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1.
Nat Commun ; 11(1): 3501, 2020 07 09.
Article in English | MEDLINE | ID: mdl-32647108

ABSTRACT

While most monogenic diseases are caused by loss or reduction of protein function, the need for technologies that can selectively increase levels of protein in native tissues remains. Here we demonstrate that antisense-mediated modulation of pre-mRNA splicing can increase endogenous expression of full-length protein by preventing naturally occurring non-productive alternative splicing and promoting generation of productive mRNA. Bioinformatics analysis of RNA sequencing data identifies non-productive splicing events in 7,757 protein-coding human genes, of which 1,246 are disease-associated. Antisense oligonucleotides targeting multiple types of non-productive splicing events lead to increases in productive mRNA and protein in a dose-dependent manner in vitro. Moreover, intracerebroventricular injection of two antisense oligonucleotides in wild-type mice leads to a dose-dependent increase in productive mRNA and protein in the brain. The targeting of natural non-productive alternative splicing to upregulate expression from wild-type or hypomorphic alleles provides a unique approach to treating genetic diseases.


Subject(s)
Alternative Splicing , Gene Expression Regulation , Oligonucleotides, Antisense/pharmacology , Alleles , Animals , Animals, Newborn , Brain/metabolism , Computational Biology , Exons , Female , Gene Expression/drug effects , HEK293 Cells , Humans , Introns , Male , Mice , Mice, Inbred C57BL , RNA, Messenger/metabolism , Transcriptional Activation/drug effects , Up-Regulation
2.
Mol Cancer Ther ; 16(8): 1680-1692, 2017 08.
Article in English | MEDLINE | ID: mdl-28428441

ABSTRACT

Increased glucocorticoid receptor (GR) expression and activity following androgen blockade can contribute to castration-resistant prostate cancer (CRPC) progression. Therefore, we hypothesized that GR antagonism will have therapeutic benefit in CRPC. However, the FDA-approved nonselective, steroidal GR antagonist, mifepristone, lacks GR specificity, reducing its therapeutic potential. Here, we report that two novel nonsteroidal and highly selective GR modulators (SGRM), CORT118335 and CORT108297, have the ability to block GR activity in prostate cancer and slow CRPC progression. In contrast to mifepristone, these novel SGRMs did not affect androgen receptor (AR) signaling, but potently inhibited GR transcriptional activity. Importantly, SGRMs decreased GR-mediated tumor cell viability following AR blockade. In vivo, SGRMs significantly inhibited CRPC progression in high GR-expressing, but not in low GR-expressing xenograft models. Transcriptome analysis following AR blockade and GR activation revealed that these SGRMs block GR-mediated proliferative gene expression pathways. Furthermore, GR-regulated proliferation-associated genes AKAP12, FKBP5, SGK1, CEBPD, and ZBTB16 are inhibited by CORT108297 treatment in vivo Together, these data suggest that GR-selective nonsteroidal SGRMs potently inhibit GR activity and prostate cancer growth despite AR pathway inhibition, demonstrating the therapeutic potential of SGRMs in GR-expressing CRPC. Mol Cancer Ther; 16(8); 1680-92. ©2017 AACR.


Subject(s)
Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/pathology , Receptors, Glucocorticoid/metabolism , Small Molecule Libraries/therapeutic use , Animals , Benzamides , Cell Line, Tumor , Cell Proliferation , Cell Survival , Gene Expression Regulation, Neoplastic , Humans , Male , Mice, Nude , Nitriles , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Phenylthiohydantoin/therapeutic use , Prostatic Neoplasms, Castration-Resistant/genetics , Receptors, Androgen/metabolism , Signal Transduction , Small Molecule Libraries/pharmacology , Transcription, Genetic
3.
Oncotarget ; 7(18): 26259-74, 2016 May 03.
Article in English | MEDLINE | ID: mdl-27036029

