Hypertonicity-induced RNF183 targets NKCC1 to protect kidney collecting duct cells but induces apoptosis in colon cells

Abstract

RING-finger protein 183 (RNF183) is a ubiquitin ligase specifically expressed in the kidney and especially in collecting ducts that are constantly exposed to hypertonic stress. The expression of RNF183 in the colon is low but is upregulated in patients with inflammatory bowel disease (IBD) and patients with colorectal cancer. However, the function and relationship of RNF183 with these diseases remain unclear. Here, we identify the Na-K-2Cl co-transporter NKCC1 as a substrate of RNF183. RNF183 promotes K63-linked ubiquitination of NKCC1, leading to its lysosomal degradation, which is enhanced under hypertonic conditions. Dysregulation of RNF183 expression increases hypertonic-induced cell death in Rnf183-knockout mIMCD-3 cells and RNF183-overexpressing Caco-2 cells, accompanied by an increase in cleaved caspase-3. Under isotonic and hypertonic conditions, intracellular Na⁺ homeostasis is disrupted in these cells. Notably, RNF183 expression exerts opposing effects in mIMCD-3 cells, which endogenously express RNF183, and in Caco-2 cells, which do not. Thus, RNF183, which targets multiple ion transporters, including NKCC1 identified in this study, may play an important role in the adaptation to hypertonic conditions in the kidney, whereas aberrant expression of RNF183 could cause the development of diseases such as IBD and colorectal cancer.

The ubiquitin ligase RNF183 regulates the ion transporter NKCC1 under hyperosmotic stress through ubiquitin-dependent mechanisms, providing new insights into osmoadaptive regulation of membrane transporters.

Introduction

Proteins are regulated in their activities, turnover, and localization by post-translational modifications such as phosphorylation, ubiquitination, and glycosylation^1–4. Ubiquitination is involved in proteasomal degradation, and the ubiquitin-proteasome pathway is responsible for 80%–90% of eukaryotic protein degradation⁵. However, ubiquitination is also involved in the regulation of various intracellular functions such as endocytosis, lysosomal degradation, signal transduction, and DNA repair^6–9. Ubiquitination is performed by a set of enzymes, including ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin ligase. The most crucial molecule responsible for the substrate selection of ubiquitination is the ubiquitin ligase. The most common of the three major types of ubiquitin ligases is the RING-finger (RNF) type^10–12. Previously, we undertook an in silico search for transmembrane domain-containing RING-type ubiquitin ligases in the human genome and identified 37 genes¹³, and elucidated that RNF183 is specifically expressed in human and mouse kidneys, especially in the renal medulla^14,15.

The kidney has a complex structure and plays a crucial role in regulating the amount of water and ion balance in the body by reabsorbing nutrients, minerals, and water. The glomerulus processes approximately 150 L of primitive urine per day by passing this through the tubules, concentrating unnecessary substances and then excreting them as urine. To efficiently concentrate urine, an osmotic gradient is created in the interstitium from the cortex to the medulla, and the renal medulla is the only organ that is constantly in a hypertonic environment. RNF183 is expressed in the inner medullary collecting ducts, a particularly severe hypertonic environment in the kidney. Therefore, we focused on the relationship between RNF183 and hypertonic stress and clarified that the transcription of RNF183 is directly induced by the transcription factor nuclear factor of activated T cells 5 (NFAT5)/tonicity-responsive enhancer binding protein¹⁶. Moreover, we identified Na,K-ATPase as a substrate for RNF183¹⁷. Although Na,K-ATPase is involved in adaptation to hypertonic stress, the role of RNF183 in the kidney remains unresolved. Currently, although RNF183 has been associated with disease, no specific association with renal disease has been reported¹⁸. Interestingly, RNF183, which is originally expressed in a kidney-specific manner, is upregulated in the colon of patients with inflammatory bowel disease (IBD) and patients with colorectal cancer^19–22. Herein, we identified the Na-K-2Cl co-transporter NKCC1 as a substrate protein for RNF183 and investigated the effects of RNF183 expression on cells that do not naturally express this ubiquitin ligase.

Results

Identification of NKCC1 as a substrate protein for RNF183

We previously identified Na,K-ATPase as a substrate protein for RNF183 from proteins detected by the BirA proximity-biotinylation technique, and also identified multiple additional candidate interacting proteins, including ion transporters¹⁷. Among them, we focused on NKCC1, which is known to be important in maintaining cell volume during short-term hypertonic stress, as is Na,K-ATPase²³.

