Molecular pathways involved in the anti-apoptotic effect of 1,25-dihydroxyvitamin D3 in primary human keratinocytes

We previously reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] protects primary human keratinocytes against ultraviolet (UV)B-induced apoptosis. Here, we confirmed the anti-apoptotic effect of 1,25(OH)2D3 in keratinocytes, using cisplatin and doxorubicin as apoptotic triggers. We further showed that 1,25(OH)2D3 activates two survival pathways in keratinocytes: the MEK/extracellular signal regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway. Activation of ERK and Akt by 1,25(OH)2D3 was transient, required a minimal dose of 10(-9) M and could be blocked by actinomycin D and cycloheximide. Moreover, inhibition of Akt or ERK activity with respectively a PI-3K inhibitor (LY294002) or MEK inhibitors (PD98059, UO126), partially or totally suppressed the anti-apoptotic capacity of 1,25(OH)2D3. Finally, 1,25(OH)2D3 changed the expression of different apoptosis regulators belonging to the Bcl-2 family. Indeed, 1,25(OH)2D3 treatment increased levels of the anti-apoptotic protein Bcl-2 and decreased levels of the pro-apoptotic proteins Bax and Bad in a time- and dose-dependent way. Induction of Bcl-2 by 1,25(OH)2D3 was further shown to be mediated by ERK and, to a lesser extent, by Akt. In conclusion, 1,25(OH)2D3 clearly protects keratinocytes against apoptosis (1) by activating the MEK/ERK and the PI-3K/Akt survival pathways and (2) by increasing the Bcl-2 to Bax and Bad ratio.     

Both src-dependent and -independent mechanisms mediate phosphatidylinositol 3-kinase regulation of colony-stimulating factor 1-activated mitogen-activated protein kinases in myeloid progenitors

Colony-stimulating factor 1 (CSF-1) supports the proliferation, survival, and differentiation of bone marrow-derived cells of the monocytic lineage. In the myeloid progenitor 32D cell line expressing CSF-1 receptor (CSF-1R), CSF-1 activation of the extracellular signal-regulated kinase (ERK) pathway is both Ras and phosphatidylinositol 3-kinase (PI3-kinase) dependent. PI3-kinase inhibition did not influence events leading to Ras activation. Using the activity of the PI3-kinase effector, Akt, as readout, studies with dominant-negative and oncogenic Ras failed to place PI3-kinase downstream of Ras. Thus, PI3-kinase appears to act in parallel to Ras. PI3-kinase inhibitors enhanced CSF-1-stimulated A-Raf and c-Raf-1 activities, and dominant-negative A-Raf but not dominant-negative c-Raf-1 reduced CSF-1-provoked ERK activation, suggesting that A-Raf mediates a part of the stimulatory signal from Ras to MEK/ERK, acting in parallel to PI3-kinase. Unexpectedly, a CSF-1R lacking the PI3-kinase binding site (DeltaKI) remained capable of activating MEK/ERK in a PI3-kinase-dependent manner. To determine if Src family kinases (SFKs) are involved, we demonstrated that CSF-1 activated Fyn and Lyn in cells expressing wild-type (WT) or DeltaKI receptors. Moreover, CSF-1-induced Akt activity in cells expressing DeltaKI is SFK dependent since Akt activation was prevented by pharmacological or genetic inhibition of SFK activity. The docking protein Gab2 may link SFK to PI3-kinase. CSF-1 induced Gab2 tyrosyl phosphorylation and association with PI3-kinase in cells expressing WT or DeltaKI receptors. However, only in DeltaKI cells are these events prevented by PP1. Thus in myeloid progenitors, CSF-1 can activate the PI3-kinase/Akt pathway by at least two mechanisms, one involving direct receptor binding and one involving SFKs.     

