Rapamycin and Starvation Mitigate Indomethacin-Induced Intestinal Damage through Preservation of Lysosomal Vacuolar ATPase Integrity.

Nonsteroidal anti-inflammatory drugs (NSAIDs) possess anti-inflammatory, antipyretic, and analgesic properties and are among the most commonly used drugs. Although the cause of NSAID-induced gastric ulcers is well understood, the mechanism behind small intestinal ulcers remains elusive. In this study, we examined the mechanism through which indomethacin (IM), a prominent NSAID, induces small intestinal ulcers, both in vitro and in vivo. In IEC6 cells, a small intestinal epithelial cell line, IM treatment elevated levels of LC3-II and p62. These expression levels remained unaltered after treatment with chloroquine or bafilomycin, which are vacuolar ATPase (V-ATPase) inhibitors. IM treatment reduced the activity of cathepsin B, a lysosomal protein hydrolytic enzyme, and increased the lysosomal pH. There was a notable increase in subcellular colocalization of LC3 with Lamp2, a lysosome marker, post IM treatment. The increased lysosomal pH and decreased cathepsin B activity were reversed by pretreatment with rapamycin (Rapa) or glucose starvation, both of which stabilize V-ATPase assembly. To validate the in vitro findings in vivo, we established an IM-induced small intestine ulcer mouse model. In this model, we observed multiple ulcerations and heightened inflammation following IM administration. However, pretreatment with Rapa or fasting, which stabilize V-ATPase assembly, mitigated the IM-induced small intestinal ulcers in mice. Coimmunoprecipitation studies demonstrated that IM binds to V-ATPase in vitro and in vivo. These findings suggest that IM induces small intestinal injury through lysosomal dysfunction, likely due to the disassembly of lysosomal V-ATPase caused by direct binding. Moreover, Rapa or starvation can prevent this injury by stabilizing the assembly. SIGNIFICANCE STATEMENT: This study elucidates the largely unknown mechanisms behind small intestinal ulceration induced by indomethacin and reveals the involvement of lysosomal dysfunction via vacuolar ATPase disassembly. The significance lies in identifying potential preventative interventions, such as rapamycin treatment or glucose starvation, offering pivotal insights that extend beyond nonsteroidal anti-inflammatory drugs-induced ulcers to broader gastrointestinal pathologies and treatments, thereby providing a foundation for novel therapeutic strategies aimed at a wide array of gastrointestinal disorders.

Differential expression of TRKB tyrosine kinase in the two histological types of parotid salivary duct carcinoma with cancer aggressiveness.

Parotid salivary duct carcinoma (SDC) is a rare and aggressive parotid gland carcinoma (PGC). SDC has two origins: de novo and ex pleomorphic adenoma (SDC ex PA); however, because of its rarity, the clinical and molecular features of the two types of SDC are not sufficiently understood. Here, we studied the differences in their clinicopathological and molecular features using clinical specimens while comparing them to those of adenoid cystic carcinoma (AdCC), an intermediate-grade PGC. Clinicopathological analysis of tissues from patients with PGC revealed significant associations between histological types and malignant phenotypes, including nodal metastasis, recurrence, vascular invasion, and neural invasion, and revealed more malignant phenotypes of de novo SDC than of SDC ex PA. The de novo SDC showed a significantly higher frequency of intra-neural invasion (intra-NI) and vascular invasion than AdCC and SDC ex PA. PGCs with high intra-NI were significantly correlated with malignant phenotypes and survival rates. Recently, we observed the overexpression of tropomyosin receptor kinase B (TRKB), a receptor tyrosine kinase, in PGC cells. Here, immunohistochemical and clinicopathological analyses showed that TRKB was highly expressed in SDC cells, particularly de novo SDC cells, and was significantly associated with poor survival and highly malignant phenotypes, including intra-NI and vascular invasion. Collectively, these data show that TRKB expression is significantly elevated in PGC, particularly in de novo SDC, and can be one of the biomarkers of their aggressiveness.

O-GlcNAcylation-induced GSK-3ß activation deteriorates pressure overload-induced heart failure via lack of compensatory cardiac hypertrophy in mice.

