TrkA signalling pathways in human airway smooth muscle cell proliferation

NGF may play a role in airway inflammation and hyperresponsiveness. We studied its possible involvement in airway remodelling and report here its proliferative effect and its receptor and signalling pathways in human airway smooth muscle cells in culture (HASMC). Proliferation of HASMC induced by NGF (0.1-10 pM) was assessed by the XTT and BrdU techniques with and without kinase inhibitors. Immunoprecipitation and Western blotting were used to study phosphorylation of TrkA and MAPK. NGF caused dose-dependent proliferation of HASMC and induced TrkA phosphorylation, both abolished by the tyrosine-kinase inhibitor K252a. PI3K and JNK inhibitors had no effect. PKC inhibitors partially inhibited NGF-induced proliferation and totally abolished p38 phosphorylation but did not affect ERK1/2 phosphorylation. The rafK inhibitor decreased NGF-induced proliferation, and totally abolished ERK1/2 phosphorylation, but did not affect p38 phosphorylation. This finding was confirmed by the decrease of NGF-induced proliferation after treatment with inhibitors of the p38 or of ERK1/2 pathways. In conclusion, NGF activation of the TrkA receptor involves two distinct signalling pathways: PKC selectively activates p38, and the ras/raf pathway selectively activates ERK1/2. Both are necessary to induce HASMC proliferation.     

VCAM-1 activates phosphatidylinositol 3-kinase and induces p120Cbl phosphorylation in human airway smooth muscle cells

VCAM-1 is a member of the Ig superfamily of receptors the expression of which is up-regulated on human airway smooth muscle (ASM) cells following stimulation with inflammatory mediators. The function of these receptors in adhesion is well known, but there is growing recognition that they also possess "outside-in" signaling functions, such as cytoskeletal reorganization, calcium mobilization, and cytokine release. The present study examined the activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase (PI3K) in ASM cells following VCAM-1 engagement. VCAM-1 ligation activated extracellular signal-regulated kinase 2 and resulted in increased expression of cyclin D1, yet there was neither p27(kip1) degradation nor an increase in smooth muscle cell DNA synthesis. VCAM-1 ligation, however, augmented the proliferative response to submitogenic concentrations of epidermal growth factor. VCAM-1 engagement also stimulated a rapid increase in PI3K activity. This was associated with phosphorylation of the adapter protein p120(Cbl) and an increase in Cbl-associated PI3K activity. These studies suggest that VCAM-1 is linked to multiple signaling pathways in human ASM cells and may function to augment growth factor-induced responses.     

ERK activation and mitogenesis in human airway smooth muscle cells

Asthmatic airways are characterized by an increase in smooth muscle mass, due mainly to hyperplasia. Many studies suggest that extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2, respectively), one group of the mitogen-activated protein (MAP) kinase superfamily, play a key role in the signal transduction pathway leading to cell proliferation. PGE(2) and forskolin inhibited mitogen-induced ERK activation. Inhibition of MAP kinase kinases 1 and 2 (MEK1 and MEK2, respectively), which are upstream from ERK, with the specific MEK inhibitor U-0126 blocked both cell proliferation and ERK activation. In addition, U-0126 inhibited mitogen-induced activation of p90 ribosomal S6 kinase and expression of c-Fos and cyclin D1, all of which are downstream from ERK in the signaling cascade that leads to cell proliferation. Antisense oligodeoxynucleotides directed to ERK1 and -2 mRNAs reduced ERK protein and cell proliferation. These results indicate that ERK is required for human airway smooth muscle cell proliferation. Thus targeting the control of ERK activation may provide a new therapeutic approach for hyperplasia seen in asthma.     

Insulin-dependent phosphatidylinositol 3-kinase/Akt and ERK signaling pathways inhibit TLR3-mediated human bronchial epithelial cell apoptosis

TLR3, one of the TLRs involved in the recognition of infectious pathogens for innate and adaptive immunity, primarily recognizes viral-associated dsRNA. Recognition of dsRNA byproducts released from apoptotic and necrotic cells is a recently proposed mechanism for the amplification of toxicity, suggesting a pivotal participation of TLR3 in viral infection, as well as in lung diseases where apoptosis plays a critical role, such as asthma and chronic obstructive pulmonary disease. In addition to metabolic control, insulin signaling was postulated to be protective by inhibiting apoptosis. Therefore, we explored the role of insulin signaling in protecting against TLR3-mediated apoptosis of human bronchial epithelial cells. Significant TLR3-mediated apoptosis was induced by polyinosinic-polycytidylic acid, a dsRNA analog, via caspase-8-dependent mechanisms. However, insulin efficiently inhibited TLR3/polyinosinic-polycytidylic acid-induced human bronchial epithelial cell apoptosis via PI3K/Akt and ERK pathways, at least in part, via upregulation of cellular FLIPs and through protein synthesis-independent mechanisms. These results indicate the significance of TLR3-mediated dsRNA-induced apoptosis in the pathogenesis of apoptosis-driven lung disease and provide evidence for a novel protective role of insulin.     

