PDZ domain-containing 1 (PDZK1) protein regulates phospholipase C-ß3 (PLC-ß3)-specific activation of somatostatin by forming a ternary complex with PLC-ß3 and somatostatin receptors.

Phospholipase C-ß (PLC-ß) is a key molecule in G protein-coupled receptor (GPCR)-mediated signaling. Many studies have shown that the four PLC-ß subtypes have different physiological functions despite their similar structures. Because the PLC-ß subtypes possess different PDZ-binding motifs, they have the potential to interact with different PDZ proteins. In this study, we identified PDZ domain-containing 1 (PDZK1) as a PDZ protein that specifically interacts with PLC-ß3. To elucidate the functional roles of PDZK1, we next screened for potential interacting proteins of PDZK1 and identified the somatostatin receptors (SSTRs) as another protein that interacts with PDZK1. Through these interactions, PDZK1 assembles as a ternary complex with PLC-ß3 and SSTRs. Interestingly, the expression of PDZK1 and PLC-ß3, but not PLC-ß1, markedly potentiated SST-induced PLC activation. However, disruption of the ternary complex inhibited SST-induced PLC activation, which suggests that PDZK1-mediated complex formation is required for the specific activation of PLC-ß3 by SST. Consistent with this observation, the knockdown of PDZK1 or PLC-ß3, but not that of PLC-ß1, significantly inhibited SST-induced intracellular Ca(2+) mobilization, which further attenuated subsequent ERK1/2 phosphorylation. Taken together, our results strongly suggest that the formation of a complex between SSTRs, PDZK1, and PLC-ß3 is essential for the specific activation of PLC-ß3 and the subsequent physiologic responses by SST.

Activation of AMP-activated protein kinase is essential for lysophosphatidic acid-induced cell migration in ovarian cancer cells.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, and survival, through LPA receptors. Among them, the motility of cancer cells is an especially important activity for invasion and metastasis. Recently, AMP-activated protein kinase (AMPK), an energy-sensing kinase, was shown to regulate cell migration. However, the specific role of AMPK in cancer cell migration is unknown. The present study investigated whether LPA could induce AMPK activation and whether this process was associated with cell migration in ovarian cancer cells. We found that LPA led to a striking increase in AMPK phosphorylation in pathways involving the phospholipase C-ß3 (PLC-ß3) and calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) in SKOV3 ovarian cancer cells. siRNA-mediated knockdown of AMPKα1, PLC-ß3, or (CaMKKß) impaired the stimulatory effects of LPA on cell migration. Furthermore, we found that knockdown of AMPKα1 abrogated LPA-induced activation of the small GTPase RhoA and ezrin/radixin/moesin proteins regulating membrane dynamics as membrane-cytoskeleton linkers. In ovarian cancer xenograft models, knockdown of AMPK significantly decreased peritoneal dissemination and lung metastasis. Taken together, our results suggest that activation of AMPK by LPA induces cell migration through the signaling pathway to cytoskeletal dynamics and increases tumor metastasis in ovarian cancer.

Human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is an energy-sensing kinase that has recently been shown to regulate the differentiation of preadipocytes and osteoblasts. However, the role of AMPK in stem cell differentiation is largely unknown. Using in vitro culture models, the present study demonstrates that AMPK is a critical regulatory factor for osteogenic differentiation. We observed that expression and phosphorylation of AMPK were increased during osteogenesis in human adipose tissue-derived mesenchymal stem cells (hAMSC). To elucidate the role of AMPK in osteogenic differentiation, we investigated the effect of AMPK inhibition or knockdown on mineralization of hAMSC. Compound C, an AMPK inhibitor, reduced mineralized matrix deposition and suppressed the expression of osteoblast-specific genes, including alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN). Knockdown of AMPK by shRNA-lentivirus infection also reduced osteogenesis. In addition, inhibition or knockdown of AMPK during osteogenesis inhibited ERK phosphorylation, which is required for osteogenesis. Interestingly, inhibition of AMPK induced adipogenic differentiation of hAMSC, even in osteogenic induction medium (OIM). These results provide a potential mechanism involving AMPK activation in osteogenic differentiation of hAMSC and suggest that commitment of hAMSC to osteogenic or adipogenic lineage is governed by activation or inhibition of AMPK, respectively.

Wedelolactone inhibits adipogenesis through the ERK pathway in human adipose tissue-derived mesenchymal stem cells.