O-GlcNAc transferase (OGT) modulates many functions of proteins via O-GlcNAcylation that adds O-linked ß-N-acetylglucosamine (O-GlcNAc) to the serine/threonine residues of proteins. However, the role of O-GlcNAcylation in cardiac remodeling and function is not fully understood. To examine the effect of O-GlcNAcylation on pressure overload-induced cardiac hypertrophy and subsequent heart failure, transverse aortic constriction (TAC) surgery was performed in wild type (WT) and Ogt transgenic (Ogt-Tg) mice. Four weeks after TAC (TAC4W), the heart function of Ogt-Tg mice was significantly lower than that of WT mice (reduced fractional shortening and increased ANP levels). The myocardium of left ventricle (LV) in Ogt-Tg mice became much thinner than that in WT mice. Moreover, compared to the heart tissues of WT mice, O-GlcNAcylation of GSK-3ß at Ser9 was increased and phosphorylation of GSK-3ß at Ser9 was reduced in the heart tissues of Ogt-Tg mice, resulting in its activation and subsequent inactivation of nuclear factor of activated T cell (NFAT) activity. Finally, the thinned LV wall and reduced cardiac function induced by TAC4W in Ogt-Tg mice was reversed by the treatment of a GSK-3ß inhibitor, TDZD-8. These results imply that augmented O-GlcNAcylation exacerbates pressure overload-induced heart failure due to a lack of compensatory cardiac hypertrophy via O-GlcNAcylation of GSK-3ß, which deprives the phosphorylation site of GSK-3ß to constantly inactivate NFAT activity to prevent cardiac hypertrophy. Our findings may provide a new therapeutic strategy for cardiac hypertrophy and subsequent heart failure.

BDNF/TRKB axis provokes EMT progression to induce cell aggressiveness via crosstalk with cancer-associated fibroblasts in human parotid gland cancer.

Parotid gland cancer (PGC) is a rare malignancy and its molecular characteristics remain poorly understood, which has precluded the development of effective drug therapies. Given the poor prognosis of many human cancers in which tropomyosin receptor kinase B (TRKB) is highly expressed, we investigated the involvement of brain-derived neurotrophic factor (BDNF)/TRKB pathway in PGC cells using clinical specimens and observed upregulation of TRKB and BDNF. In primary culture systems of patient-derived PGC cells and cancer-associated fibroblasts (CAFs), PGC cells co-cultured with CAFs exhibited significant upregulation of BDNF and epithelial-mesenchymal transition (EMT). Similar results were observed in PGC cells treated with conditioned medium from co-cultures of PGC cells with CAFs. Administration of TRK inhibitors suppressed BDNF-induced cell migration in PGC cells. Immunohistochemical and clinicopathological analyses of tumors from patients with PGC revealed that BDNF and TRKB were highly expressed in both tumor cells and stromal cells such as CAFs, and TRKB expression levels in PGC cells were significantly correlated with aggressive features, including vascular invasion, nodal metastasis, and poor prognosis. Collectively, these data suggest that the BDNF/TRKB pathway regulates PGC cell aggressiveness via crosstalk with CAFs and is a potential therapeutic target for PGC harboring invasive and metastatic features.

O-GlcNAcylation-mediated degradation of FBXL2 stabilizes FOXM1 to induce cancer progression.

O-GlcNAcylation is a dynamic and reversible post-translational modification of cytonuclear molecules that regulates cellular signaling. Elevated O-GlcNAcylation is a general property of cancer and plays a critical role in cancer progression. We previously showed that the expression of FOXM1, a critical oncogenic transcription factor widely overexpressed in solid tumors, was elevated in MKN45 cells, a human gastric cancer cell line, by the O-GlcNAcase inhibitor Thiamet G (TMG), which induces augmented O-GlcNAcylation. Here, we identified FBXL2 E3 ubiquitin ligase as a new target of O-GlcNAcylation. Consistent with the results in MKN45 cells, FOXM1 expression was increased, accompanied by its decreased ubiquitination and degradation by TMG in the other gastric cancer cell lines, including NUGC-3 cells. We found that FBXL2 ubiquitinated FOXM1, and the interaction with FBXL2 and ubiquitination of FOXM1 were reduced by TMG in NUGC-3 cells. Interestingly, FBXL2 was also ubiquitinated, which was promoted by TMG in the cells. Moreover, FOXM1 expression and cell proliferation were reduced in FBXL2-induced NUGC-3 cells, and the reductions were attenuated by TMG, indicating that FOXM1 was stabilized by O-GlcNAcylation-mediated degradation of FBXL2 to induce cancer progression. These data suggest that elevated O-GlcNAcylation contributes to cancer progression by suppressing FBXL2-mediated degradation of FOXM1.

