The MYPT2-regulated striated muscle-specific myosin light chain phosphatase limits cardiac myosin phosphorylation in vivo.

The physiological importance of cardiac myosin regulatory light chain (RLC) phosphorylation by its dedicated cardiac myosin light chain kinase has been established in both humans and mice. Constitutive RLC-phosphorylation, regulated by the balanced activities of cardiac myosin light chain kinase and myosin light chain phosphatase (MLCP), is fundamental to the biochemical and physiological properties of myofilaments. However, limited information is available on cardiac MLCP. In this study, we hypothesized that the striated muscle-specific MLCP regulatory subunit, MYPT2, targets the phosphatase catalytic subunit to cardiac myosin, contributing to the maintenance of cardiac function in vivo through the regulation of RLC-phosphorylation. To test this hypothesis, we generated a floxed-PPP1R12B mouse model crossed with a cardiac-specific Mer-Cre-Mer to conditionally ablate MYPT2 in adult cardiomyocytes. Immunofluorescence microscopy using the gene-ablated tissue as a control confirmed the localization of MYPT2 to regions where it overlaps with a subset of RLC. Biochemical analysis revealed an increase in RLC-phosphorylation in vivo. The loss of MYPT2 demonstrated significant protection against pressure overload-induced hypertrophy, as evidenced by heart weight, qPCR of hypertrophy-associated genes, measurements of myocyte diameters, and expression of ß-MHC protein. Furthermore, mantATP chase assays revealed an increased ratio of myosin heads distributed to the interfilament space in MYPT2-ablated heart muscle fibers, confirming that RLC-phosphorylation regulated by MLCP, enhances cardiac performance in vivo. Our findings establish MYPT2 as the regulatory subunit of cardiac MLCP, distinct from the ubiquitously expressed canonical smooth muscle MLCP. Targeting MYPT2 to increase cardiac RLC-phosphorylation in vivo may improve baseline cardiac performance, thereby attenuating pathological hypertrophy.

Phosphatase regulatory subunit MYPT2 knockout partially compensates for the cardiac dysfunction in mice caused by lack of myosin light chain kinase 3.

Cardiac contraction is modulated by the phosphorylation state of myosin regulatory light chain 2 (MLC-2v). The level of MLC-2v phosphorylation is dependent on the opposing activities of MLC kinases and phosphatases. The predominant MLC phosphatase found in cardiac myocytes contains Myosin Phosphatase Targeting Subunit 2 (MYPT2). Overexpression of MYPT2 in cardiac myocytes results in a decreased level of MLC phosphorylation, reduced left ventricular contraction, and induction of hypertrophy; however, the effect of knocking out MYPT2 on cardiac function is unknown. We obtained heterozygous mice containing a MYPT2 null allele from the Mutant Mouse Resource Center. These mice were produced in a C57BL/6N background which lack MLCK3, the main regulatory light chain kinase in cardiac myocytes. We found that mice null for MYPT2 were viable and had no obvious phenotypic abnormality when compared to WT mice. Additionally, we determined that WT C57BL/6N mice had a low basal level of MLC-2v phosphorylation, which was significantly increased when MYPT2 was absent. At 12-weeks, MYPT2 KO mice had smaller hearts and showed downregulation of genes involved in cardiac remodeling. Using cardiac echo, we found that 24-week-old male MYPT2 KO mice had decreased heart size with increased fractional shortening compared to their MYPT2 WT littermates. Collectively, these studies highlight the important role that MYPT2 plays in cardiac function in vivo and demonstrate that its deletion can partially compensate for the lack of MLCK3.

Role of protein kinase D1 in vasoconstriction and haemodynamics in rats.

