Cooperative regulation of GSK-3 by muscarinic and PDGF receptors is associated with airway myocyte proliferation

Muscarinic receptors and platelet-derived growth factor (PDGF) receptors synergistically induce proliferation of airway smooth muscle (ASM), but the pathways that regulate these effects are not yet completely identified. We hypothesized that glycogen synthase kinase-3 (GSK-3), a kinase that represses several promitogenic signaling pathways in its unphosphorylated form, is cooperatively inhibited by PDGF and muscarinic receptors in immortalized human ASM cell lines. PDGF or methacholine alone induced rapid GSK-3 phosphorylation. This phosphorylation was sustained only for PDGF; however, methacholine potentiated PDGF-induced sustained GSK-3 phosphorylation. Synergistic effects of methacholine also were observed on PDGF-induced retinoblastoma protein (Rb) phosphorylation and cell proliferation. Suppression of GSK-3 inhibitory function using SB 216763 also augmented PDGF-induced Rb phosphorylation and cell cycle progression; this synergy was similar in magnitude to that seen for methacholine with PDGF. GSK-3 phosphorylation induced by methacholine required PKC, since it was abolished by GF 109203X and G? 6976; however, inhibition of PKC had no effect on cell responses to PDGF. PKC inhibition also specifically abolished the synergistic effect of methacholine on PDGF-induced GSK-3 phosphorylation and cell proliferation. Collectively, these results show that GSK-3 plays a key repressive role in ASM cell proliferation. Moreover, muscarinic receptors mediate PKC-dependent GSK-3 inhibition, and this appears to be a primary mechanism underpinning augmentation of PDGF-induced cell growth.     

Protein kinase C-α upregulates sodium channel Nav1.9 in nociceptive dorsal root ganglion neurons in an inflammatory arthritis pain model of rat

Previous studies have found that increased expression of Nav1.9 and protein kinase C (PKC) contributes to pain hypersensitivity in a couple of inflammatory pain models. Here we want to observe if PKC can regulate the expression of Nav1.9 in dorsal root ganglion (DRG) in rheumatoid arthritis (RA) pain model. A chronic knee joint inflammation model was produced by intra-articular injection of the complete Freund's adjuvant (CFA) in rats. Nociceptive behaviors including mechanical, cold, and heat hyperalgesia were examined. The expression of Nav1.9 and PKCα in DRG was detected by a quantitative polymerase chain reaction, Western blot, and immunofluorescence. The in vitro and in vivo effects of a PKC activator (phorbol 12-myristate 13-acetate [PMA]) and a PKC inhibitor (GF-109203X) on the expression of Nav1.9 were examined. Moreover, the effects of PKC modulators on nociceptive behaviors were studied. Increased mechanical, heat, and cold sensitivity was observed 3 to 14 days after CFA injection. Parallel increases in messenger RNA and protein expression of Nav1.9 and PKCα were found. Immunofluorescence experiments found that Nav1.9 was preferentially colocalized with IB4+DRG neurons in RA rats. In cultured DRG neurons, PMA increased Nav1.9 expression while GF-109203X prevented the effect of PMA. PMA increased Nav1.9 expression in naïve rats while GF-109203X decreased Nav1.9 expression in RA rats. In naïve rats, PMA caused mechanical and cold hyperalgesia. On the other hand, GF-109203X attenuated mechanical and cold hyperalgesia in RA-pain model. Nav1.9 might be upregulated by PKCα in DRG, which contributes to pain hypersensitivity in CFA-induced chronic knee joint inflammation model of RA pain.     

Insulin-like growth factor I and protein kinase C activation stimulate pulmonary artery smooth muscle cell proliferation through separate but synergistic pathways

