Functional analysis of protein interactions using coupled bi-fluorescence complementation/GFP nanobody techniques.

Transcription factors (TFs) form homo- or hetero-dimeric DNA binding complexes along with associated co-regulators that can have transcriptional repressor or activator functions. Defining the specific composition of the complexes is therefore key to understanding their biological role. Here, we utilized bimolecular fluorescence complementation (BiFC) to visualize the formation of defined TF dimers and associated co-regulators derived from the activator protein-1 (AP-1) and myocyte enhancer factor 2 (MEF2) families. Firstly, BiFC signals were observed in cells co-expressing TFs tagged with complimentary combinations of the split fluorescent protein, demonstrating the engineered formation of defined dimer complexes. Next, we applied this approach and determined that defined AP-1 dimers localized at discrete sub-nuclear locations. Subsequently, a combination of BiFC coupled with GFP binding peptide (GBP)-nanotrap allowed observation of protein-protein interactions between a co-regulator, HDAC4, and defined BiFC-MEF2 engineered dimers. To determine transactivation properties of defined TF dimers in a cellular system, the Gal4-DNA binding domain fused to GBP was utilized to assess the transcriptional properties of the BiFC-TF dimers using a generically applicable Gal4/UAS luciferase reporter gene assay system. Here, we report efficacy of a BiFC/GBP-nanobody approach that allows engineering, visualization, and functional analysis of defined TF dimers.

Re-organization of nucleolar architecture in myogenic differentiation.

Myogenesis, the process of muscle differentiation, requires an extensive remodeling of the cellular transcriptome and proteome. Whereas the transcriptional program underpinning myogenesis is well characterized, the required adaptation in protein synthesis is incompletely understood. Enhanced protein synthesis necessitates ribosome biogenesis at the nucleolus. Nucleolar size and activity are inextricably linked with altered gene expression. Here, we report changes in nucleolar morphology and function during myogenic differentiation. Immunofluorescence analysis revealed alterations in nucleolar morphology that were dependent on the cellular state - proliferative or quiescent myogenic progenitors (myoblasts or reserve cells) contained multiple small nucleoli with a characteristic spherical shape, whereas multinucleated myotubes typically contained one large, often irregularly shaped nucleolus. These morphological alterations are consistent with changes to nucleolar phase separation properties. Re-organization of the nucleolar structure was correlated with enhanced rRNA production and protein translation. Inhibition of mTOR signaling with rapamycin perturbed nucleolar re-organization. Conversely, hyperactivated mTOR enhanced alterations in nucleolar morphology. These findings support the idea that there is an mTOR dependent re-organization of nucleolar structure during myogenesis, enhancing our understanding of myogenesis and possibly facilitating new approaches to therapeutic interventions in muscle pathologies.

TAZ exhibits phase separation properties and interacts with Smad7 and ß-catenin to repress skeletal myogenesis.

Hippo signaling in Drosophila and mammals is prominent in regulating cell proliferation, death and differentiation. Hippo signaling effectors (YAP and TAZ; also known as YAP1 and WWTR1, respectively) exhibit crosstalk with transforming growth factor-ß (TGF-ß)-Smad and Wnt/ß-catenin pathways. Previously, we implicated Smad7 and ß-catenin in mammalian myogenesis. Therefore, we assessed a potential role of TAZ on the Smad7-ß-catenin complex in muscle cells. Here, we document functional interactions between Smad7, TAZ and ß-catenin in mouse myogenic cells. Ectopic TAZ expression resulted in repression of the muscle-specific creatine kinase muscle (Ckm) gene promoter and its corresponding protein level. Depletion of endogenous TAZ enhanced Ckm promoter activation. Ectopic TAZ, while potently active on a TEAD reporter (HIP-HOP), repressed myogenin (Myog) and Myod1 enhancer regions and myogenin protein level. Additionally, a Wnt/ß-catenin readout (TOP flash) demonstrated TAZ-mediated inhibition of ß-catenin activity. In myoblasts, TAZ was predominantly localized in nuclear speckles, while in differentiation conditions TAZ was hyperphosphorylated at Ser89, leading to enhanced cytoplasmic sequestration. Finally, live-cell imaging indicated that TAZ exhibits properties of liquid-liquid phase separation (LLPS). These observations indicate that TAZ, as an effector of Hippo signaling, suppresses the myogenic differentiation machinery.