Inhibition of Src family kinases blocks epidermal growth factor (EGF)-induced activation of Akt, phosphorylation of c-Cbl, and ubiquitination of the EGF receptor

Stimulation of T47D cells with epidermal growth factor (EGF) results in the activation of the intrinsic tyrosine kinases of the receptor and the phosphorylation of multiple cellular proteins including the receptor, scaffold molecules such as c-Cbl, adapter molecules such as Shc, and the serine/threonine protein kinase Akt. We demonstrate that EGF stimulation of T47D cells results in the activation of the Src protein-tyrosine kinase and that the Src kinase inhibitor PP1 blocks the EGF-induced phosphorylation of c-Cbl but not the activation/phosphorylation of the EGF receptor itself. PP1 also blocks EGF-induced ubiquitination of the EGF receptor, which is presumably mediated by phosphorylated c-Cbl. Src is associated with c-Cbl, and we have previously demonstrated that the Src-like kinase Fyn can phosphorylate c-Cbl at a preferred binding site for the p85 subunit of phosphatidylinositol 3'-kinase. PP1 treatment blocks EGF-induced activation of the anti-apoptotic protein kinase Akt suggesting that Src may regulate activation of Akt, perhaps by a Src --> c-Cbl --> phosphatidylinositol 3'-kinase --> Akt pathway.     

Interferon-dependent activation of the serine kinase PI 3'-kinase requires engagement of the IRS pathway but not the Stat pathway

Several signaling pathways are activated by interferon alpha (IFNalpha) in hematopoietic cells, including the Jak-Stat and the insulin receptor substrate (IRS) pathways. It has been previously shown that IFNalpha activates the phosphatidylinositol (PI) 3'-kinase via an interaction of the p85 subunit of PI 3'-kinase with IRS proteins. Other studies have proposed that Stat-3 also functions as an adapter for p85. We sought to identify the major pathway that regulates IFNalpha activation of the PI3'-kinase in hematopoietic cells. Our data demonstrate that IFNalpha induces the interaction of p85 with IRS-1 or IRS-2, but not Stat-3, in various hematopoietic cell lines in which IRS-1 and/or IRS-2 and Stat-3 are activated by IFNalpha. In addition, inhibition of PI 3'-kinase activity by preincubation of cells with the PI 3'-kinase inhibitor LY294002 does not affect IFN-dependent formation of SIF complexes that contain Stat-3. To determine whether phosphorylation of tyrosine residues in the IFN receptor is required for activation of the PI 3'-kinase, we performed studies using mouse L929 fibroblasts transfected with mutated human IFNAR1 and/or IFNAR2 subunits of the Type I IFN receptor, lacking tyrosine phosphorylation sites. The serine kinase activity of the PI-3K was activated by human IFNalpha in these cells, suggesting that phosphorylation of the Type I IFN receptor is not essential for PI3K activation. We then determined whether IFNalpha activates the Akt kinase, a known downstream target for PI 3'-kinase that mediates anti-apoptotic signals. Akt was activated by insulin or IGF-1, but not IFNalpha, in the IFNalpha-sensitive U-266 myeloma cell line. Altogether, our data establish that the IRS pathway and not the Stat pathway, is the major pathway regulating engagement of PI 3'-kinase in hematopoietic cells. Furthermore, the selective activation of Akt by insulin/IGF-1 suggests the existence of distinct regulatory activities of PI3'-kinase in growth factor versus interferon signaling.     

Akt kinase phosphorylation of inositol 1,4,5-trisphosphate receptors

A consensus RXRXX(S/T) substrate motif for Akt kinase is conserved in the C-terminal tail of all three inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) isoforms. We have shown that IP3R can be phosphorylated by Akt kinase in vitro and in vivo. Endogenous IP3Rs in Chinese hamster ovary T-cells were phosphorylated in response to Akt activation by insulin. LnCAP cells, a prostate cancer cell line with constitutively active Akt kinase, also showed a constitutive phosphorylation of endogenous type I IP3Rs. In all cases, the IP3R phosphorylation was diminished by the addition of LY294002, an inhibitor of phosphatidylinositol 3-kinase. Mutation of IP3R serine 2681 in the Akt substrate motif to alanine (S2681A) or glutamate (S2681E) prevented IP3R phosphorylation in COS cells transfected with constitutively active Akt kinase. Analysis of the Ca2+ flux properties of these IP3R mutants expressed in COS cell microsomes or in DT40 triple knock-out (TKO) cells did not reveal any modification of channel function. However, staurosporine-induced caspase-3 activation in DT40 TKO cells stably expressing the S2681A mutant was markedly enhanced when compared with wild-type or S2681E IP3Rs. We conclude that IP3 receptors are in vivo substrates for Akt kinase and that phosphorylation of the IP3R may provide one mechanism to restrain the apoptotic effects of calcium.     