Dual effects of lysophosphatidic acid on human airway smooth muscle cell proliferation and survival

Lysophosphatidic acid (LPA) is a phospholipid growth mediator found in serum at 2-20 microM. In many cell types, including human airway smooth muscle (HASM) cells, LPA-induced proliferation occurs at 10-100 microM LPA. At these concentrations LPA forms Ca2+ precipitates. The potential involvement of Ca2+ and Ca2+ LPA precipitates in LPA-induced HASM cell mitogenesis was investigated. In the absence of extracellular Ca2+, 10 and 30 microM LPA stimulated HASM cell mitogenesis. However, with 100 microM LPA in the absence of extracellular Ca2+, HASM cells exhibited a profound shape change and loss of viability, determined to be apoptosis by both DNA staining and assessment of cytosolic nucleosomal reactivity. A bioassay based on the adenosine 3':5'-cyclic monophosphate response of C62B rat glioma cells was used to measure the bioactivity of LPA solutions prepared in Ca2+ free and Ca2+ containing medium. After 24 h, a 100 microM LPA solution in Ca2+ free medium contained markedly greater bioactivity than a 100 microM LPA solution made in Ca2+ containing medium. In summary, formation of Ca2+ LPA precipitates decreases the amount of biologically active LPA in solution, and high concentrations of bioactive LPA achieved in Ca2+ free but not in Ca2+ containing medium induce apoptosis of HASM cells.     

Synchronous activation of ERK and phosphatidylinositol 3-kinase pathways is required for collagen and extracellular matrix production in keloids

Keloid fibroproliferation appears to be influenced by epithelial-mesenchymal interactions between keloid keratinocytes (KKs) and keloid fibroblasts (KFs). Keloid and normal fibroblasts exhibit accelerated proliferation and collagen I and III production in co-culture with KKs compared with single cell culture or co-culture with normal keratinocytes. ERK and phosphatidylinositol 3-kinase (PI3K) pathway activation has been observed in excessively proliferating KFs in co-culture with KKs. We hypothesized that ERK and PI3K pathways might be involved in collagen and extracellular matrix production in KFs. To test our hypothesis, four samples of KFs were co-cultured in defined serum-free medium with KKs for 2-5 days. KF cell lysate was subjected to Western blot analysis. Compared with KF single cell culture, phospho-ERK1/2 and downstream phospho-Elk-1 showed up-regulation in the co-culture groups, as did phospho-PI3K and phospho-Akt-1, indicating ERK and PI3K pathway activation. Western blotting of the conditioned medium demonstrated increased collagen I-III, laminin beta2, and fibronectin levels. Addition of the MEK1/2-specific inhibitor U0126 or the PI3K-specific inhibitor LY294002 (but not p38 kinase and JNK inhibitors) completely nullified collagen I-III production and significantly decreased laminin beta2 and fibronectin secretion. In the presence of the MEK1/2 or PI3K inhibitor, fibronectin demonstrated changes in molecular mass reflected by faster in-gel migration. These data strongly suggest that synchronous activation of both the ERK and PI3K pathways is essential for collagen I-III and laminin beta2 production. These pathways additionally appear to affect the side chain attachments of fibronectin. Modulation of these pathways may suggest a direction for keloid therapy.     

PDGF stimulates DNA synthesis in human vascular smooth muscle cells via a novel wortmannin-insensitive phosphatidylinositol 3-kinase

The class 1(A) phosphatidylinositol 3-kinase enzymes consist of a number of heterodimeric complexes of regulatory and catalytic subunits and have been implicated in a number of cellular responses. While platelet-derived growth factor (PDGF)-induced chemotaxis of human vascular smooth muscle cells (HVSMC) is inhibited by both wortmannin and LY294002, DNA synthesis is only inhibited by LY294002. Serum-induced DNA synthesis however is inhibited by LY294002, wortmannin and rapamycin. Similarly PDGF-induced protein kinase B (PKB) activation is inhibited by LY294002 but not by wortmannin or rapamycin. In conclusion PDGF-induced DNA synthesis appears to occur through a phosphatidylinositol 3-kinase (PI3-K)-dependent, but wortmannin-insensitive, PKB/Akt pathway.     

