Dynamic phenotypic shifts and M2 receptor downregulation in bladder smooth muscle cells induced by mirabegron.

Introduction: Mirabegron is available for treatment of overactive bladder (OAB). However, mechanisms underlying symptom improvements and long-term effects on bladder smooth muscle cells are uncertain. Contractility and growth of bladder smooth muscle contribute to OAB, and depend on smooth muscle phenotypes, and on muscarinic receptor expression. Here, we examined prolonged exposure to mirabegron (20-48 h) on phenotype markers, muscarinic receptor expression, and phenotype-dependent functions in human bladder smooth muscle cells (hBSMC). Methods: Expression of markers for contractile (calponin, MYH11) and proliferative (MYH10, vimentin) phenotypes, proliferation (Ki-67), and of muscarinic receptors were assessed by RT-PCR. Proliferation, viability, actin organization and contractions in cultured hBSMC were examined by EdU, CCK-8, phalloidin staining and matrix contraction assays. Results: Calponin-1 mRNA decreased with 100 nM and 150 nM mirabegron applied for 20 h (0.56-0.6 fold of controls). Decreases were resistant to the ß3-AR antagonist L-748,337 (0.34-0.55 fold, 100-150 nM, 20 h). After 40 h, decreases occured in the presence of L-748,337, but not without L-748,337. MYH11 mRNA increased with 150 nM mirabegron (40 h, 1.9 fold). This was partly preserved with L-748,337, but not observed after 20 h mirabegron exposure. Vimentin mRNA reduced with 150 nM mirabegron after 20 h, but not after 40 h, with and without L-748,337 (0.71-0.63 fold). MYH10 mRNA expression remained unaffected by mirabegron. Exposure to 150 nM mirabegron increased Ki-67 mRNA after 20 h in the presence of, but not without L-748,337, and after 40 h without, but not with L-748,337. Proliferation rates and actin organization were stable with 50-150 nM mirabegron (24 h, 48 h). Viability increased significantly after mirabegron exposure for 20 h, and by trend after 40 h, which was fully sensitive to L-748,337. M2 mRNA was reduced by 20 h mirabegron, which was resistant to L-748,337. Carbachol (3 µM) enhanced time-dependent contractions of hBSMC, which was inhibited by mirabegron (150 nM) in late phases (24 h), but not in early phases of contractions. Conclusion: Mirabegron induces dynamic phenotype alterations and M2 downregulation in hBSMC, which is paralleled by time-shifted anticontractile effects. Phenotype transitions may be involved in improvements of storage symptoms in OAB by mirabegron.

Inhibition of prostate smooth muscle contraction and prostate stromal cell growth by the inhibitors of Rac, NSC23766 and EHT1864.

BACKGROUND AND PURPOSE: Medical therapy of lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH) targets smooth muscle contraction in the prostate, or prostate growth. However, current therapeutic options are insufficient. Here, we investigated the role of Rac in the control of smooth muscle tone in human prostates and growth of prostate stromal cells. EXPERIMENTAL APPROACH: Experiments were performed using human prostate tissues from radical prostatectomy and cultured stromal cells (WPMY-1). Expression of Rac was examined by Western blot and fluorescence staining. Effects of Rac inhibitors (NSC23766 and EHT1864) on contractility were assessed in the organ bath. The effects of Rac inhibitors were assessed by pull-down, cytotoxicity using a cell counting kit, cytoskeletal organization by phalloidin staining and cell growth using an 5-ethynyl-2'-deoxyuridine assay. KEY RESULTS: Expression of Rac1-3 was observed in prostate samples from each patient. Immunoreactivity for Rac1-3 was observed in the stroma, where it colocalized with the smooth muscle marker, calponin. NSC23766 and EHT1864 significantly reduced contractions of prostate strips induced by noradrenaline, phenylephrine or electrical field stimulation. NSC23766 and EHT1864 inhibited Rac activity in WPMY-1 cells. Survival of WPMY-1 cells ranged between 64 and 81% after incubation with NSC23766 (50 or 100 µM) or EHT1864 (25 µM) for 24 h. NSC23766 and EHT1864 induced cytoskeletal disorganization in WPMY-1 cells. Both inhibitors impaired the growth of WPMY-1 cells. CONCLUSIONS AND IMPLICATIONS: Rac may be a link connecting the control of prostate smooth muscle tone with proliferation of smooth muscle cells. Improvements in LUTS suggestive of BPH by Rac inhibitors appears possible.

