Polydimethylsiloxane (PDMS) has become a staple of the microfluidics community by virtue of its simple fabrication process and material attributes, such as gas permeability, optical transparency, and flexibility. As microfluidic systems are put toward biological problems and increasingly utilized as cell culture platforms, the material properties of PDMS must be considered in a biological context. Two properties of PDMS were addressed in this study: the leaching of uncured oligomers from the polymer network into microchannel media, and the absorption of small, hydrophobic molecules (i.e. estrogen) from serum-containing media into the polymer bulk. Uncured PDMS oligomers were detectable via MALDI-MS in microchannel media both before and after Soxhlet extraction of PDMS devices in ethanol. Additionally, PDMS oligomers were identified in the plasma membranes of NMuMG cells cultured in PDMS microchannels for 24 hours. Cells cultured in extracted microchannels also contained a detectable amount of uncured PDMS. It was shown that MCF-7 cells seeded directly on PDMS inserts were responsive to hydrophilic prolactin but not hydrophobic estrogen, reflecting its specificity for absorbing small, hydrophobic molecules; and the presence of PDMS floating in wells significantly reduced cellular response to estrogen in a serum-dependent manner. Quantification of estrogen via ELISA revealed that microchannel estrogen partitioned rapidly into the surrounding PDMS to a ratio of approximately 9:1. Pretreatments such as blocking with serum or pre-absorbing estrogen for 24 hours did not affect estrogen loss from PDMS-based microchannels. These findings highlight the importance of careful consideration of culture system properties when determining an appropriate environment for biological experiments.
Estrogen receptor alpha (ER␣), a key driver of growth in the majority of breast cancers, contains an unstructured transactivation domain (AF1) in its N terminus that is a convergence point for growth factor and hormonal activation. This domain is controlled by phosphorylation, but how phosphorylation impacts AF1 structure and function is unclear. We found that serine 118 (S118) phosphorylation of the ER␣ AF1 region in response to estrogen (agonist), tamoxifen (antagonist), and growth factors results in recruitment of the peptidyl prolyl cis/trans isomerase Pin1. Phosphorylation of S118 is critical for Pin1 binding, and mutation of S118 to alanine prevents this association. Importantly, Pin1 isomerizes the serine118-proline119 bond from a cis to trans isomer, with a concomitant increase in AF1 transcriptional activity. Pin1 overexpression promotes ligand-independent and tamoxifeninducible activity of ER␣ and growth of tamoxifen-resistant breast cancer cells. Pin1 expression correlates with proliferation in ER␣-positive rat mammary tumors. These results establish phosphorylation-coupled proline isomerization as a mechanism modulating AF1 functional activity and provide insight into the role of a conformational switch in the functional regulation of the intrinsically disordered transactivation domain of ER␣.E strogen receptor alpha (ER␣), a member of the nuclear receptor superfamily of transcription factors, mediates the actions of estrogen in normal physiology and disease (17). ER␣ is expressed in the normal mammary gland and in 70% of human breast cancers and is a key driver of breast cell proliferation (16,26,83). Directed overexpression of ER␣ in the mammary gland is sufficient to induce hyperplasia, and blockade of ER␣ activity by hormonal therapies (aromatase inhibitors, tamoxifen, and fulvestrant) reduces recurrence and improves clinical outcomes of ER␣-positive breast cancer patients (19,22). Two activation functions mediate the transcriptional activity of ER␣, a C-terminal liganddependent AF2 and an N-terminal ligand-independent AF1 (89). Regulation of ER␣ activity via the C-terminal AF2 has been wellcharacterized through biochemical and crystallographic studies and forms the basis for our understanding of hormonal therapy for breast cancer (10,34,84). In the canonical activation pathway, ligand binding initiates C-terminal structural rearrangements that facilitate downstream events, including dimerization, DNA binding, and coregulator interactions, ultimately engaging the basal transcriptional machinery to regulate gene expression. However, ER␣ can also be activated by growth factors and kinases, which phosphorylate the receptor N terminus and other domains to regulate transcription in the absence of direct ligand engagement (for reviews, see references 27, 47, 75, 94, and 95). In contrast to the C-terminal AF2 domain, biochemical and structural mechanisms that control N-terminal AF1 remain poorly understood (48,91).Multiple challenges have hindered molecular dissection of AF1 regulation. First, AF1 resid...