ABSTRACT

Enzalutamide (MDV3100) is a second generation Androgen Receptor (AR) antagonist with proven efficacy in the treatment of castration resistant prostate cancer (CRPC). The majority of treated patients, however, develop resistance and disease progression and there is a critical need to identify novel targetable pathways mediating resistance. The purpose of this study was to develop and extensively characterize a series of enzalutamide-resistant prostate cancer cell lines. Four genetically distinct AR-positive and AR-pathway dependent prostate cancer cell lines (CWR-R1, LAPC-4, LNCaP, VCaP) were made resistant to enzalutamide by long-term culture (> 6 months) in enzalutamide. Extensive characterization of these lines documented divergent in vitro growth characteristics and AR pathway modulation. Enzalutamide-resistant LNCaP and CWR-R1 cells, but not LAPC-4 and VCAP cells, demonstrated increased castration-resistant and metastatic growth in vivo. Global gene expression analyses between short-term enzalutamide treated vs. enzalutamide-resistant cells identified both AR pathway and non-AR pathway associated changes that were restored upon acquisition of enzalutamide resistance. Further analyses revealed very few common gene expression changes between the four resistant cell lines. Thus, while AR-mediated pathways contribute in part to enzalutamide resistance, an unbiased approach across several cell lines demonstrates a greater contribution toward resistance via pleiotropic, non-AR mediated mechanisms.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/physiology , Phenylthiohydantoin/analogs & derivatives , Prostatic Neoplasms, Castration-Resistant/pathology , Receptors, Androgen/metabolism , Androgen Receptor Antagonists/pharmacology , Animals , Benzamides , Cell Line, Tumor , Humans , Male , Mice , Nitriles , Phenylthiohydantoin/pharmacology , Xenograft Model Antitumor Assays
4.
Sci Transl Med ; 7(305): 305ps19, 2015 Sep 16.
Article in English | MEDLINE | ID: mdl-26378243

ABSTRACT

Steroid receptors for androgens and estrogens have essential roles in prostate and breast cancers. Recently, glucocorticoid receptor (GR) activity has also been proposed as having an important role in these cancers. Underscoring the cooperative nature of nuclear receptor activity, data now suggest that GR function in prostate and breast cancers is dependent on the tumor's concomitant androgen or estrogen receptor activity.


Subject(s)
Breast Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/metabolism , Receptors, Glucocorticoid/metabolism , Animals , Breast Neoplasms/drug therapy , Cell Survival , Estrogen Receptor alpha/metabolism , Female , Humans , Male , Prognosis , Prostatic Neoplasms/drug therapy , Receptors, Androgen/metabolism , Signal Transduction , Translational Research, Biomedical
5.
Respir Res ; 16: 45, 2015 Mar 27.
Article in English | MEDLINE | ID: mdl-25885656

ABSTRACT

BACKGROUND: Fibrosing disorders of the lung, such as idiopathic pulmonary fibrosis, are characterized by progressive extracellular matrix accumulation that is driven by myofibroblasts. The transcription factor megakaryoblastic leukemia-1 (MKL1) mediates myofibroblast differentiation in response to several profibrotic stimuli, but the role it plays in mediating pulmonary fibrosis has not been fully elucidated. In this study, we utilized mice that had a germline deletion of MKL1 (MKL1 (-,-)) to determine the role that MKL1 plays in the development of bleomycin-induced pulmonary fibrosis. METHODS: Bleomycin or normal saline were intratracheally delivered to 9 to 12 week old female MKL1 (+,+) and MKL1 (-,-) mice. Mice were assessed for weight loss and survival to 28 days. Inflammatory responses were assessed through bronchoalveolar lavage at days 3 and 7 post-treatment. The development of pulmonary fibrosis was characterized using hydroxyproline assay and histological staining. MKL1 (+,+) and MKL1 (-,-) mouse lung fibroblasts were isolated to compare morphologic, gene expression and functional differences. RESULTS: MKL1 (-,-) mice demonstrated increased survival, attenuated weight loss, and decreased collagen accumulation compared to wild-type animals 28-days after intratracheal instillation of bleomycin. Histological analysis demonstrated decreased trichrome, smooth muscle α-actin, and fibronectin staining in MKL1(-,-) mice compared to MKL1 (+,+) controls. Differential cell counts from bronchoalveolar lavage demonstrated that there was attenuated neutrophilia 3 days after bleomycin administration, but no difference at day 7. Isolated mouse lung fibroblasts from MKL1 (-,-) mice had decreased contractility and deposited less fibronectin matrix compared to wild-type controls, suggesting a defect in key remodeling functions. CONCLUSIONS: Altogether, these data demonstrate that MKL1 plays a significant role in mediating the fibrotic response to bleomycin injury. Loss of MKL1 attenuated early neutrophil influx, as well as myofibroblast-mediated remodeling. Targeting MKL1 activity may therefore be a useful strategy in treating pulmonary fibrosis.


