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The RAS/mitogen-activated protein kinase (MAPK) pathway controls a plethora of developmental and post-developmental processes. It is now clear that mutations in the RAS-MAPK pathway cause developmental diseases collectively referred to as the RASopathies. The RASopathies include Noonan syndrome, Noonan syndrome with multiple lentigines, cardiofaciocutaneous syndrome, neurofibromatosis type 1, and Costello syndrome. RASopathy patients exhibit a wide spectrum of congenital heart defects (CHD), such as valvular abnormalities and hypertrophic cardiomyopathy (HCM). Since the cardiovascular defects are the most serious and recurrent cause of mortality in RASopathy patients, it is critical to understand the pathological signaling mechanisms that drive the disease. Therapies for the treatment of HCM and other RASopathy-associated comorbidities have yet to be fully realized. Recent developments have shown promise for the use of repurposed antineoplastic drugs that target the RAS-MAPK pathway for the treatment of RASopathy-associated HCM. However, given the impact of the RAS-MAPK pathway in post-developmental physiology, establishing safety and evaluating risk when treating children will be paramount. As such insight provided by preclinical and clinical information will be critical. This review will highlight the cardiovascular manifestations caused by the RASopathies and will discuss the emerging therapies for treatment.

Purpose Noonan syndrome with multiple lentigines (NSML) is an autosomal dominant disorder presenting with hypertrophic cardiomyopathy (HCM). Up to 85% of NSML cases are caused by mutations in the PTPN11 gene that encodes for the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP2). We previously showed that low-dose dasatinib protects from the development of cardiac fibrosis in a mouse model of NSML harboring a Ptpn11Y279C mutation. This study is performed to determine the pharmacokinetic (PK) and pharmacodynamic (PD) properties of a low-dose of dasatinib in NSML mice and to determine its effectiveness in ameliorating the development of HCM. Methods Dasatinib was administered intraperitoneally into NSML mice with doses ranging from 0.05 to 0.5 mg/kg. PK parameters of dasatinib in NSML mice were determined. PD parameters were obtained for biochemical analyses from heart tissue. Dasatinib-treated NSML mice (0.1 mg/kg) were subjected to echocardiography and assessment of markers of HCM by qRT-PCR. Transcriptome analysis was performed from the heart tissue of low-dose dasatinib-treated mice. Results Low-dose dasatinib exhibited PK properties that were linear across doses in NSML mice. Dasatinib treatment of between 0.05 and 0.5 mg/kg in NSML mice yielded an exposure-dependent inhibition of c-Src and PZR tyrosyl phosphorylation and inhibited AKT phosphorylation. We found that doses as low as 0.1 mg/kg of dasatinib prevented HCM in NSML mice. Transcriptome analysis identified differentially expressed HCM-associated genes in the heart of NSML mice that were reverted to wild type levels by low-dose dasatinib administration. Conclusion These data demonstrate that low-dose dasatinib exhibits desirable therapeutic PK properties that is sufficient for effective target engagement to ameliorate HCM progression in NSML mice. These data demonstrate that low-dose dasatinib treatment may be an effective therapy against HCM in NSML patients.

Noonan syndrome with multiple lentigines (NSML) is a rare autosomal dominant disorder that presents with cardio-cutaneous-craniofacial defects. Hypertrophic cardiomyopathy (HCM) represents the major life-threatening presentation in NSML. Mutations in the PTPN11 gene that encodes for the protein tyrosine phosphatase (PTP), SHP2, represents the predominant cause of HCM in NSML. NSML-associated PTPN11 mutations render SHP2 catalytically inactive with an “open” conformation. NSML-associated PTPN11 mutations cause hypertyrosyl phosphorylation of the transmembrane glycoprotein, protein zero-related (PZR), resulting in increased SHP2 binding. Here we show that NSML mice harboring a tyrosyl phosphorylation–defective mutant of PZR (NSML/PZR(Y242F)) that is defective for SHP2 binding fail to develop HCM. Enhanced AKT/S6 kinase signaling in heart lysates of NSML mice was reversed in NSML/PZR(Y242F) mice, demonstrating that PZR/SHP2 interactions promote aberrant AKT/S6 kinase activity in NSML. Enhanced PZR tyrosyl phosphorylation in the hearts of NSML mice was found to drive myocardial fibrosis by engaging an Src/NF-κB pathway, resulting in increased activation of IL-6. Increased expression of IL-6 in the hearts of NSML mice was reversed in NSML/PZR(Y242F) mice, and PZR(Y242F) mutant fibroblasts were defective for IL-6 secretion and STAT3-mediated fibrogenesis. These results demonstrate that NSML-associated PTPN11 mutations that induce PZR hypertyrosyl phosphorylation trigger pathophysiological signaling that promotes HCM and cardiac fibrosis.