Public preferences for pharmacogenetic testing in the NHS: Embedding a discrete choice experiment within service design to better meet user needs.

AIMS: Genetic testing can be used to improve the safety and effectiveness of commonly prescribed medicines-a concept known as pharmacogenetics. This study aimed to quantify members of the UK public's preferences for a pharmacogenetic service to be delivered in primary care in the National Health Service. METHODS: Members of the UK population were surveyed via an online panel company. Respondents completed 1 of 2 survey versions, asking respondents to select their preferred pharmacogenetic testing service in the context of a presentation of low mood or pain. A conditional logit model was estimated, before the best functional form for the dataset was identified. Preference heterogeneity was identified via latent class analysis. Coefficients from the final selected models were used to estimate uptake in the context of different hypothetical pharmacogenetic services. RESULTS: Responses from 1993 individuals were included in the analysis. There were no differences observed in preference between the 2 clinical scenarios. Conditional logit analysis, using maximum likelihood estimation, indicated that respondents preferred to have noninvasive tests and wanted their data to be shared between different healthcare organizations to guide future prescribing. There was a preference for regional over national data sharing initiatives, and respondents preferred to have access to their data. Predicted uptake varied considerably, ranging from 51% to >99%, depending on design of the service. CONCLUSION: This study identifies public preferences for a pharmacogenetic testing service and demonstrates how predicted uptake can be impacted by relatively minor adaptations. This highlights areas for prioritization during development of future pharmacogenetic services.

Exploring NICU nurses' views of a novel genetic point-of-care test identifying neonates at risk of antibiotic-induced ototoxicity: A qualitative study.

AIM: To explore the views of neonatal intensive care nursing staff on the deliverability of a novel genetic point-of-care test detecting a genetic variant associated with antibiotic-induced ototoxicity. DESIGN: An interpretive, descriptive, qualitative interview study. METHODS: Data were collected using semi-structured interviews undertaken between January and November 2020. Participants were neonatal intensive care nursing staff taking part in the Pharmacogenetics to Avoid Loss of Hearing trial. RESULTS: Thematic analysis resulted in four themes: perceived clinical utility; the golden hour; point-of-care device; training and support. Recommendations were made to streamline the protocol and ongoing training and support were considered key to incorporating the test into routine care. CONCLUSION: Exploring the views of nurses involved in the delivery of the point-of-care test was essential in its implementation. By the study endpoint, all participants could see the value of routine clinical introduction of the point-of care test. IMPLICATIONS FOR THE PROFESSION AND/OR PATIENT CARE: Nurses are in a key position to support the delivery of point-of-care genetic testing into mainstream settings. This study has implications for the successful integration of other genetic point-of-care tests in acute healthcare settings. IMPACT: The study will help to tailor the training and support required for routine deployment of the genetic point-of-care test. The study has relevance for nurses involved in the development and delivery of genetic point-of-care tests in other acute hospital settings. REPORTING METHOD: This qualitative study adheres to the Standards for Reporting Qualitative Research EQUATOR guidelines and utilizes COREQ and SRQR checklists. PATIENT OR PUBLIC CONTRIBUTION: All staff working on the participating neonatal intensive care units were trained to use the genetic point-of-care test. All inpatients on the participating units were eligible to have testing via the point-of-care test. The Pharmacogenetics to Avoid Loss of Hearing Patient and Public Involvement and Engagement group provided valuable feedback. TRIAL AND PROTOCOL REGISTRATION: Registered within the University of Manchester. Ethics approval reference numbers: IRAS: 253102 REC reference: 19/NW/0400. Also registered with the ISRCTN ref: ISRCTN13704894.

