Serum amyloid A induces lipolysis by downregulating perilipin through ERK1/2 and PKA signaling pathways

Serum amyloid A (SAA) is not only an apolipoprotein, but also a member of the adipokine family with potential to enhance lipolysis. The purpose of this study was to explore how SAA facilitates lipolysis in porcine adipocytes. We found that SAA increased the phosphorylation of perilipin and hormone-sensitive lipase (HSL) after 12-h treatment and decreased perilipin expression after 24-h treatment, and these effects were prevented by extracellular signal-regulated kinase (ERK) or protein kinase A (PKA) inhibitors in primary adipocyte cell culture. SAA treatment decreased HSL and adipose triglyceride lipase (ATGL) expression. SAA treatment also activated ERK and PKA by increasing the phosphorylation of these kinases. Moreover, SAA significantly increased porcine adipocyte glycerol release and lipase activity, which was inhibited by either ERK (PD98059) or PKA (H89) inhibitors, suggesting that ERK and PKA were involved in mediating SAA enhanced lipolysis. SAA downregulated the expression of peroxisome proliferator-activated receptor γ (PPARγ) mRNA, which was reversed by the ERK inhibitor. We performed a porcine perilipin promoter assay in differentiated 3T3-L1 adipocytes and found that SAA reduced the porcine perilipin promoter specifically through the function of its PPAR response element (PPRE), and this effect was reversed by the ERK inhibitor. These findings demonstrate that SAA-induced lipolysis is a result of downregulation of perilipin and activation of HSL via ERK/PPARγ and PKA signaling pathways. The finding could lead to developing new strategies for reducing human obesity.     

Signaling pathways implicated in alpha-melanocyte stimulating hormone-induced lipolysis in 3T3-L1 adipocytes

Melanocortins, besides their central roles, have also recently been reported to regulate adipocyte metabolism. In this study, we attempted to characterize the mechanism underlying alpha-melanocyte-stimulating hormone (MSH)-induced lipolysis, and compared it with that of the adrenocorticotrophin hormone (ACTH) in 3T3-L1 adipocytes. Similar to ACTH, MSH treatment resulted in the release of glycerol into the cell supernatant. The activity of hormone-sensitive lipase, a rate-limiting enzyme, which is involved in lipolysis, was significantly increased by MSH treatment. In addition, a variety of kinases, including protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) were also phosphorylated as the result of MSH treatment, and their specific inhibitors caused a reduction in MSH-induced glycerol release and HSL activity, indicating that MSH-induced lipolysis was mediated by these kinases. These results suggest that PKA and ERK constitute the principal signaling pathways implicated in the MSH-induced lipolytic process via the regulation of HSL in 3T3-L1 adipocytes.     

Signaling pathway of magnolol-stimulated lipolysis in sterol ester-loaded 3T3-L1 preadipocyes

The aims of the present study were to examine the effect of magnolol on lipolysis in sterol ester (SE)-loaded 3T3-L1 preadipocytes and to determine the signaling mechanism involved. We demonstrate that magnolol treatment resulted in a decreased number and surface area of lipid droplets, accompanied by release of glycerol. The lipolytic effect of magnolol was not mediated by PKA based on the facts that magnolol did not induce an elevation of intracellular cAMP levels, and protein kinase A (PKA) inhibitor KT5720 did not block magnolol-induced lipolysis. Calcium/calmodulin-dependent protein kinase (CaMK) was involved in this signaling pathway, since magnolol-induced a transient rise of intracellular [Ca(2+)] and Ca(2+) influx across the plasma membrane, and CaMK inhibitor significantly abolished magnolol-induced lipolysis. Moreover, magnolol increased the relative levels of phosphorylated extracellular signal-related kinases (ERK1 and ERK2). In support of the involvement ERK, we demonstrated that magnolol-induced lipolysis was inhibited by PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK), and PD98059 reversed magnolol-induced ERK phosphorylation. Further, the relationship between CaMK and ERK was connected by the finding that CaMK inhibitor also blocked magnolol-induced ERK phosphorylation. Taken together, these findings suggest that magnolol-induced lipolysis is both CaMK- and ERK-dependent, and this lipolysis signaling pathway is distinct from the traditional PKA pathway. ERK phosphorylation is reported to enhance lipolysis by direct activation of hormone sensitive lipase (HSL), thus magnolol may likely activate HSL through ERK and increase lipolysis of adipocytes.     

