Angiotensin II-induced smooth muscle cell migration is mediated by LDL receptor-related protein 1 via regulation of matrix metalloproteinase 2 expression

Angiotensin II (Ang II), one of the main vasoactive hormones of the renin-angiotensin system, contributes to the development and progression of atherosclerosis by inducing vascular smooth muscle cells (VSMCs) migration. Although previous studies have shown that Ang II upregulates low density lipoprotein receptor-related protein 1 (LRP1) expression in VSMCs and increases VSMCs migration, the role of LRP1 in Ang II-induced VSMCs migration remains unclear. Here, we reveal a novel mechanism by which LRP1 induces the expression of matrix metalloproteinase 2 (MMP2) and thereby promotes the migration of rat aortic SMCs (RAoSMCs). Knockdown of LRP1 expression greatly decreased RAoSMCs migration, which was rescued by forced expression of a functional LRP1 minireceptor, suggesting that LRP1 is a key regulator of Ang II-induced RAoSMCs migration. Inhibition of ligand binding to LRP1 by the specific antagonist receptor-associated protein (RAP) also led to reduced RAoSMCs migration. Because MMPs play critical roles in RAoSMCs migration, we examined the expression of several MMPs and found that the expression of functional MMP2 was selectively increased by Ang II treatment and decreased in LRP1-knockdown RAoSMCs. More interestingly, reduced MMP2 expression in LRP1-knockdown cells was completely rescued by exogenous expression of mLRP4, suggesting that MMP2 is a downstream regulator of LRP1 in Ang II-induced RAoSMCs migration. Together, our data strongly suggest that LRP1 promotes the migration of RAoSMCs by regulating the expression and function of MMP2.     

Differential Auxin-Transporting Activities of PIN-FORMED Proteins in Arabidopsis Root Hair Cells