Utility of screening for silent myocardial ischaemia in diabetes with an annual electrocardiogram.

BACKGROUND AND METHODS: Asymptomatic coronary artery disease (CAD) is common in people with diabetes mellitus, but there is a lack of consensus regarding appropriate screening for the condition. We performed a 12-lead electrocardiogram (ECG) on 312 consecutive participants with diabetes mellitus attending for routine annual outpatient review in order to determine the effectiveness of a yearly ECG in screening people with diabetes for asymptomatic CAD. RESULTS: Three of 312 participants (0.96%, 95% CI 0.2%-2.78%) had a newly identified ECG abnormality. One person had newly discovered atrial fibrillation. Two people had abnormalities which prompted further investigation for asymptomatic CAD. One of these participants underwent percutaneous coronary intervention. Seventeen further participants had abnormalities on ECG which had been previously documented, the majority having been present since their diagnosis of diabetes. CONCLUSION: A low positive yield of routine annual ECG in our study does not support its use as a screening tool for asymptomatic CAD in diabetes. Our findings support advice to perform an ECG at diagnosis of diabetes and to repeat only if a person develops relevant symptoms.

Small is Powerful: Demonstration of the Impact of Nanoformed Piroxicam in a Controlled Clinical Study.

PURPOSE: New solutions are needed to enable the efficient use of poorly water-soluble drugs. Therefore, we aimed to demonstrate that decreasing particle size with a solution-to-particle method known as nanoforming can improve dissolution and thus bioavailability. METHODS: Piroxicam, a poorly water-soluble non-steroidal anti-inflammatory drug (NSAID), was used as a model compound. A Quality-by-Design (QbD) approach was used to nanoform piroxicam and a design space was established. The pharmacokinetics of piroxicam nanoparticles were compared to two marketed products in a clinical trial. RESULTS: Nanoformed tablets showed a 33% increase in exposure during the first hour after dosing (AUC0-1 h) compared with an immediate release tablet and was similar to a fast absorbing tablet incorporating complexation of piroxicam with ß-cyclodextrin. CONCLUSIONS: The results show that nanoforming enabled more rapid absorption in comparison to a typical marketed tablet and indicate that nanoforming is an alternative to complex formulation such as cyclodextrins based products. The study outcomes support the potential of nanoforming for producing fast-acting dosage forms of poorly soluble drugs.

First Genotype-Phenotype Study in TBX4 Syndrome: Gain-of-Function Mutations Causative for Lung Disease.

Rationale: Despite the increased recognition of TBX4 (T-BOX transcription factor 4)-associated pulmonary arterial hypertension (PAH), genotype-phenotype associations are lacking and may provide important insights. Objectives: To compile and functionally characterize all TBX4 variants reported to date and undertake a comprehensive genotype-phenotype analysis. Methods: We assembled a multicenter cohort of 137 patients harboring monoallelic TBX4 variants and assessed the pathogenicity of missense variation (n = 42) using a novel luciferase reporter assay containing T-BOX binding motifs. We sought genotype-phenotype correlations and undertook a comparative analysis with patients with PAH with BMPR2 (Bone Morphogenetic Protein Receptor type 2) causal variants (n = 162) or no identified variants in PAH-associated genes (n = 741) genotyped via the National Institute for Health Research BioResource-Rare Diseases. Measurements and Main Results: Functional assessment of TBX4 missense variants led to the novel finding of gain-of-function effects associated with older age at diagnosis of lung disease compared with loss-of-function effects (P = 0.038). Variants located in the T-BOX and nuclear localization domains were associated with earlier presentation (P = 0.005) and increased incidence of interstitial lung disease (P = 0.003). Event-free survival (death or transplantation) was shorter in the T-BOX group (P = 0.022), although age had a significant effect in the hazard model (P = 0.0461). Carriers of TBX4 variants were diagnosed at a younger age (P < 0.001) and had worse baseline lung function (FEV1, FVC) (P = 0.009) than the BMPR2 and no identified causal variant groups. Conclusions: We demonstrated that TBX4 syndrome is not strictly the result of haploinsufficiency but can also be caused by gain of function. The pleiotropic effects of TBX4 in lung disease may be in part explained by the differential effect of pathogenic mutations located in critical protein domains.