Cardiotrophin-1 stimulates lipolysis through the regulation of main adipose tissue lipases

Cardiotrophin-1 (CT-1) is a cytokine with antiobesity properties and with a role in lipid metabolism regulation and adipose tissue function. The aim of this study was to analyze the molecular mechanisms involved in the lipolytic actions of CT-1 in adipocytes. Recombinant CT-1 (rCT-1) effects on the main proteins and signaling pathways involved in the regulation of lipolysis were evaluated in 3T3-L1 adipocytes and in mice. rCT-1 treatment stimulated basal glycerol release in a concentration- and time-dependent manner in 3T3-L1 adipocytes. rCT-1 (20 ng/ml for 24 h) raised cAMP levels, and in parallel increased protein kinase (PK)A-mediated phosphorylation of perilipin and hormone sensitive lipase (HSL) at Ser660. siRNA knock-down of HSL or PKA, as well as pretreatment with the PKA inhibitor H89, blunted the CT-1-induced lipolysis, suggesting that the lipolytic action of CT-1 in adipocytes is mainly mediated by activation of HSL through the PKA pathway. In ob/ob mice, acute rCT-1 treatment also promoted PKA-mediated phosphorylation of perilipin and HSL at Ser660 and Ser563, and increased adipose triglyceride lipase (desnutrin) content in adipose tissue. These results showed that the ability of CT-1 to regulate the activity of the main lipases underlies the lipolytic action of this cytokine in vitro and in vivo, and could contribute to CT-1 antiobesity effects.     

Lipase-selective functional domains of perilipin A differentially regulate constitutive and protein kinase A-stimulated lipolysis

Perilipin (Peri) A is a lipid droplet-associated phosphoprotein that acts dually as a suppressor of basal (constitutive) lipolysis and as an enhancer of cyclic AMP-dependent protein kinase (PKA)-stimulated lipolysis by both hormone-sensitive lipase (HSL) and non-HSL(s). To identify domains of Peri A that mediate these multiple actions, we introduced adenoviruses expressing truncated or mutated Peri A and HSL into NIH 3T3 fibroblasts lacking endogenous perilipins and HSL but overexpressing acyl-CoA synthetase 1 and fatty acid transporter 1. We identified two lipase-selective functional domains: 1) Peri A (amino acids 1-300), which inhibits basal lipolysis and promotes PKA-stimulated lipolysis by HSL, and 2) Peri A (amino acids 301-517), which inhibits basal lipolysis by non-HSL and promotes PKA-stimulated lipolysis by both HSL and non-HSL. PKA site mutagenesis revealed that PKA-stimulated lipolysis by HSL requires phosphorylation of one or more sites within Peri 1-300 (Ser81, Ser222, and Ser276). PKA-stimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301-517 (Ser433, Ser492, and Ser517). Peri 301-517 promoted PKA-stimulated lipolysis by HSL yet did not block HSL-mediated basal lipolysis, indicating that an additional region(s) within Peri 301-517 promotes hormone-stimulated lipolysis by HSL. These results suggest a model of Peri A function in which 1) lipase-specific "barrier" domains block basal lipolysis by HSL and non-HSL, 2) differential PKA site phosphorylation allows PKA-stimulated lipolysis by HSL and non-HSL, respectively, and 3) additional domains within Peri A further facilitate PKA-stimulated lipolysis, again with lipase selectivity.     

