SUMOylation modulates the transcriptional activity of androgen receptor in a target gene and pathway selective manner.

Androgen receptor (AR) plays an important regulatory role in prostate cancer. AR's transcriptional activity is regulated by androgenic ligands, but also by post-translational modifications, such as SUMOylation. To study the role of AR SUMOylation in genuine chromatin environment, we compared androgen-regulated gene expression and AR chromatin occupancy in PC-3 prostate cancer cell lines stably expressing wild-type (wt) or doubly SUMOylation site-mutated AR (AR-K386R,K520R). Our genome-wide gene expression analyses reveal that the SUMOylation modulates the AR function in a target gene and pathway selective manner. The transcripts that are differentially regulated by androgen and SUMOylation are linked to cellular movement, cell death, cellular proliferation, cellular development and cell cycle. Fittingly, SUMOylation mutant AR cells proliferate faster and are more sensitive to apoptosis. Moreover, ChIP-seq analyses show that the SUMOylation can modulate the chromatin occupancy of AR on many loci in a fashion that parallels their differential androgen-regulated expression. De novo motif analyses reveal that FOXA1, C/EBP and AP-1 motifs are differentially enriched at the wtAR- and the AR-K386R,K520R-preferred genomic binding positions. Taken together, our data indicate that SUMOylation does not simply repress the AR activity, but it regulates AR's interaction with the chromatin and the receptor's target gene selection.

Quantitative test for sensory hand symptoms based on mechanoreceptor-specific vibrotactile thresholds.

A vibrotactile test for assessing the presence or absence of sensory symptoms in the hand has been developed from thresholds believed mediated by Merkel disks and Meissner corpuscles at the fingertips. It is constructed from the summed differences between the thresholds recorded at the fingertip of an individual and the mean values of the threshold for healthy persons at the same stimulation frequencies. The summed normalized threshold shift, TS(Sum(SD)), is shown to be related to reports by subjects of numbness and pain using three statistical tests for evaluating the significance of associations in 2x2 contingency tables. The small number of subjects (15) restricts direct calculation of a fence value for TS(Sum(SD)), t, between the presence and absence of symptoms: accordingly, interpolation between calculated t values has been performed graphically. A common range of t values can be identified that is judged significantly by each statistical test (3.3

Vibration-induced hearing loss: mechanical and physiological aspects.

HYPOTHESIS: The sensorineural hearing loss (HL) after middle ear surgery has been explained by the noise generated by drilling, without considering the vibration generated by the burr. BACKGROUND: The role of temporal bone vibration in the etiology of the HL was evaluated. METHODS: An electromagnetic shaker was used to vibrate the bony external ear canal of guinea pigs at different frequencies ranging from 32 to 1,000 Hz and at intensities ranging from 4.2 to 18.8 m/s for 15 minutes. The hearing threshold was measured with auditory evoked responses. A total of 30 animals were tested. RESULTS: After vibration, 60% of the guinea pigs developed a threshold shift (TS) exceeding 10 dB at two frequencies, with average TS of 8.8 dB across all frequencies and animals. The exposure to vibration at higher frequencies (range, 500-1,000 Hz) produced stronger TS than did the exposure to lower frequencies (range, 32-250 Hz). The vibration-induced TS showed prominent recovery so that after 7 days, TS was 2.4 dB on average and 27 of 30 animals had recovered. After 14 days, the TS was 1.3 dB. The vibration excitation measurements showed that at lower frequencies, the vibration transmission into the skull was significantly greater than at higher frequencies, at which the transmission was heavily attenuated. There were no acoustic resonances detected in the skull. The frequency of vibration and the hearing frequency in auditory brainstem response were significant determinants in the model explaining the vulnerability of vibration on hearing. Hearing loss primarily occurred at higher frequencies. The HL was mostly reversible, consistent with the results observed after human temporal bone surgery. CONCLUSION: We conclude that in the guinea pig model, the temporal bone vibration at higher frequencies produced a more severe HL than did the vibration at lower frequencies, although the vibration at higher frequencies caused less efficient transmission from the vibrating probe to the temporal bone. The guinea pig model may be useful in the development of surgical techniques and in the understanding of temporal bone pathology.

Global analysis of transcription in castration-resistant prostate cancer cells uncovers active enhancers and direct androgen receptor targets.

