The Balance of CD8-Positive T Cells and PD-L1 Expression in the Myocardium Predicts Prognosis in Lymphocytic Fulminant Myocarditis.

INTRODUCTION: The clinical significance and prognostic value of T cell involvement and programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) have not been established in lymphocytic fulminant myocarditis (FM). We investigated the prognostic impact of the number of CD4+, CD8+, FoxP3+, and PD-1+ T cells, as well as PD-L1 expression, in cardiomyocytes in lymphocytic FM. METHODS: This is a single-center observational cohort study. Myocardial tissue was obtained from 16 consecutive patients at lymphocytic FM onset. The median follow-up was 140 days. Cardiac events were defined as a composite of cardiac death and left ventricular-assist device implantation. CD4, CD8, FoxP3, PD-1, and PD-L1 immunostaining were performed on myocardial specimens. RESULTS: The median age of the patients was 52 years (seven men and nine women). There was no significant difference in the number of CD4+ cells. The number of CD8+ cells and the CD8+/CD4+ T cell ratio were higher in the cardiac event group (Event+) than in the group without cardiac events (Event-) (p = 0.048 and p = 0.022, respectively). The number of FoxP3+ T cells was higher in the Event+ group (p = 0.049). Although there was no difference in the number of PD-1+ cells, cardiomyocyte PD-L1 expression was higher in the Event+ group (p = 0.112). Event-free survival was worse in the group with a high CD8+ cell count (p = 0.012) and high PD-L1 expression (p = 0.049). When divided into three groups based on the number of CD8+ cells and PD-L1 expression (CD8highPD-L1high [n = 8], CD8lowPD-L1high [n = 1], and CD8lowPD-L1low [n = 7]), the CD8highPD-L1high group demonstrated the worst event-free survival, while the CD8lowPD-L1high group had a favorable prognosis without cardiac events (p = 0.041). CONCLUSION: High myocardial expression of CD8+ T cells and PD-L1 may predict a poor prognosis in lymphocytic FM.

Effect of eribulin on epithelial-mesenchymal transition plasticity in metastatic breast cancer: An exploratory, prospective study.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in cancer metastasis and treatment resistance, which worsens prognosis. In phase III trials, eribulin improved overall survival in metastatic breast cancer (MBC) patients. In preclinical studies, eribulin suppressed EMT. However, clinical data on the use of eribulin for MBC patients are limited. In this exploratory, prospective study, we examined the effect of eribulin on EMT in MBC patients. Twenty-two patients aged 44-82 years with recurrent breast cancer or MBC were treated with eribulin. Breast cancer tissue samples were obtained before treatment and on Day 15 ± 5 of the first cycle of eribulin treatment. EMT markers (E-cadherin, claudin-3, vimentin, and N-cadherin) were analyzed using western blotting. EMT changes were evaluated based on the ratio of epithelial to mesenchymal markers before and after treatment in individual tumors. E-cadherin/vimentin, claudin-3/vimentin, E-cadherin/N-cadherin, and claudin-3/N-cadherin ratios were significantly higher after treatment (p = .007, p = .005, p = .006, and p = .011, respectively). Based on E-cadherin/vimentin, 65.0% of tumors shifted to an epithelial phenotype, as compared to 66.7% based on claudin-3/vimentin, 84.6% based on E-cadherin/N-cadherin, and 71.4% based on claudin-3/N-cadherin ratios. Thus, our results showed that eribulin suppressed EMT in breast cancer tissues.

Nutritional control of thyroid morphogenesis through gastrointestinal hormones.

