Morphological and Biochemical Changes During Programmed Cell Death of Rat Cerebellar Granule Cells

Cultured cerebellar granule cells deprived of depolarizing concentrations of KCl and serum die by programmed cell death. Recently, it was shown that serum removal by itself can lead to oxidative stress and DNA fragmentation in these cells. We have modified the protocol which initiates cell death in such a way that only the effect of KCl withdrawal-induced cell death was observed. We have performed a series of experiments to correlate the structural and biochemical changes in this process of cell death. Significant morphological alterations occur in cell bodies and neurites during a 48-hour period of KCl removal. Cell viability dropped to 53%, 34% or 10% of control levels, respectively, as a result of 1-, 2-, or 3-day KCl removal. A series of experiments was conducted to determine the change of total protein level, protein synthesis rate, RNA synthesis rate, and mitochondrial activity during the first 48 hours of KCl removal. These studies not only provide a picture correlating the morphological and biochemical changes in the process of programmed cell death, but also serve as a reference for future studies of this complex phenomenon.     

Ethanol Differentially Inhibits Homoquinolinic Acid- and NMDA-Induced Neurotoxicity in Primary Cultures of Cerebellar Granule Cells

The potency of ethanol to inhibit N-methyl-D-aspartate (NMDA) receptor functions may depend on the subunit composition of the NMDA receptors. We used a NR2A-B subunit-selective NMDA receptor agonist, homoquinolinic acid (HQ), and a subunit-unselective agonist, NMDA, to induce neurotoxicity in cerebellar granule cells and examined the neuroprotective actions of ethanol, as well as NR2A- and NR2B-subunit selective antagonists, respectively. HQ was a more potent neurotoxic agent than NMDA, as measured by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. NR2A- and NR2B-selective NMDA receptor antagonists displayed quite similar neuroprotective potencies against the NMDA- and HQ-produced cell death, indicating that the higher potency of HQ to induce neurotoxicity cannot be simply explained by NR2A- or NR2B-subunit selectivity. As expected, ethanol (25 and 50 mM) attenuated the NMDA-induced neurotoxicity in a non-competitive manner by significantly reducing the maximum neurotoxicity produced by NMDA. By contrast, ethanol inhibited the HQ-induced neurotoxicity in a manner resembling a competitive-like interaction significantly increasing the EC₅₀ value for HQ, without reducing the maximum neurotoxicity produced by HQ. These results suggest that HQ reveals either a novel site or a not previously observed mechanism of interaction between ethanol and NMDA receptors in rat cerebellar granule cell cultures.     

Development of Benzodiazepine and Picrotoxin (t-Butylbicyclophosphorothionate) Binding Sites in Rat Cerebellar Granule Cells in Culture

The specific bindings of [3H]flunitrazepam [( 3H]FLU), [3H]CGS 8216, and t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to sites on rat cerebellar granule cells all increase from 4 to 15 days in culture, although their time courses differ. Specific [3H]FLU binding doubles, [3H]CGS 8216 binding triples, and [35S]TBPS binding increases about fourfold from 4 to 15 days in culture. Displacement studies, using the type I-selective ligand CL 218,872, indicate that at 4 days the [3H]FLU binding sites are almost entirely "type II," judging from an IC50 value near 300 nM and a pseudo-Hill number near 1. By 10 days, approximately equal numbers of type I and type II binding sites are present in the cultured cells, and this ratio remains constant thereafter (12 and 15 days). At days 10-15, both the IC50 value for CL 218,872 (near 100 nM) and the pseudo-Hill number (near 0.7) remain constant and are significantly different from the values at culture day 4. The development of specific [35S]TBPS binding parallels that of [3H]CGS 8216 binding more closely than the development of [3H]FLU binding. The [3H]CGS 8216/[3H]FLU ratio increased by a factor of 1.6 from day 4 to day 15 (p less than 0.001). Taken together, our data suggest the existence of several gamma-aminobutyric acidA (GABAA) receptor subunits, the relative proportions of which change during development. The presence of the GABA-mimetic 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridine-3-ol (THIP) in the culture medium had no apparent effect on any of the binding sites studied, although THIP was shown previously to induce low-affinity GABA binding sites.     

