RESUMEN

BACKGROUND: A buried penis (BP) is rare in which the penile body is retracted into the prepubic adipose tissue. This research focuses on differences in smooth muscle myosin heavy chain (SMMHC) isoform expressions in the dartos fascia. METHODS: A total of 82 children, 41 of whom had BPs, who applied for circumcision between May and November 2021, were included in the study. The cases were divided into four groups aged ≥6 years (NP6, n = 18) and aged ≤3 years (NP3, n = 17) with normal penile appearance, aged ≥6 years (BP6, n = 23) and aged ≤3 years (BP,n = 24) with a BP. SMMHC isoforms mRNA gene expression analyses were performed by quantitative PCR technique in dartos fascia obtained from foreskin removed by circumcision. RESULTS: Compared to the NP3 group, the SM1 mRNA expressed in the BP6 group was statistically significantly higher (p < 0.005). SM2 mRNA levels expressed in dartos fascia were considerably higher in NP6 and NP3 groups compared to BP6 and BP3 groups (p < 0.001). The SM2/SM1 ratio was 0.85 in the BP6 group and 1.46 in the NP6 group, which was statistically significant (p = 0.006) and increased from 0.87 in the BP3 group to 2.21 in the NP3 group (p < 0.001). CONCLUSION: In a buried penis, there is a difference in the expression of SMMHC isoforms. SM1 is highly expressed, while SM2 decreases, increasing the SM2/SM1 ratio. This causes increased contractility in the smooth muscle, leading to retraction of the penile body. The dartos fascia surrounding it resembles aberrant muscle tissue in boys with a BP. LEVEL OF EVIDENCE: Level III. TYPE OF STUDY: Case-control study.

Asunto(s)

Cadenas Pesadas de Miosina , Pene , Isoformas de Proteínas , Humanos , Masculino , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Niño , Preescolar , Isoformas de Proteínas/genética , Pene/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/análisis , Lactante , Circuncisión Masculina , Enfermedades del Pene/metabolismo , Enfermedades del Pene/genética , Miosinas del Músculo Liso/metabolismo , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/análisis

RESUMEN

Children with Hutchinson-Gilford Progeria Syndrome (HGPS) suffer from multiple cardiovascular pathologies due to the expression of progerin, a mutant form of the nuclear envelope protein Lamin A. Progerin expression has a dramatic effect on arterial smooth muscle cells (SMCs) and results in decreased viability and increased arterial stiffness. However, very little is known about how progerin affects SMC contractility. Here, we studied the LaminAG609G/G609G mouse model of HGPS and found reduced arterial contractility at an early age that correlates with a decrease in smooth muscle myosin heavy chain (SM-MHC) mRNA and protein expression. Traction force microscopy on isolated SMCs from these mice revealed reduced force generation compared to wild-type controls; this effect was phenocopied by depletion of SM-MHC in WT SMCs and overcome by ectopic expression of SM-MHC in HGPS SMCs. Arterial SM-MHC levels are also reduced with age in wild-type mice and humans, suggesting a common defect in arterial contractility in HGPS and normal aging.

Asunto(s)

Regulación de la Expresión Génica , Contracción Muscular/fisiología , Músculo Liso Vascular/fisiopatología , Cadenas Pesadas de Miosina/genética , Progeria/genética , Progeria/fisiopatología , Miosinas del Músculo Liso/genética , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/patología , Animales , Aorta/patología , Aorta/fisiopatología , Humanos , Ratones Endogámicos C57BL , Persona de Mediana Edad , Cadenas Pesadas de Miosina/metabolismo , Miosinas del Músculo Liso/metabolismo

RESUMEN

Muscle contraction depends on the cyclical interaction of myosin and actin filaments. Therefore, it is important to understand the mechanisms of polymerization and depolymerization of muscle myosins. Muscle myosin 2 monomers exist in two states: one with a folded tail that interacts with the heads (10S) and one with an unfolded tail (6S). It has been thought that only unfolded monomers assemble into bipolar and side-polar (smooth muscle myosin) filaments. We now show by electron microscopy that, after 4 s of polymerization in vitro in both the presence (smooth muscle myosin) and absence of ATP, skeletal, cardiac, and smooth muscle myosins form tail-folded monomers without tail-head interaction, tail-folded antiparallel dimers, tail-folded antiparallel tetramers, unfolded bipolar tetramers, and small filaments. After 4 h, the myosins form thick bipolar and, for smooth muscle myosin, side-polar filaments. Nonphosphorylated smooth muscle myosin polymerizes in the presence of ATP but with a higher critical concentration than in the absence of ATP and forms only bipolar filaments with bare zones. Partial depolymerization in vitro of nonphosphorylated smooth muscle myosin filaments by the addition of MgATP is the reverse of polymerization.

