Akt-mediated signaling is induced by cytokines and cyclic adenosine monophosphate and suppresses hepatocyte inducible nitric oxide synthase expression independent of MAPK P44/42

Cyclic AMP inhibits the expression of nitric oxide synthase (Harbrecht et al., 1995 [1]) in hepatocytes but the mechanism for this effect is incompletely understood. Cyclic AMP can activate several intracellular signaling pathways in hepatocytes including Protein Kinase A (PKA), cAMP regulated guanine nucleotide exchange factors (cAMP-GEFs), and calcium-mediated Protein Kinases. There is considerable overlap and cross-talk between many of these signaling pathways, however, and how these cascades regulate hepatocyte iNOS is not known. We hypothesized that Akt mediates the effect of cAMP on hepatocyte iNOS expression. Hepatocytes cultured with cytokines and dbcAMP increased Akt phosphorylation up to 2 h of culture. Akt phosphorylation was inhibited by the PI3K inhibitor LY294002 (10 μM), farnyltranferase inhibitor FTI-276, or transfection with a dominant negative Akt. The cyclic AMP-induced suppression of cytokine-stimulated iNOS was partially reversed by LY294002 and FTI-276. LY294002 also increased NFκB nucleus translocation by Western blot analysis in nuclear extracts. Cyclic AMP increased phosphorylation of Raf1 at serine 259 which was blocked by LY294002 and associated with decreased MAPK P44/42 phosphorylation. However, inhibition of MAPK P44/42 signaling with PD98059 failed to suppress cytokine-induced hepatocyte iNOS expression and did not enhance the inhibitory effect of dbcAMP on iNOS production. A constitutively active MAPK P44/42 plasmid had no effect on cytokine-stimulated NO production. These data demonstrate that dbcAMP regulates hepatocyte iNOS expression through an Akt-mediated signaling mechanism that is independent of MAPK P44/42.     

The luteinizing hormone-releasing hormone inhibits the anti-apoptotic activity of insulin-like growth factor-1 in pituitary alphaT3 cells by protein kinase Calpha-mediated negative regulation of Akt

The luteinizing hormone-releasing hormone (LHRH) receptor is a G protein-coupled receptor involved in the synthesis and release of pituitary gonadotropins and in the proliferation and apoptosis of pituitary cells. Insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor that has a mitogenic effect on pituitary cells. In this study, we used the alphaT3 gonadotrope cell line as a model to characterize the IGF-1R signaling pathways and to investigate whether this receptor interacts with the LHRH cascade. We found that IGF-1 activated the IGF-1R, insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase, and Akt in a time-dependent manner in alphaT3 cells. The MAPK (ERK1/2, p38, and JNK) pathways were only weakly activated by IGF-1. In contrast, LHRH strongly stimulated the MAPK pathways but had no effect on Akt activation. Cotreatment with IGF-1 and LHRH had various effects on these signaling pathways. 1) It strongly increased IGF-1-induced tyrosine phosphorylation of IRS-1 and IRS-1-associated phosphatidylinositol 3-kinase through activation of the epidermal growth factor receptor. 2) It had an additive effect on ERK1/2 activation without modifying the phosphorylation of p38 and JNK1/2. 3) It strongly reduced IGF-1 activation of Akt. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and cell cycle analysis revealed that, in addition to having an additive effect on ERK1/2 activation, cotreatment with IGF-1 and LHRH also had an additive effect on cell proliferation. The LHRH-induced inhibition of Akt stimulated by IGF-1 was completely blocked by Safingol, a protein kinase C (PKC) alpha-specific inhibitor, and by a dominant negative form of PKCalpha. Finally, we showed that the inhibitory effect of LHRH on IGF-1-induced PKCalpha-mediated Akt activation was associated with a marked reduction in Bad phosphorylation and a substantial decrease in the ability of IGF-1 to rescue alphaT3 cells from apoptosis induced by serum starvation. Our results demonstrate for the first time that several interactions take place between IGF-1 and LHRH receptors in gonadotrope cells.     