Wedelolactone is an herbal medicine that is used to treat septic shock, hepatitis and venom poisoning. Although in differentiated and cancer cells, wedelolactone has been identified as anti-inflammatory, growth inhibitory, and pro-apoptotic, the effects of wedelolactone on stem cell differentiation remain largely unknown. Here, we report that wedelolactone inhibits the adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSCs). Wedelolactone reduced the formation of lipid droplets and the expression of adipogenesis-related proteins, such as CCAAT enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein aP2 (aP2). Wedelolactone mediated this process by sustaining ERK activity. In addition, inhibition of ERK activity with PD98059 resulted in reversion of the wedelolactone-mediated inhibition of adipogenic differentiation. Taken together, these results indicate that wedelolactone inhibits adipogenic differentiation through ERK pathway and suggest a novel inhibitory effect of wedelolactone on adipogenic differentiation in hAMSCs.

Quercetin suppresses HeLa cell viability via AMPK-induced HSP70 and EGFR down-regulation.

Quercetin, an anti-oxidant flavonoid that is widely distributed in the plant kingdom, has been suggested to have chemopreventive effects on cancer cells, although the mechanism is not completely understood. In this study, we found that quercetin increased the phosphorylation of AMP-activated protein kinase (AMPK) and downstream acetyl-CoA carboxylase (ACC) and suppressed the viability of HeLa cells. AICAR, an AMPK activator, and quercetin down-regulated heat shock protein (HSP)70 and increased the activity of the pro-apoptotic effector, caspase 3. Knock-down of AMPK blocked quercetin-mediated HSP70 down-regulation. Moreover, knock-down of HSP70 enhanced quercetin-mediated caspase 3 activation. Furthermore, quercetin sustained epidermal growth factor receptor (EGFR) activation by suppressing the phosphatases, PP2a and SHP-2. Finally, quercetin increased the interaction between EGFR and Cbl, and also induced the tyrosine phosphorylation of Cbl. Together, these results suggest that quercetin may have anti-tumor effects on HeLa cells via AMPK-induced HSP70 and down-regulation of EGFR.

Curcumin stimulates glucose uptake through AMPK-p38 MAPK pathways in L6 myotube cells.

Curcumin has been shown to exert a variety of beneficial human health effects. However, mechanisms by which curcumin acts are poorly understood. In this study, we report that curcumin activated AMP-activated protein kinase (AMPK) and increased glucose uptake in rat L6 myotubes. In addition, curcumin activated the mitogen-activated protein kinase kinase (MEK)3/6-p38 mitogen-activated protein kinase (MAPK) signaling pathways in the downstream of the AMPK cascade. Moreover, inhibition of either AMPK or p38 MAPK resulted in blockage of curcumin-induced glucose uptake. Furthermore, the administration of curcumin to mice increased AMPK phosphorylation in the skeletal muscles. Taken together, these results indicate that the beneficial health effect of curcumin can be explained by its ability to activate AMPK-p38 MAPK pathways in skeletal muscles.

The effect of thoracic epidural anesthesia on pulmonary shunt fraction and arterial oxygenation during one-lung ventilation.

OBJECTIVE: To compare the effect of thoracic epidural local anesthetic, epidural opioid, and intravenous opioid on pulmonary shunt fraction, arterial oxygenation, and hemodynamic changes during one-lung ventilation (OLV) in patients undergoing thoracic surgery. DESIGN: A prospective, randomized, double-blind study. SETTING: A university hospital. PARTICIPANTS: Thirty-nine patients undergoing OLV for pulmonary resection. INTERVENTIONS: Patients were randomized into 1 of 3 groups: epidural bupivacaine (TEA-B group, n = 13), epidural sufentanil (TEA-S group, n = 13), or intravenous remifentanil (IV-R group, n = 13) during general anesthesia with propofol. A double-lumen tube was inserted, and mechanical ventilation with 100% oxygen was used in the lateral decubitus position. MEASUREMENTS AND MAIN RESULTS: Hemodynamic variables and arterial and mixed venous blood gas analysis from the radial and pulmonary artery catheter were measured and shunt fraction was calculated during two-lung ventilation (TLV), 15, 30, and 60 minutes after the initiation of OLV, and 15 minutes after the reinstitution of TLV. Although mean arterial pressures 15 and 30 minutes after OLV in the IV-R group were significantly higher than the value in TEA-S group, cardiac output and pulmonary vascular resistance were maintained. Decreases in PaO(2), SaO(2), PvO(2), and SvO(2) and an increase in the shunt fraction after OLV were not different among groups and returned to baseline value after the resumption of TLV. CONCLUSIONS: Thoracic epidural bupivacaine, epidural sufentanil, and intravenous remifentanil-combined general intravenous anesthesia have comparable effects on shunt fraction and arterial oxygenation during OLV in patients undergoing thoracic surgery.

