RESUMEN
Skeletal homeostasis is sensitive to perturbations in Wnt signaling. Beyond its role in the bone, Wnt is a major target for pharmaceutical inhibition in a wide range of diseases, most notably cancers. Numerous clinical trials for Wnt-based candidates are currently underway, and Wnt inhibitors will likely soon be approved for clinical use. Given the bone-suppressive effects accompanying Wnt inhibition, there is a need to expose alternate pathways/molecules that can be targeted to counter the deleterious effects of Wnt inhibition on bone properties. Activation of the Pi3k/Akt pathway via Pten deletion is one possible osteoanabolic pathway to exploit. We investigated whether the osteopenic effects of ß-catenin deletion from bone cells could be rescued by Pten deletion in the same cells. Mice carrying floxed alleles for Pten and ß-catenin were bred to Dmp1-Cre mice to delete Pten alone, ß-catenin alone, or both genes from the late-stage osteoblast/osteocyte population. The mice were assessed for bone mass, density, strength, and formation parameters to evaluate the potential rescue effect of Pten deletion in Wnt-impaired mice. Pten deletion resulted in high bone mass and ß-catenin deletion resulted in low bone mass. Compound mutants had bone properties similar to ß-catenin mutant mice, or surprisingly in some assays, were further compromised beyond ß-catenin mutants. Pten inhibition, or one of its downstream nodes, is unlikely to protect against the bone-wasting effects of Wnt/ßcat inhibition. Other avenues for preserving bone mass in the presence of Wnt inhibition should be explored to alleviate the skeletal side effects of Wnt inhibitor-based therapies.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Neoplasias/tratamiento farmacológico , Fosfohidrolasa PTEN/genética , beta Catenina/genética , Animales , Enfermedades Óseas Metabólicas/tratamiento farmacológico , Enfermedades Óseas Metabólicas/genética , Proliferación Celular/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Neoplasias/genética , Osteoblastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Following vascular injury medial smooth muscle cells dedifferentiate and migrate through the internal elastic lamina where they form a neointima. The goal of the current study was to identify changes in gene expression that occur before the development of neointima and are associated with the early response to injury. Vascular injury was induced in C57BL/6 mice and in Myh11-creER(T2) mTmG reporter mice by complete ligation of the left carotid artery. Reporter mice were used to visualize cellular changes in the injured vessels. Total RNA was isolated from control carotid arteries or from carotid arteries 3 days following ligation of C57BL/6 mice and analyzed by Affymetrix microarray and quantitative RT-PCR. This analysis revealed decreased expression of mRNAs encoding smooth muscle-specific contractile proteins that was accompanied by a marked increase in a host of mRNAs encoding inflammatory cytokines following injury. There was also marked decrease in molecules associated with BMP, Wnt, and Hedgehog signaling and an increase in those associated with B cell, T cell, and macrophage signaling. Expression of a number of noncoding RNAs were also altered following injury with microRNAs 143/145 being dramatically downregulated and microRNAs 1949 and 142 upregulated. Several long noncoding RNAs showed altered expression that mirrored the expression of their nearest coding genes. These data demonstrate that following carotid artery ligation an inflammatory cascade is initiated that is associated with the downregulation of coding and noncoding RNAs that are normally required to maintain smooth muscle cells in a differentiated state.