Differences in survival-promoting effects and intracellular signaling properties of BDNF and IGF-1 in cultured cerebral cortical neurons

Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) act on various neurons of the CNS as neurotrophic factors promoting neuronal differentiation and survival. We examined the survival-promoting effects of BDNF and IGF-1 on serum deprivation-induced death in cultured cerebral cortical neurons, and compared the intracellular signaling pathways stimulated by BDNF and IGF-1 in the neurons. We found that the survival-promoting effect of BDNF was much weaker than that of IGF-1 in serum deprivation-induced death of cultured cortical neurons. We found no differences in the levels of phosphatidylinositol 3-kinase (PtdIns3-K) activity or Akt (also called PKB) phosphorylation induced by BDNF and IGF-1 in the cultured cortical neurons, although many reports suggest that PtdIns3-K and Akt are involved in survival promotion. In addition, phosphorylation signals of mitogen-activated protein kinase (MAPK) and cAMP responsive element-binding protein (CREB), which have also been reported to be involved in survival promotion, were stimulated by BDNF much more potently than by IGF-1. These results show that there may be, as yet unidentified, intracellular signaling pathways other than the PtdIns3-K-Akt, MAPK and CREB signaling, to regulate survival promotion. These unidentified signaling pathways may be responsible for the distinct strengths of the survival-promoting effects of BDNF and IGF-1.     

Receptor-mediated formation of multilayer aggregates of primary cultured adult rat hepatocytes on lactose-substituted polystyrene.

We used a lactose-substituted polystyrene, poly-N-p-vinylbenzyl-D-lactonamide (PVLA), as a substratum for adult rat hepatocytes in primary culture. Spherical-shaped hepatocytes attached on PVLA substratum formed stable multilayer aggregates anchored on substratum through the stimulation of epidermal growth factor (EGF). The cells required calcium ion essentially to form the aggregates. The formation of multilayer aggregates was inhibited by colchicine, but not by cytochalasin B. The inhibition was also observed by added PVLA molecules in the culture medium and by treating surfaces of PVLA-coated dishes with allo A lectin. It was suggested that adult rat hepatocytes attached on PVLA substratum required the specific interaction between asialoglycoprotein receptors on the cell surface and PVLA substratum to form anchored multilayer aggregates.     

Abnormal insulin-like growth factor 1 signaling in human osteoarthritic subchondral bone osteoblasts

Insulin-like growth factor (IGF)-1 is a key factor in bone homeostasis and could be involved in bone tissue sclerosis as observed in osteoarthritis (OA). Here, we compare the key signaling pathways triggered in response to IGF-1 stimulation between normal and OA osteoblasts (Obs). Primary Obs were prepared from the subchondral bone of tibial plateaus of OA patients undergoing knee replacement or from normal individuals at autopsy. Phenotypic characterization of Obs was evaluated with alkaline phosphatase and osteocalcin release. The effect of IGF-1 on cell proliferation, alkaline phosphatase and collagen synthesis was evaluated in the presence or not of 50 ng/ml IGF-1, whereas signaling was studied with proteins separated by SDS-PAGE before western blot analysis. We also used immunoprecipitation followed by western blot analysis to detect interactions between key IGF-1 signaling elements. IGF-1 receptor (IGF-1R), Shc, Grb2, insulin receptor substrate (IRS)-1, and p42/44 mitogen-activated protein kinase (MAPK) levels were similar in normal and OA Obs in the presence or absence of IGF-1. After IGF-1 stimulation, the phosphorylation of IGF-1R in normal and OA Obs was similar; however, the phosphorylation of IRS-1 was reduced in OA Ob. In addition, the PI3K pathway was activated similarly in normal and OA Obs while that for p42/44 MAPK was higher in OA Obs compared to normal. p42/44 MAPK can be triggered via an IRS-1/Syp or Grb2/Shc interaction. Interestingly, Syp was poorly phosphorylated under basal conditions in normal Obs and was rapidly phosphorylated upon IGF-1 stimulation, yet Syp showed a poor interaction with IRS-1. In contrast, Syp was highly phosphorylated in OA Obs and its interaction with IRS-1 was very strong initially, yet rapidly dropped with IGF-1 treatments. The interaction of Grb2 with IRS-1 progressively increased in response to IGF-1 in OA Obs whereas this was absent in normal Ob. IGF-1 stimulation altered alkaline phosphatase in Ob, an effect reduced in the presence of PD98059, an inhibitor of p42/44 MAPK signaling, whereas neither IGF-1 nor PD98059 had any significant effect on collagen synthesis. In contrast, cell proliferation was higher in OA Obs compared to normal under basal conditions, and IGF-1 stimulated more cell proliferation in OA Obs than in normal Ob, an effect totally dependent on p42/44 MAPK activiy. The altered response of OA Obs to IGF-1 may be due to abnormal IGF-1 signaling in these cells. This is mostly linked with abnormal IRS-1/Syp and IRS-1/Grb2 interaction in these cells.     