Androgen receptor (AR) is a male sex steroid-activated transcription factor (TF) that plays a critical role in prostate cancers, including castration-resistant prostate cancers (CRPC) that typically express amplified levels of the AR. CRPC-derived VCaP cells display an excessive number of chromatin AR-binding sites (ARBs) most of which localize to distal inter- or intragenic regions. Here, we analyzed direct transcription programs of the AR in VCaP cells using global nuclear run-on sequencing (GRO-seq) and integrated the GRO-seq data with the ARB and VCaP cell-specific TF-binding data. Androgen immediately activated transcription of hundreds of protein-coding genes, including IGF-1 receptor and EGF receptor. Androgen also simultaneously repressed transcription of a large number of genes, including MYC. As functional enhancers have been postulated to produce enhancer-templated non-coding RNAs (eRNAs), we also analyzed the eRNAs, which revealed that only a fraction of the ARBs reside at functional enhancers. Activation of these enhancers was most pronounced at the sites that also bound PIAS1, ERG and HDAC3, whereas binding of HDAC3 and PIAS1 decreased at androgen-repressed enhancers. In summary, our genome-wide data of androgen-regulated enhancers and primary target genes provide new insights how the AR can directly regulate cellular growth and control signaling pathways in CPRC cells.

Vibration induced hearing loss in guinea pig cochlea: expression of TNF-alpha and VEGF.

Transcranial vibration was applied for seven animals at a frequency of 250 Hz for 15 min, and five animals were used as normal controls to investigate cellular and molecular mechanism linked to vibration-induced hearing loss in animal model. Compound action potential (CAP) thresholds were measured by round window niche electrode. The expression of tumour necrosis factor alpha (TNF-alpha) and its receptors (TNF R1, TNF R2), vascular endothelium growth factor (VEGF) and its receptors (VEGF R1, VEGF R2) were analysed by immunohistochemistry. Transcranial vibration caused expression of TNF-alpha, TNF R1 and TNF R2 in the cochlea and the expression of TNF R2 was stronger than that of TNF R1. Vibration also induced VEGF and VEGF R2 expression in the cochlea. The average immediate hearing loss was 62 dB and after three days still 48 dB. It is concluded that transcranial vibration as during temporal bone drilling produces cochlear shear stress that is connected with up-regulation of TNF-alpha and its receptors. Also VEGF and VEGF R2 are up-regulated. These responses may be linked to both the damage and repair process of the cochlea.

A new vertebrate SUMO enzyme family reveals insights into SUMO-chain assembly.

SUMO chains act as stress-induced degradation tags or repair factor-recruiting signals at DNA lesions. Although E1 activating, E2 conjugating and E3 ligating enzymes efficiently assemble SUMO chains, specific chain-elongation mechanisms are unknown. E4 elongases are specialized E3 ligases that extend a chain but are inefficient in the initial conjugation of the modifier. We identified ZNF451, a representative member of a new class of SUMO2 and SUMO3 (SUMO2/3)-specific enzymes that execute catalysis via a tandem SUMO-interaction motif (SIM) region. One SIM positions the donor SUMO while a second SIM binds SUMO on the back side of the E2 enzyme. This tandem-SIM region is sufficient to extend a back side-anchored SUMO chain (E4 elongase activity), whereas efficient chain initiation also requires a zinc-finger region to recruit the initial acceptor SUMO (E3 ligase activity). Finally, we describe four human proteins sharing E4 elongase activities and their function in stress-induced SUMO2/3 conjugation.

Global SUMOylation on active chromatin is an acute heat stress response restricting transcription.

