A mathematical model for the receptor mediated cellular regulation of the low density lipoprotein metabolism

A prototype mathematical model for Brown and Goldstein's pioneering studies on the LDL receptor mediated pathway for the regulation of the cellular content of cholesterol has been developed in this paper. In order to analyze the essential features of this complex system quantitatively and still reflect the framework of the total system, six important processes are considered in the model. They are: (1A, B) the hydrolysis and synthesis of the LDL receptor; (2) the binding of LDL to its receptors; (3) the hydrolysis of LDL; (4) the storage of cholesteryl esters; (5) the regulation of de novo synthesis of cholesterol; and (6) the efflux of free cholesterol to the external medium. All these processes form a system to let the cells take up enough cholesterol from the external medium for their utilization and yet avoid the excessive accumulation of the lipid within the cells. The validity of the model is tested by showing that it can predict many of experimental curves obtained for human fibroblasts in tissue culture studies. The main purpose of the model is to determine how the free cholesterol level in the cell is related to the external LDL concentration and the regulatory capacity of the cells to adapt to a changing LDL environment. In addition, the model reveals an important behavior of SMC, i.e., for a slowly increasing LDL concentration in the extracellular medium, the rate of intracellular degradation of LDL will first increase and then become saturated. It is proposed based on these results that the saturation of LDL degradation by SMCs and the subsequent increase in subendothelial LDL levels in regions of high macromolecular permeability might play a vital role in the formation of the early foam cell lesion.     

Differential DNA-binding abilities of estrogen receptor occupied with two classes of antiestrogens: studies using human estrogen receptor overexpressed in mammalian cells.

We have developed a transient transfection system using the Cytomegalovirus (CMV) promoter to express the human estrogen receptor (ER) at very high levels in COS-1 cells and have used it to study the interaction of agonist and antagonist receptor complexes with estrogen response element (ERE) DNA. ER can be expressed to levels of 20-40 pmol/mg or 0.2-0.3% of total soluble protein and all of the soluble receptor is capable of binding hormone. The ER binds estradiol with high affinity (Kd 0.2 nM), and is indistinguishable from native ER in that the receptor is capable of recognizing its cognate DNA response element with high affinity, and of transactivating a transgene in an estradiol-dependent manner. Gel mobility shift assays reveal interesting ligand-dependent differences in the binding of receptor complexes to ERE DNA. Receptors occupied by estradiol or the type I antiestrogen transhydroxytamoxifen bind to DNA response elements when exposed to the ligand in vitro or in vivo. Likewise, receptors exposed to the type II antiestrogen ICI 164,384 in vitro bind to ERE DNA. However, when receptor exposure to ICI 164,384 is carried out in vivo, the ER-ICI 164,384 complexes do not bind to ERE DNA, or do so only weakly. This effect is not reversed by subsequent incubation with estradiol in vitro, but is rapidly reversible by in vivo estradiol exposure of intact COS-1 cells. This suggests there may be some cellular process involved in the mechanism of antagonism by the pure antiestrogen ICI 164,384, which is not observed in cell-free extracts.     

Structural organization and regulation of the chicken estrogen receptor

We have cloned the chicken estrogen receptor (ER) from a chicken oviduct lambda gt11 library using the human ER cDNA sequence. This chicken ER sequence is virtually identical to the recently published sequence. One noteable difference is an amino acid change from glutamine to arginine located toward the central region of the sequence. The size of the ER protein predicted from the 589 amino acids is approximately 66,000 which fits well with the range of molecular weights previously published for the calf uterine and human ER (65,000-70,000). We observed the size of the chicken ER mRNA to be approximately 7.8 kilobases which is in agreement with the previously published size of 7.5 kilobases. In vivo secondary stimulation of chicken oviduct total RNA with diethylstilbestrol does not induce chicken ER mRNA. A time course following the chicken ER mRNA levels after secondary stimulation with diethylstilbestrol indicated a decrease in mRNA levels 8 h after DES administration. A similar study was performed using progesterone for the secondary stimulation. An increase in the chicken ER mRNA levels was observed 24 h after stimulation with progesterone. Two regions of very high homology were delineated by analyzing the sequence of this chicken ER cDNA and comparing it to the sequences of the human ER, human glucocorticoid, and chicken progesterone receptors and the P75-erbA fusion product of the avian erythroblastosis virus. The first concensus region is 72 amino acids in length and the second region of high homology is 62 amino acids long. Detailed comparisons of these regions for the steroid hormone receptors and v-erb A are presented. Possible functions for the individual regions of high homology are discussed.