Asunto(s)

Citoesqueleto de Actina/química , Miosina Tipo II/química , Miosinas/química , Miosinas del Músculo Liso/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestructura , Animales , Pollos , Microscopía Electrónica , Miosina Tipo II/genética , Miosina Tipo II/ultraestructura , Miosinas/genética , Miosinas/ultraestructura , Fosforilación/genética , Polimerizacion , Conformación Proteica , Pliegue de Proteína , Multimerización de Proteína/genética , Desplegamiento Proteico , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/ultraestructura

RESUMEN

Mechanical tension and humoral stimuli can induce transitions in airway smooth muscle phenotype between a synthetic inflammatory state that promotes cytokine secretion and a differentiated state that promotes the expression of smooth muscle phenotype-specific proteins. When tissues are maintained under high tension, Akt activation and eotaxin secretion are suppressed, but expression of the differentiation marker protein, smooth muscle myosin heavy chain (SmMHC), is promoted. When tissues are maintained under low tension, Akt activation and eotaxin secretion are stimulated, and the differentiated phenotype is suppressed. We hypothesized that mechanical stimuli are differentially transduced to Akt-mediated signaling pathways that regulate phenotype expression by α-parvin and ß-parvin integrin-linked kinase/PINCH/parvin (IPP) signaling complexes within integrin adhesomes. High tension or ACh triggered paxillin phosphorylation and the binding of phospho-paxillin to ß-parvin IPP complexes. This inhibited Akt activation and promoted SmMHC expression. Low tension or IL-4 did not elicit paxillin phosphorylation and triggered the binding of unphosphorylated paxillin to α-parvin IPP complexes, which promoted Akt activation and eotaxin secretion and suppressed SmMHC expression. Expression of a nonphosphorylatable paxillin mutant or ß-parvin depletion by siRNA promoted the inflammatory phenotype, whereas the depletion of α-parvin promoted the differentiated phenotype. Results demonstrate that phenotype expression is regulated by the differential interaction of phosphorylated and unphosphorylated paxillin with α-parvin and ß-parvin IPP complexes and that these complexes have opposite effects on the activation of Akt. Our results describe a novel molecular mechanism for transduction of mechanical and humoral stimuli within integrin signaling complexes to regulate phenotype expression in airway smooth muscle.

Asunto(s)

Actinina/genética , Mecanotransducción Celular , Músculo Liso/metabolismo , Paxillin/genética , Proteínas Proto-Oncogénicas c-akt/genética , Tráquea/metabolismo , Acetilcolina/farmacología , Actinina/metabolismo , Animales , Quimiocina CCL11/genética , Quimiocina CCL11/metabolismo , Perros , Femenino , Regulación de la Expresión Génica , Interleucina-4/genética , Interleucina-4/metabolismo , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/genética , Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Paxillin/metabolismo , Fenotipo , Fosforilación/efectos de los fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/metabolismo , Tráquea/efectos de los fármacos

RESUMEN

Blebbistatin is a potent and specific inhibitor of the motor functions of class II myosins, including striated muscle myosin and nonmuscle myosin-2 (NM2). However, the blebbistatin inhibition of NM2c has not been assessed and remains controversial with respect to its efficacy with smooth muscle myosin (SmM), which is highly homologous to NM2. To clarify these issues, we analyzed the effects of blebbistatin on the motor activities of recombinant SmM and three NM2s (NM2a, -2b, and -2c). We found that blebbistatin potently inhibits the actin-activated ATPase activities of SmM and NM2s with following IC50 values: 6.47 µM for SmM, 3.58 µM for NM2a, 2.30 µM for NM2b, and 1.57 µM for NM2c. To identify the blebbistatin-resistant myosin-2 mutant, we performed mutagenesis analysis of the conserved residues in the blebbistatin-binding site of SmM and NM2s. We found that the A456F mutation renders SmM and NM2s resistant to blebbistatin without greatly altering their motor activities or phosphorylation-dependent regulation, making A456F a useful mutant for investigating the cellular function of NM2s.

Asunto(s)

Proteínas Aviares/antagonistas & inhibidores , Proteínas Aviares/química , Compuestos Heterocíclicos de 4 o más Anillos/química , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/química , Miosinas del Músculo Liso/antagonistas & inhibidores , Miosinas del Músculo Liso/química , Sustitución de Aminoácidos , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Pollos , Humanos , Ratones , Mutación Missense , Miosina Tipo IIB no Muscular/genética , Miosina Tipo IIB no Muscular/metabolismo , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/metabolismo