Overexpression of Na+/H+ exchanger 1 specifically induces cell death in human iPS cells via sustained activation of the Rho kinase ROCK.

Understanding the specific properties of human induced pluripotent stem cells (iPSCs) is important for quality control of iPSCs. Having incidentally discovered that overexpression of plasma membrane Na+/H+ exchanger 1 (NHE1) induces cell death in iPSCs, we investigated the mechanism of NHE1-induced cell death. Doxycycline-induced NHE1 overexpression arrested cell growth, and nearly all cells were killed by a necrotic process within 72 h. NHE1 overexpression led to sustained activation of Rho-associated coiled-coil kinase (ROCK), accompanied by dramatic changes in cell shape, cell elongation, and swelling of peripheral cells in iPSC colonies, as well as marked stress fiber formation. The ROCK inhibitor Y27632 reduced NHE1-induced cell death. ROCK-dependent phenotypes were suppressed by a loss-of-function mutation of NHE1 and inhibited by an inhibitor of NHE1 activity, indicating that NHE1-mediated transport activity is required. Moreover, ROCK was activated by trimethylamine treatment-mediated cytosolic alkalinization and accumulated in the plasma membrane near NHE1 in peripheral iPSCs of cell colonies. By contrast, cell death did not occur in mesendoderm-like cells that had differentiated from iPSCs, indicating that the NHE1-mediated effects were specific for iPSCs. These results suggest that NHE1 overexpression specifically induces death of iPSCs via sustained ROCK activation, probably caused by an increase in local pH near NHE1. Finally, monensin, a Na+/H+ exchange ionophore, selectively killed iPSCs, suggesting that monensin could help eliminate iPSCs that remain after differentiation, a strategy that might be useful for improving regenerative medicine.

Augmented O-GlcNAcylation attenuates intermittent hypoxia-induced cardiac remodeling through the suppression of NFAT and NF-κB activities in mice.

Type 2 diabetes mellitus (T2DM) has been reported to be associated with cardiac remodeling. Although O-GlcNAcylation is known to be elevated in diabetic and ischemic hearts, the effects of O-GlcNAcylation on cardiac remodeling induced by intermittent hypoxia (IH), such as sleep apnea syndrome (SAS), remain unknown. To evaluate the effects, we induced IH in wild-type (WT) and transgenic O-GlcNAc transferase (Ogt-Tg) mice. Two weeks of IH increased O-GlcNAcylation in the heart tissues of both strains of mice, whereas O-GlcNAcylation in Ogt-Tg mice was significantly higher than that in WT mice under both normoxic and IH conditions. WT mice exhibited cardiac remodeling after IH, whereas cardiac remodeling was significantly attenuated in Ogt-Tg mice. Oxidative stress and apoptosis increased after IH in both strains of mice, whereas the rate of increase in these processes in Ogt-Tg mice was significantly lower than that in WT mice. To examine the mechanism of cardiac remodeling attenuation in Ogt-Tg mice after IH, the effects of O-GlcNAcylation on the activities of the master regulators nuclear factor of activated T cells (NFAT) and NF-κB were determined. The O-GlcNAcylation of GSK-3ß, a negative regulator of NFAT, was significantly increased in Ogt-Tg mice, whereas the phosphorylation of GSK-3ß was reciprocally reduced. The same result was observed for NF-κB p65. An in vitro reporter assay showed that the augmentation of O-GlcNAcylation by an O-GlcNAcase inhibitor suppressed NFAT and NF-κB promoter activity. These data suggest that augmented O-GlcNAcylation mitigates IH-induced cardiac remodeling by suppressing NFAT and NF-κB activities through the O-GlcNAcylation of GSK-3ß and NF-κB p65.

