Smooth Muscle Gamma-actin Promoter Research Articles

Smooth Muscle-Specific Gene Delivery in the Vasculature Based on Restriction of DNA Nuclear Import

The two currently employed approaches restricting gene delivery and/or expression to desired cell types in vivo rely on cell surface targeting or cell-specific promoters. We have developed a third approach based on cell-specific nuclear transport of the delivered plasmid DNA. We have previously shown that plasmid nuclear import in non-dividing cells is sequence-specific and have identified a set of cell-specific DNA nuclear targeting sequences that can be used to limit DNA nuclear import to desired cell types. Specifically we have identified elements of the smooth muscle gamma actin (SMGA) promoter that direct plasmid nuclear import selectively in smooth muscle cells (SMCs) in vitro (Vacik et al, 1999, Gene Therapy 6:1006-1014). In the present study, we demonstrate that the SMC-specific DNA nuclear targeting sequence from the SMGA promoter drives nuclear accumulation of plasmids and subsequent gene expression exclusively in the smooth muscle cell layer of the vessel wall in the intact vasculature of rats using electroporation mediated delivery. These results demonstrate that certain DNA nuclear targeting sequences can be used to restrict DNA nuclear import to specific cell types providing a new, novel means of cell targeting for gene therapy.

The smooth muscle gamma-actin gene is androgen responsive in prostate epithelia.

Nkx 3.1 is an evolutionarily conserved vertebrate homolog of the Drosophila Nk-3 homeodomain gene bagpipe that is expressed by a variety of cells during early mammalian development and has been shown to be a critical factor for prostate development and function. Previous studies utilizing a heterologous cell transfection strategy from our laboratory identified the smooth muscle gamma-actin (SMGA) gene as a novel molecular target of Nkx 3.1 regulatory activity. In the studies presented here, SMGA gene activity and regulation were evaluated in normal and cancerous prostate epithelial cells. SMGA transcripts were demonstrated in prostate epithelia and SMGA mRNA levels were increased in androgen-responsive LNCaP cancer and normal prostate epithelial cells. SMGA gene transcriptional activity was androgen responsive in these cells and required a segment of the human SMGA promoter containing NKE and SRF (serum response factor) binding elements. This region of the human SMGA proximal promoter is well conserved across species and is synergistically activated by coexpression of Nkx 3.1 and SRF in heterologous CV-1 cells. SMGA transcription was not responsive to steroid in PC-3 prostate epithelial cancer cells, which do not express Nkx 3.1. However, SMGA transcription was influenced by expression of androgen receptor in these cells, a situation that allows the androgen-dependent expression of Nkx 3.1. Furthermore, SMGA gene activity was influenced by direct Nkx 3.1 expression in the PC-3 cells. Thus, SMGA gene activity in prostate epithelia is due, in part, to the androgen-dependent expression of Nkx 3.1. As such, our studies provide the initial description of Nkx 3.1 target gene regulatory activity in the prostate.

The Smooth Muscle γ-Actin Gene Promoter Is a Molecular Target for the Mouse bagpipe Homologue, mNkx3-1, and Serum Response Factor

An evolutionarily conserved vertebrate homologue of the Drosophila NK-3 homeodomain gene bagpipe, Nkx3-1, is expressed in vascular and visceral mesoderm-derived muscle tissues and may influence smooth muscle cell differentiation. Nkx3-1 was evaluated for mediating smooth muscle gamma-actin (SMGA) gene activity, a specific marker of smooth muscle differentiation. Expression of mNkx3-1 in heterologous CV-1 fibroblasts was unable to elicit SMGA promoter activity but required the coexpression of serum response factor (SRF) to activate robust SMGA transcription. A novel complex element containing a juxtaposed Nkx-binding site (NKE) and an SRF-binding element (SRE) in the proximal promoter region was found to be necessary for the Nkx3-1/SRF coactivation of SMGA transcription. Furthermore, Nkx3-1 and SRF associate through protein-protein interactions and the homeodomain region of Nkx3-1 facilitated SRF binding to the complex NKE.SRE. Mutagenesis of Nkx3-1 revealed an inhibitory domain within its C-terminal segment. In addition, mNkx3-1/SRF cooperative activity required an intact Nkx3-1 homeodomain along with the MADS box of SRF, which contains DNA binding and dimerization structural domains, and the contiguous C-terminal SRF activation domain. Thus, SMGA is a novel target for Nkx3-1, and the activity of Nkx3-1 on the SMGA promoter is dependent upon SRF.

Cell-specific nuclear import of plasmid DNA.

One factor limiting the success of non-viral gene therapy vectors is the relative inability to target genes specifically to a desired cell type. To address this limitation, we have begun to develop cell-specific vectors whose specificity is at the level of the nuclear import of the plasmid DNA. We have recently shown that nuclear import of plasmid DNA is a sequence-specific event, requiring the SV40 enhancer, a region known to bind to a number of general transcription factors (Dean DA, Exp Cell Res 1997; 230: 293). From these studies we developed a model whereby transcription factor(s) bind to the DNA in the cytoplasm to create a protein-DNA complex that can enter the nucleus using the protein import machinery. Our model predicts that by using DNA elements containing binding sites for transcription factors expressed in unique cell types, we should be able to create plasmids that target to the nucleus in a cell-specific manner. Using the promoter from the smooth muscle gamma actin (SMGA) gene whose expression is limited to smooth muscle cells, we have created a series of reporter plasmids that are expressed selectively in smooth muscle cells. Moreover, when injected into the cytoplasm, plasmids containing portions of the SMGA promoter localize to the nucleus of smooth muscle cells, but remain cytoplasmic in fibroblasts and CV1 cells. In contrast, a similar plasmid carrying the SV40 enhancer is transported into the nuclei of all cell types tested. Nuclear import of the SMGA promoter-containing plasmids could be achieved when the smooth muscle specific transcription factor SRF was expressed in stably transfected CV1 cells, supporting our model for the nuclear import of plasmids. Finally, these nuclear targeting sequences were also able to promote increased gene expression in liposome- and polycation-transfected non-dividing cells in a cell-specific manner, similar to their nuclear import activity. These results provide proof of principle for the development of cell-specific non-viral vectors for any desired cell type.