Differential Effects of Lithium on Muscarinic Cholinoceptor-Stimulated CMP-Phosphatidate Accumulation in Cerebellar Granule Cells, CHO-M3 Cells, and SH-SY5Y Neuroblastoma Cells

Abstract: The ability of lithium to potentiate muscarinic cholinoceptor-stimulated CMP-phosphatidate (CMP.PA) accumulation has been examined in various cells in which muscarinic cholinoceptor agonists evoke a phosphoinositide response. Cell types examined include rat cerebellar granule cells, Chinese hamster ovary cells transfected to express the human muscarinic M3 receptor (CHO-M3 cells), and SH-SY5Y neuroblastoma cells. Neither carbachol (1 m M ) nor lithium (10 m M ) caused significant increases in CMP.PA accumulation in rat cerebellar granule cells; however, when added together for 20 min a linear 17-fold increase over basal levels was observed. The increase was dependent on the concentration of carbachol and lithium present, and the effect could be reversed by addition of exogenous myo -inositol (10 m M ). Addition of carbachol alone to CHO-M3 cells caused a five-fold increase in CMP.PA accumulation. In the presence of lithium, a 70-fold increase was observed at 20 min after carbachol plus lithium addition. This latter response was concentration dependent and could be abolished by preincubation in the presence of 10 m M myo -inositol. In contrast, whereas carbachol elicited a three-fold increase in CMP.PA accumulation in SH-SY5Y neuroblastoma cells, which reached a plateau 10 min after agonist addition, the response could neither be augmented by addition of lithium nor inhibited by addition of myo -inositol. These results emphasise that the ability of lithium to affect agonist-stimulated CMP.PA accumulation is not simply a function of stimulus strength, but is also crucially dependent on the intracellular concentration of inositol.     

Effects of Extracellular Matrix Molecules on Subepidermal Neural Crest Cell Migration in Wild Type and White Mutant (dd) Axolotl Embryos

Migration of neural crest (NC) derived pigment cells is restricted in the white mutant (dd) axolotl embryo (Ambystoma mexicanum). Transplantations between mutant and wild type embryos show that the extracellular matrix (ECM) of the white mutant is unable to support the migration of prospective pigment cells in wild type embryos (Löfberg et al., 1989, Dev. Biol. 131:168–181). In the present study, we test the effects of various purified ECM molecules on NC cell migration in the subepidermal migratory pathway of wild type (D/-) and white mutant (dd) axolotl embryos. We adsorbed the ECM molecules onto membrane microcarriers, which were then implanted under the epidermis. Fibronectin (FN), tenascin (TN), collagens I and VI, and a chick aggrecan stimulated migration in both types of embryos. Laminin-nidogen, rat chondrosarcoma aggrecan, and shark aggrecan stimulated migration in dd embryos but did not affect migration in D/- embryos. Collagen III, fibromodulin and bovine aggrecan had no effect on migration in either type of embryo. NC cells did not migrate on control micro-carriers, which lacked ECM molecules. Some cells observed contacting, and presumably migrating on, coated microcarriers could be identified as pigment cells by their ultra-structure. Enzymatic digestion in vivo with chondroitinase ABC had no effect on NC cell migration. The neutral or stimulatory effect of the aggrecans is surprising; when tested in vitro they inhibited NC cell migration. The effect of three-dimensionality and other molecules present either in the embryonic ECM or in solution may overcome the inhibitory effect of aggrecans.     