Smooth muscle cell (SMC) hyperplasia is an important component of vascular remodeling in chronic hypoxic pulmonary hypertension. The mechanisms underlying SMC proliferation in the remodeling process are poorly understood, but may involve insulin-like growth factor I (IGF-I). This study investigates the potential proliferative effects of IGF-I on SMC cultured from the pulmonary arteries (PA) of neonatal calves. We hypothesized that IGF-I stimulates PA SMC proliferation through a protein kinase C (PKC)-independent pathway, but that PKC activation would augment this proliferative response. Incorporation of 3H-thymidine was used as an index of cellular proliferation, and was correlated with subsequent changes in cell counts. Under serum-free conditions, IGF-I (100 ng/ml) induced a 6-fold increase in thymidine incorporation by quiescent PA SMC. This stimulation was not blocked by dihydrosphingosine, an inhibitor of PKC activation. Phorbol myristate acetate (PMA) (1 nM), a membrane-permeable PKC activator, induced a 12-fold increase in thymidine incorporation which was 70% inhibited by dihydrosphingosine. Co-incubation with IGF-I and PMA caused a 60-fold increase in thymidine incorporation, which was 30% inhibited by dihydrosphingosine. This synergistic increase in thymidine incorporation was associated with a subsequent significant increase in cell number. PKC-downregulated cells (1,000 nM PMA x 30 hr) proliferated in response to IGF-I but not PMA, and did not demonstrate synergism with the combination of IGF-I and PMA. The threshold concentrations of IGF-I and PMA for synergism were approximately 1 ng/ml and 1 pM, respectively. We conclude that IGF-I stimulates neonatal PA SMC proliferation via a PKC-independent pathway, and that trace amounts of PKC activators are capable of synergistically augmenting this response. We speculate that the synergistic stimulation of SMC proliferation by IGF-I and PKC activators may play an important role in hypertensive pulmonary vascular remodeling.     

The role of Na+/H+ exchanger in serotonin secretion from porcine blood platelets

This study was undertaken to evaluate whether a link exists between the activation of protein kinase C (PKC), operation of Na(+)/H(+) exchanger (NHE), cell swelling and serotonin (5-HT) secretion in porcine platelets. Activation of platelets by thrombin or phorbol 12-myristate 13-acetate (PMA), a PKC activator, initiated a rapid rise in the activity of Na(+)/H(+) exchanger and secretion of 5-HT. Both thrombin- and PMA-evoked activation of Na(+)/H(+) exchanger was less pronounced in the presence of ethyl-isopropyl-amiloride (EIPA), an NHE inhibitor, and by GF 109203X, a PKC inhibitor. Monensin (simulating the action of NHE) caused a dose-dependent release of 5-HT that was not abolished by GF 109203X or EGTA. Lack of Na(+) in the suspending medium reduced thrombin-, PMA-, and monensin-evoked 5-HT secretion. GF 109203X nearly completely inhibited 5-HT release induced by PMA-, partly that induced by thrombin, and had no effect on 5-HT release induced by monensin. EIPA partly inhibited 5-HT release induced by thrombin and nearly totally that evoked by PMA. Electronic cell sizing measurements showed an increase in mean platelet volume upon treatment of cells with monensin, PMA or thrombin. The PMA- and thrombin-evoked rise in mean platelet volume was strongly reduced in the presence of EIPA. As judged by optical swelling assay monensin and PMA produced a rapid rise in platelet volume. The swelling elicited by PMA was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Hypoosmotically evoked platelet swelling did not affect platelet aggregation but significantly potentiated thrombin-evoked release of 5-HT and ATP. Taken together, these results show that in porcine platelets PKC may promote 5-HT secretion through the activation of NHE. It is hypothesized that enhanced Na(+)/H(+) antiport may result in a rise in cell membrane tension (due to cell swelling) which in turn facilitates fusion of secretory granules with the plasma membrane leading to 5-HT secretion.     

Characterization of protein kinase pathways responsible for Ca2+ sensitization in rat ileal longitudinal smooth muscle

We investigated the protein kinases responsible for myosin regulatory light chain (LC20) phosphorylation and regulation of myosin light chain phosphatase (MLCP) activity during microcystin (phosphatase inhibitor)-induced contraction at low Ca2+ concentrations of rat ileal smooth muscle stretched in the longitudinal axis. Application of 1 microM microcystin induced LC20 diphosphorylation and contraction of beta-escin-permeabilized rat ileal smooth muscle at pCa 9. The PKC inhibitor GF-109203x, the MEK inhibitor PD-98059, and the p38 MAPK inhibitor SB-203580 significantly reduced this contraction. These inhibitory effects were abolished when the microcystin concentration was increased to 10 muM, indicating that application of these kinase inhibitors generated an increase in MLCP activity. GF-109203x and PD-98059, but not SB-203580, significantly decreased the phosphorylation level of the myosin-targeting subunit of MLCP, MYPT1, at Thr-697 (rat sequence) during microcystin-induced contraction at pCa 9. On the other hand, SB-203580, but not GF-109203x or PD-98059, significantly reduced the phosphorylation level of the PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17). A zipper-interacting protein kinase (ZIPK) inhibitor (SM1 peptide) and a Rho-associated kinase inhibitor (Y-27632) had little effect on microcystin-induced contraction at pCa 9. In conclusion, PKC, ERK1/2, and p38 MAPK pathways facilitate microcystin-induced contraction at low Ca2+ concentrations by contributing to the inhibition of MLCP activity either through phosphorylation of MYPT1 or CPI-17 [probably mediated by integrin-linked kinase (ILK)]. ILK and not ZIPK is likely to be the protein kinase responsible for LC20 diphosphorylation during microcystin-induced contraction of rat ileal smooth muscle at pCa 9, similar to its recently described role in vascular smooth muscle. The negative regulation of MLCP by PKC and MAPKs during microcystin-induced contraction at pCa 9, which is not observed in vascular smooth muscle, may be unique to phasic smooth muscle.     