To determine whether RNF183 interacts with NKCC1, we performed co-immunoprecipitation with anti-NKCC1 or anti-EGFR antibodies in HEK293 cells inducibly expressing 3 × FLAG-tagged wild-type (WT) RNF183 via a Tet-on system. Although both NKCC1 and EGFR are localized at the plasma membrane, 3 × FLAG-RNF183 was immunoprecipitated with endogenous NKCC1 but not with EGFR (Fig. 1). Conversely, when 3 × FLAG-tagged RNF183 was immunoprecipitated with anti-FLAG antibody, NKCC1 was co-precipitated with 3 × FLAG-RNF183 (Fig. S1), suggesting that RNF183 interacts with NKCC1 as well as with Na,K-ATPase. The involvement of polyubiquitination in proteasomal and lysosomal degradation pathways is well known, and we previously showed that RNF183 promotes the lysosomal degradation of Na,K-ATPase¹⁷. Therefore, to demonstrate that RNF183-mediated NKCC1 ubiquitination is involved in NKCC1 degradation, we compared the levels of NKCC1 in HEK293 cells inducibly expressing RNF183 with or without doxycycline treatment. NKCC1 levels decreased in cells expressing WT RNF183, regardless of the presence or absence of the proteasomal inhibitor MG132 (Fig. 2A, B). Treatment of these cells with chloroquine, a lysosomal degradation inhibitor, inhibited the RNF183-induced decrease in NKCC1 levels (Fig. 2A, B). The NKCC1 levels were unaffected in cells inducibly expressing the CS mutant of RNF183 deficient in ubiquitination activity (the consensus cysteines of the RING-finger domain have been substituted by serine) (Fig. 2C, D). Furthermore, the NKCC1 levels in mock and CS RNF183-expressing cells did not change at 12 and 24 h after the addition of doxycycline, whereas an induction time-dependent decrease in the levels of NKCC1 was present in WT RNF183-expressing cells (Fig. 2E, F).

Fig. 1. RNF183 interacts with NKCC1 as a substrate.

Cell lysates were prepared from HEK293 cells expressing 3 × FLAG-tagged RNF183 (with or without doxycycline induction). Immunoprecipitation was performed with anti-NKCC1 or anti-EGFR antibodies or normal rabbit IgG. Co-precipitated proteins were analyzed via western blotting using anti-FLAG, anti-NKCC1, and anti-EGFR antibodies. The asterisk indicates the signal of the light chain of antibodies.

Fig. 2. RNF183 promotes the lysosomal degradation of NKCC1.

A Western blotting analysis of the NKCC1 levels in HEK293 cells inducibly expressing 3 × FLAG-tagged RNF183. Cells were treated with doxycycline to induce 3 × FLAG-tagged RNF183 expression and then treated with chloroquine (CQ) and MG132 to inhibit protein degradation. B Quantification of relative levels of NKCC1 in (A) (mean ± SD, n = 3; *P < 0.05). C Western blotting analysis of NKCC1 levels in HEK293 cells inducibly expressing 3 × FLAG-tagged wild-type (WT) or mutant RNF183 or in mock cells, which do not express any protein with doxycycline (DOX), with or without CQ. D Quantification of relative protein levels of NKCC1 in (C) (mean ± SD, n = 4; *P < 0.05, ***P < 0.005). E Levels of NKCC1 after the induction of 3 × FLAG-tagged WT or CS RNF183. DOX induction was performed for 12 and 24 h. F Quantification of relative protein levels of NKCC1 in (E) (mean ± SD, n = 5; ***P < 0.005). (versus mock).

To examine whether RNF183 mediates the ubiquitination of NKCC1, we used TUBE-agarose beads to perform a pulldown assay in HEK293 cells inducibly expressing WT and CS RNF183. TUBE is an artificial protein containing tandem ubiquitin binding-associated domains that bind to and stabilize polyubiquitin chains with high affinity and efficiently purify ubiquitinated proteins from cell lysates²⁴. Ubiquitinated NKCC1 was clearly detected in the TUBE pull-down samples from WT RNF183-expressing cells, whereas only a faint but detectable signal was observed in the samples from cells expressing the RNF183 CS mutant (Fig. 3A). Furthermore, the treatment with the lysosomal degradation inhibitor, bafilomycin, also increased ubiquitination of NKCC1 in HEK293 cells expressing WT RNF183 but not CS RNF183 (Fig. 3A). Thus, RNF183 may promote the lysosomal degradation, rather than proteasomal degradation, of NKCC1 via ubiquitination.

Fig. 3. NKCC1 is ubiquitinated by RNF183.

A TUBE pulldown assay. Cell lysates were prepared from HEK293 cells inducibly expressing 3 × FLAG-tagged wild-type RNF183 (WT) or mutant RNF183 (CS) with doxycycline and bafilomycin. Mock cells with doxycycline were used as controls. A pulldown assay was performed with TUBE agarose. Pulldown proteins were analyzed via western blotting with anti-FLAG and anti-NKCC1 antibodies. B HEK293 cells were transfected with 3 × FLAG-tagged RNF183 and K63- or K48-linked HA-tagged ubiquitins and treated with bafilomycin for 3 h before harvest. Cell lysates were immunoprecipitated with anti-NKCC1 antibody.

TUBE exhibits a higher affinity for the K63-linked polyubiquitin chain than the K48-linked polyubiquitin chain. To further determine whether RNF183 promotes the formation of K63- or K48-linked chains, we used two types of mutant ubiquitin that can only form K63- or K48-linked chains. RNF183 formed both K63-type and K48-linked ubiquitin chains, but K63-type chains were formed more prominently (Fig. 3B). Moreover, even under proteasome-inhibited conditions (MG132 treatment), we did not observe an increase in the K48-linked ubiquitination of NKCC1, whereas robust K63-linked ubiquitination was consistently detected upon RNF183 overexpression (Fig. S2). These results suggest that RNF183 predominantly promotes K63-linked, rather than K48-linked, ubiquitin chain formation on NKCC1, thereby promoting its lysosomal targeting and degradation.