The tandem affinity purification (TAP) method: a general procedure of protein complex purification.

Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level. Prior knowledge of complex composition or function is not required. The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented. The TAP method was initially developed in yeast but can be successfully adapted to various organisms. Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration.

A dual role for BBP/ScSF1 in nuclear pre-mRNA retention and splicing.

The MSL5 gene, which codes for the splicing factor BBP/ScSF1, is essential in Saccharomyces cerevisiae, yet previous analyses failed to reveal a defect in assembly of (pre)-spliceosomes or in vitro splicing associated with its depletion. We generated 11 temperature-sensitive (ts) mutants and one cold-sensitive (cs) mutant in the corresponding gene and analyzed their phenotypes. While all mutants were blocked in the formation of commitment complex 2 (CC2) at non-permissive and permissive temperature, the ts mutants showed no defect in spliceosome formation and splicing in vitro. The cs mutant was defective in (pre)-spliceosome formation, but residual splicing activity could be detected. In vivo splicing of reporters carrying introns weakened by mutations in the 5' splice site and/or in the branchpoint region was affected in all mutants. Pre-mRNA leakage to the cytoplasm was strongly increased (up to 40-fold) in the mutants. A combination of ts mutants with a disruption of upf1, a gene involved in nonsense-mediated decay, resulted in a specific synthetic growth phenotype, suggesting that the essential function of SF1 in yeast could be related to the retention of pre-mRNA in the nucleus.

A generic protein purification method for protein complex characterization and proteome exploration.

We have developed a generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag. The TAP tag allows the rapid purification of complexes from a relatively small number of cells without prior knowledge of the complex composition, activity, or function. Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein. The TAP method has been tested in yeast but should be applicable to other cells or organisms.

Transient interaction of BBP/ScSF1 and Mud2 with the splicing machinery affects the kinetics of spliceosome assembly.

Removal of introns from pre-mRNA is an essential step of gene expression. The splicing reaction is catalyzed in a large complex termed the spliceosome. Introns are recognized during the early steps of spliceosome assembly with the formation of commitment complexes. Intron recognition is mediated by the interaction of splicing factors with conserved sequences present in the pre-mRNA. BBP/SF1 participates in this recognition by interacting with the pre-mRNA branch point in both yeast and mammals. This protein, which is essential in yeast, also interacts with the U2AF65/Mud2 splicing factor. However, its precise role in splicing complex formation is still unclear. We have now analyzed the presence of BBP and Mud2 in yeast splicing complexes using supershift and coprecipitation assays. We found that BBP is present together with Mud2 in commitment complex 2 (CC2), but is not detectable in commitment complex 1 (CC1). Furthermore, genetic and biochemical depletion of BBP demonstrated that it is required for CC2 formation. In addition we observed that BBP and Mud2 are not detectable in pre-spliceosomes. These are the first commitment complex components that are shown to be released during or immediately after pre-spliceosome formation. Interestingly, depletion of BBP or disruption of MUD2 had no significant effect on pre-spliceosome formation and splicing in vitro but led to a transient accumulation of CC1. These observations support a model in which BBP and Mud2 are recycled during transition from CC2 to pre-spliceosome.

New constructs and strategies for efficient PCR-based gene manipulations in yeast.

Gene disruption and tagging can be achieved by homologous recombination in the yeast genome. Several PCR-based methods have been described towards this end. However these strategies are often limited in their applications and/or their efficiencies and may be technically demanding. Here we describe two plasmids for C-terminal tagging of proteins with the IgG binding domain of the Staphylococcus aureus protein A. We also present simple and reliable strategies based on PCR to promote efficient integration of exogenous DNA into the yeast genome. These simple methods are not limited to specific strains or markers and can be used for any application requiring homologous recombination such as gene disruption and epitope tagging. These strategies can be used for consecutive introduction of various constructs into a single yeast strain.