KDM5B Is Essential for the Hyperactivation of PI3K/AKT Signaling in Prostate Tumorigenesis

KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer. KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to prostate cancer tumorigenesis remain unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and prostate cancer progression in cultured cells and in mouse models of prostate epithelium–specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of prostate cancer in Pten-null mice, whereas Kdm5b loss alone caused no morphologic abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared with that of Pten mice. Pathologic analysis revealed Pten/Kdm5b mice displayed mild morphologic changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high-grade prostatic intraepithelial neoplasia and prostate cancer. Mechanistically, KDM5B governed PI3K/AKT signaling in prostate cancer in vitro and in vivo. KDM5B directly bound the PIK3CA promoter, and KDM5B knockout resulted in a significant reduction of P110α and PIP3 levels and subsequent decrease in proliferation of human prostate cancer cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110α/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against prostate cancer

Spontaneous preterm labor is associated with an increase in the proinflammatory signal transducer TLR4 receptor on maternal blood monocytes

<p>Abstract</p> <p>Background</p> <p>Localized inflammation and increased expression of TLR4 receptors within the uterus has been implicated in the pathogenesis of preterm labor. It remains unclear whether intrauterine inflammatory responses activate the maternal peripheral circulatory system. Therefore we determined whether increased TLR4 expression is present in the peripheral maternal white blood cells of women with spontaneous preterm labor.</p> <p>Methods</p> <p>This is a cross-sectional study of 41 preterm labor cases and 41 non-preterm controls. For each case and control sample, RNA was purified from white blood cells and TLR4 mRNA pool size was evaluated by quantitative PCR. Protein expression levels were determined by flow cytometry. Statistical evaluation using multiple linear regressions was used to determine any significant differences between the cases and controls. The purpose was to determine association prevalence of TLR4 levels and preterm labor.</p> <p>Results</p> <p>Adjusted mean TLR4 mRNA levels of 0.788 ± 0.037 (standard error) for preterm labor and 0.348 ± 0.038 for the corresponding pregnant control women were statistically significantly different <it>(P </it>= 0.002). Using the lower 95% confidence interval of the mean expression level in PTL subjects (0.7) as a cutoff value for elevated TLR4 mRNA levels, 25/41 (60.9%) of PTL patients expressed elevated TLR4 mRNA as compared to 0/41 (0%) in control subjects. The TLR4 receptor levels in the granulocyte fraction of white blood cells from preterm labor and pregnant controls were similar. However, TLR4⁺/CD14⁺monocytes were 2.3 times more frequent (70% vs. 30%) and TLR4 also had a 2.6-fold higher density (750 vs. 280 molecules per cell) in preterm labor women compared with pregnant controls. There was no difference in the levels of TLR4 in patients at term.</p> <p>Conclusions</p> <p>Patients with preterm labor exhibited elevated levels of CD14⁺maternal blood monocytes each bearing enhanced expression of TLR4, indicating that the peripheral circulatory system is activated in patients with preterm labor. Elevated leukocyte TLR4 levels may be a useful biomarker associated with preterm labor.</p

A Large-Scale Rheumatoid Arthritis Genetic Study Identifies Association at Chromosome 9q33.2

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease affecting both joints and extra-articular tissues. Although some genetic risk factors for RA are well-established, most notably HLA-DRB1 and PTPN22, these markers do not fully account for the observed heritability. To identify additional susceptibility loci, we carried out a multi-tiered, case-control association study, genotyping 25,966 putative functional SNPs in 475 white North American RA patients and 475 matched controls. Significant markers were genotyped in two additional, independent, white case-control sample sets (661 cases/1322 controls from North America and 596 cases/705 controls from The Netherlands) identifying a SNP, rs1953126, on chromosome 9q33.2 that was significantly associated with RA (ORcommon = 1.28, trend Pcomb = 1.45E-06). Through a comprehensive fine-scale-mapping SNP-selection procedure, 137 additional SNPs in a 668 kb region from MEGF9 to STOM on 9q33.2 were chosen for follow-up genotyping in a staged-approach. Significant single marker results (Pcomb<0.01) spanned a large 525 kb region from FBXW2 to GSN. However, a variety of analyses identified SNPs in a 70 kb region extending from the third intron of PHF19 across TRAF1 into the TRAF1-C5 intergenic region, but excluding the C5 coding region, as the most interesting (trend Pcomb: 1.45E-06 → 5.41E-09). The observed association patterns for these SNPs had heightened statistical significance and a higher degree of consistency across sample sets. In addition, the allele frequencies for these SNPs displayed reduced variability between control groups when compared to other SNPs. Lastly, in combination with the other two known genetic risk factors, HLA-DRB1 and PTPN22, the variants reported here generate more than a 45-fold RA-risk differential

CD55 expression was enhanced on the surface of essentially all THP-1 cells at 24 h, following addition of LPS to 1,25-D3 pretreated cells.

