PPP1R12A is a recycling endosomal phosphatase that facilitates YAP activation.

Yes-associated protein (YAP) is a transcriptional coactivator that is essential for the malignancy of various cancers. We have previously shown that YAP activity is positively regulated by phosphatidylserine (PS) in recycling endosomes (REs). However, the mechanism by which YAP is activated by PS in REs remains unknown. In the present study, we examined a group of protein phosphatases (11 phosphatases) that we had identified previously as PS-proximity protein candidates. Knockdown experiments of these phosphatases suggested that PPP1R12A, a regulatory subunit of the myosin phosphatase complex, was essential for YAP-dependent proliferation of triple-negative breast cancer MDA-MB-231 cells. Knockdown of PPP1R12A increased the level of phosphorylated YAP, reduced that of YAP in the nucleus, and suppressed the transcription of CTGF (a YAP-regulated gene), reinforcing the role of PPP1R12A in YAP activation. ATP8A1 is a PS-flippase that concentrates PS in the cytosolic leaflet of the RE membrane and positively regulates YAP signalling. In subcellular fractionation experiments using cell lysates, PPP1R12A in control cells was recovered exclusively in the microsomal fraction. In contrast, a fraction of PPP1R12A in ATP8A1-depleted cells was recovered in the cytosolic fraction. Cohort data available from the Cancer Genome Atlas showed that high expression of PPP1R12A, PP1B encoding the catalytic subunit of the myosin phosphatase complex, or ATP8A1 correlated with poor prognosis in breast cancer patients. These results suggest that the "ATP8A1-PS-YAP phosphatase" axis in REs facilitates YAP activation and thus cell proliferation.

ROK and RSK2-kinase pathways differ between senescent human renal and mesenteric arteries.

OBJECTIVE: Small arteries from different organs vary with regard to the mechanisms that regulate vasoconstriction. This study investigated the impact of advanced age on the regulation of vasoconstriction in isolated human small arteries from kidney cortex and periintestinal mesenteric tissue. METHODS: Renal and mesenteric tissues were obtained from patients (mean age 71 ±â€Š9 years) undergoing elective surgery. Furthermore, intrarenal and mesenteric arteries from young and aged mice were studied. Arteries were investigated by small vessel myography and western blot. RESULTS: Human intrarenal arteries (h-RA) showed higher stretch-induced tone and higher reactivity to α 1 adrenergic receptor stimulation than human mesenteric arteries (h-MA). Rho-kinase (ROK) inhibition resulted in a greater decrease in Ca 2+ and depolarization-induced tone in h-RA than in h-MA. Basal and α 1 adrenergic receptor stimulation-induced phosphorylation of the regulatory light chain of myosin (MLC 20 ) was higher in h-RA than in h-MA. This was associated with higher ROK-dependent phosphorylation of the regulatory subunit of myosin light-chain-phosphatase (MLCP), MYPT1-T853. In h-RA phosphorylation of ribosomal S6-kinase II (RSK2-S227) was significantly higher than in h-MA. Stretch-induced tone and RSK2 phosphorylation was also higher in interlobar arteries (m-IAs) from aged mice than in respective vessels from young mice and in murine mesenteric arteries (m-MA) from both age groups. CONCLUSION: Vasoconstriction in human intrarenal arteries shows a greater ROK-dependence than in mesenteric arteries. Activation of RSK2 may contribute to intrarenal artery tone dysregulation associated with aging. Compared with h-RA, h-MA undergo age-related remodeling leading to a reduction of the contractile response to α 1 adrenergic stimulation.

MYPT1 inhibits the metastasis of renal clear cell carcinoma via the MAPK8/N-cadherin pathway.

Myosin phosphatase target subunit 1 (MYPT1) is a subunit of myosin phosphatase that is capable of regulating smooth muscle contraction. MYPT1 has been reported to be involved in a wide variety of tumours, but its expression and biological functions in renal clear cell carcinoma (ccRCC) remain obscure. Herein, we analysed the relationship between patient clinicopathological characteristics and MYPT1 expression levels in ccRCC patients using a tissue microarray (TMA) and data retrieved from the TCGA-KIRC dataset. MYPT1 was overexpressed or depleted using siRNA in ccRCC cells to assess the effects on migration and invasion in vitro and in vivo. Additionally, RNA-sequencing and bioinformatics analysis were performed to investigate the precise mechanism. MYPT1 expression in ccRCC tissues was observed to be lower than that in nonmalignant tissues (P < 0.05). In addition, MYPT1 downregulation was closely linked to advanced pathological stage (P < 0.05), and poor OS (overall survival; P < 0.05). Functionally, increased expression of MYPT1 suppressed ccRCC migration and invasion in vitro, and inhibited tumour metastasis in vivo. In addition, MYPT1 overexpression exerted its suppressive effects via the MAPK8/N-cadherin pathway in ccRCC.