H-89 potentiates adipogenesis in 3T3-L1 cells by activating insulin signaling independently of protein kinase A

Among four kinds of protein kinase A (PKA) inhibitors tested, H-89 exhibited a unique action to remarkably enhance adipocyte differentiation of 3T3-L1 cells, whereas the other three PKA inhibitors, PKA inhibitor Fragment 14-22 (PKI), Rp-cAMP, and KT 5720, did not enhance adipocyte differentiation. H-85, which is an inactive form of H-89, exhibited a similar enhancing effect on adipocyte differentiation. H-89 also potentiated the phosphorylation of Akt and extracellular signal-regulated kinase (ERK) 1/2 in 3T3-L1 cells, which function as downstream signaling of insulin. Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and mitogen-activated protein kinase kinase (MEK) inhibitor PD 98059 suppressed both the H-89-induced promotion of adipocyte differentiation and the H-89-induced potentiation of phosphorylation of Akt and ERK1/2. Rho kinase inhibitor Y-27632 also promoted the phosphorylation of both Akt and ERK1/2 and enhanced adipocyte differentiation, although its effect was somewhat less than that of H-89. Even when cells were treated with a mixture of Y-27632 and H-89, the additive enhancing effects on both the insulin signaling and adipocyte differentiation were not detected. Therefore, it is suggested that the major possible mechanism whereby H-89 potentiates adipocyte differentiation of 3T3-L1 cells is activation of insulin signaling that is elicited mostly by inhibiting Rho/Rho kinase pathway.     

Modulation of hormone-sensitive lipase and protein kinase A-mediated lipolysis by perilipin A in an adenoviral reconstituted system.

Perilipin (Peri) A is a phosphoprotein located at the surface of intracellular lipid droplets in adipocytes. Activation of cyclic AMP-dependent protein kinase (PKA) results in the phosphorylation of Peri A and hormone-sensitive lipase (HSL), the predominant lipase in adipocytes, with concurrent stimulation of adipocyte lipolysis. To investigate the relative contributions of Peri A and HSL in basal and PKA-mediated lipolysis, we utilized NIH 3T3 fibroblasts lacking Peri A and HSL but stably overexpressing acyl-CoA synthetase 1 (ACS1) and fatty acid transport protein 1 (FATP1). When incubated with exogenous fatty acids, ACS1/FATP1 cells accumulated 5 times more triacylglycerol (TG) as compared with NIH 3T3 fibroblasts. Adenoviral-mediated expression of Peri A in ACS1/FATP1 cells enhanced TG accumulation and inhibited lipolysis, whereas expression of HSL fused to green fluorescent protein (GFPHSL) reduced TG accumulation and enhanced lipolysis. Forskolin treatment induced Peri A hyperphosphorylation and abrogated the inhibitory effect of Peri A on lipolysis. Expression of a mutated Peri A Delta 3 (Ser to Ala substitutions at PKA consensus sites Ser-81, Ser-222, and Ser-276) reduced Peri A hyperphosphorylation and blocked constitutive and forskolin-stimulated lipolysis. Thus, perilipin expression and phosphorylation state are critical regulators of lipid storage and hydrolysis in ACS1/FATP1 cells.     

Analysis of lipolytic protein trafficking and interactions in adipocytes

This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormonesensitive lipase (HSL) to perilipin A (Plin)-containing droplets and increases the colocalization of adipose tissue triglyceride lipase (Atgl) with its coactivator, Abhd5. Imaging of live 3T3-L1 preadipocytes transfected with Aquorea victoria-based fluorescent reporters demonstrated that HSL rapidly and specifically translocates to lipid droplets (LDs) containing Plin, and that this translocation is partially dependent on Plin phosphorylation. HSL closely, if not directly, interacts with Plin, as indicated by fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) experiments. In contrast, tagged Atgl did not support FRET or BiFC with Plin, although it did modestly translocate to LDs upon stimulation. Abhd5 strongly interacted with Plin in the basal state, as indicated by FRET and BiFC. PKA activation rapidly (within minutes) decreased FRET between Abhd5 and Plin, and this decrease depended upon Plin phosphorylation. Together, these results indicate that Plin mediates hormone-stimulated lipolysis via direct and indirect mechanisms. Plin indirectly controls Atgl activity by regulating accessibility to its coactivator, Abhd5. In contrast, Plin directly regulates the access of HSL to substrate via close, if not direct, interactions. The differential interactions of HSL and Atgl with Plin and Abhd5 also explain the findings that following stimulation, HSL and Atgl are differentially enriched at specific LDs.     

