Thrombin stimulates stress fiber assembly in RPE cells by PKC/CPI-17-mediated MLCP inactivation

Ruiz-Loredo, Ariadna Yolanda; López, Edith; López-Colomé, Ana Marı́a

doi:10.1016/j.exer.2012.01.008

Cited by 11 publications

(6 citation statements)

References 67 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We also found that thrombin-induced Ca 2+ sensitization was inhibited by GF-109203X, suggesting that thrombin activates the PKC pathway in SMCs of the small airways. This notion is supported by a recent study showing that proteinase-activated receptor 1 stimulation with thrombin results in PKC activation, CPI-17 phosphorylation, inhibition of MLCP, and rMLC phosphorylation during assembly and contraction of stress fibers in retinal pigment epithelium (Ruiz-Loredo et al, 2012).…”

Section: Pkc Activation Induces Small Airway Smc Twitchingsupporting

confidence: 71%

“…Thrombin induces Ca 2+ sensitization of the contraction mediated by stress fibers in retinal pigment epithelial cells by activating PKC and thereby promoting phosphorylation of PKC-potentiated PP1 inhibitory protein of 17 kD (CPI-17) and rMLC (Ruiz-Loredo et al, 2012). Here, we investigated the role of PKC in small airway contraction by characterizing its contribution to: (a) the contractile response, (b) Ca 2+ signaling in SMCs, (c) changes in SMC Ca 2+ sensitivity, and (d) the cellular mechanisms underlying Ca 2+ oscillations and Ca 2+ sensitization.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Ca²⁺oscillations, Ca²⁺sensitization, and contraction activated by protein kinase C in small airway smooth muscle

Mukherjee¹,

Trice²,

Shinde³

et al. 2013

J Cell Biol

View full text Add to dashboard Cite

Section: Pkc Activation Induces Small Airway Smc Twitchingsupporting

confidence: 71%

Section: Introductionmentioning

confidence: 99%

Ca²⁺oscillations, Ca²⁺sensitization, and contraction activated by protein kinase C in small airway smooth muscle

Mukherjee¹,

Trice²,

Shinde³

et al. 2013

J Cell Biol

View full text Add to dashboard Cite

“…1 A). The concentration of Y-27632 was chosen based on the work of Ruiz-Loredo et al which utilized a similar stress fiber disrupting property of the inhibitor 45 . The cited work used 0.5, 1 and 5 µM of the inhibitor; however, we found that employing 2 μM of the inhibitor was sufficient to dissolve stress fibers, while at this concentration we could minimize the aspecific effects of the inhibitor.…”

Section: Resultsmentioning

confidence: 99%

Effect of allosteric inhibition of non-muscle myosin 2 on its intracellular diffusion

Horváth

Gyimesi

Várkuti

et al. 2020

Sci Rep

View full text Add to dashboard Cite

Subcellular dynamics of non-muscle myosin 2 (NM2) is crucial for a broad-array of cellular functions. To unveil mechanisms of NM2 pharmacological control, we determined how the dynamics of NM2 diffusion is affected by NM2′s allosteric inhibitors, i.e. blebbistatin derivatives, as compared to Y-27632 inhibiting ROCK, NM2′s upstream regulator. We found that NM2 diffusion is markedly faster in central fibers than in peripheral stress fibers. Y-27632 accelerated NM2 diffusion in both peripheral and central fibers, whereas in peripheral fibers blebbistatin derivatives slightly accelerated NM2 diffusion at low, but markedly slowed it at high inhibitor concentrations. In contrast, rapid NM2 diffusion in central fibers was unaffected by direct NM2 inhibition. Using our optopharmacological tool, Molecular Tattoo, sub-effective concentrations of a photo-crosslinkable blebbistatin derivative were increased to effective levels in a small, irradiated area of peripheral fibers. These findings suggest that direct allosteric inhibition affects the diffusion profile of NM2 in a markedly different manner compared to the disruption of the upstream control of NM2. The pharmacological action of myosin inhibitors is channeled through autonomous molecular processes and might be affected by the load acting on the NM2 proteins. Stress fibers are contractile actomyosin bundles in non-muscle cells, playing fundamental roles in diverse biological functions 1-4. Stress fibers and stress fiber-like structures play central roles in cell division as key components of the cleavage furrow 5 , determine cancer cell motility during metastasis 6 , influence cancer cell growth 7 , and they have been demonstrated as crucial elements in neuronal plasticity including neurite outgrowth 4,8-11. These force-generating structures consist of actin filaments and different co-assemblies of non-muscle myosin 2 (NM2) isoforms 12,13. The two major stress fiber-forming myosin isoforms in human cells are NM2A and NM2B, that together build up mixed minifilaments of various length and thickness 14. Stress fibers may be categorized as central and peripheral, based on their subcellular localization. Central stress fibers include those localized ventrally, dorsally, in transverse arcs and those associated around the nucleus. Their main roles are in cell adhesion, motility, cell division and cell shape determination 15,16. Peripheral stress fibers are located at the edge of the cell. Laser nanosurgery experiments have shown that incision of peripheral stress fibers causes them to retract significantly, suggesting that these structures are under larger mechanical strain than their central counterparts 17. Resisting loads on NM2 heads have been shown to significantly decrease the rate of ADP release from actin-bound myosin heads, resulting in slower actomyosin dissociation 18-20. NM2 minifilaments are also activated or repressed by the phosphorylation or dephosphorylation of the myosin light chains (MLC), respectively 21-24. Rho-dependent kinase (ROCK) positively regulates...