Abnormal insulin-like growth factor 1 signaling in human osteoarthritic subchondral bone osteoblasts

Insulin-like growth factor (IGF)-1 is a key factor in bone homeostasis and could be involved in bone tissue sclerosis as observed in osteoarthritis (OA). Here, we compare the key signaling pathways triggered in response to IGF-1 stimulation between normal and OA osteoblasts (Obs). Primary Obs were prepared from the subchondral bone of tibial plateaus of OA patients undergoing knee replacement or from normal individuals at autopsy. Phenotypic characterization of Obs was evaluated with alkaline phosphatase and osteocalcin release. The effect of IGF-1 on cell proliferation, alkaline phosphatase and collagen synthesis was evaluated in the presence or not of 50 ng/ml IGF-1, whereas signaling was studied with proteins separated by SDS-PAGE before western blot analysis. We also used immunoprecipitation followed by western blot analysis to detect interactions between key IGF-1 signaling elements. IGF-1 receptor (IGF-1R), Shc, Grb2, insulin receptor substrate (IRS)-1, and p42/44 mitogen-activated protein kinase (MAPK) levels were similar in normal and OA Obs in the presence or absence of IGF-1. After IGF-1 stimulation, the phosphorylation of IGF-1R in normal and OA Obs was similar; however, the phosphorylation of IRS-1 was reduced in OA Ob. In addition, the PI3K pathway was activated similarly in normal and OA Obs while that for p42/44 MAPK was higher in OA Obs compared to normal. p42/44 MAPK can be triggered via an IRS-1/Syp or Grb2/Shc interaction. Interestingly, Syp was poorly phosphorylated under basal conditions in normal Obs and was rapidly phosphorylated upon IGF-1 stimulation, yet Syp showed a poor interaction with IRS-1. In contrast, Syp was highly phosphorylated in OA Obs and its interaction with IRS-1 was very strong initially, yet rapidly dropped with IGF-1 treatments. The interaction of Grb2 with IRS-1 progressively increased in response to IGF-1 in OA Obs whereas this was absent in normal Ob. IGF-1 stimulation altered alkaline phosphatase in Ob, an effect reduced in the presence of PD98059, an inhibitor of p42/44 MAPK signaling, whereas neither IGF-1 nor PD98059 had any significant effect on collagen synthesis. In contrast, cell proliferation was higher in OA Obs compared to normal under basal conditions, and IGF-1 stimulated more cell proliferation in OA Obs than in normal Ob, an effect totally dependent on p42/44 MAPK activiy. The altered response of OA Obs to IGF-1 may be due to abnormal IGF-1 signaling in these cells. This is mostly linked with abnormal IRS-1/Syp and IRS-1/Grb2 interaction in these cells.     

Comparative signaling pathways of insulin-like growth factor-1 and brain-derived neurotrophic factor in hippocampal neurons and the role of the PI3 kinase pathway in cell survival

Insulin-like growth factor-1 (IGF-1) and brain-derived neurotrophic factor (BDNF) are trophic factors required for the viability and normal functions of various neuronal cells. However, the detailed intracellular mechanism(s) involved in these effects in neuronal cells remains to be fully elucidated. In present study, the respective intracellular signaling pathway induced by IGF-1 and BDNF and their possible role in neuronal survival were investigated. Both IGF-1 and BDNF protected hippocampal neurons from serum deprivation-induced death with IGF-1 apparently being more potent. Western blot analyses showed that both IGF-1 and BDNF induced the activation of the phosphatidylinositide 3 kinase (PI3)/Akt (protein kinase B) kinase and the mitogen-activated protein kinase (MAPK) pathways. The phosphorylation of Akt and its downstream target, FKHRL1, induced by IGF-1 was rapid and sustained while that of MAPK was transient. The reverse situation was observed for BDNF. Moreover, IGF-1 potently induced the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and its association with PI3 kinase while BDNF was weak in these assays. In contrast, the tyrosine phosphorylation of Shc proteins was dramatically stimulated by BDNF, with IGF-1 having only a minimal effect. Most interestingly, only the inhibitor of the PI3K/Akt pathway, LY294002, was able to block the survival effects of both IGF-1 and BDNF; an inhibitor of the MAPK pathway inhibitor, PD98059, being ineffective. Taken together, these data reveal that the survival properties of both IGF-1 and BDNF against serum deprivation are mediated by the activation of the PI3K/Akt, but not the MAPK, pathway in hippocampal neurons.     