The Implementation of Pharmacogenetics in the United Kingdom.

There is considerable inter-individual variability in the effectiveness and safety of pharmaceutical interventions. This phenomenon can be attributed to a multitude of factors; however, it is widely acknowledged that common genetic variation affecting drug absorption or metabolism play a substantial contributory role. This is a concept known as pharmacogenetics. Understanding how common genetic variants influence responses to medications, and using this knowledge to inform prescribing practice, could yield significant advantages for both patients and healthcare systems. Some health services around the world have introduced pharmacogenetics into routine practice, whereas others are less advanced along the implementation pathway. This chapter introduces the field of pharmacogenetics, the existing body of evidence, and discusses barriers to implementation. The chapter will specifically focus on efforts to introduce pharmacogenetics in the NHS, highlighting key challenges related to scale, informatics, and education.

Development of an Aptamer-Based Electrochemical Microfluidic Device for Viral Vaccine Quantitation.

Global deployment of vaccines poses significant challenges in the distribution and use of the accompanying immunoassays, one of the standard methods for quality control of vaccines, particularly when establishing assays in countries worldwide to support testing/release upon importation. This work describes our effort toward developing an integrated, portable device to carry out affinity assays for viral particles quantification in viral vaccines by incorporating (i) aptamers, (ii) microfluidic devices, and (iii) electrochemical detection. We generated and characterized more than eight aptamers against multiple membrane proteins of cytomegalovirus (CMV), which we used as a model system and designed and fabricated electrochemical microfluidic devices to measure CMV concentrations in a candidate vaccine under development. The aptamer-based assays provided a half maximal effective concentration, EC50, of 12 U/mL, comparable to that of an ELISA using a pair of antibodies (EC50 60 U/mL). The device measured relative CMV concentrations accurately (within ±10% bias) and precisely (11%, percent relative standard deviation). This work represents the critical first steps toward developing simple, affordable, and robust affinity assays for global deployment without the need for sensitive equipment and extensive analyst training.

The importance of biological sex in cardiac cachexia.

Cardiac cachexia is a catabolic muscle-wasting syndrome observed in approximately 1 in 10 patients with heart failure. Increased skeletal muscle atrophy leads to frailty and limits mobility, which impacts quality of life, exacerbates clinical care, and is associated with higher rates of mortality. Heart failure is known to exhibit a wide range of prevalence and severity when examined across individuals of different ages and with comorbidities related to diabetes, renal failure, and pulmonary dysfunction. It is also recognized that men and women exhibit striking differences in the pathophysiology of heart failure, as well as skeletal muscle homeostasis. Given that both skeletal muscle and heart failure physiology are in part sex-dependent, the diagnosis and treatment of cachexia in patients with heart failure may depend on a comprehensive examination of how these organs interact. In this review, we explore the potential for sex-specific differences in cardiac cachexia. We summarize advantages and disadvantages of clinical methods used to measure muscle mass and function and provide alternative measurements that should be considered in preclinical studies. In addition, we summarize sex-dependent effects on muscle wasting in preclinical models of heart failure, disuse, and cancer. Lastly, we discuss the endocrine function of the heart and outline unanswered questions that could directly impact patient care.

Patient and public involvement in hearing research: opportunities, impact and reflections with case studies from the Manchester Centre for Audiology and Deafness.