Modulation by Ionotropic Excitatory Amino Acids and Potassium of (±)-1-Aminocyclopentane-trans-1,3-Dicarboxylic Acid-Stimulated Phosphoinositide Hydrolysis in Mouse Cerebellar Granule Cells

Abstract: The effect of ionotropic excitatory amino acids and potassium on the formation of inositol phosphates elicited by the metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) was studied in mouse cerebellar granule cells. In Mg²⁺-containing buffers, NMDA (50–100 µM), α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA; 10–1,000 µM), and high potassium (10–30 mM) enhanced synergistically the response to a maximally effective concentration of 500 µMtrans-ACPD. Potentiation of the trans-ACPD response was blocked by higher concentrations of NMDA (>500 µM) and potassium (>35 mM) but not by AMPA (up to 1 mM). The potentiation by NMDA of the trans-ACPD-stimulated phosphoinositide hydrolysis was blocked by d,l -2-amino-5-phosphonopentanoic acid (APV), a competitive NMDA-receptor antagonist. Under Mg²⁺-free conditions, the accumulation of inositol phosphates in the presence of trans-ACPD alone was equal to that attained by trans-ACPD in Mg²⁺-containing buffers when costimulated with maximally enhancing concentrations of NMDA (50 µM). trans-ACPD potentiated synergistically the NMDA-evoked increases in cytosolic free-Ca²⁺ levels in Mg²⁺-containing but not in Mg²⁺-free solutions, and moreover did not enhance the AMPA-evoked increases in cytosolic free-Ca²⁺ levels. The calcium ionophore A23187 caused a dose-dependent increase in inositol phosphate accumulation but did not enhance the response stimulated by trans-ACPD alone. These results demonstrate the existence of cross talk between metabotropic and ionotropic glutamate receptors in cerebellar granule cells. The exact mechanism remains unclear but appears to involve interplay of G protein-coupled phospholipase C activation and regulated elevation of cytosolic free-Ca²⁺ levels. This study may provide a framework for future investigations at the cellular and molecular level that clarify the functional relevance and molecular mechanisms that are described.     

Effects of Depolarization and NMDA Antagonists on the Survival of Cerebellar Granule Cells: A Pivotal Role for Protein Kinase C Isoforms

Abstract: Primary cultures of cerebellar granule cells (CGCs) grown in high-K⁺ (25 mM; K25) medium progressively differentiate in vitro. Differentiation is noticeable after 3–4 days in vitro (DIV) and reach a mature stage after 8 DIV. Longer cultivation of CGCs (>13 DIV) triggers the processes of spontaneous cell death. However, if cultured in normal physiological K⁺ concentration (5 mM; K5), a significant proportion of the cells dies by the end of the first week in culture. To address the role of protein kinase C (PKC) in the development of CGCs, we measured the kinase activity as well as the protein level of the kinase isoforms. As the K25 CGC culture proceeded, the PKC activity time-dependently increased by 3.2-fold, reaching a steady state at 8 DIV. Western blot analysis using PKC isoform-specific antibodies revealed an increase in levels of PKC α, γ, μ, λ, and ι from 2 to 8 DIV. A slight increase or decrease at 4 DIV was observed for PKC ε and βII, respectively, whereas no significant change was observed for βI. The isoforms of δ, θ, η, and ζ were not detected. Comparing the 14 DIV cultures with the 10 DIV cultures, the immunoreactivities of PKC ι and ε were decreased, those of PKC α, βI, βII, γ, and λ were unchanged, whereas that of PKC μ was still increased. In K5 cultures, the immunoreactivity of each PKC isoform at 2–4 DIV was similar to that observed in K25 cells, although no remarkable differentiation features were observed. Coordinated with the appearance of cell death at 8 DIV in low-K⁺ cultures, levels of PKC α, μ, λ, and ι, but not the others, were markedly decreased. The NMDA receptor antagonists MK-801 and 2-amino-5-phosphopentanoic acid markedly prevented the age-induced apoptosis of CGCs, and the cells survived >18 DIV under these conditions. The cytoprotective effect of MK-801 was concomitant with the increases in levels of PKC γ, λ, ι, and μ at 10 and 14 DIV. In addition, the PKC ε level was increased at 14 DIV but decreased at early stages, whereas PKC α, βI, and βII levels were unchanged, as compared with K25 culture alone. Taken together, induction and up-regulation of PKC isoforms may play an important role in the maintenance of CGC survival by depolarization and MK-801.     