Engineering and Characterization of Oncolytic Vaccinia Virus Expressing Truncated Herpes Simplex Virus Thymidine Kinase.

Oncolytic viruses are a promising class of anti-tumor agents; however, concerns regarding uncontrolled viral replication have led to the development of a replication-controllable oncolytic vaccinia virus (OVV). The engineering involves replacing the native thymidine kinase (VV-tk) gene, in a Wyeth strain vaccinia backbone, with the herpes simplex virus thymidine kinase (HSV-tk) gene, which allows for viral replication control via ganciclovir (GCV, an antiviral/cytotoxic pro-drug). Adding the wild-type HSV-tk gene might disrupt the tumor selectivity of VV-tk deleted OVVs; therefore, only engineered viruses that lacked tk activity were selected as candidates. Ultimately, OTS-412, which is an OVV containing a mutant HSV-tk, was chosen for characterization regarding tumor selectivity, sensitivity to GCV, and the influence of GCV on OTS-412 anti-tumor effects. OTS-412 demonstrated comparable replication and cytotoxicity to VVtk- (control, a VV-tk deleted OVV) in multiple cancer cell lines. In HCT 116 mouse models, OTS-412 replication in tumors was reduced by >50% by GCV (p = 0.004); additionally, combination use of GCV did not compromise the anti-tumor effects of OTS-412. This is the first report of OTS-412, a VV-tk deleted OVV containing a mutant HSV-tk transgene, which demonstrates tumor selectivity and sensitivity to GCV. The HSV-tk/GCV combination provides a safety mechanism for future clinical applications of OTS-412.

Engineering and Preclinical Evaluation of Western Reserve Oncolytic Vaccinia Virus Expressing A167Y Mutant Herpes Simplex Virus Thymidine Kinase.

Viral replication of thymidine kinase deleted (tk-) vaccinia virus (VV) is attenuated in resting normal cells, enabling cancer selectivity, however, replication potency of VV-tk- appears to be diminished in cancer cells. Previously, we found that wild-type herpes simplex virus (HSV)-tk (HSV-tk) disappeared in most of the recombinant VV after multiple screenings, and only a few recombinant VV containing naturally mutated HSV-tk remained stable. In this study, VV-tk of western reserve (WR) VV was replaced by A167Y mutated HSV-tk (HSV-tk418m), to alter nucleoside selectivity from broad spectrum to purine exclusive selectivity. WOTS-418 remained stable after numerous passages. WOTS-418 replication was significantly attenuated in normal cells, but cytotoxicity was almost similar to that of wild type WR VV in cancer cells. WOTS-418 showed no lethality following a 5 × 108 PFU intranasal injection, contrasting WR VV, which showed 100% lethality at 1 × 105 PFU. Additionally, ganciclovir (GCV) but not BvdU inhibited WOTS-418 replication, confirming specificity to purine nucleoside analogs. The potency of WOTS-418 replication inhibition by GCV was > 10-fold higher than that of our previous truncated HSV-tk recombinant OTS-412. Overall, WOTS-418 demonstrated robust oncolytic efficacy and pharmacological safety which may delegate it as a candidate for future clinical use in OV therapy.

Phospholipase C-gamma1 potentiates integrin-dependent cell spreading and migration through Pyk2/paxillin activation.

Phospholipase C-gamma1 (PLC-gamma1), which generates two second messengers, namely, inositol-1, 4, 5-trisphosphate and diacylglycerol, is implicated in growth factor-mediated chemotaxis. However, the exact role of PLC-gamma1 in integrin-mediated cell adhesion and migration remains poorly understood. In this study, we demonstrate that PLC-gamma1 is required for actin cytoskeletal organization and cell motility through the regulation of Pyk2 and paxillin activation. After fibronectin stimulation, PLC-gamma1 directly interacted with the cytoplasmic tail of integrin beta1. In PLC-gamma1-silenced cells, integrin-induced Pyk2 and paxillin phosphorylation were significantly reduced and PLC-gamma1 potentiated the integrin-induced Pyk2/paxillin activation in its enzymatic activity-dependent manner. In addition, specific knock-down of PLC-gamma1 resulted in a failure to form focal adhesions dependent on fibronectin stimulation, which appeared to be caused by the suppression of Pyk2 and paxillin phosphorylation. Interestingly, PLC-gamma1 potentiated the activations of Rac, thus integrin-induced lamellipodia formation was up-regulated. Consequently, the strength of cell-substratum interaction and cell motility were profoundly up-regulated by PLC-gamma1. Taken together, these results suggest that PLC-gamma1 is a key player in integrin-mediated cell spreading and motility achieved by the activation of Pyk2/paxillin/Rac signaling.