Dual regulation of MMP-2 expression by the type 1 insulin-like growth factor receptor: the phosphatidylinositol 3-kinase/Akt and Raf/ERK pathways transmit opposing signals

The matrix metalloproteinase (MMP)-2 has been recognized as a major mediator of basement membrane degradation, angiogenesis, tumor invasion, and metastasis. The factors that regulate its expression have not, however, been fully elucidated. We previously identified the type I insulin-like growth factor (IGF-I) receptor as a regulator of MMP-2 synthesis. The objective of the present study was to investigate the signal transduction pathway(s) mediating this regulation. We show here that in Lewis lung carcinoma subline H-59 cells treated with IGF-I (10 ng/ml), the PI 3-kinase (phosphatidylinositol 3'-kinase) /protein kinase B (Akt) and C-Raf/ERK pathways were activated, and MMP-2 promoter activity, mRNA, and protein synthesis were induced. MMP-2 induction was blocked by the PI 3-kinase inhibitors LY294002 and wortmannin, by overexpression of a dominant-negative Akt or wild-type PTEN (phosphatase and tensin homologue deleted on chromosome 10), and by rapamycin. In contrast, a MEK inhibitor PD98059 failed to reduce MMP-2 promoter activation and actually increased MMP-2 mRNA and protein synthesis by up to 30%. Interestingly, suppression of PI 3-kinase signaling by a dominant-negative Akt enhanced ERK activity in cells stimulated with 10 ng/ml but not with 100 ng/ml IGF-I. Furthermore, at the higher (100 ng/ml) IGF-I concentration, C-Raf and ERK, but not PI 3-kinase activation, was enhanced, and this resulted in down-regulation of MMP-2 synthesis. This effect was reversed in cells expressing a dominant-negative ERK mutant. The results suggest that IGF-I can up-regulate MMP-2 synthesis via PI 3-kinase/Akt/mTOR (the mammalian target of rapamycin) signaling while concomitantly transmitting a negative regulatory signal via the Raf/ERK pathway. The outcome of IGF-IR (the receptor for IGF-I) activation may ultimately depend on factors, such as ligand bioavailability, that can shift the balance preferentially toward one pathway or the other.     

Cartducin stimulates mesenchymal chondroprogenitor cell proliferation through both extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways

Cartducin, a paralog of Acrp30/adiponectin, is a secretory protein produced by both chondrogenic precursors and proliferating chondrocytes, and belongs to a novel C1q family of proteins. We have recently shown that cartducin promotes the growth of both mesenchymal chondroprogenitor cells and chondrosarcoma-derived chondrocytic cells in vitro. However, the cartducin-signaling pathways responsible for the regulation of cell proliferation have not been documented. In this study, we examined whether cartducin exists in serum and further investigated the intracellular signaling pathways stimulated by cartducin in mesenchymal chondroprogenitor cells. Western blot analysis showed that, unlike Acrp30/adiponectin, cartducin was undetectable in mouse serum. Next, mesenchymal chondroprogenitor N1511 cells were stimulated with cartducin, and three major groups of mitogen-activated protein kinase (MAPK) pathways and the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway were examined. Cartducin activated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, but not c-jun N-terminal kinase (JNK) nor p38 MAPK. The MEK1/2 inhibitor, U0126, blocked cartducin-stimulated ERK1/2 phosphorylation and suppressed the DNA synthesis induced by cartducin in N1511 cells. The PI3K inhibitor, LY294002, blocked cartducin-stimulated Akt phosphorylation and a decrease in cartducin-induced DNA synthesis in N1511 cells was also observed. These data suggest that cartducin is a peripheral skeletal growth factor, and that the proliferation of mesenchymal chondroprogenitor cells stimulated by cartducin is associated with activations of the ERK1/2 and PI3K/Akt signaling pathways.     

