Aberrant Nuclear Localization of aPKCλ/ι is Associated With Poorer Prognosis in Uterine Cervical Cancer.

We previously reported that aberrant expression of atypical protein kinase C λ/ι (aPKCλ/ι) in low-grade squamous intraepithelial uterine cervix lesions was associated with an increased risk of progression to higher grade. This study aimed to investigate aPKCλ/ι expression patterns in cervical squamous cell carcinoma (SCC) and its association with disease progression. We immunohistochemically assessed aPKCλ/ι expression in 168 SCC samples and 13 normal uterine cervix samples. In 69.0% of SCC cases, aPKCλ/ι was expressed more abundantly than in normal epithelium, but there was no significant association between aPKCλ/ι intensity and disease progression (P=0.087, Cochran-Mantel-Haenszel test). aPKCλ/ι in normal cervical epithelium was confined to the cytoplasm or intercellular junctions. In contrast, aPKCλ/ι was predominantly localized within the nucleus in 36.9% of SCC samples (P<0.001, χ test), and the prevalence was significantly increased relative to advanced tumor stage (P<0.001, Cochran-Mantel-Haenszel test). Moreover, patients with SCC with aPKCλ/ι nuclear localization had worse prognoses than those with cytoplasmic localization (P<0.001, log-rank test). aPKCλ/ι localization differed between the intraepithelial lesion and adjacent invasive cancer in 40% of cases, while the expression pattern was similar between primary and matched metastatic tumors. In conclusion, aPKCλ/ι nuclear localization in cervical cancer is associated with tumor progression and worse prognosis. This is the first report to show aberrant nuclear aPKCλ/ι localization in a subgroup of cervical cancer patients and its association with worse prognosis.

AKT capture by feline leukemia virus.

Oncogene-containing retroviruses are generated by recombination events between viral and cellular sequences, a phenomenon called "oncogene capture". The captured cellular genes, referred to as "v-onc" genes, then acquire new oncogenic properties. We report a novel feline leukemia virus (FeLV), designated "FeLV-AKT", that has captured feline c-AKT1 in feline lymphoma. FeLV-AKT contains a gag-AKT fusion gene that encodes the myristoylated Gag matrix protein and the kinase domain of feline c-AKT1, but not its pleckstrin homology domain. Therefore, it differs structurally from the v-Akt gene of murine retrovirus AKT8. AKT may be involved in the mechanisms underlying malignant diseases in cats.

DAAM1 and DAAM2 are co-required for myocardial maturation and sarcomere assembly.

Wnt ligands regulate heart morphogenesis but the underlying mechanisms remain unclear. Two Formin-related proteins, DAAM1 and 2, were previously found to bind the Wnt effector Disheveled. Here, since DAAM1 and 2 nucleate actin and mediate Wnt-induced cytoskeletal changes, a floxed-allele of Daam1 was used to disrupt its function specifically in the myocardium and investigate Wnt-associated pathways. Homozygous Daam1 conditional knockout (CKO) mice were viable but had misshapen hearts and poor cardiac function. The defects in Daam1 CKO mice were observed by mid-gestation and were associated with a loss of protrusions from cardiomyocytes invading the outflow tract. Further, these mice exhibited noncompaction cardiomyopathy (NCM) and deranged cardiomyocyte polarity. Interestingly, Daam1 CKO mice that were also homozygous for an insertion disrupting Daam2 (DKO) had stronger NCM, severely reduced cardiac function, disrupted sarcomere structure, and increased myocardial proliferation, suggesting that DAAM1 and DAAM2 have redundant functions. While RhoA was unaffected in the hearts of Daam1/2 DKO mice, AKT activity was lower than in controls, raising the issue of whether DAAM1/2 are only mediating Wnt signaling. Daam1-floxed mice were thus bred to Wnt5a null mice to identify genetic interactions. The hearts of Daam1 CKO mice that were also heterozygous for the null allele of Wnt5a had stronger NCM and more severe loss of cardiac function than Daam1 CKO mice, consistent with DAAM1 and Wnt5a acting in a common pathway. However, deleting Daam1 further disrupted Wnt5a homozygous-null hearts, suggesting that DAAM1 also has Wnt5a-independent roles in cardiac development.