Phospholipase Cγ1 links inflammation and tumorigenesis in colitis-associated cancer.

Colorectal cancer (CRC) is the third diagnosed cancer and the second leading cause of cancer-related deaths in the United States. Colorectal cancer is linked to inflammation and phospholipase Cγ1 (PLCγ1) is associated with tumorigenesis and the development of colorectal cancer; however, evidence of mechanisms connecting them remains unclear. The tight junctions (TJ), as intercellular junctional complexes, have an important role for integrity of the epithelial barrier to regulate the cellular permeability. Here we found that PLCγ1 regulated colitis and tumorigenesis in intestinal epithelial cells (IEC). To induce the colitis-associated cancer (CAC), we used the AOM/DSS model. Mice were sacrificed at 100 days (DSS three cycles) and 120 days (DSS one cycle). In a CAC model, we showed that the deletion of PLCγ1 in IEC decreased the incidence of tumors by enhancing apoptosis and inhibiting proliferation during tumor development. Accordingly, the deletion of PLCγ1 in IEC reduced colitis-induced epithelial inflammation via inhibition of pro-inflammatory cytokines and mediators. The PLCγ1 pathway in IEC accelerated colitis-induced epithelial damage via regulation of TJ proteins. CONCLUSIONS: Our findings suggest that PLCγ1 is a critical regulator of colitis and colorectal cancer and could further help in the development of therapy for colitis-associated cancer.

Dynamic relocalization of NHERF1 mediates chemotactic migration of ovarian cancer cells toward lysophosphatidic acid stimulation.

NHERF1/EBP50 (Na+/H+ exchanger regulating factor 1; Ezrin-binding phosphoprotein of 50 kDa) organizes stable protein complexes beneath the apical membrane of polar epithelial cells. By contrast, in cancer cells without any fixed polarity, NHERF1 often localizes in the cytoplasm. The regulation of cytoplasmic NHERF1 and its role in cancer progression remain unclear. In this study, we found that, upon lysophosphatidic acid (LPA) stimulation, cytoplasmic NHERF1 rapidly translocated to the plasma membrane, and subsequently to cortical protrusion structures, of ovarian cancer cells. This movement depended on direct binding of NHERF1 to C-terminally phosphorylated ERM proteins (cpERMs). Moreover, NHERF1 depletion downregulated cpERMs and further impaired cpERM-dependent remodeling of the cell cortex, suggesting reciprocal regulation between these proteins. The LPA-induced protein complex was highly enriched in migratory pseudopodia, whose formation was impaired by overexpression of NHERF1 truncation mutants. Consistent with this, NHERF1 depletion in various types of cancer cells abolished chemotactic cell migration toward a LPA gradient. Taken together, our findings suggest that the high dynamics of cytosolic NHERF1 provide cancer cells with a means of controlling chemotactic migration. This capacity is likely to be essential for ovarian cancer progression in tumor microenvironments containing LPA.

Haloperidol induces calcium ion influx via L-type calcium channels in hippocampal HN33 cells and renders the neurons more susceptible to oxidative stress.

Haloperidol is a classical neuroleptic drug that is still in clinical use and can lead to abnormal motor activity following repeated administration. However, there is little knowledge of how it triggers neuronal impairment. In this study, we report that it induced calcium ion influx via L-type calcium channels and that the elevation of calcium ions induced by haloperidol appeared to render hippocampal cells more susceptible to oxidative stress. Indeed, the level of cytotoxic reactive oxygen species (ROS) and the expression of pro-apoptotic Bax increased in response to oxidative stress in haloperidol-treated cells, and these effects were inhibited by verapamil, a specific L-type calcium channel blocker, but not by the T-type calcium channel blocker, mibefradil. These findings indicate that haloperidol induces calcium ion influx via L-type calcium channels and that this calcium influx influences neuronal fate.

PPARγ Antagonist Gleevec Improves Insulin Sensitivity and Promotes the Browning of White Adipose Tissue.

