Preparation of a Nanoscaled Poly(vinyl alcohol)/Hydroxyapatite/DNA Complex Using High Hydrostatic Pressure Technology for In Vitro and In Vivo Gene Delivery

Our previous research showed that poly(vinyl alcohol) (PVA) nanoparticles incorporating DNA with hydrogen bonds obtained by high hydrostatic pressurization are able to deliver DNA without any significant cytotoxicity. To enhance transfection efficiency of PVA/DNA nanoparticles, we describe a novel method to prepare PVA/DNA nanoparticles encapsulating nanoscaled hydroxyapatites (HAps) prepared by high hydrostatic pressurization (980 MPa), which is designed to facilitate endosomal escape induced by dissolving HAps in an endosome. Scanning electron microscopic observation and dynamic light scattering measurement revealed that HAps were significantly encapsulated in PVA/HAp/DNA nanoparticles. The cytotoxicity, cellular uptake, and transgene expression of PVA/HAp/DNA nanoparticles were investigated using COS-7 cells. It was found that, in contrast to PVA/DNA nanoparticles, their internalization and transgene expression increased without cytotoxicity occurring. Furthermore, a similar level of transgene expression between plasmid DNA and PVA/HAp/DNA nanoparticles was achieved using in vivo hydrodynamic injection. Our results show a novel method of preparing PVA/DNA nanoparticles encapsulating HAp nano-crystals by using high hydrostatic pressure technology and the potential use of HAps as an enhancer of the transfection efficiency of PVA/DNA nanoparticles without significant cytotoxicity

Identification of a novel type of focal adhesion remodelling via FAK/FRNK replacement, and its contribution to cancer progression

Abstract Numerous studies have investigated the various cellular responses against genotoxic stress, including those mediated by focal adhesions. We here identified a novel type of focal adhesion remodelling that occurs under genotoxic stress conditions, which involves the replacement of active focal adhesion kinase (FAK) with FAK-related non-kinase (FRNK). FRNK stabilized focal adhesions, leading to strong cell-matrix adhesion, and FRNK-depleted cells were easily detached from extracellular matrix upon genotoxic stress. This remodelling occurred in a wide variety of cells. In vivo, the stomachs of Frnk-knockout mice were severely damaged by genotoxic stress, highlighting the protective role of FRNK against genotoxic stress. FRNK was also found to play a vital role in cancer progression, because FRNK depletion significantly inhibited cancer dissemination and progression in a mouse cancer model. Furthermore, in human cancers, FRNK was predominantly expressed in metastatic tissues and not in primary tissues. We hence conclude that this novel type of focal adhesion remodelling reinforces cell adhesion and acts against genotoxic stress, which results in the protection of normal tissues, but in turn facilitates cancer progression

Retinol Promotes In Vitro Growth of Proximal Colon Organoids through a Retinoic Acid-Independent Mechanism.

Retinol (ROL), the alcohol form of vitamin A, is known to control cell fate decision of various types of stem cells in the form of its active metabolite, retinoic acid (RA). However, little is known about whether ROL has regulatory effects on colonic stem cells. We examined in this study the effect of ROL on the growth of murine normal colonic cells cultured as organoids. As genes involved in RA synthesis from ROL were differentially expressed along the length of the colon, we tested the effect of ROL on proximal and distal colon organoids separately. We found that organoid forming efficiency and the expression level of Lgr5, a marker gene for colonic stem cells were significantly enhanced by ROL in the proximal colon organoids, but not in the distal ones. Interestingly, neither retinaldehyde (RAL), an intermediate product of the ROL-RA pathway, nor RA exhibited growth promoting effects on the proximal colon organoids, suggesting that ROL-dependent growth enhancement in organoids involves an RA-independent mechanism. This was confirmed by the observation that an inhibitor for RA-mediated gene transcription did not abrogate the effect of ROL on organoids. This novel role of ROL in stem cell maintenance in the proximal colon provides insights into the mechanism of region-specific regulation for colonic stem cell maintenance

Expression of genes involved in ROL-RA pathway in colonic epithelia.