Interleukin-9 influences chemokine release in airway smooth muscle: role of ERK

Interleukin (IL)-9 is a pleiotropic cytokine that has been proposed as a candidate gene for asthma. As IL-9 expression is correlated with airway hyperresponsiveness in animals, we examined the effects of IL-9 on cultured human airway smooth muscle (HASM) cells. IL-9 alone had no effect on IL-8 release, but at concentrations of > or =30 ng/ml, IL-9 significantly increased IL-8 release induced by TNF-alpha. IL-9 increased phosphorylation of extracellular signal-regulated protein kinase (ERK, p42 and p44) in a concentration- and time-dependent fashion, and U-0126 (10 micro M), which inhibits ERK phosphorylation, abolished the synergism between TNF-alpha and IL-9 on IL-8 release. IL-9 alone had no effect on eotaxin release into HASM cell supernatants but at concentrations of > or =10 ng/ml caused an approximately 50% increase in release of eotaxin evoked by IL-13 (10 ng/ml). U-0126 blocked the synergism between IL-9 and IL-13 on eotaxin release. IL-9 had no effect on cyclooxygenase-2 (COX-2) expression or PGE(2) release and did not augment the COX-2 expression that was induced by IL-1beta. Our results indicate that airway smooth muscle is a target for IL-9 and that IL-9 amplifies the potential for these cells to recruit eosinophils and neutrophils into the airways by a mechanism involving ERK.     

Extracellular acidosis induces neutrophil activation by a mechanism dependent on activation of phosphatidylinositol 3-kinase/Akt and ERK pathways

Inflammation in peripheral tissues is usually associated with the development of local acidosis; however, there are few studies aimed at analyzing the influence of acidosis on immune cells. We have shown previously that extracellular acidosis triggers human neutrophil activation, inducing a transient increase in intracellular Ca2+ concentration, a shape change response, the up-regulation of CD18 expression, and a delay of apoptosis. In this study, we analyzed the signaling pathways responsible for neutrophil activation. We found that acidosis triggers the phosphorylation of Akt (the main downstream target of PI3K) and ERK MAPK, but not that of p38 and JNK MAPK. No degradation of IkappaB was observed, supporting the hypothesis that NF-kappaB is not activated under acidosis. Inhibition of PI3K by wortmannin or LY294002 markedly decreased the shape change response and the induction of Ca2+ transients triggered by acidosis, whereas the inhibition of MEK by PD98059 or U0126 significantly inhibited the shape change response without affecting the induction of Ca2+ transients. We also found that acidosis not only induces a shape change response and the induction of Ca2+ transients in human neutrophils but also stimulates the endocytosis of FITC-OVA and FITC-dextran. Stimulation of endocytosis was partially prevented by inhibitors of PI3K and MEK. Together, our results support the notion that the stimulation of human neutrophils by extracellular acidosis is dependent on the activation of PI3K/Akt and ERK pathways. Of note, using mouse peritoneal neutrophils we observed that the enhancement of endocytosis induced by acidosis was associated with an improved ability to present extracellular Ags through a MHC class I-restricted pathway.     

PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways.

Phosphatidylinositol 3-kinase (PI3-kinase) activates protein kinase B (also known as Akt), which phosphorylates and activates a cyclic nucleotide phosphodiesterase 3B. Increases in cyclic nucleotide concentrations inhibit agonist-induced contraction of vascular smooth muscle. Thus we hypothesized that the PI3-kinase/Akt pathway may regulate vascular smooth muscle tone. In unstimulated, intact bovine carotid artery smooth muscle, the basal phosphorylation of Akt was higher than that in cultured smooth muscle cells. The phosphorylation of Akt decreases in a time-dependent manner when incubated with the PI3-kinase inhibitor, LY-294002. Agonist (serotonin)-, phorbol ester (phorbol 12,13-dibutyrate; PDBu)-, and depolarization (KCl)-induced contractions of vascular smooth muscles were all inhibited in a dose-dependent fashion by LY-294002. However, LY-294002 did not inhibit serotonin- or PDBu-induced increases in myosin light chain phosphorylation or total O(2) consumption, suggesting that inhibition of contraction was not mediated by reversal or inhibition of the pathways that lead to smooth muscle activation and contraction. Treatment of vascular smooth muscle with LY-294002 increased the activity of cAMP-dependent protein kinase and increased the phosphorylation of the cAMP-dependent protein kinase substrate heat shock protein 20 (HSP20). These data suggest that activation of the PI3-kinase/Akt pathway in unstimulated smooth muscle may modulate vascular smooth muscle tone (allow agonist-induced contraction) through inhibition of the cyclic nucleotide/HSP20 pathway and suggest that cyclic nucleotide-dependent inhibition of contraction is dissociated from the myosin light chain contractile regulatory pathways.     