Blocking phosphorylation of peroxisome proliferator-activated receptor (PPAR)γ at Ser(273) is one of the key mechanisms for antidiabetes drugs to target PPARγ. Using high-throughput phosphorylation screening, we here describe that Gleevec blocks cyclin-dependent kinase 5-mediated PPARγ phosphorylation devoid of classical agonism as a PPARγ antagonist ligand. In high fat-fed mice, Gleevec improved insulin sensitivity without causing severe side effects associated with other PPARγ-targeting drugs. Furthermore, Gleevec reduces lipogenic and gluconeogenic gene expression in liver and ameliorates inflammation in adipose tissues. Interestingly, Gleevec increases browning of white adipose tissue and energy expenditure. Taken together, the results indicate that Gleevec exhibits greater beneficial effects on both glucose/lipid metabolism and energy homeostasis by blocking PPARγ phosphorylation. These data illustrate that Gleevec could be a novel therapeutic agent for use in insulin resistance and type 2 diabetes.

Flightless-1, a novel transcriptional modulator of PPARγ through competing with RXRα.

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor family and plays key roles in glucose and lipid metabolism. Its transcriptional control of target genes is mediated by ligand-dependent recruitment of coactivators. In this study, we demonstrate that a novel transcriptional modulator of PPARγ, Flightless-I (FLII) binds directly to and suppresses the transcriptional activity of PPARγ. The LXXLL motif within the leucine-rich repeat (LRR) domain of FLII interacts directly with the DNA-binding domain of PPARγ. Interestingly, in the presence of PPARγ ligands, such as rosiglitazone and SR1664, this interaction was abolished in vitro. When FLII was overexpressed, both the transcriptional activity of PPARγ and adipogenesis were suppressed significantly, whereas specific knockdown of FLII reversed these effects. Furthermore, DNA occupancy of PPARγ on its target gene promoters was enhanced by FLII knockdown, and the interaction between PPARγ and retinoid X receptor α (RXRα) was blocked by FLII. Together, these findings strongly suggest that FLII functions in PPARγ activation as a molecular switch to repress transcriptional activity by interrupting formation of the PPARγ/RXRα complex, and FLII may serve as a novel therapeutic target in the treatment of adiposity-related metabolic syndromes.

Novel phosphorylation of PPARγ ameliorates obesity-induced adipose tissue inflammation and improves insulin sensitivity.

Chronic inflammation in adipose tissue is highly associated with insulin resistance. Herein, we demonstrate that a novel modification of PPARγ is strongly associated with inflammatory responses in adipose tissue. c-Src kinase directly phosphorylated PPARγ at Tyr78, and this process was reversed by protein tyrosine phosphatase-1B (PTP-1B). In adipocytes, phosphorylation of PPARγ suppressed the expression of pro-inflammatory genes as well as the secretion of chemokines and cytokines, thus reducing macrophage migration. Importantly, pharmacological inhibition of c-Src kinase aggravated insulin resistance in obese mice with a concomitant increase in the expression of pro-inflammatory genes in adipose tissue. These data strongly suggest that PPARγ phosphorylation is the key regulatory mechanism of the inflammatory response in adipose tissue, which is highly associated with glucose tolerance and insulin sensitivity. Furthermore, these data increase our understanding of the mechanical aspects of developing novel anti-diabetic drugs targeting PPARγ phosphorylation.

RhoA and Rac1 play independent roles in lysophosphatidic acid-induced ovarian cancer chemotaxis.

Lysophosphatidic acid (LPA), which is a bioactive phospholipid existing at high level in ascites and plasma of ovarian cancer patients, is known to be involved in cell survival, proliferation, adhesion, and migration. Small guanosine triphosphatases (GTPases) such as RhoA and Rac1 are intracellular signaling molecules which affect morphology and chemotactic behavior of cells. In this research, we first investigated roles of RhoA and Rac1 in the LPA-induced chemotaxis of SKOV3 human ovarian cancer cells using a multilevel microfluidic platform. The multilevel microfluidic device was fabricated by a rapid prototyping method based on soft lithography using multi-layered adhesive tapes. This platform allows us to conduct the on-chip chemotaxis assays in conventional biology laboratories without any huge and expensive equipment for fabrication and fluidic manipulation. Based on image-based analysis of single cell trajectories in the microfluidic device, the chemotaxis of SKOV3 cells could be quantitatively analyzed in two independent parameters-migration speed and directional persistence. Inhibition of the RhoA/ROCK pathways reduced the directional persistence, not the migration speed, of the cells, while only the migration speed was decreased when the activity of Rac1/PAK pathways was suppressed. These results suggest that RhoA and Rac1 signaling pathways potentially play independent roles in the chemotactic migration of SKOV3 ovarian cancer cells in the linear and stable LPA concentration gradient. Our microfluidic platform would provide a rapid, low cost, easy-to-use, and versatile way for research of cancer cell migration which is crucial for tumor metastasis.