MYPT1-PP1ß phosphatase negatively regulates both chromatin landscape and co-activator recruitment for beige adipogenesis.

Protein kinase A promotes beige adipogenesis downstream from ß-adrenergic receptor signaling by phosphorylating proteins, including histone H3 lysine 9 (H3K9) demethylase JMJD1A. To ensure homeostasis, this process needs to be reversible however, this step is not well understood. We show that myosin phosphatase target subunit 1- protein phosphatase 1ß (MYPT1-PP1ß) phosphatase activity is inhibited via PKA-dependent phosphorylation, which increases phosphorylated JMJD1A and beige adipogenesis. Mechanistically, MYPT1-PP1ß depletion results in JMJD1A-mediated H3K9 demethylation and activation of the Ucp1 enhancer/promoter regions. Interestingly, MYPT1-PP1ß also dephosphorylates myosin light chain which regulates actomyosin tension-mediated activation of YAP/TAZ which directly stimulates Ucp1 gene expression. Pre-adipocyte specific Mypt1 deficiency increases cold tolerance with higher Ucp1 levels in subcutaneous white adipose tissues compared to control mice, confirming this regulatory mechanism in vivo. Thus, we have uncovered regulatory cross-talk involved in beige adipogenesis that coordinates epigenetic regulation with direct activation of the mechano-sensitive YAP/TAZ transcriptional co-activators.

Epigallocatechine-3-gallate Inhibits the Adipogenesis of Human Mesenchymal Stem Cells via the Regulation of Protein Phosphatase-2A and Myosin Phosphatase.

Epigallocatechin-3-gallate (EGCG) has widespread effects on adipocyte development. However, the molecular mechanisms of EGCG are not fully understood. We investigate the adipogenic differentiation of human-derived mesenchymal stem cells, including lipid deposition and changes in the expression and phosphorylation of key transcription factors, myosin, protein phosphatase-2A (PP2A), and myosin phosphatase (MP). On day 6 of adipogenic differentiation, EGCG (1-20 µM) suppressed lipid droplet formation, which was counteracted by an EGCG-binding peptide for the 67 kDa laminin receptor (67LR), suggesting that EGCG acts via 67LR. EGCG decreased the phosphorylation of CCAAT-enhancer-binding protein beta via the activation of PP2A in a protein kinase A (PKA)-dependent manner, leading to the partial suppression of peroxisome proliferator-activated receptor gamma (PPARγ) and adiponectin expression. Differentiated cells exhibited a rounded shape, cortical actin filaments, and lipid accumulation. The EGCG treatment induced cell elongation, stress fiber formation, and less lipid accumulation. These effects were accompanied by the degradation of the MP target subunit-1 and increased the phosphorylation of the 20 kDa myosin light chain. Our results suggest that EGCG acts as an agonist of 67LR to inhibit adipogenesis via the activation of PP2A and suppression of MP. These events are coupled with the decreased phosphorylation and expression levels of adipogenic transcription factors and changes in cell shape, culminating in curtailed adipogenesis.

