PI3 Kinase-dependent Pathways Research Articles

Photobiomodulation induces microvesicle release in human keratinocytes: PI3 kinase-dependent pathway role

Microvesicles (MVs, 100-1000 nm diameter) are released into the extracellular environment by mammalian cells. MVs interact with near or remote cells through different mechanisms; in particular, MVs from human keratinocytes accelerate wound healing. Photobiomodulation by laser improves wound healing, but no information is available about its effects on MV release from human keratinocyte. Human-immortalized keratinocytes (human adult low-calcium high-temperature, HaCaT) were starved for 24 h and then irradiated using a 980-nm energy density of 0, 16.2, 32.5, and 48.7 J/cm2. After 24 h, MVs released in the conditioned medium were isolated, stained, and quantified using flow cytometry. MVs were distinguished from exosomes on the basis of their volume (forward scatter signals). In some experiments, phosphatidylinositol 3-kinase (PI-3K) activity, involved in MV release and stimulated by laser light, was inhibited by pre-treating cells with Wortmannin (WRT, 10 μg/mL). MVs were observed in HaCaT-conditioned medium both in basal- and laser-stimulated conditions. Photobiomodulation therapy, also known as PBMT, was able to increase MV release from human keratinocytes reaching a maximum effect at 32.5 J/cm2 with a stimulation of (148.6 ±15.1)% of basal (p<0.001). PI-3K activity inhibition strongly reduced both basal- and laser-induced MV release; but PBMT by laser still increased MV release, compared to basal values in the presence of WRT. In vitro near infrared photobiomodulation increased the releasing of MVs from human keratinocytes, while Wortmannin, a PI-3K inhibitor, negatively affects both basal- and laser-induced releasing. Laser-induced MV release could be a new effect of biostimulation on the wound healing process.

Phycocyanin alleviates ICV-STZ induced cognitive and molecular deficits via PI3-Kinase dependent pathway

In this study, the effect of Phycocyanin (Pc) to ameliorate the cognitive dysfunction in experimental model of Alzheimer's disease (AD) was evaluated. Intracerebroventricular (ICV) induction of Streptozotocin (STZ) (3 mg/kg) was done bilaterally twice in rats on alternative days. Rats were injected with Pc (50, 100 mg/kg; i. p.) for 28 days daily for behavioural and cholinergic activity assessment. As the effect was only significant at 100 mg/kg, later molecular experiments were performed using the same only. STZ induction led to increased activity of hippocampal cholinesterases and BAX and decreased activity of BCL-2 and ChAT. It enhanced TNF-α, and NF-κB in rat's brain and reduced BDNF and IGF-1 levels. Dysfunctional insulin signaling and decreased gene expressions of PI3–K, AKT was also observed. However, Pc treatment significantly prevented STZ-induced increased activity of hippocampal cholinesterases and BAX as well as increased the levels of BCL-2 and ChAT. Neuroinflammation was significantly attenuated and BDNF and IGF-1 levels were upregulated. Further, Pc also alleviated dysfunctional insulin signaling as evidenced by increased gene expression of IRS-1, PI3–K, AKT. In conclusion, our study demonstrated the immense potential of Pc in attenuating STZ-induced cognitive decline and it may be further explored as a therapeutic agent in managing AD.

Caffeic acid phenethyl ester rescued streptozotocin-induced memory loss through PI3-kinase dependent pathway