RNF183-induced NKCC1 protein degradation is enhanced under hypertonic conditions

We then investigated the effect of RNF183 expression on the subcellular localization of NKCC1. NKCC1 was predominantly localized to the plasma membrane without RNF183. On induced expression of WT RNF183, NKCC1 accumulated in puncta in the cytoplasm and colocalized with RNF183 (Fig. 4A). Concurrently, RNF183 colocalized with a lysosomal marker LAMP1 (Fig. 4B). NKCC1 signals also showed a clear overlap with LAMP1 signals, indicating lysosomal localization of NKCC1 (Fig. 4B). These results suggest that RNF183 promotes the lysosomal targeting of NKCC1. This is consistent with inhibition of RNF183-induced NKCC1 degradation by chloroquine. Conversely, when CS RNF183 expression was induced, NKCC1 remained localized at the plasma membrane. Moreover, since endogenous RNF183 expression is induced by NFAT5 in response to hypertonic stress, we examined the subcellular localization of NKCC1 under hypertonic conditions. NKCC1 was localized to the plasma membrane in mock and CS RNF183-expressing cells under hypertonic conditions as under isotonic conditions. In WT RNF183-expressing cells, NKCC1 puncta accumulation in the cytosol was enhanced under hypertonic conditions (Fig. 5A, B). Immunoprecipitation also showed that the interaction between RNF183 and NKCC1 was enhanced under hyperosmotic conditions (Fig. 5C, D). In addition, we investigated the effect of hypertonicity on NKCC1 ubiquitination and found that NKCC1 ubiquitination increased under hypertonic conditions (Fig. 5E). We therefore verified the RNF183-induced degradation of NKCC1 under hypertonic conditions. NKCC1 levels did not differ in mock and CS RNF183-expressing cells (Fig. S3A, B). However, in WT RNF183-expressing cells, NKCC1 levels were reduced to 62% after 24 h of RNF183 induction under isotonic conditions and to 33% under hypertonic conditions (Fig. 5F, G). Thus, RNF183-induced NKCC1 degradation may be enhanced under hypertonic conditions.

Fig. 4. RNF183 induces changes in localization of NKCC1 from the plasma membrane to lysosomes.

A The localization of endogenous NKCC1 (red) in HEK293 cells were observed via confocal microscopy with or without the induction of 3 × FLAG-tagged WT or CS RNF183 (green). Scale bar: 20 µm. B In the upper panels, 3 × FLAG-tagged WT RNF183 (green) was co-stained with the lysosomal marker LAMP1 (red). In the lower panels, NKCC1 (green) was co-stained with LAMP1 (red). Scale bar: 20 µm.

Fig. 5. The ubiquitination and degradation of NKCC1 by RNF183 is promoted under hypertonic conditions.

A The localization of endogenous NKCC1 (green) in HEK293 cells inducibly expressing 3×FLAG-tagged WT or CS RNF183 and mock cells was observed via confocal microscopy. The upper panel is under isotonic and the lower panel is under hypertonic conditions. Scale bar: 20 µm. B Quantification of cytosolic NKCC1 puncta shown in (A). Data are presented as mean ± SD (n = 10 cells per group; ****P < 0.0001). C Cell lysates were prepared from HEK293 cells inducibly expressing 3 × FLAG-tagged RNF183 under isotonic or hypertonic conditions. Immunoprecipitation was performed with anti-NKCC1 antibody. Co-precipitated proteins were analyzed via western blotting using anti-FLAG. D Quantification of relative protein levels of RNF183 in (C) (mean ± SD, n = 3; *P < 0.05, **P < 0.01, ***P < 0.005) E K63- and K48-linked ubiquitination of NKCC1. HEK293 cells were transfected with FLAG-tagged RNF183 and HA-tagged ubiquitin K48 or K63 mutants and treated with bafilomycin for 3 h isotonic or hypertonic conditions. Cell lysates were immunoprecipitated with anti-NKCC1 antibody. F Western blotting analysis of NKCC1 levels in HEK293 cells inducibly expressing 3 × FLAG-tagged WT RNF183 with DOX under isotonic and hypertonic conditions. G Quantification of relative protein levels of the NKCC1 in (F) (mean ± SD, n = 4; **P < 0.01). (versus isotonic).

The relationship between RNF183 and apoptotic cell death in different cell types

We previously reported that Rnf183 is involved in cell protection against hypertonicity-induced apoptosis¹⁶. mIMCD-3 cells are derived from mouse renal collecting duct under hyperosmotic conditions and are the only cultured cell line that expresses endogenous Rnf183. Therefore, we determined the protective effect of Rnf183 expression on hypertonic stress-induced cell death using WT and Rnf183-knockout mIMCD-3 cells. The LDH assay confirmed that hypertonicity-induced cell death was increased in Rnf183-knockout cells as compared with WT cells (Fig. 6A). Hypertonic stress-induced apoptosis was determined via immunostaining with anti-cleaved caspase-3 antibody. Immunoreactivity of cleaved caspase-3 was observed in Rnf183-knockout mIMCD-3 cells under hypertonic and isotonic conditions (Fig. 6B, C). Additionally, although RNF183 is specifically expressed in vivo in renal collecting ducts, increased expression of RNF183 has been reported in the colon of patients with IBD and patients with colorectal cancer. Consequently, we performed similar experiments using Caco-2 cells derived from the colon. Since Caco-2 cells do not express RNF183, we established inducible expression of 3 × FLAG-RNF183 in Caco-2 cells. NKCC1 localization in these cells changed from the plasma membrane to cytosolic puncta with RNF183-induced expression, and these puncta were increased by chloroquine treatment (Fig. 7A, B). The level of NKCC1 was decreased with RNF183 induction, and this decrease was inhibited by chloroquine treatment (Fig. S4A, B). Furthermore, hypertonic stress-induced cell death increased in Caco-2 cells expressing RNF183 (Fig. 7C), and increased amounts of cleaved caspase-3 were observed in Caco-2 cells, inducing RNF183 expression under hypertonic conditions (Fig. 7D, E). These results suggest that RNF183 acts cytoprotectively in cells that normally express RNF183, whereas abnormal expression of RNF183 induces apoptosis in cells that do not normally express it.

