Rho Kinase (ROCK) collaborates with Pak to Regulate Actin Polymerization and Contraction in Airway Smooth Muscle

Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F‐actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase inactive mutant of ROCK, ROCK‐K121G, in the tissues or by treating them with the ROCK inhibitor, H‐1152P. Our results show no role for ROCK in the regulation of non‐muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue. We find that ROCK regulates airway smooth muscle contraction by mediating activation of the serine‐threonine kinase, Pak, to promote actin polymerization. Pak catalyzes paxillin phosphorylation on Ser273 and coupling of the GIT1‐βPIX‐Pak signaling module to paxillin, which activates the GEF activity βPIX towards cdc42. Cdc42 is required for the activation of Neuronal Wiskott‐Aldrich Syndrome protein (N‐WASp), which transmits signals from cdc42 to the Arp2/3 complex for the nucleation of actin filaments. Our results demonstrate a novel molecular function for ROCK in the regulation of Pak and cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle

Basal and agonist activated calcium sensitization pathways in murine gastrointestinal smooth muscles

This dissertation has focused on two major projects; (1) characterizing the basal expression level of Ca2+ sensitization proteins in unstimulated gastrointestinal (GI) smooth muscles from regionally and functionally distinct regions and (2) addressing a fundamental question in the smooth muscle field; does exogenously applied agonist in the myobath and neural release of agonist induce similar Ca2+ sensitization signaling changes in GI smooth muscle. In carrying out these projects, contractions of GI smooth muscles were determined using standard myobath - isometric force transducer techniques and total or phospho proteins were determined by western blotting. Ca2+ sensitization is largely defined as a mechanism to regulate smooth muscle contraction independent of intracellular Ca2+ concentration. Though smooth muscle contraction is mainly achieved by Ca2+ dependent myosin light chain kinase activation, contribution of Ca2+ sensitization mechanisms by myosin light chain phosphatase (MLCP) inhibition is being increasingly recognized as these mechanisms have been shown to be altered in many pathological states of smooth muscle contraction. Similar to any other signaling pathways, activation of Ca2+ sensitization pathways is also likely to be determined by the basal expression levels of total and phospho proteins involved in the pathways. The basal expression level of total and phospho proteins is important to recognize as this could provide information on how close the basal level is to the threshold for effective stimulation of GI smooth muscles. The basal level expression will also determine how much additional activation of proteins could be achieved when the tissues are stimulated. However, only a few studies have examined the basal level expression of Ca2+ sensitization proteins in GI smooth muscles, while most of the studies have been carried out in stimulated vascular and airway smooth muscles. Additionally, many studies in vascular smooth muscles and one study in rat GI smooth muscles have shown differential expression pattern of Ca2+ sensitization proteins characteristic of phasic and tonic properties of smooth muscles. Therefore, in the first component of this dissertation, we examined the basal expression of proteins involved in Ca2+ sensitization pathways in gastric antrum (predominantly a phasic smooth muscle with some resting tone), gastric fundus (purely a tonic muscle in mouse), and proximal colon (a phasic smooth muscle). These muscles are from functionally and regionally distinct GI regions. ã-actin, total and phospho-MYPT1 (pT696, pT853), total and phospho-CPI-17 (pT38), and total and phospho-MLC20 (pS19) were determined during spontaneous contraction of these three smooth muscles. The expression levels of the LZ+ and LZ- MYPT1 isoforms, and the MLCP-anchoring protein M-RIP were also determined for the first time in GI smooth muscles. We also examined the effects of ROK inhibitor (Y27632), VDCC blocker (nicardipine), or PKC inhibitor (GF109203X) on the basal phosphorylation levels of MYPT1, CPI-17, and MLC20 in these three smooth muscles. The effects of Y27632, nicardipine, and Ca2+ free Kreb's solution on basal contraction were also measured. We found differences in the expression levels of MLC20, ROK1, ROK2, total MYPT1 and LZ+ and LZ- isoforms, M-RIP, and CPI-17 and in the phosphorylation levels of MLC20, MYPT1, and CPI-17 in gastric antrum, gastric fundus, and proximal colon smooth muscles. Actin expression was similar in all three smooth muscles. We also found differences in the sensitivity to Y27632, nicardipine, and GF109203X, from gastric antrum, gastric fundus, and proximal colon smooth muscles. Y27632 reduced pT853 in each smooth muscle, but reduced pT696 only in fundus. Y27632 reduced pT38 levels only in proximal colon smooth muscles. Nicardipine had no effect on pT38 in all three smooth muscles, while GF109203X reduced pT38 levels in fundus and proximal colon smooth muscles but not in antrum. Y27632 and nicardipine reduced pS19 levels in fundus and proximal colon smooth muscles, but had no effect on pS19 in antrum smooth muscles. Y27632 or nicardipine inhibited the spontaneous phasic contractions of antrum and proximal colon smooth muscles, but only Y27632 reduced fundus smooth muscle tone. However, zero external Ca2+ completely relaxed each smooth muscle and abolished MLC20 phosphorylation. In