Synergy between PI3K/Akt and Raf/MEK/ERK pathways in IGF-1R mediated cell cycle progression and prevention of apoptosis in hematopoietic cells

The insulin like growth factor-1 (IGF-1) receptor (R) induced PI3K/Akt signal transduction cascade has critical roles in prevention of apoptosis and regulation of cell cycle progression. Here, we discuss the effects of IGF-1R-mediated signal transduction on hematopoietic cells which normally require interleukin-3 (IL-3) for growth and prevention of apoptosis. Cytokine-dependent FDC-P1 hematopoietic cells were conditionally transformed to grow in response to overexpression of IGF-1R in the presence of IGF-1. When these cells were deprived of IL-3 or IGF-1 for 24 hrs, they exited the cell cycle, activated caspase 3 and underwent apoptosis. The effects of inhibitors which targeted the PI3K/Akt and Raf/MEK/ERK pathways were determined. When the cells were cultured with IGF-1 and either PI3K or MEK inhibitors, cell cycle progression and DNA synthesis were inhibited and caspase 3 activity and apoptosis were induced. Coinhibition of both pathways synergized to prevent cell cycle progression, inhibit DNA synthesis and induce apoptosis. These inhibitors had more apoptotic inducing effects when the cells were grown in response to IGF-1 than IL-3, indicating that IL-3 can induce additional anti-apoptotic pathways. These results demonstrate that the PI3K/Akt and Raf/MEK/ERK pathways are intimately involved in IGF-1R-mediated cell cycle progression and prevention of apoptosis in hematopoietic cells.     

Quercetin protects against cerebral ischemia/reperfusion and oxygen glucose deprivation/reoxygenation neurotoxicity

Beyond nutrition effect, quercetin is applied as a complement or an alternative for promoting human health and treating diseases. However, its complicated neuroprotective mechanisms have not yet been fully elucidated. This study provides evidence of an alternative target for quercetin, and sheds light on the mechanisms of its neuroprotection against cerebral ischemia/reperfusion (I/R) injury in Sprague–Dawley rats. Oral pretreatment using quercetin has alleviated cerebral I/R-induced neurological deficits, brain infarction, blood–brain barrier disruption, oxidative stress, TNF-α and IL-1β mRNA expression, along with apoptotic caspase 3 activity. The neuroprotective, anti-oxidative, anti-inflammatory, and anti-apoptotic effects of quercetin were replicated in rat hippocampal slice cultures and neuron/glia cultures which suffered from oxygen–glucose deprivation and reoxygenation (OGDR). Biochemical studies revealed a reduction of extracellular signal-regulated kinase (ERK) and Akt phosphorylation, along with an increase in protein tyrosine and serine/threonine phosphatase activity in cerebral I/R rat cortical tissues and OGDR hippocampal slice and neuron/glia cultures. Quercetin alleviated the changes in ERK/Akt phosphorylation and protein phosphatase activities. Inhibition of ERK or Akt alone was enough to cause apoptotic cell death and cytotoxicity in hippocampal slice cultures and neuron/glia cultures, while activators of ERK or Akt alleviated OGDR-induced cytotoxicity. Taken together, our results demonstrate that quercetin alleviated the increment of protein tyrosine and serine/threonine phosphatase activity, along with the reduction of ERK and Akt phosphorylation, which may play pivotal roles in the expansion of brain injury after cerebral I/R.     