Augmented O-GlcNAcylation alleviates inflammation-mediated colon carcinogenesis via suppression of acute inflammation.

Colon cancer prevalence is high worldwide. O-GlcNAcylation has been associated with tumor growth in various tissues, including the colon; however, its link to carcinogenesis is not fully understood. We investigated the association of O-GlcNAcylation with colon carcinogenesis using a 1,2-dimethylhydrazine/dextran sodium sulfate-induced colon carcinogenesis model in wild type and O-GlcNAc transferase-transgenic (Ogt-Tg) mice. The incidence of colon cancer was significantly lower in Ogt-Tg than in wild type mice. The colonic length was not shortened in Ogt-Tg mice, and NF-κB p65 phosphorylation was strongly suppressed, indicating that reduction of inflammation might be related to the alleviation of colon carcinogenesis. Dextran sodium sulfate-induced acute colitis mice were used to evaluate the effect of O-GlcNAcylation on inflammation at the maximal inflammation period. In Ogt-Tg mice, NF-κB p65 phosphorylation and interleukin-1ß mRNA expression were suppressed. Histochemical staining demonstrated shedding of colon epithelial cells in wild type mice a few days after dextran sodium sulfate treatment, whereas they remained essentially intact in Ogt-Tg mice. There were no significant differences on histochemical staining in the remaining epithelia between groups. These data suggest that O-GlcNAcylation could prevent colon carcinogenesis through reducing acute maximum inflammation, suggesting modulation of O-GlcNAcylation as a novel therapeutic option.

TRKB tyrosine kinase receptor is a potential therapeutic target for poorly differentiated oral squamous cell carcinoma.

It has been reported that one of the neurotrophin receptors, tropomyosin receptor kinase B (TRKB), is frequently overexpressed in various tumor tissues including oral squamous cell carcinoma (OSCC), and that its upregulation promotes tumor progression in human cancers. However, the correlation between TRKB overexpression and clinicopathological characteristics is not fully elucidated. Here, we present the correlation between the expression levels of TRKB and/or its secreted ligand, brain-derived neurotrophic factor (BDNF), and clinicopathological characteristics, especially regarding tumor differentiation, tissue invasion, and disease-free survival in patients with OSCC. The results obtained through immunohistochemical analysis of human OSCC tumor specimens showed that the expression levels of TRKB and/or BDNF, were significantly higher in moderately and poorly differentiated OSCC (MD/PD-OSCC) tumor cells than in well differentiated cells (WD-OSCC). Moreover, the OSCC tumors highly expressing TRKB and/or BDNF exhibited promotion in tissue invasion and reduction in disease-free survival in the patients. In an orthotopic transplantation mouse model of human OSCC cell lines, administration of a TRKB-specific inhibitor significantly suppressed the tumor growth and invasion in PD-OSCC-derived tumor cells, but not in WD-OSCC-derived tumor cells. Moreover, the TRKB inhibitor selectively blocked BDNF-induced tumor cell proliferation and migration accompanied with the suppression of TRKB phosphorylation in PD-OSCC but not in WD-OSCC in vitro. Taken together, these data suggest that the BDNF/TRKB signaling pathway may regulate tumor progression in poorly differentiated OSCC. Expression levels of signal molecules may be an accurate prognosis marker for tumor aggressiveness, and the molecules may be an attractive target for new OSCC therapies.

Elevated O-GlcNAcylation stabilizes FOXM1 by its reduced degradation through GSK-3ß inactivation in a human gastric carcinoma cell line, MKN45 cells.

