Inn-Oc Han

Syndecan-2 Mediates Adhesion and Proliferation of Colon Carcinoma Cells

Syndecan-2 is a transmembrane heparan sulfate proteoglycan whose function at the cell surface is unclear. In this study, we examined the function of syndecan-2 in colon cancer cell lines. In several colon cancer cell lines, syndecan-2 was highly expressed compared with normal cell lines. In contrast, syndecan-1 and -4 were decreased. Cell biological studies using the extracellular domain of recombinant syndecan-2 (2E) or spreading assay with syndecan-2 antibody-coated plates showed that syndecan-2 mediated adhesion and cytoskeletal organization of colon cancer cells. This interaction was critical for the proliferation of colon carcinoma cells. Blocking with 2E or antisense syndecan-2 cDNA induced G0/G1 cell cycle arrest with concomitantly increased expression of p21, p27, and p53. Furthermore, blocking of syndecan-2 through antisense syndecan-2 cDNA significantly reduced tumorigenic activity in colon carcinoma cells. Therefore, increased syndecan-2 expression appears to be a critical for colon carcinoma cell behavior, and syndecan-2 regulates tumorigenic activity through regulation of adhesion and proliferation in colon carcinoma cells.

Ionizing radiation induces astrocyte gliosis through microglia activation.

The aim of this study was to investigate the role of microglia in radiation-induced astrocyte gliosis. We found that a single dose of 15 Gy radiation to a whole rat brain increased immunostaining of glial fibrillary acidic protein in astrocytes 6 h later, and even more so 24 h later, indicating the initiation of gliosis. While irradiation of cultured rat astrocytes had little effect, irradiation of microglia-astrocyte mixed-cultures displayed altered astrocyte phenotype into more processed, which is another characteristic of gliosis. Experiments using microglia-conditioned media indicated this astrocyte change was due to factors released from irradiated microglia. Irradiation of cultured mouse microglial cells induced a dose-dependent increase in mRNA levels for cyclooxygenase-2 (COX-2), interleukin (IL)-1beta, IL-6, IL-18, tumor necrosis factor-alpha and interferon-gamma-inducible protein-10, which are usually associated with microglia activation. Consistent with these findings, irradiation of microglia activated NF-kappaB, a transcription factor that regulates microglial activation. Addition of prostaglandin E2 (PGE2: a metabolic product of the COX-2 enzyme) to primary cultured rat astrocytes resulted in phenotypic changes similar to those observed in mixed-culture experiments. Therefore, it appears that PGE(2) released from irradiated microglia is a key mediator of irradiation-induced gliosis or astrocyte phenotype change. These data suggest that radiation-induced microglial activation and resultant production of PGE2 seems to be associated with an underlying cause of inflammatory complications associated with radiation therapy for malignant gliomas.

An N-terminal region of Sp1 targets its proteasome-dependent degradation in vitro

The transcription factor Sp1 is important for the expression of many cellular genes. Previously, it was shown that reduced O-glycosylation of Sp1 is associated with increased proteasome susceptibility. Sp1 undergoes proteasome-dependent degradation in cells stressed with glucose deprivation and adenylate cyclase activation, and this process is blocked in cells treated with glucosamine. In this study, using a reconstituted in vitro system, we identified the principal structural determinant in Sp1 that targets Sp1 for proteasome-dependent degradation. We found by using deletion analysis that the N-terminal 54 amino acids of Sp1 is required for Sp1 degradation. This element can act as an independent processing signal by directing degradation of an unrelated protein. Recognition of this Sp1 element by the proteasome-dependent system is saturable, and ubiquitination of this element is not required for recognition. Time course experiments revealed that Sp1 degradation is a two-step process. First, a discrete endoproteolytic cleavage occurs downstream of the target region immediately C-terminal to Leu56. The Sp1 sequence C-terminal to the cleavage site is subsequently degraded, whereas the N-terminal peptide remains intact. The identification of this Sp1 degradation-targeting signal will facilitate the identification of the critical proteins involved in the control of Sp1 proteasome-dependent degradation and the role of OGlcNAc in this process.

Promoter hypermethylation downregulates RUNX3 gene expression in colorectal cancer cell lines.

