Eileen R. Mulvihill

A significant lag in the induction of ovalbumin messenger RNA by steroid hormones: A receptor translocation hypothesis

Although ovalbumin and conalbumin mRNA accumulate in the same tubular gland cells of the chick oviduct in response to estrogen or progesterone treatment, the kinetics of induction are markedly different. Conalbumin mRNA begins to accumulate within 30 min after estrogen administration, whereas there is a lag of approximately 3 hr before ovalbumin mRNA begins to accumulate, as measured by three independent assays. The kinetics of estrogen-receptor binding to chromatin indicate that these sites are saturated within 15 min of estrogen administration to the chicks, demonstrating that the lag is not due to slow uptake of the steroid. Suboptimal doses of estrogen produce the same lag, but the resultant rate of ovalbumin mRNA accumulation is lower than with an optimal dose. Partial induction of ovalbumin mRNA by a low dose of estrogen does not shorten the lag with an optimal dose. With progesteone, there is a lag of about 2 hr before either ovalbumin or conalbumin mRNA begins to accumulate. Treatment of chicks with hydroxyurea shortens the lag for ovalbumin induction with either hormone. Inhibition of protein synthesis with emetine does not prevent the accumulation of either ovalbumin or conalbumin mRNA. With cycloheximide, however, ovalbumin mRNA accumulation can be prevented. The existence of a lag suggests that there are intermediate steps between the binding of steroid receptors to chromatin and the induction of ovalbumin mRNA. There are basically two models to explain these delays in response: one involving the accumulation of an essential intermediate, and the other involving a rate-limiting translocation of steroid receptors from initial nonproductive chromatin-binding sites to productive sites. Several aspects of the kinetics of ovalbumin mRNA induction are more consistent with the latter model.

α1-Proteinase Inhibitor, α1-Antichymotrypsin, and α2-Macroglobulin Are the Antiapoptotic Factors of Vascular Smooth Muscle Cells

Serum depletion induces cell death. Whereas serum contains growth factors and adhesion molecules that are important for survival, serum is also likely to have antiapoptotic factor(s). We show here that the plasma proteinase inhibitors α1-proteinase inhibitor, α1-antichymotrypsin, and α2-macroglobulin function as critical antiapoptotic factors for human vascular smooth muscle cells. Cell survival was assured when serum-free medium was supplemented with any one or all of the above serine proteinase inhibitors. In contrast, the cells were sensitive to apoptosis when cultured in medium containing serum from which the proteinase inhibitors were removed. The antiapoptotic effect conferred by the proteinase inhibitors was proportional to proteinase inhibitory activity. Without proteinase inhibitors, the extracellular matrix was degraded, and cells could not attach to the matrix. Cell survival was dependent on the intact extracellular matrix. In the presence of the caspase inhibitor z-VAD, the cells detached but did not die. The activity of caspases was elevated without proteinase inhibitors; in contrast, caspases were not activated when medium was supplemented with one of the proteinase inhibitors. In conclusion, the plasma proteinase inhibitors prevent degradation of extracellular matrix by proteinases derived from cells. Presumably an intact cell-matrix interaction inhibits caspase activation and supports cell survival.

Atherosclerotic Plaque Smooth Muscle Cells Have a Distinct Phenotype

Objective—The present study addresses the question, “Are plaque smooth muscles cells (SMCs) genetically distinct from medial SMCs as reflected by the ability to maintain a distinctive expression phenotype in vitro?” Methods and Results—Multiple cell strains were developed from carotid endarcterectomy specimens, and quadruplicate array hybridizations were completed for each sample. A new normalization protocol was developed and used to analyze the data. Permutation analysis suggests that most of the significant differences in expression could not have occurred by chance. A broad pattern of significant expression differences, consisting of almost 5% of the genes probed, was detected. Quantitative polymerase chain reaction (QPCR) confirmation was found in 70% of a subset of genes selected for validation. Conclusions—The SMC cultures were nearly indistinguishable by morphological features, population doubling time, and sensitivity to cell death induced by Fas cross-linking. Surprisingly, array expression analysis identified differences so extensive that we conclude that plaque and medial SMCs are distinctly different SMC cell types.