<p>(A) Crypts isolated from the entire length of the colon were cultured for 6 days. Organoids were then dissociated into single cells and further cultured in the presence of vehicle alone, or indicated concentrations of ROL. Phase-contrast images were acquired on Day 5 of culture. Representative images of three independent experiments are shown. Scale bar, 100 μm. (B) Total RNA was extracted from the whole colon or its epithelial compartment isolated as crypts. Semi-quantitative PCR was performed for the indicated genes. Representative data are shown for three independent experiments.</p

ROL-dependent growth promotion of proximal colon organoids involves an RA-independent mechanism.

<p>(A) Proximal and distal colon organoids were separately subjected to single-cell passage, and the following culture was initiated in the absence (vehicle) or presence of ROL (1 μM). Total RNA was extracted from the isolated cells before (pre) or after the culture (post). Semi-quantitative PCR was performed for <i>Lgr5</i>, a marker gene of colonic stem cells, and <i>Gapdh</i>. (B) After single-cell passage, proximal colon organoids were cultured either with ROL (1 μM), RAL (1 μM) or RA (1 μM) or left untreated (vehicle). Total RNA was extracted after the culture, and semi-quantitative PCR was performed for <i>Lgr5</i>, <i>Rarβ</i>, and <i>Gapdh</i>. (C) Proximal colon cells were cultured as in (B). Representative phase-contrast images on Day 5 are shown. Scale bar, 100 μm. (D) Proximal colon cells were cultured as in (B) either with different concentrations (1, 3, or 10 μM) of ROL or RA, or left untreated (vehicle). Total RNA was extracted on Day 5 of culture and semi-quantitative PCR was performed for <i>MUC2</i>, <i>Lgr5</i>, <i>Rarβ</i> and <i>Gapdh</i>. (E) Proximal colon cells were cultured in the absence (vehicle) or presence of 1 μM ROL. ROL-treated cells were co-treated with different concentrations of RA (1, 3, or 10 μM) or left untreated. Semi-quantitative PCR was performed as described in (B) for <i>Lgr5</i>, <i>Rarβ</i> and <i>Gapdh</i>. (F) Proximal colon cells cultured as in (B) were left untreated (vehicle) or treated with either ROL, ROL+AGN193109, RA, RA+AGN193109 all at a concentration of 1 μM. On Day 5 of culture, total RNA was extracted after the culture, and semi-quantitative PCR was performed for <i>Lgr5</i>, <i>Rarβ</i> and <i>Gapdh</i>. (G) Proximal cells were cultured as in (F) and phase-contrast images on Day 5 were acquired. Scale bar, 100 μm. Experiments for A-G were performed more than twice independently and representative images are shown.</p

TNFAIP3 promotes survival of CD4 T cells by restricting MTOR and promoting autophagy

<p>Autophagy plays important roles in metabolism, differentiation, and survival in T cells. TNFAIP3/A20 is a ubiquitin-editing enzyme that is thought to be a negative regulator of autophagy in cell lines. However, the role of TNFAIP3 in autophagy remains unclear. To determine whether TNFAIP3 regulates autophagy in CD4 T cells, we first analyzed <i>Tnfaip3</i>-deficient naïve CD4 T cells in vitro. We demonstrated that <i>Tnfaip3</i>-deficient CD4 T cells exhibited reduced MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) puncta formation, increased mitochondrial content, and exaggerated reactive oxygen species (ROS) production. These results indicate that TNFAIP3 promotes autophagy after T cell receptor (TCR) stimulation in CD4 T cells. We then investigated the mechanism by which TNFAIP3 promotes autophagy signaling. We found that TNFAIP3 bound to the MTOR (mechanistic target of rapamycin) complex and that <i>Tnfaip3</i>-deficient cells displayed enhanced ubiquitination of the MTOR complex and MTOR activity. To confirm the effects of enhanced MTOR activity in <i>Tnfaip3</i>-deficient cells, we analyzed cell survival following treatment with Torin1, an MTOR inhibitor. <i>Tnfaip3</i>-deficient CD4 T cells exhibited fewer cell numbers than the control cells in vitro and in vivo. In addition, the impaired survival of <i>Tnfaip3</i>-deficient cells was ameliorated with Torin1 treatment in vitro and in vivo. The effect of Torin1 was abolished by <i>Atg5</i> deficiency. Thus, enhanced MTOR activity regulates the survival of <i>Tnfaip3</i>-deficient CD4 T cells. Taken together, our findings illustrate that TNFAIP3 restricts MTOR signaling and promotes autophagy, providing new insight into the manner in which MTOR and autophagy regulate survival in CD4 T cells.</p