It was recently reported that RUNX3 gene expression is significantly downregulated in human gastric cancer cells due to hypermethylation of its promoter region or hemizygous deletion (Cell, 109, 2002). To verify the genetic alterations and methylation status of the RUNX3 gene in colorectal carcinogenesis, we analysed for mutations, loss of heterozygosity (LOH), and RUNX3 gene promoter hypermethylation, in 32 colorectal cancer cell lines. RT–PCR analysis showed undetectable or low RUNX3 expression in 16 cell lines, and no mutations were found in the RUNX3 gene by PCR-SSCP analysis. Of these 16 cell lines, hypermethylation of the RUNX3 promoter was confirmed in 12. The following observations were made: (i) RUNX3 was re-expressed after 5-aza-2′-deoxycytidine treatment, (ii) the RUNX3 promoter was found to be methylated by MS-PCR, and (iii) hypermethylation of the RUNX3 promoter was confirmed by direct sequencing analysis after sodium bisulfite modification in the above 12 cell lines. RUNX3 was neither methylated nor expressed in four cell lines. Of these four, microsatellite instability (MSI) at the RUNX3 locus was found in three, SNU-61 (D1S246), SNU-769A, and SNU-769B (D1S199). This study suggests that transcriptional repression of RUNX3 is caused by promoter hypermethylation of the RUNX3 CpG island in colorectal cancer cell lines, and the results of these experiments may contribute to an understanding of the role of RUNX3 inactivation in the pathogenesis of colorectal cancers.

IL-1β, an immediate early protein secreted by activated microglia, induces iNOS/NO in C6 astrocytoma cells through p38 MAPK and NF-κB pathways

In the present study we sought to examine cell–cell interactions by investigating the effects of factors released by stimulated microglia on inducible nitric oxide (NO) synthase (iNOS) induction in astrocytoma cells. After examining the temporal profiles of proinflammatory molecules induced by lipopolysaccharide (LPS) stimulation in BV2 microglial cells, iNOS and IL‐1β were observed to be the first immediate‐response molecules. Removal of LPS after 3 hr stimulation abrogated NO release, whereas a full induction of IL‐1β was retained in BV2 cells. We observed consistently that conditioned medium (CM) from activated microglia resulted in the induction of iNOS in C6 cells, and IL‐1β was shown to be a key regulator of iNOS induction. An IL‐1β‐neutralizing antibody diminished NO induction. Incubation with recombinant IL‐1β stimulated NO release to a lesser extent compared to microglial CM; co‐treatment of LPS and IL‐1β had a potent, synergistic effect on NO release from C6 cells. Transient transfection with MEK kinase 1 (MEKK1) or nuclear factor‐kappa B (NF‐κB) expression plasmids induced iNOS, and IL‐1β further enhanced the MEKK1 response. Furthermore, IL‐1β‐mediated NO release from C6 cells was significantly suppressed by inhibition of p38 mitogen activated protein kinase (MAPK) or NF‐κB by specific chemical inhibitors. Both IL‐1β and MEKK1 stimulated p38 and JNK MAPKs, as well as the NF‐κB pathway, to induce iNOS in C6 cells. Microglia may represent an anti‐tumor response in the central nervous system, which is potentiated by the local secretion of immunomodulatory factors that in turn affects astrocytoma (glioma) cells. A better understanding of microglia–glioma or microglia–astrocyte interactions will help in the design of novel immune‐based therapies for brain tumors or neuronal diseases.

Transmembrane Domain-induced Oligomerization Is Crucial for the Functions of Syndecan-2 and Syndecan-4

The syndecans are known to form homologous oligomers that may be important for their functions. We have therefore determined the role of oligomerization of syndecan-2 and syndecan-4. A series of glutathione S-transferase-syndecan-2 and syndecan-4 chimeric proteins showed that all syndecan constructs containing the transmembrane domain formed SDS-resistant dimers, but not those lacking it. SDS-resistant dimer formation was hardly seen in the syndecan chimeras where each transmembrane domain was substituted with that of platelet-derived growth factor receptor (PDGFR). Increased MAPK activity was detected in HEK293T cells transfected with syndecan/PDGFR chimeras in a syndecan transmembrane domain-dependent fashion. The chimera-induced MAPK activation was independent of both ligand and extracellular domain, implying that the transmembrane domain is sufficient to induce dimerization/oligomerization in vivo. Furthermore, the syndecan chimeras were defective in syndecan-4-mediated focal adhesion formation and protein kinase Cα activation or in syndecan-2mediated cell migration. Taken together, these data suggest that the transmembrane domains are sufficient for inducing dimerization and that transmembrane domain-induced oligomerization is crucial for syndecan-2 and syndecan-4 functions.

Carbonic anhydrase IX (CA9) modulates tumor-associated cell migration and invasion.