Inhibitory Effects of Alcohols on the Activity of Human Matrix Metalloproteinase 7 (Matrilysin)

Aliphatic alcohols inhibited the activity of human matrix metalloproteinase 7 (matrilysin) competitively with K_i of 6.1–19.4% (v/v) or 0.66–4.80 M. From the relationship between the structures of alcohols and their K_i values, alcohols are considered to bind the hydrophobic S1′ subsite most plausibly, and the size of the pocket was estimated to be large enough to accommodate the length of 1-butanol (4-carbon chain) and the bulk of tertiary alcohols. Alcohols might be suitable probes for exploring the active-site geometry of enzymes.     

Lipopolysaccharide (LPS) Stimulates the Production of Tumor Necrosis Factor (TNF)-α and Expression of Inducible Nitric Oxide Synthase (iNOS) by Osteoclasts (OCL) in Murine Bone Marrow Cell Culture

Osteoclasts (OCL) resorb bone. They are essential for the development of normal bones and the repair of impaired bones. The function of OCL is presumed to be supported by cytokines and other biological mediators, including tumor necrosis factor (TNF)-α and nitric oxide (NO). Bacterial lipopolysaccharide (LPS) is a potent inducer of TNF-α and inducible nitric oxide synthase (iNOS), which is the specific enzyme for synthesizing NO from L-arginine. To obtain direct evidence on LPS-induced TNF-α production and iNOS expression by OCL, OCL-enriched cultures were prepared by 7-day cocultures of bone marrow cells of adult BALB/c mice and osteoblastic cells (OBs) derived from calvaria of newborn BALB/c mice, and the generation of TNF-α and iNOS in OCL stimulated with LPS was examined immunocytochemically. When the cultured cells were stimulated with 100 ng/ml of LPS, OCL clearly showed TNF-α and iNOS expression. Without LPS-stimulation, no expression was observed. TNF activity in the culture supernatants of the OCL-enriched cultures in the presence of LPS was also detected by cytotoxic assay that used TNF-sensitive L929 cells. The dentin resorption activity of OCL was estimated by area and number of pits formed on dentin slices, which were covered by the OCL fraction and cultured in the presence or absence of LPS, sodium nitroprusside (SNP; a NO generating compound), N^G-monomethyl L-arginine acetate (L-NMMA; a competitive inhibitor of NO synthase (NOS)), or LPS plus L-NMMA. Pit formation was obviously inhibited in the presence of SNP and slightly inhibited in the presence of L-NMMA, but it was not affected in the presence of LPS or LPS plus L-NMMA. These findings indicate that OCL produces TNF and expresses iNOS in response to LPS, but the LPS-activation of OCL scarcely affects pit formation by them.     

Single and Compound Knock-outs of MicroRNA (miRNA)-155 and Its Angiogenic Gene Target CCN1 in Mice Alter Vascular and Neovascular Growth in the Retina via Resident Microglia

The response of the retina to ischemic insult typically leads to aberrant retinal neovascularization, a major cause of blindness. The epigenetic regulation of angiogenic gene expression by miRNAs provides new prospects for their therapeutic utility in retinal neovascularization. Here, we focus on miR-155, a microRNA functionally important in inflammation, which is of paramount importance in the pathogenesis of retinal neovascularization. Whereas constitutive miR-155-deficiency in mice results in mild vascular defects, forced expression of miR-155 causes endothelial hyperplasia and increases microglia count and activation. The mouse model of oxygen-induced retinopathy, which recapitulates ischemia-induced aberrant neovessel growth, is characterized by increased expression of miR-155 and localized areas of microglia activation. Interestingly, miR-155 deficiency in mice reduces microglial activation, curtails abnormal vessel growth, and allows for rapid normalization of the retinal vasculature following ischemic insult. miR-155 binds to the 3′-UTR and represses the expression of the CCN1 gene, which encodes an extracellular matrix-associated integrin-binding protein that both promotes physiological angiogenesis and harnesses growth factor-induced abnormal angiogenic responses. Single CCN1 deficiency or double CCN1 and miR-155 knock-out in mice causes retinal vascular malformations typical of faulty maturation, mimicking the vascular alterations of miR-155 gain of function. During development, the miR-155/CCN1 regulatory axis balances the proangiogenic and proinflammatory activities of microglia to allow for their function as guideposts for sprout fusion and anastomosis. Under ischemic conditions, dysregulated miR-155 and CCN1 expression increases the inflammatory load and microglial activation, prompting aberrant angiogenic responses. Thus, miR-155 functions in tandem with CCN1 to modulate inflammation-induced vascular homeostasis and repair.     