A Marfan syndrome gene expression phenotype in cultured skin fibroblasts

BackgroundMarfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the fibrillin-1 gene. This syndrome constitutes a significant identifiable subtype of aortic aneurysmal disease, accounting for over 5% of ascending and thoracic aortic aneurysms.ResultsWe used spotted membrane DNA macroarrays to identify genes whose altered expression levels may contribute to the phenotype of the disease. Our analysis of 4132 genes identified a subset with significant expression differences between skin fibroblast cultures from unaffected controls versus cultures from affected individuals with known fibrillin-1 mutations. Subsequently, 10 genes were chosen for validation by quantitative RT-PCR.ConclusionDifferential expression of many of the validated genes was associated with MFS samples when an additional group of unaffected and MFS affected subjects were analyzed (p-value < 3 × 10-6 under the null hypothesis that expression levels in cultured fibroblasts are unaffected by MFS status). An unexpected observation was the range of individual gene expression. In unaffected control subjects, expression ranges exceeding 10 fold were seen in many of the genes selected for qRT-PCR validation. The variation in expression in the MFS affected subjects was even greater.

A link between smooth muscle cell death and extracellular matrix degradation during vascular atrophy

OBJECTIVE High blood flow induces neointimal atrophy in polytetrafluoroethylene (PTFE) aortoiliac grafts and a tight external PTFE wrap of the iliac artery induces medial atrophy. In both nonhuman primate models, atrophy with loss of smooth muscle cells and extracellular matrix (ECM) begins at ≤4 days. We hypothesized that matrix loss would be linked to cell death, but the factors and mechanisms involved are not known. The purpose of this study was to determine commonly regulated genes in these two models, which we hypothesized would be a small set of genes that might be key regulators of vascular atrophy. METHODS DNA microarray analysis (Sentrix Human Ref 8; Illumina, San Diego, Calif; ∼23,000 genes) was performed on arterial tissue from the wrap model (n = 9) and graft neointima from the graft model (n = 5) 1 day after wrapping or the switch to high flow, respectively. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was also performed. Expression of this vascular atrophy gene set was also studied after Fas ligand-induced cell death in cultured smooth muscle cells and organ cultured arteries. RESULTS Microarray analysis showed 15 genes were regulated in the same direction in both atrophy models: 9 upregulated and 6 downregulated. Seven of nine upregulated genes were confirmed by qRT-PCR in both models. Upregulated genes included the ECM-degrading enzymes ADAMTS4, tissue plasminogen activator (PLAT), and hyaluronidase 2; possible growth regulatory factors, including chromosome 8 open reading frame 4 and leucine-rich repeat family containing 8; a differentiation regulatory factor (musculoskeletal embryonic nuclear protein 1); a dead cell removal factor (ficolin 3); and a prostaglandin transporter (solute carrier organic anion transporter family member 2A1). Five downregulated genes were confirmed but only in one or the other model. Of the seven upregulated genes, ADAMTS4, PLAT, hyaluronidase 2, solute carrier organic anion transporter family member 2A1, leucine-rich repeat family containing 8, and chromosome 8 open reading frame 4 were also upregulated in vitro in cultured smooth muscle cells or cultured iliac artery by treatment with FasL, which causes cell death. However, blockade of caspase activity with Z-VAD inhibited FasL-mediated cell death, but not gene induction. CONCLUSION Seven gene products were upregulated in two distinctly different in vivo nonhuman primate vascular atrophy models. Induction of cell death by FasL in vitro induced six of these genes, including the ECM-degrading factors ADAMTS4, hyaluronidase 2, and PLAT, suggesting a mechanism by which the program of tissue atrophy coordinately removes extracellular matrix as cells die. These genes may be key regulators of vascular atrophy.