The present study was undertaken to elucidate the role of PI3-kinase signaling in memory enhancing potential of caffeic acid phenethyl ester (CAPE) against cognitive defects in rats after centrally administered streptozotocin as a model of Alzheimer’s disease. The Morris water maze and elevated plus maze paradigms showed profound loss of memory in adult Wistar rats (180–200 g) injected with streptozotocin (3 mg/kg) bilaterally (STZ-ICV) on day 1 and 3. Intraperitoneal administration of CAPE (6 mg/kg, i.p., 28 days) attenuated STZ-ICV triggered memory loss in rats. Treatment with PI3-kinase inhibitor (wortmannin, 5 μg/rat, ICV) or NOS blocker (L-NAME, 20 mg/kg, i.p., 28 days) interfered with memory restorative function of CAPE in STZ treated rats. In biochemical analysis markers of oxidative stress (TBARS, GSH, SOD, CAT), nitrite, AChE, TNF-α, eNOS and NFκB were measured in brain of rats on day 28. Interestingly, L-Arginine (100 mg/kg, i.p., 28 days) group exhibited moderate (p > 0.05) decline in memory functions. The brain oxidative stress, TNF-α, AChE activity and NFκB levels were elevated, and eNOS level was lowered by STZ-ICV treatment. Administration of CAPE lowered oxidative stress, AChE, nitrite and TNF-α levels in brain of rats. The eNOS level was enhanced and NFκB level was decreased by CAPE in STZ treated rats. Wortmannin injection elevated the brain oxidative stress, AChE activity and TNF-α levels, and decreased the nitrite, eNOS and NFκB level. Rise of brain oxidative stress parameters, AChE activity, TNF-α, eNOS and NFκB levels, and decline in brain nitrite content was observed in L-NAME treated group. L-Arginine administration showed modest effects (p > 0.05) on oxidative stress parameters. Brain nitrite content was enhanced although eNOS, NFκB levels, and AChE activity was decimated by L-Arginine treatment. It can be concluded that PI3-kinase mediated nitric oxide facilitation is an essential feature of CAPE action in STZ-ICV treated rats.

Enhanced expression of Giα proteins contributes to the hyperproliferation of vascular smooth muscle cells from spontaneously hypertensive rats via MAP kinase- and PI3 kinase-independent pathways.

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit hyperproliferation, enhanced MAP kinase (MAPK) activity, and overexpression of Giα proteins. This study was undertaken to examine whether the overexpression of Giα proteins contributes to the hyperproliferation of VSMC of SHR through MAPK signaling. The hyperproliferation of VSMC from SHR in the absence and presence of angiotensin II was restored towards those in Wistar-Kyoto (WKY) rats levels by pertussis toxin (PT) treatment. In addition, siRNA knockdown of Giα proteins also resulted in the attenuation of hyperproliferation towards control levels. The overexpression of Giα proteins was also inhibited by MAPK and PI3 kinase (PI3K) inhibitors. In addition, the hyperproliferation and enhanced phosphorylation of ERK1/2 and Akt in VSMC from SHR were attenuated towards WKY levels by the inhibitors of MAPK, PI3K, c-Src, and antioxidants, whereas PT was unable to attenuate the enhanced phosphorylation of ERK1/2 and Akt. Furthermore, 8Br-cAMP and forskolin also attenuated the hyperproliferation of VSMC from SHR. These results suggest that the hyperproliferation of VSMC from SHR may be attributed to the enhanced expression of Giα proteins and increased activation of MAPK and PI3 kinase. However, Giα-mediated hyperproliferation may not be mediated through MAPK- and PI3 kinase-dependent pathways and may involve decreased levels of intracellular cAMP.

The Promigratory Activity of the Matricellular Protein Galectin-3 Depends on the Activation of PI-3 Kinase

Expression of galectin-3 is associated with sarcoma progression, invasion and metastasis. Here we determined the role of extracellular galectin-3 on migration of sarcoma cells on laminin-111. Cell lines from methylcholanthrene-induced sarcomas from both wild type and galectin-3−/− mice were established. Despite the presence of similar levels of laminin-binding integrins on the cell surface, galectin-3−/− sarcoma cells were more adherent and less migratory than galectin-3+/+ sarcoma cells on laminin-111. When galectin-3 was transiently expressed in galectin-3−/− sarcoma cells, it inhibited cell adhesion and stimulated the migratory response to laminin in a carbohydrate-dependent manner. Extracellular galectin-3 led to the recruitment of SHP-2 phosphatase to focal adhesion plaques, followed by a decrease in the amount of phosphorylated FAK and phospho-paxillin in the lamellipodia of migrating cells. The promigratory activity of extracellular galectin-3 was inhibitable by wortmannin, implicating the activation of a PI-3 kinase dependent pathway in the galectin-3 triggered disruption of adhesion plaques, leading to sarcoma cell migration on laminin-111.