The Arabidopsis (Arabidopsis thaliana) genome includes eight PIN-FORMED (PIN) members that are molecularly diverged. To comparatively examine their differences in auxin-transporting activity and subcellular behaviors, we expressed seven PIN proteins specifically in Arabidopsis root hairs and analyzed their activities in terms of the degree of PIN-mediated root hair inhibition or enhancement and determined their subcellular localization. Expression of six PINs (PIN1–PIN4, PIN7, and PIN8) in root hair cells greatly inhibited root hair growth, most likely by lowering auxin levels in the root hair cell by their auxin efflux activities. The auxin efflux activity of PIN8, which had not been previously demonstrated, was further confirmed using a tobacco (Nicotiana tabacum) cell assay system. In accordance with these results, those PINs were localized in the plasma membrane, where they likely export auxin to the apoplast and formed internal compartments in response to brefeldin A. These six PINs conferred different degrees of root hair inhibition and sensitivities to auxin or auxin transport inhibitors. Conversely, PIN5 mostly localized to internal compartments, and its expression in root hair cells rather slightly stimulated hair growth, implying that PIN5 enhanced internal auxin availability. These results suggest that different PINs behave differentially in catalyzing auxin transport depending upon their molecular activity and subcellular localization in the root hair cell.Auxin plays a critical role in plant development and growth by forming local concentration gradients. Local auxin gradients, created by the polar cell-to-cell movement of auxin, are implicated in primary axis formation, root meristem patterning, lateral organ formation, and tropic movements of shoots and roots (for recent review, see Vanneste and Friml, 2009). The cell-to-cell movement of auxin is achieved by auxin influx and efflux transporters such as AUXIN-RESISTANT1 (AUX1)/LIKE-AUX1 for influx and PIN-FORMED (PIN) and the P-glycoprotein (PGP) of ABCB (ATP-binding cassette-type transporter subfamily B) for efflux. Since diffusive efflux of the natural auxin indole-3-acetic acid (IAA; pKa = 4.75) is not favorable and PINs are localized in the plasma membrane in a polar manner, PINs act as rate-limiting factors for cellular auxin efflux and polar auxin transport through the plant body. These PINs'' properties explain why representative physiological effects of auxin transport are associated with PINs.Auxin flows from young aerial parts all the way down to the root tip columella in which an auxin maximum is formed for root stem cell maintenance and moves up toward the root differentiation zone through root epidermal cells, where a part of it travels back to the root tip via cortical cells (Blilou et al., 2005). This directional auxin flow is supported by the polar localization of PINs: PIN1, PIN3, and PIN7 at the basal side of stele cells (Friml et al., 2002a, 2002b; Blilou et al., 2005), PIN4 at the basal side in root stem cells (Friml et al., 2002a), and PIN2 at the upper side of root epidermis and at the basal side of the root cortex (Luschnig et al., 1998; Müller et al., 1998). Another interesting aspect of PIN-mediated auxin transport is the dynamics in directionality of auxin flow due to environmental stimuli-directed changes of subcellular PIN polarity, as exemplified for PIN3, whose subcellular localization changes in response to the gravity vector (Friml et al., 2002b).An intriguing question is how different PIN proteins have different subcellular polarities, which might be attributable to PIN-specific molecular properties, cell-type-specific factors, or both. The different PIN subcellular polarities in different cell types seemingly indicate that cell-type-specific factors are involved in polarity. In the case of PIN1, however, both classes of factors appear to affect its subcellular localization because when expressed under the PIN2 promoter, PIN1 localizes to the upper or basal side of root epidermal cells, depending on the GFP insertion site of the protein (Wiśniewska et al., 2006). A recent study demonstrated that the polar targeting of PIN proteins is modulated by phosphorylation/dephosphorylation of the central hydrophilic loop of PINs, which is mediated by PINOID (PID; a Ser/Thr protein kinase)/PP2A phosphatase (Michniewicz et al., 2007). The central hydrophilic domain of PINs might provide the molecule-specific cue for PIN polarity, together with as yet unknown cell-specific factors. Different recycling behaviors of PINs, which show variable sensitivities to brefeldin A (BFA), also imply different molecular characters among PIN species. Most PIN1 proteins are internalized by BFA treatment, whereas considerable amounts of PIN2 remain in the plasma membrane in addition to internal accumulation after BFA treatment. Recycling and basal polar targeting of PIN1 is dependent on the BFA-sensitive guanine nucleotide exchange