RESUMEN

Diversity of smooth muscle within the vascular system is generated by alternative splicing of exons, yet there is limited understanding of its timing or control mechanisms. We examined splicing of myosin phosphatase regulatory subunit (Mypt1) exon 24 (E24) in relation to smooth muscle myosin heavy chain (Smmhc) and smoothelin (Smtn) alternative exons (Smmhc E6 and Smtn E20) during maturation of mouse mesenteric artery (MA) smooth muscle. The role of transformer 2ß (Tra2ß), a master regulator of splicing in flies, in maturation of arterial smooth muscle was tested through gene inactivation. Splicing of alternative exons in bladder smooth muscle was examined for comparative purposes. MA smooth muscle maturation began after postnatal week 2 and was complete at maturity, as indicated by switching to Mypt1 E24+ and Smtn E20- splice variants and 11-fold induction of Smmhc. Similar changes in bladder were complete by postnatal day 3. Splicing of Smmhc E6 was temporally dissociated from Mypt1 E24 and Smtn E20 and discordant between arteries and bladder. Tamoxifen-induced smooth muscle-specific inactivation of Tra2ß within the first week of life but not in maturity reduced splicing of Mypt1 E24 in MAs. Inactivation of Tra2ß causing a switch to the isoform of MYPT1 containing the COOH-terminal leucine zipper motif (E24-) increased arterial sensitivity to cGMP-mediated relaxation. In conclusion, maturation of mouse MA smooth muscle begins postnatally and continues until sexual maturity. TRA2ß is required for specification during this period of maturation, and its inactivation alters the contractile properties of mature arterial smooth muscle.

Asunto(s)

Empalme Alternativo , Diferenciación Celular , Exones , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Quinasa de Cadena Ligera de Miosina/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Edad , Animales , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Regulación del Desarrollo de la Expresión Génica , Genotipo , Técnicas In Vitro , Masculino , Arterias Mesentéricas/enzimología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/genética , Fosfatasa de Miosina de Cadena Ligera , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Fenotipo , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-Arginina , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/metabolismo , Vasodilatación , Vasodilatadores/farmacología

RESUMEN

OBJECTIVE: The goal of the present study was to identify novel mechanisms that regulate smooth muscle cell (SMC) differentiation marker gene expression. APPROACH AND RESULTS: We demonstrate that the CArG-containing regions of many SMC-specific promoters are imbedded within CpG islands. A previously identified GC repressor element in the SM myosin heavy chain (MHC) promoter was highly methylated in cultured aortic SMC but not in the aorta, and this difference was inversely correlated with SM MHC expression. Using an affinity chromatography/mass spectroscopy-based approach, we identified the multifunctional Notch transcription factor, recombination signal binding protein for immunoglobulin κ J region (RBPJ), as a methylated GC repressor-binding protein. RBPJ protein levels and binding to the endogenous SM MHC GC repressor were enhanced by platelet-derived growth factor-BB treatment. A methylation mimetic mutation to the GC repressor that facilitated RBPJ binding inhibited SM MHC promoter activity as did overexpression of RBPJ. Consistent with this, knockdown of RBPJ in phenotypically modulated human aortic SMC enhanced endogenous SMC marker gene expression, an effect likely mediated by increased recruitment of serum response factor and Pol II to the SMC-specific promoters. In contrast, the depletion of RBPJ in differentiated transforming growth factor-ß-treated SMC inhibited SMC-specific gene activation, supporting the idea that the effects of RBPJ/Notch signaling are context dependent. CONCLUSIONS: Our results indicate that methylation-dependent binding of RBPJ to a GC repressor element can negatively regulate SM MHC promoter activity and that RBPJ can inhibit SMC marker gene expression in phenotypically modulated SMC. These results will have important implications on the regulation of SMC phenotype and on Notch-dependent transcription.

Asunto(s)

Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Cadenas Pesadas de Miosina/genética , Regiones Promotoras Genéticas , Miosinas del Músculo Liso/genética , Animales , Secuencia de Bases , Becaplermina , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Islas de CpG , Metilación de ADN , Secuencia Rica en GC , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/deficiencia , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-sis/metabolismo , Receptores Notch/metabolismo , Transducción de Señal

RESUMEN

All class II myosins have the conserved amino acid sequence Pro-Leu-Leu at their head-tail junctions. We systematically altered this sequence in smooth muscle heavy meromyosin (HMM) by site-directed mutagenesis and examined the effects of these mutations on actin-myosin interactions. Deletion of the proline and second leucine did not cause any noticeable change in either actin-activated ATPase activity or actin-sliding velocity. In contrast, deletion of the two leucine residues and substitution of the first leucine with alanine resulted in a 14-fold and 5-fold decrease, respectively, in actin-activated ATPase activity. However, both these mutations did not appreciably affect actin-sliding velocity, which was consistent with a result that there was no considerable change in the ADP release rate from acto-HMM in the deletion mutant. In contrast to double-headed HMM, a single-headed subfragment-1 (S1) with a Leu-Leu deletion mutation exhibited actin activated ATPase activity similar to that by wild type S1. Our results suggest that the first leucine of the conserved Leu-Leu sequence at the head-tail junction profoundly affects the cooperativity between the two heads involved in the actin activated ATPase activity of myosin II.