OBJECTIVE: Patient and public involvement (PPI) in research improves relevance to end users and improves processes including recruitment participants. PPI in our research has gone from being non-existent to ubiquitous over a few years. We provide critical reflections on the benefits and challenges of PPI. DESIGN: Case studies are reported according to a modified GRIP2 framework; the aims, methodology, impact of PPI and critical reflections on each case and our experiences with PPI in general. STUDY SAMPLE: We report five UK projects that included PPI from teenagers, families, people living with dementia, autistic people, and people from South Asian and d/Deaf communities. RESULTS: Our experience has progressed from understanding the rationale to grappling methodologies and integrating PPI in our research. PPI took place at all stages of research, although commonly involved input to design including recruitment and development of study materials. Methodologies varied between projects, including PPI co-investigators, advisory panels and online surveys. CONCLUSION: On-going challenges include addressing social exclusion from research for people that lack digital access following increasing on-line PPI and involvement from underserved communities. PPI was initially motivated by funders; however the benefits have driven widespread PPI, ensuring our research is relevant to people living with hearing loss.

TNAP limits TGF-ß-dependent cardiac and skeletal muscle fibrosis by inactivating the SMAD2/3 transcription factors.

Fibrosis is associated with almost all forms of chronic cardiac and skeletal muscle diseases. The accumulation of extracellular matrix impairs the contractility of muscle cells contributing to organ failure. Transforming growth factor ß (TGF-ß) plays a pivotal role in fibrosis, activating pro-fibrotic gene programmes via phosphorylation of SMAD2/3 transcription factors. However, the mechanisms that control de-phosphorylation of SMAD2 and SMAD3 (SMAD2/3) have remained poorly characterized. Here, we show that tissue non-specific alkaline phosphatase (TNAP, also known as ALPL) is highly upregulated in hypertrophic hearts and in dystrophic skeletal muscles, and that the abrogation of TGF-ß signalling in TNAP-positive cells reduces vascular and interstitial fibrosis. We show that TNAP colocalizes and interacts with SMAD2. The TNAP inhibitor MLS-0038949 increases SMAD2/3 phosphorylation, while TNAP overexpression reduces SMAD2/3 phosphorylation and the expression of downstream fibrotic genes. Overall our data demonstrate that TNAP negatively regulates TGF-ß signalling and likely represents a mechanism to limit fibrosis.

New insights into the clinical and molecular spectrum of the novel CYFIP2-related neurodevelopmental disorder and impairment of the WRC-mediated actin dynamics.

PURPOSE: A few de novo missense variants in the cytoplasmic FMRP-interacting protein 2 (CYFIP2) gene have recently been described as a novel cause of severe intellectual disability, seizures, and hypotonia in 18 individuals, with p.Arg87 substitutions in the majority. METHODS: We assembled data from 19 newly identified and all 18 previously published individuals with CYFIP2 variants. By structural modeling and investigation of WAVE-regulatory complex (WRC)-mediated actin polymerization in six patient fibroblast lines we assessed the impact of CYFIP2 variants on the WRC. RESULTS: Sixteen of 19 individuals harbor two previously described and 11 novel (likely) disease-associated missense variants. We report p.Asp724 as second mutational hotspot (4/19 cases). Genotype-phenotype correlation confirms a consistently severe phenotype in p.Arg87 patients but a more variable phenotype in p.Asp724 and other substitutions. Three individuals with milder phenotypes carry putative loss-of-function variants, which remain of unclear pathogenicity. Structural modeling predicted missense variants to disturb interactions within the WRC or impair CYFIP2 stability. Consistent with its role in WRC-mediated actin polymerization we substantiate aberrant regulation of the actin cytoskeleton in patient fibroblasts. CONCLUSION: Our study expands the clinical and molecular spectrum of CYFIP2-related neurodevelopmental disorder and provides evidence for aberrant WRC-mediated actin dynamics as contributing cellular pathomechanism.