factor for adenosyl ribosylation factors (ARF GEFs), GNOM, which is the major target of BFA. In contrast, apical targeting and recycling of PIN2 is independent of GNOM and controlled by BFA-resistant ARF GEFs (Geldner et al., 2003; Kleine-Vehn and Friml, 2008).In contrast to their distinct subcellular localizations, the differential auxin-transporting activities of PINs remain to be studied. The divergent primary structures of PIN proteins are not only indicative of differential subcellular polarity, but also would represent their differential catalytic activities for auxin transport. The auxin efflux activities of Arabidopsis (Arabidopsis thaliana) PINs have been demonstrated using Arabidopsis and heterologous systems: PIN1 and PIN5 in Arabidopsis cells (Petrásek et al., 2006; Mravec et al., 2009); PIN2, PIN3, PIN4, PIN6, and PIN7 in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells (Lee and Cho, 2006; Petrásek et al., 2006; Mravec et al., 2008); PIN1, PIN2, PIN5, and PIN7 in yeast (Saccharomyces cerevisiae) cells (Petrásek et al., 2006; Blakeslee et al., 2007; Mravec et al., 2009; Yang and Murphy, 2009); and PIN1, PIN2, and PIN7 in HeLa cells (Petrásek et al., 2006; Blakeslee et al., 2007). Among the eight Arabidopsis PIN members, PIN1, PIN2, PIN3, PIN4, PIN6, and PIN7, which share a similar molecular structure in terms of the presence of a long central loop (hereafter called long-looped PINs; Fig. 1A; Supplemental Fig. S1), have been shown to catalyze auxin efflux at the cellular level. On the other hand, PIN5 and PIN8 possess a very short putative central loop (hereafter called short-looped PINs). Although PIN5 was recently shown to be localized in the endoplasmic reticulum (ER) and proposed to transport auxin metabolites into the ER lumen, its cellular function regarding its intracellular auxin-transporting activity has not been shown, and the auxin-transporting activity of PIN8 has yet to be demonstrated. In spite of the same transport directionality (auxin efflux) and similar molecular structures, the long-looped PINs exhibit sequence divergence not only in their central loop, but also in certain residues of the transmembrane domains. This structural divergence of long-looped PINs might be indicative of their differential auxin-transporting activities, which have not yet been quantitatively compared.Open in a separate windowFigure 1.Differential activities of PINs in the Arabidopsis root hair. A, Two distinctive PIN groups with different central hydrophilic loop sizes. Topology of PIN proteins was predicted by four different programs as described in Supplemental Figure S1. Numbers above indicate the number of transmembrane helices for each N- and C-terminal region, and numbers below indicate the number of amino acid residues of the central hydrophilic domain. B, Representative root images of control (Cont; Columbia-0) and root-hair-specific PIN-overexpressing (PINox; ProE7:PIN-GFP or ProE7:PIN [−]) plants. Bar = 100 μm for all. C, Root hair lengths of control and PINox plants. Six to 12 independent transgenic lines (average = 8.3), and 42 to 243 roots (average = 86.8) and 336 to 2,187 root hairs (average = 727.8) per construct, were observed for the estimation of root hair length. Data represent means ± se. The root hair lengths of PIN5ox lines were significantly longer than those of the control (P = 0.016 for PIN5ox; P < 0.0001 for PIN5-GFP1ox and PIN5-GFP2ox).To comparatively assess the cytological behaviors and molecular activities of different PIN members, it would be favorable to use a single assay system that provides a consistent cellular environment and enables quantitative estimation of PIN activity. In previous studies, we adopted the root hair single cell system to quantitatively assay auxin-transporting or regulatory activities of PINs, PGPs, AUX1, and PID (Lee and Cho, 2006; Cho et al., 2007a). Root hair growth is proportional to internal auxin levels in the root hair cell. Therefore, auxin efflux inhibits and auxin influx enhances root hair growth (Cho et al., 2007b; Lee and Cho, 2008). In addition, the use of a root-hair-specific promoter (Cho and Cosgrove, 2002; Kim et al., 2006) for expression of auxin transporters enables the transporters'' biological effect to be pinpointed to only the root hair cell, thus excluding probable non-cell-autonomous effects that could be caused by the general expression of auxin transporters.In this study, we expressed five long-looped PINs (PIN1, PIN2, PIN3, PIN4, and PIN7) and two short-looped PINs (PIN5 and PIN8) in root hair cells and compared their auxin-transporting activities and cytological dynamics. To directly measure the radiolabeled auxin-transporting activities of PIN5 and PIN8, we used an additional assay system, tobacco suspension cells. Our data revealed that PINs have differential molecular activities and pharmacological responses and that the short-looped and long-looped PINs have different subcellular localizations.     

