EGFR-mediated epidermal stem cell motility drives skin regeneration through COL17A1 proteolysis.

Skin regenerative capacity declines with age, but the underlying mechanisms are largely unknown. Here we demonstrate a functional link between epidermal growth factor receptor (EGFR) signaling and type XVII collagen (COL17A1) proteolysis on age-associated alteration of keratinocyte stem cell dynamics in skin regeneration. Live-imaging and computer simulation experiments predicted that human keratinocyte stem cell motility is coupled with self-renewal and epidermal regeneration. Receptor tyrosine kinase array identified the age-associated decline of EGFR signaling in mouse skin wound healing. Culture experiments proved that EGFR activation drives human keratinocyte stem cell motility with increase of COL17A1 by inhibiting its proteolysis through the secretion of tissue inhibitor of metalloproteinases 1 (TIMP1). Intriguingly, COL17A1 directly regulated keratinocyte stem cell motility and collective cell migration by coordinating actin and keratin filament networks. We conclude that EGFR-COL17A1 axis-mediated keratinocyte stem cell motility drives epidermal regeneration, which provides a novel therapeutic approach for age-associated impaired skin regeneration.

NUAK2 localization in normal skin and its expression in a variety of skin tumors with YAP.

BACKGROUND: NUAK2 is a critical gene that participates in the carcinogenesis of various types of cancers including melanomas. However, the expression patterns of NUAK2 in normal skin and in various types of skin tumors have not been fully elucidated to date. OBJECTIVES: To elucidate the distribution and localization of NUAK2 expression in normal skin, and characterize the expression patterns of NUAK2 and YAP in various types of skin tumors. METHODS: In this study, we characterized the expression of NUAK2 in tissues by developing a novel NUAK2-specific monoclonal antibody and using that to determine NUAK2 expression patterns in normal skin and in 155 cases of various types of skin tumors, including extramammary Paget's disease (EMPD), squamous cell carcinoma (SCC), Bowen's disease (BD), actinic keratosis (AK), basal cell carcinoma (BCC) and angiosarcoma (AS). Further, we analyzed the expression patterns of YAP and p-Akt in those tumors. RESULTS: Our analyses revealed that NUAK2 is expressed at high frequencies in EMPD, SCC, BD, AK, BCC and AS. The expression of p-Akt was positively correlated with tumor size in EMPD (P = 0.001). Importantly, the expression of NUAK2 was significantly correlated with YAP in SCC (P = 0.012) and in BD (P = 0.009). CONCLUSIONS: Our results suggest that the YAP-NUAK2 axis has critical importance in the tumorigenesis of SCC and BD, and that therapeutic modalities targeting the YAP-NUAK2 axis may be an effective approach against skin tumors including SCC and BD.

Neuronal stimulation induces autophagy in hippocampal neurons that is involved in AMPA receptor degradation after chemical long-term depression.

Many studies have reported the roles played by regulated proteolysis in synaptic plasticity and memory, but the role of autophagy in neurons remains unclear. In mammalian cells, autophagy functions in the clearance of long-lived proteins and organelles and in adaptation to starvation. In neurons, although autophagy-related proteins (ATGs) are highly expressed, autophagic activity markers, autophagosome (AP) number, and light chain protein 3-II (LC3-II) are low compared with other cell types. In contrast, conditional knock-out of ATG5 or ATG7 in mouse brain causes neurodegeneration and behavioral deficits. Therefore, this study aimed to test whether autophagy is especially regulated in neurons to adapt to brain functions. In cultured rat hippocampal neurons, we found that KCl depolarization transiently increased LC3-II and AP number, which was partially inhibited with APV, an NMDA receptor (NMDAR) inhibitor. Brief low-dose NMDA, a model of chemical long-term depression (chem-LTD), increased LC3-II with a time course coincident with Akt and mammalian target of rapamycin (mTOR) dephosphorylation and degradation of GluR1, an AMPA receptor (AMPAR) subunit. Downstream of NMDAR, the protein phosphatase 1 inhibitor okadaic acid, PTEN inhibitor bpV(HOpic), autophagy inhibitor wortmannin, and short hairpin RNA-mediated knockdown of ATG7 blocked chem-LTD-induced autophagy and partially recovered GluR1 levels. After chem-LTD, GFP-LC3 puncta increased in spines and in dendrites when AP-lysosome fusion was blocked. These results indicate that neuronal stimulation induces NMDAR-dependent autophagy through PI3K-Akt-mTOR pathway inhibition, which may function in AMPAR degradation, thus suggesting autophagy as a contributor to NMDAR-dependent synaptic plasticity and brain functions.