Shuxuetong injection and its peptides enhance angiogenesis after hindlimb ischemia by activating the MYPT1/LIMK1/Cofilin pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuxuetong (SXT) injection is formulated by leech and earthworm, has been widely used in the treatment of thrombotic cardiovascular and cerebrovascular diseases with remarkable clinical efficacy. AIM OF THE STUDY: The purpose of this study is to investigate the protective mechanism of SXT injection on the mice model of hindlimb ischemia, and to evaluate the angiogenic effects of SXT injection and its main active substances. MATERIALS AND METHODS: Hindlimb ischemia was induced by left femoral artery ligation. After operation, the mice were injected with saline, 10 mg/kg/d cilostazol, 37.5 mg/kg/d SXT injection, 75 mg/kg/d SXT injection and 150 mg/kg/d SXT injection via tail vein for 4 weeks. Ischemia severity was assessed using laser Doppler perfusion imaging system. Tissue recovery and capillary density were evaluated by histological and immunofluorescent staining. Vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor (PDGF-BB) expression were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses. Human umbilical vein endothelial cells (HUVECs) proliferation was measured using a BrdU kit and the viability of HUVECs was performed by MTT assay. Migration of HUVECs was performed by the wound healing method and a modified transwell assay. Capillary tube formation by HUVECs was examined by using Matrigel assay. Western blotting was used to detect the expressions of p-Cofilin, p-MYPT1, and p-LIMK1. RESULTS: SXT injection treatment significantly restored the blood flow and reduced tissue injury in mouse gastrocnemius muscle. SXT injection treatment increased capillary density and promoted angiogenesis in hindlimb ischemia. Moreover, SXT injection enhanced the expression of VEGF-A and PDGF-BB at both mRNA and protein levels in ischemic tissue of mice. SXT injection and its main active peptides dramatically increased the migration and capillary tube formation of HUVECs. SXT injection and its peptides enhanced protein expressions of the phosphorylation of MYPT1, Cofilin, and LIMK1. DSYVGDEAQSKR, YNELRVAPEEHP, and IQFLPEGSPVTM may act as the active components of SXT injection. CONCLUSION: SXT injection promoted angiogenesis and improved function recovery in hindlimb ischemia mice by regulation of VEGF-A/PDGF-BB. Moreover, SXT injection and its active peptides induced cell migration and tube formation in HUVECs through activating the MYPT1/LIMK1/Cofilin pathway. This study provided experimental basis for SXT injection in the treatment of ischemic diseases and revealed the effective substance of SXT injection in regulating angiogenesis, providing better evidence for the clinical application of SXT injection.

MYPT1/PP1-Mediated EZH2 Dephosphorylation at S21 Promotes Epithelial-Mesenchymal Transition in Fibrosis through Control of Multiple Families of Genes.

The methyltransferase EZH2 plays an important role in regulating chromatin conformation and gene transcription. Phosphorylation of EZH2 at S21 by AKT kinase suppresses its function. However, protein phosphatases responsible for the dephosphorylation of EZH2-S21 remain elusive. Here, it is demonstrated that EZH2 is highly expressed in the ocular lens, and AKT-EZH2 axis is important in TGFß-induced epithelial-mesenchymal transition (EMT). More importantly, it is identified that MYPT1/PP1 dephosphorylates EZH2-S21 and thus modulates its functions. MYPT1 knockout accelerates EMT, but expression of the EZH2-S21A mutant suppresses EMT through control of multiple families of genes. Furthermore, the phosphorylation status and gene expression modulation of EZH2 are implicated in control of anterior subcapsular cataracts (ASC) in human and mouse eyes. Together, the results identify the specific phosphatase for EZH2-S21 and reveal EZH2 dephosphorylation control of several families of genes implicated in lens EMT and ASC pathogenesis. These results provide important novel information in EZH2 function and regulation.

Smoothelin-Like Protein 1 Regulates the Thyroid Hormone-Induced Homeostasis and Remodeling of C2C12 Cells via the Modulation of Myosin Phosphatase.

The pathological elevation of the active thyroid hormone (T3) level results in the manifestation of hyperthyroidism, which is associated with alterations in the differentiation and contractile function of skeletal muscle (SKM). Myosin phosphatase (MP) is a major cellular regulator that hydrolyzes the phosphoserine of phosphorylated myosin II light chain. MP consists of an MYPT1/2 regulatory and a protein phosphatase 1 catalytic subunit. Smoothelin-like protein 1 (SMTNL1) is known to inhibit MP by directly binding to MP as well as by suppressing the expression of MYPT1 at the transcriptional level. Supraphysiological vs. physiological concentration of T3 were applied on C2C12 myoblasts and differentiated myotubes in combination with the overexpression of SMTNL1 to assess the role and regulation of MP under these conditions. In non-differentiated myoblasts, MP included MYPT1 in the holoenzyme complex and its expression and activity was regulated by SMTNL1, affecting the phosphorylation level of MLC20 assessed using semi-quantitative Western blot analysis. SMTNL1 negatively influenced the migration and cytoskeletal remodeling of myoblasts measured by high content screening. In contrast, in myotubes, the expression of MYPT2 but not MYPT1 increased in a T3-dependent and SMTNL1-independent manner. T3 treatment combined with SMTNL1 overexpression impeded the activity of MP. In addition, MP interacted with Na+/K+-ATPase and dephosphorylated its inhibitory phosphorylation sites, identifying this protein as a novel MP substrate. These findings may help us gain a better understanding of myopathy, muscle weakness and the disorder of muscle regeneration in hyperthyroid patients.