Subject(s)
Bleomycin , Fibroblasts/metabolism , Lung/metabolism , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/metabolism , Trans-Activators/metabolism , Animals , Bronchoalveolar Lavage Fluid/chemistry , Cell Shape , Cells, Cultured , Collagen/metabolism , Female , Fibroblasts/pathology , Fibronectins/metabolism , Genotype , Germ-Line Mutation , Inflammation Mediators/metabolism , Lung/pathology , Mice, Inbred C57BL , Mice, Knockout , Neutrophil Infiltration , Phenotype , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/prevention & control , Signal Transduction , Time Factors , Trans-Activators/deficiency , Trans-Activators/genetics
6.
J Transl Sci ; 1(1)2015 Jul.
Article in English | MEDLINE | ID: mdl-26753099

ABSTRACT

The function and clinical utility of stem cell markers in metastatic castration-resistant prostate cancer (mCRPC) remains unresolved, and their expression may confer important therapeutic opportunities for staging and therapy. In the adult human prostate, CD133 (PROM1) expression identifies infrequent prostate epithelial progenitor cells and putative cancer stem cells. Previous work demonstrated an association with CD133 and cancer cell proliferation using in vitro model systems. The primary objective here was to investigate the expression of CD133 in circulating tumor cells (CTCs) from patients with mCRPC and to test the hypothesis that patients with mCRPC had CD133-positive CTCs associated with increased cell proliferation, changes in the androgen receptor (AR) protein expression, or AR nuclear co-localization. We utilized ImageStreamX technology, which combines flow cytometry and fluorescence microscopy, to capture and analyze CD45-negative/EpCAM-positive CTCs for CD133, Ki-67, and AR. All patient samples (20/20) contained CD133-positive populations of CTCs, and on average 50.9 ± 28.2% (range of 18.2% to 100%) of CTCs were CD133-positive. CD133-positive CTCs have increased Ki-67 protein expression compared to CD133-negative CTCs, implying that CD133-positive CTCs may have greater proliferative potential when compared to their CD133-negative counterparts. CD133-positive and CD133-negative CTCs have similar levels of AR protein expression and cellular co-localization with nuclear markers, implying that CD133 expression is independent of AR pathway activity and an AR-independent marker of mCRPC proliferation. These studies demonstrate the presence of CD133-positive populations in CTCs from mCRPC with increased proliferative potential.