<p>THP-1 cells were grown on coverslips, pretreated with 100 nM 1,25-D3 for 72 h and then stimulated with either vehicle or LPS for 24 h, as indicated. Serial 0.5 µm optical sections were obtained by confocal microscopy (40 × magnification). A, sections containing either a central (internal Z-stack) or the apical region of the cells (cell surface Z-stack) and the entire 3D image of LPS treated cells are shown. White represents CD55 staining; nucleic acid staining was omitted for clarity. B, shows a graphical representation of mean (±SD) CD55 expression levels (normalized to nucleic acid content) for three independent experiments with two fields quantified per experiment (n = 6).</p

Protein expression profiles for CD55, CD14, and CD11b following addition of LPS (1 µg/ml) to THP-1 cells pretreated with or without 100 nM 1,25-D3 (72 h).

<p>A multiplexed fluorescence-based immunoblot approach was used to determine corresponding protein expression profiles from cells whose mRNA profiles are shown in Fig. 2. A, the temporal protein induction pattern of CD55, CD11b and β-actin following a LPS-challenge (without pretreatment). CD14 levels are not shown because the apparent 50-fold induction remained below the threshold of detection (nd). B, the relative band intensities for CD55 and CD11b, normalized to β-actin, are graphically presented as the average fold-change (relative to time zero) of three independent experiments (± SD). In some instances the SD are covered by the data point symbol. C and D, the multiplex immunoblot of LPS-induced protein expression in cells pretreated for 72 h with 1,25-D3 is shown in panel C; the fold induction is graphically represented in panel D as the average fold-change (relative to time zero) of three independent experiments (± SD). In some instances the SD are covered by the data point symbol. The apparent 100-fold induction of CD14 following 1,25-D3 pretreatment but before LPS (time = 0) was within the immunoblot detection limits.</p

Pretreatment with 1,25-D3 for 72 h sustained the subsequent LPS-induced expression of CD55 mRNA in THP-1 cells, compared to cells pretreated with vehicle for 72 h.

<p>THP-1 cells were first preincubated in complete culture media with vehicle or with 100 nM 1,25-D3. Seventy-two h later (t = 0 on A–C), either vehicle or 1 µg/ml LPS was added and the incubation continued for an additional 72 h. Cells were harvested at the indicated times and quantitative PCR (n = 3) was used to determine CD55 (A), CD14 (B) and CD11b (C) mRNA pool sizes using the absolute copy number method. Mean levels are expressed in arbitrary units normalized to 18S rRNA (± SD). Differences in mRNA pool size were determined using a two tailed student’s t test. Shaded data points indicate statistical differences from corresponding controls.</p

CD55 mRNA and protein expression was not altered during the early response phase (0–24 h) following a 1,25-D3 preincubation period of 72 h with human THP-1 monocytes.

<p>THP-1 cells were cultured in complete media in the absence or presence of 100 nM 1,25-D3 (added at time = 0) and harvested at the indicated times. A, CD55, CD14 and CD11b mRNA pool sizes were determined by quantitative PCR analysis using 18S rRNA as a reference. Fold-induction is expressed relative to vehicle treated controls. Values shown are the mean +/− SD of 3 independent experiments. In some instances, the SD bars are covered by the data point symbol. Shaded data points indicate statistical differences from control values. P<0.05 was considered significant. B, protein expression was analyzed in cells at various times following addition of 1,25-D3, using a fluorescence-based immunoblot approach. Corresponding controls showed no difference over the same time period.</p

NF-κB activation was required for LPS-induced CD55 expression in 1,25-D3 pretreated THP-1 monocytes.

<p>As illustrated under panel A, at the end of the 72 h pretreatment phase with 100 nM 1,25-D3, either vehicle or one of two NF-kB inhibitors [50 µM MG132 (MG) or 30 µm parthenolide (Pa)] was added and the incubation continued for an additional 1 h. Then LPS (1 µg/ml) or vehicle was added. Cells were ultimately harvested at 2 h (mRNA) or 24 h (protein) post addition of LPS or vehicle. Quantitative PCR was used to determine CD55 (A) and CD11b (D) mRNA levels assessed at 2 h. Differences between mRNA pool sizes were determined using a two tailed student’s t test. Asterisks (Fig. 5A) indicate statistical differences between indicated treatments; *P<0.05, **P<0.01. CD55 and CD11b protein expression was assessed at 24 h following a vehicle or LPS challenge by immunoblotting (B) and quantification (C and E), respectively. Because of the semi-qualitative nature of the protein determinations, a statistical analysis was not performed on these data. Values represent means +/− SD (n = 3).</p