BACKGROUND: Cells have developed many ways to cope with external stress. One distinctive feature in acute proteotoxic stresses, such as heat shock (HS), is rapid post-translational modification of proteins by SUMOs (small ubiquitin-like modifier proteins; SUMOylation). While many of the SUMO targets are chromatin proteins, there is scarce information on chromatin binding of SUMOylated proteins in HS and the role of chromatin SUMOylation in the regulation of transcription. RESULTS: We mapped HS-induced genome-wide changes in chromatin occupancy of SUMO-2/3-modified proteins in K562 and VCaP cells using ChIP-seq. Chromatin SUMOylation was further correlated with HS-induced global changes in transcription using GRO-seq and RNA polymerase II (Pol2) ChIP-seq along with ENCODE data for K562 cells. HS induced a rapid and massive rearrangement of chromatin SUMOylation pattern: SUMOylation was gained at active promoters and enhancers associated with multiple transcription factors, including heat shock factor 1. Concomitant loss of SUMOylation occurred at inactive intergenic chromatin regions that were associated with CTCF-cohesin complex and SETDB1 methyltransferase complex. In addition, HS triggered a dynamic chromatin binding of SUMO ligase PIAS1, especially onto promoters. The HS-induced SUMOylation on chromatin was most notable at promoters of transcribed genes where it positively correlated with active transcription and Pol2 promoter-proximal pausing. Furthermore, silencing of SUMOylation machinery either by depletion of UBC9 or PIAS1 enhanced expression of HS-induced genes. CONCLUSIONS: HS-triggered SUMOylation targets promoters and enhancers of actively transcribed genes where it restricts the transcriptional activity of the HS-induced genes. PIAS1-mediated promoter SUMOylation is likely to regulate Pol2-associated factors in HS.

Nuclear mobility and activity of FOXA1 with androgen receptor are regulated by SUMOylation.

Forkhead box (FOX) protein A1 has been dubbed a pioneer transcription factor because it binds target sites in DNA, thereby displacing nucleosomes to loosen chromatin and facilitating steroid receptor DNA binding nearby. FOXA1 is an important regulator of prostate development, collaborating with androgen receptor (AR). Post-translational modifications regulating FOXA1 are thus far poorly understood. SUMOylation, post-translational modification of proteins by small ubiquitin-like modifier (SUMO) proteins, has emerged as an important regulatory mechanism in transcriptional regulation. In this work, we show by SUMOylation assays in COS-1 cells that the FOXA1 is modified at least in two of its three lysines embedded in SUMOylation consensus, K6 and K389, in proximity to its transactivation domains and K267 proximal to its DNA-binding domain. We also provide evidence for SUMO-2/3 modification of endogenous FOXA1 in LNCaP prostate cancer cells. Based on fluorescence recovery after photobleaching assays with mCherry-fused FOXA1 and EGFP-fused AR in HEK293 cells, the presence of FOXA1 retards the nuclear mobility of agonist-bound AR. Interestingly, mutation of the FOXA1 SUMOylation sites slows down the mobility of the pioneer factor, further retarding the nuclear mobility of the AR. Chromatin immunoprecipitation and gene expression assays suggest that the mutation enhances FOXA1's chromatin occupancy as well as its activity on AR-regulated prostate-specific antigen (PSA) locus in LNCaP cells. Moreover, the mutation altered the ability of FOXA1 to influence proliferation of LNCaP cells. Taken together, these results strongly suggest that the SUMOylation can regulate the transcriptional activity of FOXA1 with the AR.

Prostaglandin 15d-PGJ(2) inhibits androgen receptor signaling in prostate cancer cells.

Androgen signaling, in particular overexpression of the androgen receptor (AR), is critical for the growth and progression of prostate cancer. Because the AR is amenable to targeting by small-molecule inhibitors, it remains the major druggable target for the advanced disease. Inflammation has also been implicated in the cancerous growth in the prostate. Here we show that 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), an endogenously produced antiinflammatory prostaglandin, targets the AR and acts as a potent AR inhibitor, rapidly repressing AR target genes, such as FKBP51 and TMPRSS2 in prostate cancer cells. However, exposure of prostate cancer cells to 15d-PGJ(2) does not simply evoke a general inhibition of nuclear receptor activity or transcription because under the same conditions, peroxisome proliferator-activated receptor-γ is activated by 15d-PGJ(2). Moreover, 15d-PGJ(2) rapidly triggers modifications of AR by small ubiquitin-related modifier-2/3 (SUMO-2/3), which may modulate the repressing effect of 15d-PGJ(2) on AR-dependent transcription. Chromatin immunoprecipitation assays indicate that the inhibitory effect of 15d-PGJ(2) on FKBP51 and TMPRSS2 expression occurs in parallel with the inhibition of the AR binding to the regulatory regions of these genes. However, the DNA-binding activity is not the only AR function targeted by 15d-PGJ(2) because the prostaglandin also blunted the androgen-dependent interaction between the AR amino and carboxy termini. In conclusion, our results identify 15d-PGJ(2) as a potent and direct inhibitor of androgen signaling, suggesting novel possibilities in restricting the AR activity in prostate cancer cells.