Hepatitis B virus X protein activates the p38 mitogen-activated protein kinase pathway in dedifferentiated hepatocytes

Hepatitis B virus X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. Employing a cellular model linked to pX-mediated transformation, we investigated the role of the previously reported Stat3 activation by pX in hepatocyte transformation. Our model is composed of a differentiated hepatocyte (AML12) 3pX-1 cell line that undergoes pX-dependent transformation and a dedifferentiated hepatocyte (AML12) 4pX-1 cell line that does not exhibit transformation by pX. We report that pX-dependent Stat3 activation occurs only in non-pX-transforming 4pX-1 cells and conclude that Stat3 activation is not linked to pX-mediated transformation. Maximum Stat3 transactivation requires Ser727 phosphorylation, mediated by mitogenic pathway activation. Employing dominant negative mutants and inhibitors of mitogenic pathways, we demonstrate that maximum, pX-dependent Stat3 transactivation is inhibited by the p38 mitogen-activated protein kinase (MAPK)-specific inhibitor SB 203580. Using transient-transreporter and in vitro kinase assays, we demonstrate for the first time that pX activates the p38 MAPK pathway only in 4pX-1 cells. pX-mediated Stat3 and p38 MAPK activation is Ca(2+) and c-Src dependent, in agreement with the established cellular action of pX. Importantly, pX-dependent activation of p38 MAPK inactivates Cdc25C by phosphorylation of Ser216, thus initiating activation of the G(2)/M checkpoint, resulting in 4pX-1 cell growth retardation. Interestingly, pX expression in the less differentiated hepatocyte 4pX-1 cells activates signaling pathways known to be active in regenerating hepatocytes. These results suggest that pX expression in the infected liver effects distinct mitogenic pathway activation in less differentiated versus differentiated hepatocytes.     

Multiple mechanisms are responsible for transactivation of the epidermal growth factor receptor in mammary epithelial cells

The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.     

Growth factors stimulate kidney proximal tubule cell migration independent of augmented tyrosine phosphorylation of focal adhesion kinase

Migration of human proximal tubule cells (HKC-5) was stimulated by epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). Integrin signaling via phosphorylation of focal adhesion kinase (FAK) appears to play a central role in cell migration. Once stimulated, FAK undergoes autophosphorylation at tyrosine (Y) 397, followed by phosphorylation of several sites including Y576/Y577 which increases FAK's kinase activity, as well as at Y407, Y861, and Y925. EGF, HGF, and IGF-1 stimulate FAK phosphorylation in various cells. We showed that endothelin stimulated phosphorylation of Y397 in fibroblasts but not HKC-5 cells. After EGF stimulation, HKC-5 cells showed no change in tyrosine phosphorylation at FAK Y397, 407, 576, 861, or 925. Similarly, HGF and IGF-1 did not stimulate the phosphorylation of FAK Y397 in HKC-5 cells. Further, after inhibition of FAK expression by siRNA, cell migration was similar to cells treated with non-target siRNA and responded to EGF with increased migration. Thus, in proximal tubule cells, stimulation of cell migration by growth factors was independent of augmented FAK tyrosine phosphorylation.     