Asunto(s)

Miosinas del Músculo Liso/metabolismo , Actinas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Pollos , Secuencia Conservada , Leucina/genética , Leucina/metabolismo , Mutación , Prolina/genética , Prolina/metabolismo , Miosinas del Músculo Liso/genética

RESUMEN

Allergen challenges induce airway hyperresponsiveness (AHR) and increased airway smooth muscle (ASM) mass in the sensitized rat. Whether the remodeled ASM changes its phenotype is uncertain. We examined, in sensitized Brown Norway rats, the effects of multiple ovalbumin (Ova) challenges on ASM remodeling and phenotype and the role of the epidermal growth factor receptor (EGFR) in these processes. Rats were sensitized with Ova and challenged three times at 5-day intervals with phosphate-buffered saline or Ova and pretreated with the EGFR inhibitor AG-1478 (5 mg/kg) or its vehicle dimethyl sulfoxide. Ova challenges increased ASM mass in all-sized airways and in large airway mRNA expression of smooth muscle myosin heavy chain (sm-MHC), assessed by laser capture. Myosin light chain kinase and the fast myosin isoform SM-B mRNA expressions were not affected. Ova induced AHR to methacholine, and, based on the constant-phase model, this was largely attributable to the small airways and lung derecruitment at 48 h that recovered by 1 wk. The EGFR ligands amphiregulin and heparin-binding epidermal growth factor (HB-EGF) were increased in bronchoalveolar lavage fluid at 48 h after Ova exposure. AG-1478 inhibited AHR and prevented ASM growth. Epithelial gene expression of EGFR, HB-EGF, matrix metalloproteinase (MMP)-9, Gro-α, and transforming growth factor-ß was unaffected by Ova challenges. We conclude that EGFR drives remodeling of ASM, which results from repeated Ova challenge. Furthermore, the latter results in excessive small airway and, to a lesser degree, large airway narrowing to methacholine, and large airway gene expression of contractile protein is conserved.

Asunto(s)

Bronquios/patología , Receptores ErbB/genética , Músculo Liso/patología , Hipersensibilidad Respiratoria/patología , Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Alérgenos/inmunología , Alérgenos/farmacología , Anfirregulina , Animales , Bronquios/efectos de los fármacos , Bronquios/inmunología , Líquido del Lavado Bronquioalveolar/química , Familia de Proteínas EGF , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/genética , Glicoproteínas/inmunología , Factor de Crecimiento Similar a EGF de Unión a Heparina , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/inmunología , Masculino , Cloruro de Metacolina/farmacología , Músculo Liso/efectos de los fármacos , Músculo Liso/inmunología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/inmunología , Ovalbúmina/inmunología , Ovalbúmina/farmacología , Quinazolinas/farmacología , Ratas , Hipersensibilidad Respiratoria/inducido químicamente , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/prevención & control , Transducción de Señal/efectos de los fármacos , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/inmunología , Tirfostinos/farmacología

RESUMEN

The role of SMA and SMB smooth muscle myosin heavy chain (MHC) isoforms in tonic and phasic contractions was studied in phasic (longitudinal ileum and stomach circular antrum) and tonic (stomach circular fundus) smooth muscle tissues of SMB knockout mice. Knocking out the SMB MHC gene eliminated SMB MHC protein expression and resulted in upregulation of the SMA MHC protein without altering the total MHC protein level. Switching from SMB to SMA MHC protein expression decreased the rate of the force transient and increased the sustained tonic force in SMB((-/-)) ileum and antrum with high potassium (KPSS) but not with carbachol (CCh) stimulation. The increased tonic contraction under the depolarized condition was not through changes in second messenger signaling pathways (PKC/CPI-17 or Rho/ROCK signaling pathway) or LC(20) phosphorylation. Biochemical analyses showed that the expression of contractile regulatory proteins (MLCK, MLCP, PKCδ, and CPI-17) did not change significantly in tissues tested except for PKCα protein expression being significantly decreased in the SMB((-/-)) antrum. However, specifically activating PKCα with phorbol dibutyrate (PDBu) was not significantly different in knockout and wild-type tissues, with total force being a fraction of the force generation with KPSS or CCh stimulation in SMB((-/-)) ileum and antrum. Taken together, these data show removing the SMB MHC protein expression with a compensatory increase in the SMA MHC protein results in enhanced sustained KPSS-induced tonic contraction with a reduced rate of force generation in these phasic tissues.