Contractions induce phosphorylation of the AMPK site Ser565 in hormone-sensitive lipase in muscle

Intramyocellular triglyceride is an important energy store which is related to insulin resistance. Mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by epinephrine via PKA and by contractions via PKC and ERK. 5' AMP-activated protein kinase (AMPK) is an intracellular fuel gauge which regulates metabolism. In this study we incubated rat soleus muscle to investigate if AMPK influences HSL during 5min of repeated tetanic contractions. An eightfold increase in AMPK activity was accompanied by a 2.5-fold increase in phosphorylation of the AMPK-site Ser(565) in HSL (p<0.05). Inhibition of PKC by Calphostin C abolished the contraction-mediated HSL activation while HSL-Ser(565) phosphorylation was not reduced. The study indicates that during contractions AMPK phosphorylates HSL in Ser(565), but this phosphorylation is not directly responsible for the contraction-induced activation of HSL.     

Regulation of HSL serine phosphorylation in skeletal muscle and adipose tissue

Hormone-sensitive lipase (HSL) is important for the degradation of triacylglycerol in adipose and muscle tissue, but the tissue-specific regulation of this enzyme is not fully understood. We investigated the effects of adrenergic stimulation and AMPK activation in vitro and in circumstances where AMPK activity and catecholamines are physiologically elevated in humans in vivo (during physical exercise) on HSL activity and phosphorylation at Ser(563) and Ser(660), the PKA regulatory sites, and Ser(565), the AMPK regulatory site. In human experiments, skeletal muscle, subcutaneous adipose and venous blood samples were obtained before, at 15 and 90 min during, and 120 min after exercise. Skeletal muscle HSL activity was increased by approximately 80% at 15 min compared with rest and returned to resting rates at the cessation of and 120 min after exercise. Consistent with changes in plasma epinephrine, skeletal muscle HSL Ser(563) and Ser(660) phosphorylation were increased by 27% at 15 min (P < 0.05), remained elevated at 90 min, and returned to preexercise values postexercise. Skeletal muscle HSL Ser(565) phosphorylation and AMPK signaling were increased at 90 min during, and after, exercise. Phosphorylation of adipose tissue HSL paralleled changes in skeletal muscle in vivo, except HSL Ser(660) was elevated 80% in adipose compared with 35% in skeletal muscle during exercise. Studies in L6 myotubes and 3T3-L1 adipocytes revealed important tissue differences in the regulation of HSL. AMPK inhibited epinephrine-induced HSL activity in L6 myotubes and was associated with reduced HSL Ser(660) but not Ser(563) phosphorylation. HSL activity was reduced in L6 myotubes expressing constitutively active AMPK, confirming the inhibitory effects of AMPK on HSL activity. Conversely, in 3T3-L1 adipocytes, AMPK activation after epinephrine stimulation did not prevent HSL activity or glycerol release, which coincided with maintenance of HSL Ser(660) phosphorylation. Taken together, these data indicate that HSL activity is maintained in the face of AMPK activation as a result of elevated HSL Ser(660) phosphorylation in adipose tissue but not skeletal muscle.     

Bacterial Peptidoglycan Stimulates Adipocyte Lipolysis via NOD1

Obesity is associated with inflammation that can drive metabolic defects such as hyperlipidemia and insulin resistance. Specific metabolites can contribute to inflammation, but nutrient intake and obesity are also associated with altered bacterial load in metabolic tissues (i.e. metabolic endotoxemia). These bacterial cues can contribute to obesity-induced inflammation. The specific bacterial components and host receptors that underpin altered metabolic responses are emerging. We previously showed that Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) activation with bacterial peptidoglycan (PGN) caused insulin resistance in mice. We now show that PGN induces cell-autonomous lipolysis in adipocytes via NOD1. Specific bacterial PGN motifs stimulated lipolysis in white adipose tissue (WAT) explants from WT, but not NOD1^−/− mice. NOD1-activating PGN stimulated mitogen activated protein kinases (MAPK),protein kinase A (PKA), and NF-κB in 3T3-L1 adipocytes. The NOD1-mediated lipolysis response was partially reduced by inhibition of ERK1/2 or PKA alone, but not c-Jun N-terminal kinase (JNK). NOD1-stimulated lipolysis was partially dependent on NF-κB and was completely suppressed by inhibiting ERK1/2 and PKA simultaneously or hormone sensitive lipase (HSL). Our results demonstrate that bacterial PGN stimulates lipolysis in adipocytes by engaging a stress kinase, PKA, NF-κB-dependent lipolytic program. Bacterial NOD1 activation is positioned as a component of metabolic endotoxemia that can contribute to hyperlipidemia, systemic inflammation and insulin resistance by acting directly on adipocytes.     