Developing animals absorb nutrients either through the placenta or from ingested food; however, the mechanisms by which embryos use external nutrients for individual organ morphogenesis remain to be elucidated. In this study, we assessed nutrient-dependent thyroid follicle morphogenesis in Xenopus laevis and investigated the role of secreted gastrointestinal (GI) hormones post-feeding. We found that feeding triggers thyroid follicle formation, and the thyroid cells showed transient inactivation of cell proliferation after feeding. In addition, the thyroid cells with multi-lumina were frequently observed in the fed tadpoles. The expression of the particular GI hormone incretin, glucose-dependent insulinotropic polypeptide (GIP), responded to feeding in the intestines of Xenopus tadpoles. Inhibition of dipeptidyl peptidase 4 (Dpp4), a degradative enzyme of incretin, increased the size of the thyroid follicles by facilitating follicular lumina connection, whereas inhibition of the sodium-glucose cotransporter (SGLT) reversed the effects of Dpp4 inhibition. Furthermore, injection of GIP peptide in unfed tadpoles initiated thyroid follicle formation-without requiring feeding-and injection of an incretin receptor antagonist suppressed follicle enlargement in the fed tadpoles. Lastly, GIP receptor knockout in neonatal mice showed smaller follicles in the thyroid, suggesting that the GI hormone-dependent thyroid morphogenesis is conserved in mammals. In conclusion, our study links external nutrients to thyroid morphogenesis and provides new insights into the function of GI hormone as a regulator of organ morphology in developing animals.

A new primate model of hypophyseal dysfunction.

For pituitary regenerative medicine, the creation of a hypophyseal model in monkeys is necessary to conduct future preclinical studies; however, previous studies reported that hypophysectomy in monkeys is not always safe or satisfactory. This study aimed to create a hypophyseal dysfunction model in a cynomolgus monkey using a safer surgical technique and establish the protocol of pituitary hormone replacement therapy for this model. Surgical resection of the pituitary gland of a 7.8-year-old healthy adult cynomolgus male monkey weighing 5.45 kg was performed to create a hypophyseal dysfunction model for future regenerative studies. Endoscopic transoral transsphenoidal surgery was used to perform hypophysectomy under navigation support. These procedures were useful for confirming total removal of the pituitary gland without additional bone removal and preventing complications such as cerebrospinal fluid leakage. Total removal was confirmed by pathological examination and computed tomography. Hypopituitarism was verified with endocrinological examinations including stimulation tests. Postoperatively, the monkey's general condition of hypopituitarism was treated with hormone replacement therapy, resulting in long-term survival. The success of a minimally invasive and safe surgical method and long-term survival indicate the creation of a hypophyseal dysfunction model in a cynomolgus monkey; hence, this protocol can be employed in the future.

The Daple-CK1ε complex regulates Dvl2 phosphorylation and canonical Wnt signaling.

The canonical Wnt signaling pathway plays a crucial role in embryonic development, tissue homeostasis and cancer progression. The binding of Wnt ligands to their cognate receptors, the Frizzled (Fzd) family of proteins, recruits Dishevelled segment polarity protein (Dvl) to the plasma membrane and induces its phosphorylation via casein kinase 1 (CK1), which leads to the activation of ß-catenin. Previous studies showed that Dishevelled-associating protein with a high frequency of leucine residues (Daple) is an important component of the Wnt signaling pathway and essential for Dvl phosphorylation. However, the mechanism by which Daple promotes CK1-mediated phosphorylation of Dvl is not fully understood. In this study, we found that Daple overexpression induced CK1ε-mediated Dvl2 phosphorylation at threonine 224 (Thr224). A Daple mutant (Daple ΔGCV) that lacks a carboxyl-terminal motif to associate with Dvl, retained the ability to interact with CK1ε, but did not induce Dvl phosphorylation, suggesting the importance of the Daple/Dvl/CK1ε trimeric protein complex. We further found that Thr224 phosphorylation of Dvl was required for full activation of ß-catenin transcriptional activity. Consistent with this, wild-type Daple promoted ß-catenin transcriptional activity, following dissociation of ß-catenin and axin. Finally, Wnt3a stimulation increased the membrane localization of Daple and its association with Dvl, and Daple knockdown attenuated Wnt3a-mediated ß-catenin transcriptional activity. Collectively, these data suggested a essential role of spatial Daple localization in CK1ε-mediated activation of Dvl in the canonical Wnt signaling pathway.

Hydrogen water alleviates obliterative airway disease in mice.