7.
Horm Cancer ; 5(2): 72-89, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24615402

ABSTRACT

Despite new treatments for castrate-resistant prostate cancer (CRPC), the prognosis of patients with CRPC remains bleak due to acquired resistance to androgen receptor (AR)-directed therapy. The glucocorticoid receptor (GR) and AR share several transcriptional targets, including the anti-apoptotic genes serum and glucocorticoid-regulated kinase 1 (SGK1) and Map kinase phosphatase 1 (MKP1)/dual specificity phosphatase 1 (DUSP1). Because GR expression increases in a subset of primary prostate cancer (PC) following androgen deprivation therapy, we sought to determine whether GR activation can contribute to resistance to AR-directed therapy. We studied CWR-22Rv1 and LAPC4 AR/GR-expressing PC cell lines following treatment with combinations of the androgen R1881, AR antagonist MDV3100, GR agonist dexamethasone, GR antagonists mifepristone and CORT 122928, or the SGK1 inhibitor GSK650394. Cell lines stably expressing GR (NR3C1)-targeted shRNA or ectopic SGK1-Flag were also studied in vivo. GR activation diminished the effects of the AR antagonist MDV3100 on tumor cell viability. In addition, GR activation increased prostate-specific antigen (PSA) secretion and induced SGKI and MKP1/DUSP gene expression. Glucocorticoid-mediated cell viability was diminished by a GR antagonist or by co-treatment with the SGK1 inhibitor GSK650394. In vivo, GR depletion delayed castrate-resistant tumor formation, while SGK1-Flag-overexpressing PC xenografts displayed accelerated castrate-resistant tumor initiation, supporting a role for SGK1 in GR-mediated CRPC progression. We studied several PC models before and following treatment with androgen blockade and found that increased GR expression and activity contributed to tumor-promoting PC cell viability. Increased GR-regulated SGK1 expression appears, at least in part, to mediate enhanced PC cell survival. Therefore, GR and/or SGK1 inhibition may be useful adjuncts to AR blockade for treating CRPC.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Drug Resistance, Neoplasm , Prostatic Neoplasms/drug therapy , Receptors, Androgen/metabolism , Receptors, Glucocorticoid/metabolism , Androgen Receptor Antagonists/administration & dosage , Androgen Receptor Antagonists/pharmacology , Animals , Benzamides , Benzoates/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Dexamethasone/administration & dosage , Dexamethasone/pharmacology , HEK293 Cells , Humans , Immediate-Early Proteins/antagonists & inhibitors , Immediate-Early Proteins/genetics , Immediate-Early Proteins/metabolism , Immunoblotting , Male , Metribolone/administration & dosage , Metribolone/pharmacology , Mice , Mice, Nude , Microscopy, Fluorescence , Mifepristone/administration & dosage , Mifepristone/pharmacology , Nitriles , Phenylthiohydantoin/administration & dosage , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA Interference , Receptors, Androgen/genetics , Receptors, Glucocorticoid/antagonists & inhibitors , Receptors, Glucocorticoid/genetics , Reverse Transcriptase Polymerase Chain Reaction , Xenograft Model Antitumor Assays
8.
J Biol Chem ; 289(11): 7505-13, 2014 Mar 14.
Article in English | MEDLINE | ID: mdl-24492608

ABSTRACT

Myofibroblast differentiation is a key process in the pathogenesis of fibrotic disease. We have shown previously that differentiation of myofibroblasts is regulated by microtubule polymerization state. In this work, we examined the potential antifibrotic effects of the antitussive drug, noscapine, recently found to bind microtubules and affect microtubule dynamics. Noscapine inhibited TGF-ß-induced differentiation of cultured human lung fibroblasts (HLFs). Therapeutic noscapine treatment resulted in a significant attenuation of pulmonary fibrosis in the bleomycin model of the disease. Noscapine did not affect gross microtubule content in HLFs, but inhibited TGF-ß-induced stress fiber formation and activation of serum response factor without affecting Smad signaling. Furthermore, noscapine stimulated a rapid and profound activation of protein kinase A (PKA), which mediated the antifibrotic effect of noscapine in HLFs, as assessed with the PKA inhibitor, PKI. In contrast, noscapine did not activate PKA in human bronchial or alveolar epithelial cells. Finally, activation of PKA and the antifibrotic effect of noscapine in HLFs were blocked by the EP2 prostaglandin E2 receptor antagonist, PF-04418948, but not by the antagonists of EP4, prostaglandin D2, or prostacyclin receptors. Together, we demonstrate for the first time the antifibrotic effect of noscapine in vitro and in vivo, and we describe a novel mechanism of noscapine action through EP2 prostaglandin E2 receptor-mediated activation of PKA in pulmonary fibroblasts.