Fig. 6. Rnf183 protects cells from apoptosis in mIMCD-3 cells.

A WT and Rnf183-knockout mIMCD-3 cells treated with 150 mM NaCl were analyzed via LDH assays (mean ± SD, n = 3; *P < 0.05). B Immunostaining analysis of cleaved caspase-3 (red) of WT and Rnf183-knockout mIMCD-3 cells under isotonic and hypertonic (75 mM NaCl) conditions. Overlays represent merged images with DNA (blue). Scale bar: 20 µm. C Quantification of cleaved caspase-3 clusters relative to total cell number in the cells (mean ± SD, n = 3; **P < 0.01, ***P < 0.005). Total cell numbers for each condition from three independent experiments: n = 20,516, WT, isotonic; n = 18,470, WT, hypertonic; n = 11,664, KO, isotonic; n = 11,715, KO, hypertonic.

Fig. 7. RNF183 contributes to apoptosis induction in Caco-2 cells.

A The localization of endogenous NKCC1 (red) in Caco-2 cells was observed via confocal microscopy with or without the induction of 3×FLAG-tagged RNF183 (green). The bottom panel is cells treated with DOX and CQ. Scale bar: 10 µm. B Quantification of cytosolic NKCC1 puncta in (A). (mean ± SD, n = 10 cells per group; **P < 0.01), C Caco-2 cells inducibly expressing 3×FLAG-tagged RNF183 treated with 150 mM NaCl were analyzed via LDH assays (mean ± SD, n = 5; *P < 0.05). D Immunostaining analysis of cleaved caspase-3 (red) of Caco-2 cells inducibly expressing 3 × FLAG-tagged RNF183 with and without doxycycline treatment and under isotonic and hypertonic (75 mM NaCl) conditions. Overlays represent merged images with DNA (blue). Scale bar: 10 µm. E Quantification of cleaved caspase-3 clusters relative to total cell number in the cells (mean ± SD, n = 3; *P < 0.05). Total cell numbers for each condition from three independent experiments: n = 39,265, without doxycycline, isotonic; n = 28,442, without doxycycline, hypertonic; n = 31,216, with doxycycline, isotonic; n = 22784, with doxycycline, hypertonic.

The effect of RNF183 on the homeostasis of intracellular Na⁺ levels

This study therefore identified NKCC1, as an additional ion transporter to the previously identified Na,K-ATPase, as a substrate protein for RNF183. Consequently, we measured intracellular Na⁺ levels to investigate the effects of RNF183 expression on Na⁺ homeostasis. In WT mIMCD-3 cells, intracellular Na⁺ levels did not change under hypertonic conditions, indicating that cells were adapted to hypertonic conditions. However, Na⁺ homeostasis was disrupted in Rnf183-knockout mIMCD-3 cells under isotonic conditions compared with that in WT mIMCD-3 cells, with greater disruption present in Rnf183-knockout mIMCD-3 cells under hypertonic conditions (Fig. 8A). In Caco-2 cells not expressing RNF183, the Na⁺ homeostasis was retained regardless of whether conditions were isotonic or hypertonic, whereas in cells overexpressing RNF183 the Na⁺ homeostasis was disrupted even under isotonic conditions with greater disruption under hypertonic conditions (Fig. 8B), suggesting that abnormal expression of RNF183 in cells that do not originally express RNF183 induces disruption of cellular Na⁺ homeostasis.

Fig. 8. The relationship between RNF183 expression and intracellular Na⁺ homeostasis.

Intracellular Na⁺ levels were measured in A mIMCD-3 cells in WT and Rnf183-knockout or B Caco-2 cells inducibly expressing 3 × FLAG-tagged RNF183. (mean ± SD, (A) n = 6, (B) n = 8; **P < 0.01, ***P < 0.005).

Discussion

Herein, we identified NKCC1 as a substrate for RNF183 by validating its binding, ubiquitination, and protein levels. Ubiquitination of NKCC1 was verified using TUBE and K63-specific ubiquitin.

In this study, we showed that RNF183 mediated the formation of K63-linked polyubiquitin chains on NKCC1 (Fig. 3A, B). In our previous study, we demonstrated that DR5 is modified by RNF183 with K63-linked polyubiquitin chains²⁰. Taken together, these findings suggest that RNF183 predominantly functions as an E3 ligase that generates K63-linked polyubiquitin chains rather than K48-linked chains. Furthermore, RNF183-mediated K63-linked ubiquitination may contribute to the endocytosis or lysosomal sorting of substrate proteins, as K63-linked ubiquitin chains have been reported to promote endocytosis and lysosomal targeting.