Gene expression results from the coordinated actions of transcription factor proteins and coregulators. Estrogen receptor alpha (ER␣) is a ligand-activated transcription factor that can both activate and repress the expression of genes. Activation of transcription by estrogen-bound ER␣ has been studied in detail, as has antagonist-induced repression, such as that which occurs by tamoxifen. How estrogen-bound ER␣ represses gene transcription remains unclear. In this report, we identify a new mechanism of estrogen-induced transcriptional repression by using the ER␣ gene, ESR1. Upon estrogen treatment, ER␣ is recruited to two sites on ESR1, one distal (ENH1) and the other at the proximal (A) promoter. Coactivator proteins, namely, p300 and AIB1, are found at both ER␣-binding sites. However, recruitment of the Sin3A repressor, loss of RNA polymerase II, and changes in histone modifications occur only at the A promoter. Reduction of Sin3A expression by RNA interference specifically inhibits estrogen-induced repression of ESR1. Furthermore, an estrogen-responsive interaction between Sin3A and ER␣ is identified. These data support a model of repression wherein actions of ER␣ and Sin3A at the proximal promoter can overcome activating signals at distal or proximal sites and ultimately decrease gene expression.Downregulation of receptors by their ligands is a fundamental process by which cells control sensitivity to stimuli. For steroid hormones, this involves lipophilic ligands binding to intracellular receptors to induce a decline in receptor number. Regulation of estrogen (E2) receptor alpha (ER␣) by E2 is one example. The E2-induced decline in ER␣ is, in part, mediated through direct regulation of the protein. It is well documented that decreases in ER␣ protein levels in response to E2 occur via the ubiquitin-proteasome pathway (1, 42). The mRNA levels of ER␣ also decrease, but the mechanism responsible for E2-induced repression of the ER␣ gene, ESR1, is not established (5, 49, 52).ER␣ is a ligand-activated transcription factor that mediates the effects of E2 by regulating gene expression. Activation by ER␣ has been studied in detail, but little is understood about how E2-bound ER␣ represses transcription. E2-induced repression is, however, of significant biological importance. Microarray analyses of E2-treated breast cancer cell lines show that the number of repressed genes is greater than or near the number of activated genes (10,19,29,32). Yet, there are limited reports investigating E2-induced repression, and no generalized mechanism has emerged (6,13,22,25,43,47,59,60,71,74). Antagonistinduced repression by selective ER modulators involves conformational changes that prevent coactivator binding to ER␣ (55). Such a conformational blockade does not occur with agonist binding and thus cannot account for E2-induced gene repression.Many repressive complexes exist to restrict gene expression in response to cellular signals. One example is the Sin3 complex, which was identified in yeast but is conserved in mammals (41, ...
Estrogen receptor-alpha (ERα) is an important biomarker used to classify and direct therapy decisions in breast cancer. Both ERα protein and its transcript, ESR1, are used to predict response to tamoxifen therapy, yet certain tumors have discordant levels of ERα protein and ESR1, which is currently unexplained. Cellular ERα protein levels can be controlled post-translationally by the ubiquitin-proteasome pathway (UPP) through a mechanism that depends on phosphorylation at residue S118. Phospho-S118 (pS118-ERα) is a substrate for the peptidyl prolyl isomerase, Pin1, which mediates cis-trans isomerization of the pS118-P119 bond to enhance ERα transcriptional function. Here, we demonstrate that Pin1 can increase ERα protein without affecting ESR1 transcript levels by inhibiting proteasome-dependent receptor degradation. Pin1 disrupts ERα ubiquitination by interfering with receptor interactions with the E3 ligase, E6AP, which also is shown to bind pS118-ERα. Quantitative in situ assessments of ERα protein, ESR1, and Pin1 in human tumors from a retrospective cohort show that Pin1 levels correlate with ERα protein but not to ESR1 levels. These data show that ERα protein is post-translationally regulated by Pin1 in a proportion of breast carcinomas. Since Pin1 impacts both ERα protein levels and transactivation function, these data implicate Pin1 as a potential surrogate marker for predicting outcome of ERα-positive breast cancer.
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