Asunto(s)
Arterias Carótidas/patología , Desdiferenciación Celular , Inflamación/patología , Músculo Liso Vascular/patología , Animales , Citocinas/metabolismo , Regulación hacia Abajo/genética , Inflamación/genética , Mediadores de Inflamación/metabolismo , Ligadura , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismo , Modelos Biológicos , Contracción Muscular/genética , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Transducción de Señal/genética , Regulación hacia Arriba/genéticaRESUMEN
The mylk1 gene encodes a 220-kDa nonmuscle myosin light chain kinase (MLCK), a 130-kDa smooth muscle MLCK (smMLCK), as well as the non-catalytic product telokin. Together, these proteins play critical roles in regulating smooth muscle contractility. Changes in their expression are associated with many pathological conditions; thus, it is important to understand the mechanisms regulating expression of mylk1 gene transcripts. Previously, we reported a highly conserved CArG box, which binds serum response factor, in intron 15 of mylk1. Because this CArG element is near the promoter that drives transcription of the 130-kDa smMLCK, we examined its role in regulating expression of this transcript. Results show that deletion of the intronic CArG region from a ß-galactosidase reporter gene abolished transgene expression in mice in vivo. Deletion of the CArG region from the endogenous mylk1 gene, specifically in smooth muscle cells, decreased expression of the 130-kDa smMLCK by 40% without affecting expression of the 220-kDa MLCK or telokin. This reduction in 130-kDa smMLCK expression resulted in decreased phosphorylation of myosin light chains, attenuated smooth muscle contractility, and a 24% decrease in small intestine length that was associated with a significant reduction of Ki67-positive smooth muscle cells. Overall, these data show that the CArG element in intron 15 of the mylk1 gene is necessary for maximal expression of the 130-kDa smMLCK and that the 130-kDa smMLCK isoform is specifically required to regulate smooth muscle contractility and small intestine smooth muscle cell proliferation.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Contracción Muscular/genética , Miocitos del Músculo Liso/metabolismo , Quinasa de Cadena Ligera de Miosina/genética , Proteínas Represoras/genética , Animales , Proliferación Celular , Regulación de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Intrones/genética , Antígeno Ki-67/metabolismo , Ratones , Quinasa de Cadena Ligera de Miosina/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Represoras/metabolismo , Factor de Respuesta Sérica/metabolismoRESUMEN
Smooth muscle cells (SMCs) modulate their phenotype from a quiescent contractile state to a dedifferentiated, proliferative and migratory state during the pathogenesis of many diseases, including intestinal pseudoobstruction. Understanding how smooth muscle gene expression is regulated in these different phenotypic states is critical for unraveling the pathogenesis of these diseases. In the current study we examined the specific roles of Foxf1 in visceral SMC differentiation. Data show that Foxf1 is specifically required for expression of several contractile and regulatory proteins such as telokin, smooth muscle γ-actin, and Cav1.2b in visceral SMCs. Mechanistically, Foxf1 directly binds to and activates the telokin promoter. Foxf1 also directly binds to serum response factor (SRF) and myocardin-related transcription factors (MRTFs). Unlike Foxo4 and Foxq1, which bind to MRTFs and block their interaction with SRF, Foxf1 acts synergistically with these proteins to regulate telokin expression. Knock-out of Foxf1 specifically in SMCs results in neonatal lethality, with mice exhibiting GI tract abnormalities. Mice heterozygous for Foxf1 in SMC exhibited impaired colonic contractility and decreased expression of contractile proteins. These studies together with previous studies, suggest that different forkhead proteins can regulate gene expression in SMCs through modulating the activity of the SRF-myocardin axis to either promote or inhibit differentiation and proliferation thereby altering gastrointestinal contractility and development.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Factor de Respuesta Sérica/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Vísceras/citología , Secuencia Rica en At/genética , Animales , Animales Recién Nacidos , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Colon/citología , Proteínas Contráctiles/genética , Proteínas Contráctiles/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Contracción Muscular/genética , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/genéticaRESUMEN