12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) induces cell growth and improves barrier function through BLT2 interaction in intestinal epithelial Caco-2 cell cultures.

12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) is an unusual product of the cyclooxygenase pathway that is an endogenous ligand of the low-affinity receptor for leukotriene 4 (LTB4), BLT2. Recent findings suggested that BLT2 possibly plays an important role in the healing of intestinal lesions and the regulation of barrier function. Here, we studied the role of 12-HHT on intestinal epithelial cell growth and the paracellular permeability of intestinal epithelium using Caco-2 cell cultures as experimental model. Our results demonstrated that 12-HHT stimulates intestinal epithelial Caco-2 cell growth through 12-HHT-BLT2-p38-PKC axis and improves paracellular permeability in differentiated Caco-2 cell cultures through the regulation of tight junction elements such as myosin light chain phosphorylation through 12-HHT-BLT2-p38-PKC-MYPT1 axis. Thus, 12-HHT-BLT2 interaction can be involved in intestinal epithelial cell growth and consequently in the epithelium regeneration/repair processes, together with an interesting improvement on the paracellular permeability. These effects appoint that 12-HHT/BLT2 axis may be a suitable strategy for treating wound healing epithelium and barrier-disrupted intestinal processes.

MYPT1, regulated by miR-19b-3p inhibits the progression of non-small cell lung cancer via inhibiting the activation of wnt/ß-catenin signaling.

AIMS: Myosin phosphatase targeting protein 1 (MYPT1) was identified to function as a tumor suppressor in several kinds of cancers, but its role and the molecular mechanisms in non-small cell lung cancer (NSCLC) remain undiscovered. Herein, we aimed to reveal MYPT1 expression pattern and role in NSCLC, and investigate the underlying mechanisms. MAIN METHODS: Sixty-eight paired NSCLC tissues and the adjacent normal tissues were included in this study. Western blotting and quantitative reverse transcription-polymerase chain (qPCR) technologies were applied for protein and RNA detection. CCK-8, colony formation, flow cytometry, wound healing, transwell chambers coated with Matrigel and in vivo experiments were applied to detect cell viability, colony formation, apoptosis, migration, invasiveness and tumorigenesis, respectively. KEY FINDINGS: MYPT1 expressed at a lower level in NSCLC tissues as compared with the adjacent normal tissues, which predicted advanced clinic process and poor prognosis. Overexpression of MYPT1 resulted in obvious inhibitions in cell viability, colony formation, migration, invasiveness and tumorigenesis, and induced cell apoptotic rates, as well as decreased the expression levels of ß-catenin and TCF4. Besides, overexpression of ß-catenin weakened the above roles of MYPT1. In addition, the luciferase gene reporter assay verified that MYPT1 was a target of miR-19b-3p. Further experiments showed that miR-19b-3p promoted cell viability, invasiveness and migration and repressed cell apoptosis by targeting MYPT1. SIGNIFICANCE: In conclusion, this study demonstrates that MYPT1, regulated by miR-19b-3p, inhibits the progression of NSCLC via inhibiting the activation of wnt/ß-catenin signaling.

Cadmium-induced hypertension is associated with renal myosin light chain phosphatase inhibition via increased T697 phosphorylation and p44 mitogen-activated protein kinase levels.

Dietary intake of the heavy metal cadmium (Cd2+) is implicated in hypertension, but potassium supplementation reportedly mitigates hypertension. This study aims to elucidate the hypertensive mechanism of Cd2+. Vascular reactivity and protein expression were assessed in Cd2+-exposed rats for 8 weeks to determine the calcium-handling effect of Cd2+ and the possible signaling pathways and mechanisms involved. Cd2+ induced hypertension in vivo by significantly (p < 0.001) elevating systolic blood pressure (160 ± 2 and 155 ± 1 vs 120 ± 1 mm Hg), diastolic blood pressure (119 ± 2 and 110 ± 1 vs 81 ± 1 mm Hg), and mean arterial pressure (133 ± 2 and 125 ± 1 vs 94 ± 1 mm Hg) (SBP, DBP, and MAP, respectively), while potassium supplementation protected against elevation of these parameters. The mechanism involved augmentation of the phosphorylation of renal myosin light chain phosphatase targeting subunit 1 (MYPT1) at threonine 697 (T697) (2.58 ± 0.36 vs 1 ± 0) and the expression of p44 mitogen-activated protein kinase (MAPK) (1.78 ± 0.20 vs 1 ± 0). While acetylcholine (ACh)-induced relaxation was unaffected, 5 mg/kg b.w. Cd2+ significantly (p < 0.001) attenuated phenylephrine (Phe)-induced contraction of the aorta, and 2.5 mg/kg b.w. Cd2+ significantly (p < 0.05) augmented sodium nitroprusside (SNP)-induced relaxation of the aorta. These results support the vital role of the kidney in regulating blood pressure changes after Cd2+ exposure, which may be a key drug target for hypertension management. Given the differential response to Cd2+, it is apparent that its hypertensive effects could be mediated by myosin light chain phosphatase (MLCP) inhibition via phosphorylation of renal MYPT1-T697 and p44 MAPK. Further investigation of small arteries and the Rho-kinase/MYPT1 interaction is recommended.