Asunto(s)

Íleon/fisiología , Contracción Muscular/fisiología , Fuerza Muscular/fisiología , Músculo Liso/fisiología , Cadenas Pesadas de Miosina/fisiología , Antro Pilórico/fisiología , Miosinas del Músculo Liso/fisiología , Animales , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Íleon/citología , Íleon/efectos de los fármacos , Ratones , Ratones Noqueados , Contracción Muscular/efectos de los fármacos , Fuerza Muscular/efectos de los fármacos , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Pesadas de Miosina/genética , Forbol 12,13-Dibutirato/farmacología , Fosforilación , Potasio/farmacología , Proteína Quinasa C-alfa/biosíntesis , Proteína Quinasa C-alfa/fisiología , Antro Pilórico/citología , Antro Pilórico/efectos de los fármacos , Sistemas de Mensajero Secundario/efectos de los fármacos , Sistemas de Mensajero Secundario/genética , Sistemas de Mensajero Secundario/fisiología , Miosinas del Músculo Liso/biosíntesis , Miosinas del Músculo Liso/genética

RESUMEN

Ca(2+) sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr(697) and/or Thr(855) (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser(696) prevents phosphorylation at Thr(697). However, the effects of Ser(854) and dual Ser(696)-Thr(697) and Ser(854)-Thr(855) phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser(696), Thr(697), Ser(854), and Thr(855)), Ser phosphorylation events (Ser(696)/Ser(854)) and dual Ser/Thr phosphorylation events (Ser(696)-Thr(697) and Ser(854)-Thr(855)). Dual phosphorylation at Ser(696)-Thr(697) and Ser(854)-Thr(855) by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr(697) and Thr(855) by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser(696), Thr(697), Ser(854), and Thr(855) in rat caudal artery, whereas U46619 induced Thr(697) and Thr(855) phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser(696)-Thr(697) and Ser(854)-Thr(855) inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.

Asunto(s)

AMP Cíclico/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteína Fosfatasa 1/metabolismo , Sistemas de Mensajero Secundario/fisiología , Miosinas del Músculo Liso/metabolismo , Quinasas Asociadas a rho/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Sustitución de Aminoácidos , Animales , Colforsina/farmacología , AMP Cíclico/genética , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Mutación Missense , Miocitos del Músculo Liso/citología , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 1/genética , Ratas , Ratas Sprague-Dawley , Sistemas de Mensajero Secundario/efectos de los fármacos , Miosinas del Músculo Liso/genética , Vasoconstrictores/farmacología , Quinasas Asociadas a rho/genética , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo

RESUMEN

OBJECTIVE: Smad4 is a central mediator of transforming growth factor-ß/bone morphogenetic protein signaling that controls numerous developmental processes as well as homeostasis in the adult. The present studies sought to understand the function of Smad4 expressed in vascular smooth muscle cells (VSMC) in vascular development and the underlying mechanisms. METHODS AND RESULTS: Breeding of Smad4(flox/flox) mice with SM22α-Cre mice resulted in no viable offspring with SM22α-Cre;Smad4(flox/flox) genotype in a total of 165 newborns. Subsequent characterization of 301 embryos between embryonic day (E) 9.5 and E14.5 demonstrated that mice with SM22α-Cre;Smad4(flox/flox) genotype died between E12.5 and E14.5 because of decreased cell proliferation and increased apoptosis in the embryonic heart and arteries. Additionally, deletion of Smad4 more specifically in SMC with the inducible smooth muscle myosin heavy chain (SMMHC)-Cre mice, in which decreased cell proliferation was observed only in the artery but not the heart, also caused lethality of the knockout embryos at E12.5 and E14.5. The Smad4-deficient VSMC lacked smooth muscle α-actin filaments, decreased expression of SMC-specific gene markers, and markedly reduced cell proliferation, migration, and attachment. Using specific pharmacological inhibitors and small interfering RNAs, we demonstrated that inhibition of transforming growth factor-ß signaling and its regulatory Smad 2/3 decreased VSMC proliferation, migration, and expression of SMC-specific gene markers, whereas inhibition of bone morphogenetic protein signaling only affected VSMC migration. CONCLUSIONS: SMC-specific deletion of Smad4 results in vascular defects that lead to embryonic lethality in mice, which may be attributed to decreased VSMC differentiation, proliferation, migration, as well as cell attachment and spreading. The transforming growth factor-ß signaling pathway contributes to VSMC differentiation and function, whereas the bone morphogenetic protein signaling pathway regulates VSMC migration. These studies provide important insight into the role of Smad4 and its upstream Smads in regulating SMC function and vascular development of mice.

