Pentabromopseudilin: a myosin V inhibitor suppresses TGF-<b>β</b> activity by recruiting the type II TGF-<b>β</b> receptor to lysosomal degradation

<p>Pentabromopseudilin (PBrP) is a marine antibiotic isolated from the marine bacteria <i>Pseudomonas bromoutilis</i> and <i>Alteromonas luteoviolaceus</i>. PBrP exhibits antimicrobial, anti-tumour, and phytotoxic activities. In mammalian cells, PBrP is known to act as a reversible and allosteric inhibitor of myosin Va (MyoVa). In this study, we report that PBrP is a potent inhibitor of transforming growth factor-β (TGF-β) activity. PBrP inhibits TGF-β-stimulated Smad2/3 phosphorylation, plasminogen activator inhibitor-1 (PAI-1) protein production and blocks TGF-β-induced epithelial–mesenchymal transition in epithelial cells. PBrP inhibits TGF-β signalling by reducing the cell-surface expression of type II TGF-β receptor (TβRII) and promotes receptor degradation. Gene silencing approaches suggest that MyoVa plays a crucial role in PBrP-induced TβRII turnover and the subsequent reduction of TGF-β signalling. Because, TGF-β signalling is crucial in the regulation of diverse pathophysiological processes such as tissue fibrosis and cancer development, PBrP should be further explored for its therapeutic role in treating fibrotic diseases and cancer.</p

Number of miRNAs and mRNAs up- or down-regulated for each chromosome in GCKO testes on P18. Data for miRNA and mRNA are expressed on a per chromosome basis and reported for those probe sets with intensities that could be scored as increased, decreased or no change in GCKO when compared to WT testes.

<p>Number of miRNAs and mRNAs up- or down-regulated for each chromosome in GCKO testes on P18. Data for miRNA and mRNA are expressed on a per chromosome basis and reported for those probe sets with intensities that could be scored as increased, decreased or no change in GCKO when compared to WT testes.</p

Meiotic spreads show deletion of <i>Dicer1</i> disrupts progression of meiosis I.

<p>(A) It was possible to identify the 5 sub-stages of meiosis I by combining the reactivity patterns of two antibodies against synaptonemal complex protein 3 (SYCP3) and γH2AX. SYCP3 is a protein essential for synapsis of homologous chromosomes and γH2AX localizes to double-strand breaks and XY bodies during meiosis. A total of 200 spreads were counted in testis cell preparations from WT and GCKO mice at P22. (B) Chi square analysis shows GCKO testes contained significantly higher numbers of germ cell spreads at the leptotene and zygotene stages of meiosis I and fewer spreads at pachytene, diplotene and metaphase I stages (<i>P</i><0.001), suggesting that the loss of <i>Dicer1</i> lead to disruptions in progression through meiosis I.</p

Levels of <i>Dicer1</i> transcripts and DICER1 protein in GCKO testis samples are significantly reduced by P18.

<p>(A) In comparison to WT testes, real-time qRT-PCR shows a 6-fold reduction in <i>Dicer1</i> RNase III endonuclease transcripts in GCKO testes by P18 (*<i>P</i>  = 0.018). (B) Western blot analysis using rabbit antibody against the N-terminal helicase domain of DICER1 and HRP-conjugated goat anti-rabbit IgG antibody shows reduced protein expression in GCKO testes by P18. The arrows point to the 217 kDa DICER1 protein and to the equivalent loading of 50 kDa protein from WT and GCKO testes. (C) Immunofluorescence detection of DICER1 protein in WT and GCKO testes at P18 using the same antibody used for western blotting. In WT sections, DICER1 localizes to the cytoplasm of most cell types populating the P18 testis, including Sertoli cells, spermatogonia and spermatocytes. At this developmental time point, secondary spermatocytes and spermatids are not present. In the P18 GCKO testis sections, DICER1 appears primarily in the cytoplasm of Sertoli cells located near the basement membrane with scant detection of DICER1 protein in the cytoplasm of spermatogonia and pachytene spermatocytes (magnification  = 40x). (D) Cross sections of WT and GCKO testes on P18 stained with HE show no major differences in cellular composition suggesting that the changes in in <i>Dicer1</i> transcript and protein levels in the GCKO testes were not explained by differences in cell populations.</p

Histology comparing WT and GCKO seminiferous tubules and epididymides at 5, 8 and 10 weeks.