Aberrant Expression of the Cell Polarity Regulator aPKCλ/ι is Associated With Disease Progression in Cervical Intraepithelial Neoplasia (CIN): A Possible Marker for Predicting CIN Prognosis.

Atypical protein kinase C λ/ι (aPKCλ/ι) is a regulator of epithelial cellular polarity. It is also overexpressed in several cancers and functions in cell proliferation and invasion. Therefore, we hypothesized that aPKCλ/ι may be involved in development and progression of cervical intraepithelial neoplasia (CIN), the precancerous disease of cervical cancer induced by human papillomavirus. To do this, we investigated the relationship between aPKCλ/ι expression and CIN. aPKCλ/ι expression level and subcellular localization were assessed in 192 CIN biopsy samples and 13 normal epithelial samples using immunohistochemistry. aPKCλ/ι overexpression (normal epithelium, 7.7%; CIN1, 41.7%; CIN2/3, 76.4%) and aPKCλ/ι nuclear localization (normal epithelium, 0.0%; CIN1, 36.9%; CIN2/3, 78.7%) were higher in CIN samples than normal samples (P<0.05), suggesting that CIN grade is related to aPKCλ/ι overexpression and nuclear localization. Then, 140 CIN cases were retrospectively analyzed for 4-yr cumulative disease progression and regression rates using the Cox proportional hazards model. CIN1 cases with aPKCλ/ι overexpression or aPKCλ/ι nuclear localization had a higher progression rate than CIN1 cases with normal aPKCλ/ι expression levels or cytoplasmic localization (62.5% vs. 9.7% and 63.1% vs. 9.4%, respectively; P<0.001). Multivariate analysis indicated that human papillomavirus types 16 and 18, aPKCλ/ι overexpression (hazard ratio=4.26; 95% confidence interval, 1.50-12.1; P=0.007), and aPKCλ/ι nuclear localization (hazard ratio=3.59; 95% confidence interval, 1.24-10.4; P=0.019) were independent risk factors for CIN1 progression. In conclusion, aPKCλ/ι could be useful for the therapeutic management of patients with CIN, particularly those with non-human papillomavirus 16/18 types.

Planar polarity of multiciliated ependymal cells involves the anterior migration of basal bodies regulated by non-muscle myosin II.

Motile cilia generate constant fluid flow over epithelial tissue, and thereby influence diverse physiological processes. Such functions of ciliated cells depend on the planar polarity of the cilia and on their basal bodies being oriented in the downstream direction of fluid flow. Recently, another type of basal body planar polarity, characterized by the anterior localization of the basal bodies in individual cells, was reported in the multiciliated ependymal cells that line the surface of brain ventricles. However, little is known about the cellular and molecular mechanisms by which this polarity is established. Here, we report in mice that basal bodies move in the apical cell membrane during differentiation to accumulate in the anterior region of ependymal cells. The planar cell polarity signaling pathway influences basal body orientation, but not their anterior migration, in the neonatal brain. Moreover, we show by pharmacological and genetic studies that non-muscle myosin II is a key regulator of this distribution of basal bodies. This study demonstrates that the orientation and distribution of basal bodies occur by distinct mechanisms.

Impact of adrenomedullin on dextran sulfate sodium-induced inflammatory colitis in mice: insights from in vitro and in vivo experimental studies.