fundus and antrum, but not proximal colon smooth muscles, the level of MLC20 phosphorylation does not appear to correlate with the spontaneous contractile activity. The second component of this dissertation tested if the exogenously applied synthetic acetylcholine (ACh); carbachol (CCh) in the myobath, and neural release of ACh induce similar Ca2+ sensitization signaling changes in GI smooth muscle. Furthermore, we tested how removal of interstitial cells of Cajal (ICC) would affect activation of Ca2+ sensitization pathways during neural release of ACh. So far activation of Ca2+ sensitization pathways in smooth muscles have been mostly studied by exogenous application of neurotransmitters (NT) or agonists in the myobath which may not represent the signaling changes triggered by in-vivo neural release of neurotransmitter. Exogenously applied NT can potentially activate any cell types in the given tissue preparation whereas neurally released NT is more likely to be more specific to its immediate cell target. To address this question, we contracted gastric fundus smooth muscles by exogenously applied 1ìM or 10ìM CCh or by cholinergic nerve stimulation (1, 5, 10, and 20 Hz). During the contraction induced by either method, smooth muscle strips were frozen at different phases of the contractile trace (upstroke, peak and plateau) and analyzed for the increase in CPI-17, MYPT1 and MLC20-phosphorylation. Key findings of the study were; (1) bath application of carbachol (CCh) increased phosphorylation of CPI-17 (pT38) and MYPT1 (pT853 and pT696) whereas cholinergic nerve stimulation (NS) increased only pT38CPI-17 indicating Rho kinse (ROK) may not be activated sufficiently to phosphorylate MYPT1 in the nerve stimulation (NS) mode. (2) 1 ìM CCh/1min stimulation resulted in maximal CPI-17 and MYPT1 phosphorylation in the bath applied mode whereas 10 Hz/ 5 sec stimulation resulted in maximal CPI-17 phosphorylation in NS mode. Interestingly 1 min time point in CCh contractile trace and 5 sec time point in NS contractile trace referred to the peak contraction in the respective traces. (3) Neither CCh stimulation nor NS appeared to increase MLC20 phosphorylation despite strong contractions elicited by both stimulation modes. (4) NS and CCh stimulated increase in CPI-17 phosphorylation was sensitive to PKC and L-type voltage dependent Ca2+ channel (VDCC) inhibitors indicating need of Ca2+ influx and PKC activation into the fundus smooth muscle for inducing CPI-17 phosphorylation in both stimulations. (5) The CCh stimulated increase in pT853 was highly sensitive to ROK inhibitor but not to PKC inhibitors or VDCC antagonist whereas the increase pT696 was sensitive to PKC, ROK, and VDCC inhibitors. (6) NS in the presence of neostigmine further potentiated contractile force and CPI-17 phosphorylation and induced MYPT1 phosphorylation at Thr-853. (7) In W/Wv gastric fundus smooth muscles that lack ICCs, NS alone increased pT38 and pT853, and neostigmine augmented the contractions and further increased pT38 and pT853. Thus it is clear from these results that ACh released from the nerve varicosities is not normally enough to elicit ROK activation in fundus smooth muscles. But once ACh concentration is increased at the neuroeffector junction or a barrier (ICC) between nerve varicosities and smooth muscle cells is removed, MYPT1 phosphorylation increases mimicking the effect of bath applied CCh. Taken together, these results indicate that ACh availability at the neuromuscular junction determines whether only PKC activation and hence CPI-17 phosphorylation or both PKC and ROK activation and hence CPI-17, and MYPT1 phosphorylation increases. In this study, using gastric fundus smooth muscle we have shown that bath applied agonist (which has remained a standard method of smooth muscle stimulation) does not recapitulate Ca2+ sensitization pathways activation triggered by neural release of agonist. Additionally, for the past two decades in the field of neurogastroenterology there is a debate going on regarding whether NT released from enteric motor neurons directly regulates SMC or through ICC. Whether it is for or against the role of ICC in enteric motor neurotransmission, the conclusions have been made mostly based on structural and electrophysiological evidence from wild type or ICC knock out GI tissues. Here we took a molecular approach to address this debate. Our results indicate that most likely cholinergic neurotransmission occurs through ICC as neural release of ACh does not seem to directly stimulate SMC but stimulate ICC to set electrical coupling with SMC. This study opens a door for taking similar approach to address ICC role in other types of neurotransmission in the GI tract. In summary, gastric antrum, gastric fundus and proximal colon smooth muscles have different expression pattern of Ca2+ sensitization proteins and tissue specific Ca2+ sensitization mechanisms for generating spontaneous contractions. Furthermore, NS activates the PKC-CPI-17 Ca2+ sensitization pathway whereas bath applied CCh activates the Rho kinase-MYPT1 and PKC-CPI-17 Ca2+ sensitization pathways. Acetyl cholinesterase inhibition (neostigmine present) or the absence of ICCs allows NS to activate both the Rho kinase-MYPT1 and the PKC-CPI-17 Ca2+ sensitization pathways in gastric fundus smooth muscles. These findings provide evidence that due considerations should be given that signaling changes triggered by exogenously applied NT may not recapitulate signaling triggered by in-vivo release of NT from nerve terminals in smooth muscles

