Transcription factor Sp1 is involved in expressional regulation of coxsackie and adenovirus receptor in cancer cells.

Coxsackie and adenovirus receptor (CAR) was first known as a virus receptor. Recently, it is also known to have tumor suppressive activity such as inhibition of cell proliferation, migration, and invasion. It is important to understand how CAR expression can be regulated in cancers. Based on an existence of putative Sp1 binding site within CAR promoter, we investigated whether indeed Sp1 is involved in the regulation of CAR expression. We observed that deletion or mutation of Sp1 binding motif (-503/-498) prominently impaired the Sp1 binding affinity and activity of CAR promoter. Histone deacetylase inhibitor (TSA) treatment enhanced recruitment of Sp1 to the CAR promoter in ChIP assay. Meanwhile, Sp1 binding inhibitor suppressed the recruitment. Exogenous expression of wild-type Sp1 increased CAR expression in CAR-negative cells; meanwhile, dominant negative Sp1 decreased the CAR expression in CAR-positive cells. These results indicate that Sp1 is involved in regulation of CAR expression.

Altered expression of prohibitin in psoriatic lesions and its cellular implication.

Psoriasis is characterized by excessive proliferation of keratinocytes accompanying acanthosis and incomplete differentiation. Prohibitin was investigated by examining its function of HaCaT as well as psoriasis. Psoriatic involved skin revealed high level of prohibitin in the basal layer. Prohibitin was analyzed by applying RNAi (PHBi) with HaCaT, which demonstrated increased S-phase. PHBi showed enhanced sensitivity to anthralin-mediated cell death due to enhanced loss of mitochondrial membrane potential, suggesting a protective role of prohibitin against apoptosis. Collectively, prohibitin plays a role both in cell cycle regulation and in maintaining mitochondrial integrity, implying its association with pathogenesis of psoriasis.

Coxsackievirus B3 modulates cell death by downregulating activating transcription factor 3 in HeLa cells.

Activating transcription factor 3 (ATF3) is an early-induced gene involved in diverse cellular functions in response to various stresses including viral infection. Here we observed marked reduction of ATF3 by coxsackievirus B3 (CVB3) infection and investigated the regulation and functional role of ATF3 in HeLa cells for the understanding of biological significance of ATF3 downregulation. CVB3 infection markedly reduced ATF3 expression at mRNA and protein levels in parallel with p53 degradation, and preservation of p53 expression rescued CVB3 infection-induced ATF3 downregulation. ATF3 overexpression stimulated apoptotic cell death following CVB3 infection, accompanying with augmentation of CVB3 infection-induced eIF2alpha phosphorylation. However, ATF3 overexpression did not affect viral protein production but promoted virus progeny release. Taken together, our results suggest that ATF3 is under control of p53 in part and that the ATF3 downregulation via p53 degradation may contribute to effective viral production as a modulation mechanism of CVB3 infection-induced cell death.

Expression of short hairpin RNAs against the coxsackievirus B3 exerts potential antiviral effects in Cos-7 cells and in mice.

Chemically synthesized small interfering RNA (siRNA) has been used as an anti-coxsackievirus B3 (CVB3) agent. Herein, we investigated whether vector-derived short hairpin RNAs (shRNA) targeting CVB3 can exert antiviral activities, prior to their further application to viral vector system for efficient in vivo administration. Employing transient transfection assays to in vivo mouse models as well as to in vitro Cos-7 cell cultures, we directly demonstrated the potential antiviral activity of shRNAs following challenges with infectious CVB3. Of the six shRNAs that we designed, three prevented cell death from CVB3 infection by suppressing viral replication and viral production in Cos-7 cells. These were shRNA 2, which targeted the capsid protein VP1, and shRNAs 4 and 5, which targeted two different regions of the RNA-dependent RNA polymerase 3D. Furthermore, shRNAs 2 and 5 also exerted strong antiviral effects in viral replication in vivo, accompanied by attenuated pancreatic tissue damage. Through this direct evaluation system we addressed the development and application of vector-derived shRNAs as an anti-CVB3 agent, revealing new target sequences.

Analysis of T cell receptor alpha-chain variable region (Valpha) usage and CDR3alpha of T cells infiltrated into lesions of psoriasis patients.