Grb10 and active Raf-1 kinase promote Bad-dependent cell survival

The proapoptotic protein Bad is a key player in cell survival decisions, and is regulated post-translationally by several signaling networks. We expressed Bad in mouse embryonic fibroblasts to sensitize them to apoptosis, and tested cell lines derived from knock-out mice to establish the significance of the interaction between the adaptor protein Grb10 and the Raf-1 protein kinase in anti-apoptotic signaling pathways targeting Bad. When compared with wild-type cells, both Grb10 and Raf-1-deficient cells exhibit greatly enhanced sensitivity to apoptosis in response to Bad expression. Structure-function analysis demonstrates that, in this cellular model, the SH2, proline-rich, and pleckstrin homology domains of Grb10, as well as its Akt phosphorylation site and consequent binding by 14-3-3, are all necessary for its anti-apoptotic functions. As for Raf-1, its kinase activity, its ability to be phosphorylated by Src on Tyr-340/341 and the binding of its Ras-associated domain to the Grb10 SH2 domain are all necessary to promote cell survival. Silencing the expression of either Grb10 or Raf-1 by small interfering RNAs as well as mutagenesis of specific serine residues on Bad, coupled with signaling inhibitor studies, all indicate that Raf-1 and Grb10 are required for the ability of both the phosphatidylinositol 3-kinase/Akt and MAP kinase pathways to modulate the phosphorylation and inactivation of Bad. Because total Raf-1, ERK, and Akt kinase activities are not impaired in the absence of Grb10, we propose that this adapter protein creates a subpopulation of Raf-1 with specific anti-apoptotic activity.     

Interleukin-6 inhibits transforming growth factor-beta-induced apoptosis through the phosphatidylinositol 3-kinase/Akt and signal transducers and activators of transcription 3 pathways.

The multifunctional cytokine interleukin-6 (IL-6) regulates growth and differentiation of many cell types and induces production of acute-phase proteins in hepatocytes. Here we report that IL-6 protects hepatoma cells from apoptosis induced by transforming growth factor-beta (TGF-beta), a well known apoptotic inducer in liver cells. Addition of IL-6 blocked TGF-beta-induced activation of caspase-3 while showing no effect on the induction of plasminogen activator inhibitor-1 and p15(INK4B) genes, indicating that IL-6 interferes with only a subset of TGF-beta activities. To further elucidate the mechanism of this anti-apoptotic effect of IL-6, we investigated which signaling pathway transduced by IL-6 is responsible for this effect. IL-6 stimulation of hepatoma cells induced a rapid tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and its kinase activity followed by the activation of Akt. Inhibition of PI 3-kinase by wortmannin or LY294002 abolished the protection of IL-6 against TGF-beta-induced apoptosis. A dominant-negative Akt also abrogated this anti-apoptotic effect. Dominant-negative inhibition of STAT3, however, only weakly attenuated the IL-6-induced protection. Finally, inhibition of both STAT3 and PI 3-kinase by treating cells overexpressing the dominant-negative STAT3 with LY294002 completely blocked IL-6-induced survival signal. Thus, concomitant activation of the PI 3-kinase/Akt and the STAT3 pathways mediates the anti-apoptotic effect of IL-6 against TGF-beta, with the former likely playing a major role in this anti-apoptosis.     

β-Arrestin prevents cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt pathways

Our previous studies have shown that β-arrestin 2 plays an anti-apoptotic effect. However, the mechanisms by which β-arrestin contribute to anti-apoptotic role remain unclear. In this study, we show that a deficiency of either β-arrestin 1 or β-arrestin 2 significantly increases serum deprivation (SD)-induced percentage of apoptotic cells. β-arrestin 2 deficient-induced apoptosis was inhibited by transfection with β-arrestin 2 full-length plasmid, revealing that SD-induced apoptosis is dependent on β-arrestin 2. Furthermore, in the absence of either β-arrestin 1 or β-arrestin 2 significantly enhances SD-induced the level of pro-apoptotic proteins, including cleaved caspase-3, extracellular-signal regulated kinase 1/2 (ERK1/2) and p38, members of mitogen-activated protein kinases (MAPKs). In addition, a deficiency of either β-arrestin 1 or β-arrestin 2 inhibits phosphorylation of Akt. The SD-induced changes in cleaved caspase-3, ERK1/2 and p38 MAPKs, Akt, and apoptotic cell numbers could be blocked by double knockout of β-arrestin 1/2. Our study thus demonstrates that β-arrestin inhibits cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt signaling pathways.     

