LPA5-Dependent Signaling Regulates Regeneration of the Intestinal Epithelium Following Irradiation.

Lysophosphatidic acid (LPA) is a bioactive lipid molecule that regulates a wide array of cellular functions, including proliferation, differentiation, and survival, via activation of cognate receptors. The LPA5 receptor is highly expressed in the intestinal epithelium, but its function in restoring intestinal epithelial integrity following injury has not been examined. Here, we use a radiation-induced injury model to study the role of LPA5 in regulating intestinal epithelial regeneration. Control mice (Lpar5f/f) and mice with an inducible, epithelial cell-specific deletion of Lpar5 in the small intestine (Lpar5IECKO) were subjected to 10 Gy total body X-ray irradiation and analyzed during recovery. Repair of the intestinal mucosa was delayed in Lpar5IECKO mice, with reduced epithelial proliferation and increased crypt cell apoptosis. These effects were accompanied by reduced numbers of OLFM4+ intestinal stem cells (ISCs). The effects of LPA5 on ISCs were corroborated by studies using organoids derived from Lgr5-lineage tracking reporter mice with deletion of Lpar5 in Lgr5+-stem cells (Lgr5Cont or Lgr5ΔLpar5). Irradiation of organoids resulted in fewer numbers of Lgr5ΔLpar5 organoids retaining Lgr5+-derived progenitor cells compared to Lgr5Cont organoids. Finally, we observed that impaired regeneration in Lpar5IECKO mice was associated with reduced numbers of Paneth cells and decreased expression of YAP, a critical factor for intestinal epithelial repair. Our study highlights a novel role for LPA5 in regeneration of the intestinal epithelium following irradiation and its effect on the maintenance of Paneth cells that support the stem cell niche.

Regulation of the intestinal Na+/H+ exchanger NHE3 by AMP-activated kinase is dependent on phosphorylation of NHE3 at S555 and S563.

Electroneutral NaCl transport by Na+/H+ exchanger 3 (NHE3, SLC9A3) is the major Na+ absorptive mechanism in the intestine and decreased NHE3 activity contributes to diarrhea. Patients with diabetes often experience gastrointestinal adverse effects and medications are often a culprit for chronic diarrhea in type 2 diabetes (T2D). We have shown previously that metformin, the most widely prescribed drug for the treatment of T2D, induces diarrhea by inhibition of Na+/H+ exchanger 3 (NHE3) in rodent models of T2D. Metformin was shown to activate AMP-activated protein kinase (AMPK), but AMPK-independent glycemic effects of metformin are also known. The current study is undertaken to determine whether metformin inhibits NHE3 by activation of AMPK and the mechanism by which NHE3 is inhibited by AMPK. Inhibition of NHE3 by metformin was abolished by knockdown of AMPK-α1 or AMPK-α2. AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) phosphorylated NHE3 at S555. S555 is the primary site of phosphorylation by protein kinase A (PKA), but AMPK phosphorylated S555 independently of PKA. Using Mass spectrometry, we found S563 as a newly recognized phosphorylation site in NHE3. Altering either S555 or S563 to Ala was sufficient to block the inhibition of NHE3 activity by AMPK. NHE3 inhibition is dependent on ubiquitination by the E3 ubiquitin ligase Nedd4-2 and metformin was shown to induce NHE3 internalization via Nedd4-2-mediated ubiquitination. AICAR did not increase NHE3 ubiquitination when S555 or S563 was mutated. We conclude that AMPK activation inhibits NHE3 activity and NHE3 inhibition is associated with phosphorylation of NHE3 at S555 and S563.NEW & NOTEWORTHY We show that AMP-activated protein kinase (AMPK) phosphorylates NHE3 at S555 and S563 to inhibit NHE3 activity in intestinal epithelial cells. Phosphorylation of NHE3 by AMPK is necessary for ubiquitination of NHE3.

Inhibition of protein kinase C-α and activation of ezrin by Lactobacillus acidophilus restore Na+/H+ exchange activity and fluid absorption in db/db mice.