Subject(s)
Antitussive Agents/pharmacology , Cyclic AMP-Dependent Protein Kinases/metabolism , Noscapine/pharmacology , Pulmonary Fibrosis/drug therapy , Receptors, Prostaglandin E/metabolism , Animals , Antineoplastic Agents/pharmacology , Bleomycin/pharmacology , Cell Line, Tumor , DNA/metabolism , Fibroblasts/metabolism , Fibrosis , Gene Expression Regulation , Humans , Hydroxyproline/chemistry , Luciferases/metabolism , Lung/metabolism , Mice , Mice, Inbred C57BL , Microtubules/metabolism , Myofibroblasts/cytology , Neoplasms/metabolism , Pulmonary Fibrosis/metabolism , Pulmonary Fibrosis/pathology
9.
Am J Physiol Lung Cell Mol Physiol ; 305(10): L693-701, 2013 Nov 15.
Article in English | MEDLINE | ID: mdl-24077945

ABSTRACT

T cell migration toward sites of antigen exposure is mediated by G protein signaling and is a key function in the development of immune responses. Regulators of G protein signaling (RGS) proteins modulate G protein signaling; however, their role in the regulation of adaptive immune responses has not been thoroughly explored. Herein we demonstrated abundant expression of the Gi/Gq-specific RGS3 in activated T cells, and that diminished RGS3 expression in a T cell thymoma increased cytokine-induced migration. To examine the role of endogenous RGS3 in vivo, mice deficient in the RGS domain (RGS3(ΔRGS)) were generated and tested in an experimental model of asthma. Compared with littermate controls, the inflammation in the RGS3(ΔRGS) mice was characterized by increased T cell numbers and the striking development of perivascular lymphoid structures. Surprisingly, while innate inflammatory cells were also increased in the lungs of RGS3(ΔRGS) mice, eosinophil numbers and Th2 cytokine production were equivalent to control mice. In contrast, T cell numbers in the draining lymph nodes (dLN) were reduced in the RGS3(ΔRGS), demonstrating a redistribution of T cells from the dLN to the lungs via increased RGS3(ΔRGS) T cell migration. Together these novel findings show a nonredundant role for endogenous RGS3 in controlling T cell migration in vitro and in an in vivo model of inflammation.


Subject(s)
Cell Movement , Inflammation/etiology , RGS Proteins/physiology , Respiratory Mucosa/immunology , T-Lymphocytes/immunology , Th2 Cells/immunology , Animals , Apoptosis , Blotting, Western , Cell Differentiation , Cell Proliferation , Disease Models, Animal , Female , Flow Cytometry , Inflammation/metabolism , Inflammation/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Pyroglyphidae/pathogenicity , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Th2 Cells/metabolism , Th2 Cells/pathology
10.
J Biol Chem ; 288(22): 15466-73, 2013 May 31.
Article in English | MEDLINE | ID: mdl-23580645