OBJECTIVE: Bronchiolitis obliterans syndrome arising from chronic airway inflammation is a leading cause of death following lung transplantation. Several studies have suggested that inhaled hydrogen can protect lung grafts from ischemia-reperfusion injury via anti-inflammatory and -oxidative mechanisms. We investigated whether molecular hydrogen-saturated water can preserve lung allograft function in a heterotopic tracheal allograft mouse model of obliterative airway disease METHODS: Obliterative airway disease was induced by heterotopically transplanting tracheal allografts from BALB/c donor mice into C57BL/6 recipient mice, which were subsequently administered hydrogen water (10 ppm) or tap water (control group) (n = 6 each) daily without any immunosuppressive treatment. Histological and immunohistochemical analyses were performed on days 7, 14, and 21. RESULTS: Hydrogen water decreased airway occlusion on day 14. No significant histological differences were observed on days 7 or 21. The cluster of differentiation 4/cluster of differentiation 3 ratio in tracheal allografts on day 14 was higher in the hydrogen water group than in control mice. Enzyme-linked immunosorbent assay performed on day 7 revealed that hydrogen water reduced the level of the pro-inflammatory cytokine interleukin-6 and increased that of forkhead box P3 transcription factor, suggesting an enhancement of regulatory T cell activity. CONCLUSIONS: Hydrogen water suppressed the development of mid-term obliterative airway disease in a mouse tracheal allograft model via anti-oxidant and -inflammatory mechanisms and through the activation of Tregs. Thus, hydrogen water is a potential treatment strategy for BOS that can improve the outcome of lung transplant patients.

Meflin-Positive Cancer-Associated Fibroblasts Inhibit Pancreatic Carcinogenesis.

Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. Recent observations in genetically engineered mouse models and clinical studies have suggested that there may exist at least two functionally different populations of CAFs, that is, cancer-promoting CAFs (pCAF) and cancer-restraining CAFs (rCAF). Although various pCAF markers have been identified, the identity of rCAFs remains unknown because of the lack of rCAF-specific marker(s). In this study, we found that Meflin, a glycosylphosphatidylinositol-anchored protein that is a marker of mesenchymal stromal/stem cells and maintains their undifferentiated state, is expressed by pancreatic stellate cells that are a source of CAFs in pancreatic ductal adenocarcinoma (PDAC). In situ hybridization analysis of 71 human PDAC tissues revealed that the infiltration of Meflin-positive CAFs correlated with favorable patient outcome. Consistent herewith, Meflin deficiency led to significant tumor progression with poorly differentiated histology in a PDAC mouse model. Similarly, genetic ablation of Meflin-positive CAFs resulted in poor differentiation of tumors in a syngeneic transplantation model. Conversely, delivery of a Meflin-expressing lentivirus into the tumor stroma or overexpression of Meflin in CAFs suppressed the growth of xenograft tumors. Lineage tracing revealed that Meflin-positive cells gave rise to α-smooth muscle actin-positive CAFs that are positive or negative for Meflin, suggesting a mechanism for generating CAF heterogeneity. Meflin deficiency or low expression resulted in straightened stromal collagen fibers, which represent a signature for aggressive tumors, in mouse or human PDAC tissues, respectively. Together, the data suggest that Meflin is a marker of rCAFs that suppress PDAC progression. SIGNIFICANCE: Meflin marks and functionally contributes to a subset of cancer-associated fibroblasts that exert antitumoral effects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5367/F1.large.jpg.

Mouse NC/Jic strain provides novel insights into host genetic factors for malaria research.

Malaria is caused by Plasmodium parasites and is one of the most life-threatening infectious diseases in humans. Infection can result in severe complications such as cerebral malaria, acute lung injury/acute respiratory distress syndrome, and acute renal injury. These complications are mainly caused by P. falciparum infection and are major causes of death associated with malaria. There are a few species of rodent-infective malaria parasites, and mice infected with such parasites are now widely used for screening candidate drugs and vaccines and for studying host immune responses and pathogenesis associated with disease-related complications. We found that mice of the NC/Jic strain infected with rodent malarial parasites exhibit distinctive disease-related complications such as cerebral malaria and nephrotic syndrome, in addition to a rapid increase in parasitemia. Here, we focus on the analysis of host genetic factors that affect malarial pathogenesis and describe the characteristic features, utility, and future prospects for exploitation of the NC/Jic strain as a novel mouse model for malaria research.

Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections.