Antidepressant Drugs Diversely Affect Autophagy Pathways in Astrocytes and Neurons—Dissociation from Cholesterol Homeostasis

In the search for antidepressants' (ADs') mechanisms of action beyond their influence on monoaminergic neurotransmission, we analyzed the effects of three structurally and pharmacologically different ADs on autophagic processes in rat primary astrocytes and neurons. Autophagy has a significant role in controlling protein turnover and energy supply. Both, the tricyclic AD amitriptyline (AMI) and the selective serotonin re-uptake inhibitor citalopram (CIT) induced autophagy as mirrored by pronounced upregulation and cellular redistribution of the marker LC3B-II. Redistribution was characterized by formation of LC3B-II-positive structures indicative of autophagosomes, which associated with AVs in a time-dependent manner. Deletion of Atg5, representing a central mediator of autophagy in MEFs, led to abrogation of AMI-induced LC3B-I/II conversion. By contrast, VEN, a selective serotonin and noradrenaline reuptake inhibitor, did not promote autophagic processes in either cell type. The stimulatory impact of AMI on autophagy partly involved class-III PI3 kinase-dependent pathways as 3-methyladenine slightly diminished the effects of AMI. Autophagic flux as defined by autophagosome turnover was vastly undisturbed, and degradation of long-lived proteins was augmented upon AMI treatment. Enhanced autophagy was dissociated from drug-induced alterations in cholesterol homeostasis. Subsequent to AMI- and CIT-mediated autophagy induction, neuronal and glial viability decreased, with neurons showing signs of apoptosis. In conclusion, we report that distinct ADs promote autophagy in neural cells, with important implications on energy homeostasis.

Thrombin and Collagen Induce a Feedback Inhibitory Signaling Pathway in Platelets Involving Dissociation of the Catalytic Subunit of Protein Kinase A from an NFκB-IκB Complex

Protein kinase A (PKA) activation by cAMP phosphorylates multiple target proteins in numerous platelet inhibitory pathways that have a very important role in maintaining circulating platelets in a resting state. Here we show that in thrombin- and collagen-stimulated platelets, PKA is activated by cAMP-independent mechanisms involving dissociation of the catalytic subunit of PKA (PKAc) from an NFkappaB-IkappaBalpha-PKAc complex. We demonstrate mRNA and protein expression for most of the NFkappaB family members in platelets. From resting platelets, PKAc was co-immunoprecipitated with IkappaBalpha, and conversely, IkappaBalpha was also co-immunoprecipitated with PKAc. This interaction was significantly reduced in thrombin- and collagen-stimulated platelets. Stimulation of platelets with thrombin- or collagen-activated IKK, at least partly by PI3 kinase-dependent pathways, leading to phosphorylation of IkappaBalpha, disruption of an IkappaBalpha-PKAc complex, and release of free, active PKAc, which phosphorylated VASP and other PKA substrates. IKK inhibitor inhibited thrombin-stimulated IkBalpha phosphorylation, PKA-IkBalpha dissociation, and VASP phosphorylation, and potentiated integrin alphaIIbbeta3 activation and the early phase of platelet aggregation. We conclude that thrombin and collagen not only cause platelet activation but also appear to fine-tune this response by initiating downstream NFkappaB-dependent PKAc activation, as a novel feedback inhibitory signaling mechanism for preventing undesired platelet activation.

Lenalidomide treatment promotes CD154 expression on CLL cells and enhances production of antibodies by normal B cells through a PI3-kinase–dependent pathway

AbstractChronic lymphocytic leukemia (CLL) involves a profound humoral immune defect and tumor-specific humoral tolerance that directly contribute to disease morbidity and mortality. CD154 gene therapy can reverse this immune defect, but attempts to do this pharmacologically have been unsuccessful. The immune-modulatory agent lenalidomide shows clinical activity in CLL, but its mechanism is poorly understood. Here, we demonstrate that lenalidomide induces expression of functional CD154 antigen on CLL cells both in vitro and in vivo. This occurs via enhanced CD154 transcription mediated by a Nuclear Factor of Activated T cells c1 (NFATc1)/Nuclear Factor-κB (NF-κB) complex and also through phosphoinositide-3 (PI3)–kinase pathway-dependent stabilization of CD154 mRNA. Importantly, CD154-positive CLL cells up-regulate BID, DR5, and p73, become sensitized to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–mediated apoptosis, and promote costimulatory activation of normal B cells to produce antibodies. In CLL patients receiving lenalidomide, similar evidence of CD154 activation is observed including BID, DR5, and p73 induction and also development of anti-ROR1 tumor-directed antibodies. Our data demonstrate that lenalidomide promotes CD154 expression on CLL cells with subsequent activation phenotype, and may therefore reverse the humoral immune defect observed in this disease. This study is registered at http://clinicaltrials.gov as NCT00466895.