The development of Wnt-based osteoanabolic agents has progressed rapidly in recent years, given the potent effects of Wnt modulation on bone homeostasis. Simultaneous pharmacologic inhibition of the Wnt antagonists sclerostin and Dkk1 can be optimized to create potentiated effects in the cancellous bone compartment. We looked for other candidates that might be co-inhibited along with sclerostin to potentiate the effects in the cortical compartment. Sostdc1 (Wise), like sclerostin and Dkk1, also binds and inhibits Lrp5/6 coreceptors to impair canonical Wnt signaling, but Sostdc1 has greater effects in the cortical bone. To test this concept, we deleted Sostdc1 and Sost from mice and measured the skeletal effects in cortical and cancellous compartments individually. Sost deletion alone produced high bone mass in all compartments, whereas Sostdc1 deletion alone had no measurable effects on either envelope. Mice with codeletion of Sostdc1 and Sost had high bone mass and increased cortical properties (bone mass, formation rates, mechanical properties), but only among males. Combined administration of sclerostin antibody and Sostdc1 antibody in wild-type female mice produced potentiation of cortical bone gain despite no effect of Sostdc1 antibody alone. In conclusion, Sostdc1 inhibition/deletion can work in concert with sclerostin deficiency to improve cortical bone properties. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
Asunto(s)
Glicoproteínas , Péptidos y Proteínas de Señalización Intercelular , Masculino , Femenino , Animales , Ratones , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Glicoproteínas/metabolismo , Huesos/metabolismo , Hueso Cortical/metabolismo , Hueso Esponjoso/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
Age-associated low bone mass disease is a growing problem in the US. Development of osteoanabolic therapies for treating skeletal fragility has lagged behind anti-catabolic therapies, but several bone-building molecules are clinically available. We reported previously that antibody-based neutralization of the Lrp5/Lrp6 inhibitor Dkk1 has minimal effects on bone gain, but can potentiate the already potent osteoanabolic effects of sclerostin inhibition (another Lrp5/Lrp6 inhibitor highly expressed by osteocytes). In this communication, we test whether an optimized ratio of sclerostin and Dkk1 antibodies (Scl-mAb and Dkk1-mAb, respectively), administered at low doses, can maintain the same bone-building effects as higher dose Scl-mAb, in adult (6 months of age) and aged (20 months of age) wild-type mice. A 3:1 dose of Scl-mAb:Dkk1-mAb at 12.5 mg/kg was equally efficacious as 25 mg/kg of Scl-mAb in both age groups, using radiographic (DXA, µCT), biomechanical, (3-point bending tests), and histological (fluorochrome-based bone formation parameters) outcome measures. For some bone properties, including trabecular thickness and bone mineral density in the spine, and endocortical bone formation rates in the femur, the 3:1 treatment was associated with significantly improved skeletal properties compared to twice the dose of Scl-mAb. Cortical porosity in aged mice was also reduced by both Scl-mAb and low-dose 3:1 treatment. Overall, both treatments were efficacious in the mature adult (6 mo.) and aged (20 mo.) skeletons, suggesting Wnt targeting is a viable strategy for improving skeletal fragility in the very old. Further, the data suggest that low dose of combination therapy can be at least equally efficacious as higher doses of Scl-mAb monotherapy.
RESUMEN
Sclerostin antibody (romosozumab) was recently approved for clinical use in the United States to treat osteoporosis. We and others have explored Wnt-based combination therapy to disproportionately improve the anabolic effects of sclerostin inhibition, including cotreatment with sclerostin antibody (Scl-mAb) and Dkk1 antibody (Dkk1-mAb). To determine the optimal ratio of Scl-mAb and Dkk1-mAb for producing maximal anabolic action, the proportion of Scl-mAb and Dkk1-mAb were systematically varied while holding the total antibody dose constant. A 3:1 mixture of Scl-mAb to Dkk1-mAb produced two to three times as much cancellous bone mass as an equivalent dose of Scl-mAb alone. Further, a 75% reduction in the dose of the 3:1 mixture was equally efficacious to a full dose of Scl-mAb in the distal femur metaphysis. The Scl-mAb/Dkk1-mAb combination approach was highly efficacious in the cancellous bone mass, but the cortical compartment was much more subtly affected. The osteoanabolic effects of Wnt pathway targeting can be made more efficient if multiple antagonists are simultaneously targeted. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.
RESUMEN
Wnt signaling plays a vital role in the cell biology of skeletal patterning, differentiation, and maintenance. Notum is a secreted member of the α/ß-hydrolase superfamily that hydrolyzes the palmitoleoylate modification on Wnt proteins, thereby disrupting Wnt signaling. As a secreted inhibitor of Wnt, Notum presents an attractive molecular target for improving skeletal health. To determine the cell type of action for Notum's effect on the skeleton, we generated mice with Notum deficiency globally (Notum-/- ) and selectively (Notumf/f ) in limb bud mesenchyme (Prx1-Cre) and late osteoblasts/osteocytes (Dmp1-Cre). Late-stage deletion induced increased cortical bone properties, similar to global mutants. Notum expression was enhanced in response to sclerostin inhibition, so dual inhibition (Notum/sclerostin) was also investigated using a combined genetic and pharmacologic approach. Co-suppression increased cortical properties beyond either factor alone. Notum suppressed Wnt signaling in cell reporter assays, but surprisingly also enhanced Shh signaling independent of effects on Wnt. Notum is an osteocyte-active suppressor of cortical bone formation that is likely involved in multiple signaling pathways important for bone homeostasis © 2021 American Society for Bone and Mineral Research (ASBMR).
