Splicing of HDAC7 modulates the SRF-myocardin complex during stem-cell differentiation towards smooth muscle cells

Histone deacetylases (HDACs) have a central role in the regulation of gene expression. Here we investigated whether HDAC7 has an impact on embryonic stem (ES) cell differentiation into smooth muscle cells (SMCs). ES cells were seeded on collagen-IV-coated flasks and cultured in the absence of leukemia inhibitory factor in differentiation medium to induce SMC differentiation. Western blots and double-immunofluorescence staining demonstrated that HDAC7 has a parallel expression pattern with SMC marker genes. In ex vivo culture of embryonic cells from SM22-LacZ transgenic mice, overexpression of HDAC7 significantly increased beta-galactosidase-positive cell numbers and enzyme activity, indicating its crucial role in SMC differentiation during embryonic development. We found that HDAC7 undergoes alternative splicing during ES cell differentiation. Platelet-derived growth factor enhanced ES cell differentiation into SMCs through upregulation of HDAC7 splicing. Further experiments revealed that HDAC7 splicing induced SMC differentiation through modulation of the SRF-myocardin complex. These findings suggest that HDAC7 splicing is important for SMC differentiation and vessel formation in embryonic development.

Sp1-dependent activation of HDAC7 is required for platelet-derived growth factor-BB-induced smooth muscle cell differentiation from stem cells

We have previously demonstrated that histone deacetylase 7 (HDAC7) expression and splicing play an important role in smooth muscle cell (SMC) differentiation from embryonic stem (ES) cells, but the molecular mechanisms of increased HDAC7 expression during SMC differentiation are currently unknown. In this study, we found that platelet-derived growth factor-BB (PDGF-BB) induced a 3-fold increase in the transcripts of HDAC7 in differentiating ES cells. Importantly, our data also revealed that PDGF-BB regulated HDAC7 expression not through phosphorylation of HDAC7 but through transcriptional activation. By dissecting its promoters with progressive deletion analysis, we identified the sequence between -343 and -292 bp in the 5'-flanking region of the Hdac7 gene promoter as the minimal PDGF-BB-responsive element, which contains one binding site for the transcription factor, specificity protein 1 (Sp1). Mutation of the Sp1 site within this PDGF-BB-responsive element abolished PDGF-BB-induced HDAC7 activity. PDGF-BB treatment enhanced Sp1 binding to the Hdac7 promoter in differentiated SMCs in vivo as demonstrated by the chromatin immunoprecipitation assay. Moreover, we also demonstrated that knockdown of Sp1 abrogated PDGF-BB-induced HDAC7 up-regulation and SMC differentiation gene expression in differentiating ES cells, although enforced expression of Sp1 alone was sufficient to increase the activity of the Hdac7 promoter and expression levels of SMC differentiation genes. Importantly, we further demonstrated that HDAC7 was required for Sp1-induced SMC differentiation of gene expression. Our data suggest that Sp1 plays an important role in the regulation of Hdac7 gene expression in SMC differentiation from ES cells. These findings provide novel molecular insights into the regulation of HDAC7 and enhance our knowledge in SMC differentiation and vessel formation during embryonic development.

Functional expression of KCNQ (Kv7) channels in guinea-pig bladder smooth muscle and their contribution to spontaneous activity

Background and Purpose: The aim of the study was to determine whether KCNQ channels are functionally expressed in bladder smooth muscle cells (SMC) and to investigate their physiological significance in bladder contractility. 

Experimental Approach: KCNQ channels were examined at the genetic, protein, cellular and tissue level in guinea pig bladder smooth muscle using RT-PCR, immunofluorescence, patch-clamp electrophysiology, calcium imaging, detrusor strip myography, and a panel of KCNQ activators and inhibitors. 

Key Results: KCNQ subtypes 1-5 are expressed in bladder detrusor smooth muscle. Detrusor strips typically displayed TTX-insensitive myogenic spontaneous contractions that were increased in amplitude by the KCNQ channel inhibitors XE991, linopirdine or chromanol 293B. Contractility was inhibited by the KCNQ channel activators flupirtine or meclofenamic acid (MFA). The frequency of Ca2+-oscillations in SMC contained within bladder tissue sheets was increased by XE991. Outward currents in dispersed bladder SMC, recorded under conditions where BK and KATP currents were minimal, were significantly reduced by XE991, linopirdine, or chromanol, and enhanced by flupirtine or MFA. XE991 depolarized the cell membrane and could evoke transient depolarizations in quiescent cells. Flupirtine (20M) hyperpolarized the cell membrane with a simultaneous cessation of any spontaneous electrical activity. 

Conclusions and Implications: These novel findings reveal the role of KCNQ currents in the regulation of the resting membrane potential of detrusor SMC and their important physiological function in the control of spontaneous contractility in the guinea pig bladder.

