PMID: 7723369;Abstract:
A putative explanation of the effect of sulindac on adenomatous colon and duodenal polyps from clinical observations and related in vitro experiments is presented. In cells with mutant APC genes, persistent high prostaglandin content of polyps leads to desensitization, downregulation of adenylate cyclase, uncoupling of cAMP synthesis from prostaglandin, and inactivation of protein kinase A (PKA). It is suggested that in normal cells, (APC) protein binds to catenins and microtubules to maintain structure and contribute to cell-cell communication, adherence, and the dephosphorylated state, a necessary condition for such functions. Cells with mutant APC product become isolated, deprived of communication and adhesion to other epithelial cells, overphosphorylated, and without corrective capability. The latter is largely due tn downregulation of cAMP synthesis and protein kinase A activity secondary tn high prostaglandin. Three main biochemical defects ensue: (1) the restrictive influence of PKA catalyzed phosphorylation of Raf-1 kinase and resultant effects on the MAP kinase cascade and transcription is lost, (2) the transcription of immediate early genes, including cyclooxygenase is stimulated, and (3) the stimulation of protein tyrosine phosphatase (PTPase) by PKA is in abeyance. These putative abnormalities are reversed by inhibition of cyclooxygenase-1 by sulindac. cAMP synthesis and PKA activity return to normal. PKA catalyzed phosphorylations block Raf-1 kinase at the confluence of the Ras and protein kinase C pathways. The MAP kinase cascade is inhibited as is transcription of immediate early genes. At the same time PKA stimulates PTPase, which dephosphorylates the cytoskeleton and restores cell-cell communication, adherence, and structure. The transformed phenotype is circumvented by adjustment of the phosphorylation state and mutant cells rejoin the epithelial community. The redox state of cytoplasm in mutant cells may be shifted toward reduction.
This review has highlighted several topics in the study of steroid hormone action. The unanswered questions regarding the mechanism of ligand-controlled LRF activity, the extent of evolutionary conservation and specificity of DNA binding, and the validity of various models of transcriptional regulation mediated through gene networks point to the future direction of research in this field. Steroid hormones are used extensively in clinical treatments, especially glucocorticoids. Our laboratory is attempting to determine which gene networks are responsible for some of these clinical phenotypes. Figure 5 points out that the study of glucocorticoid action holds a unique position because it spans both the basic sciences and the field of applied molecular biology. Now that we have a fundamental knowledge of the necessary elements required for steroid-dependent regulation of gene expression, we can better investigate the clinical responses to steroid therapy (which include devastating side effects) by isolating and characterizing the important target gene(s). In this author's opinion, future directions in the study of steroid responsiveness will have to include a systematic approach toward deciphering a variety of these LRF-regulated gene networks in experimentally feasible systems. Hopefully, work in this area may be revealing and perhaps beneficial to ongoing clinical studies. In addition, the study of mechanisms of transcriptional induction and repression, using the model system of LRFs, could be applicable to many gene regulatory systems which are controlled by such processes as development and differentiation.
PMID: 21971482;PMCID: PMC3210400;Abstract:
To better understand the mechanism of de novo lipid biosynthesis in blood fed Aedes aegypti mosquitoes, we quantitated acetyl-CoA carboxylase (ACC) and fatty acid synthase 1 (FAS1) transcript levels in blood fed mosquitoes, and used RNAi methods to generate ACC and FAS1 deficient mosquitoes. Using the ketogenic amino acid 14C-leucine as a metabolic precursor of 14C-acetyl-CoA, we found that 14C-triacylglycerol and 14C-phospholipid levels were significantly reduced in both ACC and FAS1 deficient mosquitoes, confirming that ACC and FAS1 are required for de novo lipid biosynthesis after blood feeding. Surprisingly however, we also found that ACC deficient mosquitoes, but not FAS1 deficient mosquitoes, produced defective oocytes, which lacked an intact eggshell and gave rise to inviable eggs. This severe phenotype was restricted to the 1st gonotrophic cycle, suggesting that the eggshell defect was due to ACC deficiencies in the follicular epithelial cells, which are replaced after each gonotrophic cycle. Consistent with lower amounts of de novo lipid biosynthesis, both ACC and FAS1 deficient mosquitoes produced significantly fewer eggs than control mosquitoes in both the 1st and 2nd gonotrophic cycles. Lastly, FAS1 deficient mosquitoes, but not ACC deficient mosquitoes, showed delayed blood meal digestion, suggesting that a feedback control mechanism may coordinate rates of fat body lipid biosynthesis and midgut digestion during feeding. We propose that decreased ACC and FAS1 enzyme levels lead to reduced lipid biosynthesis and lower fecundity, whereas altered levels of the regulatory metabolites acetyl-CoA and malonyl-CoA account for the observed defects in eggshell formation and blood meal digestion, respectively. © 2011 Elsevier Ltd.
PMID: 2178510;Abstract:
The recent isolation and characterization of steroid receptor coding sequences has revolutionized the field of steroid hormone action. These studies have revealed that steroid receptors are members of a much larger nuclear receptor 'super family'. The ligand and DNA binding domains have been shown to be molecular components that functionally interact to transform the steroid-receptor complex into a highly specific gene regulator that induces or represses the expression of cell-specific target genes. Molecular genetic approaches have been used to study structure-function relationships of several steroid receptor proteins, the most extensive analysis has been that of the glucocorticoid receptor. Several breakthroughs in the study of steroid hormone action include the construction of novel chimeric steroid receptor proteins, functional expression of steroid receptors in yeast, and the development of sensitive cloning techniques designed to isolate low abundance, hormonally regulated transcripts.
The WEHI7.2 thymoma cell line undergoes apoptotic cell death when exposed to glucocorticoids and agents that increase intracellular cAMP. Several lines of evidence indicate that calcium may play an important role in events culminating in lymphocyte apoptosis. In these studies, calbindin-D28K was stably overexpressed in WEHI7.2 cells to determine if increasing the Ca(2+)-binding capacity of the cell interferes with the apoptotic pathway. Indeed, stable expression of calbindin-D28K decreased the apoptotic effects of dexamethasone and forskolin, and the level of resistance to these agents correlated with the relative amount of calbindin expressed in each line. Overexpression of calbindin also increased cell survival in the presence of the calcium ionophore A23187. The stably expressed calcium-binding protein appeared to exert its protective effect subsequent to transcriptional activation, since glucocorticoid- and cAMP-induced gene expression were not affected. These data support the proposal that calcium fluxes are involved in apoptosis and suggest that high level expression of proteins that buffer calcium fluxes can effectively suppress death in apoptosis-susceptible cells.