AIMS: Protein kinase D (PKD), once considered an effector of protein kinase C (PKC), now plays many pathophysiological roles in various tissues. However, little is known about role of PKD in vascular function. We investigated the role of PKD in contraction of rat aorta and human aortic smooth muscle cells (HASMCs) and in haemodynamics in rats. METHODS AND RESULTS: Isometric tension of rat aortic was measured to examine norepinephrine-induced contraction in the presence of PKD, PKC and Rho-kinase inhibitors. Phosphorylation of PKD1, myosin targeting subunit-1 (MYPT1), myosin light chain (MLC), CPI-17 and heat-shock protein 27 (HSP27), and actin polymerization were measured in the aorta. Phosphorylation of MYPT1 and MLC was also measured in HASMCs knocked down with specific siRNAs of PKD 1, 2 and 3. Intracellular calcium concentrations and cell shortening were measured in HASMCs. Norepinephrine-induced aortic contraction was accompanied by increased phosphorylation of PKD1, MYPT1 and MLC and actin polymerization, all of which were attenuated with PKD inhibitor CRT0066101. PKD1 phosphorylation was not inhibited by PKC inhibitor, chelerythrine or Rho kinase inhibitor, fasudil. In HASMCs, the phosphorylation of MYPT1 and MLC was attenuated by PKD1, but not PKD2, 3 knockdown. In HASMCs, CRT0066101 inhibited norepinephrine-induced cell shortening without affecting calcium concentration. Administration of CRT0066101 decreased systemic vascular resistance and blood pressure without affecting cardiac output in rats. CONCLUSIONS: PKD1 may play roles in aorta contraction and haemodynamics via phosphorylation of MYPT1 and actin polymerization in a calcium-independent manner.

Myosin light chain phosphatase catalytic subunit dephosphorylates cardiac myosin via mechanisms dependent and independent of the MYPT regulatory subunits.

Cardiac muscle myosin regulatory light chain (RLC) is constitutively phosphorylated at ∼0.4 mol phosphate/mol RLC in normal hearts, and phosphorylation is maintained by balanced activities of dedicated cardiac muscle-specific myosin light chain kinase and myosin light chain phosphatase (MLCP). Previously, the identity of the cardiac-MLCP was biochemically shown to be similar to the smooth muscle MLCP, which is a well-characterized trimeric protein comprising the regulatory subunit (MYPT1), catalytic subunit PP1cß, and accessory subunit M20. In smooth muscles in vivo, MYPT1 and PP1cß co-stabilize each other and are both necessary for normal smooth muscle contractions. In the cardiac muscle, MYPT1 and MYPT2 are both expressed, but contributions to physiological regulation of cardiac myosin dephosphorylation are unclear. We hypothesized that the main catalytic subunit for cardiac-MLCP is PP1cß, and maintenance of RLC phosphorylation in vivo is dependent on regulation by striated muscle-specific MYPT2. Here, we used PP1cß conditional knockout mice to biochemically define cardiac-MLCP proteins and developed a cardiac myofibrillar phosphatase assay to measure the direct contribution of MYPT-regulated and MYPT-independent phosphatase activities toward phosphorylated cardiac myosin. We report that (1) PP1cß is the main isoform expressed in the cardiac myocyte, (2) cardiac muscle pathogenesis in PP1cß knockout animals involve upregulation of total PP1cα in myocytes and non-muscle cells, (3) the stability of cardiac MYPT1 and MYPT2 proteins in vivo is not dependent on the PP1cß expression, and (4) phosphorylated myofibrillar cardiac myosin is dephosphorylated by both myosin-targeted and soluble MYPT-independent PP1cß activities. These results contribute to our understanding of the cardiac-MLCP in vivo.

Loss-of-Function Variants in PPP1R12A: From Isolated Sex Reversal to Holoprosencephaly Spectrum and Urogenital Malformations.

In two independent ongoing next-generation sequencing projects for individuals with holoprosencephaly and individuals with disorders of sex development, and through international research collaboration, we identified twelve individuals with de novo loss-of-function (LoF) variants in protein phosphatase 1, regulatory subunit 12a (PPP1R12A), an important developmental gene involved in cell migration, adhesion, and morphogenesis. This gene has not been previously reported in association with human disease, and it has intolerance to LoF as illustrated by a very low observed-to-expected ratio of LoF variants in gnomAD. Of the twelve individuals, midline brain malformations were found in five, urogenital anomalies in nine, and a combination of both phenotypes in two. Other congenital anomalies identified included omphalocele, jejunal, and ileal atresia with aberrant mesenteric blood supply, and syndactyly. Six individuals had stop gain variants, five had a deletion or duplication resulting in a frameshift, and one had a canonical splice acceptor site loss. Murine and human in situ hybridization and immunostaining revealed PPP1R12A expression in the prosencephalic neural folds and protein localization in the lower urinary tract at critical periods for forebrain division and urogenital development. Based on these clinical and molecular findings, we propose the association of PPP1R12A pathogenic variants with a congenital malformations syndrome affecting the embryogenesis of the brain and genitourinary systems and including disorders of sex development.

FAT10 differentially stabilizes MYPT2 isoforms.