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is phosphorylated by both receptor and non-receptor tyrosine kinases at tyrosine 1798. Omori et al. developed site- and phosphorylation status-specific antibodies against human girdin at tyrosine-1798 (pY1798), which specifically bind to phosphorylated tyrosine-1798, but not to unphosphorylated tyrosine-1798. pY1798 antibodies have been used to specifically label tuft cells (TCs) that are present in mammalian gastrointestinal tissues, but the function of these cells is unclear. This protocol allows the robust visualization of TCs in the jejunum using pY1798 antibodies and immunofluorescence. To ensure successful and simple TC visualization, this protocol includes two histological techniques: production of free-floating cryosections from gelatin-filled jejunum tissue, and low-temperature antigen retrieval at 50 °C for 3 h. Filling the jejunum with gelatin maintains the shape of free-floating sections throughout the staining procedure, whereas low-temperature antigen retrieval ensures robust signals from TCs. Successful use of this protocol results in pY1798 staining of TCs distributed from villus tip to crypt. Stained TCs have a spool-shaped soma and fluorescent signals condense at the lumenal tip, which corresponds to the protruding 'tuft.' Phalloidin staining colocalized with pY1798-positive TCs at the thickened brush border, and corresponds to a rootlet mass extending from the TC tuft. This protocol could be used to examine TCs in human biopsy samples collected with gastrointestinal endoscopes. Furthermore, TCs were recently reported to accumulate following parasite infection in mice, suggesting that this protocol could have applications for diagnosis of parasite infections in the human gut.

Development of a method to preliminarily embed tissue samples using low melting temperature fish gelatin before sectioning: A technical note.

Embedding of tissue samples that maintains a desired orientation is critical for preparing sections suitable for diagnosis and study objectives. Methods to prepare tissue sections include: (i) paraffin embedding or snap-freezing followed by microtome or cryostat sectioning; and (ii) agarose embedding followed by cutting on a vibrating microslicer. Although these methods are useful for routine laboratory work, preparation of small and fragile tissues such as mouse organs, small human biopsy samples, and cultured floating spheres is difficult and requires special skills. In particular, tissue specimen orientation can be lost during embedding in molds and subsequent sectioning. Here, we developed a method using low melting temperature (LM) gelatin either alone or mixed with agarose to preliminarily embed collected tissues that are either prefixed or unfixed, followed by conventional fixation, paraffin embedding, freezing, and sectioning. The advantage of the method is that the LM gelatin and its mixture with agarose can be handled at room temperature but quickly hardens at 4°C, which allows embedding, trimming, and arranging of small and fragile tissues in a desired orientation and are compatible with traditional stainings. Thus, this method can have various laboratory applications and can be modified according to the needs of each laboratory.

Significance of perivascular tumour cells defined by CD109 expression in progression of glioma.

In the progression of glioma, tumour cells often exploit the perivascular microenvironment to promote their survival and resistance to conventional therapies. Some of these cells are considered to be brain tumour stem cells (BTSCs); however, the molecular nature of perivascular tumour cells has not been specifically clarified because of the complexity of glioma. Here, we identified CD109, a glycosylphosphatidylinositol-anchored protein and regulator of multiple signalling pathways, as a critical regulator of the progression of lower-grade glioma (World Health Organization grade II/III) by clinicopathological and whole-genome sequencing analysis of tissues from human glioma. The importance of CD109-positive perivascular tumour cells was confirmed not only in human lower-grade glioma tissues but also in a mouse model that recapitulated human glioma. Intriguingly, BTSCs isolated from mouse glioma expressed high levels of CD109. CD109-positive BTSCs exerted a proliferative effect on differentiated glioma cells treated with temozolomide. These data reveal the significance of tumour cells that populate perivascular regions during glioma progression, and indicate that CD109 is a potential therapeutic target for the disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Daple Coordinates Planar Polarized Microtubule Dynamics in Ependymal Cells and Contributes to Hydrocephalus.

Motile cilia in ependymal cells, which line the cerebral ventricles, exhibit a coordinated beating motion that drives directional cerebrospinal fluid (CSF) flow and guides neuroblast migration. At the apical cortex of these multi-ciliated cells, asymmetric localization of planar cell polarity (PCP) proteins is required for the planar polarization of microtubule dynamics, which coordinates cilia orientation. Daple is a disheveled-associating protein that controls the non-canonical Wnt signaling pathway and cell motility. Here, we show that Daple-deficient mice present hydrocephalus and their ependymal cilia lack coordinated orientation. Daple regulates microtubule dynamics at the anterior side of ependymal cells, which in turn orients the cilial basal bodies required for the directional cerebrospinal fluid flow. These results demonstrate an important role for Daple in planar polarity in motile cilia and provide a framework for understanding the mechanisms and functions of planar polarization in the ependymal cells.