Asunto(s)
Hueso Cortical , Esterasas/genética , Osteogénesis , Vía de Señalización Wnt , Animales , Hueso Cortical/metabolismo , Ratones , Ratones Noqueados , Osteoblastos/metabolismo , Osteocitos/metabolismoRESUMEN
Serum response factor (SRF) is a widely expressed protein that plays a key role in the regulation of smooth muscle differentiation, proliferation, migration, and apoptosis. It is generally accepted that one mechanism by which SRF regulates these diverse functions is through pathway-specific cofactor interactions. A novel SRF cofactor, chromodomain helicase DNA binding protein 8 (CHD8), was isolated from a yeast two-hybrid screen using SRF as bait. CHD8 is highly expressed in adult smooth muscle tissues. Coimmunoprecipitation assays from A10 smooth muscle cells demonstrated binding of endogenous SRF and CHD8. Data from GST-pulldown assays indicate that the NH(2)-terminus of CHD8 can interact directly with the MADS domain of SRF. Adenoviral-mediated knockdown of CHD8 in smooth muscle cells resulted in attenuated expression of SRF-dependent, smooth muscle-specific genes. Knockdown of CHD8, SRF, or CTCF, a previously described binding partner of CHD8, in A10 VSMCs also resulted in a marked induction of apoptosis. Mechanistically, apoptosis induced by CHD8 knockdown was accompanied by attenuated expression of the anti-apoptotic proteins, Birc5, and CARD10, whereas SRF knockdown attenuated expression of CARD10 and Mcl-1, but not Birc5, and CTCF knockdown attenuated expression of Birc5. These data suggest that CHD8 plays a dual role in smooth muscle cells modulating SRF activity toward differentiation genes and promoting cell survival through interactions with both SRF and CTCF to regulate expression of Birc5 and CARD10.
Asunto(s)
Apoptosis/fisiología , Proteínas de Unión al ADN/metabolismo , Miocitos del Músculo Liso/fisiología , Factor de Respuesta Sérica/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Factor de Unión a CCCTC , Células Cultivadas , Proteínas de Unión al ADN/genética , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Miocitos del Músculo Liso/citología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor de Respuesta Sérica/genética , Survivin , Distribución Tisular , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND AND AIMS: The transcription factors FOXF1 and FOXF2 have been implicated in the development of the gastrointestinal tract but their role in adults or in gastrointestinal diseases is poorly understood. We have recently shown that expression of serum response factor (SRF), a transcription factor whose activity is modulated by FOXF proteins, is decreased in the stomach muscularis of patients with gastroparesis. The aim of the current study was to determine whether FOXF expression is decreased in gastroparesis patients and whether loss of FOXF1 and/or FOXF2 from adult smooth muscle is sufficient to impair gastric emptying in mice. METHODS: Full-thickness stomach biopsy samples were collected from control subjects and from patients with gastroparesis. mRNA was isolated from the muscularis externa, and FOXF mRNA expression levels were determined by quantitative reverse transcriptase (RT)-PCR. Foxf1 and Foxf2 were knocked out together and separately from smooth muscle cells in adult mice, and the subsequent effect on liquid gastric emptying and contractile protein expression was determined. KEY RESULTS: Expression of FOXF1 and FOXF2 is decreased in smooth muscle tissue from gastroparesis patients. Knockout of Foxf1 and Foxf2 together, but not alone, from mouse smooth muscle resulted in delayed liquid gastric emptying. Foxf1/2 double knockout mice had decreased expression of smooth muscle contractile proteins, SRF, and myocardin in stomach muscularis. CONCLUSIONS AND INFERENCES: Our findings suggest that decreased expression of FOXF1 and FOXF2 may be contributing to the impaired gastric emptying seen in gastroparesis patients.