Previously, we identified Na,K-ATPase as a substrate for RNF183¹⁷. Both NKCC1 and Na,K-ATPase are known to be involved in regulating cell volume^23,25. When cells are exposed to hypertonic stress, expression of ion transporters, including NKCC1 and Na,K-ATPase is increased^26,27. NKCC1 promotes the influx of Na⁺, K⁺, and Cl⁻, leading to osmotic water entry and restoration of cell volume²⁸. In parallel, Na,K-ATPase maintains low intracellular Na⁺ and high intracellular K⁺ concentrations, although it does not directly mediate net ion influx²⁹. This electrochemical gradient is essential for sustained NKCC1 activity, thereby indirectly supporting ion accumulation and cell volume recovery under hypertonic conditions. However, during prolonged hypertonic stress, these ionic osmolytes are changed to uncharged small organic osmolytes to avoid prolonging high ionic strength in the cell³⁰. In the adaptation to long-term hypertonic conditions, the transcription factor NFAT5 induces the expression of the sodium-coupled taurine transporter (TauT/SLC6A12)³¹, the myo-inositol transporter (SMIT/SLC5A3)³², the sodium-coupled betaine transporter (BGT1/SLC6A13)^33,34 and aldose reductase (AR)³⁵ to accumulate compatible organic osmolytes such as taurine, myo-inositol, betaine, or sorbitol, respectively. The expression of RNF183 is also induced by NFAT5 under hypertonic conditions. This suggested that RNF183 is involved in regulating the expression level of NKCC1 and Na,K-ATPase in the renal collecting ducts, where RNF183 is specifically expressed and is constantly in a hypertonic environment. A time-course analysis of endogenous RNF183 and NKCC1 expression under acute versus chronic hypertonic stress would be valuable to test whether NFAT5-mediated RNF183 upregulation contributes to chronic adaptation via the degradation of NKCC1, potentially facilitating the transition from ionic to organic osmolyte regulation; we plan to address this in future studies. RNF183 may also recognize ion transporters other than NKCC1 and Na,K-ATPase as substrates.

Moreover, we found that hypertonic conditions enhanced the degradation of NKCC1 by RNF183 (Fig. 5F, G). The enhancement of E3–substrate interactions through the phosphorylation of the substrate has been reported in the ubiquitination-dependent degradation of several membrane proteins, such as EGFR by Cbl and E-cadherin by Hakai, where phosphorylation creates or stabilizes binding interfaces for E3 ligases^36,37. Under hypertonic conditions, intracellular Cl⁻ depletion activates WNK kinases, leading to the phosphorylation of NKCC1 via the SPAK/OSR1 pathway³⁸. Such phosphorylation can induce conformational changes or generate recognition motifs that enhance interactions between NKCC1 and RNF183. The phosphorylation of NKCC1 under hypertonic conditions may strengthen its binding to RNF183. To address this, we analyzed NKCC1 phosphorylation under our experimental conditions using a commercially available antibody that recognizes Thr212/Thr217-phosphorylated NKCC1. Using this antibody, we detected the basal phosphorylation of NKCC1 under isotonic conditions (Fig. S5). However, we did not observe a further increase in phosphorylation upon NaCl-induced hypertonic stress. Multiple phosphorylation sites, including Thr212 and Thr217, are involved in the regulation of NKCC1 transport activity³⁹. However, phosphorylation sites specifically associated with NKCC1 endocytosis or lysosomal targeting have not yet been identified. Therefore, it is possible that hypertonicity induces phosphorylation at other, currently uncharacterized sites, which could enhance the interaction between NKCC1 and RNF183.

This study demonstrated that RNF183 binds to NKCC1 and induces its degradation via K63-linked ubiquitination. Although our findings are based on in vitro mechanistic analyses, they suggest possible physiological implications for epithelial tissues of the kidney and intestine. For example, NKCC1 deficiency in colorectal cancer cells has been reported to impair epithelial barrier function by increasing the expression of claudin-2, a pore-forming tight junction protein⁴⁰. This raises the possibility that increased RNF183 expression contributes to intestinal barrier dysfunction via enhanced degradation of NKCC1, which may be relevant to the pathophysiology of IBD.

The renal medulla is chronically exposed to a highly hypertonic environment (approximately 600–1200 mOsm), wherein epithelial cells must finely balance acute volume recovery and long-term adaptation. Acute hypertonic stress activates NKCC1 via the WNK–SPAK/OSR1 pathway in response to intracellular Cl⁻ depletion, promoting rapid cell volume recovery³⁸. Based on our in vitro findings, we propose that RNF183 may participate in a subsequent adaptive phase by promoting K63-linked ubiquitination and lysosomal targeting of NKCC1, thereby potentially preventing prolonged intracellular ionic overload during sustained hypertonic stress.