Spatiotemporal Expression of tmie in the Inner Ear of Rats during Postnatal Development

The circling (cir/cir) mouse is a murine model for human nonsyndromic deafness DFNB6. Transmembrane inner ear (tmie) is the causative gene and its mutation through deletion of a 40-kilobase genomic region including tmie leads to deafness. The function of Tmie is unknown. To better understand the function of Tmie, we focused on the spatiotemporal expression of tmie in the rat cochlea by using a Tmie-specific antibody. Results showed that tmie expression was prominent in early postnatal rat cochleas in the stereocilia bundles of hair cells. The Tmie signal spread from the stereocilia to the hair cell body region and on to organ of Corti cells. No Tmie signal was observed in cell nuclei; Tmie was localized to the cytoplasm. Because Tmie is predicted to have 1 or 2 transmembrane domains, we postulate that it is localized to membrane-based organelles or the plasma membrane. Our results imply that Tmie exists in the cytoplasm and may have a key role in the maturation and structure of stereocilia bundles in developing hair cells. After hair cell maturation, Tmie is thought to be involved in the maintenance of organ of Corti cells.Circling is often observed in mouse and rat deafness mutants and is commonly suggested to be a consequence of inner ear defects that impair vestibular systems.^3,12,14 The circling (cir/cir) mouse is a murine model for human nonsyndromic deafness DFNB6; these mice have abnormal circling behavior, suggesting a balance disorder, and profound deafness.^6,7 The most notable pathologic phenotypes of circling mice are the almost completely degenerated cochlea and remarkably reduced cellularity in spiral ganglion neurons. The causative gene for circling is transmembrane inner ear (tmie), with a 40-kilobase genomic deletion including tmie.¹ tmie is also the causative gene of the spinner (sr/sr) mouse, which has phenotypes similar to circling mice, although the mutation patterns are different.⁸ Spinner mice also show circling behavior, hearing loss, imbalance, and swimming inability. In addition, spinner mice have 2 mutations in the tmie gene: the 40-kb genomic deletion including tmie and a point mutation that leads to a truncated protein.⁸In humans, 7 different homozygous recessive mutations in TMIE currently are known to exist in affected members of consanguineous families segregating severe-to-profound prelingual deafness, consistent with linkage to DFNB6.^9,10 Although the functions of murine Tmie and human TMIE are unknown, this protein appears to be important for normal hearing and vestibular function.In a previous study, we produced transgenic mice overexpressing tmie that resulted in phenotypic rescue of circling.¹¹ Normal expression of transgenic tmie induced phenotypic rescue in circling homozygous mutants, although some mice did not show amelioration of abnormal behavior, hearing ability, or tissue morphology in the inner ear. Therefore the Tmie protein is required for normal inner ear function in mouse.¹¹To better understand the function of Tmie, we focused on the spatiotemporal expression of tmie. Knowing when, where, and to what extent this protein is produced in the developing inner ear will provide important clues to protein function. In adult mouse and rat, tmie is expressed in various tissues.^2,13 Whether Tmie plays an important role in those tissues is uncertain, because circling mice that lack the entire tmie gene have no noteworthy problems in any tissues except those of the inner ear systems.⁶In this study, we were interested in the postnatal stages before and after the onset of hearing (around postnatal day [P] 12) in rats; therefore, the postnatal period P0 to19 was studied. Although all the cells that form the mature cochlea are present at birth, important conformational changes occur during this period, including the formation of the tunnel of Corti and the establishment or retraction of neuronal connections. The expression pattern of tmie in the developing inner ear during early postnatal development has not been investigated previously. Here we document our use of a Tmie-specific antibody to elucidate the spatial and temporal expression of tmie in the rat inner ear during postnatal development.     

Two novel ubiquitin-fold modifier 1 (Ufm1)-specific proteases, UfSP1 and UfSP2

Ubiquitin-fold modifier 1 (Ufm1) is a recently identified new ubiquitin-like protein, whose tertiary structure displays a striking resemblance to ubiquitin. Similar to ubiquitin, it has a Gly residue conserved across species at the C-terminal region with extensions of various amino acid sequences that need to be processed in vivo prior to conjugation to target proteins. Here we report the isolation, cloning, and characterization of two novel mouse Ufm1-specific proteases, named UfSP1 and UfSP2. UfSP1 and UfSP2 are composed of 217 and 461 amino acids, respectively, and they have no sequence homology with previously known proteases. UfSP2 is present in most, if not all, of multicellular organisms including plant, nematode, fly, and mammal, whereas UfSP1 could not be found in plant and nematode upon data base search. UfSP1 and UfSP2 cleaved the C-terminal extension of Ufm1 but not that of ubiquitin or other ubiquitin-like proteins, such as SUMO-1 and ISG15. Both were also capable of releasing Ufm1 from Ufm1-conjugated cellular proteins. They were sensitive to inhibition by sulfhydryl-blocking agents, such as N-ethylmaleimide, and their active site Cys could be labeled with Ufm1-vinylmethylester. Moreover, replacement of the conserved Cys residue by Ser resulted in a complete loss of the UfSP1 and UfSP2 activities. These results indicate that UfSP1 and UfSP2 are novel thiol proteases that specifically process the C terminus of Ufm1.     

New fermentation technique for complete digestion of soybean protein

The aim of this study was to develop a new fermentation method in order to improve the digestion of soybean protein, and to promote normal fermentation of soybean. A proximate composition, such as moisture, pH, and reducing sugar, of fermented soybeans by the new fermentation was similar to those of controls. Neutral protease activity, the most important factor for fermented soybean products, was the highest, having about 636 U/g at 54 h fermentation. The content of total free amino acid was almost 3-18 times higher than controls. The three-step fermented soybeans can be used as a functional food ingredient for human consumption, with higher protein digestibility.     