Asunto(s)
Factores de Transcripción Forkhead/genética , Gastroparesia/genética , Adulto , Animales , Biopsia , Complicaciones de la Diabetes/genética , Femenino , Vaciamiento Gástrico , Gastroparesia/patología , Regulación de la Expresión Génica , Humanos , Masculino , Ratones Noqueados , Persona de Mediana Edad , Miocitos del Músculo Liso/metabolismo , Estómago/patologíaRESUMEN
BACKGROUND: Gastric emptying is impaired in patients with gastroparesis whereas it is either unchanged or accelerated in obese individuals. The goal of the current study was to identify changes in gene expression in the stomach muscularis that may be contributing to altered gastric motility in idiopathic gastroparesis and obesity. METHODS: Quantitative real time RT-PCR and whole transcriptome sequencing were used to compare the transcriptomes of lean individuals, obese individuals and either lean or obese individuals with idiopathic gastroparesis. RESULTS: Obesity leads to an increase in mRNAs associated with muscle contractility whereas idiopathic gastroparesis leads to a decrease in mRNAs associated with PDGF BB signaling. Both obesity and idiopathic gastroparesis were also associated with similar alterations in pathways associated with inflammation. CONCLUSIONS: Our findings show that obesity and idiopathic gastroparesis result in overlapping but distinct changes in the gastric muscularis transcriptome. Increased expression of mRNAs encoding smooth muscle contractile proteins may be contributing to the increased gastric motility observed in obese subjects, whereas decreased PDGF BB signaling may be contributing to the impaired motility seen in subjects with idiopathic gastroparesis.
Asunto(s)
Gastroparesia/complicaciones , Gastroparesia/genética , Perfilación de la Expresión Génica , Músculo Liso/metabolismo , Estómago/fisiopatología , Índice de Masa Corporal , Fibroblastos/metabolismo , Fibroblastos/patología , Gastroparesia/metabolismo , Gastroparesia/fisiopatología , Humanos , Contracción Muscular , Músculo Liso/fisiopatología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de SeñalRESUMEN
Mechanical stimulation is a key regulator of bone mass, maintenance, and turnover. Wnt signaling is a key regulator of mechanotransduction in bone, but the role of ß-catenin-an intracellular signaling node in the canonical Wnt pathway-in disuse mechanotransduction is not defined. Using the ß-catenin exon 3 flox (constitutively active [CA]) mouse model, in conjunction with a tamoxifen-inducible, osteocyte-selective Cre driver, we evaluated the effects of degradation-resistant ß-catenin on bone properties during disuse. We hypothesized that if ß-catenin plays an important role in Wnt-mediated osteoprotection, then artificial stabilization of ß-catenin in osteocytes would protect the limbs from disuse-induced bone wasting. Two disuse models were tested: tail suspension, which models fluid shift, and botulinum-toxin (botox)-induced muscle paralysis, which models loss of muscle force. Tail suspension was associated with a significant loss of tibial bone mass and density, reduced architectural properties, and decreased bone formation indices in uninduced (control) mice, as assessed by dual-energy X-ray absorptiometry (DXA), micro-computed tomography (µCT), and histomorphometry. Activation of the ßcatCA allele in tail-suspended mice resulted in little to no change in those properties; ie, these mice were protected from bone loss. Similar protective effects were observed among botox-treated mice when the ßcatCA was activated. RNAseq analysis of altered gene regulation in tail-suspended mice yielded 35 genes, including Wnt11, Gli1, Nell1, Gdf5, and Pgf, which were significantly differentially regulated between tail-suspended ß-catenin stabilized mice and tail-suspended nonstabilized mice. Our findings indicate that selectively targeting/blocking of ß-catenin degradation in bone cells could have therapeutic implications in mechanically induced bone disease. © 2019 American Society for Bone and Mineral Research.