BACKGROUND: Although adrenomedullin (AM) is known to ameliorate inflammatory processes, few data exist regarding the effect of AM on inflammatory colitis. Therefore, we examined the effect of AM on inflammatory response in vitro and in vivo colitis model. METHODS: In mice experimental colitis induced by 3% dextran sulfate sodium (DSS) in drinking water for 7 days, AM with 225-900 µg/kg in 0.5 ml of saline or saline alone were given intraperitoneally once a day. In the in vitro experiment, we determined the cytokine response in THP-1 cell activated by lipopolysaccharide with or without AM of 10 nM. Additionally, we performed wound healing assay in Caco-2 cell interfered by DSS with or without AM of 100 nM. RESULTS: In the colitis model, AM significantly reduced the disease activity index, histological score, and local production of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in accordance with reduction of serum amyloid A levels. Secretion of TNF-α in lipopolysaccharide-stimulated THP-1 cells was significantly reduced in the presence of AM. The distance of wound healing interfered by 0.25% DSS was significantly improved in the presence of AM of 100 nM. CONCLUSIONS: These results demonstrate that AM could ameliorate DSS-induced experimental colitis possibly through suppression of systemic and local production of cytokines such as TNF-α, associated with acceleration of ulcer reepithelialization and colon tissue regeneration.

Placental defects lead to embryonic lethality in mice lacking the Formin and PCP proteins Daam1 and Daam2.

The actin cytoskeleton plays a central role in establishing cell polarity and shape during embryonic morphogenesis. Daam1, a member of the Formin family of actin cytoskeleton regulators, is a Dvl2-binding protein that functions in the Wnt/Planar Cell Polarity (PCP) pathway. To examine the role of the Daam proteins in mammalian development, we generated Daam-deficient mice by gene targeting and found that Daam1, but not Daam2, is necessary for fetal survival. Embryonic development of Daam1 mutants was delayed most likely due to functional defects in the labyrinthine layer of the placenta. Examination of Daam2 and Daam1/2 double mutants revealed that Daam1 and Daam2 are functionally redundant during placental development. Of note, neural tube closure defects (NTD), which are observed in several mammalian PCP mutants, are not observed in Wnt5a or Daam1 single mutants, but arise in Daam1;Wnt5a double mutants. These findings demonstrate a unique function for Daam genes in placental development and are consistent with a role for Daam1 in the Wnt/PCP pathway in mammals.

Shank2 Binds to aPKC and Controls Tight Junction Formation with Rap1 Signaling during Establishment of Epithelial Cell Polarity.

Epithelial cells establish apicobasal polarity by forming tight junctions (TJs) at the apical-lateral boundary, which play fundamental roles in physiological functions. An evolutionarily conserved atypical protein kinase C (aPKC)-partitioning defective (PAR) complex functions as a platform for TJ assembly during cell polarity establishment. However, how this complex converts the spatial cues into a subsequent active unit is unclear. Here, we identify an epithelial isoform of Shank2 as a mediator of the aPKC-PAR complex. Shank2 binds to and colocalizes with aPKC at apical junctional regions of polarized epithelial cells. Shank2 knockdown results in defects in TJ formation. Mechanistically, we find that the N-terminal SPN domain is required for the junctional localization of Shank2 and binds to the active form of Rap1 small GTPase, which is involved in TJ formation. Our findings suggest that a close physical and functional relationship between aPKC and Shank2-active Rap1 signaling serves as the platform for TJ assembly to regulate epithelial cell polarity.

Expression of cAMP response element-binding protein in the duct system of the mouse submandibular gland.

The submandibular gland (SMG) of mice shows a marked sexual dimorphism in which a duct portion called the granular convoluted tubule (GCT) is developed preferentially in males during puberty. The administration of testosterone to female mice causes the conversion of striated duct (SD) cells into GCT cells, but the underlying molecular mechanisms are unclear. Cyclic AMP response element-binding protein (CREB) is a transcription factor functioning downstream of a variety of signal transduction pathways. In the present study, we examined the expression, activation and cellular localization of CREB in the mouse SMG using Western blotting and immunohistochemistry. Both total CREB (t-CREB) and phosphorylated CREB (p-CREB) were significantly more abundant in the female than in the male gland and were localized to the nuclei of intercalated duct cells and a subpopulation of SD cells. In contrast, the GCT cells in males were negative for t- and p-CREB. The levels of CREB in the SMG were increased by castration in males and decreased by repeated administration of testosterone to females or castrated males. From 3 h after a single administration of testosterone to females, many SD cells temporarily gained nuclear immunoreactivity for both t- and p-CREB, which was lost as the cells were converted to GCT cells by 24 h. These results suggest the involvement of CREB in the androgen-dependent differentiation of the duct system in the mouse SMG.

