRP1 regulates the sodium-chloride cotransporter by a PKC-independent pathway

Abstract

The sodium-chloride cotransporter (NCC) is the principal salt-absorptive pathway in the mammalian distal convoluted tubule (DCT) and is the site of action of one of the most effective classes of antihypertensive medications, thiazide diuretics. We developed a cell model system to assess NCC function in a mammalian cell line that natively expresses NCC, the mouse DCT (mDCT) cell line. We used this system to study the complex regulation of NCC by the phorbol ester (PE) 12-O-tetradecanoylphorbol-13-acetate (TPA), a diacylglycerol (DAG) analog. It has generally been thought that PEs mediate their effects on transporters through the activation of PKC. However, there are at least five other DAG/PE targets. Here we describe how one of those alternate targets of DAG/PE effects, Ras guanyl-releasing protein 1 (RasGRP1), mediates the PE-induced suppression of function and the surface expression of NCC. Functional assessment of NCC by using thiazide-sensitive 22 Na ؉ uptakes revealed that TPA completely suppresses NCC function. Biotinylation experiments demonstrated that this result was primarily because of decreased surface expression of NCC. Although inhibitors of PKC had no effect on this suppression, MAPK inhibitors completely prevented the TPA effect. RasGRP1 activates the MAPK pathway through activation of the small G protein Ras. Gene silencing of RasGRP1 prevented the PE-mediated suppression of NCC activity, the activation of the H-Ras isoform of Ras, and the activation of ERK1/2 MAPK. This finding confirmed the critical role of RasGRP1 in mediating the PE-induced suppression of NCC activity through the stimulation of the MAPK pathway.diacylglycerol ͉ distal convoluted tubule ͉ NaCl cotransporter

WNK4 Enhances the Degradation of NCC through a Sortilin-Mediated Lysosomal Pathway

“…A recent study also showed that WNK4 increases the association of NCC with AP3 to promote NCC delivery to the lysosomal compartment for degradation, 40 which is consistent with our conclusion. Our findings further support the notion that NCC is regulated through affecting its forward trafficking in addition to other mechanisms such as altering its abundance, [41][42][43] glycosylation, 10 and phosphorylation. 44,45 In the kidney, NCC is exclusively expressed in DCT and responsible for 5 to 7% of sodium reabsorption.…”

“…46 It is likely that, under normal physiologic conditions, NCC constantly traffics to both the plasma membrane via a secretory pathway and the lysosome for degradation to maintain its basal steady protein level, which is subject to regulation by many mediators. 3,6,10,[41][42][43][44][45] Renal DCT cells contain endogenous WNK4 kinase. 2 In the presence of WNK4, the rate of NCC targeting to the lysosomal compartment is much higher than that of NCC without WNK4.…”

“…mDCT cells were a gift of Peter Friedman (University of Pittsburgh, Pittsburgh, PA). 43,54 mDCT cells were maintained in DMEM/ F-12 medium containing 5% FCS, 1% penicillin, 1% streptomycin, and 1% neomycin. All other media and components were purchased from Invitrogen (Carlsbad, CA).…”

The sodium chloride cotransporter SLC12A3: new roles in sodium, potassium, and blood pressure regulation

“…One example is the regulation of NCC by phorbol esters [54]. This 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 12 effect is mediated through Ras guanyl-releasing protein 1 and ERK1/2, which stimulates ubiquitylation and endocytosis of NCC [55].…”

Phosphorylation Decreases Ubiquitylation of the Thiazide-sensitive Cotransporter NCC and Subsequent Clathrin-mediated Endocytosis

“…A WNK4-induced decrease in NCC membrane abundance has been shown to occur via alteration of NCC forward trafficking (exocytosis) (10,11). Ko et al (12,13) demonstrated that treatment of DCT cells with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate reduced NCC membrane abundance via a Ras-GRP1 (Ras-guanyl nucleotide-releasing protein 1)-dependent ERK1/2-MAPK pathway (12) that increased ubiquitylation and dynamin-dependent internalization of NCC (13). However, the exact mechanisms behind the regulated endocytosis of NCC and whether, similarly to the water channel aquaporin 2 (AQP2) (14) and the sodium potassium chloride cotransporter NKCC2 (15), constitutive endocytosis of NCC plays a role in steady-state surface expression of NCC are not known.…”