The Inhibitory Effect of C-phycocyanin Containing Protein Extract (C-PC Extract) on Human Matrix Metalloproteinases (MMP-2 and MMP-9) in Hepatocellular Cancer Cell Line (HepG2)

Spirulina platensis :have been studied for several biological activities. In the current study C-phycocyanin containing protein extract (C-PC extract) of Spirulina platensis have been studied for its effect on human matrix metalloproteinases (MMP-1, MMP-2 and MMP-9) and tissue inhibitors of MMPs (TIMP-1 and TIMP-2). In the present study, breast cancer cell line (MDA-MB 231) and hepatocellular cancer cell line (HepG2) were examined for inhibition of MMPs at different levels of expression after C-PC extract treatment. Herein, we have demonstrated that C-PC extract significantly reduced activity of MMP-2 by 55.13% and MMP-9 by 57.9% in HepG2 cells at 15 μg concentration. Additionally, the treatment has reduced mRNA expression of MMP-2 and MMP-9 at 20 μg concentration by 1.65-folds and 1.66-folds respectively. The C-PC extract treatment have also downregulated a mRNA expression of TIMP-2 by 1.12 folds at 20 μg concentration in HepG2 cells. Together, these results indicate that C-PC, extract successfully inhibited MMP-2 and -9 at different levels of expression and TIMP-2 at a mRNA expression level; however, extract did not have any effect on MMP-1 expressed in MDA-MB231 and TIMP-1 expressed in HepG2 cells as well as the exact mechanism of inhibition of MMP-2, MMP-9 and TIMP-2 remained unclear.     

Inhibitory Effects of Maleimide Derivatives from the Mycelia of the Fungus Antrodia cinnamomea BCRC 36799 on Nitric Oxide Production in Lipopolysaccharide (LPS)‐Activated RAW264.7 Macrophages

Four new maleimide derivatives, antrocinnamomins E–H ( 1 – 4 , resp.), together with (3S,4R)‐1‐hydroxy‐3‐(4‐hydroxyphenyl)‐4‐(2‐methylpropyl)pyrrolidine‐2,5‐dione ( 5 ) and ergosterol were isolated from the mycelia of Antrodia cinnamomea BCRC 36799. The structures were elucidated by 1D‐ and 2D‐NMR spectroscopy, and mass spectrometry. Compounds 1 – 5 were evaluated for their inhibitory effects on nitric oxide (NO) production by macrophages. Compounds 2 and 4 showed stronger inhibition of NO production than the positive control quercetin.     

Inhibitory effect of the human liver-derived antimicrobial peptide hepcidin 20 on biofilms of polysaccharide intercellular adhesin (PIA)-positive and PIA-negative strains of Staphylococcus epidermidis

Staphylococcus epidermidis plays a major role in biofilm-related medical device infections. Herein the anti-biofilm activity of the human liver-derived antimicrobial peptide hepcidin 20 (hep20) was evaluated against polysaccharide intercellular adhesin (PIA)-positive and PIA-negative clinical isolates of S. epidermidis. Hep20 markedly inhibited biofilm formation and bacterial cell metabolism of PIA-positive and PIA-negative strains, but the decrease in biofilm biomass only partially correlated with a decrease in viable bacteria. Confocal microscope images revealed that, in the presence of hep20, both PIA-positive and PIA-negative strains formed biofilms with altered architectures and reduced amounts of extracellular matrix. Co-incubation of hep20 with vancomycin produced no synergistic effect, evaluated as number of viable cells, both in preventing biofilm formation and in treating preformed biofilms. In contrast, biofilms obtained in the presence of hep20, and then exposed to vancomycin, displayed an increased susceptibility to vancomycin. These results suggest that hep20 may inhibit the production/accumulation of biofilm extracellular matrix.     