Significance of low mTORC1 activity in defining the characteristics of brain tumor stem cells.

Background: The significance of mammalian target of rapamycin complex 1 (mTORC1) activity in the maintenance of cancer stem cells (CSCs) remains controversial. Previous findings showed that mTORC1 activation depleted the population of leukemia stem cells in leukemia, while maintaining the stemness in pancreatic CSCs. The purpose of this study was to examine the currently unknown role and significance of mTORC1 activity in brain tumor stem cells (BTSCs). Methods: Basal mTORC1 activity and its kinetics were investigated in BTSC clones isolated from patients with glioblastoma and their differentiated progenies (DIFFs). The effects of nutrient deprivation and the mTORC1 inhibitors on cell proliferation were compared between the BTSCs and DIFFs. Tissue sections from patients with brain gliomas were examined for expression of BTSC markers and mTORC1 activity by immunohistochemistry. Results: BTSCs presented lower basal mTORC1 activity under each culture condition tested and a more rapid decline of mTORC1 activity after nutrient deprivation than observed in DIFFs. The self-renewal capacity of BTSCs was unaffected by mTORC1 inhibition, whereas it effectively suppressed DIFF proliferation. In agreement, immunohistochemical staining of glioma tissues revealed low mTORC1 activity in tumor cells positive for BTSC markers. In in vitro culture, BTSCs exhibited resistance to the antitumor agent temozolomide. Conclusions: Our findings indicated the importance of low mTORC1 activity in maintaining the undifferentiated state of BTSCs, implicating the relevance of manipulating mTORC1 activity when developing future strategies that target BTSCs.

Tyrosine Phosphorylation of an Actin-Binding Protein Girdin Specifically Marks Tuft Cells in Human and Mouse Gut.

Tuft cells (TCs) are minor components of gastrointestinal epithelia, characterized by apical tufts and spool-shaped somas. The lack of reliable TC-markers has hindered the elucidation of its role. We developed site-specific and phosphorylation-status-specific antibodies against Girdin at tyrosine-1798 (pY1798) and found pY1798 immunostaining of mouse jejunum clearly depicted epithelial cells closely resembling TCs. This study aimed to validate pY1798 as a TC-marker. Double-fluorescence staining of intestines was performed with pY1798 and known TC-markers, for example, hematopoietic-prostaglandin-D-synthase (HPGDS), or doublecortin-like kinase 1 (DCLK1). Odds ratios (ORs) were calculated from cell counts to determine whether two markers were attracting (OR<1) or repelling (OR>1). In consequence, pY1798 signals strongly attracted those of known TC-markers. ORs for HPGDS in mouse stomach, small intestine, and colon were 0 for all, and 0.08 for DCLK1 in human small intestine. pY1798-positive cells in jejunum were distinct from other minor epithelial cells, including goblet, Paneth, and neuroendocrine cells. Thus, pY1798 was validated as a TC-marker. Interestingly, apoptosis inducers significantly increased relative TC frequencies despite the absence of proliferation at baseline. In conclusion, pY1798 is a novel TC-marker. Selective tyrosine phosphorylation and possible resistance to apoptosis inducers implied the activation of certain kinase(s) in TCs, which may become a clue to elucidate the enigmatic roles of TCs. .

High-fat diet feeding promotes stemness and precancerous changes in murine gastric mucosa mediated by leptin receptor signaling pathway.