The RASopathies are a group of genetic disorders that result from germline pathogenic variants affecting RAS-mitogen activated protein kinase (MAPK) pathway genes. RASopathies share RAS/MAPK pathway dysregulation and share phenotypic manifestations affecting numerous organ systems, causing lifelong and at times life-limiting medical complications. RASopathies may benefit from precision medicine approaches. For this reason, the Sixth International RASopathies Symposium focused on exploring precision medicine. This meeting brought together basic science researchers, clinicians, clinician scientists, patient advocates, and representatives from pharmaceutical companies and the National Institutes of Health. Novel RASopathy genes, variants, and animal models were discussed in the context of medication trials and drug development. Attempts to define and measure meaningful endpoints for treatment trials were discussed, as was drug availability to patients after trial completion. ispartof: AMERICAN JOURNAL OF MEDICAL GENETICS PART A vol:182 issue:3 pages:597-606 ispartof: location:United States status: published

Protein tyrosine phosphatases (PTPs) play a crucial role in the regulation of human health and it is now clear that PTP dysfunction is causal to a variety of human diseases. Research in the PTP field has accelerated dramatically over the last decade fueled by cutting-edge technologies in genomic and proteomic techniques. This system-wide non-biased approach when applied to the discovery of PTP function has led to the elucidation of new and unanticipated roles for the PTPs. These discoveries, driven by genomic and proteomic approaches, have uncovered novel PTP findings that range from those that describe fundamental cell signaling mechanisms to implications for PTPs as novel therapeutic targets for the treatment of human disease. This review will discuss how new PTP functions have been uncovered through studies that have utilized genomic and proteomic technologies and strategies.

The mitogen-activated protein kinases (MAPKs) have been shown to regulate skeletal muscle function. Previously, we showed that MAPK phosphatase-5 (MKP-5) negatively regulates myogenesis and regeneration of skeletal muscle through inhibition of p38 MAPK and c-Jun N-terminal kinase (JNK). However, the identity and contribution of MKP-5-regulated MAPK targets in the control of skeletal muscle function and regenerative myogenesis have not been established. To identify MKP-5-regulated MAPK substrates in skeletal muscle, we performed a global differential phospho-MAPK substrate screen in regenerating skeletal muscles of wild type and MKP-5-deficient mice. We discovered a novel MKP-5-regulated MAPK substrate called guanine nucleotide exchange factor for Rab3A (GRAB) that was hyperphosphorylated on a phospho-MAPK motif in skeletal muscle of MKP-5-deficient mice. GRAB was found to be phosphorylated by JNK on serine 169. Myoblasts overexpressing a phosphorylation-defective mutant of GRAB containing a mutation at Ser-169 to Ala-169 (GRAB-S169A) inhibited the ability of C2C12 myoblasts to differentiate. We found that GRAB phosphorylation at Ser-169 was required for the secretion of the promyogenic cytokine interleukin 6 (IL-6). Consistent with this observation, MKP-5-deficient mice exhibited increased circulating IL-6 expression as compared with wild type mice. Collectively, these data demonstrate a novel mechanism whereby MKP-5-mediated regulation of JNK negatively regulates phosphorylation of GRAB, which subsequently controls secretion of IL-6. These data support the notion that MKP-5 serves as a negative regulator of MAPK-dependent signaling of critical skeletal muscle signaling pathways.

Abundantly expressed in fetal tissues and adult muscle, the developmentally regulated H19 long noncoding RNA (lncRNA) has been implicated in human genetic disorders and cancer. However, how H19 acts to regulate gene function has remained enigmatic, despite the recent implication of its encoded miR-675 in limiting placental growth. We noted that vertebrate H19 harbors both canonical and noncanonical binding sites for the let-7 family of microRNAs, which plays important roles in development, cancer, and metabolism. Using H19 knockdown and overexpression, combined with in vivo crosslinking and genome-wide transcriptome analysis, we demonstrate that H19 modulates let-7 availability by acting as a molecular sponge. The physiological significance of this interaction is highlighted in cultures in which H19 depletion causes precocious muscle differentiation, a phenotype recapitulated by let-7 overexpression. Our results reveal an unexpected mode of action of H19 and identify this lncRNA as an important regulator of the major let-7 family of microRNAs.

Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease with dismal prognosis and no cure. The potential role of the ubiquitously expressed SH2 domain-containing tyrosine phosphatase-2 (SHP2) as a therapeutic target has not been studied in IPF.To determine the expression, mechanistic role, and potential therapeutic usefulness of SHP2 in pulmonary fibrosis.The effects of SHP2 overexpression and inhibition on fibroblast response to profibrotic stimuli were analyzed in vitro in primary human and mouse lung fibroblasts. In vivo therapeutic effects were assessed in the bleomycin model of lung fibrosis by SHP2-lentiviral administration and transgenic mice carrying a constitutively active SHP2 mutation.SHP2 was down-regulated in lungs and lung fibroblasts obtained from patients with IPF. Immunolocalization studies revealed that SHP2 was absent within fibroblastic foci. Loss of SHP2 expression or activity was sufficient to induce fibroblast-to-myofibroblast differentiation in primary human lung fibroblasts. Overexpression of constitutively active SHP2 reduced the responsiveness of fibroblasts to profibrotic stimuli, including significant reductions in cell survival and myofibroblast differentiation. SHP2 effects were mediated through deactivation of fibrosis-relevant tyrosine kinase and serine/threonine kinase signaling pathways. Mice carrying the Noonan syndrome-associated gain-of-function SHP2 mutation (SHP2Our data suggest that SHP2 is an important regulator of fibroblast differentiation, and its loss as observed in IPF facilitates profibrotic phenotypic changes. Augmentation of SHP2 activity or expression should be investigated as a novel therapeutic strategy for IPF.

Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host-pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.

Since detergent-resistant lipid rafts play important roles in the signal transduction for myogenesis, their comprehensive proteomic analysis could provide new insights to understand their function in myotubes. Here, the detergent-resistant lipid rafts were isolated from C2C12 myotubes and analyzed by capillary RPLC/MS/MS. Among the 327 proteins (or protein groups) identified, 28% were categorized to the plasma membrane or raft proteins, 29% to mitochondria, 20% to microsomal proteins, 10% to other proteins, and 13% to unknown proteins. The localization of oxidative phosphorylation (OXPHOS) complexes in the sarcolemma lipid rafts was further confirmed from C2C12 myotubes by cellular fractionation, surface-biotin labeling, immunofluorescence, and lipid raft fractionation. After adding exogenous cytochrome c, the sarcolemma isolated from myotubes had an ability to consume oxygen in the presence of NADH or succinate. The generation of NADH-dependent extracellular superoxide was increased by inhibiting or downregulating OXPHOS I, III, and IV in myotubes, indicating that OXPHOS proteins are major sources for extracellular ROS in skeletal muscle. With all these data, we can conclude that OXPHOS proteins are associated with the sarcolemma lipid rafts during C2C12 myogenesis to generate extracellular ROS.

Lipid rafts are plasma membrane platforms mediating signal transduction pathways for cellular proliferation, differentiation and apoptosis. Here, we show that membrane fluidity was increased in HeLa cells following treatment with ginsenoside Rh2 (Rh2), as determined by cell staining with carboxy-laurdan (C-laurdan), a two-photon dye designed for measuring membrane hydrophobicity. In the presence of Rh2, caveolin-1 appeared in non-raft fractions after sucrose gradient ultracentrifugation. In addition, caveolin-1 and GM1, lipid raft landmarkers, were internalized within cells after exposure to Rh2, indicating that Rh2 might disrupt lipid rafts. Since cholesterol overloading, which fortifies lipid rafts, prevented an increase in Rh2-induced membrane fluidity, caveolin-1 internalization and apoptosis, lipid rafts appear to be essential for Rh2-induced apoptosis. Moreover, Rh2-induced Fas oligomerization was abolished following cholesterol overloading, and Rh2-induced apoptosis was inhibited following treatment with siRNA for Fas. This result suggests that Rh2 is a novel lipid raft disruptor leading to Fas oligomerization and apoptosis.