O-GlcNAcylation is a dynamic post-translational modification of cytonuclear proteins for intracellular signaling. Elevated O-GlcNAcylation is a general feature of cancer and contributes to cancer progression, and recent studies indicate the contribution to increasing incidence of various types of cancer in diabetic patients. However, the role of O-GlcNAcylation in tumor progression is not fully elucidated. Forkhead box M1 (FOXM1), a master mitotic transcription factor, has been implicated in all major hallmarks of cancer, and is wildly expressed in solid tumors. Given that FOXM1 expression was reported to be elevated in gastric cancer, we examined the effect of high glucose or an inhibitor of O-GlcNAc hydrolase, Thiamet G (TMG), on FOXM1 protein expression in a human gastric cancer cell line, MKN45 cells, and confirmed that FOXM1 protein level and the cell proliferation were upregulated. To investigate the molecular mechanisms by which FOXM1 protein expression is regulated by O-GlcNAcylation, the effect of high glucose and TMG on FOXM1 ubiquitination was examined in MKN45 cells. As a result, the ubiquitination and degradation of FOXM1 protein were both suppressed by high glucose and TMG treatment. However, the O-GlcNAcylation was not detected on FOXM1 but not on GSK-3ß. High glucose and TMG treatment increased phospho-serine 9 GSK-3ß, an inactive form, and the degradation of FOXM1 protein was suppressed by treatment of GSK-3ß inhibitors in MKN45 cells. Taken together, we suggest that high glucose and elevated O-GlcNAcylation stabilize FOXM1 protein by its reduced degradation via GSK-3ß inactivation in MKN45 cells, suggesting that the higher risk of gastric cancer in diabetic patients could be partially due to O-GlcNAcylation-mediated FOXM1 stabilization.

W9 peptide enhanced osteogenic differentiation of human adipose-derived stem cells.

W9 is a peptide that abrogates osteoclast differentiation via blockade of nuclear factor-κB ligand (RANKL)-RANK signaling, which activates bone formation. However, W9 stimulated osteogenesis in osteoblasts and mesenchymal stem cells. The present study demonstrated that the W9 peptide promoted osteogenic differentiation of human adipose-derived stem cells (hAdSCs) even under non-osteogenic differentiation culture conditions. W9-treated hAdSCs exhibited several osteocalcin-expressing cells and great mineralization compared to the BMP2-treated hAdSCs, which suggests that the W9 peptide had potent osteogenic potential in hAdSCs. W9 treatment also markedly enhanced the phosphorylation of p38, JNK, Erk1/2, and Akt, and BMP2 treatment only enhanced the phosphorylation of p38 and Erk1/2 in hAdSCs. hAdSCs did not express the RANKL gene, but W9 treatment upregulated Runx2, Collagen type 1A1 and TGF receptor genes and increased Akt phosphorylation. These results suggest that the W9-induced potent osteogenic induction was attributed to activation of TGF and the PI3 kinase/Akt signaling pathway in hAdSCs.

Augmented O-GlcNAcylation of AMP-activated kinase promotes the proliferation of LoVo cells, a colon cancer cell line.

Increasing incidence of various cancers has been reported in diabetic patients. O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins at serine/threonine residues (O-GlcNAcylation) is an essential post-translational modification that is upregulated in diabetic patients and has been implicated in tumor growth. However, the mechanisms by which O-GlcNAcylation promotes tumor growth remain unclear. Given that AMP-activated kinase (AMPK) has been thought to play important roles in suppressing tumor growth, we evaluated the involvement of AMPK O-GlcNAcylation on the growth of LoVo cells, a human colon cancer cell line. Results revealed that treatment with Thiamet G (TMG), an inhibitor of O-GlcNAc hydrolase, increased both anchorage-dependent and -independent growth of the cells. O-GlcNAc transferase overexpression also increased the growth. These treatments increased AMPK O-GlcNAcylation in a dose-dependent manner, which led to reduced AMPK phosphorylation and mTOR activation. Chemical inhibition or activation of AMPK led to increased or decreased growth, respectively, which was consistent with the data with genetic inhibition of AMPK. In addition, TMG-mediated acceleration of tumor growth was abolished by both chemical and genetic inhibition of AMPK. To examine the effects of AMPK O-GlcNAcylation in vivo, the LoVo cells were s.c. transplanted onto the backs of BALB/c-nu/nu mice. Injection of TMG promoted the growth and enhanced O-GlcNAcylation of the tumors of the mice. Consistent with in vitro data, AMPK O-GlcNAcylation was increased, which reduced AMPK phosphorylation and resulted in activation of mTOR. Collectively, the higher colon cancer risk of diabetic patients could be due to O-GlcNAcylation-mediated AMPK inactivation and subsequent activation of mTOR.