Mechanisms involved in desensitization of particulate guanylyl cyclase in human airway smooth muscle: the role of protein kinase C

We recently showed that cultured human airway smooth muscle cells (HASMC) express both soluble and particulate guanylyl cyclases (GC) and that long term treatment with atrial natriuretic peptide (ANP) causes homologous desensitization of particulate GC. Here we determine if protein kinase C (PKC) activation would desensitize particulate GC and probe the role of PKC in particulate GC desensitization. Pretreatment of HASMC with phorbol 12-myristate 13-acetate (PMA), a PKC activator led to time and concentration-dependent desensitization of ANP-stimulated cGMP accumulation. GF109203X, a selective PKC inhibitor, blocked the PMA-induced desensitization, but did not block ANP-induced desensitization. In addition, desensitization by PMA and ANP showed an additive effect. These results suggest that PKC activation can desensitize particulate GC but that the desensitization induced by ANP is PKC-independent.     

Role of protein kinase C in alpha(1)-adrenergic regulation of a(Na)(i) in guinea pig ventricular myocytes

We investigated the role of protein kinase C (PKC) in alpha(1)-adrenergic regulation of intracellular Na(+) activity (a(Na)(i)) in single guinea pig ventricular myocytes. a(Na)(i) and membrane potentials were measured with the Na(+)-sensitive indicator sodium-binding benzofuran isophthalate and conventional microelectrodes, respectively, at room temperature (24-26 degrees C) while myocytes were stimulated at a rate of 0.25-0.3 Hz. The PKC activator 4beta-phorbol 12-myristate 13-acetate (PMA) decreased a(Na)(i) in a concentration-dependent manner. PMA (100 nM) produced a maximal decrease in a(Na)(i) of 1.5 mM from 6.5 +/- 0.4 to 5.0 +/- 0.4 mM (means +/- SE, n = 12, P < 0.01). The PMA concentration required for a half-maximal decrease in a(Na)(i) was 0.46 +/- 0.13 nM (n = 3, P < 0.01). An inactive phorbol, 4alpha-phorbol 12-myristate 13-acetate, did not decrease a(Na)(i). The decrease caused by PMA could be blocked by the PKC inhibitors staurosporine and bisindolylmaleimide I (GF-109203X). Stimulation of the alpha(1)-adrenoceptor with 50 microM phenylephrine decreased a(Na)(i) from 6.1 +/- 0.3 to 4.6 +/- 0.3 mM (n = 11, P < 0.01). The decrease in a(Na)(i) produced by phenylephrine was blocked by pretreatment with staurosporine, GF-109203X, or PMA. The decrease in a(Na)(i) produced by PMA was not prevented by pretreatment with tetrodotoxin but was blocked by pretreatment with strophanthidin or high extracellular K(+) concentration. The results suggest that alpha(1)-adrenergic receptor activation results in a decrease in a(Na)(i) via PKC-induced stimulation of the Na(+)-K(+) pump in cardiac myocytes.     

缺氧对体外培养的肺动脉平滑肌细胞形态及增殖的影响

本实验应用形态学、免疫组化染色,＾３Ｈ－ＴｄＲ掺入、流式细胞等技术探讨缺氧（〈１％Ｏ２和２．５％Ｏ２）对肺动脉平滑肌细胞（ＰＡＳＭ）形态及增殖的影响。结果表明：缺氧２４ｈ使ＰＡＳＭ表型从收缩型向合成型转换,胞浆内ＳＭ－ａ ａｃｔｉｎ减少,线粒体和粗面内质网增多,缺氧４８ｈ以后线粒体肿胀,空泡化,内质网扩张,胞浆内出现髓鞘样结构。流式细胞分析显示缺氧ＰＡＳＭ Ｇ２／Ｍ期细胞比例明显增多（Ｐ〈０．００     

Salidroside inhibits platelet-derived growth factor–induced proliferation and migration of airway smooth muscle cells