Expression of matrix metalloproteinases in the neurogenic niche of the adult monkey hippocampus after ischemia.

Matrix metalloproteinases (MMPs), zinc-dependent endopeptidases capable of remodeling extracellular matrix and regulating cellular signals, have been implicated in various neurological functions and diseases. However, the role of MMPs in the adult neurogenesis still remains to be clarified, particularly in the primate. Here, we studied differential expression of MMP9/2 in the neurogenic niche of the hippocampal dentate gyrus (DG) after transient global brain ischemia in young adult macaque monkeys. Zymography demonstrated biphasic upregulation of MMP9 in acute (Days 1-3) and delayed (Days 7-15) phases of postischemic reaction, whereas the level of MMP2 was elevated only in the delayed phase. Immunofluorescence histochemistry showed that MMP9 and MMP2 colabeled with markers of endothelial cells, astrocytes, and newborn neurons in the subgranular zone (SGZ) of the DG, and also that the percentage of coexpression significantly increased in the delayed postischemic phase, as compared with controls. However, colabeling with different cell selective markers reached its peak at different time points, i.e., with endothelial cells on Day 7, whereas with astrocytes and newborn neurons on Day 15, respectively. MMPs were localized both in the perikarya and dendrites in the newborn neurons. In conclusion, MMP9/2 expression was regulated in a cell- and time-specific manner in hippocampal neurogenic niche of adult primates.

Expression of free fatty acid receptor GPR40 in the neurogenic niche of adult monkey hippocampus.

Polyunsaturated free fatty acids (PUFAs) are known to play critical roles for the development, maintenance, and function of the brain. Recently, we reported that G-protein coupled receptor 40 (GPR40), one type of PUFA receptors, is expressed throughout the adult primate central nervous system including the hippocampus. This opens a possibility that PUFA might act as extracellular signaling molecules at the GPR40 receptor to regulate neuronal function. Here we studied protein expression of GPR40 in the neurogenic niche of the adult monkey hippocampus under normal and postischemic conditions. Confocal laser microscope analysis of immunostained sections revealed GPR40 immunoreactivity in neural progenitors, immature neurons, astrocytes and endothelial cells of the subgranular zone (SGZ) of the dentate gyrus (DG); a well-known neurogenic niche within the adult brain. Immunoblotting analysis showed that the GPR40 protein increased significantly in the second week after global cerebral ischemia as compared with the control. This was compatible with the postischemic increment of GPR40-positive cells in the SGZ as detected by immunofluorescence imaging. Taken together with our previous findings of the SGZ progenitor cell upregulation after ischemia, the present data suggest that PUFA such as docosahexaenoic acid may act via GPR40 to regulate adult hippocampal neurogenesis in primates.

Coexpression of menin and JunD during the duct cell differentiation in mouse submandibular gland.

