Michael A. Mancini

Synthetic rexinoids effectively suppress both estrogen receptor-positive and estrogen receptor-negative mammary tumors in animal models, which makes them prime candidates for a novel class of cancer-preventive agents. When used in combination with chemotherapy for non–small-cell lung cancer, the rexinoid bexarotene was most effective for patients who developed hypertriglyceridemia as a side effect. Although serum triglycerides originate from the liver, the effect of bexarotene on lipogenesis in breast epithelial cells is not known. Gene expression studies with normal mammary epithelial cells indicated that rexinoids modulate lipid metabolism, particularly enzymes involved in triglyceride synthesis. High-content analysis revealed dose-dependent accumulation of neutral lipids within adipocyte differentiation-related protein-associated cytoplasmic lipid droplets after long-term bexarotene treatment. Bexarotene also induced mRNA and protein levels for peroxisome proliferator-activated receptor (PPAR) γ, whereas selective knockdown of PPARγ attenuated the induction of both lipid droplets and adipocyte differentiation-related protein. Pharmacological activation of PPARγ, but not PPARα or retinoic acid receptors, effectively induced lipid accumulation. Furthermore, the combination of the PPARγ agonist rosiglitazone with bexarotene synergistically suppressed the growth of human mammary epithelial cells and revealed a strong, nonlinear, inverse correlation of cell growth with lipid droplet accumulation in the cell population. These findings indicate that rexinoids activate a lipogenic program in mammary epithelial cells through a retinoid X receptor/PPARγ-mediated mechanism. It is noteworthy that combining low doses of bexarotene with the PPARγ agonist rosiglitazone provides effective growth suppression of mammary epithelial cells, potentially dissociating systemic adverse effects associated with standard bexarotene treatment from the antiproliferative effects on mammary epithelium.

Resistance to tamoxifen treatment occurs in approximately 50% of the estrogen receptor (ER)α-positive breast cancer patients. Resistant patients would benefit from treatment with other available antiestrogens. Arzoxifene is an effective growth inhibitor of ERα-positive breast cancer cells, including tamoxifen-resistant tumors. In this study, we show that overexpression of a regular component of the ERα transcription factor complex, cyclin D1, which occurs in approximately 40% of breast cancer patients, renders cells resistant to a new promising antiestrogen, arzoxifene. Overexpression of cyclin D1 alters the conformation of ERα in the presence of arzoxifene. In this altered conformation, ERα still recruits RNA polymerase II to an estrogen response element-containing promoter, inducing transcription of an ERα-dependent reporter gene and of endogenous pS2, and promoting arzoxifene-stimulated growth of MCF-7 cells. Arzoxifene is then converted from an ERα antagonist into an agonist. This can be explained by a stabilization of the ERα/steroid receptor coactivator-1 complex in the presence of arzoxifene, only when cyclin D1 is overexpressed. These results indicate that subtle changes in the conformation of ERα upon binding to antiestrogen are at the basis of resistance to antiestrogens.

Estrogen receptor-α (ER) transcription function is regulated in a ligand-dependent (e.g., estradiol, E2) or ligand-independent (e.g., growth factors) manner. Our laboratory seeks to understand these two modes of action. Using a cell line that contains a visible prolactin enhancer/promoter array (PRL-HeLa) regulated by ER, we analyzed ER response to E2 and EGF by quantifying image-based results. Data show differential recruitment of GFP-ER to the array, with the AF1 domain playing a vital role in EGF-mediated responsiveness. Temporal analyses of large-scale chromatin dynamics, and accumulation of array-localized reporter mRNA over 24 hours showed that the EGF response consists of a single pulse of reporter mRNA accumulation concomitant with transient increase in array decondensation. Estradiol induced a novel cyclical pattern of mRNA accumulation with a sustained increase in array decondensation. Collectively, our work shows that there is a stimuli-specific pattern of large-scale chromatin modification and transcript levels by ER.

Using manual and automated high throughput microscopy (HTM), ligand‐dependent trafficking of green fluorescent protein‐androgen receptor (GFP‐AR) was analyzed in fixed and living cells to determine its spatial distribution, solubility, mobility, and co‐activator interactions. Within minutes, addition of the agonist R1881 resulted translocation of GFP‐AR from the cytoplasm to the nucleus, where it displayed a hyperspeckled pattern and extraction resistance in low expressing cells. AR antagonists (Casodex, hydroxyflutamide) also caused nuclear translocation, however, the antagonist‐bound GFP‐AR had a more diffuse nuclear distribution, distinct from the agonist‐bound GFP‐AR, and was completely soluble; overexpressed GFP‐AR in treated cells was extraction resistant, independent of ligand type. To more dramatically show the different effects of ligand on AR distribution, we utilized an AR with a mutation in the DNA binding domain (ARC619Y) that forms distinct foci upon exposure to agonists but retains a diffuse nuclear distribution in the presence of antagonists. Live‐cell imaging of this mutant demonstrated that cytoplasmic foci formation occurs immediately upon agonist but not antagonist addition. Fluorescence recovery after photobleaching (FRAP) revealed that agonist‐bound GFP‐AR exhibited reduced mobility relative to unliganded or antagonist‐bound GFP‐AR. Importantly, agonist‐bound GFP‐AR mobility was strongly affected by protein expression levels in transiently transfected cells, and displayed reduced mobility even in slightly overexpressing cells. Cyan fluorescent protein‐AR (CFP‐AR) and yellow fluorescent protein‐CREB binding protein (YFP‐CBP) in the presence of agonists and antagonists were used to demonstrate that CFP‐AR specifically co‐localizes with YFP‐CBP in an agonist dependent manner. Dual FRAP experiments demonstrated that CBP mobility mirrored AR mobility only in the presence of agonist. HTM enabled simultaneous studies of the sub‐cellular distribution of GFP‐AR and ARC619Y in response to a range of concentrations of agonists and antagonists (ranging from 10⁻¹² to 10⁻⁵) in thousands of cells. These results further support the notion that ligand specific interactions rapidly affect receptor and co‐factor organization, solubility, and molecular dynamics, and each can be aberrantly affected by mutation and overexpression. J. Cell. Biochem. 98: 770–788, 2006.

