Simvastatin inhibits TNFalpha-induced invasion of human cardiac myofibroblasts via both MMP-9-dependent and -independent mechanisms.

Statins can reduce adverse myocardial remodeling independently of their cholesterol-lowering ability. We have previously reported that simvastatin inhibits tumor necrosis factor-alpha (TNFalpha)-induced cardiac myofibroblast invasion and MMP-9 secretion, key events in this remodeling process. The aim of the present study was to investigate the mechanisms underlying this effect. Selective MMP-9 gene silencing with siRNA oligonucleotides revealed that myofibroblast invasion through a Matrigel barrier (Boyden chamber assay) was MMP-9-dependent. In contrast, cell migration (in the absence of Matrigel) was MMP-9-independent. Simvastatin, a commonly prescribed statin, inhibited both invasion and migration of myofibroblasts and disrupted the actin cytoskeleton as determined by confocal microscopy of rhodamine-phalloidin staining. All these effects of simvastatin were mimicked by the Rho-kinase inhibitor Y27632. TNFalpha activated the ERK-1/2, p38 MAPK, PI-3-kinase and NF-kappaB pathways but not the JNK pathway, as determined by immunoblotting with phospho-specific antibodies. Quantitative RT-PCR revealed that TNFalpha-induced MMP-9 mRNA expression was substantially reduced by pharmacological inhibitors of the ERK-1/2, PI-3-kinase and NF-kappaB pathways. However, none of the signal transduction pathways studied was influenced by simvastatin treatment. Moreover, despite reducing MMP-9 secretion, simvastatin had no effect on MMP-9 promoter activity (luciferase reporter assay) and actually increased MMP-9 mRNA levels. In summary, simvastatin reduces TNFalpha-induced invasion of human cardiac myofibroblasts through two distinct mechanisms: (i) by attenuating cell migration via Rho-kinase inhibition and subsequent cytoskeletal disruption, and (ii) by decreasing MMP-9 secretion via a post-transcriptional mechanism.

Selective gene silencing of either MMP-2 or MMP-9 inhibits invasion of human saphenous vein smooth muscle cells.

Activation of matrix metalloproteinases (MMPs) facilitates smooth muscle cell (SMC) invasion, an important event in the development of intimal hyperplasia in saphenous vein (SV) bypass grafts. In this study, we performed selective gene silencing using small inhibitory RNA (siRNA) oligonucleotides to examine the relative contributions of MMP-2 and MMP-9 to the invasiveness of cultured human SV-SMCs. Cultures were established from human SV obtained from patients undergoing coronary artery bypass surgery. Transfection of SV-SMCs with MMP-2 siRNA selectively reduced MMP-2 secretion and inhibited invasion through a Matrigel barrier. Supplementation of medium with recombinant MMP-2 overcame these effects. Similarly, transfection of SV-SMCs with MMP-9 siRNA selectively reduced MMP-9 secretion and subsequent invasion, effects reversed by recombinant MMP-9 supplementation. Neither MMP-2 nor MMP-9 siRNA inhibited SV-SMC migration in the absence of a Matrigel barrier. Our data demonstrate that selective gene-silencing of either MMP-2 or MMP-9 markedly reduces the invasive capacity of cultured human SV-SMCs, indicating that these MMPs play distinct non-overlapping roles in SV-SMC invasion in vitro. Specific manipulation of either MMP-2 or MMP-9 may therefore provide a valuable strategy for prevention of SV graft stenosis in man.

The herpesvirus saimiri Rta gene autostimulates via binding to a non-consensus response element.