Myosin phosphatase (MP) is an enzyme complex that regulates muscle contraction and plays important roles in various physiological and pathological conditions. Myosin phosphatase targeting subunit (MYPT) 2, a subunit of MP, interacts with protein phosphatase 1c to regulate its phosphatase activity. MYPT2 exists in various isoforms that differ in the composition of essential motifs that contribute to its function. However, regulatory mechanisms underlying these isoforms are poorly understood. Human leukocyte antigen-F adjacent transcript 10 (FAT10) is a ubiquitin-like modifier that not only targets proteins for proteasomal degradation but also stabilizes its interacting proteins. In this study, we investigated the effect of the interaction between FAT10 and MYPT2 isoform a (the canonical full-length form of MYPT2) or MYPT2 isoform f (the natural truncated form of MYPT2). FAT10 interacted with both MYPT2 isoforms a and f; however, only MYPT2 isoform f was increased by FAT10, whereas MYPT2 isoform a remained unaffected by FAT10. We further confirmed that, in contrast to MYPT2 isoform a, MYPT2 isoform f undergoes rapid degradation via the ubiquitin-proteasome pathway and that FAT10 stabilizes MYPT2 isoform f by inhibiting its ubiquitination. Therefore, our findings suggest that the interaction between FAT10 and MYPT2 isoforms leads to distinct stabilization effects on each isoform, potentially modulating MP activity.

Rho kinase inhibitors ameliorate cognitive impairment in a male mouse model of methamphetamine-induced schizophrenia.

Schizophrenia (SCZ) is a severe psychiatric disorder characterized by positive symptoms, negative symptoms, and cognitive deficits. Current antipsychotic treatment in SCZ improves positive symptoms but has major side effects and little impact on negative symptoms and cognitive impairment. The pathoetiology of SCZ remains unclear, but is known to involve small GTPase signaling. Rho kinase, an effector of small GTPase Rho, is highly expressed in the brain and plays a major role in neurite elongation and neuronal architecture. This study used a touchscreen-based visual discrimination (VD) task to investigate the effects of Rho kinase inhibitors on cognitive impairment in a methamphetamine (METH)-treated male mouse model of SCZ. Systemic injection of the Rho kinase inhibitor fasudil dose-dependently ameliorated METH-induced VD impairment. Fasudil also significantly suppressed the increase in the number of c-Fos-positive cells in the infralimbic medial prefrontal cortex (infralimbic mPFC) and dorsomedial striatum (DMS) following METH treatment. Bilateral microinjections of Y-27632, another Rho kinase inhibitor, into the infralimbic mPFC or DMS significantly ameliorated METH-induced VD impairment. Two proteins downstream of Rho kinase, myosin phosphatase-targeting subunit 1 (MYPT1; Thr696) and myosin light chain kinase 2 (MLC2; Thr18/Ser19), exhibited increased phosphorylation in the infralimbic mPFC and DMS, respectively, after METH treatment, and fasudil inhibited these increases. Oral administration of haloperidol and fasudil ameliorated METH-induced VD impairment, while clozapine had little effect. Oral administration of haloperidol and clozapine suppressed METH-induced hyperactivity, but fasudil had no effect. These results suggest that METH activates Rho kinase in the infralimbic mPFC and DMS, which leads to cognitive impairment in male mice. Rho kinase inhibitors ameliorate METH-induced cognitive impairment, perhaps via the cortico-striatal circuit.

Inhibition of Rho-kinase ameliorates decreased spine density in the medial prefrontal cortex and methamphetamine-induced cognitive dysfunction in mice carrying schizophrenia-associated mutations of the Arhgap10 gene.

Copy-number variations in the ARHGAP10 gene encoding Rho GTPase-activating protein 10 are associated with schizophrenia. Model mice (Arhgap10 S490P/NHEJ mice) that carry "double-hit" mutations in the Arhgap10 gene mimic the schizophrenia in a Japanese patient, exhibiting altered spine density, methamphetamine-induced cognitive dysfunction, and activation of RhoA/Rho-kinase signaling. However, it remains unclear whether the activation of RhoA/Rho-kinase signaling due to schizophrenia-associated Arhgap10 mutations causes the phenotypes of these model mice. Here, we investigated the effects of fasudil, a brain permeable Rho-kinase inhibitor, on altered spine density in the medial prefrontal cortex (mPFC) and on methamphetamine-induced cognitive impairment in a touchscreen­based visual discrimination task in Arhgap10 S490P/NHEJ mice. Fasudil (20 mg/kg, intraperitoneal) suppressed the increased phosphorylation of myosin phosphatase-targeting subunit 1, a substrate of Rho-kinase, in the striatum and mPFC of Arhgap10 S490P/NHEJ mice. In addition, daily oral administration of fasudil (20 mg/kg/day) for 7 days ameliorated the reduced spine density of layer 2/3 pyramidal neurons in the mPFC. Moreover, fasudil (3-20 mg/kg, intraperitoneal) rescued the methamphetamine (0.3 mg/kg)-induced cognitive impairment of visual discrimination in Arhgap10 S490P/NHEJ mice. Our results suggest that Rho-kinase plays significant roles in the neuropathological changes in spine morphology and in the vulnerability of cognition to methamphetamine in mice with schizophrenia-associated Arhgap10 mutations.