Augmented TME O-GlcNAcylation Promotes Tumor Proliferation through the Inhibition of p38 MAPK.

O-GlcNAcylation is a dynamic O-linked glycosylation event that plays a crucial role in regulating cellular signaling. Recent studies indicate that increased O-GlcNAcylation is a general feature in cancer and contributes to various cancer phenotypes, including cell proliferation, survival, invasion, metastasis, and energy metabolism. However, the role of O-GlcNAcylation in the tumor microenvironment (TME) is not fully elucidated. Here, B16 melanoma cells were subcutaneously transplanted into O-GlcNAc transferase transgenic (Ogt-Tg) mice exhibiting elevated O-GlcNAcylation to examine the effect of O-GlcNAcylation in the TME on tumor progression. In this model system, B16 tumor growth was significantly higher in Ogt-Tg/+ mice compared with wild-type (WT) mice. The tumors grown in Ogt-Tg/+ mice showed significant downregulation of p38 MAPK activity and upregulation of the ERK1/2 signaling pathway. In addition, proinflammatory cytokine production was significantly lower in the tumor tissues from Ogt-Tg/+ mice than in those from WT mice. Activation of NF-κB, a key regulator in the cytokine production, was downregulated in the macrophages of the tumor tissues grown in Ogt-Tg/+ mice. These data reveal that elevated O-GlcNAcylation in the TME reduces the production of inflammatory cytokines and promotes cancer progression through downregulation of p38 MAPK activity and subsequent upregulation of the ERK1/2 signaling pathway.Implications: The reduced production of inflammatory cytokines by augmented O-GlcNAcylation in the TME, mainly macrophages, promotes tumor proliferation through the inhibition of p38 MAPK and suggests a possible cause of increased morbidity and mortality rates for various cancers in diabetic patients. Mol Cancer Res; 15(9); 1287-98. ©2017 AACR.

Caveolae-specific activation loop between CaMKII and L-type Ca2+ channel aggravates cardiac hypertrophy in α1-adrenergic stimulation.

Activation of CaMKII induces a myriad of biological processes and plays dominant roles in cardiac hypertrophy. Caveolar microdomain contains many calcium/calmodulin-dependent kinase II (CaMKII) targets, including L-type Ca2+ channel (LTCC) complex, and serves as a signaling platform. The location of CaMKII activation is thought to be critical; however, the roles of CaMKII in caveolae are still elusive due to lack of methodology for the assessment of caveolae-specific activation. Our aim was to develop a novel tool for the specific analysis of CaMKII activation in caveolae and to determine the functional role of caveolar CaMKII in cardiac hypertrophy. To assess the caveolae-specific activation of CaMKII, we generated a fusion protein composed of phospholamban and caveolin-3 (cPLN-Cav3) and GFP fusion protein with caveolin-binding domain fused to CaMKII inhibitory peptide (CBD-GFP-AIP), which inhibits CaMKII activation specifically in caveolae. Caveolae-specific activation of CaMKII was detected using phosphospecific antibody for PLN (Thr17). Furthermore, adenoviral overexpression of LTCC ß2a-subunit (ß2a) in NRCMs showed its constitutive phosphorylation by CaMKII, which induces hypertrophy, and that both phosphorylation and hypertrophy are abolished by CBD-GFP-AIP expression, indicating that ß2a phosphorylation occurs specifically in caveolae. Finally, ß2a phosphorylation was observed after phenylephrine stimulation in ß2a-overexpressing mice, and attenuation of cardiac hypertrophy after chronic phenylephrine stimulation was observed in nonphosphorylated mutant of ß2a-overexpressing mice. We developed novel tools for the evaluation and inhibition of caveolae-specific activation of CaMKII. We demonstrated that phosphorylated ß2a dominantly localizes to caveolae and induces cardiac hypertrophy after α1-adrenergic stimulation in mice.NEW & NOTEWORTHY While signaling in caveolae is thought to be important in cardiac hypertrophy, direct evidence is missing due to lack of tools to assess caveolae-specific signaling. This is the first study to demonstrate caveolae-specific activation of CaMKII signaling in cardiac hypertrophy induced by α1-adrenergic stimulation using an originally developed tool.