Herpesvirus saimiri ORF 50a protein expression is sufficient to reactivate the entire lytic-replication cycle. ORF 50a functions as a sequence-specific transactivator that is capable of activating delayed-early gene expression via direct binding to an ORF 50 response element (RE) within the respective promoter. Here, it is shown that ORF 50a is capable of transactivating its own promoter. Deletion analysis of the ORF 50a promoter showed that the ORF 50-responsive element is contained within an 80 bp fragment, situated 293-373 bp from the transcription initiation site. Gel-retardation analysis further mapped the RE to a 34 bp fragment that was able to confer ORF 50 responsiveness to an enhancerless SV40 minimal promoter. Sequence analysis showed that this RE has no direct similarity to previously identified ORF 50 REs. Therefore, it is concluded that ORF 50a is capable of stimulating its own promoter via a novel RE.

The herpesvirus saimiri open reading frame (ORF) 50 (Rta) protein contains an at hook required for binding to the ORF 50 response element in delayed-early promoters.

The herpesvirus saimiri open reading frame (ORF) 50 encodes two proteins, which activate transcription directly, following interactions with delayed-early (DE) promoters containing a specific motif. In this report, we demonstrate that ORF 50 contains a DNA binding domain that has homology to an AT hook DNA binding motif. Deletion analysis of this domain reduces ORF 50-mediated transactivation of the DE ORF 6 and ORF 57 promoters by 100 and 90%, respectively. Furthermore, gel retardation experiments demonstrated that the AT hook motif is required for binding the ORF 50 response element in the promoters of DE genes. Single site-directed mutagenesis of the AT hook revealed that mutation of the glycine residue at position 408 to an alanine reduces ORF 50 transactivation of the ORF 57 promoter by 40%. Moreover, the mutation of multiple basic residues in conjunction with the glycine residue within the core element of the AT hook abolishes ORF 50-mediated transactivation. In addition, p50GFPDeltaAT-hook is capable of functioning as a trans-dominant mutant, leading to a reduction in virus production of approximately 50% compared to that for wild-type ORF 50.

The herpesvirus saimiri ORF73 gene product interacts with host-cell mitotic chromosomes and self-associates via its C terminus.

The herpesvirus saimiri (HVS) ORF73 gene product shares limited homology with the ORF73 protein of Kaposi's sarcoma-associated herpesvirus (KSHV). ORF73 is expressed in an in vitro model of HVS latency, where the genome persists as a non-integrated circular episome. This suggests it may have a similar role to KSHV ORF73 in episomal maintenance, by tethering viral genomes to host-cell chromosomes. Here, the association of ORF73 with host mitotic chromosomes is described. Deletion analysis demonstrates that the distal 123 aa of the ORF73 protein are required for mitotic chromosomal localization and for self-association. Moreover, deletion of the extreme C terminus disrupts both self-association and host mitotic chromosome colocalization. This suggests that HVS ORF73 has a similar role to KSHV ORF73 in episomal maintenance and that association of ORF73 to host mitotic chromosomes is dependent on its ability to form multimers.

The herpesvirus saimiri ORF 73 regulatory region provides long-term transgene expression in human carcinoma cell lines.

Herpesvirus saimiri (HVS) is capable of establishing a persistent infection in a variety of human carcinoma cell lines, by virtue of episomal maintenance. Moreover, the viral episome provides expression of a transgene in both in vitro and in vivo environments. At present, HVS vectors utilize heterologous promoters such as the IE hCMV promoter. However, this promoter maybe unsuitable for long-term expression in vivo, as promoter silencing has been observed in this and other herpesvirus-based vector systems. Ideal regulatory regions would be functional when the herpesvirus genome is maintained as a latent episome. We have previously shown that gene expression in an HVS-persistently-infected human carcinoma cell line is limited to an adjacent set of genes encoding ORFs 71-73. These genes are transcribed as a polycistronic mRNA species from a common regulatory region upstream of the ORF 73 gene. In this report, we assess the potential of the ORF 73 regulatory region to provide heterologous gene expression in a wide variety of human cancer cell lines. We demonstrate, utilizing transient transfection assays, that the ORF 73 regulatory region can provide transgene expression in a variety of human carcinoma cell lines, although levels of transgene expression are not as high as achieved under the control of heterologous promoters such as the IE hCMV promoter. Furthermore, incorporation of the minimal ORF 73 regulatory region in a recombinant HVS-based vector provides sustained expression of the green fluorescent protein in both in vitro and in vivo environments. These results suggest that the ORF 73 regulatory region may be suitable for use in HVS-based cancer gene therapy applications.