Gastrointestinal (GI) complications, including diarrhea, constipation, and gastroparesis, are common in patients with diabetes. Dysregulation of the Na+/H+ exchanger NHE3 in the intestine is linked to diarrhea and constipation, and recent studies showed that NHE3 expression is reduced in type 1 diabetes and metformin causes diarrhea in the db/db mouse model of type 2 diabetes (T2D) via inhibition of NHE3. In this study, we investigated whether NHE3 expression is altered in type 2 diabetic intestine and the underlying mechanism that dysregulates NHE3. NHE3 expression in the brush border membrane (BBM) of the intestine of diabetic mice and humans was decreased. Protein kinase C (PKC) activation is associated with pathologies of diabetes, and immunofluorescence (IF) analysis revealed increased BBM PKCα abundance. Inhibition of PKCα increased NHE3 BBM abundance and NHE3-mediated intestinal fluid absorption in db/db mice. Previous studies have shown that Lactobacillus acidophilus (LA) stimulates intestinal ion transporters. LA increased NHE3 BBM expression and mitigated metformin-mediated inhibition of NHE3 in vitro and in vivo. To understand the underlying mechanism of LA-mediated stimulation of NHE3, we used Caco-2bbe cells overexpressing PKCα that mimic the elevated state of PKCα in T2D. LA diminished PKCα BBM expression, increased phosphorylation of ezrin, and the interaction of NHE3 with NHE regulatory factor 2 (NHERF2). In addition, inhibition of PKCι blocked phosphorylation of ezrin and activation of NHE3 by LA. These findings demonstrate that NHE3 is downregulated in T2D, and LA restores NHE3 expression via regulation of PKCα, PKCι, and ezrin.NEW & NOTEWORTHY We used mouse models of type 2 diabetes (T2D) and human patient-derived samples to show that Na+/H+ exchanger 3 (NHE3) expression is decreased in T2D. We show that protein kinase C-α (PKCα) is activated in diabetes and inhibition of PKCα increased NHE3 expression and mitigates diarrhea. We show that Lactobacillus acidophilus (LA) stimulates NHE3 via inhibition of PKCα and phosphorylation of ezrin.

Compensatory Upregulation of LPA2 and Activation of the PI3K-Akt Pathway Prevent LPA5-Dependent Loss of Intestinal Epithelial Cells in Intestinal Organoids.

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.

Metformin Inhibits Na+/H+ Exchanger NHE3 Resulting in Intestinal Water Loss.

Glycemic control is the key to the management of type 2 diabetes. Metformin is an effective, widely used drug for controlling plasma glucose levels in diabetes, but it is often the culprit of gastrointestinal adverse effects such as abdominal pain, nausea, indigestion, vomiting, and diarrhea. Diarrhea is a complex disease and altered intestinal transport of electrolytes and fluid is a common cause of diarrhea. Na+/H+ exchanger 3 (NHE3, SLC9A3) is the major Na+ absorptive mechanism in the intestine and our previous study has demonstrated that decreased NHE3 contributes to diarrhea associated with type 1 diabetes. The goal of this study is to investigate whether metformin regulates NHE3 and inhibition of NHE3 contributes to metformin-induced diarrhea. We first determined whether metformin alters intestinal water loss, the hallmark of diarrhea, in type 2 diabetic db/db mice. We found that metformin decreased intestinal water absorption mediated by NHE3. Metformin increased fecal water content although mice did not develop watery diarrhea. To determine the mechanism of metformin-mediated regulation of NHE3, we used intestinal epithelial cells. Metformin inhibited NHE3 activity and the effect of metformin on NHE3 was mimicked by a 5'-AMP-activated protein kinase (AMPK) activator and blocked by pharmacological inhibition of AMPK. Metformin increased phosphorylation and ubiquitination of NHE3, resulting in retrieval of NHE3 from the plasma membrane. Previous studies have demonstrated the role of neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2) in regulation of human NHE3. Silencing of Nedd4-2 mitigated NHE3 inhibition and ubiquitination by metformin. Our findings suggest that metformin-induced diarrhea in type 2 diabetes is in part caused by reduced Na+ and water absorption that is associated with NHE3 inhibition, probably by AMPK.

Survival of Stem Cells and Progenitors in the Intestine Is Regulated by LPA5-Dependent Signaling.

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 ß-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.

Nedd4-2-dependent Ubiquitination Potentiates the Inhibition of Human NHE3 by Cholera Toxin and Enteropathogenic Escherichia coli.