Identification of the Most Important Subset of Doppler, Laboratory, and Clinical Parameters for Serial TIPS Evaluation.

OBJECTIVE. The purpose of the present study was to identify the subset of a wide range of serial Doppler, laboratory, and clinical parameters most predictive (both individually and in combination) of TIPS dysfunction in a large patient sample. MATERIALS AND METHODS. The medical records of 189 patients who had undergone TIPS procedures were analyzed. The patients (mean age, 52 years; 62% of whom were men) had undergone 1139 Doppler studies and 323 portovenograms. Laboratory parameters included model for end-stage liver disease (MELD) scores, serum albumin levels, presence of ascites, and time since last intervention. Doppler parameters included intrashunt velocities, temporal change in intrashunt velocities, main portal vein velocity, direction of flow in the left portal hepatic vein, and venous pulsatility index. Statistical analysis used ROC, univariate, and multivariate regression models to assess the parameters both individually and in combination. Shunt dysfunction was defined by a portosystemic gradient of more than 12 mm Hg. RESULTS. The laboratory and clinical parameters of greatest predictive value included the MELD score and the time since the last intervention. The Doppler parameters that were of greatest predictive value included the change in velocity at the hepatic venous end and the left portal vein flow direction. Multivariate models produced an AUC of 0.74. Differences between functional and dysfunctional shunts were also statistically significant for absolute velocity at the hepatic venous end, the change in velocity within the stent, and the temporal change in the mid shunt velocity. CONCLUSION. The subset of serial parameters most predictive of TIPS dysfunction are the temporal change in the velocity at the hepatic venous end, the absolute velocity at the hepatic venous end, the direction of flow in the left portal venous branch, and changes in the MELD score.

Using a biomarker acutely to identify babies at risk of serious adverse effects from antibiotics: where is the 'Terrible Moral and Medical Dilemma'?

We thank Parker and Wright for engaging in this roundtable debate in such a spirited way. The 'Pharmacogenetic [test] to Avoid Loss of Hearing' (PALOH) Trial is the first time a genetic point of care test has been applied in the acute neonatal setting; therefore, it is not surprising that questions have been raised which require debate, discussion and clarification. Parker and Wright misattribute several assumptions to the roundtable authors, which we would like to clarify here. Since they raise wider questions about the PALOH trial itself, several of the roundtable discussants have made a joint response.

FMRP recruitment of ß-catenin to the translation pre-initiation complex represses translation.

Canonical Wnt/ß-catenin signaling is an essential regulator of various cellular functions throughout development and adulthood. Aberrant Wnt/ß-catenin signaling also contributes to various pathologies including cancer, necessitating an understanding of cell context-dependent mechanisms regulating this pathway. Since protein-protein interactions underpin ß-catenin function and localization, we sought to identify novel ß-catenin interacting partners by affinity purification coupled with tandem mass spectrometry in vascular smooth muscle cells (VSMCs), where ß-catenin is involved in both physiological and pathological control of cell proliferation. Here, we report novel components of the VSMC ß-catenin interactome. Bioinformatic analysis of the protein networks implies potentially novel functions for ß-catenin, particularly in mRNA translation, and we confirm a direct interaction between ß-catenin and the fragile X mental retardation protein (FMRP). Biochemical studies reveal a basal recruitment of ß-catenin to the messenger ribonucleoprotein and translational pre-initiation complex, fulfilling a translational repressor function. Wnt stimulation antagonizes this function, in part, by sequestering ß-catenin away from the pre-initiation complex. In conclusion, we present evidence that ß-catenin fulfills a previously unrecognized function in translational repression.

Mutations in PCYT2 disrupt etherlipid biosynthesis and cause a complex hereditary spastic paraplegia.