In the submandibular gland (SMG) of mice, a duct portion called the granular convoluted tubule (GCT) is developed preferentially in males with puberty. This sexual dimorphism is androgen-dependent, but the underlying molecular mechanisms are unclear. We have demonstrated that the expression of a transcription factor JunD is regulated in association with the androgen-induced differentiation of GCT cells from striated duct (SD) cells. Menin, a nuclear protein encoded by the MEN1 tumor-suppressor gene, is known to bind JunD, thereby inhibiting its activity. In the present study, we examined the expression of menin in the mouse SMG by use of Northern blotting, Western blotting, and immunohistochemistry. Immunoreactivity for menin was higher in the female than male gland, and localized to the nuclei of intercalated duct cells and a subpopulation of SD cells. In contrast, GCT cells in males appeared negative for menin. The levels of menin in the SMG were increased with castration in males and decreased by repeated administration of testosterone to females or to castrated males. After a single administration of testosterone to females, many SD cells newly gained nuclear menin, which was lost as the cells converted to GCT cells by 48 hrs. These patterns of the expression and localization of menin were quite similar to those of JunD. Furthermore, the coimmunoprecipitation analysis of the SMG homogenates indicated that menin binds JunD in vivo. The present study suggests that the JunD-menin complex plays significant roles in the androgen-dependent differentiation of the duct system in the mouse SMG.

Cytoplasmic localization of GRHL3 upon epidermal differentiation triggers cell shape change for epithelial morphogenesis.

Epithelial cell shape change is a pivotal driving force for morphogenesis of complex three-dimensional architecture. However, molecular mechanisms triggering shape changes of epithelial cells in the course of growth and differentiation have not been entirely elucidated. Grhl3 plays a crucial role as a downstream transcription factor of Wnt/ß-catenin in epidermal differentiation. Here, we show Grhl3 induced large, mature epidermal cells, enriched with actomyosin networks, from embryoid bodies in vitro. Such epidermal cells were apparently formed by the simultaneous activation of canonical and non-canonical Wnt signaling pathways. A nuclear transcription factor, GRHL3 is localized in the cytoplasm and cell membrane during epidermal differentiation. Subsequently, such extranuclear GRHL3 is essential for the membrane-associated expression of VANGL2 and CELSR1. Cytoplasmic GRHL3, thereby, allows epidermal cells to acquire mechanical properties for changes in epithelial cell shape. Thus, we propose that cytoplasmic localization of GRHL3 upon epidermal differentiation directly triggers epithelial morphogenesis.

Publisher Correction: Cytoplasmic localization of GRHL3 upon epidermal differentiation triggers cell shape change for epithelial morphogenesis.

The original version of this Article contained an error in the labelling of Fig. 4. In panel i, the sixth column was incorrectly labelled as NSC23766 negative, and should have been NSC23766 positive. This has now been corrected in both the PDF and HTML versions of the Article.

Oral Ingestion of Collagen Hydrolysate Leads to the Transportation of Highly Concentrated Gly-Pro-Hyp and Its Hydrolyzed Form of Pro-Hyp into the Bloodstream and Skin.

Collagen hydrolysate is a well-known dietary supplement for the treatment of skin aging; however, its mode of action remains unknown. Previous studies have shown that the oral ingestion of collagen hydrolysate leads to elevated levels of collagen-derived peptides in the blood, but whether these peptides reach the skin remains unclear. Here, we analyzed the plasma concentration of collagen-derived peptides after ingestion of high tripeptide containing collagen hydrolysate in humans. We identified 17 types of collagen-derived peptides transiently, with a particular enrichment in Gly-Pro-Hyp. This was also observed using an in vivo mouse model in the plasma and skin, albeit with a higher enrichment of Pro-Hyp in the skin. Interestingly, this Pro-Hyp enrichment in the skin was derived from Gly-Pro-Hyp hydrolysis, as the administration of pure Gly-Pro-Hyp peptide led to similar results. Therefore, we propose that functional peptides can be transferred to the skin by dietary supplements of collagen.

Function of atypical protein kinase C lambda in differentiating photoreceptors is required for proper lamination of mouse retina.