Akirin1 ( Mighty), a novel promyogenic factor regulates muscle regeneration and cell chemotaxis

Akirin1 ( Mighty) is a downstream target gene of myostatin and has been shown to be a promyogenic factor. Although expressed in many tissues, akirin1 is negatively regulated by myostatin specifically in skeletal muscle tissue. In this manuscript we have characterized the possible function of akirin1 in postnatal muscle growth. Molecular and immunohistological analyses indicated that while low levels of akirin1 are associated with quiescent satellite cells (SC), higher levels of akirin1 are detected in activated proliferating SC indicating that akirin1 could be associated with satellite cell activation. In addition to SC, macrophages also express akirin1, and increased expression of akirin1 resulted in more efficient chemotaxis of both macrophages and myoblasts. Akirin1 appears to regulate chemotaxis of both macrophages and myoblasts by reorganising actin cytoskeleton, leading to more efficient lamellipodia formation via a PI3 kinase dependent pathway. Expression analysis during muscle regeneration also indicated that akirin1 expression is detected very early (day 2) in regenerating muscle, and expression gradually peaks to coincide the nascent myotube formation stage of muscle regeneration. Based on these results we propose that akirin1 could be acting as a transducer of early signals of muscle regeneration. Thus, we speculate that myostatin regulates key steps of muscle regeneration including chemotaxis of inflammatory cells, SC activation and migration through akirin1.

Effects of advanced glycation end products‐inductor glyoxal and hydrogen peroxide as oxidative stress factors on rat retinal organ cultures and neuroprotection by UK‐14,304

Retinal ganglion cell degeneration is supposed to be mediated by reactive oxygen species (ROS) and advanced glycation end products (AGEs). The alpha2-adrenergic agonist, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (brimonidine; UK-14,304), is said to exert a neuroprotective effect. To investigate these mechanisms in detail, we exposed rat whole mounts to glyoxal or H(2)O(2) and treated them with either UK-14,304 alone or additionally with the phosphatidylinositide 3 kinase (PI3) kinase inhibitor, 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (Ly 294002). The accumulation of Nepsilon-[carboxymethyl] lysine (CML) was assessed immunohistochemically and changes in intracellular pH (pHi), mitochondrial transmembrane potential (MTMP) and ROS production in cell bodies of multipolar ganglion cell layer were studied by intravital fluorescence microscopy and confocal laser scanning microscopy. Ultrastructural changes in mitochondria of multipolar ganglion cell layer cell bodies were determined by transmission electron microscopy. We found that glyoxal and H(2)O(2) increased accumulation of CML-modified proteins and ROS production and decreased pHi and MTMP in cell bodies of multipolar ganglion cell layer. UK-14,304 could prevent production of ROS, accumulation of CML-modified proteins, ameliorate acidification, preserve MTMP and attenuate ultrastructural damages of ganglion cell mitochondria. Ly 294002 reversed the UK-14,304-mediated attenuation of CML and ROS production. We conclude that the protective effects of UK-14,304 seem partly to be mediated by PI3 kinase-dependent pathways.

Adrenomedullin protects neurons against oxygen glucose deprivation stress in an autocrine and paracrine manner

The understanding of mechanisms involved in ischaemic brain tolerance may provide new therapeutical targets for stroke. In vivo genomic studies revealed an up-regulation of adrenomedullin expression by hypoxic pre-conditioning. Furthermore, adrenomedullin reduced ischaemia-induced brain damage in rodents. However, whether adrenomedullin is involved in hypoxic pre-conditioning-induced tolerance and whether adrenomedullin protects directly neurons against ischaemia remain unknown. Using a neuronal model of hypoxic pre-conditioning and oxygen glucose deprivation (OGD), we showed that 0.1% or 0.5% of O2 pre-conditioning reduced the OGD-induced neuronal death, whereas 1% or 2% of O2 pre-treatment did not induce neuroprotection. Adrenomedullin expression increased following the hypoxic period, and following OGD only in pre-conditioned (0.1% or 0.5% of O2) neurons. Adrenomedullin pre-treatment and post-treatment reduced the OGD-induced neuronal death, partly through PI3kinase-dependent pathway. However, adrenomedullin antagonism during hypoxic pre-conditioning failed to inhibit the neuroprotection whereas adrenomedullin antagonism following OGD abolished the hypoxic pre-conditioning-induced neuroprotection. Finally, we showed that adrenomedullin is involved in neuroprotection induced by endothelial cells and microglia. In contrast, neuroprotection induced by astrocytes occurred through adrenomedullin-independent mechanisms. Altogether, our results suggest that adrenomedullin is an effector of the hypoxic pre-conditioning-induced neuronal tolerance and a potent autocrine and paracrine neuroprotective factor during cerebral ischaemia.