Target chromosomes of inducible deletion by a Cre/inverted loxP system in mouse embryonic stem cells.

Chromosomal deletions are widely involved in serious genetic diseases and in the pathogenesis of cancers. These deletions often generate loss of heterozygosity (LOH) of one of the alleles of a tumor suppressor gene. Because of the technical difficulty inherent in genetic manipulation studies of a chromosome-wide deficiency, it has not been experimentally determined whether chromosome deletions could be a trigger for cancer development. Using the Cre/inverted loxP system, we have developed a chromosome elimination cassette (CEC) that Cre-dependently induces whole or partial deletions of the CEC-tagged chromosomes. Most deletions are usually fatal, but diploid cells carrying small deletions have been obtained from mouse embryonic stem cells carrying a CEC transgene (CEC-ESC). Here, we further isolated various CEC-ESC clones and analyzed CEC integration sites using the fluorescence in-situ hybridization method. In 17 CEC-ESC clones possessing normal chromosome sets, 13 types of chromosomes out of 20 pairs of mouse chromosomes were tagged by CEC. Each CEC-tagged chromosome could become a future target for the creation of a Cre-inducible LOH by a combination of in vitro and in vivo genetic mutation.

Rapid induction of large chromosomal deletions by a Cre/inverted loxP system in mouse ES Cell hybrids.

Loss of heterozygosity by whole or partial loss of chromosomal regions is crucial to genetic disorders, cancers and diseases. It is difficult to analyze the mechanisms of pathogenesis caused by large-scale chromosomal abnormalities due to the extreme rarity of this mutagenesis. Using a Cre/inverted loxP system, we have generated a chromosome elimination cassette (CEC) that induces a selective loss of embryonic-stem-cell-derived chromosomes in undifferentiated embryonic stem cell-somatic cell hybrids. Here, due to the increased expression of Cre, rapid formation of Cre recombination products and immediate loss of CEC-tagged chromosomes were detected by fluorescence in situ hybridization. Cre also initiated intrachromosomal recombination between identical short sequences outside loxP, leading to large chromosomal deletions of CEC-tagged regions. The Cre-mediated antiparallel synapses likely act as a scaffold to bring the identical short sequences into close proximity for recombination. This CEC technology might allow better understanding of the modulator sequences responsible for the tangled structure formation and its solution mechanism, inducing mitotic recombination leading to chromosomal deletions.

Cell fusion-mediated nuclear reprogramming of somatic cells.

Pluripotential embryonic stem cells (ESC) possess a unique property of being able to carry out nuclear reprogramming of somatic nuclei, as shown after cell fusion. The nuclear reprogramming activity has been applied for producing pluripotential stem cells from personal somatic cells through several new technologies, including cytoplasmic cell fusion and ES cell factor introduction. Targeted elimination of ESC-derived chromosome(s) following cell fusion-mediated reprogramming of somatic chromosomes is one of the new technologies for producing personalized stem cells. A universal chromosome elimination cassette (CEC) has been developed that confers drug resistance and GFP (green fluorescent protein) fluorescence, flanked by oppositely orientated loxP sites, to induce sister chromatid recombination and targeted chromosome loss. GFP-positive ESC generated with a CEC-integrated chromosome were hybridized with adult thymocytes and then exposed to Cre recombinase. This led to loss of GFP expression and elimination of the CEC-tagged chromosome. Targeted elimination of a pair of ESC-derived chromosome 6s, which are key chromosomes for maintaining pluripotency, demonstrated that the reprogrammed somatic factors are sufficient for the continued pluripotentiality of hybrid cells. Targeted chromosome elimination technology therefore offers a means for developing major histocompatibility complex-personalized or completely personalized pluripotential stem cell populations for use in a range of therapeutic applications.

Uptake and retention of the photosensitizer mono-L-asparthyl chlorine e6 in experimental malignant glioma.