The Herpesvirus Saimiri open reading frame 73 gene product interacts with the cellular protein p32.

The role of the gamma-2 herpesvirus open reading frame (ORF) 73 gene product has become the focus of considerable interest. It has recently been shown that the Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is expressed during a latent infection and can modulate both viral and cellular gene expression. The herpesvirus saimiri (HVS) ORF 73 gene product has some sequence homology to LANA; however, the role of HVS ORF 73 is unknown. We have previously demonstrated that HVS ORF73 is expressed in a stably transduced human carcinoma cell line, where HVS genomes persist as nonintegrated circular episomes. This implies that there may be some functional homology between these proteins. To further investigate the role of the HVS ORF 73 protein, the yeast two-hybrid system was employed to identify interacting cellular proteins. We demonstrate that ORF 73 interacts with the cellular protein p32 and triggers the accumulation of p32 in the nucleus. Using reporter gene-based transient-transfection assays, we demonstrate that ORF 73 can transactivate a number of heterologous promoter constructs and also upregulate its own promoter. Moreover, ORF 73 and p32 act synergistically to transactivate these promoters. The binding of ORF 73 to p32 is mediated by an amino-terminal arginine-rich domain, which contains two functionally distinct nuclear localization signals. The p32 binding domains are required for ORF 73 transactivating abilities and for ORF 73 to induce nuclear accumulation of p32. These results suggest that ORF 73 can function as a regulator of gene expression and that p32 is involved in ORF 73-dependent transcriptional activation.

Characterization of the herpesvirus saimiri ORF73 gene product.

The herpesvirus saimiri (HVS) gene product encoded by ORF73 shares a limited homology with the ORF73 encoded protein of Kaposi's sarcoma-associated herpesvirus (KSHV). It has recently been shown that the KSHV ORF73 protein is expressed during a latent infection and co-localizes with host cell chromosomes, suggesting that it plays a role in episomal maintenance by tethering viral genomes to host cell chromosomes. At present the role of the HVS ORF73 gene product is unknown. However, the expression of HVS ORF73 in a stably transduced human carcinoma cell line, where the HVS genome persists as a non-integrated circular episome, has recently been shown. In this report, the characterization of the HVS ORF73 protein and the mapping of its functional domains are described. The results suggest that the HVS ORF73 gene encodes a 64 kDa nuclear protein. Moreover, the amino terminus contains two functional nuclear localization signals, whereas the carboxy terminus is required for the distinctive speckled nuclear distribution pattern as observed with both the HVS and KSHV ORF73 proteins.

The carboxy terminus of the herpesvirus saimiri ORF 57 gene contains domains that are required for transactivation and transrepression.

Herpesvirus saimiri (HVS) ORF 57 is homologous to genes identified in all classes of herpesviruses. We have previously shown that ORF 57 encodes a multifunctional protein, responsible for both transactivation and repression of viral gene expression at a post-transcriptional level. This suggests that the ORF 57 protein shares some functional similarities with the herpes simplex virus IE63/ICP27 and Epstein-Barr virus Mta proteins. However, little is known about the functional domains responsible for the properties of ORF 57 due to the limited homology shared between these proteins. In this report, we have identified the functional domains responsible for transactivation and repression by the ORF 57 protein. We demonstrate that the carboxy terminus is required for ORF 57 transactivation, repression and an intense SC-35 nuclear spotting. This region contains two highly conserved motifs amongst its homologues, a zinc finger-like motif and a highly hydrophobic domain. We further show that the hydrophobic domain is required for transactivation and is also involved in nuclear localization of the ORF 57 protein, whereas the zinc finger-like domain is required for transactivation, repression and the intense SC-35 nuclear spotting.