Tetraspanin CD9 regulates β1 integrin activation and enhances cell motility to fibronectin via a PI-3 kinase-dependent pathway

Tetraspanin CD9 regulates cell motility and other adhesive processes in a variety of tissue types. Using transfected Chinese Hamster Ovary cells as our model system, we examined the cellular pathways critical for CD9 promoted cell migration. α5β1 integrin was directly involved as CD9 enhanced migration was abolished by the α5β1 blocking antibody PB1. Furthermore, the ligand mimetic peptide RGDS, significantly upregulated the expression of a β1 ligand induced binding site (LIBS) demonstrating for the first time that CD9 expression potentiates β1 integrin high affinity conformation states. CD9 promoted cell motility was significantly blocked by phosphatidylinositol-3 kinase (PI-3K) inhibitors, wortmannin and LY294002, whereas inhibitors targeting protein kinase C or mitogen-activated protein kinase had no effect. PI-3K dominant/negative cDNA transfections confirmed that PI-3K was an essential component. CD9 enhanced the phosphorylation of the PI-3K substrate, Akt, in response to cell adhesion on FN. CD9 expression also upregulated p130Cas phosphorylation and total protein levels; however, p130Cas siRNA knockdown did not alter the motile phenotype. CD9 enhanced migration was also unaffected by serum deprivation suggesting that growth factors were not critical. Our studies demonstrate that CD9 upregulates β1 LIBS, and in concert with α5β1, enhances cell motility to FN via a PI-3K dependent mechanism.

Inhibition of ADP-induced platelet adhesion to immobilised fibrinogen by nitric oxide: Evidence for cGMP-independent mechanisms

Nitric oxide (NO) is an established regulator of platelet function, although the processes by which NO modulates platelet adhesion are unclear. We studied the importance of Ca 2+ and phosphoinositol-3-kinase (PI3kinase) as targets for NO signalling, in the physiological context of platelet adhesion using adenosine diphosphate (ADP)-stimulated adhesion to immobilised fibrinogen. DPTA-NONOate induced a time and concentration-dependent inhibition of adhesion, and reduced protein tyrosine phosphorylation. The action of NO was cGMP-independent despite activation of the cGMP-signalling cascade, as evidenced by VASP phosphorylation. Furthermore, the cGMP-independent mechanism did not involve PKA. Platelet activation by ADP requires Ca 2+ and PI3kinase-dependent signalling pathways. We examined the effect of NO on these pathways using two approaches. Firstly, we dissected the signalling pathways using the P2Y 1-receptor antagonist A3P5P, and secondly, directly inhibited Ca 2+ mobilisation and PI3kinase activity. ADP-induced adhesion was reduced but not abolished by A3P5P, suggesting signalling from P2Y 12 can induce adhesion. NO further reduced adhesion in the presence of A3P5P, indicating that NO inhibited adhesion independently of any effects on Ca 2+ mobilisation. Dimethyl bis-( o-aminophenoxy) ethane-tetraacetic acid (BAPTA) and wortmannin both partially inhibited ADP-induced adhesion, but completely abolished adhesion when used in combination, demonstrating that ADP-induced adhesion requires Ca 2+ and PI3kinase-regulated pathways. Combination of either dimethyl-BAPTA or wortmannin with DPTA-NONOate enhanced inhibition of both the Ca 2+ and PI3kinase-dependent pathways when compared to the levels of inhibition with either agent alone. Thus, we demonstrate that NO inhibits α IIbβ 3-mediated adhesion, by targeting both Ca 2+ and PI3kinase pathways in a cGMP-independent manner.