In the kidneys, dehydration increases the osmolality of the interstitium surrounding the collecting duct through vasopressin-mediated antidiuretic responses⁴¹. NKCC1 expression has been reported to be upregulated in the outer medulla of rats under hypertonic conditions and following vasopressin treatment²⁷. In this context, RNF183 induction under hypertonic stress may protect collecting duct cells by preventing a prolonged increase in intracellular ionic strength through the lysosomal degradation of NKCC1, representing a potential mechanism for long-term adaptation to sustained hypertonicity.

However, since the present study was limited to in vitro experimental systems, further in vivo studies will be required to validate the physiological relevance of RNF183-mediated NKCC1 regulation.

RNF183 has been reported in relation to IBD^18–22. In this study, the overexpression of RNF183 in Caco-2 cells disrupted intracellular Na⁺ homeostasis, showing the opposite effect seen in mIMCD-3 cells that express endogenous Rnf183. The epithelium of the colon has a barrier function, and a connection between the loss of barrier function due to apoptosis of colonic epithelial cells and IBD has been reported⁴². Several reports suggest that disruption of intracellular Na⁺ homeostasis induces apoptosis^43,44. Additionally, we previously reported that Rnf183 expression is elevated earlier than that of inflammatory markers in the colon of mice treated with dextran sodium sulfate for the generation of mouse models of IBD²⁰. These reports and the present study suggest that the increased expression of RNF183 in the colon due to hypertonicity and other factors could disrupt intracellular Na⁺ homeostasis, inducing apoptosis and contributing to the development of IBD.

Abnormal RNF183 expression in cells and tissues other than the colon that do not normally express RNF183 may also disrupt Na⁺ homeostasis in the cells. Recent reports have documented the association of RNF183 with the progression of bladder cancer⁴⁵, and prognosis of uterine corpus endometrial cancer⁴⁶. In cancer cells, the expression of NFAT5 under hypertonic conditions promotes migration and proliferation with disruption of intracellular Na⁺ homeostasis^47–49. Although RNF183 is induced by the transcription factor NFAT5 under hypertonic conditions in the kidney, whether RNF183 is induced in other tissues by NFAT5 or in other pathways remains unclear. A more detailed study of RNF183 expression in other tissues than the kidney may improve understanding of the relationship between RNF183 and diseases, leading to the elucidation of pathophysiology and the development of therapeutic strategies.

Methods

Cell culture

HEK293 or mIMCD-3 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan; Cat. No. 044-29765) or DMEM/Ham’s F-12 Nutrient Mixture (FUJIFILM Wako Pure Chemical Corporation; Cat. No. 048-29785) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; FUJIFILM Wako Pure Chemical Corporation; Cat. No. 553-41945), and 100 U/mL penicillin and 100 μg/mL streptomycin (FUJIFILM Wako Pure Chemical Corporation; Cat. No. 168-23191). Caco-2 cells were maintained in Eagle’s minimal essential medium (FUJIFILM Wako Pure Chemical Corporation; Cat. No. 051-07615) supplemented with 10% FBS, nonessential amino acids solution (Nacalai Tesque, Kyoto, Japan, Cat. No. 06344-56), and GlutaMAX (Life Technologies, Carlsbad, CA; Cat. No. 35050061). HEK293 cells and Caco-2 cells were obtained from RIKEN BRC, and mIMCD-3 cells was obtained from ATCC. The cell lines were incubated at 37 °C in a 5% CO₂ humidified air atmosphere. HEK293 and Caco-2 cells are listed as commonly misidentified according to the ICLAC database. Both were obtained from RIKEN BRC, which authenticates cell lines using STR profiling prior to distribution. To induce hyperosmotic stress, cells received equal volumes of 75 mM NaCl in media for the indicated time. To inhibit protein degradation, cells were treated with 10 µM MG132 (MedChemExpress, Monmouth Junction, NJ; Cat. No. HY-13259) for 6 h, 100 µM chloroquine (Sigma-Aldrich, St. Louis, MO; Cat. No. C6628) for 6 h, and 100 nM bafilomycin A1 (AdipoGen life sciences, Basel, Switzerland; Cat. No. AG-CN2-2001) for 3 h.

Plasmids and cell lines

Inducible HEK293 cells for expression of 3 × FLAG-tagged (WT) RNF183 or CS mutant RNF183 were constructed by transfection of pcDNA5/FRT/TO-3×FLAG-RNF183 or pcDNA5/FRT/TO-3×FLAG-CS-RNF183 into the Flp-In^TM-293 cells (Thermo Fisher Scientific, Waltham, MA; Cat. No. R75007). pcDNA5/FRT/TO-3×FLAG-RNF183 and pcDNA5/FRT/TO-3×FLAG-CS-RNF183 were constructed according to the manufacturer’s protocol using a pcDNA5/FRT/TO^© TOPO^® TA Expression Kit (Thermo Fisher Scientific; Cat. No. K651020). 3×FLAG-RNF183 and 3×FLAG-CS-RNF183 fragments were amplified from pcDNA6.2/3×FLAG-RNF183 and pcDNA6.2/3×FLAG-CS-RNF183¹⁴.