Dynamic SUMOylation is linked to the activity cycles of androgen receptor in the cell nucleus.

Despite of the progress in the molecular etiology of prostate cancer, the androgen receptor (AR) remains the major druggable target for the advanced disease. In addition to hormonal ligands, AR activity is regulated by posttranslational modifications. Here, we show that androgen induces SUMO-2 and SUMO-3 (SUMO-2/3) modification (SUMOylation) of the endogenous AR in prostate cancer cells, which is also reflected in the chromatin-bound receptor. Although only a small percentage of AR is SUMOylated at the steady state, AR SUMOylation sites have an impact on the receptor's stability, intranuclear mobility, and chromatin interactions and on expression of its target genes. Interestingly, short-term proteotoxic and cell stress, such as hyperthermia, that detaches the AR from the chromatin triggers accumulation of the SUMO-2/3-modified AR pool which concentrates into the nuclear matrix compartment. Alleviation of the stress allows rapid reversal of the SUMO-2/3 modifications and the AR to return to the chromatin. In sum, these results suggest that the androgen-induced SUMOylation is linked to the activity cycles of the holo-AR in the nucleus and chromatin binding, whereas the stress-induced SUMO-2/3 modifications sustain the solubility of the AR and protect it from proteotoxic insults in the nucleus.

Analysis of androgen receptor SUMOylation.

Androgen receptor (AR) is a ligand-controlled transcription factor that is deregulated and therefore targeted in prostate cancer. In addition to androgens, AR is regulated by post-translational modifications (PTMs). SUMOylation, conjugation of small ubiquitin-related modifier (SUMO) protein 1, 2, or 3, is a bulky PTM regulating several important physiological processes. We have shown that AR is modified by SUMO-1 at two conserved lysine residues in its N-terminal domain. This agonist-enhanced modification represses the transcriptional activity of the receptor in a reversible and target gene-selective fashion. Acceptor sites for SUMOs are also found in several other nuclear receptors. Since the cellular steady-state level of SUMO modifications of most substrates, including AR, is very low, transfection- and SUMO overexpression-based protocols are often needed to render the modifications clearly detectable. This chapter describes protocols for analyzing AR SUMOylation in cultured cells by immunoblotting, gel mobility shift assays, and immunoprecipitation. These methodologies are generally applicable for determining whether a particular protein is SUMOylated and for identifying the lysine residue(s) modified.

Hand-arm vibration syndrome with use of anti-vibration chain saws: 19-year follow-up study of forestry workers.

OBJECTIVES: Hand-arm vibration syndrome (HAVS) consists of vascular and neurological component. Musculoskeletal component has not been delineated yet. In the present follow-up study, we evaluated the prevalence of HAVS and the cumulative exposure to vibration among a cohort of forestry workers. Special interest was given to numbness and musculoskeletal disorders of upper extremity and neck in forestry workers. METHODS: A follow-up study starting from 1976 was conducted among forestry workers in Suomussalmi in Finland. Total exposure of hand-arm vibration was recorded during 11 cross-sectional surveys. The last study was carried out in 1995. The lifetime dose of vibration energy was calculated. A cohort of 52 forest workers participated to all 11 cross-sectional surveys 1976-1995. HAVS and musculoskeletal disorders were evaluated. RESULTS: The prevalence of active vibration white finger (VWF) decreased from 13 to 4% in the cross-sectional study. In the cohort VWF decreased from 17 to 8% and numbness increased from 23 to 40%. Rotator cuff syndrome (P=0.034) and epicondylitis (P=0.004) associated with numbness. Regional neck pain was diagnosed in 38% of workers and associated with low back pain. In modeling VWF, the lifelong vibration energy (OR 1.03, CI 1.01-1.05), and smoking (OR 7.36, CI 1.07-50.76) were significant. Numbness was modeled by pain in upper extremities (OR 12.43, CI 2.42-63.80) and neck pain (5.97, CI 1.25-28.39), not by lifelong vibration energy. Right rotator cuff syndrome was modeled by age (OR 2.58, CI 1.04-6.41) and lifelong vibration energy (OR 1.04, CI 1.00-1.07). CONCLUSIONS: The prevalence of VWF constantly decreased. Numbness did not follow the vibration exposure profile. Numbness also associated with upper extremity musculoskeletal disorders. Hand-arm vibration associated with the right rotator cuff syndrome in forestry workers.