O-GlcNAcase is essential for embryonic development and maintenance of genomic stability.

Dysregulation of O-GlcNAc modification catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) contributes to the etiology of chronic diseases of aging, including cancer, cardiovascular disease, type 2 diabetes, and Alzheimer's disease. Here we found that natural aging in wild-type mice was marked by a decrease in OGA and OGT protein levels and an increase in O-GlcNAcylation in various tissues. Genetic disruption of OGA resulted in constitutively elevated O-GlcNAcylation in embryos and led to neonatal lethality with developmental delay. Importantly, we observed that serum-stimulated cell cycle entry induced increased O-GlcNAcylation and decreased its level after release from G2/M arrest, indicating that O-GlcNAc cycling by OGT and OGA is required for precise cell cycle control. Constitutively, elevated O-GlcNAcylation by OGA disruption impaired cell proliferation and resulted in mitotic defects with downregulation of mitotic regulators. OGA loss led to mitotic defects including cytokinesis failure and binucleation, increased lagging chromosomes, and micronuclei formation. These findings suggest an important role for O-GlcNAc cycling by OGA in embryonic development and the regulation of the maintenance of genomic stability linked to the aging process.

Subtype-specific role of phospholipase C-beta in bradykinin and LPA signaling through differential binding of different PDZ scaffold proteins.

Among phospholipase C (PLC) isozymes (beta, gamma, delta, epsilon, zeta and eta), PLC-beta plays a key role in G-protein coupled receptor (GPCR)-mediated signaling. PLC-beta subtypes are often overlapped in their distribution, but have unique knock-out phenotypes in organism, suggesting that each subtype may have the different role even within the same type of cells. In this study, we examined the possibility of the differential coupling of each PLC-beta subtype to GPCRs, and explored the molecular mechanism underlying the specificity. Firstly, we found that PLC-beta1 and PLC-beta 3 are activated by bradykinin (BK) or lysophosphatidic acid (LPA), respectively. BK-triggered phosphoinositides hydrolysis and subsequent Ca(2+) mobilization were abolished specifically by PLC-beta1 silencing, whereas LPA-triggered events were by PLC-beta 3 silencing. Secondly, we showed the evidence that PDZ scaffold proteins is a key mediator for the selective coupling between PLC-beta subtype and GPCR. We found PAR-3 mediates physical interaction between PLC-beta1 and BK receptor, while NHERF2 does between PLC-beta 3 and LPA(2) receptor. Consistently, the silencing of PAR-3 or NHERF2 blunted PLC signaling induced by BK or LPA respectively. Taken together, these data suggest that each subtype of PLC-beta is selectively coupled to GPCR via PDZ scaffold proteins in given cell types and plays differential role in the signaling of various GPCRs.

Retinoic acid leads to cytoskeletal rearrangement through AMPK-Rac1 and stimulates glucose uptake through AMPK-p38 MAPK in skeletal muscle cells.

Retinoic acid (RA) is one of the major components of vitamin A. In the present study, we found that retinoic acid activated AMP-activated protein kinase (AMPK). RA induced Rac1-GTP formation and phosphorylation of its downstream target, p21-activated kinase (PAK), whereas the inhibition of AMPK blocked RA-induced Rac1 activation. Moreover, cofilin, an actin polymerization regulator, was activated when incubated with RA. We then showed that inhibition of AMPK by compound C, a selective inhibitor of AMPK, or small interfering RNA of AMPK alpha1 blocked RA-induced cofilin phosphorylation. Additionally, we found that retinoic acid-stimulated glucose uptake in differentiated C2C12 myoblast cells and activated p38 mitogen-activated protein kinase (MAPK). Finally, the inhibition of AMPK and p38 MAPK blocked retinoic acid-induced glucose uptake. In summary, our results suggest that retinoic acid may have cytoskeletal roles in skeletal muscle cells via stimulation of the AMPK-Rac1-PAK-cofillin pathway and may also have beneficial roles in glucose metabolism via stimulation of the AMPK-p38 MAPK pathway.