Flt1/VEGFR1 heterozygosity causes transient embryonic edema.

Vascular endothelial growth factor-A is a major player in vascular development and a potent vascular permeability factor under physiological and pathological conditions by binding to a decoy receptor Flt1 and its primary receptor Flk1. In this study, we show that Flt1 heterozygous (Flt1(+/-)) mouse embryos grow up to adult without life-threatening abnormalities but exhibit a transient embryonic edema around the nuchal and back regions, which is reminiscent of increased nuchal translucency in human fetuses. Vascular permeability is enhanced and an intricate infolding of the plasma membrane and huge vesicle-like structures are seen in Flt1(+/-) capillary endothelial cells. Flk1 tyrosine phosphorylation is elevated in Flt1(+/-) embryos, but Flk1 heterozygosity does not suppress embryonic edema caused by Flt1 heterozygosity. When Flt1 mutants are crossed with Aspp1(-/-) mice which exhibit a transient embryonic edema with delayed formation and dysfunction of lymphatic vessels, only 5.7% of Flt1(+/-); Aspp1(-/-) mice survive, compared to expected ratio (25%). Our results demonstrate that Flt1 heterozygosity causes a transient embryonic edema and can be a risk factor for embryonic lethality in combination with other mutations causing non-lethal vascular phenotype.

The sphingosine-1-phosphate transporter Spns2 expressed on endothelial cells regulates lymphocyte trafficking in mice.

The bioactive lysophospholipid mediator sphingosine-1-phosphate (S1P) promotes the egress of newly formed T cells from the thymus and the release of immature B cells from the bone marrow. It has remained unclear, however, where and how S1P is released. Here, we show that in mice, the S1P transporter spinster homolog 2 (Spns2) is responsible for the egress of mature T cells and immature B cells from the thymus and bone marrow, respectively. Global Spns2-KO mice exhibited marked accumulation of mature T cells in thymi and decreased numbers of peripheral T cells in blood and secondary lymphoid organs. Mature recirculating B cells were reduced in frequency in the bone marrow as well as in blood and secondary lymphoid organs. Bone marrow reconstitution studies revealed that Spns2 was not involved in S1P release from blood cells and suggested a role for Spns2 in other cells. Consistent with these data, endothelia-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global Spns2-KO mice. These data suggest that Spns2 functions in ECs to establish the S1P gradient required for T and B cells to egress from their respective primary lymphoid organs. Furthermore, Spns2 could be a therapeutic target for a broad array of inflammatory and autoimmune diseases.

Claudin-2-deficient mice are defective in the leaky and cation-selective paracellular permeability properties of renal proximal tubules.

Claudin-2 is highly expressed in tight junctions of mouse renal proximal tubules, which possess a leaky epithelium whose unique permeability properties underlie their high rate of NaCl reabsorption. To investigate the role of claudin-2 in paracellular NaCl transport in this nephron segment, we generated knockout mice lacking claudin-2 (Cldn2(-/-)). The Cldn2(-/-) mice displayed normal appearance, activity, growth, and behavior. Light microscopy revealed no gross histological abnormalities in the Cldn2(-/-) kidney. Ultrathin section and freeze-fracture replica electron microscopy revealed that, similar to those of wild types, the proximal tubules of Cldn2(-/-) mice were characterized by poorly developed tight junctions with one or two continuous tight junction strands. In contrast, studies in isolated, perfused S2 segments of proximal tubules showed that net transepithelial reabsorption of Na(+), Cl(-), and water was significantly decreased in Cldn2(-/-) mice and that there was an increase in paracellular shunt resistance without affecting the apical or basolateral membrane resistances. Moreover, deletion of claudin-2 caused a loss of cation (Na(+)) selectivity and therefore relative anion (Cl(-)) selectivity in the proximal tubule paracellular pathway. With free access to water and food, fractional Na(+) and Cl(-) excretions in Cldn2(-/-) mice were similar to those in wild types, but both were greater in Cldn2(-/-) mice after i.v. administration of 2% NaCl. We conclude that claudin-2 constitutes leaky and cation (Na(+))-selective paracellular channels within tight junctions of mouse proximal tubules.