show abstract

“…It is a PKC-activated phosphatase inhibitor with a relative molecular weight of 17 kDa, and is widely expressed in vascular and visceral smooth muscle cells ( 11 ). Studies have demonstrated that PKC phosphorylates CPI-17 at Thr-38, then phosphorylated CPI-17 binds with the 38 kDa MLCP catalytic subunit (the main unit responsible for enzymatic activity), which causes a 1,000-fold inhibition of MLCP, thus increasing calcium sensitivity and smooth muscle contraction ( 12 – 14 ). Additionally, nitric oxide (NO) binds with soluble guanylate cyclase in smooth muscle cells, which increases cyclic guanosine monophosphate (cGMP) levels; this activates a cGMP-dependent kinase, which then dephosphorylates CPI-17 and inhibits the calcium sensitization mediated by phosphorylated CPI-17; thus, this pathway reduces the Ca 2+ sensitivity of smooth muscle cells ( 15 ).…”

Section: Discussionmentioning

confidence: 99%

“…The PKC/PKC-potentiated phosphatase inhibitor protein of 17 kDa (CPI-17) signaling pathway serves a critical role in the calcium sensitization of smooth muscle. CPI-17, a key substrate of PKC, is a PKC-dependent phosphatase inhibitor that inhibits the enzymatic activity of myosin light chain phosphatase (MLCP) ( 14 , 15 ). Su et al ( 16 ) silenced CPI-17 gene expression in bronchial smooth muscle using RNA interference technology, which decreased bronchial smooth muscle calcium sensitization, contraction frequency and contractility, suggesting that CPI-17 affects the contraction of smooth muscle.…”

Section: Introductionmentioning

confidence: 99%

Roles and mechanisms of TRPC3 and the PLCγ/PKC/CPI-17 signaling pathway in regulating parturition

et al. 2017

View full text Add to dashboard Cite

The aim of the current study was to investigate the role of phospholipase C (PLC)γ/protein kinase C (PKC)/C-kinase-activated protein phosphatase-1 (CPI-17) signaling pathways in uterine smooth muscle during parturition. Samples of uterine tissue were collected from pregnant patients who underwent a caesarean section for preterm delivery, full-term delivery with labor onset, full-term delivery without labor onset, and from a non-pregnant control group undergoing surgery for cervical intraepithelial neoplasia III. Immunohistochemistry, and western blotting were used to assess the association between TRPC3 levels and parturition and the influence of calcium ion channels. In addition, pregnant mice were used to explore the effect of uterine canonical transient receptor potential 3 (TRPC3) expression on the parturition-triggering mechanism and PLCγ/PKC/CPI-17 signaling pathways. Pregnant mouse uterine smooth muscle cells were cultivated, with and without TRPC3 silencing, and the expression levels of PLCγ, PKC and CPI-17, the upstream and downstream factors of the TRPC3 pathway, were measured in pregnant mouse uterine smooth muscle cells, in order to provide a theoretical basis for the prevention and treatment of premature labor. In the preterm and full-term without labor onset patient groups, the TRPC3 gene expression in the mSMCs was significantly overexpressed when compared with the non-pregnant group (P<0.05); however, TRPC3 expression was not elevated in the full-term with labor onset group, exhibiting no significant difference compared with the non-pregnant group (P>0.05). During pregnancy, compared with the non-pregnant controls, Cav1.2, Cav3.1 and Cav3.2 gene expression levels were markedly increased (P<0.05) in mSMCs from the preterm delivery group and the full-term with labor onset group, however were non-significantly increased in the full-term without labor onset group. The level of TRPC3 was highest in the preterm group, while the levels of Cav1.2, Cav3.1 and Cav3.2 were highest in the full-term with labor onset group. In the preterm, LPS-treated preterm and full-term groups, TRPC3, MAPK, ERK1/2, P-ERK, Cav3.2, Cav3.1 and Cav1.2 were all expressed at higher levels than in the unfertilized group. In the LPS-treated preterm group, the levels of TRPC3, MAPK, ERK1/2, P-ERK, Cav3.2, Cav3.1 and Cav1.2 were increased compared with the preterm group. Furthermore, following transfection of small interfering TRPC3 (siTRPC3) into cells, it was demonstrated that the levels of TRPC3, PLCγ, PKC, CPI-17, P-CPI-17, Cav1.2, Cav3.1 and Cav3.2 expression were lower in the LPS siTRPC3 group when compared with that of the LPS-treated untransfected control group.

show abstract

Thrombin stimulates stress fiber assembly in RPE cells by PKC/CPI-17-mediated MLCP inactivation

Cited by 11 publications

References 67 publications

Ca²⁺oscillations, Ca²⁺sensitization, and contraction activated by protein kinase C in small airway smooth muscle

Ca²⁺oscillations, Ca²⁺sensitization, and contraction activated by protein kinase C in small airway smooth muscle

Effect of allosteric inhibition of non-muscle myosin 2 on its intracellular diffusion

Roles and mechanisms of TRPC3 and the PLCγ/PKC/CPI-17 signaling pathway in regulating parturition

Contact Info

Product

Resources

About

Thrombin stimulates stress fiber assembly in RPE cells by PKC/CPI-17-mediated MLCP inactivation

Cited by 11 publications

References 67 publications

Ca2+oscillations, Ca2+sensitization, and contraction activated by protein kinase C in small airway smooth muscle

Ca2+oscillations, Ca2+sensitization, and contraction activated by protein kinase C in small airway smooth muscle

Effect of allosteric inhibition of non-muscle myosin 2 on its intracellular diffusion

Roles and mechanisms of TRPC3 and the PLCγ/PKC/CPI-17 signaling pathway in regulating parturition

Contact Info

Product

Resources

About

Ca²⁺oscillations, Ca²⁺sensitization, and contraction activated by protein kinase C in small airway smooth muscle

Ca²⁺oscillations, Ca²⁺sensitization, and contraction activated by protein kinase C in small airway smooth muscle