Differential activation of the PI 3-kinase effectors AKT/PKB and p70 S6 kinase by compound 48/80 is mediated by PKCα

The secretagogue compound 48/80 (c48/80) is a well known activator of calcium mediated processes and PKCs, and is a potent inducer of mast cell degranulation. As the latter process is a phosphoinositide 3-kinase (PI 3-kinase) mediated event, we wished to address whether or not c48/80 was an activator of PI 3-kinases. The data presented here reveal that c48/80 is an effective activator of PI 3-kinases as judged by the increased phosphorylation of PKB and p70 S6K in fibroblasts in a PI 3-kinase dependent fashion. Compound 48/80 effectively translocates PKB to the plasma membrane and induces phosphorylation at serine 473 (S473), detected by fluorescence imaging of fixed cells. At higher concentrations the secretagogue is inhibitory towards PKB phosphorylation on S473. Conversely, p70 S6K phosphorylation on T389 is unaffected at high doses. We provide evidence that the differential effect on the two PI 3-kinase effectors is due to activation of PKCα by c48/80, itself a PI 3-kinase dependent process. We conclude that compound 48/80 is an effective activator of PI 3-kinase dependent pathways, leading to the activation of effectors including PKB/Akt, p70 S6K and PKCα. The latter is only activated by higher doses of c48/80 resulting in an inhibition of the c48/80 induced PKB phosphorylation, thus explaining the observed biphasic activation profile for PKB in response to this secretagogue.

Protein tyrosine phosphatase 1B regulates TGFβ1-induced Smad2 activation through PI3 kinase-dependent pathway

Insulin is known to modulate transforming growth factor-β (TGFβ) signaling. In this report, by using the IN Cell Analyzer 1000, the fluorescence cell imaging instrument, we demonstrated that protein tyrosine phosphatase 1B (PTP1B) could regulate TGFβ1-induced Smad2 activation in a PI3 kinase-dependent manner. By using the CHO cells stably expressing EGFP-Smad2, we showed that TGFβ1 effectively stimulated Smad2 nuclear translocation in CHO cells. When pretreated with insulin, TGFβ1-induced Smad2 nuclear entry was dramatically decreased. Furthermore, both the PI3K inhibitor LY294002 and the dominant negative AKT (DN-AKT) abolished the inhibitory effects of insulin, demonstrating that the inhibition of Smad2 activation by insulin was PI3K/AKT dependent. Since PTP1B negatively modulates insulin signaling, we further addressed the effects of PTP1B on insulin-mediated inhibition of Smad2 activation. Our data showed that overexpression of PTP1B effectively attenuated insulin-induced inhibition of Smad2 stimulation. Moreover, the PTP1B inhibitor, 3-(3,5-dibromo-4-hydroxy-benzoyl)-2-ethyl-benzofuran-6-sulfonicacid-(4-(thiazol-2-ylsulfamyl)-phenyl)-amide (Compound-2), recovered insulin inhibition of Smad2 activation. In conclusion, our data revealed the insulin inhibitory effects on TGFβ1-induced Smad2 activation and the regulation role of PTP1B in the inhibition events.

B-RAF and PI-3 kinase signaling protect melanoma cells from anoikis

A hallmark feature of cancer is resistance to anoikis, apoptosis induced when cells either lose contact with or encounter an inappropriate extracellular matrix. Melanoma is inherently associated with a high degree of resistance to apoptosis. Mutations in B-RAF are prevalent in melanoma and promote constitutive MEK-ERK1/2 signaling and cell transformation. Acquisition of B-RAF mutations correlates with vertical phase growth when melanoma cells invade into the dermis, a collagen-rich environment that also contains fibronectin matrix. In addition, alterations in phosphoinositide-3 kinase (PI-3 kinase) signaling that lead to activation of AKT are detected in advanced melanomas. Here we show that knockdown of B-RAF expression by siRNA or pharmacological inhibition of MEK rendered melanoma cells susceptible to anoikis. Furthermore, adhesion to fibronectin but not collagen protected melanoma cells from anoikis through a PI-3 kinase-dependent pathway. Therefore, melanoma cells require either B-RAF or PI-3 kinase activation for protection from anoikis. Notably, AKT signaling in melanoma cells is substrate specific. These findings demonstrate that melanoma cells utilize multiple signaling pathways to provide resistance to apoptosis.