<p>(A) In comparison to WT seminiferous tubules at 5 weeks, GCKO sections show tubules with increased lumen diameters and few elongating and elongated spermatids (*). By 8 weeks, cross sections from GCKO testes show prominent pynotic cells (arrows) and reduced numbers of elongating and elongated spermatids. By 10 weeks, cross sections of GCKO testes show further enlargement of tubule lumens and absence of elongating spermatids in the majority of tubules. (B) Epididymides of WT mice at 5 weeks and GCKO mice at 5, 8 and 10 weeks showing reduced sperm numbers in epididymides of knockout mice by 5 weeks and the absence of mature spermatozoa in GCKO epididymides at the 8 and 10 week timepoints.</p

Failure of meiotic sex chromosome inactivation (MSCI) in P18 <i>Dicer1</i> GCKO testes.

<p>A) Pie charts show the number of genes that are normally silenced during MSCI that were detected by array overlaid with the percentages of X- and Y-genes in GCKO testes with expression levels that were either significantly up-regulated (83.8% and 100%, respectively) or not changed (16.2 and 0%, respectively. B) Bar graph showing the number of overexpressed (>1.5 RFC) X- and Y-genes with or without binding sites for miRNAs shown to be deregulated in P18 GCKO testes. The majority of overexpressed genes (102/123 = 82.9%) contain recognition sites for deregulated miRNAs.</p

GCKO mice show reduced testis weight and sperm number, abnormal sperm morphology and infertility.

<p>(A) Body and testis weights were measured in WT and GCKO males at P22, 5 and 8 weeks (at each time point, n = 5 for WT and n = 5 for GCKO). In comparison to WT, GCKO males had no change in body weight over 8 weeks, yet showed significant reductions in testis weight as early as 5 weeks and remained so at 8 weeks (*both <i>P</i>≤0.004). Values represent the mean ± SEM. (B) In comparison to WT, GCKO epididymal sperm counts revealed significant reductions in sperm number by 8 weeks (**<i>P  = </i>0.0003). Approximately 80% of WT and less than 10% of GCKO epididymal sperm showed normal head and tail morphologies as represented in the photomicrographs taken at 40x. (C) Fertility testing showed that 4–6 month old GCKO males were unable to sire litters in comparison to littermate controls when mated 3–6 times with 12-week fertile B6CBAF1/J females.</p

Preferential down-regulation of miRNAs and overexpression of XY genes in GCKO testis samples by P18.

<p>A) Febit miRNA arrays show 96.1% (296/308) of miRNAs encoded on all chromosomes except the Y were predominantly down regulated in GCKO testes by P18, with a small remainder (12/308 = 3.9%) upregulated. B) Validation of Febit miRNA array fold changes by qRT-PCR and primers amplifying miR-34b-3p, -15b-5p, -19a and -191. In comparison to WT (n  = 3; RFC  = 1.0), significant reductions in GCKO miRNA expression were observed using qRT-PCR and P18 RNA from GCKO testes (n = 3). C) Affymetrix ST 1.0 gene arrays showed dysregulation of 36.7% (7700/20985) of gene expression in GCKO testes with 55.7% (4290/7700) showing increased and 44.3% (3410/7700) showing decreased expression for autosomal genes. In contrast, X- and Y-linked genes were preferentially overexpressed in P18 GCKO testis samples (44.3% and 77.8%, respectively). D) Validation of Affymetrix mRNA array fold changes by qRT-PCR and primers amplifying genes highly expressed in somatic (<i>Sox9</i>) and germ cells (<i>Stra8, Adad1, Bcl2l11, Kitl</i>), and linked to X (<i>Vsig4, Pgrmc1, Lamp2</i>) and Y (<i>Ube1Y1</i>) chromosomes.</p