Asunto(s)
Mecanotransducción Celular , Osteocitos/metabolismo , Osteogénesis , Tibia/metabolismo , beta Catenina/metabolismo , Animales , Densidad Ósea , Ratones , Ratones Transgénicos , Osteocitos/patología , Tibia/diagnóstico por imagen , Tibia/patología , Microtomografía por Rayos X , beta Catenina/genéticaRESUMEN
Wnt signaling plays a key role in regulating bone remodeling. In vitro studies suggest that sclerostin's inhibitory action on Lrp5 is facilitated by the membrane-associated receptor Lrp4. We generated an Lrp4 R1170W knockin mouse model (Lrp4KI), based on a published mutation in patients with high bone mass (HBM). Lrp4KI mice have an HBM phenotype (assessed radiographically), including increased bone strength and formation. Overexpression of a Sost transgene had osteopenic effects in Lrp4-WT but not Lrp4KI mice. Conversely, sclerostin inhibition had blunted osteoanabolic effects in Lrp4KI mice. In a disuse-induced bone wasting model, Lrp4KI mice exhibit significantly less bone loss than wild-type (WT) mice. In summary, mice harboring the Lrp4-R1170W missense mutation recapitulate the human HBM phenotype, are less sensitive to altered sclerostin levels, and are protected from disuse-induced bone loss. Lrp4 is an attractive target for pharmacological targeting aimed at increasing bone mass and preventing bone loss due to disuse.
RESUMEN
A novel approach with chimeric SM22alpha/telokin promoters was used to identify gene regulatory modules that are required for regulating the expression of genes in distinct smooth muscle tissues. Conventional deletion or mutation analysis of promoters does not readily distinguish regulatory elements that are required for basal gene expression from those required for expression in specific smooth muscle tissues. In the present study, the mouse telokin gene was isolated, and a 370-bp (-190 to 180) minimal promoter was identified that directs visceral smooth muscle-specific expression in vivo in transgenic mice. The visceral smooth muscle-specific expression of the telokin promoter transgene is in marked contrast to the reported arterial smooth muscle-specific expression of a 536-bp minimal SM22alpha (-475 to 61) promoter transgene. To begin to identify regulatory elements that are responsible for the distinct tissue-specific expression of these promoters, a chimeric promoter in which a 172-bp SM22alpha gene fragment (-288 to -116) was fused to the minimal telokin promoter was generated and characterized. The -288 to -116 SM22alpha gene fragment significantly increased telokin promoter activity in vascular smooth muscle cells in vitro and in vivo. Conversely, a fragment of the telokin promoter (-94 to -49) increased the activity of the SM22alpha promoter in visceral smooth muscle cells of the bladder. Together, these data demonstrate that both vascular- and visceral smooth muscle-specific regulatory modules direct gene expression in subsets of smooth muscle tissues.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Secuencia Rica en At/fisiología , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Marcación de Gen , Genes Reporteros , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Músculo Liso/citología , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Quinasa de Cadena Ligera de Miosina , Especificidad de Órganos , Fragmentos de Péptidos , Péptidos , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ADN , Transfección , Transgenes , Vejiga Urinaria/metabolismo , Vísceras/metabolismoRESUMEN
BACKGROUND: The origins of neointimal smooth muscle cells that arise following vascular injury remains controversial. Studies have suggested that these cells may arise from previously differentiated medial vascular smooth muscle cells, resident stem cells or blood born progenitors. In the current study we examined the contribution of the previously differentiated vascular smooth muscle cells to the neointima that forms following carotid artery ligation. METHODS: We utilized transgenic mice harboring a cre recombinase-dependent reporter gene (mTmG). These mice express membrane targeted tandem dimer Tomato (mTomato) prior to cre-mediated excision and membrane targeted EGFP (mEGFP) following excision. The mTmG mice were crossed with transgenic mice expressing either smooth muscle myosin heavy chain (Myh11) or smooth muscle α-actin (Acta2) driven tamoxifen regulated cre recombinase. Following treatment of adult mice with tamoxifen these mice express mEGFP exclusively in differentiated smooth muscle cells. Subsequently vascular injury was induced in the mice by carotid artery ligation and the contribution of mEGFP positive cells to the neointima determined. RESULTS: Analysis of the cellular composition of the neointima that forms following injury revealed that mEGFP positive cells derived from either Mhy11 or Acta2 tagged medial vascular smooth muscle cells contribute to the majority of neointima formation (79 ± 17% and 81 ± 12%, respectively). CONCLUSION: These data demonstrate that the majority of the neointima that forms following carotid ligation is derived from previously differentiated medial vascular smooth muscle cells.