To address whether mitochondrial biogenesis is essential for skeletal myogenesis, C2C12 myogenesis was investigated after knockdown of NADH dehydrogenase (ubiquintone) flavoprotein 1 (NDUFV1), which is an oxidative phosphorylation complex I subunit that is the first subunit to accept electrons from NADH. The NDUFVI knockdown enhanced C2C12 myogenesis by decreasing the NAD(+)/NADH ratio and subsequently inactivating SIRT1 and SIRT1 activators (pyruvate, SRT1720, and resveratrol) abolished the NDUFV1 knockdown-induced myogenesis enhancement. However, the insulin-elicited activation of insulin receptor β (IRβ) and insulin receptor substrate-1 (IRS-1) was reduced with elevated levels of protein-tyrosine phosphatase 1B after NDUFV1 knockdown in C2C12 myotubes. The NDUFV1 knockdown-induced blockage of insulin signaling was released by protein-tyrosine phosphatase 1B knockdown in C2C12 myotubes, and we found that NDUFV1 or SIRT1 knockdown did not affect mitochondria biogenesis during C2C12 myogenesis. Based on these data, we can conclude that complex I dysfunction-induced SIRT1 inactivation leads to myogenesis enhancement but blocks insulin signaling without affecting mitochondria biogenesis.

The striated muscle-specific mitsugumin 53 (MG53) is a novel E3 ligase that induces the ubiquitination of insulin receptor substrate 1 (IRS-1) during skeletal myogenesis, negatively regulating insulin-like growth factor and insulin signaling. Here we show that focal adhesion kinase (FAK) is the second target of MG53 during skeletal myogenesis. The FAK protein level gradually decreased, whereas its mRNA level was constant during myogenesis in C2C12 cells and MyoD-overexpressing mouse embryonic fibroblasts. The FAK protein was associated with the E2 enzyme UBE2H and the E3 enzyme MG53 in endogenous and exogenous immunoprecipitation experiments. FAK ubiquitination and degradation was induced by MG53 overexpression in myoblasts but abolished by MG53 or UBE2H knockdown in myotubes. Because RING-disrupted MG53 mutants (C14A and ΔR) did not induce FAK ubiquitination and degradation, the RING domain was determined to be required for MG53-induced FAK ubiquitination. Taken together, these data indicate that MG53 induces FAK ubiquitination with the aid of UBE2H during skeletal myogenesis.

Mitsugumin 53 (MG53) negatively regulates skeletal myogenesis by targeting insulin receptor substrate 1 (IRS-1). Here, we show that MG53 is an ubiquitin E3 ligase that induces IRS-1 ubiquitination with the help of an E2-conjugating enzyme, UBE2H. Molecular manipulations that disrupt the E3-ligase function of MG53 abolish IRS-1 ubiquitination and enhance skeletal myogenesis. Skeletal muscles derived from the MG53 � / � mice show an elevated IRS-1 level with enhanced insulin signalling, which protects the MG53 � / � mice from developing insulin resistance when challenged with a high-fat/high-sucrose diet. Muscle samples derived from human diabetic patients and mice with insulin resistance show normal expression of MG53, indicating that altered MG53 expression does not serve as a causative factor for the development of metabolic disorders. Thus, therapeutic interventions that target the interaction between MG53 and IRS-1 may be a novel approach for the treatment of metabolic diseases that are associated with insulin resistance.

Since detergent-resistant lipid rafts play important roles in multidrug resistance (MDR), their comprehensive proteomics could provide new insights to understand the underlying molecular mechanism of MDR in cancer cells. In the present work, lipid rafts were isolated from MCF-7 and adriamycin-resistant MCF-7/ADR cells and their proteomes were analyzed by label-free quantitative proteomics. Polymerase I and transcript release factor (PTRF)/cavin-1 was measured to be upregulated along with multidrug-resistant P-glycoprotein, caveolin-1, and serum deprivation protein response/cavin-2 in the lipid rafts of MCF-7/ADR cells. PTRF knockdown led to reduction in the amount of lipid rafts on the surface of MCF7/ADR cells as determined by cellular staining with lipid raft-specific dyes such as S-laurdan2 and FITC-conjugated cholera toxin B. PTRF knockdown also reduced MDR in MCF-7/ADR cells. These data indicate that PTRF is necessary for MDR in cancer cells via the fortification of lipid rafts.

We previously demonstrated that the receptor for the complement component C1q (gC1qR) is a lipid raft protein that is indispensable for adipogenesis and insulin signaling. Here, we provide the first report that gC1qR is an essential component of lamellipodia in human lung carcinoma A549 cells. Cell-surface gC1qR was concentrated in the lamellipodia along with CD44, monosialoganglioside, actin, and phosphorylated focal adhesion kinase in cells stimulated with insulin, IGF-1, EGF, or serum. The growth factor-induced lamellipodia formation and cell migration were significantly decreased in gC1qR-depleted cells, with a concomitant blunt activation of the focal adhesion kinase and the respective receptor tyrosine kinases. Moreover, the gC1qR-depleted cells exhibited a reduced proliferation rate in culture as well as diminished tumorigenic and metastatic activities in grafted mice. We therefore conclude that cell-surface gC1qR regulates lamellipodia formation and metastasis via receptor tyrosine kinase activation.