The role of the cyclic GMP-inhibited cyclic AMP-specific phosphodiesterase (PDE3) in regulating clonal BRIN-BD11 insulin secreting cell survival

We report here that the cyclic GMP-inhibited cyclic AMP specific phosphodiesterase (PDE3B) is expressed as a membrane-bound protein in clonal insulin-secreting BRIN-BD11 cells. This was shown using SKF94836 (PDE3 inhibitor) which maximally inhibited membrane-bound cyclic AMP PDE activity by approximately 25-30% and by RT-PCR. We also demonstrated that insulin growth factor-1 (IGF-1) activates PDE3B in BRIN-BD11 cells. We therefore evaluated the role of phosphoinositide 3-kinase (PI3K) and p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) pathways in regulating this enzyme. We report here that the PI3K inhibitor, wortmannin, prevented the IGF-1-dependent stimulation of PDE3B activity. In contrast, the inhibitor of MEK-1 activation, PD098059 (which reduced IGF-1-stimulated p42/p44 MAPK phosphorylation), had no effect on PDE3B activation. Furthermore, IGF-1-dependent stimulation of p42/p44 MAPK and PDE3B was abolished in serum-deprived cells and this was associated with apoptosis. We propose that the deregulation of the PI3K/PDE3B pathway might result in increased intracellular cyclic AMP accumulation, which promotes apoptosis. This was supported by the finding that the adenylyl cyclase activator, forskolin, also induced apoptosis. Finally, we found that orthovanadate (a phosphotyrosine phosphatase inhibitor) fully restored the activation of p42/p44 MAPK in serum-deprived cells, but had only a small effect on PDE activity. This confirmed that p42/p44 MAPK is on a separate pathway to PDE3B. Therefore, IGF-1-dependent regulation of PDE3B may be linked to cell survival through PI3K and not p42/p44 MAPK.     

PI3 kinase/Akt activation mediates estrogen and IGF-1 nigral DA neuronal neuroprotection against a unilateral rat model of Parkinson's disease

Recently, using the medial forebrain bundle (MFB) 6-hydroxydopmaine (6-OHDA) lesion rat model of Parkinson's disease (PD), we have demonstrated that blockade of central IGF-1 receptors (IGF-1R) attenuated estrogen neuroprotection of substantia nigra pars compacta (SNpc) DA neurons, but exacerbated 6-OHDA lesions in IGF-1 only treated rats (Quesada and Micevych [2004]: J Neurosci Res 75:107-116). This suggested that the IGF-1 system is a central mechanism through which estrogen acts to protect the nigrostriatal DA system. Moreover, these results also suggest that IGF-1R-induced intracellular signaling pathways are involved in the estrogen mechanism that promotes neuronal survival. In vitro, two convergent intracellular signaling pathways used by estrogen and IGF-1, the mitogen-activated protein kinase (MAPK/ERK), and phosphatidyl-inositol-3-kinase/Akt (PI3K/Akt), have been demonstrated to be neuroprotective. Continuous central infusions of MAPK/ERK and PI3K/Akt inhibitors were used to test the hypothesis that one or both of these signal transduction pathways mediates estrogen and/or IGF-1 neuroprotection of SNpc DA neurons after a unilateral administration of 6-OHDA into the MFB of rats. Motor behavior tests and tyrosine hydroxylase immunoreactivity revealed that the inhibitor of the PI3K/Akt pathway (LY294002) blocked the survival effects of both estrogen and IGF-1, while an inhibitor of the MAPK/ERK signaling (PD98059) was ineffective. Western blot analyses showed that estrogen and IGF-1 treatments increased PI3K/Akt activation in the SN; however, MAPK/ERK activation was decreased in the SN. Indeed, continuous infusions of inhibitors blocked phosphorylation of PI3K/Akt and MAPK/ERK. These findings indicate that estrogen and IGF-1-mediated SNpc DA neuronal protection is dependent on PI3K/Akt signaling, but not on the MAPK/ERK pathway.     

Insulin-like growth factor-1 receptor transactivation modulates the inflammatory and proliferative responses of neurotensin in human colonic epithelial cells