BACKGROUND & AIMS: Diarrhea is one of the most common illnesses and is often caused by bacterial infection. Recently, we have shown that human Na+/H+ exchanger NHE3 (hNHE3), but not non-human NHE3s, interacts with the E3 ubiquitin ligase Nedd4-2. We hypothesize that this property of hNHE3 contributes to the increased severity of diarrhea in humans. METHODS: We used humanized mice expressing hNHE3 in the intestine (hNHE3int) to compare the contribution of hNHE3 and mouse NHE3 to diarrhea induced by cholera toxin (CTX) and enteropathogenic Escherichia coli (EPEC). We measured Na+/H+ exchange activity and fluid absorption. The role of Nedd4-2 on hNHE3 activity and ubiquitination was determined by knockdown in Caco-2bbe cells. The effects of protein kinase A (PKA), the primary mediator of CTX-induced diarrhea, on Nedd4-2 and hNHE3 phosphorylation and their interaction were determined. RESULTS: The effects of CTX and EPEC were greater in hNHE3int mice than in control wild-type (WT) mice, resulting in greater inhibition of NHE3 activity and increased fluid accumulation in the intestine, the hallmark of diarrhea. Activation of PKA increased ubiquitination of hNHE3 and enhanced interaction of Nedd4-2 with hNHE3 via phosphorylation of Nedd4-2 at S342. S342A mutation mitigated the Nedd4-2-hNHE3 interaction and blocked PKA-induced inhibition of hNHE3. Unlike non-human NHE3s, inhibition of hNHE3 by PKA is independent of NHE3 phosphorylation, suggesting a distinct mechanism of hNHE3 regulation. CONCLUSIONS: The effects of CTX and EPEC on hNHE3 are amplified, and the unique properties of hNHE3 may contribute to diarrheal symptoms occurring in humans.

Control of Intestinal Epithelial Permeability by Lysophosphatidic Acid Receptor 5.

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.

Ubiquitin-specific peptidase 7 (USP7) and USP10 mediate deubiquitination of human NHE3 regulating its expression and activity.

Na+ /H+ exchanger NHE3 of human or primates differs from NHE3 of other animals by having a PY motif, which mediates interaction with the E3 ubiquitin (Ub) ligase Nedd4-2. Ub-conjugation of human NHE3 by Nedd4-2 regulates endocytosis of NHE3 but does not affect its cellular expression. Because Ub-conjugation is a reversible process, the aim of this study is to identify deubiquitinating enzyme (DUB) regulating the post-endosomal fate of human NHE3. Using an activity-based chemical screening, we identified ubiquitin specific protease-7 (USP7) and USP10 that bind NHE3. The roles of DUBs in regulation of NHE3 were studied by determining the effects of silencing of USP7 and USP10. Knockdown of USP7 or USP10 resulted in increased NHE3 ubiquitination and decreased NHE3 expression at the surface membrane and cellular level. The endocytic retrieval of NHE3 was promoted by depletion of USP7 or USP10, with increased association of NHE3 with Rab5a and Rab7. Inhibition of USP7 and USP10 by chemical inhibitors or knockdown had an additive effect on NHE3. In addition, NHE3 half-life was reduced accounting for decreased NHE3 protein abundance. NHE3 is inhibited by protein kinase A. Activation of PKA by forskolin decreased the binding of USP7 and USP10 to NHE3, while increasing ubiquitination of NHE3. Knockdown of USP10 had an additive effect on PKA-dependent inhibition of NHE3. These findings demonstrate that USP7 and USP10 are DUBs that regulate NHE3 ubiquitination and expression, and reveal a new mechanism of NHE3 inhibition involving DUBs.

Lysophosphatidic Acid and Autotaxin-associated Effects on the Initiation and Progression of Colorectal Cancer.

The intestinal epithelium interacts dynamically with the immune system to maintain its barrier function to protect the host, while performing the physiological roles in absorption of nutrients, electrolytes, water and minerals. The importance of lysophosphatidic acid (LPA) and its receptors in the gut has been progressively appreciated. LPA signaling modulates cell proliferation, invasion, adhesion, angiogenesis, and survival that can promote cancer growth and metastasis. These effects are equally important for the maintenance of the epithelial barrier in the gut, which forms the first line of defense against the milieu of potentially pathogenic stimuli. This review focuses on the LPA-mediated signaling that potentially contributes to inflammation and tumor formation in the gastrointestinal tract.

Autotaxin determines colitis severity in mice and is secreted by B cells in the colon.