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.

Novel circular RNA circSOBP governs amoeboid migration through the regulation of the miR-141-3p/MYPT1/p-MLC2 axis in prostate cancer.

BACKGROUND: Metastatic prostate cancer is a fatal disease despite multiple new approvals in recent years. Recent studies revealed that circular RNAs (circRNAs) can be involved in cancer metastasis. Defining the role of circRNAs in prostate cancer metastasis and discovering therapeutic targets that block cancer metastasis is of great significance for the treatment of prostate cancer. METHODS: The circSOBP levels in prostate cancer (PCa) were determined by qRT-PCR. We evaluated the function of circSOBP using a transwell assay and nude mice lung metastasis models. Immunofluorescence assay and electron microscopic assay were applied to determine the phenotypes of prostate cancer cells' migration. We used fluorescence in situ hybridization assay to determine the localization of RNAs. Dual luciferase and rescue assays were applied to verify the interactions between circSOBP, miR-141-3p, MYPT1, and phosphomyosin light chain (p-MLC2). RESULTS: We observed that circSOBP level was significantly lower in PCa specimens compared with adjacent noncancerous prostate specimens, and was correlated with the grade group of PCa. Overexpression of circSOBP suppressed PCa migration and invasion in vitro and metastasis in vivo. CircSOBP depletion increased migration and invasion and induced amoeboid migration of PCa cells. Mechanistically, circSOBP bound miR-141-3p and regulated the MYPT1/p-MLC2 axis. Moreover, the depletion of MYPT1 reversed the inhibitory effect of circSOBP on the migration and invasion of PCa cells. Complementary intronic Alu elements induced but were not necessary for the formation of circSOBP. The nuclear export of circSOBP was mediated by URH49. CONCLUSION: Our results suggest that circSOBP suppresses amoeboid migration of PCa cells and inhibits migration and invasion through sponging miR-141-3p and regulating the MYPT1/p-MLC2 axis.

Activation of Myosin Phosphatase by Epigallocatechin-Gallate Sensitizes THP-1 Leukemic Cells to Daunorubicin.

BACKGROUND: The Myosin Phosphatase (MP) holoenzyme is composed of a Protein Phosphatase type 1 (PP1) catalytic subunit and a regulatory subunit termed Myosin Phosphatase Target subunit 1 (MYPT1). Besides dephosphorylation of myosin, MP has been implicated in the control of cell proliferation via dephosphorylation and activation of the tumor suppressor gene products, retinoblastoma protein (pRb) and merlin. Inhibition of MP was shown to attenuate the drug-induced cell death of leukemic cells by chemotherapeutic agents, while activation of MP might have a sensitizing effect. OBJECTIVE: Recently, Epigallocatechin-Gallate (EGCG), a major component of green tea, was shown to activate MP by inducing the dephosphorylation of MYPT1 at phospho-Thr696 (MYPT1pT696), which might confer enhanced chemosensitivity to cancer cells. METHODS: THP-1 leukemic cells were treated with EGCG and Daunorubicin (DNR) and cell viability was analyzed. Phosphorylation of tumor suppressor proteins was detected by Western blotting. RESULTS: EGCG or DNR (at sub-lethal doses) alone had moderate effects on cell viability, while the combined treatment caused a significant decrease in the number of viable cells by enhancing apoptosis and decreasing proliferation. EGCG plus DNR decreased the phosphorylation level of MYPT1pT696, which was accompanied by prominent dephosphorylation of pRb. In addition, significant dephosphorylation of merlin was observed when EGCG and DNR were applied together. CONCLUSION: Our results suggest that EGCG-induced activation of MP might have a regulatory function in mediating the chemosensitivity of leukemic cells via dephosphorylation of tumor suppressor proteins.