Further Elucidation of the Role of Protein Kinase-C and Rho Kinase in the Mechanism of Action of Bitter Melon Extract on Human Metastatic Breast Cancer Cells

Introduction: It is well documented in the literature that Bitter Melon Extract (BME) inhibits the growth and proliferation of MCF-7 breast cancer cells. While the exact mechanism of this inhibition is unknown, it is possible that Protein Kinase C (PKC) and Rho Kinase (ROK) play a role in this mechanistic pathway due to their known critical roles in cell migration, division, and survival. Previous research has shown that ROK inhibition results in decreased cell viability and decreased MYPT1 (Myosin phosphatase targeting subunit-1) expression, while PKC inhibition alone does not have a significant effect. Aims: The aim of this research is to investigate the cytotoxic mechanism of action enacted by BME on MCF-7 cells and how PKC and ROK might be involved in this mechanism by building on past research to create more robust data and a better understanding of the metabolic state of MCF-7 cells under the set conditions. Methods: Glucose assays were run via a GM-100 glucometer to test for cell metabolism as a measure of cell viability on BME dose (0%, 0.5%, 1%, 2%, 5%, and 10% BME (v/v)) study samples, PKC inhibitor (GF109203x; 0.5 uM) samples, and ROK inhibitor (H-1152; 1 uM) samples. The samples used in each of the three conditions were secured from past research. Results & Discussion: BME dose-dependently increased glucose remaining in the culture media. The sample sizes for different doses of BME were as follows; at 0% (n=4), 0.5% (n=5), and 1% (n=5), while 2% (n=2), 5% (n=3), and 10% (n=3). The data suggests two possibilities; (i) with increasing BME dose, there is an increasing effect on the MCF-7 cell’s metabolism of glucose, and that this decrease in ability to metabolize glucose when in the presence of increasing [BME] could play a role in the cytotoxic mechanism of BME on MCF-7 cells. (ii) The BME kills cells and hence glucose is not used as much thus leaving plenty of glucose in the medium. Our results further showed that neither PKC inhibitor samples (n= 7) nor ROK inhibitor samples (n=11) showed significant differences in [glucose]. This suggests that the measured effect in glucose metabolism in the BME dose-dependent study samples is independent of PKC and ROK mechanistic pathways

Role of Telokin in Regulating Murine Gastric Fundus Smooth Muscle Tension

Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from telokin(-/-) mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/W-V mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that telokin S13 is basally phosphorylated and that 8-BromocGMP and SNP increased basal telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased telokin phosphorylation and reduced MLC phosphorylation. In telokin(-/-) gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted telokin(-/-) gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8Bromo- cGMP, SNP, and nitrergic neurotransmission. However, basal telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased telokin Ser13 phosphorylation in both wild-type and W/W-V gastric fundus smooth muscles. Our findings indicate that telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism to augment gastric fundus mechanical responses to inhibitory neurotransmission

Telokin phosphorylation in non-contracted gastric fundus smooth muscles is not increased by nitrergic neurotransmission.

<p>A. Representative recording of the relaxation responses evoked by 30 sec of 5Hz or 10Hz EFS of muscles incubated in 1μM atropine, 1μM phentolamine, and 1μM propranolol. Representative western blots of the phosphorylation of telokin S13 (B), CPI-17 T38 (C), MYPT1 T853 and T696 (D), and MLC S19 (E) evoked by 30 sec of 5Hz or 10Hz EFS of muscles incubated in 1μM atropine, 1μM phentolamine, and 1μM propranolol. F. Average ratios ± SD of pS13:telokin, pT38:CPI-17, pT696:MYPT1, pT853:MYPT1, and pS19:MLC evoked by 30 sec of 5Hz or 10Hz EFS of muscles incubated in 1μM atropine, 1μM phentolamine, and 1μM propranolol (<i>P</i>>0.05, n = 6).</p

Telokin phosphorylation is increased by incubation of non-contracted gastric fundus smooth muscles with compounds that elevate cGMP.