Psoriasis is a common inflammatory skin disease and is considered as T cell-mediated immune response. In this study, we analyzed T cell receptor alpha-chain variable region (TCR Valpha) usage in the lesions of psoriasis patients using 5'-RACE. As the results, Valpha1, -2, -7, -8, -10, -11, -12, and -23 were commonly detected in psoriatic lesions and comparison of expressions of these Valpha types between psoriasis patients and healthy individuals showed that Valpha1, -7, -11, and -12 were highly increased in psoriasis patients than in healthy individuals. Compared with atopy dermatitis patients, the expressions of Valpha1 and Valpha7 were increased in psoriasis patients. Then, to identify CDR3alpha of T cells infiltrated in psoriatic lesions, we examined which type of J gene segment was rearranged with Valpha1 or Valpha7, which the expressions was specifically increased in psoriatic lesions. The result showed that the V-J rearrangements between the examined patients were not equivalent and their frequencies were diverse, however, several common rearrangements such as Valpha1-Jalpha13, -23, -27, or -34 and Valpha7-Jalpha12, -33 were detected. The results in this study might provide the clue to define the characteristics of T cells associated with the pathogenesis of psoriasis.

TGF-beta1 induces cardiac hypertrophic responses via PKC-dependent ATF-2 activation.

Several reports have suggested that the TAK1-MKK3/6-p38MAPK signaling axis is important for TGF-beta-related cardiac hypertrophy. Despite this, the effects of exogenous TGF-beta on cardiac hypertrophy and associated signaling mechanisms have not been demonstrated directly. Moreover, the roles of the signaling mechanisms involved in cardiac hypertrophy (TAK1 upstream and p38MAPK downstream) remain unclear. In this study, we investigated the potential involvement of protein kinase C and activating transcription factor-2 in TGF-beta1-induced cardiac hypertrophic responses in cultured neonatal rat ventricular cardiomyocytes. TGF-beta1 treatment resulted in upregulation of mRNA expression or promoter activities of beta-myosin heavy chain, atrial natriuretic factor, and brain natriuretic peptide, and increased myocyte protein content, cell size, and sarcomeric organization. These are all characteristic hallmarks of cardiac hypertrophy. PKC was found to be involved throughout the signaling system, and it was shown that it acts by mediating upstream TAK1 activation and leads to ATF-2 activation. PKC-dependent ATF-2 activation was shown to be involved in TGF-beta1-induced cardiac hypertrophic responses. The PKC inhibitors, GO6976 and GF109203X, completely blocked TGF-beta1-induced TAK1 kinase activity and subsequent downstream signaling pathways including ATF-2 phosphorylation, leading to suppression of ATF-2 transcriptional activity. This inhibitory effect was reflected in cardiac hypertrophic responses such as inhibitions of beta-MHC gene induction and ANF promoter activity. Our results suggest that PKC is involved in TGF-beta1-induced cardiac hypertrophic responses in our cell culture system and that ATF-2 activation plays a role.

Coxsackievirus B3 infection induces cyr61 activation via JNK to mediate cell death.

Coxsackievirus B3 (CVB3), an enterovirus in the Picornavirus family, is the most common human pathogen associated with myocarditis and idiopathic dilated cardiomyopathy. We found upregulation of the cysteine-rich protein gene (cyr61) after CVB3 infection in HeLa cells with a cDNA microarray approach, which is confirmed by Northern blot analysis. It is also revealed that the extracellular amount of Cyr61 protein was increased after CVB3 infection in HeLa cells. cyr61 is an early-transcribed gene, and the Cyr61 protein is secreted into the extracellular matrix. Its function is related to cell adhesion, migration, and neuronal cell death. Here, we show that activation of the cyr61 promoter by CVB3 infection is dependent on JNK activation induced by CVB3 replication and viral protein expression in infected cells. To explore the role of Cyr61 protein in infected HeLa cells, we transiently overexpressed cyr61 and infected HeLa cells with CVB3. This increased CVB3 growth in the cells and promoted host cell death by viral infection, whereas down-expression of cyr61 with short interfering RNA reduced CVB3 growth and showed resistance to cell death by CVB3 infection. In conclusion, we have demonstrated a new role for cyr61 in HeLa cells infected with CVB3, which is associated with the cell death induced by virus infection. These data thus expand our understanding of the physiological functions of cyr61 in virus-induced cell death and provide new insights into the cellular factors involved.