Cell surface antigens on rat neural progenitors and characterization of the CD3 (+)/CD3 (-) cell populations

While the hematopoietic lineage has been extensively studied using cluster of differentiation (CD) antibodies, very few data are available on the extracellular epitopes expressed by rat neural progenitors (rNPC) and their derivatives. In the present study, we used flow cytometry to screen 47 cell surface antigens, initially known as immune markers. The quantitative analyses were performed on rat neurospheres and compared with primary cultures of astroglial cells or cerebellar neurons. Several antigens such as CD80 or CD86 were clearly undetectable while others, like CD26 or CD161, showed a weak expression. Interestingly, 10% and 15% of the cells were immunopositive for CD172a and CD200, two immunoglobulin superfamily members preferentially expressed by glial or neuronal cells, respectively. Over 40% of the cells were immunopositive for CD3, CD71, or MHCI. The biological significance of the latter markers in rNPC remains to be determined but analyses of the CD3(-)/CD3(+) populations isolated by magnetic cell separation revealed differences in their cell fate. Indeed, CD3(+) cells did not establish neurospheres and differentiated mostly into GFAP(+) cells while CD3(-) cells were able to generate neurospheres upon mitogen treatment and gave rise to GFAP(+), A2B5(+), Tuj-1(+), and RIP(+) cells under differentiating conditions. In contrast, CD71(-)/CD71(+) cells did not show any significant difference in their proliferating and differentiating potentials. Finally, it is worth noting that an subpopulation of cells in rat neurospheres exhibit an immunoreactivity against anti-CD25 (IL2 receptor) and anti-CD62L (L-selectin) antibodies. The results reveal particular surface antigen profiles, giving new perspectives on the properties of rat brain-derived cells.     

铝胁迫对小麦胚诱导及超微结构的影响

目的：铝毒害在酸性土壤中普遍存在,已经成为影响小麦产量和品质的重要因素,因此小麦铝毒害与耐铝分子机制的研究逐渐成为了小麦抗逆研究的热点内容之一。从细胞水平上阐明铝毒害的机理。方法：设置了3种铝浓度梯度(100、200 300μmol/1),通过组织培养及透射电镜的方法观察了铝对铝敏感材料Scout66,耐铝材料Adas66及湖北主栽品种EM12胚发生阶段及超微结构的影响。结果：铝降低了胚的诱导率,抑制了胚的进一步分化,其中幼胚的诱导率从对照的94．0%降到了25．3% (300μmaol/1),成熟胚的诱导率从对照的75．0%降到了17．3%(300μtmol/1);铝严重破坏了小麦根尖的超微结构,与对照相比,最明显的变化是细胞核结构变得不完整,同时空泡增多,线粒体数量增加,脊的结构变模糊甚至消失。结论：以上结果说明铝破坏了细胞的超微结构,特别是细胞核和线粒体结构的破坏阻止了细胞的进一步分裂和分化。     

A Novel Matrix Protein p10 from the Nacre of Pearl Oyster (Pinctada fucata) and Its Effects on Both CaCO3 Crystal Formation and Mineralogenic Cells

A novel matrix protein, designated as p10 because of its apparent molecular mass of 10 kDa, was isolated from the nacreous layer of pearl oyster (Pinctada fucata) by reverse-phase high-performance liquid chromatography. In vitro crystallization experiments showed that p10 could accelerate the nucleation of calcium carbonate crystals and induce aragonite formation, suggesting that it might play a key role in nacre biomineralization. As nacre is known to contain osteogenic factors, two mineralogenic cell lines, MRC-5 fibroblasts and MC3T3-E1 preosteoblasts, were used to investigate the biological activity of p10. The results showed that p10 could increase alkaline phosphatase activity, an early marker of osteoblast differentiation, while the viability of MRC-5 and MC3T3-E1 remained unchanged after treatment of p10. Taken together, the findings led to identification of a novel matrix protein from the nacre of P. fucata that plays a role in both the mineral phase and in the differentiation of the cells involved in biomineralization.     