Long-Term Experimental Hyperglycemia Does Not Impair Macrovascular Endothelial Barrier Integrity and Function in vitro.

Hyperglycemia is a hallmark of type 2 diabetes implicated in vascular endothelial dysfunction and cardiovascular complications. Many in vitro studies identified endothelial apoptosis as an early outcome of experimentally modeled hyperglycemia emphasizing cell demise as a significant factor of vascular injury. However, endothelial apoptosis has not been observed in vivo until the late stages of type 2 diabetes. Here, we studied the long-term (up to 4 weeks) effects of high glucose (HG, 30 mM) on human umbilical vein endothelial cells (HUVEC) in vitro. HG did not alter HUVEC monolayer morphology, ROS levels, NO production, and exerted minor effects on the HUVEC apoptosis markers. The barrier responses to various clues were indistinguishable from those by cells cultured in physiological glucose (5 mM). Tackling the key regulators of cytoskeletal contractility and endothelial barrier revealed no differences in the histamine-induced intracellular Ca2+ responses, nor in phosphorylation of myosin regulatory light chain or myosin light chain phosphatase. Altogether, these findings suggest that vascular endothelial cells may well tolerate HG for relatively long exposures and warrant further studies to explore mechanisms involved in vascular damage in advanced type 2 diabetes.

Phosphorylated Peptide Derived from the Myosin Phosphatase Target Subunit Is a Novel Inhibitor of Protein Phosphatase-1.

Identification of specific protein phosphatase-1 (PP1) inhibitors is of special importance regarding the study of its cellular functions and may have therapeutic values in diseases coupled to signaling processes. In this study, we prove that a phosphorylated peptide of the inhibitory region of myosin phosphatase (MP) target subunit (MYPT1), R690QSRRS(pT696)QGVTL701 (P-Thr696-MYPT1690-701), interacts with and inhibits the PP1 catalytic subunit (PP1c, IC50 = 3.84 µM) and the MP holoenzyme (Flag-MYPT1-PP1c, IC50 = 3.84 µM). Saturation transfer difference NMR measurements established binding of hydrophobic and basic regions of P-Thr696-MYPT1690-701 to PP1c, suggesting interactions with the hydrophobic and acidic substrate binding grooves. P-Thr696-MYPT1690-701 was dephosphorylated by PP1c slowly (t1/2 = 81.6-87.9 min), which was further impeded (t1/2 = 103 min) in the presence of the phosphorylated 20 kDa myosin light chain (P-MLC20). In contrast, P-Thr696-MYPT1690-701 (10-500 µM) slowed down the dephosphorylation of P-MLC20 (t1/2 = 1.69 min) significantly (t1/2 = 2.49-10.06 min). These data are compatible with an unfair competition mechanism between the inhibitory phosphopeptide and the phosphosubstrate. Docking simulations of the PP1c-P-MYPT1690-701 complexes with phosphothreonine (PP1c-P-Thr696-MYPT1690-701) or phosphoserine (PP1c-P-Ser696-MYPT1690-701) suggested their distinct poses on the surface of PP1c. In addition, the arrangements and distances of the surrounding coordinating residues of PP1c around the phosphothreonine or phosphoserine at the active site were distinct, which may account for their different hydrolysis rate. It is presumed that P-Thr696-MYPT1690-701 binds tightly at the active center but the phosphoester hydrolysis is less preferable compared to P-Ser696-MYPT1690-701 or phosphoserine substrates. Moreover, the inhibitory phosphopeptide may serve as a template to synthesize cell permeable PP1-specific peptide inhibitors.

Persistent Müllerian duct syndrome associated with genetic defects in the regulatory subunit of myosin phosphatase.