Asunto(s)

Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Transducción de Señal , Proteína Smad4/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Apoptosis , Arterias/embriología , Arterias/metabolismo , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Pérdida del Embrión , Regulación del Desarrollo de la Expresión Génica , Marcadores Genéticos , Genotipo , Edad Gestacional , Corazón/embriología , Integrasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/embriología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Fenotipo , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Proteína Smad4/deficiencia , Proteína Smad4/genética , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/metabolismo , Transfección , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

Diabetes mellitus (DM) is a quite common chronic disease, and the prevalence of erectile dysfunction (ED) is three times higher in this large population. Although diabetes-related ED has been studied extensively, the actin-myosin contractile apparatus was not examined. The mRNAs encoding smooth muscle myosin (SMM) heavy chains (MHC) and essential light chains (LC(17)) exist as several different alternatively spliced isoforms with distinct contractile properties. Recently, we provided novel data that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed corpus cavernosum smooth muscle (CCSM). In this study, we examine whether diabetes alters SMM expression, alternative splicing, and/or functional activities, including sensitivity to BLEB. By using streptozotocin (STZ)-induced 2-mo diabetic rats, functional activities were tested in vivo by intracavernous pressure (ICP) recording during cavernous nerve stimulation and in vitro via organ bath contractility studies. SMM isoform composition was analyzed by competitive RT-PCR and total SMM, myocardin, and embryonic SMM (SMemb) expression by real-time RT-PCR. Results revealed that the blood glucose level of STZ rats was 407.0 vs. 129.5 mg/dl (control). STZ rats exhibited ED confirmed by significantly increased CCSM contractile response to phenylephrine and decreased ICP response. For STZ rats, SM-B, LC(17a) and SM2 isoforms, total SMM, and myocardin expression increased, whereas SM-A, LC(17b), and SM1 isoforms were decreased, with SMemb unchanged. BLEB was significantly more effective in relaxing STZ CCSM both in vitro and in vivo. Thus we demonstrated a novel diabetes-specific effect on alternative splicing of the SMM heavy chain and essential light chain genes to a SMM isoform composition favoring a heightened contractility and ED. A switch to a more contractile phenotype was supported further by total SMM expression increase. Moreover, the change in CCSM phenotype was associated with an increased sensitivity to BLEB, which may serve as a novel pharmacotherapy for ED.

Asunto(s)

Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatología , Disfunción Eréctil/metabolismo , Disfunción Eréctil/fisiopatología , Pene/metabolismo , Pene/fisiopatología , Miosinas del Músculo Liso/metabolismo , Empalme Alternativo , Animales , Diabetes Mellitus Tipo 1/complicaciones , Resistencia a Medicamentos , Inhibidores Enzimáticos/farmacología , Disfunción Eréctil/complicaciones , Disfunción Eréctil/tratamiento farmacológico , Regulación de la Expresión Génica , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Músculo Liso/fisiopatología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Miosina Tipo II/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pene/efectos de los fármacos , Pene/inervación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Miosinas del Músculo Liso/genética , Estreptozocina/toxicidad , Transactivadores/genética , Transactivadores/metabolismo

RESUMEN

We hypothesized that ablation of smooth muscle α-actin (SM α-A), a contractile-cytoskeletal protein expressed in airway smooth muscle (ASM) cells, abolishes ASM shortening capacity and decreases lung stiffness. In both SM α-A knockout and wild-type (WT) mice, airway resistance (Raw) determined by the forced oscillation technique rose in response to intravenous methacholine (Mch). However, the slope of Raw (cmH(2)O·ml(-1)·s) vs. log(2) Mch dose (µg·kg(-1)·min(-1)) was lower (P = 0.007) in mutant (0.54 ± 0.14) than in WT mice (1.23 ± 0.19). RT-PCR analysis performed on lung tissues confirmed that mutant mice lacked SM α-A mRNA and showed that these mice had robust expressions of both SM γ-A mRNA and skeletal muscle (SKM) α-A mRNA, which were not expressed in WT mice, and an enhanced SM22 mRNA expression relative to that in WT mice. Compared with corresponding spontaneously breathing mice, mechanical ventilation-induced lung mechanical strain increased the expression of SM α-A mRNA in WT lungs; in mutant mice, it augmented the expressions of SM γ-A mRNA and SM22 mRNA and did not alter that of SKM α-A mRNA. In mutant mice, the expression of SM γ-A mRNA in the lung during spontaneous breathing and its enhanced expression following mechanical ventilation are consistent with the likely possibility that in the absence of SM α-A, SM γ-A underwent polymerization and interacted with smooth muscle myosin to produce ASM shortening during cholinergic stimulation. Thus our data are consistent with ASM in mutant mice experiencing compensatory mechanisms that modulated its contractile muscle capacity.