Isolation of a nuclear ribonucleoprotein fraction from chick oviduct containing ovalbumin messenger RNA sequences

Abstract A method was developed for the isolation of a ribonucleoprotein fraction from chick oviduct nuclei that contains 70% of the pulse-labeled RNA. These fractions also contain about 1% of the nuclear DNA and have an average RNA to DNA ratio of about 4:1. The major nuclear RNP proteins of 32,000 M r are present along with many additional proteins including histories. However, polysomal proteins and major oviduct cytoplasmic proteins are absent. Nuclei from fully stimulated chick oviduct contain about 3000 copies of ovalbumin messenger RNA sequences of which about 200 are in the RNP complexes: these complexes have sedimentation coefficients of 30 to 350 S and are resistant to disruption by EDTA. The level of ovalbumin mRNA sequences in these complexes reflects the overall rate of synthesis of this RNA. Withdrawal of estrogen leads to a parallel decline of nuclear estrogen receptors and ovalbumin mRNA sequences in the RNP complexes and a subsequent loss of cytoplasmic ovalbumin mRNA about three hours later. The 300-fold decrease in the level of ovalbumin mRNA sequences in these complexes and the eightfold decrease in stability of cytoplasmic ovalbumin mRNA account for the 2500-fold decrease in the level of cytoplasmic ovalbumin mRNA observed during withdrawal. Upon stimulation with estrogen, the kinetics of reappearance of ovalbumin mRNA sequences in the RNP complexes apparently accounts for the accumulation of cytoplasmic ovalbumin mRNA. Thus the nuclear RNP has some of the properties expected of nascent RNP complexes. The levels of ovalbumin and conalbumin mRNA sequences increase in the nuclear RNP with markedly different kinetics: conalbumin mRNA sequences reach half maximum by 1.5 hours, whereas ovalbumin mRNA sequences in these complexes reach half maximum at about eight hours. In the analysis in the accompanying Appendix, we show that the immediate increase of conalbumin mRNA sequences in the nuclear RNP may be accounted for by interaction of the hormone receptor complex with a single regulatory site, whereas the delayed increase of ovalbumin mRNA sequences in the RNP may be due to a requirement for interaction of the hormone receptor complex with multiple regulatory sites.

Inhibition of versican expression by siRNA facilitates tropoelastin synthesis and elastic fiber formation by human SK-LMS-1 leiomyosarcoma smooth muscle cells in vitro and in vivo.

Versican is an extracellular matrix (ECM) molecule that interacts with other ECM components to influence ECM organization, stability, composition, and cell behavior. Versican is known to increase in a number of cancers, but little is known about how versican influences the amount and organization of the ECM components in the tumor microenvironment. In the present study, we modulated versican expression using siRNAs in the human leiomyosarcoma (LMS) smooth muscle cell line SK-LMS-1, and observed the formation of elastin and elastic fibers in vitro and also in vivo in a nude mouse tumor model. Constitutive siRNA-directed knockdown of versican in LMS cells resulted in increased levels of elastin, as shown by immunohistochemical staining of the cells in vitro, and by mRNA and protein analyses. Moreover, versican siRNA LMS cells, when injected into nude mice, generated smaller tumors that had significantly greater immunohistochemical and histochemical staining for elastin when compared to control tumors. Additionally, microarray analyses were used to determine the influence of versican isoform modulation on gene expression profiles, and to identify genes that influence and relate to the process of elastogenesis. cDNA microarray analysis and TaqMan low density array validation identified previously unreported genes associated with downregulation of versican and increased elastogenesis. These results highlight an important role for the proteoglycan versican in regulating the expression and assembly of elastin and the phenotype of LMS cells.

KINETICS OF OVALBUMIN AND CONALBUMIN mRNA INDUCTION BY ESTROGEN AND PROGESTERONE

ABSTRACT Both ovalbumin and conalbumin are induced in the same tubular gland cells of the oviduct when estrogen is administered as a secondary stimulation to immature chicks. Conalbumin mRNA begins to accumulate within a few minutes of estrogen administration, whereas there is a lag of about 3 hr before ovalbumin mRNA begins to accumulate. The kinetics of estrogen receptor binding to chromatin indicate that these sites are saturated within 20 min, indicating that the lag is not due to slow uptake of steroid into the nucleus. Both the lag phase and the rate of ovalbumin mRNA accumulation can be modulated experimentally. These results suggest that there is another step between the binding of receptors in the nucleus and the induction of specific mRNA. A model of receptor translocation along chromatin is proposed to explain the lag.