Inducible Caco-2 cells that expressed 3 × FLAG-tagged WT RNF183 were established using Sleepingbeauty-based transposon vectors from Addgene^50,51. 3 × FLAG-RNF183 was amplified from pcDNA6.2/3×FLAG-RNF183 with the following primer sets: 5ʹ-CTCGAAAGGCCTCTGCACCATGGACTACAAGGACCAC-3ʹ (forward) and 5ʹ-GAAGCTTGGCCTGACTTATCCCACGCCCCACAGGAAC-3ʹ (reverse) and was cloned into pSBTet-Pur (#60507) (digested with SfiI) using the In-Fusion system (TaKaRa Bio, Shiga, Japan). This plasmid and the expression plasmid for a mutant form of transposase, SB100X (pCMV(CAT)T7-SB100: #34879), were transfected into Caco-2 cells at a 4:1 ratio. Integrated cells were selected with puromycin (10 μg/mL).

Nuclease plasmids for the disruption of Rnf183 were constructed as follows. The design of the guide RNAs was performed using the CRISPRdirect (https://crispr.dbcls.jp/) to minimize potential off-target effects. Oligos 5ʹ-CACCGTGGGGCTCTAGGCGGAGCA-3ʹ and 5ʹ-AAACTGCTCCGCCTAGAGCCCCAC-3ʹ were annealed and ligated into the BbsI site of pSpCas9(BB)-2A-Puro (PX459) V2.0, which was a gift from Dr. Feng Zhang (Addgene plasmid #62988). Clonal cell lines were isolated by limiting dilution culture following transfection mIMCD-3 cells.

All transfection procedures were performed using ScreenFect A (FUJIFILM Wako Pure Chemical; Cat. No. 297-73204). 3×FLAG-tagged WT RNF183 or CS mutant RNF183 were induced with 1 μg/mL of doxycycline (TaKaRa Bio; Cat. No. Z1311N).

Antibodies

Antibodies were obtained for β-actin (C4, mouse monoclonal; sc-47778, Santa Cruz Biotechnology, Dallas, TX; WB: 1:5,000); FLAG tag (M2, mouse monoclonal; F1084, Sigma-Aldrich; WB: 1:1,000; IF: 1:100; IP: 1:500); HA tag (rabbit polyclonal; 51064-2-AP, Proteintech Group, inc, Rosemont, IL; WB: 1:1,000); NKCC1 (D13A9, rabbit monoclonal; #8351, Cell Signaling Technology, Danvers, MA; WB: 1:1,000; IF: 1:100; IP: 1:500); p-NKCC1 (Thr212/Thr217) (rabbit polyclonal; ABS1104, Sigma-Aldrich; WB: 1:1,000); EGFR (D38B1, rabbit monoclonal; #4267, Cell Signaling Technology; WB: 1:1,000; IP: 1:500); LAMP1 (D2D11, rabbit monoclonal; #9091, Cell Signaling Technology; IF: 1:100); cleaved caspase-3 (polyclonal; #9661, Cell Signaling Technology; WB: 1:1,000); mouse IgG1 isotype control (G3A1, mouse monoclonal; #5415, Cell Signaling Technology; IP: 1:500); rabbit IgG isotype control (DA1E, rabbit monoclonal; #3900, Cell Signaling Technology; IP: 1:500); goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 568 (A-11011, Thermo Fisher Scientific); and goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 488 (A-11001, Thermo Fisher Scientific).

Immunoprecipitation

HEK293 cells expressing 3 × Flag-tagged RNF183 by Tet-On system were lysed in lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 100 μM MG132 (MedChemExpress, Monmouth Junction, NJ) and protease inhibitor cocktail Set V (EDTA free; FUJIFILM Wako Pure Chemical; Cat. No. 168-26033)] for 15 min on ice. Lysed cells were centrifuged at 15,000 rpm for 10 min, and the supernatants were incubated with anti-NKCC1, anti-EGFR, or anti-FLAG antibodies at 4 °C overnight or for 4 h, followed by incubation with Protein G Sepharose 4 Fast Flow beads (GE Healthcare Bio-Sciences AB, Uppsala, Sweden; Cat. No. 17061801) for 1 h. Subsequently, the beads were rinsed three times with IP wash buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, and 0.1% Triton X-100). Immunoprecipitates were boiled at 50 °C for 30 min with Laemmli sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer and analyzed via western blotting.

Tandem ubiquitin-binding entities (TUBEs) pulldown assay

HEK293 cells expressing 3 × FLAG-tagged RNF183 in the Tet-On system were lysed in lysis buffer containing 4 µM bortezomib (FUJIFILM Wako Pure Chemical Corporation; Cat. No. 027-18903), 1 mM N-ethylmaleimide (FUJIFILM Wako Pure Chemical Corporation; Cat. No. 058-02061), 1 µM PR619 (Cayman Chemical Company, Ann Arbor, MI; Cat. No. 16276), and 5 mM EDTA on ice for 15 min, and then quenched with 0.1% cysteine. Supernatants were incubated with 20 µL of TUBE1-agarose resin (Life Sensors, Malvern, PA; Cat. No. UM-0401-1000). Then, sample preparation as described for immunoprecipitation was performed.

Ubiquitination assay

HEK293 cells were transfected with 3 × FLAG-tagged RNF183 and K63- or K48-linked HA-ubiquitins, which can only form K63 or K48 polyubiquitin chains with other lysines mutated to arginines. Cells were incubated for 48 h and treated with 100 nM bafilomycin for 3 h before harvest. Sample preparation was then performed as described for immunoprecipitation.