Expression of carbonic anhydrase IX (CA9) was shown to be strongly involved in high incidences of metastasis and poor prognosis in various human tumors. In this study, we investigated the possible role for CA9 in tumor metastases in vitro, using a gene transfection tool in the human cervical carcinoma cell line C33A. Gene expression profiling of CA9-transfected cells (C33A/CA9) and vector-transfected cells (C33A/Mock) was investigated by DNA microarray. The biological functions of differentially expressed genes between the C33A/CA9 and C33A/Mock cells included cell growth, regulation of cell–cell and cell–extracellular matrix adhesion and cytoskeletal organization. Immunofluorescent stain and Matrigel culture showed cytoskeletal remodeling, disassembled focal adhesion, weakened cell-cell adhesion and increased motility in C33A/CA9 cells. These invasive and metastatic phenotypes were associated with Rho-GTPase-related epithelial-mesenchymal transition. Inhibition of the Rho/Rho kinase pathway by a ROCK inhibitor (Y27632) and si-Rho (short interference RNA against RhoA) showed that Rho-GTPase signaling was involved in cellular morphologic and migratory changes. The effect of CA9 on Rho-GTPase signaling was also confirmed by silencing CA9 expression. Our results suggest that CA9 overexpression induces weakening of cell adhesions and augmented cell motility by aberrant Rho-GTPase signal transduction. Our study shows an underlying mechanism of CA9-related enhanced metastatic potential of tumor cells.

Glucosamine exerts a neuroprotective effect via suppression of inflammation in rat brain ischemia/reperfusion injury.

We investigated the neuroprotective effect of glucosamine (GlcN) in a rat middle cerebral artery occlusion model. At the highest dose used, intraperitoneal GlcN reduced infarct volume to 14.3% ± 7.4% that of untreated controls and afforded a reduction in motor impairment and neurological deficits. Neuroprotective effects were not reproduced by other amine sugars or acetylated‐GlcN, and GlcN suppressed postischemic microglial activation. Moreover, GlcN suppressed lipopolysaccharide (LPS)‐induced upregulation of proinflammatory mediators both in vivo and in culture systems using microglial or macrophage cells. The anti‐inflammatory effects of GlcN were mainly attributable to its ability to inhibit nuclear factor kappaB (NF‐κB) activation. GlcN inhibited LPS‐induced nuclear translocation and DNA binding of p65 to both NF‐κB consensus sequence and NF‐κB binding sequence of inducible nitric oxide synthase promoter. In addition, we found that GlcN strongly repressed p65 transactivation in BV2 cells using Gal4‐p65 chimeras system. P65 displayed increased O‐GlcNAcylation in response to LPS; this effect was also reversed by GlcN. The LPS‐induced increase in p65 O‐GlcNAcylation was paralleled by an increase in interaction with O‐GlcNAc transferase, which was reversed by GlcN. Finally, our results suggest that GlcN or its derivatives may serve as novel neuroprotective or anti‐inflammatory agents.

p38 mitogen-activated protein kinase mediates lipopolysaccharide, not interferon-γ, -induced inducible nitric oxide synthase expression in mouse BV2 microglial cells

The present study examined the role of mitogen - activated protein kinases (MAPKs) in inducible nitric oxide synthase (iNOS) induction by lipopolysaccharide (LPS) or interferon - gamma (IFN - gamma) in the murine microglial cell line BV2 cells. LPS rapidly (<2 h) induced iNOS mRNA expression whereas IFN - gamma did not within 8 h after stimulation. LPS activated three MAPK subtypes, extracellular signal - regulated kinase (ERK), p38 and c - Jun N - terminal kinase (JNK) as early as 15 min after stimulation. In contrast, IFN - gamma activated only ERK after 6 h of stimulation and did not alter the activity of p38 and JNK. LPS-induced iNOS expression was markedly decreased by the p38 inhibitor SB203580, but not by the MAPK or ERK kinase inhibitor PD98059. IFN - gamma - induced nitric oxide (NO) generation was partially inhibited by PD98059 and SB203580 only in combination. The results demonstrate that MAPKs differentially mediate NO production by LPS - and IFN - gamma in BV2 cells.

Novel thiazolidinedione derivatives with anti-obesity effects: Dual action as PTP1B inhibitors and PPAR-γ activators

Benzylidene-2,4-thiazolidinedione derivatives with substitutions at both the ortho and para-positions of the phenyl group were synthesized as PTP1B inhibitors with IC(50) values in a low micromolar range. Compound 18l, the lowest, bore an IC(50) of 1.3 μM. In a peroxisome proliferator-activated receptor-γ (PPAR-γ) promoter reporter gene assay, 18l was found to activate the transcription of the reporter gene with potencies comparable to those of troglitazone, rosiglitazone, and pioglitazone. In vivo efficacy of 18l as an anti-obesity and hypoglycemic agent was evaluated in a mouse model system. Compound 18l significantly suppressed weight gain and significantly improved blood parameters such as TG, total cholesterol and NEFA without overt toxic effects.