The NHERF1 PDZ1 domain and IRBIT interact and mediate the activation of Na+/H+ exchanger 3 by ANG II.

Na(+)/H(+) exchanger (NHE)3, a major Na(+) transporter in the luminal membrane of the proximal tubule, is subject to ANG II regulation in renal Na(+)/fluid absorption and blood pressure control. We have previously shown that inositol 1,4,5-trisphosphate receptor-binding protein released with inositol 1,4,5-trisphosphate (IRBIT) mediates ANG II-induced exocytosis of NHE3 in cultured proximal tubule epithelial cells. In searching for scaffold protein(s) that coordinates with IRBIT in NHE3 trafficking, we found that NHE regulatory factor (NHERF)1, NHE3, and IRBIT proteins were coexpressed in the same macrocomplexes and that loss of ANG II type 1 receptors decreased their expression in the renal brush-border membrane. We found that NHERF1 was required for ANG II-mediated forward trafficking and activation of NHE3 in cultured cells. ANG II induced a concomitant increase of NHERF1 interactions with NHE3 and IRBIT, which were abolished when the NHERF1 PDZ1 domain was removed. Overexpression of a yellow fluorescent protein-NHERF1 construct that lacks PDZ1, but not PDZ2, failed to exaggerate the ANG II-dependent increase of NHE3 expression in the apical membrane. Moreover, exogenous expression of PDZ1 exerted a dominant negative effect on NHE3 activation by ANG II. We further demonstrated that IRBIT was indispensable for the ANG II-provoked increase in NHERF1-NHE3 interactions and that phosphorylation of IRBIT at Ser(68) was necessary for the assembly of the NHEF1-IRBIT-NHE3 complex. Taken together, our findings suggest that NHERF1 mediates ANG II-induced activation of renal NHE3, which requires coordination between IRBIT and the NHERF1 PDZ1 domain in binding and transporting NHE3.

HIF1α-Induced by Lysophosphatidic Acid Is Stabilized via Interaction with MIF and CSN5.

Macrophage migration inhibitory factor (MIF) is a cytokine that has broad effects on immune system and inflammatory response. A growing body of evidence implicates the role of MIF in tumor growth and metastasis. Lysophosphatidic acid (LPA), a bioactive lipid mediator, regulates colon cancer cell proliferation, invasion, and survival through LPA2 receptor. Loss of LPA2 results in decreased expression of MIF in a rodent model of colon cancer, but the mechanism of MIF regulation by LPA is yet to be determined. In this study, we show that LPA transcriptionally regulates MIF expression in colon cancer cells. MIF knockdown decreased LPA-mediated proliferation of HCT116 human adenocarcinoma cells without altering the basal proliferation rates. Conversely, extracellular recombinant MIF stimulated cell proliferation, suggesting that the effect of MIF may in part be mediated through activation of surface receptor. We have shown recently that LPA increases hypoxia-inducible factor 1α (HIF1α) expression. We found that MIF regulation by LPA was ablated by knockdown of HIF1α, indicating that MIF is a transcriptional target of HIF1α. Conversely, knockdown of MIF ablated an increase in HIF1α expression in LPA-treated cells, suggesting a reciprocal relationship between HIF1α and MIF. LPA stimulated co-immunoprecipitation of HIF1α and MIF, indicating that their association is necessary for stabilization of HIF1α. It has been shown previously that CSN9 signalosome subunit 5 (CSN5) interacts with HIF1α to stabilize HIF1α under aerobic conditions. We found that LPA did not alter expression of CSN5, but stimulated its interaction with HIF1α and MIF. Depletion of CSN5 mitigated the association between HIF1α and MIF, indicating that CSN5 acts as a physical link. We suggest that HIF1α, MIF, and CSN5 form a ternary complex whose formation is necessary to prevent degradation of HIF1α under aerobic conditions.

Regulation of NHE3 by lysophosphatidic acid is mediated by phosphorylation of NHE3 by RSK2.