STUDY QUESTION: Can mutations of genes other than AMH or AMHR2, namely PPP1R12A coding myosin phosphatase, lead to persistent Müllerian duct syndrome (PMDS)? SUMMARY ANSWER: The detection of PPP1R12A truncation mutations in five cases of PMDS suggests that myosin phosphatase is involved in Müllerian regression, independently of the anti-Müllerian hormone (AMH) signaling cascade. WHAT IS KNOWN ALREADY: Mutations of AMH and AMHR2 are detectable in an overwhelming majority of PMDS patients but in 10% of cases, both genes are apparently normal, suggesting that other genes may be involved. STUDY DESIGN, SIZE, DURATION: DNA samples from 39 PMDS patients collected from 1990 to present, in which Sanger sequencing had failed to detect biallelic AMH or AMHR2 mutations, were screened by massive parallel sequencing. PARTICIPANTS/MATERIALS, SETTING, METHODS: To rule out the possibility that AMH or AMHR2 mutations could have been missed, all DNA samples of good quality were analyzed by targeted next-generation sequencing. Twenty-four samples in which the absence of AMH or AMHR2 biallelic mutations was confirmed were subjected to whole-exome sequencing with the aim of detecting variants of other genes potentially involved in PMDS. MAIN RESULTS AND THE ROLE OF CHANCE: Five patients out of 24 (21%) harbored deleterious truncation mutations of PP1R12A, the gene coding for the regulatory subunit of myosin phosphatase, were detected. In addition to PMDS, three of these patients presented with ileal and one with esophageal atresia. The congenital abnormalities associated with PMDS in our patients are consistent with those described in the literature for PPP1R12A variants and have never been described in cases of AMH or AMHR2 mutations. The role of chance is therefore extremely unlikely. LIMITATIONS, REASONS FOR CAUTION: The main limitation of the study is the lack of experimental validation of the role of PPP1R12A in Müllerian regression. Only circumstantial evidence is available, myosin phosphatase is required for cell mobility, which plays a major role in Müllerian regression. Alternatively, PPP1R12A mutations could affect the AMH transduction pathway. WIDER IMPLICATIONS OF THE FINDINGS: The study supports the conclusion that failure of Müllerian regression in males is not necessarily associated with a defect in AMH signaling. Extending the scope of molecular analysis should shed light upon the mechanism of the initial steps of male sex differentiation. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by la Fondation Maladies Rares, GenOmics 2021_0404 and la Fondation pour la Recherche Médicale, grant EQU201903007868. The authors report no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Somatostatin Primes Endothelial Cells for Agonist-Induced Hyperpermeability and Angiogenesis In Vitro.

Somatostatin is an inhibitory peptide, which regulates the release of several hormones, and affects neurotransmission and cell proliferation via its five Gi protein-coupled receptors (SST1-5). Although its endocrine regulatory and anti-tumour effects have been thoroughly studied, little is known about its effect on the vascular system. The aim of the present study was to analyse the effects and potential mechanisms of somatostatin on endothelial barrier function. Cultured human umbilical vein endothelial cells (HUVECs) express mainly SST1 and SST5 receptors. Somatostatin did not affect the basal HUVEC permeability, but primed HUVEC monolayers for thrombin-induced hyperpermeability. Western blot data demonstrated that somatostatin activated the phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt) and p42/44 mitogen-activated protein kinase (MAPK) pathways by phosphorylation. The HUVEC barrier destabilizing effects were abrogated by pre-treating HUVECs with mitogen-activated protein kinase kinase/extracellular signal regulated kinase (MEK/ERK), but not the Akt inhibitor. Moreover, somatostatin pre-treatment amplified vascular endothelial growth factor (VEGF)-induced angiogenesis (3D spheroid formation) in HUVECs. In conclusion, the data demonstrate that HUVECs under quiescence conditions express SST1 and SST5 receptors. Moreover, somatostatin primes HUVECs for thrombin-induced hyperpermeability mainly via the activation of MEK/ERK signalling and promotes HUVEC proliferation and angiogenesis in vitro.

O-GlcNAcylation of myosin phosphatase targeting subunit 1 (MYPT1) dictates timely disjunction of centrosomes.