Autotaxin (ATX or ENPP2) is a secreted lysophospholipase D that produces lysophosphatidic acid (LPA), a pleiotropic lipid mediator acting on specific GPCRs. ATX and LPA have been implicated in key (patho)physiologic processes, including embryonic development, lymphocyte homing, inflammation, and cancer progression. Using LPA receptor knockout mice, we previously uncovered a role for LPA signaling in promoting colitis and colorectal cancer. Here, we examined the role of ATX in experimental colitis through inducible deletion of Enpp2 in adult mice. ATX expression was increased upon induction of colitis, whereas ATX deletion reduced the severity of inflammation in both acute and chronic colitis, accompanied by transient weight loss. ATX expression in lymphocytes was strongly reduced in Rag1-/- and µMT mice, suggesting B cells as a major ATX-producing source, which was validated by immunofluorescence and biochemical analyses. ATX secretion by B cells from control, but not Enpp2 knockout, mice led to ERK activation in colorectal cancer cells and promoted T cell migration. We conclude that ATX deletion suppresses experimental colitis and that B cells are a major source of ATX in the colon. Our study suggests that pharmacological inhibition of ATX could be a therapeutic strategy in colitis.-Lin, S., Haque, A., Raeman, R., Guo, L., He, P., Denning, T. L., El-Rayes, B., Moolenaar, W. H., Yun, C. C. Autotaxin determines colitis severity in mice and is secreted by B cells in the colon.

Inhibition of autotaxin alleviates inflammation and increases the expression of sodium-dependent glucose cotransporter 1 and Na+/H+ exchanger 3 in SAMP1/Fc mice.

Crohn's disease (CD) is a chronic, relapsing, inflammatory disease that is often associated with malnutrition because of inflammation in the small intestine. Autotaxin (ATX) is a secreted enzyme that produces extracellular lysophosphatidic acid. Increasing evidence suggests that ATX is upregulated during inflammation, and inhibition of ATX has been effective in attenuating chronic inflammatory conditions, such as arthritis and pulmonary fibrosis. This study aims to determine whether inhibition of ATX alleviates CD-associated inflammation and malnutrition by using SAMP1/Fc mice, a model of CD-like ileitis. SAMP1/Fc mice were treated the ATX inhibitor PF-8380 for 4 wk. Inhibition of ATX led to increased weight gain in SAMP1/Fc mice, decreased T helper 2 cytokine expression, including IL-4, IL-5, and IL-13, and attenuated immune cell migration. SAMP1/Fc mice have low expression of Na+-dependent glucose transporter 1 (SGLT1), suggesting impaired nutrient absorption associated with ileitis. PF-8380 treatment significantly enhanced SGLT1 expression in SAMP1/Fc mice, which could reflect the increased weight changes. However, IL-4 or IL-13 did not alter SGLT1 expression in Caco-2 cells, ruling out their direct effects on SGLT1 expression. Immunofluorescence analysis showed that the expression of sucrase-isomaltase, a marker for intestinal epithelial cell (IEC) differentiation, was decreased in inflamed regions of SAMP1/Fc mice, which was partially restored by PF-8380. Moreover, expression of Na+/H+ exchanger 3 was also improved by PF-8380, suggesting that suppression of inflammation by PF-8380 enhanced IEC differentiation. Our study therefore suggests that ATX is a potential target for treating intestinal inflammation and restoration of the absorptive function of the intestine. NEW & NOTEWORTHY This study is the first, to our knowledge, to determine whether autotoxin (ATX) inhibition improves inflammation and body weights in SAMP1/Fc mice, a mouse model of ileitis. ATX inhibition increased body weights of SAMP1/Fc mice and increased Na+-dependent glucose transporter 1 (SGLT1) expression. Increased SGLT1 expression in the inflamed regions was not a direct effect of cytokines but an indirect effect of increased epithelial cell differentiation upon ATX inhibition.

Expression of lysophosphatidic acid receptor 5 is necessary for the regulation of intestinal Na+/H+ exchanger 3 by lysophosphatidic acid in vivo.