Asunto(s)

Actinas/genética , Actinas/metabolismo , Pulmón/metabolismo , Pulmón/fisiología , Contracción Muscular/fisiología , Músculo Liso/metabolismo , Músculo Liso/fisiología , Resistencia de las Vías Respiratorias/efectos de los fármacos , Resistencia de las Vías Respiratorias/genética , Resistencia de las Vías Respiratorias/fisiología , Animales , Proteínas Contráctiles/genética , Proteínas Contráctiles/metabolismo , Pulmón/efectos de los fármacos , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Contracción Muscular/efectos de los fármacos , Contracción Muscular/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/fisiología , ARN Mensajero/genética , Respiración Artificial/métodos , Miosinas del Músculo Liso/efectos de los fármacos , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/metabolismo , Miosinas del Músculo Liso/fisiología

RESUMEN

Isoforms of the smooth muscle (SM) myosin motor domain differ in the presence or absence of a seven amino acid insert in a flexible surface loop spanning the nucleotide-binding pocket known as Loop 1. The presence of this insert leads to a two-fold increase in actin sliding velocity and ADP release rate between these isoforms, although the effect of Loop 1 on the kinetics of ADP release remains unclear. To further investigate the role of the Loop 1 insert in modulating ADP release in SM myosin we have inserted a single tryptophan residue into Loop 1 of both isoforms as a probe of local structural dynamics. By monitoring the dynamics of Loop 1 in relation to the release of ADP we have observed a unique movement of Loop 1 in the inserted isoform, preceding nucleotide release, which is absent in the non-inserted isoform. This movement is sequence dependent as alanine replacement of the insert residues abolishes the transition and slows ADP release. Thus movement of Loop 1 is a critical factor in increasing the ADP release rate in the inserted faster isoform of SM myosin.

Asunto(s)

Actinas/metabolismo , Músculo Liso/metabolismo , Miosinas del Músculo Liso , Adenosina Difosfato/metabolismo , Algoritmos , Secuencia de Aminoácidos , Animales , Pollos , Transferencia Resonante de Energía de Fluorescencia/métodos , Cinética , Modelos Moleculares , Contracción Muscular/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes de Fusión , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/metabolismo

RESUMEN

Bone marrow-derived mesenchymal stem cells are multipotent stem cells, an attractive resource for regenerative medicine. Accumulating evidence suggests that all-trans retinoic acid plays a key role in the development and differentiation of smooth muscle cells. In the present study, we demonstrate, for the first time, that rabbit bone marrow-derived mesenchymal stem cells differentiate into smooth muscle cells upon the treatment with all-trans retinoic acid. All-trans retinoic acid increased the expression of myocardin, caldesmon, 22-kDa smooth muscle cell-specific protein (SM22alpha), and SM-myosin heavy chains in rabbit bone marrow-derived mesenchymal stem cells, as detected by reverse transcription polymerase chain reaction (PCR). Immunostaining of SM22alpha and SM-myosin heavy chains using monoclonal antibodies also indicated smooth muscle cell differentiation of rabbit bone marrow-derived mesenchymal stem cells following the treatment with all-trans retinoic acid. In addition, more than 47% of bone marrow-derived mesenchymal stem cells demonstrated the contractile phenotype of smooth muscle cells. Western blot results showed that SM-1 and SM-2 were highly expressed in the differentiated cells. These results suggest that all-trans retinoic acid may serve as a potent agent for functional smooth muscle cell differentiation in tissue engineering.

Asunto(s)

Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Tretinoina/farmacología , Animales , Secuencia de Bases , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cartilla de ADN/genética , Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Células Madre Mesenquimatosas/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Células Madre Multipotentes/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/metabolismo , Transactivadores/genética , Transactivadores/metabolismo

RESUMEN

To understand the importance of selected regions of the regulatory light chain (RLC) for phosphorylation-dependent regulation of smooth muscle myosin (SMM), we expressed three heavy meromyosins (HMMs) containing the following RLC mutants; K12E in a critical region of the phosphorylation domain, GTDP(95-98)/AAAA in the central hinge, and R160C a putative binding residue for phosphorylated S19. Single-turnover actin-activated Mg(2+)-ATPase (V(max) and K(ATPase)) and in vitro actin-sliding velocities were examined for both unphosphorylated (up-) and phosphorylated (p-) states. Turnover rates for the up-state (0.007-0.030 s(-1)) and velocities (no motion) for all constructs were not significantly different from the up-wild type (WT) indicating that they were completely turned off. The apparent binding constants for actin in the presence of ATP (K(ATPase)) were too weak to measure as expected for fully regulated constructs. For p-HMM containing GTDP/AAAA, we found that both ATPase and motility were normal. The data suggest that the native sequence in the central hinge between the two lobes of the RLC is not required for turning the HMM off and on both kinetically and mechanically. For p-HMM containing R160C, all parameters were normal, suggesting that R160C is not involved in coordination of the phosphorylated S19. For p-HMM containing K12E, the V(max) was 64% and the actin-sliding velocity was approximately 50% of WT, suggesting that K12 is an important residue for the ability to sense or to promote the conformational changes required for kinetic and mechanical activation.