The role of O-linked N-acetylglucosamine (O-GlcNAc) modification in the cell cycle has been enigmatic. Previously, both O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) disruptions have been shown to derail the mitotic centrosome numbers, suggesting that mitotic O-GlcNAc oscillation needs to be in concert with mitotic progression to account for centrosome integrity. Here, using both chemical approaches and biological assays with HeLa cells, we attempted to address the underlying molecular mechanism and observed that incubation of the cells with the OGA inhibitor Thiamet-G strikingly elevates centrosomal distances, suggestive of premature centrosome disjunction. These aberrations could be overcome by inhibiting Polo-like kinase 1 (PLK1), a mitotic master kinase. PLK1 inactivation is modulated by the myosin phosphatase targeting subunit 1 (MYPT1)-protein phosphatase 1cß (PP1cß) complex. Interestingly, MYPT1 has been shown to be abundantly O-GlcNAcylated, and the modified residues have been detected in a recent O-GlcNAc-profiling screen utilizing chemoenzymatic labeling and bioorthogonal conjugation. We demonstrate here that MYPT1 is O-GlcNAcylated at Thr-577, Ser-585, Ser-589, and Ser-601, which antagonizes CDK1-dependent phosphorylation at Ser-473 and attenuates the association between MYPT1 and PLK1, thereby promoting PLK1 activity. We conclude that under high O-GlcNAc levels, PLK1 is untimely activated, conducive to inopportune centrosome separation and disruption of the cell cycle. We propose that too much O-GlcNAc is equally deleterious as too little O-GlcNAc, and a fine balance between the OGT/OGA duo is indispensable for successful mitotic divisions.

Myosin Phosphatase Is Implicated in the Control of THP-1 Monocyte to Macrophage Differentiation.

Monocyte to macrophage differentiation is characterized by the activation of various signal transduction pathways, which may be modulated by protein phosphorylation; however, the impact of protein kinases and phosphatases is not well understood yet. It has been demonstrated that actomyosin rearrangement during macrophage differentiation is dependent on Rho-associated protein kinase (ROCK). Myosin phosphatase (MP) target subunit-1 (MYPT1) is one of the major cellular substrates of ROCK, and MP is often a counter enzyme of ROCK; therefore, MP may also control macrophage differentiation. Changes in MP activity and the effects of MP activation were studied on PMA or l,25(OH)2D3-induced differentiation of monocytic THP-1 cells. During macrophage differentiation, phosphorylation of MYPT1 at Thr696 and Thr853 increased significantly, resulting in inhibition of MP. The ROCK inhibitor H1152 and the MP activator epigallocatechin-3-gallate (EGCG) attenuated MYPT1 phosphorylation and concomitantly decreased the extent of phosphorylation of 20 kDa myosin light chain. H1152 and EGCG pretreatment also suppressed the expression of CD11b and weakened the PMA-induced adherence of the cells. Our results indicate that MP activation/inhibition contributes to the efficacy of monocyte to macrophage differentiation, and this enzyme may be a target for pharmacological interventions in the control of disease states that are affected by excessive macrophage differentiation.

Mypt1 regulates Bmp signaling to promote embryonic exocrine pancreas growth in zebrafish.

Myosin phosphatase targeting subunit 1 (Mypt1) is the regulatory subunit of myosin phosphatase which dephosphorylates the light chain of myosin II to inhibit its contraction. Although biochemical properties of Mypt1 have been characterized in detail, its biological functions in organisms are not well understood. The zebrafish mypt1 sq181 allele was found defective in the ventral pancreatic bud and extrapancreatic duct development, resulting in dysplasia of exocrine pancreas. In mypt1 sq181 mutant, the early growth of the ventral pancreatic bud was initiated but failed to expand due to impaired cell proliferation and increased cell apoptosis. As Mypt1 is essential for cell migration, the loss-of-function of Mypt1 in the mutant disrupted the lateral plate mesoderm migration during gut looping, therefore, altering the Bmp2a expression pattern within it, and eventually leading to impaired Bmp signaling in the adjacent exocrine pancreas. Overexpression of bmp2a could rescue the development of exocrine pancreas, suggesting that the impaired Bmp2a signaling is responsible for the pancreatic development defects. Bmp2a has been reported to promote the early specification of the ventral pancreatic bud, and our study reveals that it continues to serve as a cell proliferation/survival signal to ensure pancreatic bud growth properly in zebrafish.

Rho-kinase pathway activation and apoptosis in circulating leucocytes in patients with heart failure with reduced ejection fraction.