Phosphorylation of serine 43 is not required for inhibition of c-Raf kinase by the cAMP-dependent protein kinase

The activity of the serine/threonine kinase c-Raf (Raf) is inhibited by increased intracellular cAMP. This is believed to require phosphorylation with the cAMP-dependent protein kinase (PKA), although the mechanism by which PKA inhibits Raf is controversial. We investigated the requirement for PKA phosphorylation using Raf mutants expressed in HEK293 or NIH 3T3 cells. Phosphopeptide mapping of (32)P-labeled Raf (WT) or a mutant lacking a putative PKA phosphorylation site (serine to alanine, S43A) confirmed that serine 43 (Ser(43)) was the major cAMP (forskolin)-stimulated phosphorylation site in vivo. Interestingly, the EGF-stimulated Raf kinase activity of the S43A mutant was inhibited by forskolin equivalently to that of the WT Raf. Forskolin also inhibited the activation of an N-terminal deletion mutant Delta5-50 Raf completely lacking this phosphorylation site. Although WT Raf was phosphorylated by PKA, phosphorylation did not inhibit Raf catalytic activity in vitro, nor did forskolin treatment inhibit the activity of an N-terminally truncated Raf protein (Raf 22W) or a full-length Raf protein (Raf-CAAX) expressed in NIH 3T3 cells. In contrast, forskolin inhibited the EGF-dependent activation of a Raf isoform (B-Raf), lacking an analogous phosphorylation site to Ser(43). Thus, these results demonstrate that PKA exerts its inhibitory effects independently of direct Raf phosphorylation and suggests instead that PKA prevents an event required for the EGF-dependent activation of Raf.     

Maximal beta3-adrenergic regulation of lipolysis involves Src and epidermal growth factor receptor-dependent ERK1/2 activation

Catecholamine-stimulated lipolysis is primarily a beta-adrenergic and cAMP-dependent event. In previous studies we established that the beta(3)-adrenergic receptor (beta(3)AR) in adipocytes utilizes a unique mechanism to stimulate extracellular signal-regulated kinases 1 and 2 (ERK) by direct recruitment and activation of Src kinase. Therefore, we investigated the role of the ERK pathway in adipocyte metabolism and found that the beta(3)AR agonist CL316,243 regulates lipolysis through both cAMP-dependent protein kinase (PKA) and ERK. Inhibition of PKA activity completely eliminated lipolysis at low (subnanomolar) CL316,243 concentrations and by 75-80% at higher nanomolar concentrations. The remaining 20-25% of PKA-independent lipolysis, as well as ERK activation, was abolished by inhibiting the activity of either Src (PP2 or small interfering RNA), epidermal growth factor receptor (EGFR with AG1478 or small interfering RNA), or mitogen-activated protein kinase kinase 1 or 2 (MKK1/2 with PD098059). PD098059 inhibited lipolysis by 53% in mice as well. Finally, the effect of estradiol, a reported acute activator of ERK and lipolysis, was also totally prevented by PP2, AG1478, and PD098059. These results suggest that ERK activation by beta(3)AR depends upon Src and epidermal growth factor receptor kinase activities and is responsible for the PKA-independent portion of the lipolytic response. Together these results illustrate the distinct and complementary roles for PKA and ERK in catecholamine-stimulated lipolysis.     