Abnormal proliferation and migration of airway smooth muscle cells (ASMCs) have been found to be important for the airway remodeling during the pathogenesis of asthma. Salidroside a bioactive glucoside that exerts antitumor activity via inhibiting the cell proliferation and migration of cancer cells. The aim of the current study was to evaluate the effects of salidroside on the proliferation and migration of ASMCs. Our results showed that salidroside inhibited the proliferation and migration of ASMCs in response to platelet-derived growth factor (PDGF) stimulation. Salidroside markedly attenuated the PDGF-induced production of matrix metalloproteinase 2 (MMP-2) and MMP-9 in ASMCs. The levels of contractile phenotype markers including smooth muscle α-actin and calponin were reduced in response to PDGF stimulation, which was attenuated by salidroside pretreatment. Salidroside diminished the increase in the expression levels of type I collagen and fibronectin in PDGF-stimulated ASMCs. Furthermore, salidroside blocked the PDGF-induced activation of the nuclear factor-κB (NF-κB) pathway in ASMCs. The results suggested that salidroside functionally regulated the proliferation, migration, phenotype plasticity, and extracellular matrix deposition in PDGF-induced ASMCs and the NF-κB pathway might be implicated in the effects of salidroside on ASMCs induced by PDGF.     

A role for platelet-derived growth factor beta-receptor in a newborn rat model of endothelin-mediated pulmonary vascular remodeling

Newborn rats exposed to 60% O2 for 14 days develop endothelin (ET)-1-dependent pulmonary hypertension with vascular remodeling, characterized by increased smooth muscle cell (SMC) proliferation and medial thickening of pulmonary resistance arteries. Using immunohistochemistry and Western blot analyses, we examined the effect of exposure to 60% O2 on expression in the lung of receptors for the platelet-derived growth factors (PDGF), which are implicated in the pathogenesis of arterial smooth muscle hyperplasia. We observed a marked O2-induced upregulation of PDGF-alpha and -beta receptors (PDGF-alphaR and -betaR) on arterial smooth muscle. This led us to examine pulmonary vascular PDGF receptor expression in 60% O2-exposed rats given SB-217242, a combined ET receptor antagonist, which we found prevented the O2-induced upregulation of PDGF-betaR, but not PDGF-alphaR, on arterial smooth muscle. PDGF-BB, a major PDGF-betaR ligand, was found to be a potent in vitro inducer of hyperplasia and DNA synthesis in cultured pulmonary artery SMC from infant rats. A critical role for PDGF-betaR ligands in arterial SMC proliferation was confirmed in vivo using a truncated soluble PDGF-betaR intervention, which attenuated SMC proliferation induced by exposure to 60% O2. Collectively, these data are consistent with a major role for PDGF-betaR-mediated SMC proliferation, acting downstream of increased ET-1 in a newborn rat model of 60% O2-induced pulmonary hypertension.     

Phorbol ester downregulates PDGFbeta receptor via PKCbeta1 in vascular smooth muscle cells

The role of protein kinase C (PKC) and their isoforms in cell growth regulation remains elusive. Here we showed that in cultured human vascular smooth muscle cells (SMC), the PKC stimulator phorbol 12-myristate 13-acetate (PMA) inhibited [(3)H]thymidine incorporation in response to the growth factor PDGF associated with downregulation of PDGFbeta (but not alpha) receptors, which was recovered to normal level after PKC was depleted. The changes in PDGFbeta receptor were inversely correlated with PKCbeta1 protein levels regulated by PMA. The downregulation of PDGFbeta receptor by PMA was fully prevented by the PKCbeta inhibitor LY379196, however, without recovery of [(3)H]thymidine incorporation to PDGF. In contrast, [(3)H]thymidine incorporation was fully recovered after depletion of PKCs. These results indicate that in human SMC PKCbeta1 mediates PDGFbeta receptor downregulation. Other PKC isoforms activated by phorbol ester also contribute to the inhibitory effects on cell growth.     

Induction of ANGPTL4 expression in human airway smooth muscle cells by PMA through activation of PKC and MAPK pathways

In this study, we demonstrate that protein kinase C (PKC) activators, including phorbol-12-myristate-13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol (DOG), and platelet-derived growth factor α are potent inducers of angiopoietin-like protein 4 (ANGPTL4) expression in several normal lung cell types and carcinoma cell lines. In human airway smooth muscle (HASM) cells induction of ANGPTL4 expression is observed as early as 2 h after the addition of PMA. PMA also increases the level of ANGPTL4 protein released in the medium. PKC inhibitors Ro31-8820 and Gö6983 greatly inhibit the induction of ANGPTL4 mRNA by PMA suggesting that this up-regulation involves activation of PKC. Knockdown of several PKCs by corresponding siRNAs suggest a role for PKCα. PMA does not activate MAPK p38 and p38 inhibitors have little effect on the induction of ANGPTL4 indicating that p38 is not involved in the regulation of ANGPTL4 by PMA. In contrast, treatment of HASM by PMA induces phosphorylation and activation of Ra, MEK1/2, ERK1/2, JNK, Elk-1, and c-Jun. The Ras inhibitor manumycin A, the MEK1/2 inhibitor U0126, and the JNK inhibitor SP600125, greatly reduce the increase in ANGPTL4 expression by PMA. Knockdown of MEK1/2 and JNK1/2 expression by corresponding siRNAs inhibits the induction of ANGPTL4. Our observations suggest that the induction of ANGPTL4 by PMA in HASM involves the activation of PKC, ERK, and JNK pathways. This induction may play a role in tissue remodeling during lung injury and be implicated in several lung pathologies.     

Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells

Proliferation of vascular smooth muscle cells (VSMC) contributes to the pathogenesis of atherosclerosis, and glycated serum albumin (GSA, Amadori adduct of albumin) might be a mitogen for VSMC proliferation, which may further be associated with diabetic vascular complications. In this study, we investigated the involvement of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and protein kinase C (PKC), in GSA-stimulated mitogenesis, as well as the functional relationship between these factors. VSMC stimulation with GSA resulted in a marked activation of ERK. The MAPK kinase (MEK) inhibitor, PD98059, blocked GSA-stimulated MAPK activation and resulted in an inhibition of GSA-stimulated VSMC proliferation. GSA also increased PKC activity in VSMC in a dose-dependent manner. The inhibition of PKC by the PKC inhibitors, GF109203X and Rottlerin (PKCdelta specific inhibitor), as well as PKC downregulation by phorbol 12-myristate 13-acetate (PMA), inhibited GSA-induced cell proliferation and blocked ERK activation. This indicates that phorbol ester-sensitive PKC isoforms including PKCdelta are involved in MAPK activation. Thus, we show that the MAPK cascade is required for GSA-induced proliferation, and that phorbol ester-sensitive PKC isoforms contribute to cell activation and proliferation in GSA-stimulated VSMC.     

Platelet-derived growth factor and lysophosphatidic acid inhibit growth hormone binding and signaling via a protein kinase C-dependent pathway

Growth hormone (GH) regulates body growth and metabolism. GH exerts its biological action by stimulating JAK2, a GH receptor (GHR)-associated tyrosine kinase. Activated JAK2 phosphorylates itself and GHR, thus initiating multiple signaling pathways. In this work, we demonstrate that platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA) down-regulate GH signaling via a protein kinase C (PKC)-dependent pathway. PDGF substantially reduces tyrosyl phosphorylation of JAK2 induced by GH but not interferon-gamma or leukemia inhibitory factor. PDGF, but not epidermal growth factor, decreases tyrosyl phosphorylation of GHR (by approximately 90%) and the amount of both total cellular GHR (by approximately 80%) and GH binding (by approximately 70%). The inhibitory effect of PDGF on GH-induced tyrosyl phosphorylation of JAK2 and GHR is abolished by depletion of 4beta-phorbol 12-myristate 13-acetate (PMA)-sensitive PKCs with chronic PMA treatment and is severely inhibited by GF109203X, an inhibitor of PKCs. In contrast, extracellular signal-regulated kinases 1 and 2 and phosphatidylinositol 3-kinase appear not to be involved in this inhibitory effect of PDGF. LPA, a known activator of PKC, also inhibits GH-induced tyrosyl phosphorylation of JAK2 and GHR and reduces the number of GHR. We propose that ligands that activate PKC, including PDGF, LPA, and PMA, down-regulate GH signaling by decreasing the number of cell surface GHR through promoting GHR internalization and degradation and/or cleavage of membrane GHR and release of the extracellular domain of GHR.     

Regulation of 5-hydroxytryptamine-induced signal transduction in canine cultured aorta smooth muscle cells by phorbol ester.

Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine aorta smooth muscle cells (ASMCs). Stimulation of ASMCs by 5-hydroxytryptamine (5-HT) led to IPs formation and caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of ASMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min almost abolished the 5-HT-induced IPs formation and Ca2+ mobilization. This inhibition was reduced after long-term incubating the cells with PMA. Prior treatment of ASMCs with staurosporine or GF109203X, PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the 5-HT-induced IP formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of ASMCs with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta isozymes from the cytosol to the membrane was seen after 5-min, 30-min, 2-h, and 4-h treatment. However, 24-h treatment caused a partial down-regulation of these PKC isozymes. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta induced by PMA caused an attenuation of 5-HT-induced IPs accumulation and Ca2+ mobilization in ASMCs.