<p>A. Representative recordings of the relaxation responses evoked by 10μM SNP in the absence or presence of 1μM ODQ (n = 6, each), or 50μM 8Br-cGMP (n = 6). B. Representative western blots of telokin S13 phosphorylation in untreated control muscles and muscles incubated with 50μM 8Br-cGMP for 5 min (n = 6) or 10μM SNP (n = 6) for 1 min. C. Average ratios ± SD of pS13:telokin in the absence or presence of 50μM 8Br-cGMP (n = 6) or 10μM SNP. *<i>P</i><0.01 compared to untreated controls.</p

The contractile responses of telokin^-/- gastric fundus smooth muscles are increased compared to wild-type controls.

<p>A. Representative recordings of the contractile responses of wild-type and telokin^-/- gastric fundus smooth muscles to 1μM CCh (n = 4). B. Representative recordings of the contractile responses of wild-type and telokin^-/- gastric fundus smooth muscles to 15mM, 30mM, and 60mM KCl. C. Average integrals and peak amplitudes of contraction ± SD of wild-type and telokin^-/- gastric fundus smooth muscles to 15mM, 30mM, and 60mM KCl (^#<i>P</i><0.05 compared to wild-type, n = 8). D. Representative recordings of the contractile responses of wild-type and telokin^-/- gastric fundus smooth muscles incubated with 100μM LNNA and 1μM MRS2500 to 30 sec of 1Hz, 5Hz, 10Hz, or 20Hz EFS. E. Average integrals and peak amplitudes of contraction ± SD of wild-type and telokin^-/- gastric fundus smooth muscles incubated with 100μM LNNA and 1μM MRS2500 to 30 sec of 1Hz, 5Hz, 10Hz, or 20Hz EFS KCl (^#<i>P</i><0.05 compared to wild-type, n = 4). F. Representative western blots of phosphorylated and non-phosphorylated MLC in wild-type and telokin^-/- gastric fundus smooth muscles.</p

SNP dose-dependently increases telokin S13 phosphorylation in non-pre-contracted gastric fundus smooth muscles.

<p>A. Representative recording of the relaxation responses and representative western blot of phosphorylated and non-phosphorylated MLC evoked by 10Hz EFS, 0.1μM, 1μM, or 10μM SNP (n = 4). B. Average ratios ± SD of peak relaxation amplitudes evoked by 10Hz EFS, 0.1μM, 1μM, or 10μM SNP (*<i>P</i><0.01, ^#<i>P</i><0.05, n = 4). C. Average ratios ± SD and representative western blots of pS13:telokin evoked by 0.1μM, 1μM, or 10μM SNP (^#<i>P</i><0.05 compared to 0.1μM SNP or 1μM SNP, n = 4).</p

Telokin S13 phosphorylation is not increased by nitrergic neurotransmission in <i>W/W</i>^<i>V</i> gastric fundus smooth muscles.

<p>Representative recordings of the relaxation responses evoked by 30 sec of 5Hz EFS and 10Hz EFS (A), or 10μM SNP (B) from wild-type and <i>W/W</i>^<i>V</i> fundus muscles. B. Representative western blot of phosphorylated and non-phosphorylated MLC from wild-type and <i>W/W</i>^<i>V</i> fundus muscles treated with 10μM SNP. C. Average ratios ± SD of pS13:telokin and representative western blots of phosphorylated telokin S13 and telokin from wild-type and <i>W/W</i>^<i>V</i> muscles stimulated by 10Hz/30 sec EFS, or incubated with 10μM SNP. *<i>P</i><0.01 compared to untreated wild-type or <i>W/W</i>^<i>V</i> fundus muscle controls, n = 4.</p

The relaxation responses of pre-contracted telokin^-/- gastric fundus smooth muscles are reduced compared to wild-type controls.

<p>Representative recordings of the relaxation responses of wild-type and telokin^-/- gastric fundus smooth muscles to (A) 10μM SNP, (B) 50μM 8Br-cGMP, and (C) 30 sec of 5Hz or 10Hz EFS. Representative recordings of the relaxation responses of wild-type and telokin^-/- gastric fundus smooth muscles evoked by 10μM SNP in the presence of (D) 1μM CCh or (E) 30mM KCl. F. Representative recording of the relaxation responses evoked by 30 sec of 5Hz or 10Hz EFS in wild-type and telokin^-/- gastric fundus smooth muscles incubated with 30mM KCl and 1μM atropine, 1μM phentolamine, and 1μM propranolol.</p