CTP:phosphoethanolamine cytidylyltransferase (ET), encoded by PCYT2, is the rate-limiting enzyme for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. Phosphatidylethanolamine is one of the most abundant membrane lipids and is particularly enriched in the brain. We identified five individuals with biallelic PCYT2 variants clinically characterized by global developmental delay with regression, spastic para- or tetraparesis, epilepsy and progressive cerebral and cerebellar atrophy. Using patient fibroblasts we demonstrated that these variants are hypomorphic, result in altered but residual ET protein levels and concomitant reduced enzyme activity without affecting mRNA levels. The significantly better survival of hypomorphic CRISPR-Cas9 generated pcyt2 zebrafish knockout compared to a complete knockout, in conjunction with previously described data on the Pcyt2 mouse model, indicates that complete loss of ET function may be incompatible with life in vertebrates. Lipidomic analysis revealed profound lipid abnormalities in patient fibroblasts impacting both neutral etherlipid and etherphospholipid metabolism. Plasma lipidomics studies also identified changes in etherlipids that have the potential to be used as biomarkers for ET deficiency. In conclusion, our data establish PCYT2 as a disease gene for a new complex hereditary spastic paraplegia and confirm that etherlipid homeostasis is important for the development and function of the brain.

Genetic testing in the acute setting: a round table discussion.

Genetic testing has historically been performed in the context of chronic disease and cancer diagnostics. The timelines for these tests are typically measured in days or weeks, rather than in minutes. As such, the concept that genetic information might be generated and then used to alter management in the acute setting has, thus far, not been feasible. However, recent advances in genetic technologies have the potential to allow genetic information to be generated significantly quicker. The m.1555A>G genetic variant is present in one in 500 individuals and predisposes to profound hearing loss following the administration of aminoglycoside antibiotics. These antibiotics are used frequently in cases of neonatal sepsis and it is estimated that approximately 180 neonates in the UK are at risk of antibiotic induced hearing loss each year because of this genetic change. Knowledge of this variant in the acute setting would allow clinicians to prescribe alternative antibiotics. The Pharmacogenetics to Avoid Loss of Hearing study will implement a genetic point of care test (POCT) for the m.1555A>G variant within two major UK based neonatal intensive care units. This represents the first trial of a genetic POCT aimed at altering management in the acute setting. This round table discussion outlines the novel ethical issues faced in the development of this trial and the legal barriers to implementation. We ask five stakeholders to provide their opinions on this trial and their perspectives on the concept of genetic testing in the acute setting.Trial registration numberISRCTN-13704894.

Maintenance of the Undifferentiated State in Myogenic Progenitor Cells by TGFß Signaling is Smad Independent and Requires MEK Activation.

Transforming growth factor ß (TGFß) is a pluripotent cytokine and regulates a myriad of biological processes. It has been established that TGFß potently inhibits skeletal muscle differentiation; however, the molecular mechanism is not clearly defined. Previously, we reported that inhibition of the TGFß canonical pathway by an inhibitory Smad, Smad7, does not reverse this effect on differentiation, suggesting that activation of receptor Smads (R-Smads) by TGFß is not responsible for repression of myogenesis. In addition, pharmacological blockade of Smad3 activation by TGFß did not reverse TGFß's inhibitory effect on myogenesis. In considering other pathways, we observed that TGFß potently activates MEK/ERK, and a pharmacological inhibitor of MEK reversed TGFß's inhibitory effect on myogenesis, as indicated by a myogenin promoter-reporter gene, sarcomeric myosin heavy chain accumulation, and phenotypic myotube formation. Furthermore, we found that c-Jun, a known potent repressor of myogenesis, which is coincidently also a down-stream target of MEK/ERK signaling, was phosphorylated and accumulates in the nucleus in response to TGFß activation. Taken together, these observations support a model in which TGFß activates a MEK/ERK/c-Jun pathway to repress skeletal myogenesis, maintaining the pluripotent undifferentiated state in myogenic progenitors.