Increasing production in Korean shrimp farms with white-spot syndrome virus PCR-negative brood stock

White-spot syndrome virus (WSSV) is a devastating, infectious virus affecting shrimp. Although sensitive techniques involving PCR have been developed to assist farmers in screening shrimp (brood stock) for WSSV prior to stocking ponds, such practices have not yet been applied in Korea. Despite the rationality of implementing screening, there has been some doubt as to whether the stocking of WSSV-PCR-negative fry epidemiologically decreases white-spot disease outbreaks. Here, we report a retrospective analysis of data from shrimp farms in the western coast of Korea where WSSV-PCR-negative brood stocks were used to stock rearing ponds. A total of 366 shrimp from Heuksan Island were sampled for WSSV with PCR. Of the tested shrimp, 7.2% (28 brood stocks) were identified as WSSV positive; only WSSV-PCR-negative shrimp were used for brood stocks. Total unit production (final shrimp production/ the area of the ponds) was higher, at 1.96, in ponds where WSSV-PCR-negative shrimp were used, as compared with 1.02 in other ponds in Korea in 2004. This retrospective analysis of WSSV in Korea may be useful to the shrimp aquaculture industry, suggesting a testable hypothesis that may contribute to the eventual control of WSSV outbreaks.     

Induction of Egr-1 is associated with anti-metastatic and anti-invasive ability of beta-lapachone in human hepatocarcinoma cells

beta-lapachone, a quinone compound obtained from the bark of the lapacho tree (Tabebuia avellanedae), was reported to have anti-inflammatory and anti-cancer activities. In this study, we investigated novel functions of beta-lapachone in terms of anti-metastasis and anti-invasion abilities using human hepatocarcinoma cell lines, HepG2 and Hep3B. beta-lapachone dose-dependently inhibited cell viability and migration of both HepG2 and Hep3B cells, as determined by methylthiazoletetrazolium (MTT) assay and wound healing assay. RT-PCR and Western blot data revealed that beta-lapachone dramatically increased the levels of protein, as well as mRNA expression of early growth response gene-1 (Egr-1) and throbospondin-1 (TSP-1) at an early point in time, and then decreased in a time-dependent manner. In addition, down-regulation of Snail and up-regulation of E-cadherin expression were observed in beta-lapachone-treated HepG2 and Hep3B cells, and this the associated with decreased invasive ability as measured by matrigel invasion assay. Taken together, our results strongly suggest that beta-lapachone may be expected to inhibit the progression and metastasis of hepatoma cells, at least in part by inhibiting the invasive ability of the cells via up-regulation of the expression of the Egr-1, TSP-1, and E-cadherin.     

Protective effect of anthocyanin-rich extract from bilberry (Vaccinium myrtillus L.) against myelotoxicity induced by 5-fluorouracil

The toxicities associated with 5-fluorouracil (5-FU), a potent broad-spectrum chemotherapeutic agent, can not only affect the morbidity and the efficacy of chemotherapy but also limit its clinical use. The objective of this study is to investigate the effects of a commercial anthocyanin-rich extract from bilberry (AREB) against 5-FU-induced myelotoxicity in vivo, and against chemosensitivity to 5-FU in vitro. A single injection of 5-FU at 200 mg/kg induced severe peripheral erythrocytopenia, thrombocytopenia and leucopenia as well as hypocellularity of the spleen and bone marrow in C57BL/6 mice. Oral administration of 500 mg/kg of AREB for 10 days significantly increased the number of red blood cells, neutrophils, and monocytes in peripheral blood to 1.2-fold, 9-fold, and 6-fold, respectively, compared with those seen after treatment with 5-FU alone (p< 0.05-0.001). The hypocellularity of the spleen and bone marrow caused by 5-FU was also distinctly alleviated in the AREB-treated group. Furthermore, AREB treatment with 50 and 100 microg/ml as a monomeric anthocyanin did not interfere with, but rather enhanced the chemotherapeutic efficacy of 5-FU in vitro. These results suggest that AREB may have protective potential against 5-FU-induced myelotoxiciy and/or the ability to enhance the chemotherapeutic effectiveness of 5-FU.     

Stereoselective synthesis of 1'-functionalized-4'-thionucleosides

Stereoselective functionalization of the 1'-position of 4'-thionucleosides was achieved using a stereoselective S(N)2 reaction controlled by 5-membered ring coordination.