ABSTRACT

Myofibroblast differentiation plays a critical role in wound healing and in the pathogenesis of fibrosis. We have previously shown that myofibroblast differentiation is mediated by the activity of serum response factor (SRF), which is tightly controlled by the actin polymerization state. In this study, we investigated the role of the microtubule cytoskeleton in modulating myofibroblast phenotype. Treatment of human lung fibroblasts with the microtubule-destabilizing agent, colchicine, resulted in a formation of numerous stress fibers and expression of myofibroblast differentiation marker proteins. These effects of colchicine were independent of Smad signaling but were mediated by Rho signaling and SRF, as they were attenuated by the Rho kinase inhibitor, Y27632, or by the SRF inhibitor, CCG-1423. TGF-ß-induced myofibroblast differentiation was not accompanied by gross changes in the microtubule polymerization state. However, microtubule stabilization by paclitaxel attenuated TGF-ß-induced myofibroblast differentiation. Paclitaxel had no effect on TGF-ß-induced Smad activation and Smad-dependent gene transcription but inhibited actin polymerization, nuclear accumulation of megakaryoblastic leukemia-1 protein, and SRF activation. The microtubule-associated formin, mDIA2, localized to actin stress fibers upon treatment with TGF-ß, and paclitaxel prevented this localization. Treatment with the formin inhibitor, SMI formin homology 2 domain, inhibited stress fiber formation and myofibroblast differentiation induced by TGF-ß, without affecting Smad-phosphorylation or microtubule polymerization. Together, these data suggest that (a) TGF-ß promotes association of mDia2 with actin stress fibers, which further drives stress fiber formation and myofibroblast differentiation, and (b) microtubule polymerization state controls myofibroblast differentiation through the regulation of mDia2 localization.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Differentiation/physiology , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Myofibroblasts/metabolism , Amides/pharmacology , Anilides/pharmacology , Benzamides/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Colchicine/pharmacology , DNA-Binding Proteins/metabolism , Enzyme Inhibitors/pharmacology , Formins , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Humans , Myofibroblasts/cytology , Oncogene Proteins, Fusion/metabolism , Paclitaxel/pharmacology , Protein Transport/drug effects , Protein Transport/physiology , Pyridines/pharmacology , Serum Response Factor/metabolism , Smad Proteins/genetics , Smad Proteins/metabolism , Stress Fibers/metabolism , Trans-Activators , Transforming Growth Factor beta/metabolism , Tubulin Modulators/pharmacology
11.
Am J Physiol Lung Cell Mol Physiol ; 304(11): L757-64, 2013 Jun 01.
Article in English | MEDLINE | ID: mdl-23585227

ABSTRACT

Myofibroblast differentiation induced by transforming growth factor-ß (TGF-ß) is characterized by the expression of smooth muscle α-actin (SMA) and extracellular matrix proteins. We and others have previously shown that these changes are regulated by protein kinase A (PKA). Adrenomedullin (ADM) is a vasodilator peptide that activates cAMP/PKA signaling through the calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMP). In this study, we found that recombinant ADM had little effect on cAMP/PKA in quiescent human pulmonary fibroblasts, whereas it induced a profound activation of cAMP/PKA signaling in differentiated (by TGF-ß) myofibroblasts. In contrast, the prostacyclin agonist iloprost was equally effective at activating PKA in both quiescent fibroblasts and differentiated myofibroblasts. TGF-ß stimulated a profound expression of CRLR with a time course that mirrored the increased PKA responses to ADM. The TGF-ß receptor kinase inhibitor SB431542 abolished expression of CRLR and attenuated the PKA responses of cells to ADM but not to iloprost. CRLR expression was also dramatically increased in lungs from bleomycin-treated mice. Functionally, ADM did not affect initial differentiation of quiescent fibroblasts in response to TGF-ß but significantly attenuated the expression of SMA, collagen-1, and fibronectin in pre-differentiated myofibroblasts, which was accompanied by decreased contractility of myofibroblasts. Finally, sensitization of ADM signaling by transgenic overexpression of RAMP2 in myofibroblasts resulted in enhanced survival and reduced pulmonary fibrosis in the bleomycin model of the disease. In conclusion, differentiated pulmonary myofibroblasts gain responsiveness to ADM via increased CRLR expression, suggesting the possibility of using ADM for targeting pathological myofibroblasts without affecting normal fibroblasts.