Lysophosphatidic acid (LPA) is a bioactive lipid molecule, which regulates a broad range of pathophysiological processes. Recent studies have demonstrated that LPA modulates electrolyte flux in the intestine, and its potential as an antidiarrheal agent has been suggested. Of six LPA receptors, LPA5 is highly expressed in the intestine. Recent studies by our group have demonstrated activation of Na+/H+ exchanger 3 (NHE3) by LPA5. However, much of what has been elucidated was achieved using colonic cell lines that were transfected to express LPA5. In the current study, we engineered a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC, and investigated the role of LPA5 in NHE3 regulation and fluid absorption in vivo. The intestine of Lpar5ΔIEC mice appeared morphologically normal, and the stool frequency and fecal water content were unchanged compared with wild-type mice. Basal rates of NHE3 activity and fluid absorption and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5. NHE3 activation involves trafficking of NHE3 from the terminal web to microvilli, and this mobilization of NHE3 by LPA was abolished in Lpar5ΔIEC mice. Dysregulation of NHE3 was specific to LPA, and insulin and cholera toxin were able to stimulate and inhibit NHE3, respectively, in both wild-type and Lpar5ΔIEC mice. The current study for the first time demonstrates the necessity of LPA5 in LPA-mediated stimulation of NHE3 in vivo. NEW & NOTEWORTHY This study is the first to assess the role of LPA5 in NHE3 regulation and fluid absorption in vivo using a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC. Basal rates of NHE3 activity and fluid absorption, and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5.

Lysophosphatidic Acid Receptor 1 Is Important for Intestinal Epithelial Barrier Function and Susceptibility to Colitis.

Intestinal epithelial cells form a barrier that is critical in protecting the host from the hostile luminal environment. Previously, we showed that lysophosphatidic acid (LPA) receptor 1 regulates proliferation of intestinal epithelial cells, such that the absence of LPA1 mitigates the epithelial wound healing process. This study provides evidence that LPA1 is important for the maintenance of epithelial barrier integrity. The epithelial permeability, determined by fluorescently labeled dextran flux and transepithelial resistance, is increased in the intestine of mice with global deletion of Lpar1, Lpar1-/- (Lpa1-/-). Serum liposaccharide level and bacteria loads in the intestinal mucosa and peripheral organs were elevated in Lpa1-/- mice. Decreased claudin-4, caudin-7, and E-cadherin expression in Lpa1-/- mice further suggested defective apical junction integrity in these mice. Regulation of LPA1 expression in Caco-2 cells modulated epithelial permeability and the expression levels of junctional proteins. The increased epithelial permeability in Lpa1-/- mice correlated with increased susceptibility to an experimental model of colitis. This resulted in more severe inflammation and increased mortality compared with control mice. Treatment of Caco-2 cells with tumor necrosis factor-α and interferon-γ significantly increased paracellular permeability, which was blocked by cotreatment with LPA, but not LPA1 knockdown cells. Similarly, orally given LPA blocked tumor necrosis factor-mediated intestinal barrier defect in mice. LPA1 plays a significant role in maintenance of epithelial barrier in the intestine via regulation of apical junction integrity.

Development of CXCR4 modulators by virtual HTS of a novel amide-sulfamide compound library.

CXCR4 plays a crucial role in recruitment of inflammatory cells to inflammation sites at the beginning of the disease process. Modulating CXCR4 functions presents a new avenue for anti-inflammatory strategies. However, using CXCR4 antagonists for a long term usage presents potential serious side effect due to their stem cell mobilizing property. We have been developing partial CXCR4 antagonists without such property. A new computer-aided drug design program, the FRESH workflow, was used for anti-CXCR4 lead compound discovery and optimization, which coupled both compound library building and CXCR4 docking screens in one campaign. Based on the designed parent framework, 30 prioritized amide-sulfamide structures were obtained after systemic filtering and docking screening. Twelve compounds were prepared from the top-30 list. Most synthesized compounds exhibited good to excellent binding affinity to CXCR4. Compounds Ig and Im demonstrated notable in vivo suppressive activity against xylene-induced mouse ear inflammation (with 56% and 54% inhibition). Western blot analyses revealed that Ig significantly blocked CXCR4/CXCL12-mediated phosphorylation of Akt. Moreover, Ig attenuated the amount of TNF-α secreted by pathogenic E. coli-infected macrophages. More importantly, Ig had no observable cytotoxicity. Our results demonstrated that FRESH virtual high throughput screening program of targeted chemical class could successfully find potent lead compounds, and the amide-sulfamide pharmacophore was a novel and effective framework blocking CXCR4 function.

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.

Transgenic Expression of Human Lysophosphatidic Acid Receptor LPA2 in Mouse Intestinal Epithelial Cells Induces Intestinal Dysplasia.