Neurotensin (NT) is a gastrointestinal neuropeptide that modulates intestinal inflammation and healing by binding to its high-affinity receptor NTR1. The dual role of NT in inflammation and healing is demonstrated in models of colitis induced by Clostridium difficile toxin A and dextran sulfate sodium, respectively, and involves NF-κB-dependent IL-8 expression and EGF receptor-mediated MAPK activation in human colonocytes. However, the detailed signaling pathways involved in these responses remain to be elucidated. We report here that NT/NTR1 coupling in human colonic epithelial NCM460 cells activates tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) in a time- and dose-dependent manner. NT also rapidly induces Src tyrosine phosphorylation, whereas pretreatment of cells with the Src inhibitor PP2 before NT exposure decreases NT-induced IGF-1R phosphorylation. In addition, inhibition of IGF-1R activation by either its specific antagonist AG1024 or siRNA against IGF-1 significantly reduces NT-induced IL-8 expression and NF-κB-dependent reporter gene expression. Pretreatment with AG1024 also inhibits Akt activation and apoptosis induced by NT. Silencing of Akt expression by siRNA also substantially attenuates NT-induced IL-8 promoter activity and NF-κB-dependent reporter gene expression. This is the first report to indicate that NT transactivates IGF-1R and that this response is linked to Akt phosphorylation and NF-κB activation, contributing to both pro-inflammatory and tissue repair signaling pathways in response to NT in colonic epithelial cells. We propose that IGF-1R activation represents a previously unrecognized key pathway involved in the mechanisms by which NT and NTR1 modulate colonic inflammation and inflammatory bowel disease.     

Growth hormone-stimulated insulin-like growth factor-1 expression in rainbow trout (Oncorhynchus mykiss) hepatocytes is mediated by ERK, PI3K-AKT, and JAK-STAT

Growth hormone (GH) initiates many of its growth-promoting actions by binding to GH receptors (GHR) and stimulating the synthesis and secretion of insulin-like growth factor-1 (IGF-1) from the liver and other sites. In this study, we used hepatocytes isolated from rainbow trout as a model system in which to determine the molecular signaling events of GH in fish. GH directly stimulated the phosphorylation of ERK, protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K), JAK2, and STAT5 in hepatocytes incubated in vitro. Activation of ERK, Akt, JAK2, and STAT5 was rapid, occurring within 5-10 min, and was concentration dependent. GH-induced ERK activation was completely blocked by the ERK pathway inhibitor, U0126, and the JAK2 inhibitor, 1,2,3,4,5,6-hexabromocyclohexane (Hex), and was partially blocked by the PI3K inhibitor LY294002. GH-stimulated Akt activation was completely blocked by LY294002 and Hex, but was not affected by U0126; whereas, STAT5 activation by GH was blocked only by Hex, and was not affected by either U0126 or LY294002. GH stimulated hepatic expression of IGF-1 mRNA as well as the secretion of IGF-1, effects that were partially or completely blocked by U0126, LY294002, and Hex. These results indicate that GHR linkage to the ERK, PI3K/Akt, or STAT pathways in trout liver cells requires activation of JAK2, and that GH-stimulated IGF-1 synthesis and secretion is mediated through the ERK, PI3K/Akt, and JAK-STAT pathways.     

Angiotensin II activation of focal adhesion kinase and pp60c-Src in relation to mitogen-activated protein kinases in hepatocytes

We have investigated signaling pathways leading to angiotensin II (Ang II) activation of mitogen-activated protein kinase (MAPK) in hepatocytes. MAPK activation by Ang II was abolished by the Ang II type 1 (AT1) receptor antagonist losartan, but not by the Ang II type 2 (AT2) receptor antagonist PD123319. Ang II (100 nM) induced a rapid phosphorylation of Src (peak approximately 2 min) and focal adhesion kinase (FAK, peak approximately 5 min) followed by a decrease to basal levels in 30 min. An increased association between FAK and Src in response to Ang II was detected after 1 min, which declined to basal levels after 30 min. Treatment with the Src kinase inhibitor PP-1 inhibited FAK phosphorylation. Downregulation of PKC, intracellular Ca2+ chelator BAPTA or inhibitors of PKC, Src kinase, MAPK kinase (MEK), Ca2+/calmodulin dependent protein kinase, phosphatidylinositol 3-kinase all blocked Ang II-induced MAPK phosphorylation. In contrast to other cells, there was no evidence for the role of EGF receptor transactivation in the activation of MAPK by Ang II. However, PDGF receptor phosphorylation is involved in the Ang II stimulated MAPK activation. Furthermore, Src/FAK and Ca/CaM kinase activation serve as potential links between the Ang II receptor and MAPK activation. These studies offer insight into the signaling network upstream of MAPK activation by AT1 receptor in hepatocytes.     

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.