Obesity increases the risk for gastric cancers. However, the occurrence and mechanisms of precancerous atrophic gastritis induced by high-fat diet (HFD) remain unclear. Here, we show that HFD-associated lipotoxicity induces precancerous lesions that are accompanied by the disruption of organelle homeostasis, tissue integrity, and deregulated expression of stemness genes in the gastric epithelium mediated by leptin receptor (ObR) signaling. Following HFD feeding, ectopic fat accumulated and expression of LAMP2A in lysosome and COX IV in mitochondria increased in the gastric mucosa. HFD feeding also led to enhanced expression of activated-Notch1 and stem cell markers Lgr5, CD44, and EpCAM. In addition, HFD-fed mice showed intracellular ß-catenin accumulation in the gastric mucosa with increased expression of its target genes, Nanog, Oct4, and c-Myc. These observations were abrogated in the leptin-deficient ob/ob mice and ObR-mutated db/db mice, indicating that these HFD-induced changes were responsible for effects downstream of the ObR. Consistent with this, the expression of the Class IA and III PI3Ks was increased following ObR activation in the gastric mucosa of HFD-fed mice. Together, these results suggest that HFD-induced lipotoxicity and deregulated organelle biosynthesis confer cancer stem cell-like properties to the gastric mucosa via signaling pathway mediated by leptin, PI3K and ß-catenin.

Desmin phosphorylation by Cdk1 is required for efficient separation of desmin intermediate filaments in mitosis and detected in murine embryonic/newborn muscle and human rhabdomyosarcoma tissues.

Desmin is a type III intermediate filament (IF) component protein expressed specifically in muscular cells. Desmin is phosphorylated by Aurora-B and Rho-kinase specifically at the cleavage furrow from anaphase to telophase. The disturbance of this phosphorylation results in the formation of unusual long bridge-like IF structures (IF-bridge) between two post-mitotic (daughter) cells. Here, we report that desmin also serves as an excellent substrate for the other type of mitotic kinase, Cdk1. Desmin phosphorylation by Cdk1 loses its ability to form IFs in vitro. We have identified Ser6, Ser27, and Ser31 on murine desmin as phosphorylation sites for Cdk1. Using a site- and phosphorylation-state-specific antibody for Ser31 on desmin, we have demonstrated that Cdk1 phosphorylates desmin in entire cytoplasm from prometaphase to metaphase. Desmin mutations at Cdk1 sites exhibit IF-bridge phenotype, the frequency of which is significantly increased by the addition of Aurora-B and Rho-kinase site mutations to Cdk1 site mutations. In addition, Cdk1-induced desmin phosphorylation is detected in mitotic muscular cells of murine embryonic/newborn muscles and human rhabdomyosarcoma specimens. Therefore, Cdk1-induced desmin phosphorylation is required for efficient separation of desmin-IFs and generally detected in muscular mitotic cells in vivo.

Role for Daple in non-canonical Wnt signaling during gastric cancer invasion and metastasis.

In gastric cancer, the non-canonical Wnt signaling pathway is activated by Wnt5a, which has a critical role in disease outcome. Previous studies have shown that Wnt5a mediates the expression of the extracellular matrix protein laminin γ2 through Rac and JNK activation to promote gastric cancer progression. However, the mechanism of this regulatory pathway has not been completely addressed. The scaffold protein Dvl is a major component of the Wnt signaling pathway. Here, we show that Dvl-associating protein with a high frequency of leucine residues (Daple) mediates Wnt5a-induced laminin γ2 expression. Immunohistochemical analysis showed marked expression of Daple in advanced clinical stages of gastric cancer, where it highly correlated with Wnt5a/b and laminin γ2 expression, the depth of wall invasion, and the frequency of lymph node metastasis. In cultured cancer cells, Daple depletion led to the suppression of Wnt5a-induced Rac and JNK activation, laminin γ2 expression, and cell migration and invasion. Accordingly, Daple depletion also suppressed liver metastasis in a mouse xenograft model of gastric cancer. These results suggest that the non-canonical Wnt signaling pathway contributes to gastric cancer progression at least in part via Daple, which provides a new therapeutic opportunity for the treatment of the disease.

Akt-Girdin signaling in cancer-associated fibroblasts contributes to tumor progression.