RESUMEN
Plasma membrane cholesterol accumulation has been implicated in cellular insulin resistance. Given the role of the hexosamine biosynthesis pathway (HBP) as a sensor of nutrient excess, coupled to its involvement in the development of insulin resistance, we delineated whether excess glucose flux through this pathway provokes a cholesterolgenic response induced by hyperinsulinemia. Exposing 3T3-L1 adipocytes to physiologically relevant doses of hyperinsulinemia (250pM-5000pM) induced a dose-dependent gain in the mRNA/protein levels of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR). These elevations were associated with elevated plasma membrane cholesterol. Mechanistically, hyperinsulinemia increased glucose flux through the HBP and O-linked ß-N-acetylglucosamine (O-GlcNAc) modification of specificity protein 1 (Sp1), known to activate cholesterolgenic gene products such as the sterol response element-binding protein (SREBP1) and HMGR. Chromatin immunoprecipitation demonstrated that increased O-GlcNAc modification of Sp1 resulted in a higher binding affinity of Sp1 to the promoter regions of SREBP1 and HMGR. Luciferase assays confirmed that HMGR promoter activity was elevated under these conditions and that inhibition of the HBP with 6-diazo-5-oxo-l-norleucine (DON) prevented hyperinsulinemia-induced activation of the HMGR promoter. In addition, both DON and the Sp1 DNA-binding inhibitor mithramycin prevented the hyperinsulinemia-induced increases in HMGR mRNA/protein and plasma membrane cholesterol. In these mithramycin-treated cells, both cortical filamentous actin structure and insulin-stimulated glucose transport were restored. Together, these data suggest a novel mechanism whereby increased HBP activity increases Sp1 transcriptional activation of a cholesterolgenic program, thereby elevating plasma membrane cholesterol and compromising cytoskeletal structure essential for insulin action.
Asunto(s)
Colesterol/biosíntesis , Hexosaminas/biosíntesis , Insulina/farmacología , Células 3T3-L1 , Animales , Vías Biosintéticas/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , ADN/metabolismo , Diazooxonorleucina/farmacología , Glucosa/metabolismo , Glicosilación/efectos de los fármacos , Humanos , Hidroximetilglutaril-CoA Reductasas/genética , Hiperinsulinismo/fisiopatología , Resistencia a la Insulina , Ratones , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Transporte de Proteínas/efectos de los fármacos , Factor de Transcripción Sp1/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genéticaRESUMEN
Mouse telokin and SM22alpha promoters have previously been shown to direct smooth muscle cell-specific expression of transgenes in vivo in adult mice. However, the activity of these promoters is highly dependent on the integration site of the transgene. In the current study, we found that the ectopic expression of telokin promoter transgenes could be abolished by flanking the transgene with insulator elements from the H19 gene. However, the insulator elements did not increase the proportion of mouse lines that exhibited consistent, detectable levels of transgene expression. In contrast, when transgenes were targeted to the hprt locus, both telokin and SM22alpha promoters resulted in reproducible patterns and levels of transgene expression in all lines of mice examined. Telokin promoter transgene expression was restricted to smooth muscle tissues in adult and embryonic mice. As reported previously, SM22alpha transgenes were expressed at high levels specifically in arterial smooth muscle cells; however, in contrast to randomly integrated transgenes, the hprt-targeted SM22alpha transgenes were also expressed at high levels in smooth muscle cells in veins, bladder, and gallbladder. Using hprt-targeted transgenes, we further analyzed elements within the telokin promoter required for tissue specific activity in vivo. Analysis of these transgenes revealed that the CArG element in the telokin promoter is required for promoter activity in all tissues and that the CArG element and adjacent AT-rich region are sufficient to drive transgene expression in bladder but not intestinal smooth muscle cells.
