Renewal of the intestinal epithelium is orchestrated by regenerative epithelial proliferation within crypts. Recent studies have shown that lysophosphatidic acid (LPA) can maintain intestinal epithelial renewal in vitro and conditional deletion of Lpar5 (Lpar5iKO) in mice ablates the intestinal epithelium and increases morbidity. In contrast, constitutive Lpar5 deletion (Lpar5cKO) does not cause a defect in intestinal crypt regeneration. In this study, we investigated whether another LPA receptor (LPAR) compensates for constitutive loss of LPA5 function to allow regeneration of intestinal epithelium. In Lpar5cKO intestinal epithelial cells (IECs), Lpar2 was upregulated and blocking LPA2 function reduced proliferation and increased apoptosis of Lpar5cKO IECs. Similar to Lpar5cKO mice, the absence of Lpar2 (Lpar2−/−) resulted in upregulation of Lpar5 in IECs, indicating that LPA2 and LPA5 reciprocally compensate for the loss of each other. Blocking LPA2 in Lpar5cKO enteroids reduced phosphorylation of Akt, indicating that LPA2 maintains the growth of Lpar5cKO enteroids through activation of the PI3K-Akt pathway. The present study provides evidence that loss of an LPAR can be compensated by another LPAR. This ability to compensate needs to be considered in studies aimed to define receptor functions or test the efficacy of a LPAR-targeting drug using genetically engineered animal models.
Recent studies have shown that accessory proteins that interact with the apical Na+/H+ exchanger NHE3 are a vital part of the dynamic nature of the Na+/H+ exchanger regulation. We have identified MAST205, a microtubule-associated serine/threonine kinase with a molecular mass of 205 kDa that interacts with NHE3. MAST205 contains a S/T kinase domain and a PDZ domain that mediates interaction with NHE3. Northern blot analysis showed that MAST205 is highly expressed in human and rat kidney. Expression in opossum kidney (OK) cells showed that MAST205 is predominantly expressed in the apical membrane of the cells. Immunohistochemical studies demonstrated the presence of MAST205 at the apical region of the renal proximal tubules. Heterologous expression of MAST205 in OK cells inhibited endogenous NHE3 activity, and this inhibition required the presence of the kinase domain of MAST205, since deletion of the kinase domain or a dominant-negative mutant of MAST205 did not affect the activity of NHE3. Consistent with these results, we found that MAST205 phosphorylated NHE3 under in vitro conditions. However, overexpression of MAST205 did not affect expression of NHE3 proteins, suggesting that the effect of MAST205 was not mediated by a decrease in NHE3 expression. These findings suggest that MAST205 regulates NHE3 activity and, although the precise mechanism is yet to be determined, MAST205 appears to inhibit NHE3 activity through a phosphorylation-dependent mechanism.
Background & Aims: Epithelial cells form a monolayer at mucosal surface that functions as a highly selective barrier. Lysophosphatidic acid (LPA) is a bioactive lipid that elicits a broad range of biological effects via cognate G protein-coupled receptors. LPA receptor 5 (LPA5) is highly expressed in intestinal epithelial cells, but its role in the intestine is not well-known. Here we determined the role of LPA5 in regulation of intestinal epithelial barrier. Methods: Epithelial barrier integrity was determined in mice with intestinal epithelial cell (IEC)-specific LPA5 deletion, Lpar5ΔIEC. LPA was orally administered to mice, and intestinal permeability was measured. Dextran sulfate sodium (DSS) was used to induce colitis. Human colonic epithelial cell lines were used to determine the LPA5-mediated signaling pathways that regulate epithelial barrier. Results: We observed increased epithelial permeability in Lpar5ΔIEC mice with reduced claudin-4 expression. Oral administration of LPA decreased intestinal permeability in wild-type mice, but the effect was greatly mitigated in Lpar5ΔIEC mice. Serum lipopolysaccharide level and bacterial loads in the intestine and liver were elevated in Lpar5ΔIEC mice. Lpar5ΔIEC mice developed more severe colitis induced with DSS. LPA5 transcriptionally regulated claudin-4, and this regulation was dependent on transactivation of the epidermal growth factor receptor, which induced localization of Rac1 at the cell membrane. LPA induced the translocation of Stat3 to the cell membrane and promoted the interaction between Rac1 and Stat3. Inhibition of Stat3 ablated LPA-mediated regulation of claudin-4. Conclusions: This study identifies LPA5 as a regulator of the intestinal barrier. LPA5 promotes claudin-4 expression in IECs through activation of Rac1 and Stat3.
Background/Aims
The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO3− movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO3 cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction.
Methods: Results
In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1.
Conclusion
Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO3 cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed.
Na+/H+ exchanger regulatory factors, NHERF1 and NHERF2, are structurally related proteins and highly expressed in epithelial cells. These proteins are initially identified as accessory proteins in the regulation of Na+/H+ exchanger isoform 3, NHE3. In addition to regulation of NHE3, recent studies demonstrate the importance of NHERF1 and NHERF2 in recycling and localization of membrane receptors, ion channels and transporters. Recent studies show that serum- and glucocorticoid-induced kinase 1 (SGK1) specifically interacts with NHERF2 but not with NHERF1, adding to the growing number of differences between the two proteins. The association of SGK1 with NHERF2 is necessary for stimulation of NHE3 activity by glucocorticoids. In addition, SGK1 together with NHERF2 stimulates the K+ channel ROMK1, suggesting a broader role of SGK1 in regulation of ion transport.