Phosphorylation regulates KSR1 stability, ERK activation, and cell proliferation

Kinase suppressor of Ras (KSR) is a molecular scaffold that interacts with the components of the Raf/MEK/ERK kinase cascade and positively regulates ERK signaling. Phosphorylation of KSR1, particularly at Ser(392), is a critical regulator of KSR1 subcellular localization and ERK activation. We examined the role of phosphorylation of both Ser(392) and Thr(274) in regulating ERK activation and cell proliferation. We hypothesized that KSR1 phosphorylation is involved in generating signaling specificity through the Raf/MEK/ERK kinase cascade in response to stimulation by different growth factors. In fibroblasts, platelet-derived growth factor stimulation induces sustained ERK activation and promotes S-phase entry. Treatment with epidermal growth factor induces transient ERK activation but fails to drive cells into S phase. Mutation of Ser(392) and Thr(274) (KSR1.TVSA) promotes sustained ERK activation and cell cycle progression with either platelet-derived growth factor or epidermal growth factor treatment. KSR1(-/-) mouse embryo fibroblasts expressing KSR1.TVSA proliferate two times faster and grow to a higher density than cells expressing the same level of wild-type KSR1. In addition, KSR1.TVSA is more stable than wild-type KSR1. These data demonstrate that phosphorylation and stability of the molecular scaffold KSR1 are critical regulators of growth factor-specific responses that promote cell proliferation.     

Oncogenic Ras leads to Rho activation by activating the mitogen-activated protein kinase pathway and decreasing Rho-GTPase-activating protein activity

Transformation by oncogenic Ras requires signaling through Rho family proteins including RhoA, but the mechanism(s) whereby oncogenic Ras regulates the activity of RhoA is (are) unknown. We examined the effect of Ras on RhoA activity in NIH 3T3 cells either stably transfected with H-Ras(V12) under control of an inducible promoter or transiently expressing the activated H-Ras. Using a novel method to quantitate enzymatically the GTP bound to Rho, we found that expression of the oncogenic Ras increased Rho activity approximately 2-fold. Increased Rho activity was associated with increased plasma membrane binding of RhoA and decreased activity of the Rho/Ras-regulated p21(WAF1/CIP1) promoter. RhoA activation by oncogenic Ras could be explained by a decrease in cytosolic p190 Rho-GAP activity and translocation of p190 Rho-GAP from the cytosol to a detergent-insoluble cytoskeletal fraction. Pharmacologic inhibition of the Ras/Raf/MEK/ERK pathway prevented Ras-induced activation of RhoA and translocation of p190 Rho-GAP; expression of constitutively active Raf-1 kinase or MEK was sufficient to induce p190 Rho-GAP translocation. We conclude that in NIH 3T3 cells oncogenic Ras activates RhoA through the Raf/MEK/ERK pathway by decreasing the cytosolic activity and changing the subcellular localization of p190 Rho-GAP.     

Possible mechanisms by which adipocyte lipolysis is enhanced in exercise-trained rats

A possible mechanism(s) behind exercise training-enhanced lipolysis was investigated in rat adipocytes. Exercise training (9 weeks; running) enhanced the activity of cAMP-dependent protein kinase (PKA) and the protein expressions of PKA subunits (catalytic, RII alpha, and RII beta) in P(40) fraction (sedimenting at 40,000g), but not in I(40) fraction (infranatant of 40,000g) of adipocyte homogenate. The expression of PKA-anchoring protein 150 (AKAP150) in P(40) fraction was greater in exercise-trained (TR) than in control (C) rats. Hormone-sensitive lipase (HSL) activities in both fractions were also greater in TR. On the other hand, stimulated lipolysis was accompanied by increased activities of HSL in P(40) but not in I(40) fraction. The decreases in stimulated lipolysis due to St-Ht31 were greater in TR rats. Thus, the mechanisms behind exercise training-enhanced adipocyte lipolysis could involve the increased activities of PKA and HSL with enhanced expressions of AKAP150 and some subunits of PKA, all of which may be compartmentalized within adipocytes.     

Myostatin regulates preadipocyte differentiation and lipid metabolism of adipocyte via ERK1/2

Myostatin is known as an inhibitor of muscle development, but its role in adipogenesis and lipid metabolism is still unclear, especially the underlying mechanisms. Here, we demonstrated that myostatin inhibited 3T3-L1 preadipocyte differentiation into adipocyte by suppressing C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ (peroxisome-proliferator-activated receptor γ), also activated ERK1/2 (extracellular-signal-regulated kinase 1/2). Furthermore, myostatin enhanced the phosphorylation of HSL (hormone-sensitive lipase) and ACC (acetyl-CoA carboxylase) in fully differentiated adipocytes, as well as ERK1/2. Besides, we noted that myostatin markedly raised the levels of leptin and adiponectin release and mRNA expression during preadipocyte differentiation, but the levels were inhibited by myostatin treatments in fully differentiated adipocytes. These results suggested that myostatin suppressed 3T3-L1 preadipocyte differentiation and regulated lipid metabolism of mature adipocyte, in part, via activation of ERK1/2 signalling pathway.     

