Je Kyung Seong

β-Catenin regulates expression of cyclooxygenase-2 in articular chondrocytes ☆

Pro-inflammatory cytokine such as interleukin (IL)-1beta causes inflammation of articular cartilage via induction of cyclooxygenase (COX)-2 expression. We investigated in this study the role of beta-catenin in the IL-1beta regulation of COX-2 expression in articular chondrocytes. IL-1beta increased expression of COX-2 and induced accumulation and nuclear translocation of transcriptionally competent beta-catenin. Inhibition of beta-catenin degradation by the treatment of cells with LiCl or proteasome inhibitor stimulated expression of COX-2, indicating that transcriptionally active beta-catenin is sufficient to induce COX-2 expression. This was demonstrated further by the observation that ectopic expression of transcriptionally competent beta-catenin stimulated expression of COX-2. Levels of beta-catenin and COX-2 protein were increased in osteoarthritic and rheumatoid arthritic cartilage, suggesting that beta-catenin may play a role in the inflammatory responses of arthritic cartilage. Taken together, our data suggest that accumulation of transcriptionally active beta-catenin contributes to the expression of COX-2 in articular chondrocytes.

Cholesterol Biosynthesis from Lanosterol MOLECULAR CLONING, TISSUE DISTRIBUTION, EXPRESSION, CHROMOSOMAL LOCALIZATION, AND REGULATION OF RAT 7-DEHYDROCHOLESTEROL REDUCTASE, A SMITH-LEMLI-OPITZ SYNDROME-RELATED PROTEIN

The cDNA encoding the 471-amino acid rat 7-dehydrocholesterol reductase (DHCR), an enzyme that has been implicated in both cholesterol biosynthesis and developmental abnormalities (e.g. Smith-Lemli-Opitz syndrome) in mammals, has been cloned and sequenced, and the primary structure of the enzyme has been deduced. The DHCR gene was mapped to chromosome 8q2.1 by fluorescence in situ hybridization. Rat DHCR, calculated molecular mass of 54.15-kDa polypeptide, shares a close amino acid identity with mouse and human DHCRs (96 and 87%, respectively) as compared with its other related proteins (e.g. fungal sterol Δ14-reductase) and exhibits high hydrophobicity (>68%) with 9 transmembrane domains. Five putative sterol-sensing domains were predicted to be localized in transmembrane domains 4–8, which are highly homologous to those found in 3-hydroxymethylglutaryl-CoA reductase, sterol regulatory element-binding protein cleavage-activating protein, and patched protein. The polypeptide encoded by DHCR cDNA was expressed in yeast as a 55.45-kDa myc-tagged fusion protein, which was recognized with anti-myc monoclonal antibody 9E10 and shown to possess full DHCR activity with respect to dependence on NADPH and sensitivity to DHCR inhibitors. Northern blot analysis indicates that the highest expression of DHCR mRNA was detected in liver, followed by kidney and brain. In rat brains, the highest level of mRNA encoding DHCR was detected in the midbrain, followed by the spinal cord and medulla. Feeding rats 5% cholestyramine plus 0.1% lovastatin in chow resulted in both approximately a 3-fold induction of DHCR mRNA and a 5-fold increase of the enzymic activity in the liver. When rats were fed 0.1% (w/w) AY-9944 (in chow) for 14-days, a complete inhibition of DHCR activity and a significant reduction in serum total cholesterol level were observed. However, the level of hepatic DHCR mRNA fell only slightly, suggesting that AY-9944 may act more rapidly at the protein level than at the level of transcription of the DHCR gene under these conditions.

Pharmacological characterization of adenosine receptors in PGT‐β mouse pineal gland tumour cells

The adenosine receptor in mouse pinealocytes was identified and characterized using pharmacological and physiological approaches. Expression of the two adenosine receptor subtypes A2B and A3 was detected in mouse pineal glands and PGT‐β cells by polymerase chain reaction and nucleotide sequencing. Adenosine and 5′‐N‐ethylcarboxamidoadenosine (NECA) evoked cyclic AMP generation but the A2A‐selective agonist 2‐(4‐(2‐carboxyethyl)phenylethylamino)adenosine‐5′‐N‐ethylcarboxamideadenosine (CGS 21680) and the A1‐specific agonists R‐N6‐(2‐phenylisopropyl)adenosine (R‐PIA) and N6‐cyclopentyladenosine (CPA) had little effect on intracellular cyclic AMP levels. The A2B receptor selective antagonists alloxazine and enprofylline completely blocked NECA‐mediated cyclic AMP accumulation. Treatment of cells with the A3‐selective agonist N6‐(3‐iodobenzyl)‐5′‐(N‐methylcarbamoyl)adenosine (IB‐MECA) inhibited the elevation of the cyclic AMP level induced by NECA or isoproterenol in a concentration‐dependent manner with maximal inhibition of 40 – 50%. These responses were blocked by the specific A3 adenosine receptor antagonist MRS 1191. Pretreatment of the cells with pertussis toxin attenuated the IB‐MECA‐induced responses, suggesting that this effect occurred via the pertussis toxin‐sensitive inhibitory G proteins. IB‐MECA also caused a concentration‐dependent elevation in [Ca2+]i and IP3 content. Both the responses induced by IB‐MECA were attenuated by treatment with U73122 or phorbol 12‐myristate 13‐acetate. These data suggest the presence of both A2B and A3 adenosine receptors in mouse pineal tumour cells and that the A2B receptor is positively coupled to adenylyl cyclase whereas the A3 receptor is negatively coupled to adenylyl cyclase and also coupled to phospholipase C.