Effect and mechanism of the Ang-(1-7) on human mesangial cells injury induced by low density lipoprotein

Hyperlipidemia is an independent risk factor for renal disease, and lipid deposition is associated with glomerulosclerosis. The angiotensin converting enzyme 2-angiotensin-(1-7)-Mas axis (ACE2-Ang-(1-7)-Mas axis) has been reported to participate in lipid metabolic regulation but its mechanism remains unclear. We hypothesized Ang-(1-7) would reduce lipid uptake in human mesangial cells (HMCs) by regulating the low density lipoprotein receptor–sterol regulatory element binding proteins 2–SREBP cleavage activating protein (LDLr–SREBP2–SCAP) negative feedback system, and improve glomerulosclerosis by regulating the transforming growth factor-β1 (TGF-β1). In this study we found that ACE2 was undetected in HMCs. The administration of LDL caused normal LDLr–SREBPs–SCAP negative feedback effect. Exogenous Ang-(1-7) enhanced this negative feedback effect via down-regulating LDLr, SREBP2, and SCAP expression, and effectively inhibited LDL-induced lipid deposition and cholesterol increases. This enhanced inhibitory effect was reversed by the Mas receptor antagonist A-779. Meanwhile, Ang-(1-7) significantly decreased the high LDL-induced production of TGF-β1, an effect blocked by A-779. Interestingly, HMCs treated with Ang-(1-7) alone activated the TGF-β1 expression. Our results suggested that Ang-(1-7) inhibits LDL accumulation and decreases cholesterol levels via modulating the LDLr–SREBPs–SCAP negative feedback system through the Mas receptor. Moreover, Ang-(1-7) exhibits a dual regulatory effect on TGF-β1 in HMCs.     

The tumor-associated antigen 90K/Mac-2-binding protein secreted by human colon carcinoma cells enhances extracellular levels of promatrilysin and is a novel substrate of matrix metalloproteinases-2, -7 (matrilysin) and -9: Implications of proteolytic cleavage

Background

The tumor-associated antigen 90K (TAA90K)/Mac-2-binding protein is expressed at elevated level in cancerous tissues and associated with poor prognosis. Since TAA90K has been implicated in the restructuring of the extracellular matrix, we examined the functional relationship between colon cancer cell-derived TAA90K and the matrix metalloproteinase (MMP) promatrilysin (proMMP-7), and also tested whether TAA90K is a novel substrate for MMPs-2, -7 and -9.

Methods

The effect of TAA90K on proMMP-7 levels in HT-29 conditioned media was quantified by enzyme-linked immunosorbent assays. Binding of TAA90K to MMPs, extracellular matrix proteins and galectin-3 was measured by solid-phase binding assays. Proteolytic cleavage of TAA90K by MMPs was documented by SDS-PAGE and protein sequencing analysis.

Results

TAA90K enhanced extracellular levels of proMMP-7 in HT-29 cells. In addition, TAA90K was cleaved by MMPs-2, -7 and -9. MMP-7-mediated cleavage of TAA90K did not affect its binding to MMP-7, laminin-1, collagen IV and galectin-3 but reduced its interaction with fibronectin and laminin-10, and lowered the levels of proMMP-7 in the HT-29 medium.

Conclusion

TAA90K is a novel substrate for MMPs-2, -7 and -9 and modulates proMMP-7 levels in colon cancer cells.

General significance

Proteolytic cleavage of TAA90K may have functional implications in colon cancer.