Differential signaling mechanisms of HNP-induced IL-8 production in human lung epithelial cells and monocytes

Human neutrophil peptides (HNP) kill microorganisms but also modulate immune responses through upregulation of the chemokine IL-8 by activation of the nucleotide P2Y(6) receptor. However, the intracellular signaling mechanisms remain yet to be determined. Human lung epithelial cells (A549) and monocytes (U937) were stimulated with HNP in the absence and presence of the specific kinase inhibitors for Src, extracellular signal-regulated kinase-1 and -2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinases (JNK), and Akt. HNP induced a rapid phosphorylation of the kinases in both cell types associated with a dose-dependent, selective production of IL-8 among 10 cytokines assayed. The HNP-induced IL-8 production was blocked by the Src tyrosine kinase inhibitor PP2, MEK1/2 inhibitor U0126, and the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002, but not by the JNK inhibitor SP600125 in both cell types. Treatment with the p38 inhibitor SB203580 attenuated the HNP-induced IL-8 production only in monocytes. Blockade of Src kinase blunted HNP-induced phosphorylation of the ERK1/2 and Akt but not p38 in monocytes. In contrast, Src inhibition had no effect on phosphorylation of the other kinases in the lung epithelial cells. We conclude that the activation of ERK1/2 and PI3K/Akt pathways is required for HNP-induced IL-8 release which occurs in a Src-independent manner in lung epithelial cells, while is Src-dependent in monocytes.     

JNK and PI3k/Akt signaling pathways are required for establishing persistent SARS-CoV infection in Vero E6 cells

Persistence was established after most of the SARS-CoV-infected Vero E6 cells died. RNA of the defective interfering virus was not observed in the persistently infected cells by Northern blot analysis. SARS-CoV diluted to 2 PFU failed to establish persistence, suggesting that some particular viruses in the seed virus did not induce persistent infection. Interestingly, a viral receptor, angiotensin converting enzyme (ACE)-2, was down-regulated in persistently infected cells. G418-selected clones established from parent Vero E6 cells, which were transfected with a plasmid containing the neomycin resistance gene, were infected with SARS-CoV, resulting in a potential cell population capable of persistence in Vero E6 cells. Our previous studies demonstrated that signaling pathways of extracellular signal-related kinase (ERK1/2), c-Jun N-terminal protein kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3'-kinase (PI3K)/Akt were activated in SARS-CoV-infected Vero E6 cells. Previous studies also showed that the activation of p38 MAPK by viral infection-induced apoptosis, and a weak activation of Akt was not sufficient to protect from apoptosis. In the present study, we showed that the inhibitors of JNK and PI3K/Akt inhibited the establishment of persistence, but those of MAPK/ERK kinase (MEK; as an inhibitor for ERK1/2) and p38 MAPK did not. These results indicated that two signaling pathways of JNK and PI3K/Akt were important for the establishment of persistence in Vero E6 cells.     

Activation of extracellular signal-regulated kinase couples platelet-activating factor-induced adhesion and delayed apoptosis of human neutrophils

Platelet-activating factor (PAF) promotes adhesion of neutrophil granulocytes to the endothelium, which is also linked to neutrophil survival. Here we report that PAF can prolong neutrophil survival by suppressing spontaneous apoptosis. PAF induced concurrent activation of the Ras/Raf-1/mitogen-activated protein kinase kinase (MAPKK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt pathways. ERK activation tightly correlated with up-regulation of CD11b/CD18 expression and beta(2)-integrin-dependent homotypic adhesion. These actions of PAF were markedly attenuated by the MAPKK/ERK inhibitor PD98059, but not by the phosphatidylinositol 3-kinase inhibitor wortmannin. By contrast, concurrent activation of ERK and Akt was required to inhibit caspase-3 activation and consequently to delay apoptosis. Consistently, pharmacological inhibition of either ERK or Akt partially reversed the anti-apoptotic action of PAF; however, they did not produce additive inhibition. These results indicate that PAF-induced activation of ERK contributes to both the expression of the pro-adhesive phenotype and repression of neutrophil apoptosis, thereby amplifying the inflammatory response.     