Na(+)/H(+) exchange by Na(+)/H(+) exchanger 3 (NHE3) is a major route of sodium absorption in the intestine and kidney. We have shown previously that lysophosphatidic acid (LPA), a small phospholipid produced ubiquitously by all types of cells, stimulates NHE3 via LPA5 receptor. Stimulation of NHE3 activity by LPA involves LPA5 transactivating EGF receptor (EGFR) in the apical membrane. EGFR activates proline-rich tyrosine kinase 2 (Pyk2) and ERK, both of which are necessary for NHE3 regulation. However, Pyk2 and ERK are regulated by EGFR via independent pathways and appear to converge on an unidentified intermediate that ultimately targets NHE3. The p90 ribosomal S6 kinase (RSK) family of Ser/Thr protein kinases is a known effector of EGFR and ERK. Hence, we hypothesized that RSK may be the convergent effector of Pyk2 and ERK although it is not known whether Pyk2 regulates RSK. In this study, we show that Pyk2 is necessary for the maintenance of phosphoinositide-dependent kinase 1 (PDK1) autophosphorylation, and knockdown of Pyk2 or PDK1 mitigated LPA-induced phosphorylation of RSK and stimulation of NHE3 activity. Additionally, we show that RSK2, but not RSK1, is responsible for NHE3 regulation. RSK2 interacts with NHE3 at the apical membrane domain, where it phosphorylates NHE3. Alteration of S663 of NHE3 ablated LPA-induced phosphorylation of NHE3 and stimulation of the transport activity. Our study identifies RSK2 as a new kinase that regulates NHE3 activity by direct phosphorylation.

Krüppel-like factor 5 incorporates into the ß-catenin/TCF complex in response to LPA in colon cancer cells.

Lysophosphatidic acid (LPA) is a simple phospholipid with potent mitogenic effects on various cells including colon cancer cells. LPA stimulates proliferation of colon cancer cells by activation of ß-catenin or Krüppel-like factor 5 (KLF5), but the functional relationship between these two transcription factors is not clear. Hence, we sought to investigate the mechanism of ß-catenin activation by LPA and the role of KLF5 in the regulation of ß-catenin by LPA. We found that LPA and Wnt3 additively activated the ß-catenin/TCF (T cell factor) reporter activity in HCT116 cells. In addition to phosphorylating glycogen synthase kinase 3ß (GSK-3ß) at Ser9, LPA resulted in phosphorylation of ß-catenin at Ser552 and Ser675. Mutation of Ser552 and Ser675 ablated LPA-induced ß-catenin/TCF transcriptional activity. Knockdown of KLF5 significantly attenuated activation of ß-catenin/TCF reporter activity by LPA but not by Wnt3. However, nuclear accumulation of ß-catenin by LPA was not altered by knockdown of KLF5. ß-catenin, TCF, and KLF5 were present in a 250-300kDa macro-complex, and their presence was enhanced by LPA. LPA simulated the interaction of ß-catenin with TCF4, and depletion of KLF5 decreased ß-catenin-TCF4 association and the transcriptional activity. In summary, LPA activates ß-catenin by multiple pathways involving phosphorylation of GSK-3 and ß-catenin, and enhancing ß-catenin interaction with TCF4. KLF5 plays a critical role in ß-catenin activation by increasing the ß-catenin-TCF4 interaction.

Unique regulation of human Na+/H+ exchanger 3 (NHE3) by Nedd4-2 ligase that differs from non-primate NHE3s.

Na(+)/H(+) exchanger NHE3 expressed in the intestine and kidney plays a major role in NaCl and HCO3 (-) absorption that is closely linked to fluid absorption and blood pressure regulation. The Nedd4 family of E3 ubiquitin ligases interacts with a number of transporters and channels via PY motifs. A comparison of NHE3 sequences revealed the presence of PY motifs in NHE3s from human and several non-human primates but not in non-primate NHE3s. In this study we evaluated the differences between human and non-primate NHE3s in ubiquitination and interaction with Nedd4-2. We found that Nedd4-2 ubiquitinated human NHE3 (hNHE3) and altered its expression and activity. Surprisingly, rat NHE3 co-immunoprecipitated Nedd4-2, but its expression and activity were not altered by silencing of Nedd4-2. Ubiquitination by Nedd4-2 rendered hNHE3 to undergo internalization at a significantly greater rate than non-primate NHE3s without altering protein stability. Insertion of a PY motif in rabbit NHE3 recapitulated the interaction with Nedd4-2 and enhanced internalization. Thus, we propose a new model where disruption of Nedd4-2 interaction elevates hNHE3 expression and activity.

Regulation of hypoxia-inducible factor 1α (HIF-1α) by lysophosphatidic acid is dependent on interplay between p53 and Krüppel-like factor 5.