Extracellular signal-regulated kinase-dependent proliferation is mediated through the protein kinase A/B-Raf pathway in human uveal melanoma cells

Mutated B-Raf-mediated constitutive activation of ERK1/2 is involved in about 66% of cutaneous melanoma. By contrast, activating mutations in B-RAF are rare in ocular melanoma. This study aimed to determine the role of wild-type B-Raf ((WT)B-Raf) in uveal melanoma cell growth. We used cell lines derived from primary tumors of uveal melanoma to assess the role of (WT)B-Raf in cell proliferation and to characterize its upstream regulators and downstream effectors. Melanoma cell lines expressing (WT)B-Raf and (WT)Ras grew with similar proliferation rates, showed constitutive activation of ERK1/2, and had similar levels of B-Raf expression and B-Raf kinase activity as melanoma cell lines expressing the activating V600E mutation ((V600E)B-Raf). They were equally as sensitive to pharmacological inhibition of MEK1/2 for cell proliferation and transformation as (V600E)B-Raf cells. siRNA-mediated depletion of Raf-1 did not affect either ERK1/2 activation, whereas siRNA-mediated depletion of B-Raf reduced cell proliferation by up to 65% through the inhibition of ERK1/2 activation, irrespective of the mutational status of B-Raf. Pharmacological inhibition of cAMP-dependent protein kinase (PKA) and siRNA-mediated depletion of PKA greatly reduced B-Raf activity, ERK1/2 activation, and cell proliferation in (WT)B-Raf cells, whereas it did not affect (V600E)B-Raf cells, demonstrating a key role of PKA in mediating (WT)B-Raf/ERK signaling for uveal melanoma cell growth. Moreover, inactivation or depletion of PKA did not affect Rap-1 activity, and Rap-1 depletion did not affect either B-Raf activity or ERK1/2 activation. This ruled out a role for Rap1 in the PKA-mediated B-Raf/ERK activation in (WT)B-Raf cells. Finally, we demonstrated the importance of cyclin D1 in mediating PKA/(WT)B-Raf signaling for cell proliferation. Altogether, our results suggest that the PKA/B-Raf pathway is a potential target for therapeutic strategies against (WT)B-Raf-expressing uveal melanoma.     

Involvement of 3-phosphoinositide-dependent protein kinase-1 in the MEK/MAPK signal transduction pathway

The phosphatidylinositide-3-OH kinase/3-phospho-inositide-dependent protein kinase-1 (PDK1)/Akt and the Raf/mitogen-activated protein kinase (MAPK/ERK) kinase (MEK)/mitogen-activated protein kinase (MAPK) pathways have central roles in the regulation of cell survival and proliferation. Despite their importance, however, the cross-talk between these two pathways has not been fully understood. Here we report that PDK1 promotes MAPK activation in a MEK-dependent manner. In vitro kinase assay revealed that the direct targets of PDK1 in the MAPK pathway were the upstream MAPK kinases MEK1 and MEK2. The identified PDK1 phosphorylation sites in MEK1 and MEK2 are Ser222 and Ser226, respectively, and are known to be essential for full activation. To date, these sites are thought to be phosphorylated by Raf kinases. However, PDK1 gene silencing using small interference RNA demonstrates that PDK1 is associated with maintaining the steady-state phosphorylated MEK level and cell growth. The small interference RNA-mediated down-regulation of PDK1 attenuated maximum MEK and MAPK activities but could not prolong MAPK signaling duration. Stable and transient expression of constitutively active MEK1 overcame these effects. Our results suggest a novel cross-talk between the phosphatidylinositide-3-OH kinase/PDK1/Akt pathway and the Raf/MEK/MAPK pathway.