Lipid rafts have been known to be platforms to initiate cellular signal transduction of insulin-like growth factor (IGF) inducing skeletal muscle differentiation and hypertrophy. Here, tripartite motif 72 (TRIM72), with a really interesting new gene (RING)-finger domain, a B-box, two coiled-coil domains, and a SPRY (SPla and RYanodine receptor) domain, was revealed to be predominantly expressed in the sarcolemma lipid rafts of skeletal and cardiac muscles. Adenoviral TRIM72 overexpression prevented but RNAi-mediated TRIM72 silencing enhanced C2C12 myogenesis by modulating the IGF-induced insulin receptor substrate-1 (IRS-1) activation through the molecular association of TRIM72 with IRS-1. Furthermore, myogenic activity was highly enhanced with increased IGF-induced Akt activation in the satellite cells of TRIM72(-/-) mice, compared to those of TRIM72+/+ mice. Because TRIM72 promoter analysis shows that two proximal E-boxes in TRIM72 promoter were essential for MyoD- and Akt-dependent TRIM72 transcription, we can conclude that TRIM72 is a novel antagonist of IRS-1, and is essential as a negative regulator of IGF-induced muscle differentiation.

Tripartite motif-containing (TRIM) family proteins consist of multimodular domains including a relatively conserved N-terminal RBCC domain consisting of a RING finger for E3 ubiquitin ligase activity, a zinc-bound B-box for protein–protein interaction, one or two coiledcoil domains for oligomerization, and a variable C-terminal domain. In some cases, however, TRIM proteins have a PRY-SPRY domain (PRY segment followed SPRY domain identified in a Dictyostelium discoidueum kinase splA and mammalian Ca-release channels ryanodine receptors) at their C-terminus, which has been identified as a targeting module.1,2 More than 70 members of this family have been identified and characterized, and show a very similar domain architecture; however, their cellular functions are extremely diverse, and include roles in cell proliferation, differentiation, development, oncogenesis, apoptosis, and retroviral replication.1,2 The E3 ligase activity of several TRIM proteins has been previously demonstrated, as they usually harbor a RING domain at the N-terminal region. Each TRIM protein interacts with distinct targets, which are critical in the aforementioned cellular processes.3–8 Therefore, relatively newly and incompletely characterized C-terminal domains, including the PRY-SPRY domain, are believed to be a central mediator for selective interaction with their partners. Well-studied members of the TRIM family include the following: TRIM1, TRIM5a, TRIM19, and TRIM22, which target retroviruses and prevent their replication inside cells3,6; TRIM18/MID1 and TRIM20/pyrin, which are linked to Opitz G/BBB syndrome and familial Mediterranean fever, respectively2,9; TRIM21/Ro52, which is a major autoantigen in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and Sjorgen’s syndrome10,11; and TRIM63/Murf1, TRIM55/ Murf2, TRIM41, and TRIM32, which function in muscle cells.1,12 Recently, another TRIM family protein, TRIM72/MG53 has been shown to be expressed specifically in skeletal muscle and heart, and also been demonstrated to perform a critical function in membrane repair following acute muscle injury.13–15 Human TRIM72 consists of 477 amino acid residues with the standard domain organization of the TRIM family [Fig. 1(A)]. By way of contrast with the RBCC domain, which is predictive of its molecular function, very little is currently known regarding the PRY-SPRY domain, or how it has evolved to mediate diverse functions in each TRIM protein. Thus far, two structures of the SPRY domain have been determined in the complex state; however, their interac

An aminoacyl-tRNA synthetase subunit, p43, was previously demonstrated to be released from mammalian cells, and to function as an extracellular regulator of both angiogenesis and inflammatory responses (Ko et al., [2001] J Biol Chem, 276; 23028; Park et al.[2002], J Biol Chem 277; 45243). Here, we report that p43 is internalized to the endothelial cells via lipid rafts. Exogenous p43 was co-localized on bovine aorta endothelial cells with cholera toxin B (CTB), which binds to cholesterol-enriched lipid rafts. The p43 was rapidly internalized to the cells, as early as 5 min after binding to the surfaces of the cells. p43 bound to the isolated lipid rafts, and its interaction with the lipid rafts, was prevented by high salt content, but not by detergent. This suggests that ionic bonds are involved in the molecular association of p43 with the lipid rafts. Taken together, we conclude that p43 binds to the endothelial cell surface via lipid rafts.