Abstract-We recently identified heat shock protein 27 (HSP27) as an estrogen receptor beta (ER␤)-associated protein and noted its role as a biomarker for atherosclerosis. The current study tests the hypothesis that HSP27 is protective against the development of atherosclerosis. HSP27 overexpressing (HSP27 o/e ) mice were crossed to apoE Ϫ/Ϫ mice that develop atherosclerosis when fed a high-fat diet. Aortic en face analysis demonstrated a 35% reduction (PՅ0.001) in atherosclerotic lesion area in apoE Ϫ/Ϫ HSP27 o/e mice compared to apoE Ϫ/Ϫ mice, but primarily in females. Serum HSP27 levels were Ͼ10-fold higher in female apoE Ϫ/Ϫ HSP27 o/e mice compared to males, and there was a remarkable inverse correlation between circulating HSP27 levels and lesion area in both male and female mice (r 2 ϭ0.78, PՅ0.001). Mechanistic in vitro studies showed upregulated HSP27 expression and secretion in macrophages treated with estrogen or acLDL. Moreover, exogenous HSP27 added to culture media inhibited macrophage acLDL uptake and competed for the scavenger receptor A (SR-A)-an effect that was abolished with the SR-A competitive ligand fucoidan and absent in macrophages from SR-A Ϫ/Ϫ mice. Furthermore, extracellular HSP27 decreased acLDL-induced release of the proinflammatory cytokine IL-1␤ and increased the release of the antiinflammatory cytokine IL-10. HSP27 is atheroprotective, perhaps because of its ability to compete for the uptake of atherogenic lipids or attenuate inflammation.

Summary The androgen receptor (AR) is required for castration resistant prostate cancer (CRPC) progression, but the function and disease relevance of AR-bound enhancers remain unclear. Here, we identify a group of AR-regulated enhancer RNAs (e.g. PSA eRNA) that are upregulated in CRPC cells, patient-derived xenografts (PDX) and patient tissues. PSA eRNA binds to CYCLIN T1, activates P-TEFb and promotes cis and trans target gene transcription by increasing serine-2 phosphorylation of RNA polymerase II (Pol II-Ser2p). We define an HIV-1 TAR RNA-like (TAR-L) motif in PSA eRNA that is required for CYCLIN T1 binding. Using TALEN-mediated gene editing we further demonstrate that this motif is essential for increased Pol II-Ser2p occupancy levels and CRPC cell growth. We have uncovered a P-TEFb activation mechanism and reveal altered eRNA expression that is related to abnormal AR function and may potentially be a therapeutic target in CRPC.

Inactivating mutations of the SOST (sclerostin) gene are associated with overgrowth and sclerosis of the skeleton. To determine mechanisms by which increased amounts of calcium and phosphorus are accreted to enable enhanced bone mineralization in the absence of sclerostin, we measured concentrations of calciotropic and phosphaturic hormones, and urine and serum calcium and inorganic phosphorus in mice in which the sclerostin (sost) gene was replaced by the β-D-galactosidase (lacZ) gene in the germ line. Knockout (KO) (sost −/− ) mice had increased bone mineral density and content, increased cortical and trabecular bone thickness, and greater net bone formation as a result of increased osteoblast and decreased osteoclast surfaces compared with wild-type (WT) mice. β-Galactosidase activity was detected in osteocytes of sost KO mice but was undetectable in WT mice. Eight-week-old, male sost KO mice had increased serum 1α,25-dihydroxyvitamin D, decreased 24,25-dihydroxyvitamin D, decreased intact fibroblast growth factor 23, and elevated inorganic phosphorus concentrations compared with age-matched WT mice. 25-Hydroxyvitamin D 1α-hydroxylase cytochrome P450 (cyp27B1) mRNA was increased in kidneys of sost KO mice compared with WT mice. Treatment of cultured proximal tubule cells with mouse recombinant sclerostin decreased cyp27B1 mRNA transcripts. Urinary calcium and renal fractional excretion of calcium were decreased in sost KO mice compared with WT mice. Sost KO and WT mice had similar serum calcium and parathyroid hormone concentrations. The data show that sclerostin not only alters bone mineralization, but also influences mineral metabolism by altering concentrations of hormones that regulate mineral accretion.S clerostin, an osteocyte-derived, secreted, cystine-knot protein inhibits bone formation by interacting with and altering the activity of bone morphogenetic proteins (BMPs), low-density lipoprotein-receptor-related protein 5 (LRP 5/6), cysteine-rich protein 61, the receptor tyrosine kinase v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (erb B3), among other proteins (1-12). Inactivating mutations in the human sclerostin gene (SOST) are associated with sclerosteosis (Mendelian Inheritance in Man, MIM 269500) or van Buchem disease (MIM 239100) (6), both of which are characterized by progressive overgrowth and sclerosis of the axial and appendicular skeleton and an increase in bone mineral density (6,13,14). These results are supported by data from sost knockout mice, which have markedly increased bone density (15-17).The physiological adaptations that occur in human sclerosteosis and mouse models of the disease that permit the increased accretion of calcium (Ca) and phosphorus (P) required for bone formation are unknown. For example, it is unknown whether sclerostin influences vitamin D metabolite concentrations, the concentrations of phosphaturic peptides such as fibroblast growth factor 23 (FGF-23), and the renal handling of calcium and phosphorus. It is important to understand the adaptations ...