Lysophosphatidic acids (LPA) exert growth factor-like effects through specific G protein-coupled receptors. The presence of different LPA receptors often determines the specific signaling mechanisms and the physiological consequences of LPA in different environments. Among the four members of the LPA receptor family, LPA2 has been shown to be overexpressed in colon cancer suggesting that the signaling by LPA2 may potentiate growth and survival of tumor cells. In this study, we examined the effect of LPA on survival of colon cancer cells using Caco-2 cells as a cell model system. LPA rescued Caco-2 cells from apoptosis elicited by the chemotherapeutic drug, etoposide. This protection was accompanied by abrogation of etoposide-induced stimulation of caspase activity via a mechanism dependent on Erk and PI3K. In contrast, perturbation of cellular signaling mediated by the LPA2 receptor by knockdown of a scaffold protein NHERF2 abrogated the protective effect of LPA. Etoposide decreased the expression of Bcl-2, which was reversed by LPA. Etoposide decreased the phosphorylation level of the proapoptotic protein Bad in an Erk-dependent manner, without changing Bad expression. We further show that LPA treatment resulted in delayed activation of Erk. These results indicate that LPA protects Caco-2 cells from apoptotic insult by a mechanism involving Erk, Bad, and Bcl-2.
In this study, we examined the tissue-specific expression of two electroneutral Na/HCO3 cotransporter (NBCn1) variants that differ from each other by the presence of the N-terminal 123 amino acids (cassette II). A rat Northern blot with the probe to nucleotides encoding cassette II detected a 9 kb NBCn1 mRNA strongly in the heart and weakly in skeletal muscles, but absent from most of the tissues including kidney, brain, and pancreas. In the rat heart, PCR with primers flanking cassette II preferentially amplified a DNA fragment that lacked cassette II. However, in the human heart, PCR preferentially amplified a fragment that contained cassette II. This larger PCR product was found virtually in all regions of the human cardiovascular system with strong amplification in the apex, atrium, and atrioventricular nodes. These findings indicate that the variant containing cassette II is almost absent in tissues including brain, kidney, and pancreas, where NBCn1 has been extensively examined.
Background & Aims: Regeneration of the epithelium by stem cells in the intestine is supported by intrinsic and extrinsic factors. Lysophosphatidic acid (LPA), a bioactive lipid mediator, regulates many cellular functions, including cell proliferation, survival, and cytokine secretion. Here, we identify LPA5 receptor as a potent regulator of the survival of stem cells and transit-amplifying cells in the intestine. Methods: We have used genetic mouse models of conditional deletion of Lpar5, Lpar5f/f;Rosa-CreERT (Lpar5KO), and intestinal epithelial cell–specific Lpar5f/f;AhCre (Lpar5IECKO) mice. Mice were treated with tamoxifen or β-naphthoflavone to delete Lpar5 expression. Enteroids derived from these mice were used to determine the effect of Lpar5 loss on the apoptosis and proliferation of crypt epithelial cells. Results: Conditional loss of Lpar5 induced ablation of the intestinal mucosa, which increased morbidity of Lpar5KO mice. Epithelial regeneration was compromised with increased apoptosis and decreased proliferation of crypt epithelial cells by Lpar5 loss. Interestingly, intestinal epithelial cell–specific Lpar5 loss did not cause similar phenotypic defects in vivo. Lpar5 loss reduced intestinal stem cell marker gene expression and reduced lineage tracing from Lgr5+ ISCs. Lpar5 loss induced CXCL10 expression which exerts cytotoxic effects on intestinal stem cells and progenitors in the intestinal crypts. By co-culturing Lpar5KO enteroids with wild-type or Lpar5KO splenocytes, we demonstrated that lymphocytes protect the intestinal crypts via a LPA5-dependent suppression of CXCL10. Conclusions: LPA5 is essential for the regeneration of intestinal epithelium. Our findings reveal a new finding that LPA5 regulates survival of stem cells and transit-amplifying cells in the intestine.
Na+/H+ exchanger NHE3 expressed in the intestine and kidney plays a major role in NaCl and HCO3T 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 coimmunoprecipitated 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.
Lysophosphatidic acid (LPA) is a lipid mediator that mediates several effects that promote cancer progress. The LPA receptor type 2 (LPA2) expression is often elevated in several types of cancers, including colorectal cancer (CRC). In this study, we investigated the role of LPA2 in the development of intestinal adenomas by comparing ApcMin/+ mice with ApcMin/+/Lpar2−/− mice. There were 50% fewer intestinal adenomas in ApcMin/+/Lpar2−/− mice than ApcMin/+ mice. Smaller-size adenomas (<1 mm) were found at higher frequencies in ApcMin/+/Lpar2−/− mice compared with ApcMin/+ mice at the two age groups examined. The expression level of LPA2 correlated with increased size of intestinal adenomas. Reduced tumor multiplicity and size in ApcMin/+/Lpar2−/− mice correlated with decreased proliferation of intestinal epithelial cells. ApcMin/+/Lpar2−/− mice showed an increased level of apoptosis, suggesting that LPA2-mediated signaling stimulates intestinal tumor development and progress by regulating both cell proliferation and survival. In addition, the expression levels of Krüpple-like factor 5 (KLF5), β-catenin, cyclin D1, c-Myc, and hypoxia-inducible factor-1α (HIF-1α) were significantly altered in ApcMin/+/Lpar2−/− mice compared with ApcMin/+ mice. In vitro studies using HCT116 cells showed that LPA induced cyclin D1, c-Myc, and HIF-1α expression, which was attenuated by knockdown of LPA2. In summary, intestinal tumor initiated by Apc mutations is altered by LPA2-mediated signaling, which regulates tumor growth and survival by altering multiple targets.