Caveolin and GLT‐1 gene expression is reciprocally regulated in primary astrocytes: Association of GLT‐1 with non‐caveolar lipid rafts

Caveolae represent membrane microdomains acting as integrators of cellular signaling and functional processes. Caveolins are involved in the biogenesis of caveolae and regulate the activity of caveolae-associated proteins. Although caveolin proteins are found in the CNS, the regulation of caveolins in neural cells is poorly described. In the present study, we investigated different modes and mechanisms of caveolin gene regulation in primary rat astrocytes. We demonstrated that activation of cAMP-dependent signaling pathways led to a marked reduction in protein levels of caveolin-1/-2 in cortical astrocytes. Application of transforming growth factor-alpha (TGF-alpha) also resulted in a decrease of caveolin-1/-2 expression. Decreased caveolin protein levels were mirrored by diminished caveolin gene transcription. The repressive effect of TGF-alpha on caveolin-1 expression was MAP kinase-independent and partly mediated through the PI3-kinase pathway. Further downstream, inhibition of histone deacetylases abrogated TGF-alpha effects, suggesting that chromatin remodeling processes could contribute to caveolin-1 repression. Intriguingly, alterations of caveolin gene expression in response to cAMP or TGF-alpha coincided with reciprocal and brain-region specific changes in glial glutamate transporter GLT-1 expression. The reciprocal regulation of caveolin-1 and GLT-1 expression might be gated through a common PI3-kinase dependent pathway triggered by TGF-alpha. Finally, we showed that GLT-1 is located in non-caveolar lipid rafts of cortical astrocytes. In conclusion, this study highlights the occurrence of the reciprocal regulation of caveolin and GLT-1 expression during processes such as astrocyte differentiation via common signaling pathways. We also provide strong evidence that GLT-1 itself is concentrated in lipid rafts, inferring an important role for glial glutamate transporter function.

Cell surface receptors activate p21-activated kinase 1 via multiple Ras and PI3-kinase-dependent pathways

p21-activated kinases (PAKs) were the first identified mammalian members of a growing family of Ste20-like serine–threonine protein kinases. In this study, we show that PAK1 can be stimulated by carbachol, lysophosphatidic acid (LPA), epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) by multiple independent and overlapping pathways. Dominant-negative Ras, Rac, and Cdc42 inhibited PAK1 activation by all of these agonists, while active Rac1 and Cdc42 were sufficient to maximally activate PAK1 in the absence of any treatment. Active Ras induced only a weak activation of PAK1 that could be potentiated by muscarinic receptor stimulation. Studies using inhibitors of the EGF receptor tyrosine kinase, phosphatidylinositol 3-kinase (PI3-kinase) and protein kinase C (PKC) revealed that all of the cell surface agonists could activate PAK1 through pathways independent of PKC, that EGF stimulated a PI3-kinase dependent pathway to stimulate PAK1, and that muscarinic receptor stimulation of PAK1 was predominantly mediated through this EGF-R-dependent mechanism. Activation of PAK1 by LPA was independent of PI3-kinase and the EGF receptor, but was inhibited by dominant-negative RhoA. These results identify multiple Ras-dependent pathways to activation of PAK1.

Baclofen reduces emesis and gastroesophageal reflux in neurologically impaired children with gastroesophageal reflux disaese

the potential involvement of histamine receptors was also examined in current studies. Both cimetidine (1 raM) and diphenhydramine (0.1raM), H2 and H1 histamine receptor antagonists respectively, had no effect on inhibition of 36C1 uptake by TDC. In conclusion, our studies, provide direct evidence for inhibition of human intestinal luminal CI'/OH (HCO~) exchange activity by bile acids. The results of our studies demonstrate that bile acid effects on the apical CI/OH exchange in Caco2 cells are not mediated via histamine receptors and involve a cAMP-independent but Ca + +, PKC and PI3 kinase-dependent pathways.

FcεRI-mediated activation of human mast cells (HuMCs) is regulated by an early PI-3 kinase independent, and a delayed PI-3 kinase dependent pathway

FcεRI-mediated activation of human mast cells (HuMCs) is regulated by an early PI-3 kinase independent, and a delayed PI-3 kinase dependent pathway