LIM domain-containing adaptor, leupaxin, localizes in focal adhesion and suppresses the integrin-induced tyrosine phosphorylation of paxillin.

Focal adhesion (FA) consists of multiple cellular proteins including paxillin and serves as a center for adhesion-mediated signaling. The assembly and disassembly of FAs is regulated by locally produced intracellular signals, and tyrosine phosphorylation of paxillin has been implicated in this process. A Lin-11 Isl-1 Mec-3 (LIM) domain-containing adaptor protein, leupaxin, a member of the paxillin family, is expressed in leukocytes as well as in certain cancer cells, and shares overall structural characteristics with paxillin. However, it remains unknown whether leupaxin and paxillin cooperate with or antagonize each other in integrin signaling. Here we show that leupaxin potently represses the tyrosine phosphorylation of paxillin. When expressed in mouse thymoma BW5147 cells bound to ICAM-1, leupaxin accumulated in FA-like patches in the cell periphery. When expressed in NIH3T3 and HEK293T cells, leupaxin localized to FAs upon cell adhesion to fibronectin and strongly suppressed the integrin-induced tyrosine phosphorylation of paxillin. In integrin-stimulated HEK293T cells, leupaxin's LIM3 domain appeared essential for selective FA localization and the suppression of paxillin tyrosine phosphorylation. Leupaxin's LD3 motif, which is critical for stable association with FAK, was dispensable for leupaxin's suppressive ability. In addition, leupaxin reduced the spreading of NIH3T3 cells on fibronectin, which required both the LD3 motif and LIM3 domain. When expressed in human leukocytic K562 cells, leupaxin significantly suppressed integrin alpha5beta1-mediated cell adhesion to fibronectin and the tyrosine phosphorylation of paxillin. These findings indicate that leupaxin functions as a paxillin counterpart that potently suppresses the tyrosine phosphorylation of paxillin during integrin signaling.

The role of claudin-based tight junctions in morphogenesis.

In morphogenetic events during developmental processes, the hydrostatic pressure caused by fluid accumulation is thought to be an important factor determining the shape of epithelial structures. Recent studies using loss of function of claudins have demonstrated that tight junctions, by their control of fluid accumulation, have crucial roles in morphogenesis. Interestingly, both the barrier and the channel functions of tight junctions appear to contribute to morphogenesis.

Megaintestine in claudin-15-deficient mice.

BACKGROUND & AIMS: Claudins, the major components of tight junction (TJ) strands, which form paracellular barriers, consist of 24 family members, the combination of which determines the properties of TJ-based paracellular barriers. Here, we generated claudin-15-deficient (Cldn15(-/-)) mice to examine the ubiquitously expressed functions of claudin-15. METHODS: We generated Cldn15(-/-) mice by the conventional gene-targeting strategy. Because the upper small intestine was enlarged in Cldn15(-/-) mice, we analyzed the phenotype from various angles regarding histology, physiology, and cell biology. RESULTS: Cldn15(-/-) mice were born and grew normally with an enlarged upper small intestinal phenotype, megaintestine. Deficiency of claudin-15 did not cause a compensatory increase in the background expression of other types of claudins, claudin-1, -2, -3, -4, -7, -12, -18, -20, and -23, in the small intestine. Cldn15(-/-) mice showed enhanced proliferation of normal cryptic cells after weaning without diseased states such as polyps or cancer, resulting in megaintestine, in which the upper small intestine was approximately 2 times larger than normal in length and diameter. The number of transit-amplifying cells in crypts increased approximately 2-fold. Freeze-fracture electron microscopy revealed that deficiency of claudin-15 decreased the number of TJ strands, although the electric conductance was decreased in distal segments in Cldn15(-/-) jejunum, as compared with Cldn15(+/+) littermates. CONCLUSIONS: Based on the specific roles of claudins in paracellular barrier formation without any direct role in cell proliferation, as previously shown in cultured epithelial cells, we propose that claudin-15-based formation of TJs to organize the microenvironment including ion conductance is important for normal-sized morphogenesis of the small intestine.