PI3K-Akt signaling is critical for the development, progression, and metastasis of malignant tumors, but its role in the tumor microenvironment has been relatively little studied. Here, we report that the Akt substrate Girdin, an actin-binding protein that regulates cell migration, is expressed and activated by Akt phosphorylation in cancer-associated fibroblasts (CAF) and blood vessels within the tumor microenvironment. Lewis lung tumors grafted into mice defective in Akt-mediated Girdin phosphorylation (SA transgenic mice) exhibited a decrease in both CAF infiltration and tumor growth, compared with wild-type (WT) host control animals. Contrasting with the findings of other studies, we found that Akt-dependent phosphorylation of Girdin was not a rate-limiting step in the growth of endothelial cells. In addition, Lewis lung tumors displayed limited outgrowth when cotransplanted with CAF derived from tumor-bearing SA transgenic mice, compared with CAF derived from tumor-bearing WT mice. Collectively, our results revealed a role for Akt-mediated Girdin phosphorylation in CAF during tumor progression, highlighting the need to inhibit Akt function in both tumor cells and cells that comprise the tumor microenvironment.

Girdin is phosphorylated on tyrosine 1798 when associated with structures required for migration.

The mammalian protein Girdin interacts with several key molecules such as actin, and it functions as a regulator of the cytoskeleton. Silencing of Girdin mRNA results in defective migration in a variety of cultured cells. Moreover, knockout of Girdin causes phenotypes related to defective migration, including hypoplasia of olfactory bulbs and a widened rostral migratory stream (RMS) in mice. To elucidate the molecular basis underlying cellular migration, we generated site- and phosphorylation state-specific antibodies against human Girdin peptides carrying four putative phosphorylation sites (serine1386 [S1386], S1416, tyrosine1764 [Y1764] and Y1798) that had been identified by mutagenesis analyses or mass spectrometric studies. We found that these residues were phosphorylated in an epidermal growth factor (EGF)-dependent manner. Among the four antibodies we developed, the antibody that targeted Girdin when phosphorylated at Y1798 (pY1798) worked well for immunohistochemistry of paraffin-embedded tissues as well as for cultured cells. Immunocytochemistry of HEK293FT cells transfected with an EGF receptor expression plasmid exhibited punctate signals with pY1798. These signals colocalized with those of endocytosed EGF receptors after EGF stimulation. Signals from pY1798 were also observed on lamellipodia, filopodia, focal adhesion and stress fibers in NIH3T3 cells under conventional culture conditions. Immunohistochemistry of paraffin-embedded mouse brain at P14 using anti-pY1798 antibody displayed signals at the hilum-side (internal side) of the dentate gyrus of the hippocampus, the RMS, the accessory olfactory bulb and the olfactory bulb in which Girdin expression was detected. Primary culture of RMS neurons showed punctate signals of pY1798 at the tips of leading processes as well as in the cytoplasm, whereas no signals were observed when neurons were treated with Src inhibitor, PP2. Our data revealed the changes in the phosphorylation status of Y1798 in Girdin when it associated with migration-related structures in vitro and in vivo.

The REV7 subunit of DNA polymerase ζ is essential for primordial germ cell maintenance in the mouse.

REV7 (also known as MAD2L2 and MAD2B) is involved in DNA repair, cell cycle regulation, gene expression, and carcinogenesis. In vitro studies show that REV7 interacts with several proteins and regulates their function. It has been reported that human REV7 is highly expressed in the adult testis by Northern blot analysis. However, the significance of REV7 in mammalian development has not been elucidated. Here, we present analyses of REV7-deficient (Rev7(-/-)) mice to clarify the significance of Rev7 in mouse development. In WT mice (Rev7(+/+)), Rev7 expression was ubiquitously observed in the embryo and confined to germ cells in the testes after birth. Rev7(-/-) mice exhibited growth retardation and a partial embryonic lethal phenotype. Mice that survived to adulthood were infertile in both sexes and showed germ cell aplasia in the testes and ovaries. Analyses of Rev7(-/-) embryos revealed that primordial germ cells (PGCs) were present at embryonic day 8.5 (E8.5). However, progressive loss of PGCs was observed during migration, and PGCs were absent in the genital ridges at E13.5. An increase of apoptotic cells was detected not only among PGCs but also in the forebrain of the Rev7(-/-) embryo, whereas cell proliferation was unaffected. Moreover, DNA damage accumulation and increased levels of histone methylation were detected in Rev7(-/-) embryos, and expression of Oct4 and Nanog was deregulated by REV7 deficiency at E8.5. These findings indicate that Rev7 is essential for PGC maintenance by prevention of apoptotic cell death in the mouse.