Asunto(s)
Marcación de Gen/métodos , Hipoxantina Fosforribosiltransferasa/genética , Ratones Transgénicos/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Músculo Liso/fisiología , Quinasa de Cadena Ligera de Miosina/metabolismo , Péptidos/metabolismo , Regiones Promotoras Genéticas/genética , Animales , Hipoxantina Fosforribosiltransferasa/metabolismo , Ratones , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Quinasa de Cadena Ligera de Miosina/genética , Especificidad de Órganos/genética , Fragmentos de Péptidos , Péptidos/genética , Distribución Tisular/genética , Transgenes/genéticaRESUMEN
The 130-kDa smooth muscle myosin light chain kinase (smMLCK) is a Ca2+/CaM-regulated enzyme that plays a pivotal role in the initiation of smooth muscle contraction and regulation of cellular migration and division. Despite the critical importance of smMLCK in these processes, little is known about the mechanisms regulating its expression. In this study, we have identified the proximal promoter of smMLCK within an intron of the mouse mylk gene. The mylk gene encodes at least two isoforms of MLCK (130 and 220 kDa) and telokin. Luciferase reporter gene assays demonstrated that a 282-bp fragment (-167 to +115) of the smMLCK promoter was sufficient for maximum activity in A10 smooth muscle cells and 10T1/2 fibroblasts. Deletion of the 16 bp between -167 and -151, which included a CArG box, resulted in a nearly complete loss of promoter activity. Gel mobility shift assays and chromatin immunoprecipitation assays demonstrated that serum response factor (SRF) binds to this CArG box both in vitro and in vivo. SRF knockdown by short hairpin RNA decreased endogenous smMLCK expression in A10 cells. Although the SRF coactivator myocardin induced smMLCK expression in 10T1/2 cells, myocardin activated the promoter only two- to fourfold in reporter gene assays. Addition of either intron 1 or 6 kb of the 5' upstream sequence did not lead to any further activation of the promoter by myocardin. The proximal smMLCK promoter also contains a consensus GATA-binding site that bound GATA-6. GATA-6 binding to this site decreased endogenous smMLCK expression, inhibited promoter activity in smooth muscle cells, and blocked the ability of myocardin to induce smMLCK expression. Altogether, these data suggest that SRF and SRF-associated factors play a key role in regulating the expression of smMLCK.
Asunto(s)
Proteínas de Unión al ADN/genética , Quinasa de Cadena Ligera de Miosina/genética , Regiones Promotoras Genéticas , Miosinas del Músculo Liso/genética , Transcripción Genética , Animales , Secuencia de Bases , Western Blotting , Inmunoprecipitación de Cromatina , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Factor de Transcripción GATA6/genética , Factor de Transcripción GATA6/metabolismo , Regulación de la Expresión Génica , Ratones , Datos de Secuencia Molecular , Músculo Liso/fisiología , Proteínas Nucleares/metabolismo , Reacción en Cadena de la Polimerasa , Factor de Respuesta Sérica/genética , Factor de Respuesta Sérica/metabolismo , Transactivadores/metabolismoRESUMEN
Transcription of the telokin gene is restricted to smooth muscle cells throughout development, making this gene an excellent model for unraveling the mechanisms that regulate gene expression in smooth muscle tissues. To identify proteins that bind to the telokin promoter, the AT-rich/CArG core of the promoter was used as a probe to perform a Southwestern screen of a mouse bladder cDNA library. Four clones corresponding to two distinct isoforms of mouse thyrotroph embryonic factor (TEFalpha and TEFbeta) were identified from this screen. The two TEF isoforms differ from each other at their amino termini and result from alternative promoter usage. An RNase protection assay showed that both TEF isoforms are expressed at high levels in mouse lung, bladder, kidney, gut, and brain. Gel mobility shift assays demonstrated that purified TEF protein can specifically bind to an AT-rich region within the core of the telokin promoter. Furthermore, when overexpressed in 10T1/2 cells, TEF significantly increased the activity of a telokin promoter-reporter gene; this activation was further augmented by elevated intracellular calcium levels. In contrast, overexpression of TEF had no effect on reporter genes driven by SM22alpha, smooth muscle alpha-actin, or smooth muscle myosin heavy chain promoters. Consistent with these results, overexpression of TEFalpha and TEFbeta in A10 cells, using adenoviral vectors, increased expression of endogenous telokin without altering expression of myosin light chain 20, SM22alpha, smooth muscle alpha-actin, or calponin. These findings suggest that TEF factors contribute to the activation of the telokin promoter in smooth muscle cells in a calcium-dependent manner. These data also suggest that distinct transcription factors are required to control the expression of different smooth muscle genes in a single tissue.