Transforming growth factor-beta1 regulates the fate of cultured spinal cord-derived neural progenitor cells

Abstract. Objectives: We have evaluated the physiological roles of transforming growth factor‐β1 (TGF‐β1) on differentiation, migration, proliferation and anti‐apoptosis characteristics of cultured spinal cord‐derived neural progenitor cells. Methods: We have used neural progenitor cells that had been isolated and cultured from mouse spinal cord tissue, and we also assessed the relevant reaction mechanisms using an activin‐like kinase (ALK)‐specific inhibitory system including an inhibitory RNA, and found that it involved potential signalling molecules such as phosphatidylinositol‐3‐OH kinase (PI3K)/Akt and mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK1/2). Results and Conclusions: Transforming growth factor‐β1‐mediated cell population growth was activated after treatment and was also effectively blocked by an ALK41517‐synthetic inhibitor (4‐(5‐benzo(1,3) dioxol‐5‐yl‐4‐pyridine‐2‐yl‐1H‐imidazole‐2‐yl) benzamide (SB431542) and ALK siRNA, thereby indicating the involvement of SMAD2 in the TGF‐β1‐mediated growth and migration of these neural progenitors cells (NPC). In the present study, TGF‐β1 actively induced NPC migration in vitro. Furthermore, TGF‐β1 demonstrated extreme anti‐apoptotic behaviour against hydrogen peroxide‐mediated apoptotic cell death. At low dosages, TGF‐β1 enhanced (by approximately 76%) cell survival against hydrogen peroxide treatment via inactivation of caspase‐3 and ‐9. TGF‐β1‐treated NPCs down‐regulated Bax expression and cytochrome c release; in addition, the cells showed up‐regulated Bcl‐2 and thioredoxin reductase 1. They also had increased p38, Akt and ERK1/2 phosphorylation, showing the involvement of both the PI3K/Akt and MAPK/ERK1/2 pathways in the neuroprotective effects of TGF‐β1. Interestingly, these effects operate on specific subtypes of cells, including neurones, neural progenitor cells and astrocytes in cultured spinal cord tissue‐derived cells. Lesion sites of spinal cord‐overexpressing TGF‐β1‐mediated prevention of cell death, cell growth and migration enhancement activity have been introduced as a possible new basis for therapeutic strategy in treatment of neurodegenerative disorders, including spinal cord injuries.     

Analysis of Ras-dependent signals that prevent caspase-3 activation and apoptosis induced by cytokine deprivation in hematopoietic cells

In hematopoietic cells, Ras has been implicated in signaling pathways that prevent apoptosis triggered by deprivation of cytokines, such as interleukin-3 (IL-3). However, the mechanism whereby Ras suppresses cell death remains incompletely understood. We have investigated the role of Ras in IL-3 signal transduction by using the cytokine-dependent BaF3 cell line. Herein, we show that the activation of the pro-apoptotic protease caspase-3 upon IL-3 removal is suppressed by expression of activated Ras, which eventually prevents cell death. For caspase-3 suppression, the Raf/extracellular signal-regulated kinase (ERK)- or phosphatidylinositol 3-kinase (PI3-K)/Akt-mediated signaling pathway downstream of Ras was required. However, inhibition of both pathways did not block activated Ras-dependent suppression of cell death-associated phenotypes, such as nuclear DNA fragmentation. Thus, a pathway that is independent of both Raf/ERK and PI3-K/Akt pathways may function downstream of Ras, preventing activated caspase-3-initiated apoptotic processes. Conditional activation of c-Raf-1 also suppressed caspase-3 activation and subsequent cell death without affecting Akt activity, providing further evidence for a PI3-K/Akt-independent mechanism.     

Free fatty acids inhibit serum deprivation-induced apoptosis through GPR120 in a murine enteroendocrine cell line STC-1

Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules. FFAs are known to exert a variety of physiological responses via their G protein-coupled receptors (GPCRs), such as the GPR40 family. Recently, we identified a novel FFA receptor, GPR120, that promotes secretion of glucagon-like peptide-1 (Hirasawa, A., Tsumaya, K., Awaji, T., Katsuma, S., Adachi, T., Yamada, M., Sugimoto, Y., Miyazaki, S., and Tsujimoto, G. (2005) Nat. Med. 11, 90-94). Here we showed that FFAs inhibit serum deprivation-induced apoptosis of murine enteroendocrine STC-1 cells, which express two types of GPCRs, GPR120 and GPR40, for unsaturated long chain FFA. We first found that linolenic acid potently activated ERK and Akt/protein kinase B (Akt) in STC-1 cells. ERK kinase inhibitors significantly reduced the anti-apoptotic effects of linolenic acid. Inhibitors for phosphatidylinositol 3-kinase (PI3K), a major target of which is Akt, significantly reduced the anti-apoptotic effects. Transfection of STC-1 cells with the dominant-negative form of Akt also inhibited the anti-apoptotic effect. These results suggested that the activation of ERK and PI3K-Akt pathways is required for FFA-induced anti-apoptotic effects on STC-1 cells. Transient transfection of STC-1 cells with GPR120 cDNA, but not GPR40 cDNA, enhanced inhibition of caspase-3 activation. RNA interference experiments showed that reduced expression of GPR120, but not GPR40, resulted in reduced ERK activation and reduced effects of FFAs on caspase-3 inhibition. Collectively, these results demonstrated that FFAs promote the activation of ERK and PI3K-Akt pathways mainly via GPR120, leading to the anti-apoptotic effect of STC-1 cells.     

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.     

Nerve growth factor regulates the expression of the cholinergic locus and the high-affinity choline transporter via the Akt/PKB signaling pathway

Nerve growth factor (NGF) is a trophic and survival factor for cholinergic neurons, and it induces the expression of several genes that are essential for synthesis and storage of acetylcholine (ACh), specifically choline acetyltransferase, vesicular ACh transporter (VAChT), and choline transporter. We have found previously that the phosphatidylinositol 3'-kinase pathway, but not the MEK/MAPK pathway, is the mediator of NGF-induced cholinergic differentiation. Here we demonstrate, in the rat pheochromocytoma cell line PC12 and in primary mouse neuronal cultures, that NGF-evoked up-regulation of these three cholinergic-specific genes is mediated by the anti-apoptotic signaling molecule Akt/protein kinase B. Inhibition of Akt activation by the pharmacological inhibitor 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO), or by a peptide fragment derived from the proto-oncogene TLC1, eliminated NGF-stimulated increases in cholinergic gene expression, as demonstrated by RT-PCR and reporter gene assays. Moreover, treatment with HIMO reversed NGF-evoked increases in choline acetyltransferase activity and ACh production. In co-transfection assays with the reporter construct, a dominant-negative Akt plasmid and Akt1-specific small interfering RNA also attenuated NGF-induced cholinergic promoter activity. Our data indicate that, in addition to its well-described role in promoting neuronal survival, Akt can also mediate signals necessary for neurochemical differentiation.     

IRS-4 mediates protein kinase B signaling during insulin stimulation without promoting antiapoptosis

Insulin receptor substrate (IRS) proteins are tyrosine phosphorylated and mediate multiple signals during activation of the receptors for insulin, insulin-like growth factor 1 (IGF-1), and various cytokines. In order to distinguish common and unique functions of IRS-1, IRS-2, and IRS-4, we expressed them individually in 32D myeloid progenitor cells containing the human insulin receptor (32D(IR)). Insulin promoted the association of Grb-2 with IRS-1 and IRS-4, whereas IRS-2 weakly bound Grb-2; consequently, IRS-1 and IRS-4 enhanced insulin-stimulated mitogen-activated protein kinase activity. During insulin stimulation, IRS-1 and IRS-2 strongly bound p85alpha/beta, which activated phosphatidylinositol (PI) 3-kinase, protein kinase B (PKB)/Akt, and p70(s6k), and promoted the phosphorylation of BAD. IRS-4 also promoted the activation of PKB/Akt and BAD phosphorylation during insulin stimulation; however, it weakly bound or activated p85-associated PI 3-kinase and failed to mediate the activation of p70(s6k). Insulin strongly inhibited apoptosis of interleukin-3 (IL-3)-deprived 32D(IR) cells expressing IRS-1 or IRS-2 but failed to inhibit apoptosis of cells expressing IRS-4. Consequently, 32D(IR) cells expressing IRS-4 proliferated slowly during insulin stimulation. Thus, the activation of PKB/Akt and BAD phosphorylation might not be sufficient to inhibit the apoptosis of IL-3-deprived 32D(IR) cells unless p85-associated PI 3-kinase or p70(s6k) are strongly activated.