Subject(s)
Adrenomedullin/pharmacology , Cell Differentiation/drug effects , Myofibroblasts/cytology , Pulmonary Fibrosis/physiopathology , Actins/metabolism , Adrenomedullin/therapeutic use , Animals , Bleomycin , Calcitonin Receptor-Like Protein/biosynthesis , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Humans , Iloprost/pharmacology , Mice , Myofibroblasts/drug effects , Myofibroblasts/physiology , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/drug therapy , Receptor Activity-Modifying Protein 2/genetics , Signal Transduction/drug effects , Transforming Growth Factor beta/pharmacology
12.
Am J Physiol Heart Circ Physiol ; 303(1): H19-35, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22542620

ABSTRACT

Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAP) for various Gα subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate the magnitude and duration of G-protein-coupled receptor signaling and are often referred to as fine tuners of G-protein signaling. Increasing evidence suggests that RGS proteins themselves are regulated through multiple mechanisms, which may provide an even finer tuning of G-protein signaling and crosstalk between G-protein-coupled receptors and other signaling pathways. This review summarizes the current data on the control of RGS function through regulated expression, intracellular localization, and covalent modification of RGS proteins, as related to cell function and the pathogenesis of diseases.


Subject(s)
GTP-Binding Proteins/physiology , RGS Proteins/physiology , Animals , Arginine/metabolism , Gene Expression Regulation/physiology , Humans , RGS Proteins/biosynthesis , RGS Proteins/genetics , Receptor Cross-Talk/physiology , Receptors, G-Protein-Coupled/physiology , Signal Transduction/genetics , Signal Transduction/physiology , Stress, Physiological , Sumoylation/physiology , Ubiquitination/physiology
13.
Am J Physiol Lung Cell Mol Physiol ; 301(5): L656-66, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21856814

ABSTRACT

Myofibroblast differentiation induced by transforming growth factor-ß (TGF-ß) and characterized by de novo expression of smooth muscle (SM)-specific proteins is a key process in wound healing and in the pathogenesis of fibrosis. We have previously shown that TGF-ß-induced expression and activation of serum response factor (SRF) is required for this process. In this study, we examined the signaling mechanism for SRF activation by TGF-ß as it relates to pulmonary myofibroblast differentiation. TGF-ß stimulated a profound, but delayed (18-24 h), activation of Rho kinase and formation of actin stress fibers, which paralleled SM α-actin expression. The translational inhibitor cycloheximide blocked these processes without affecting Smad-dependent gene transcription. Inhibition of Rho kinase by Y-27632 or depolymerization of actin by latrunculin B resulted in inhibition TGF-ß-induced SRF activation and SM α-actin expression, having no effect on Smad signaling. Conversely, stabilization of actin stress fibers by jasplakinolide was sufficient to drive these processes in the absence of TGF-ß. TGF-ß promoted a delayed nuclear accumulation of the SRF coactivator megakaryoblastic leukemia-1 (MKL1)/myocardin-related transcription factor-A, which was inhibited by latrunculin B. Furthermore, TGF-ß also induced MKL1 expression, which was inhibited by latrunculin B, by SRF inhibitor CCG-1423, or by SRF knockdown. Together, these data suggest a triphasic model for myofibroblast differentiation in response to TGF-ß that involves 1) initial Smad-dependent expression of intermediate signaling molecules driving Rho activation and stress fiber formation, 2) nuclear accumulation of MKL1 and activation of SRF as a result of actin polymerization, and 3) SRF-dependent expression of MKL1, driving further myofibroblast differentiation.


Subject(s)
Lung/metabolism , Muscle, Smooth/metabolism , Myofibroblasts/metabolism , Pulmonary Fibrosis , Signal Transduction , Stress Fibers/metabolism , Transforming Growth Factor beta1/pharmacology , Actins/genetics , Actins/metabolism , Adenoviridae , Blotting, Western , Cell Differentiation/drug effects , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fluorescent Antibody Technique , Gene Expression/drug effects , Genes, Reporter , Humans , Luciferases/analysis , Lung/drug effects , Lung/pathology , Lung/physiopathology , Muscle, Smooth/drug effects , Muscle, Smooth/pathology , Muscle, Smooth/physiopathology , Myofibroblasts/drug effects , Myofibroblasts/pathology , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Primary Cell Culture , Pulmonary Fibrosis/metabolism , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/physiopathology , Real-Time Polymerase Chain Reaction , Serum Response Factor/genetics , Serum Response Factor/metabolism , Signal Transduction/drug effects , Smad Proteins/genetics , Smad Proteins/metabolism , Stress Fibers/drug effects , Stress Fibers/pathology , Trans-Activators , Transduction, Genetic , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolism
14.
J Biol Chem ; 284(49): 33789-94, 2009 Dec 04.
Article in English | MEDLINE | ID: mdl-19776005