T- and L-type Ca2+ currents in freshly dispersed smooth muscle cells from the human proximal urethra

The purpose of the present study was to characterise Ca2+ currents in smooth muscle cells isolated from biopsy samples taken from the proximal urethra of patients undergoing surgery for bladder or prostate cancer. Cells were studied at 37 degreesC using the amphotericin B perforated-patch configuration of the patch-clamp technique. Currents were recorded using Cs+-rich pipette solutions to block K+ currents. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca2+ currents, were present in these cells. When steady-state inactivation curves for the L current were fitted with a Boltzmann equation, this yielded a V-1/2 of -45 +/- 5 mV. In contrast, the T current inactivated with a V-1/2 of -80 +/- 3 mV. The L currents were reduced in a concentration-dependent manner by nifedipine (ED50 = 159 +/- 54 nm) and Ni2+ (ED50 = 65 +/- 16 muM) but were enhanced when external Ca2+ was substituted with Ba2+. The T current was little affected by TTX, reduction in external Na+, application of nifedipine at concentrations below 300 nm or substitution of external Ca2+ with Ba2+, but was reduced by Ni2+ with an ED50 of 6 +/- 1 mum. When cells were stepped from -100 to -30 mV in Ca2+-free conditions, small inward currents could be detected. These were enhanced 40-fold in divalent-cation-free solution and blocked in a concentration-dependent manner by Mg2+ with an ED50 of 32 +/- 16 mum. These data support the idea that human urethral myocytes possess currents with electrophysiological and pharmacological properties typical of T- and L-type Ca2+ currents.

PH-sauvagine from the skin secretion of Phyllomedusa hypochondrialis: a novel CRF-like peptide with smooth muscle contraction activity

Amphibian skin, and particularly that of south/Central American phyllomedusine frogs, is supposed to be "a huge factory and store house of a variety of active peptides". The 40 amino acid amphibian CRF-like peptide, sauvagine, is a prototype member of a unique family of these Phyllomedusa skin peptides. In this study, we describe for the first time the structure of a mature novel peptide from the skin secretion of the South American orange-legged leaf frog, Phyllomedusa hypochondrialis, which belongs to the amphibian CRF/sauvagine family. Partial amino acid sequence from the N-terminal was obtained by automated Edman degradation with the following structure: pGlu-GPPISIDLNMELLRNMIEI-. The biosynthetic precursor of this novel sauvagine peptide, consisted of 85 amino acid residues and was deduced from cDNA library constructed from the same skin secretion. Compared with the standard sauvagine from the frog, Phyllomedusa sauvagei, this novel peptide was found to exert similar contraction effects on isolated guinea-pig colon and rat urinary bladder smooth muscle preparations.

Autonomic modification of intestinal smooth muscle contractility

Intestinal smooth muscle contracts rhythmically in the absence of nerve and hormonal stimulation because of the activity of pacemaker cells between and within the muscle layers. This means that the autonomic nervous system modifies rather than initiates intestinal contractions. The practical described here gives students an opportunity to observe this spontaneous activity and its modification by agents associated with parasympathetic and sympathetic nerve activity. A section of the rabbit small intestine is suspended in an organ bath, and the use of a pressure transducer and data-acquisition software allows the measurement of tension generated by the smooth muscle of intestinal walls. The application of the parasympathetic neurotransmitter ACh at varying concentrations allows students to observe an increase in intestinal smooth muscle tone with increasing concentrations of this muscarinic receptor agonist. Construction of a concentration-effect curve allows students to calculate an EC50 value for ACh and consider some basic concepts surrounding receptor occupancy and activation. Application of the hormone epinephrine to the precontracted intestine allows students to observe the inhibitory effects associated with sympathetic nerve activation. Introduction of the drug atropine to the preparation before a maximal concentration of ACh is applied allows students to observe the inhibitory effect of a competitive antagonist on the physiological response to a receptor agonist. The final experiment involves the observation of the depolarizing effect of K+ on smooth muscle. Students are also invited to consider why the drugs atropine, codeine, loperamide, and botulinum toxin have medicinal uses in the management of gastrointestinal problems.

Analysis of Histone Deacetylase 7 (HDAC7) Alternative Splicing and Its Role in Embryonic Stem Cell Differentiation Toward Smooth Muscle Lineage

Histone deacetylases (HDACs) have a central role in the regulation of gene expression, which undergoes alternative splicing during embryonic stem cell (ES) cell differentiation. Alternative splicing gives rise to vast diversity over gene information, arousing public concerns in the last decade. In this chapter, we describe a strategy to detect HDAC7 alternative splicing and analyze its function on ES cell differentiation.