The objective of the study was to investigate the potential of mono-L-aspartyl chlorine e6 (NPe6), a water-soluble photosensitizer derived from chlorophyll, for use in photodynamic diagnosis (PDD) of malignant brain tumor. A C6 glioma cell line was transplanted in the SD rat brain to create a brain tumor model. Five days after transplantation, NPe6 was administrated via the tail vein at concentrations ranging from 1.25 to 10 mg/kg; then the skull was opened in the rat brain, the site of tumor transplant was irradiated with a diode laser beam at 664 nm, and the time-course intensity and distribution of emerging fluorescence were observed. Furthermore, the correlation between fluorescence distribution and histopathological findings was investigated in the removed brain. Fluorescence was observed in the site of brain tumor transplant from 5 min after injection, and stable fluorescence was recognized at the site until 4 h after administration. No differences were noted in fluorescence intensity at NPe6 doses of 2.5 mg/kg or more; therefore, it was possible to estimate the optimal dose range. Fluorescence distribution had a clear correlation with tumor cell density, and it was possible to capture the margin of tumor cell invasion with fluorescence. The photosensitizer NPe6 is capable of assessing tumor cell density in malignant glioma tissue in terms of differences in fluorescence intensity. The usefulness of PDD using 5-aminoleveulinic acid during surgery for malignant glioma has been recognized in recent years. The results of the present study suggested the potential of NPe6 as a promising photosensitizer for use in PDD for accurate grasp of the extent of removal during the course of malignant glioma surgery.

Photodynamic therapy of C6-implanted glioma cells in the rat brain employing second-generation photosensitizer talaporfin sodium.

OBJECT: The usefulness of photodynamic therapy (PDT) as a local therapy for malignant glioma was evaluated by investigating histological changes in a rat C6 glioma model treated with a combination of talaporfin sodium, a water-soluble photosensitizer derived from chlorophyll and exposure to a diode laser. METHODS: Glioma cells (C6) at the confluence stage were transplanted stereotactically into the right frontal lobe of SD rats. Five days later, the rats underwent right frontal craniotomy and intravenous administration of talaporfin sodium. One hour after talaporfin sodium administration, each rat was irradiated by a 664 nm diode laser beam. The brain was removed 1, 3 or 6h after laser irradiation for histological examination of tumor-affected brain tissue and surrounding normal brain tissue. RESULTS: In addition to the tumor mass, tumor cells invading surrounding edematous brain tissue were seen in untreated rats, ranging from the brain surface to a depth of 2mm. One hour after PDT, coagulation necrosis as well as disappearance of indication of cell viability such as disappearance of tumor cell processes and foamy changes of cytoplasm were noted in the tumor tissue at a depth of 0.5mm, accompanied by reduction of cytoplasmic glial fibrillary acidic protein (GFAP) expression and appearance of granular M30 cytodeath positivity. Three hours later, the cytoplasm of the residual tumor cells showed disappearance of GFAP expression and increased expression of M30 cytodeath. Six hours later, the foamy cytoplasm of swollen tumor cells demonstrated strong positivity for M30 cytodeath. CONCLUSION: PDT using talaporfin sodium induced coagulation necrosis and apoptosis in rats with C6 glioma.

[Surgical treatment of proximal middle cerebral artery (M1) aneurysms at the origin of the lenticulostriate artery].

In contrast to aneurysms of the middle cerebral artery at the bifurcation, aneurysms at the origin of the lenticulostriate arteries (LSA) are uncommon. Six surgically treated patients (34 to 70 year-old; 3 men, 3 women) were reviewed. 5 patients presented with subarachnoid hemorrhage (H&H grade 2:3, 3:1, 4:1; Fisher type 2:1, 3:3, 4:1) and 2 patients had multiple aneurysms. All aneurysms arose from the postero-superior surface of the M1. Although neck clipping was achieved in every patient, re-application of the clip was necessary during surgery in 3 patients because the tip of the blade extended to the other perforators that ran parallel to the M1. Results were as follows: GR 3, MD1, SD 1, D1. Apart from a 70 year-old patient who died of vasospasm (H & H 4), fair results in two patients were accompanied by ischemic complications of the LSA. All 3 patients who required re-application of the clip during surgery showed a lacunar infarct of perforating arteries on post-operative CT. Special care of perforating arteries not only around the neck (the LSA) but also behind the aneurysm is essential for successful neck-clipping of aneurysms at this location.