Polymorphisms of tumor necrosis factor (TNF) alpha and beta genes in Korean patients with psoriasis.

To evaluate the association of TNF-alpha (TNFA) and TNF-beta (TNFB) polymorphisms with psoriasis in the Korean population, we investigated TNF-alpha -238 and -308 promoter region and TNF-beta NcoI polymorphism using PCR-RFLP in 103 Korean psoriasis patients and 125 normal controls. The carriage and allele frequencies of TNFB*2 were significantly increased in patients with psoriasis compared with normal controls. However, TNFB*1/1 homozygote and TNFB*1 allele were significantly decreased in the patients. There were no significant differences in the polymorphism of TNF-alpha promoter -238 and -308 between the patients and controls. We also analyzed the frequencies of TNFB alleles according to the clinical characteristics of the psoriasis patients, but no significant differences were found. However, female patients with early-onset psoriasis showed an association with the TNFB*2 allele. In conclusion, our results suggest that polymorphisms of the TNFB gene may contribute to a predisposition to psoriasis in the Korean population.

Regulatory effect of SOCS on NF-kappaB activity in murine monocytes/macrophages.

The suppressor of cytokine signaling (SOCS) group of proteins has been implicated in regulation of various cytokine signaling and in a negative crosstalk between distinct signaling pathways. Interleukin-10 (IL-10) and LPS were known to induce expression of SOCS-3 in neutrophils and monocytes/macrophages. IL-10 was also reported to inhibit a proinflammatory signal-induced NF-kappaB activation in monocytes and peripheral T lymphocytes. The effects of increased SOCS-3 expression upon IL-10 regulation of NF-kappaB activation have not yet been demonstrated. Here we examined the effects of SOCS-3 on NF-kappaB activity. SOCS-3 did not induce any alterations in NF-kappaB activity induced by LPS or TNF-alpha. However, it enhanced RelA-dependent kappaB promoter activity when cotransfected with RelA. Similar results were observed with SOCS-1. In contrast, SOCS-2 did not show any regulatory effects on RelA activity. Analysis of C-terminal truncation mutants of SOCS-1 and SOCS-3 demonstrated that the SOCS box and its N-terminal region, a less well-conserved linker region were important for SOCS-3 activation of RelA. In contrast, the SOCS box itself was critical for SOCS-1 to activate RelA. These results suggest that SOCS proteins can enhance the effects of NF-kappaB/Rel proteins, and therefore, further modulate immune and inflammatory responses.

Identification of a commonly used CDR3 region of infiltrating T cells expressing Vbeta13 and Vbeta15 derived from psoriasis patients.

Psoriasis is a common inflammatory skin disease that is thought to be mediated by activated T cells. In this study, the complementarity-determining region 3 (CDR3) in T cell receptors was examined for a common sequence motif among the T cells infiltrated in psoriatic lesional skin. A common specific CDR3 motif (Vbeta13-DWTSGV-Jbeta2.7) in lesions from psoriasis patients was identified by polymerase-chain-reaction-based spectratyping analysis and DNA sequencing. In addition, VDJ rearrangement with highly homologous amino acid composition in the CDR3 was observed in Vbeta15 of T cell receptors in lesions derived from psoriatic patients. Remarkably, T cell receptors containing the Vbeta13-DWTSGV-Jbeta2.7 were also found in the clinically normal skin from the psoriasis patients, which might seem to be responsible for the artificial production of psoriatic lesions. The identified CDR3 motif was highly expressed in cutaneous lymphocyte antigen (CLA+) cells of peripheral blood mononuclear cells from psoriasis patients compared with the expression in healthy individuals. This result showed that the infiltrated CLA+ T cells with the Vbeta13-DWTSGV-Jbeta2.7 motif in peripheral blood mononuclear cells from psoriasis patients might be involved in the development of psoriatic lesions. In addition, the results in this study suggest that the infiltrated T cells with the Vbeta13-DWTSGV-Jbeta2.7 motif in psoriatic lesions may be involved in the pathogenesis of psoriasis.