BACKGROUND: Increased Rho-kinase activity in circulating leucocytes is observed in heart failure with reduced ejection fraction (HFrEF). However, there is little information in HFrEF regarding other Rho-kinase pathway components an on the relationship between Rho-kinase and apoptosis. Here, Rho-kinase activation levels and phosphorylation of major downstream molecules and apoptosis levels were measured for the first time both in HFrEF patients and healthy individuals. METHODS: Cross-sectional study comparing HFrEF patients (n = 20) and healthy controls (n = 19). Rho-kinase activity in circulating leucocytes (peripheral blood mononuclear cells, PBMCs) was determined by myosin light chain phosphatase 1 (MYPT1) and ezrin-radixin-moesin (ERM) phosphorylation. Rho-kinase cascade proteins phosphorylation p38-MAPK, myosin light chain-2, JAK and JNK were also analysed along with apoptosis. RESULTS: MYPT1 and ERM phosphorylation were significantly elevated in HFrEF patients, (3.9- and 4.8-fold higher than in controls, respectively). JAK phosphorylation was significantly increased by 300% over controls. Phosphorylation of downstream molecules p38-MAPK and myosin light chain-2 was significantly higher by 360% and 490%, respectively, while JNK phosphorylation was reduced by 60%. Catecholamine and angiotensin II levels were significantly higher in HFrEF patients, while angiotensin-(1-9) levels were lower. Apoptosis in circulating leucocytes was significantly increased in HFrEF patients by 2.8-fold compared with controls and significantly correlated with Rho-kinase activation. CONCLUSION: Rho-kinase pathway is activated in PMBCs from HFrEF patients despite optimal treatment, and it is closely associated with neurohormonal activation and with apoptosis. ROCK cascade inhibition might induce clinical benefits in HFrEF patients, and its assessment in PMBCs could be useful to evaluate reverse remodelling and disease regression.

TIMAP inhibits endothelial myosin light chain phosphatase by competing with MYPT1 for the catalytic protein phosphatase 1 subunit PP1cß.

Transforming growth factor-ß membrane associated protein (TIMAP) is an endothelial cell (EC)-predominant PP1 regulatory subunit and a member of the myosin phosphatase target (MYPT) protein family. The MYPTs preferentially bind the catalytic protein phosphatase 1 subunit PP1cß, forming myosin phosphatase holoenzymes. We investigated whether TIMAP/PP1cß could also function as a myosin phosphatase. Endogenous PP1cß, myosin light chain 2 (MLC2), and myosin IIA heavy chain coimmunoprecipitated from EC lysates with endogenous TIMAP, and endogenous MLC2 colocalized with TIMAP in EC projections. Purified recombinant GST-TIMAP interacted directly with purified recombinant His-MLC2. However, TIMAP overexpression in EC enhanced MLC2 phosphorylation, an effect not observed with a TIMAP mutant that does not bind PP1cß. Conversely, MLC2 phosphorylation was reduced in lung lysates from TIMAP-deficient mice and upon silencing of endogenous TIMAP expression in ECs. Ectopically expressed TIMAP slowed the rate of MLC2 dephosphorylation, an effect requiring TIMAP-PP1cß interaction. The association of MYPT1 with PP1cß was profoundly reduced in the presence of excess TIMAP, leading to proteasomal MYPT1 degradation. In the absence of TIMAP, MYPT1-associated PP1cß readily bound immobilized microcystin-LR, an active-site inhibitor of PP1c. By contrast, TIMAP-associated PP1cß did not interact with microcystin-LR, indicating that the active site of PP1cß is blocked when it is bound to TIMAP. Thus, TIMAP inhibits myosin phosphatase activity in ECs by competing with MYPT1 for PP1cß and blocking the PP1cß active site.

p90 ribosomal S6 kinase (RSK) phosphorylates myosin phosphatase and thereby controls edge dynamics during cell migration.

Cell migration is essential to embryonic development, wound healing, and cancer cell dissemination. Cells move via leading-edge protrusion, substrate adhesion, and retraction of the cell's rear. The molecular mechanisms by which extracellular cues signal to the actomyosin cytoskeleton to control these motility mechanics are poorly understood. The growth factor-responsive and oncogenically activated protein extracellular signal-regulated kinase (ERK) promotes motility by signaling in actin polymerization-mediated edge protrusion. Using a combination of immunoblotting, co-immunoprecipitation, and myosin-binding experiments and cell migration assays, we show here that ERK also signals to the contractile machinery through its substrate, p90 ribosomal S6 kinase (RSK). We probed the signaling and migration dynamics of multiple mammalian cell lines and found that RSK phosphorylates myosin phosphatase-targeting subunit 1 (MYPT1) at Ser-507, which promotes an interaction of Rho kinase (ROCK) with MYPT1 and inhibits myosin targeting. We find that by inhibiting the myosin phosphatase, ERK and RSK promote myosin II-mediated tension for lamella expansion and optimal edge dynamics for cell migration. These findings suggest that ERK activity can coordinately amplify both protrusive and contractile forces for optimal cell motility.