Lysophosphatidic acid (LPA) acts on LPA2 receptor to mediate multiple pathological effects that are associated with tumorigenesis. The absence of LPA2 attenuates tumor progression in rodent models of colorectal cancer, but whether overexpression of LPA2 alone can lead to malignant transformation in the intestinal tract has not been studied. In this study, we expressed human LPA2 in intestinal epithelial cells (IECs) under control of the villin promoter. Less than 4% of F1-generation mice had germline transmission of transgenic (TG) human LPA2; as such only 3 F1 mice out of 72 genotyped had TG expression. These TG mice appeared anemic with hematochezia and died shortly after birth. TG mice were smaller in size compared with the wild type mouse of the same age and sex. Morphological analysis showed that TG LPA2 colon had hyper-proliferation of IECs resulting in increased colonic crypt depth. Surprisingly, TG small intestine had villus blunting and decreased IEC proliferation and dysplasia. In both intestine and colon, TG expression of LPA2 compromised the terminal epithelial differentiation, consistent with epithelial dysplasia. Furthermore, we showed that epithelial dysplasia was observed in founder mouse intestine, correlating LPA2 overexpression with epithelial dysplasia. The current study demonstrates that overexpression of LPA2 alone can lead to intestinal dysplasia.

Deletion of Na+/H+ exchanger regulatory factor 2 represses colon cancer progress by suppression of Stat3 and CD24.

The Na(+)/H(+) exchanger regulatory factor (NHERF) family of proteins is scaffolds that orchestrate interaction of receptors and cellular proteins. Previous studies have shown that NHERF1 functions as a tumor suppressor. The goal of this study is to determine whether the loss of NHERF2 alters colorectal cancer (CRC) progress. We found that NHERF2 expression is elevated in advanced-stage CRC. Knockdown of NHERF2 decreased cancer cell proliferation in vitro and in a mouse xenograft tumor model. In addition, deletion of NHERF2 in Apc(Min/+) mice resulted in decreased tumor growth in Apc(Min/+) mice and increased lifespan. Blocking NHERF2 interaction with a small peptide designed to bind the second PDZ domain of NHERF2 attenuated cancer cell proliferation. Although NHERF2 is known to facilitate the effects of lysophosphatidic acid receptor 2 (LPA2), transcriptome analysis of xenograft tumors revealed that NHERF2-dependent genes largely differ from LPA2-regulated genes. Activation of ß-catenin and ERK1/2 was mitigated in Apc(Min/+);Nherf2(-/-) adenomas. Moreover, Stat3 phosphorylation and CD24 expression levels were suppressed in Apc(Min/+);Nherf2(-/-) adenomas. Consistently, NHERF2 knockdown attenuated Stat3 activation and CD24 expression in colon cancer cells. Interestingly, CD24 was important in the maintenance of Stat3 phosphorylation, whereas NHERF2-dependent increase in CD24 expression was blocked by inhibition of Stat3, suggesting that NHERF2 regulates Stat3 phosphorylation through a positive feedback mechanism between Stat3 and CD24. In summary, this study identifies NHERF2 as a novel oncogenic protein and a potential target for cancer treatment. NHERF2 potentiates the oncogenic effects in part by regulation of Stat3 and CD24.

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.

Restoration of Na+/H+ exchanger NHE3-containing macrocomplexes ameliorates diabetes-associated fluid loss.

Diarrhea is one of the troublesome complications of diabetes, and the underlying causes of this problem are complex. Here, we investigated whether altered electrolyte transport contributes to diabetic diarrhea. We found that the expression of Na+/H+ exchanger NHE3 and several scaffold proteins, including NHE3 regulatory factors (NHERFs), inositol trisphosphate (IP3) receptor-binding protein released with IP3 (IRBIT), and ezrin, was decreased in the intestinal brush border membrane (BBM) of mice with streptozotocin-induced diabetes. Treatment of diabetic mice with insulin restored intestinal NHE3 activity and fluid absorption. Molecular analysis revealed that NHE3, NHERF1, IRBIT, and ezrin form macrocomplexes, which are perturbed under diabetic conditions, and insulin administration reconstituted these macrocomplexes and restored NHE3 expression in the BBM. Silencing of NHERF1 or IRBIT prevented NHE3 trafficking to the BBM and insulin-dependent NHE3 activation. IRBIT facilitated the interaction of NHE3 with NHERF1 via protein kinase D2-dependent phosphorylation. Insulin stimulated ezrin phosphorylation, which enhanced the interaction of ezrin with NHERF1, IRBIT, and NHE3. Additionally, oral administration of lysophosphatidic acid (LPA) increased NHE3 activity and fluid absorption in diabetic mice via an insulin-independent pathway. Together, these findings indicate the importance of NHE3 in diabetic diarrhea and suggest LPA administration as a potential therapeutic strategy for management of diabetic diarrhea.