Human neutrophil-derived elastase induces airway smooth muscle cell proliferation

Neutrophils and their derived elastase are abundant in chronic inflammatory responses of asthma. This study aimed to investigate the mitogenic effect of elastase on airway smooth muscle (ASM) cells and the implicated signal transduction pathway. Near confluent cultured human ASM cells were treated with human neutrophil elastase (HNE, 0.01 to 0.5 microg/ml) or vehicle for 24 hours with or without extracellular signal-regulated kinase (ERK) inhibitor (PD98059, 30 microM), p38 kinase inhibitor (SB203580, 10 microM) or elastase inhibitor II (100 microg/ml). The ASM cell numbers were counted by a hemocytometer and DNA synthesis was assessed by flowcytometry. Western blots analysis for the expression of ERK, p38 and cyclin D1 was determined. HNE dose-dependently increased ASM cell numbers and the percentage of cells entering S-phase of cell cycle. This response was abolished by neutrophil elastase inhibitors and attenuated by PD98059, but not SB203580. HNE increased ERK phosphorylation and cyclin D1 expression. Pretreatment with PD98059 significantly inhibited elastase-induced cyclin D1 activity. The increased ASM cellular gap and cell shape change by proteolytic activity of HNE may be contributory to ERK activation and therefore cell proliferation. Our results demonstrate that HNE is mitogenic for ASM cells by increasing cyclin D1 activity through ERK signaling pathway.     

Enteric neuroblasts require the phosphatidylinositol 3-kinase pathway for GDNF-stimulated proliferation

The enteric nervous system (ENS) develops from neural crest cells that enter the gut, migrate, proliferate, and differentiate into neurons and glia. The growth factor glial-derived neurotrophic factor (GDNF) stimulates the proliferation and survival of enteric crest-derived cells. We investigated the intracellular signaling pathways activated by GDNF and their involvement in proliferation. We found that GDNF stimulates the phosphorylation of both the PI 3-kinase downstream substrate Akt and the MAP kinase substrate ERK in cultures of immunoaffinity-purified embryonic avian enteric crest-derived cells. The selective PI 3-kinase inhibitor LY-294002 blocked GDNF-stimulated Akt phosphorylation in purified crest cells, and reduced proliferation in cultures of dissociated quail gut. The ERK kinase (MEK) inhibitors PD 98059 and UO126 did not reduce GDNF-stimulated proliferation, although PD 98059 blocked GDNF-stimulated phosphorylation of ERK. We conclude that the PI 3-kinase pathway is necessary for the GDNF-stimulated proliferation of enteric neuroblasts.     

Friction in airway smooth muscle: mechanism, latch, and implications in asthma

Fredberg, J. J., K. A. Jones, M. Nathan, S. Raboudi,Y. S. Prakash, S. A. Shore, J. P. Butler, and G. C. Sieck. Friction in airway smooth muscle: mechanism, latch, andimplications in asthma. J. Appl.Physiol. 81(6): 2703-2712, 1996.In muscle,active force and stiffness reflect numbers of actin-myosin interactions and shortening velocity reflects their turnover rates, but the molecular basis of mechanical friction is somewhat less clear. Tobetter characterize molecular mechanisms that govern mechanical friction, we measured the rate of mechanical energy dissipation and therate of actomyosin ATP utilization simultaneously in activated canineairway smooth muscle subjected to small periodic stretches as occur inbreathing. The amplitude of the frictional stress is proportional toE, where E is the tissue stiffness defined by the slope of theresulting force vs. displacement loop and  is the hysteresivitydefined by the fatness of that loop. From contractile stimulus onset,the time course of frictional stress amplitude followed a biphasicpattern that tracked that of the rate of actomyosin ATP consumption.The time course of hysteresivity, however, followed a differentbiphasic pattern that tracked that of shortening velocity. Takentogether with an analysis of mechanical energy storage and dissipationin the cross-bridge cycle, these results indicate, first, that likeshortening velocity and the rate of actomyosin ATP utilization,mechanical friction in airway smooth muscle is also governed by therate of cross-bridge cycling; second, that changes in cycling rateassociated with conversion of rapidly cycling cross bridges to slowlycycling latch bridges can be assessed from changes of hysteresivity ofthe force vs. displacement loop; and third, that steady-state forcemaintenance (latch) is a low-friction contractile state. This lastfinding may account for the unique inability of asthmatic patients to reverse spontaneous airways obstruction with a deep inspiration.