ABSTRACT

The contractile phenotype of smooth muscle (SM) cells is controlled by serum response factor (SRF), which drives the expression of SM-specific genes including SM alpha-actin, SM22, and others. Myocardin is a cardiac and SM-restricted coactivator of SRF that is necessary for SM gene transcription. Growth factors inducing proliferation of SM cells inhibit SM gene transcription, in a manner dependent on the activation of extracellular signal-regulated kinases ERK1/2. In this study, we found that ERK1/2 phosphorylates mouse myocardin (isoform B) at four sites (Ser(812), Ser(859), Ser(866), and Thr(893)), all of which are located within the transactivation domain of myocardin. The single mutation of each site either to alanine or to aspartate has no effect on the ability of myocardin to activate SRF. However, the phosphomimetic mutation of all four sites to aspartate (4xD) significantly impairs activation of SRF by myocardin, whereas the phosphodeficient mutation of all four sites to alanine (4xA) has no effect. This translates to a reduced ability of the 4xD (but not of 4xA) mutant of myocardin to stimulate expression of SM alpha-actin and SM22, as assessed by corresponding promoter, mRNA, or protein assays. Furthermore, we found that phosphorylation of myocardin at these sites impairs its interaction with acetyltransferase, cAMP response element-binding protein-binding protein, which is known to promote the transcriptional activity of myocardin. In conclusion, we describe a novel mode of modulation of SM gene transcription by ERK1/2 through a direct phosphorylation of myocardin.


Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation , Nuclear Proteins/physiology , Trans-Activators/physiology , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cricetulus , Cyclic AMP Response Element Modulator/metabolism , Enzyme Activation , Mice , Mitogen-Activated Protein Kinase 3/metabolism , Molecular Sequence Data , Muscle, Smooth/metabolism , Nuclear Proteins/metabolism , Phenotype , Phosphorylation , Sequence Homology, Amino Acid , Trans-Activators/metabolism
15.
J Neurosci ; 26(19): 5265-75, 2006 May 10.
Article in English | MEDLINE | ID: mdl-16687519

ABSTRACT

Planar cell polarity (PCP) is a process in which cells develop with uniform orientation within the plane of an epithelium. To begin to elucidate the mechanisms of PCP in vertebrates, the localization of the protein Vangl2 (Van Gogh-like) was determined during the development of the mammalian cochlea. Results indicate that Vangl2 becomes asymmetrically localized to specific cell-cell boundaries along the axis of polarization and that this asymmetry is lost in PCP mutants. In addition, PDZ2 (postsynaptic density/Discs large/zona occludens 1), PDZ3, and PDZ4 of the PCP protein Scrb1 (Scribble) are shown to bind to the C-terminal PDZ binding domain of Vangl2, suggesting that Scrb1 plays a direct role in asymmetric targeting of Vangl2. Finally, Fz3 (Frizzled), a newly demonstrated mediator of PCP, is also asymmetrically localized in a pattern that matches that of Vangl2. The presence and asymmetry of Fz3 at the membrane is shown to be dependent on Vangl2. This result suggests a role for Vangl2 in the targeting or anchoring of Fz3, a hypothesis strengthened by the existence of a physical interaction between the two proteins. Together, our data support the idea that protein asymmetry plays an important role in the development of PCP, but the colocalization and interaction of Fz3 and Vangl2 suggests that novel PCP mechanisms exist in vertebrates.


Subject(s)
Cochlea/cytology , Cochlea/metabolism , Frizzled Receptors/metabolism , Nerve Tissue Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Cell Polarity/physiology , Cells, Cultured , Mice , Tissue Distribution
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