Objective-We recently identified HSP27 as an atheroprotective protein that acts extracellularly to prevent foam cell formation and atherogenesis in female but not male mice, where serum levels of HSP27 were increased and inversely correlated with degree of lesion burden. In the current study we sought to determine whether estrogens are required for the observed atheroprotective benefits of HSP27 as well as its extracellular release. Methods and Results-In vitro estrogens prompted the release of HSP27 from macrophages in an ER␤ specific manner that involved exosomal trafficking. Ovariectomy nullified the previously recognized attenuation in aortic lesion area in HSP27 o/e apoE Ϫ/Ϫ mice compared to apoE Ϫ/Ϫ mice. Supplementation with 17␤-estradiol resulted in a Ͼ15ϫ increase in uterine weight and attenuation of atherogenesis in all mice, although HSP27o/e apoE Ϫ/Ϫ had 34% less lesion burden compared to apoE Ϫ/Ϫ mice. Mice treated with the ER␤-specific agonist, DPN had no effect on uterine weight but a 28% decrease in aortic lesion area in HSP27o/e apoE Ϫ/Ϫ compared to apoE Ϫ/Ϫ mice. HSP27 serum levels showed a similar gradual increase with E2 and DPN replacement treatment but did not change in untreated mice. Key Words: heat shock Ⅲ proteins Ⅲ estrogen Ⅲ receptors Ⅲ athersclerosis P reviously, we showed in human coronary arteries that Heat Shock Protein 27 (HSP27) expression is lower in atherosclerotic plaques compared to lesion-free arterial segments, suggesting higher levels of this protein may confer protection from disease. 1 More recently, we demonstrated that HSP27 is an atheroprotective protein that acts in the extracellular space to reduce foam cell formation and atherogenesis by binding scavenger receptor-A on the surface of macrophages and preventing the uptake of acLDL as well as inflammatory cytokine release. Moreover, when fed a highfat diet for 4 weeks apoE null (apoE Ϫ/Ϫ ) mice that overexpress HSP27 (HSP27 o/e apoE Ϫ/Ϫ ) show a reduction in aortic atherosclerotic plaque area-however, only in female and not male mice. 2 Interestingly, female HSP27 overexpressing mice have significantly higher levels of serum HSP27 than do their male counterparts, and serum HSP27 levels show a strong inverse correlation with atherosclerotic lesion area. These sex-specific effects of HSP27 hint that the function or release of this intriguing protein may in some way be hormonally modulated. Conclusions-TheGiven our previous observations that estrogens cause HSP27 secretion, and that HSP27 physically associates with ER␤ but not ER␣, 3 we sought to determine the role for these receptors in the release of HSP27 both in vitro and in vivo. As described herein, we now report that the release of HSP27 from macrophages is preferentially induced via specific ER␤ stimulation (not ER␣) and induction of HSP27 release in vivo via the ER␤-specific modulator DPN increases serum HSP27 levels to a level comparable to 17␤-estradiol (E2)-yet unlike E2 attenuates atherogenesis without causing unwanted uterine hypertrophy. Materials and Me...