Asunto(s)

Cadenas Ligeras de Miosina/fisiología , Miosinas del Músculo Liso/fisiología , Sustitución de Aminoácidos , Animales , Cinética , Proteínas Motoras Moleculares/genética , Cadenas Ligeras de Miosina/genética , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/fisiología , Fosforilación , Estructura Terciaria de Proteína , Miosinas del Músculo Liso/genética

RESUMEN

The physiological relevance of smooth muscle myosin isoforms SM1 and SM2 has not been understood. In this study we generated a mouse model specifically deficient in SM2 myosin isoform but expressing SM1, using an exon-specific gene targeting strategy. The SM2 homozygous knockout (SM2(-/-)) mice died within 30 days after birth, showing pathologies including segmental distention of alimentary tract, retention of urine in renal pelvis, distension of bladder, and the development of end-stage hydronephrosis. In contrast, the heterozygous (SM2(+/-)) mice appeared normal and reproduced well. In SM2(-/-) bladder smooth muscle the loss of SM2 myosin was accompanied by a concomitant down-regulation of SM1 and a reduced number of thick filaments. However, muscle strips from SM2(-/-) bladder showed increased contraction to K(+) depolarization or in response to M3 receptor agonist Carbachol. An increase of contraction was also observed in SM2(-/-) aorta. However, the SM2(-/-) bladder was associated with unaltered regulatory myosin light chain (MLC20) phosphorylation. Moreover, other contractile proteins, such as alpha-actin and tropomyosin, were not altered in SM2(-/-) bladder. Therefore, the loss of SM2 myosin alone could have induced hypercontractility in smooth muscle, suggesting that distinctly from SM1, SM2 may negatively modulate force development during smooth muscle contraction. Also, because SM2(-/-) mice develop lethal multiorgan dysfunctions, we propose this regulatory property of SM2 is essential for normal contractile activity in postnatal smooth muscle physiology.

Asunto(s)

Miosinas del Músculo Liso/fisiología , Animales , Animales Recién Nacidos , Carbacol/farmacología , Femenino , Heterocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Muscular/efectos de los fármacos , Fosforilación , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/metabolismo

RESUMEN

We examined adenomas and cancers from hereditary non-polyposis colorectal cancer (HNPCC) syndrome patients for the presence of frameshift mutations in the smooth-muscle myosin gene, MYH11. Our results show that mutations in MYH11 occur more frequently in cancers than adenomas (P=0.008) and are dependent on microsatellite instability (MSI+).

Asunto(s)

Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Cadenas Pesadas de Miosina/genética , Miosinas del Músculo Liso/genética , Adenoma/genética , Mutación del Sistema de Lectura , Humanos , Inestabilidad de Microsatélites

RESUMEN

A recent study described a recessive ATPase activating germ-line mutation in smooth-muscle myosin (smmhc/myh11) underlying the zebrafish meltdown (mlt) phenotype. The mlt zebrafish develops intestinal abnormalities reminiscent of human Peutz-Jeghers syndrome (PJS) and juvenile polyposis (JP). To examine the role of MYH11 in human intestinal neoplasia, we searched for MYH11 mutations in patients with colorectal cancer (CRC), PJS and JP. We found somatic protein-elongating frameshift mutations in 55% of CRCs displaying microsatellite instability and in the germ-line of one individual with PJS. Additionally, two somatic missense mutations were found in one microsatellite stable CRC. These two missense mutations, R501L and K1044N, and the frameshift mutations were functionally evaluated. All mutations resulted in unregulated molecules displaying constitutive motor activity, similar to the mutant myosin underlying mlt. Thus, MYH11 mutations appear to contribute also to human intestinal neoplasia. Unregulated MYH11 may affect the cellular energy balance or disturb cell lineage decisions in tumor progenitor cells. These data challenge our view on MYH11 as a passive differentiation marker functioning in muscle contraction and add to our understanding of intestinal neoplasia.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Neoplasias Intestinales/genética , Mutación , Cadenas Pesadas de Miosina/genética , Miosina Tipo II/genética , Miosinas del Músculo Liso/genética , Poliposis Adenomatosa del Colon/genética , Secuencia de Aminoácidos , Estudios de Casos y Controles , Neoplasias Colorrectales/genética , Análisis Mutacional de ADN , Humanos , Datos de Secuencia Molecular , Síndrome de Peutz-Jeghers/genética , Miosinas del Músculo Liso/fisiología