Statins Suppress TGF-ß2-Mediated MMP-2 and MMP-9 Expression and Activation Through RhoA/ROCK Inhibition in Astrocytes of the Human Optic Nerve Head.

Purpose: Matrix metalloproteinases (MMPs) are involved in extracellular matrix (ECM) maintenance and remodeling. The present study aimed to determine whether transforming growth factor (TGF)-ß2 regulates MMP-2 and MMP-9 levels and activities in astrocytes derived from the optic nerve head (ONH) and the role of statins in such modulation. Methods: Primary astrocytes cultured from the lamina cribrosa of human donor ONHs were incubated with three types of statins (5 µg/mL) for 1 hour followed by recombinant TGF-ß2 (5 ng/mL) for various periods to test their effects. Levels and activities of MMP-2 and MMP-9 in astrocytes in vitro were determined by western blotting and zymography, respectively. Levels of phosphorylated myosin phosphatase target subunit 1 (MYPT1) in astrocyte lysates were determined by western blotting, and those of phosphorylated myosin light chain (MLC) were determined by western blotting and immunocytochemistry. Results: MMP-2 and MMP-9 levels were upregulated by TGF-ß2 in human ONH astrocytes. Prior incubation with simvastatin, lovastatin, and atorvastatin inhibited TGF-ß2-mediated MMP-2 and MMP-9 expression and activities. Prior incubation with statins downregulated the TGF-ß2-induced phosphorylation of MYPT1 and MLC, which are downstream substrates of RhoA and ROCKs. Conclusions: Statins inhibited the TGF-ß2-mediated regulation of MMP-2 and MMP-9 by inhibiting the RhoA/ROCK signaling pathway. Considering the role of MMP in ECM remodeling, the present findings support the notion that statins positively impact ECM remodeling within the ONH.

Chromogranin A (CGA)-derived polypeptide (CGA47-66) inhibits TNF-α-induced vascular endothelial hyper-permeability through SOC-related Ca2+ signaling.

CGA1-78 (Vasostatin-1, VS-1) a N-terminal Chromogranin A (CGA)-derived peptide, has been shown to have a protective effect against TNF-α-induced impairment of endothelial cell integrity. However, the mechanisms of this effect have not yet been clarified. CGA47-66 (Chromofungin, CHR) is an important bioactive fragment of CGA1-78. The present study aims to explore the protective effects of CHR on the vascular endothelial cell barrier response to TNF-α and its related Ca2+ signaling mechanisms. EA.hy926 cells were used as a vascular endothelial culture model. The synthetic peptides CHR and CGA4-16 were assessed for their ability to suppress TNF-α-induced EA.hy926 cells hyper-permeability through Transwell® and TEER assays. Changes in [Ca2+]i were measured through confocal laser scanning microscopy. SOC channel currents (Isoc) were measured via patch-clamp analysis. RT-PCR and western blot were used to analyze mRNA and protein expression of the transient receptor potential channels TRPC1 and TRPC4, respectively. FITC and rhodamine-phalloidin fluorescence were used to assess cell morphology and the distribution of MyPT-1 and F-actin. Compared to untreated cells, TNF-α increased the permeability of EA.hy926 cells that was inhibited by pre-treatment with CHR (10-1000 nM) in concentration-dependent manner, and the effect was most obvious at 100 nM, but CGA4-16 (100 nM) had no effect. TNF-α treatment increased the phosphorylation of MyPT-1 and stress fiber formation. CHR (10-1000 nM) pretreatment inhibited the cytoskeletal rearrangements and increased [Ca2+]i in response to TNF-α treatment. CHR also reduced TRPC1 expression following TNF-α induction. Similar to SOC inhibitor 2-APB, CHR suppressed IP3 mediated SOC activation. These findings suggest that CHR inhibits TNF-α-induced Ca2+ influx and protects the barrier function of vascular endothelial cells, and that these effects are related to the inhibition of SOC and Ca2+ signaling by CHR.

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.

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-associated kinase and zipper-interacting protein kinase, but not myosin light chain kinase, are involved in the regulation of myosin phosphorylation in serum-stimulated human arterial smooth muscle cells.

Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30-120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.