Pit‐1, a POU‐class nuclear DNA‐binding transcription factor, specifies three of the parenchymal cell types in anterior pituitary ontogeny. Using fluorescent fusions and live cell imaging, we have compared the dynamic behavior of wild‐type and inactivating Pit‐1 point mutations. Fluorescence recovery after photobleaching (FRAP) and real‐time extraction data indicate that wild‐type Pit‐1 has a dynamic mobility profile, with t_1/2s ∼ 5–7 s when expressed from low to high amounts, respectively. Biochemically, Pit‐1 is ∼50% retained according to direct observation during extraction, indicating a dynamic interaction with nuclear structure. An analysis of transiently expressed Pit‐1 carrying two different debilitating mutations reveals that they translocate normally to the nucleus, but exhibit two different levels of mobility, both clearly distinguishable from wild‐type Pit‐1. At low expression levels, the t_1/2s of Pit_W261C and Pit_A158P are extremely rapid (0.3 and 0.6 s t_1/2s, respectively). At higher expression levels, unlike wild‐type Pit‐1, both mutant proteins become immobilized and insoluble, and fractionate completely with the insoluble nuclear matrix. Relative to wild‐type, over expression of mutated Pit‐1 elicits a nuclear stress response indicated by increased levels of heat shock inducible heat shock protein 70 (Hsp70), and reorganization of heat shock factor‐1. The decreased mobility of Pit_A158P relative to Pit_W261C at low expression levels correlates with its ability to partially activate when expressed at low levels and its ability to bind cognate DNA. At high expression levels, lower Pit_A158P activation correlates with its immobilization and insolubility. These data suggest a link between specific rates of intranuclear mobility and Pit‐1 transcription function, perhaps to insure sufficient interactions with chromatin, or in the case of non‐DNA binding Pit‐1, interaction as a repressor (Scully and Rosenfeld [2002]: Science 295:2231–2235). These data imply inactivating mutations can lead to an intranuclear sorting away from transcription related pathways, and at least in part to a misfolded protein pathway. Taken together, caution is suggested when interpreting point (or other) mutational analyses of transactivator function, as new compartmentation, especially in the context of expression levels, may cloud the distinction between defining functional molecular domains and intranuclear processing of misfolded proteins.

Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor α (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-taggedlac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integratedlac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP–SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP–SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP–SRC-1, while antagonist additions diminish YFP–SRC-1–CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER–SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP–SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.

At a simplistic level, the nucleus can be thought of as singular organelle with a nuclear envelope designed to isolate the biochemical reactions required for gene transcription and DNA replication from the cytoplasm. It has become increasingly clear, however, that many higher levels of organization exist within the nucleus. A functional consequence of this organization is that nuclear processes that include transcription, RNA processing, and DNA synthesis are isolated to specific intranuclear domains to ensure efficiency. With the advent of GFP technologies and increasingly sophisticated instrumentation, we have continued to dissect the relationship between organization and function, in particular using live cells and ligand-dependent steroid receptors as a model system. These new opportunities have provided further insight into receptor function and the dependence upon intranuclear dynamics that take place within minutes of hormone addition. J. Cell. Biochem. Suppl. 35:99–106, 2000.

Spinal bulbar muscular atrophy is a neurodegenerative disorder caused by a polyglutamine expansion in the androgen receptor (AR). We show in transiently transfected HeLa cells that an AR containing 48 glutamines (ARQ48) accumulates in a hormone-dependent manner in both cytoplasmic and nuclear aggregates. Electron microscopy reveals both types of aggregates to have a similar ultrastructure. ARQ48 aggregates sequester mitochondria and steroid receptor coactivator 1 and stain positively for NEDD8, Hsp70, Hsp90 and HDJ-2/HSDJ. Co-expression of HDJ-2/HSDJ significantly represses aggregate formation. ARQ48 aggregates also label with antibodies recognizing the PA700 proteasome caps but not 20S core particles. These results suggest that ARQ48 accumulates due to protein misfolding and a breakdown in proteolytic processing. Furthermore, the homeostatic disturbances associated with aggregate formation may affect normal cell function.