The photoreceptor is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. To investigate the mechanisms of polarity formation of the photoreceptor cells, we conditionally knocked out atypical protein kinase Clambda (aPKClambda), which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptor cells using the Cre-loxP system. In aPKClambda conditional knock-out (CKO) mice, the photoreceptor cells displayed morphological defects and failed to form ribbon synapses. Intriguingly, lack of aPKClambda in differentiating photoreceptors led to severe laminar disorganization not only in the photoreceptor layer but also in the entire retina. Cell fate determination was not affected by total laminar disorganization. After Cre recombinase began to be expressed in the developing photoreceptors at embryonic day 12.5, both the immature photoreceptors and mitotic progenitors were dispersed throughout the CKO retina. We detected that adherens junction formation between the immature photoreceptors and the progenitors was lost in the CKO retina, whereas it was maintained between the progenitors themselves. These results indicate that the expression of aPKClambda in differentiating photoreceptors is required for total retinal lamination. Our data suggest that properly polarized photoreceptors anchor progenitors at the apical edge of the neural retina, which may be essential for building correct laminar organization of the retina.

Identification and comparative expression analyses of Daam genes in mouse and Xenopus.

During vertebrate embryogenesis, secreted Wnt molecules regulate cell fates by signaling through the canonical pathway mediated by beta-catenin, and regulate planar cell polarity (PCP) and convergent extension movements through alternative pathways. The phosphoprotein Dishevelled (Dsh/Dvl) is a Wnt signal transducer thought to function in all Wnt signaling pathways. A recently identified member of the Formin family, Daam (Dishevelled--associated activator of morphogenesis), regulates the morphogenetic movements of vertebrate gastrulation in a Wnt-dependent manner through direct interactions with Dsh/Dvl and RhoA. We describe two mouse Daam cDNAs, mDaam1 and mDaam2, which encode proteins characterized by highly conserved formin homology domains and which are expressed in complementary patterns during mouse development. Cross-species comparisons indicate that the expression domains of Xenopus Daam1 (XDaam1) mirror mDaam1 expression. Our results demonstrate that Daams are expressed in tissues known to require Wnts and are consistent with Daams being effectors of Wnt signaling during vertebrate development.

Wnt3a links left-right determination with segmentation and anteroposterior axis elongation.

The alignment of the left-right (LR) body axis relative to the anteroposterior (AP) and dorsoventral (DV) axes is central to the organization of the vertebrate body plan and is controlled by the node/organizer. Somitogenesis plays a key role in embryo morphogenesis as a principal component of AP elongation. How morphogenesis is coupled to axis specification is not well understood. We demonstrate that Wnt3a is required for LR asymmetry. Wnt3a activates the Delta/Notch pathway to regulate perinodal expression of the left determinant Nodal, while simultaneously controlling the segmentation clock and the molecular oscillations of the Wnt/beta-catenin and Notch pathways. We provide evidence that Wnt3a, expressed in the primitive streak and dorsal posterior node, acts as a long-range signaling molecule, directly regulating target gene expression throughout the node and presomitic mesoderm. Wnt3a may also modulate the symmetry-breaking activity of mechanosensory cilia in the node. Thus, Wnt3a links the segmentation clock and AP axis elongation with key left-determining events, suggesting that Wnt3a is an integral component of the trunk organizer.

Whole cell segmentation in solid tissue sections.

BACKGROUND: Understanding the cellular and molecular basis of tissue development and function requires analysis of individual cells while in their tissue context. METHODS: We developed software to find the optimum border around each cell (segmentation) from two-dimensional microscopic images of intact tissue. Samples were labeled with a fluorescent cell surface marker so that cell borders were brighter than elsewhere. The optimum border around each cell was defined as the border with an average intensity per unit length greater that any other possible border around that cell, and was calculated using the gray-weighted distance transform. Algorithm initiation requiring the user to mark two points per cell, one approximately in the center and the other on the border, ensured virtually 100% correct segmentation. Thereafter segmentation was automatic. RESULTS: The method was highly robust, because intermittent labeling of the cell borders, diffuse borders, and spurious signals away from the border do not significantly affect the optimum path. Computer-generated cells with increasing levels of added noise showed that the approach was accurate provided the cell could be detected visually. CONCLUSIONS: We have developed a highly robust algorithm for segmenting images of surface-labeled cells, enabling accurate and quantitative analysis of individual cells in tissue.