Downregulation of MYPT1 increases tumor resistance in ovarian cancer by targeting the Hippo pathway and increasing the stemness.

BACKGROUND: Ovarian cancer is one of the most common and malignant cancers, partly due to its late diagnosis and high recurrence. Chemotherapy resistance has been linked to poor prognosis and is believed to be linked to the cancer stem cell (CSC) pool. Therefore, elucidating the molecular mechanisms mediating therapy resistance is essential to finding new targets for therapy-resistant tumors. METHODS: shRNA depletion of MYPT1 in ovarian cancer cell lines, miRNA overexpression, RT-qPCR analysis, patient tumor samples, cell line- and tumorsphere-derived xenografts, in vitro and in vivo treatments, analysis of data from ovarian tumors in public transcriptomic patient databases and in-house patient cohorts. RESULTS: We show that MYPT1 (PPP1R12A), encoding myosin phosphatase target subunit 1, is downregulated in ovarian tumors, leading to reduced survival and increased tumorigenesis, as well as resistance to platinum-based therapy. Similarly, overexpression of miR-30b targeting MYPT1 results in enhanced CSC-like properties in ovarian tumor cells and is connected to the activation of the Hippo pathway. Inhibition of the Hippo pathway transcriptional co-activator YAP suppresses the resistance to platinum-based therapy induced by either low MYPT1 expression or miR-30b overexpression, both in vitro and in vivo. CONCLUSIONS: Our work provides a functional link between the resistance to chemotherapy in ovarian tumors and the increase in the CSC pool that results from the activation of the Hippo pathway target genes upon MYPT1 downregulation. Combination therapy with cisplatin and YAP inhibitors suppresses MYPT1-induced resistance, demonstrating the possibility of using this treatment in patients with low MYPT1 expression, who are likely to be resistant to platinum-based therapy.

Rho kinase cascade activation in circulating leukocytes in patients with diabetes mellitus type 2.

BACKGROUND: The intracellular ROCK signaling pathway is an important modulator of blood pressure and of cardiovascular and renal remodeling when Rho-kinase activity is increased. Besides, in preclinical models of diabetes, ROCK activation has also a role in abnormal glucose metabolism as well as in subsequent vascular and myocardial dysfunction. In humans, there are a few data assessing ROCK activation in patients with type 2 diabetes mellitus (T2D) and no studies assessing upstream/downstream components of the ROCK pathway. We assessed here levels of ROCK activation and some of the RhoA/ROCK cascade molecules in peripheral blood mononuclear cells (PBMCs) in T2D patients under current treatment. METHODS: Cross-sectional observational study comparing 28 T2D patients under current antidiabetic treatment with 31 consecutive healthy subjects, matched by age and gender. Circulating levels of malondialdehyde, angiotensin II and inflammatory cytokines IL-6 and IL-8 were determined in all subjects. ROCK activation in PMBCs, upstream and downstream cascade proteins, and levels of the proinflammatory molecules VCAM, ICAM-1 and IL-8 were determined in their PMBCs by Western blot. RESULTS: Compared to healthy controls, ROCK activation in T2D patients measured by 2 direct ROCK targets in PBMCs was increased by 420 and 570% (p < 0001) and it correlated significantly with serum glucose levels. p38 MAPK phosphorylation (downstream from ROCK) and JAK-2 (upstream from ROCK) were significantly higher in the T2D patients by 580% and 220%, respectively. In T2D patients, significantly increased PBMC levels of the proinflammatory molecules VCAM-1, ICAM-1 and IL-8 were observed compared to control subjects (by 180%, 360% and 260%, respectively). Circulating levels of Ang II and MDA were significantly higher in T2D patients by 29 and 63%, respectively. CONCLUSIONS: T2D patients under treatment with glucose-lowering drugs, antihypertensive treatment as well as with statins have significantly increased ROCK activation in their circulating leukocytes along with higher phosphorylation of downstream cascade proteins despite pharmacologic treatment, along with increased plasma angiotensin II and MDA levels. ROCK inhibition might have an additional role in the prevention and treatment of T2D.