Hypoxia-inducible factor 1α (HIF-1α) and p53 are pivotal regulators of tumor growth. Lysophosphatidic acid (LPA) is a lipid mediator that functions as a mitogen by acting through LPA receptors. We have shown previously that LPA stimulates HIF-1α expression in colon cancer cells. To determine the mechanism of HIF-1α induction by LPA, we compared the effect of LPA on HIF-1α in several colon cancer cell lines. LPA transcriptionally induced HIF-1α in colon cancer cells. HIF-1α induction was observed in cells expressing WT p53, where LPA decreased p53 expression. However, LPA failed to induce HIF-1α when the p53 gene was mutated. A decrease in p53 expression was dependent on induction of p53-specific E3 ubiquitin ligase Mdm2 by LPA. Krüppel-like factor 5 (KLF5) is an effector of LPA-induced proliferation of colon cancer cells. Because HIF-1α was necessary for LPA-induced growth of colon cancer cells, we determined the relationship between KLF5 and HIF-1α by a loss-of-function approach. Silencing of KLF5 inhibited LPA-induced HIF-1α induction, suggesting that KLF5 is an upstream regulator of HIF-1α. KLF5 and p53 binding to the Hif1α promoter was assessed by ChIP assay. LPA increased the occupancy of the Hif1α promoter by KLF5, while decreasing p53 binding. Transfection of HCT116 cells with KLF5 or p53 attenuated the binding of the other transcription factor. These results identify KLF5 as a transactivator of HIF-1α and show that LPA regulates HIF-1α by dynamically modulating its interaction with KLF5 and p53.

Human intestinal epithelial cell line SK-CO15 is a new model system to study Na(+)/H(+) exchanger 3.

The Caco-2 cell line represents absorptive polarized intestinal epithelial cells that express multiple forms of Na(+)/H(+) exchanger (NHE) in their plasma membranes. Caco-2 cells express the major apical NHE isoform NHE3, but low NHE3 expression together with inefficient transfection often hamper intended studies. In this study, we examined whether SK-CO15 cells could be used to study NHE3 regulation. SK-CO15 cells grown on Transwell inserts developed polarized epithelial cells with microvilli. The transfection efficiency of SK-CO15 cells was markedly higher compared with Caco-2 cells, an advantage in gene transfer and knockout. SK-CO15 cells expressed NHE1, NHE2, and NHE3. NHE3 expression was significantly greater in these cells than Caco-2, and NHE3 comprised more than half of total NHE activity. Apical expression of NHE3 in SK-CO15 cells was confirmed by confocal immunofluorescence and surface biotinylation. NHE regulatory factors NHERF1 and NHERF2, which are important for regulation of NHE3 activity, were expressed in these cells. Stimulatory response of NHE3 in SK-CO15 cells was assessed by dexamethasone and lysophosphatidic acid (LPA). Treatment with dexamethasone for 24-48 h increased NHE3 expression and activity. Similarly to Caco-2 cells, SK-CO15 cells lacked the expression of the LPA receptor LPA(5,) but exogenous expression of LPA(5) resulted in acute stimulation of NHE3. Forskolin acutely inhibited NHE3 activity in SK-CO15 cells, further attesting the validity of these cells. We conclude that SK-CO15 cells with the amenity for transfection and high endogenous NHE3 expression are a new and better cell model for NHE3 regulatory investigation than widely used Caco-2 cells.

Transient concentration of a gamma-tubulin-related protein with a pericentrin-related protein in the formation of basal bodies and flagella during the differentiation of Naegleria gruberi.

The distribution of two proteins in Naegleria gruberi, N-gammaTRP (Naegleria gamma-tubulin-related protein) and N-PRP (Naegleria pericentrin-related protein), was examined during the de novo formation of basal bodies and flagella that occurs during the differentiation of N. gruberi. After the initiation of differentiation, N-gammaTRP and N-PRP began to concentrate at the same site within cells. The percentage of cells with a concentrated region of N-gammaTRP and N-PRP was maximal (68%) at 40 min when the synthesis of tubulin had just started but no assembled microtubules were visible. When concentrated tubulin became visible (60 min), the region of concentrated N-gammaTRP and N-PRP was co-localized with the tubulin spot and then flagella began to elongate from the region of concentrated tubulin. When cells had elongated flagella, the concentrated N-gammaTRP and N-PRP were translocated to the opposite end of the flagellated cells and disappeared. The transient concentration of N-gammaTRP coincided with the transient formation of an F-actin spot at which N-gammaTRP and alpha-tubulin mRNA were co-localized. The concentration of N-gammaTRP and formation of the F-actin spot occurred without the formation of microtubules but were inhibited by cytochalasin D. These observations suggest that the regional concentration of N-gammaTRP and N-PRP is mediated by actin filaments and might provide a site of microtubule nucleation for the assembly of newly synthesized tubulins into basal bodies and flagella.