Syndecan-2 Functions as a Docking Receptor for Pro-matrix Metalloproteinase-7 in Human Colon Cancer Cells

Although elevated syndecan-2 expression is known to be crucial for the tumorigenic activity in colon carcinoma cells, how syndecan-2 regulates colon cancer is unclear. In human colon adenocarcinoma tissue samples, we found that both mRNA and protein expression of syndecan-2 were increased, compared with the neighboring normal epithelium, suggesting that syndecan-2 plays functional roles in human colon cancer cells. Consistent with this notion, syndecan-2-overexpressing HT-29 colon adenocarcinoma cells showed enhanced migration/invasion, anchorage-independent growth, and primary tumor formation in nude mice, paralleling their morphological changes into highly tumorigenic cells. In addition, our experiments revealed that syndecan-2 enhanced both expression and secretion of matrix metalloproteinase-7 (MMP-7), directly interacted with pro-MMP-7, and potentiated the enzymatic activity of pro-MMP-7 by activating its processing into the active MMP-7. Collectively, these data strongly suggest that syndecan-2 functions as a docking receptor for pro-MMP-7 in colon cancer cells.

Expression of MUC5AC mRNA in the Goblet Cells of Human Nasal Mucosa

Objectives/Hypothesis Mucus hypersecretion is a common feature in chronic sinusitis with polyps. Because mucus hypersecretion is commonly accompanied by goblet cell hyperplasia, it is important to identify which mucin gene mRNAs are expressed in the goblet cells of the surface epithelium in the human airway. This study aims to investigate the pattern of expression of MUC5AC messenger RNA (mRNA) in the goblet cells of human nasal mucosa.

Cholesterol biosynthesis from lanosterol: molecular cloning, chromosomal localization, functional expression and liver-specific gene regulation of rat sterol delta8-isomerase, a cholesterogenic enzyme with multiple functions.

Sterol Delta(8)-isomerase (SI) (EC 5.3.3.5), also known as emopamil binding protein or sigma receptor, catalyses the conversion of the 8-ene isomer into the 7-ene isomer in the cholesterol biosynthetic pathway in mammals. Recently, mutations of SI have been found to be associated with Conradi-Hünermann syndrome in humans. To investigate the in vitro and in vivo modes of molecular regulation of SI and its role in cholesterol biosynthesis in mammals, we isolated a full-length cDNA encoding rat SI. The deduced amino-acid sequence of rat SI predicts a 230-residue protein (26737 Da) with 87% and 80% amino-acid identity to mouse and human counterparts. The rat SI gene was mapped to chromosome 12q1.2 using fluorescence in situ hybridization (FISH). The biological function of the cloned rat SI cDNA was verified by overexpressing recombinant Myc-SI in Saccharomyces cerevisiae. It showed a characteristic pattern of inhibition on exposure to trans-2-[4-(1,2-diphenylbuten-1-yl)phenoxy]-N,N-dimethylethylamine (tamoxifen; IC(50)=11.2 microM) and 3beta-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A; IC(50)=4.2 microM), two well known potent inhibitors of SI. Northern-blot analysis of 3-week-old rats compared with 2-year-old rats showed that SI mRNA expression in both age groups was restricted to liver, where a 70% reduction in mRNA levels was observed in 2-year-old rats. The FISH studies revealed ubiquitous expression of SI mRNA in rat hepatocytes. The in vitro studies showed that the SI mRNA was highly suppressed by 25-hydroxycholesterol in H4IIE cells. Treatment of H4IIE cells grown in medium supplemented with fetal bovine serum with tamoxifen for 24 h resulted in a dose-dependent induction of SI mRNA, with a concomitant suppression of sterol regulatory element binding protein-1 mRNA. Interestingly, this effect was not seen in emopamil-treated cells. The in vivo experiments also indicate that both mRNA expression and enzymic activity of SI in liver were induced approx. 3-fold in rats fed 5% (w/w) cholestyramine plus 0.1% (w/w) lovastatin in normal chow for 2 weeks. With this newly cloned rat SI cDNA, it becomes possible to gain molecular understanding of previously unknown and tamoxifen-mediated gene regulation of SI that is involved in cholesterol metabolism, ischaemia and genetic diseases.

Micrometer resolution imaging using unmonochromatized synchrotron x-rays : Phantom, human breast tissue, and live animal imaging studies

A new 5C1 beam line was recently built on Pohang Light Source (PLS) in Korea for possible medical applications. An imaging system on the beam line consisted of silicon wafer attenuators, tungsten slit, CdWO4 scintillator, CCD camera, and personal computer. Both spatial resolution test pattern and glass capillary filled with air bubbles were imaged to evaluate resolution and phase-contrast effects for synchrotron imaging system, respectively. Mammographic accreditation phantom, cancerous human breast specimens, and fish and nude mouse were also imaged. Both spatial resolution and image quality of the synchrotron imaging system were compared with those of conventional mammography system. The images of resolution test pattern showed a few tens of micrometer spatial resolution on synchrotron imaging system. The images of capillary filled with air bubbles revealed phase contrast effect. Mammographic accreditation phantom, human breast specimen, and animal images with synchrotron imaging system showed much higher resolution with great details of object and higher enhanced contrast compared to those with conventional mammography system. We successfully implemented and tested a micrometer resolution imaging system on PLS 5C1 beam line. Using a simple and inexpensive synchrotron imaging system on PLS 5C1 beam line, micrometer resolution images for resolution test pattern, mammographic accreditation phantom, human breast specimens, and live animals were acquired and visually evaluated.