Western blotting

Western blotting analysis was performed as described previously¹⁶. Secondary antibodies were goat anti-mouse IgG (H + L) horseradish peroxidase (HRP) conjugated antibody (Cat. No. 1031-05, Southern Biotech, Birmingham, AL; 1:5,000) and goat anti-rabbit IgG (H + L) HRP conjugated antibody (Cat. No. 4050-05, Southern Biotech; 1:5,000). Images were obtained using a ChemiDoc Touch (Bio-Rad Laboratories, Inc., Hercules, CA).

Immunostaining and image analysis

Immunostaining was performed as described previously^16,17. Briefly, HEK293 and Caco-2 cells were grown on collagen-coated coverslips (Matsunami Glass, Osaka, Japan) while mIMCD-3 cells were grown on noncollagen-coated coverslips. Cells were fixed with 4% paraformaldehyde at room temperature for 15 min. Fixed cells were blocked with PBS(-) containing 5% normal goat serum and 0.3% Triton X-100 at room temperature for 1 h and incubated with primary antibody in 1% BSA/0.3% Triton X-100/PBS(-) at 4 °C overnight. Subsequently, cells were incubated with Alexa Fluor-conjugated secondary antibodies (1:2,000) at room temperature for 1 h. Nuclei were stained with DAPI (Dojind, Kumamoto, Japan; 300 nM). ProLong Diamond Antifade Mountant (Thermo Fisher Scientific; Cat. No. P36970) was used to mount slips on glass slides, and samples were observed under a Zeiss LSM710 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) or all-in-one fluorescence microscopy BZ-X710 (Keyence, Osaka, Japan). Apoptotic cells were counted in nine different fields of view in three different experiments, and the ratio of the number of cleaved caspase 3-positive cells to that of DAPI-stained cells was calculated. Cell counting was performed using an Analysis Application Hybrid Cell Count BZ-H3C (Keyence).

Cell cytotoxicity assay

Cell cytotoxicity assay was performed using a Cytotoxicity LDH Assay Kit-WST (Dojindo; Cat. No. CK12). Briefly, cells were exposed to either 150 mM NaCl or doxycycline or both of these for 18 h. Cell culture supernatants were then incubated with LDH assay buffer for 30 min, and the absorbance measured at 492 nm using a multiplate reader (Awareness Technology, Westport, CT).

Measurement of intracellular Na⁺

The level of intracellular Na⁺ was measured using the fluorescent probe sodium-binding benzofuran isophthalate (SBFI) (Abcam, Cambridge, UK; Cat. No. ab142800) as previously described with slight variations⁵². mIMCD-3 cells were treated with 10 μM SBFI (from a 5 mM stock solution in DMSO) with 0.1% wt/vol Pluronic F-127 (Thermo Fisher Scientific; Cat. No. P6866) while Caco-2 cells were treated 5 μM SBFI with 0.05% wt/vol Pluronic F-127 for 3 h at 37 °C. Measurements were made by exciting SBFI at 340 nm, where fluorescence is particularly sensitive to the ion concentration of interest, and at 380 nm, which is near the isosbestic point. SBFI was excited at these wavelengths (340/380 nm) and the fluorescence was monitored at 500 nm using a multimode microplate reader, Cytation3 (BioTek Instruments, Rancho Cordova, CA) to determine the ratio of the fluorescence intensity obtained.

Image analysis

All images were analyzed offline using ImageJ software (Fiji). Fluorescence signals were quantified using Excel (Microsoft) and JMP (JMP Statistical Discovery LLC). All data are presented as mean ± SD.

To analyze cytosolic NKCC1 recruitment, fluorescence images of NKCC1 were subjected to threshold-based segmentation to identify punctate structures in the cytoplasm. A consistent threshold was applied to all images within each experiment to segment the cytosolic NKCC1 puncta. The total area of segmented cytosolic NKCC1 puncta was measured for each cell.

Statistics and reproducibility

The exact sample size (n) for each experimental group is indicated in the corresponding figure legends. Measurements were taken from distinct biological replicates unless otherwise stated. All data are presented as mean ± standard deviation (SD). Statistical comparisons were performed using two-sided t-tests or one-way ANOVA followed by two-tailed Tukey’s post hoc test. Statistical significance was set at P < 0.05. All statistical analyses were performed using statistical software, JMP Student Edition 19 (ver. 19.0.1, SAS Institute Inc., Cary, N.C., USA).

Materials availability

The cell lines generated in this study are available from the corresponding author upon reasonable request. No restrictions apply to their use and distribution for research purposes.

Author contributions

T.O., Y.H., M.T., and S.S. developed the tools for experiments and performed experiments. M.K., K.M., and K.I. supervised the project. T.O., Y.H., M.T., K.M., and M.K. designed the experiments, analyzed data, and wrote the manuscript. A.S., I.O., Y.K., and K.I. provided input into conceptualizing the project and writing the manuscript. All authors reviewed the manuscript.

Peer review

Peer review information

Communications Biology thanks Kamel Laghmani and the other anonymous reviewer(s) for their contribution to the peer review of this work. Primary handling editor: Dario Ummarino. A peer review file is available.

Data availability

Source data are provided with this paper as Supplementary Data file. Uncropped full-length western blot images corresponding to all western blotting results presented in this paper are provided in the Supplementary Information file. Additional data are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-026-09766-w.