Blockade Of Extracellular Signal-regulated Kinase Research Articles

Limonin, a natural ERK2 agonist, protects against ischemic acute kidney injury.

Acute kidney injury (AKI) is a refractory clinical syndrome with limited effective treatments. Amid AKI, activation of the extracellular signal-regulated kinase (ERK) cascade plays a critical role in promoting kidney repair and regeneration. However, a mature ERK agonist in treating kidney disease remains lacking. This study identified limonin, a member of the class of compounds known as furanolactones, as a natural ERK2 activator. Employing a multidisciplinary approach, we systemically dissected how limonin mitigates AKI. Compared to vehicles, pretreatment of limonin significantly preserved kidney functions after ischemic AKI. We revealed that ERK2 is a significant protein linked to the limonin's active binding sites through structural analysis. The molecular docking study showed a high binding affinity between limonin and ERK2, which was confirmed by the cellular thermal shift assay and microscale thermophoresis. Mechanistically, we further validated that limonin promoted tubular cell proliferation and reduced cell apoptosis after AKI by activating ERK signaling pathway in vivo. In vitro and ex vivo, blockade of ERK abolished limonin's capacity of preventing tubular cell death under hypoxia stress. Our results indicated that limonin is a novel ERK2 activator with strong translational potential in preventing or mitigating AKI.

Active Transition of Fear Memory Phase from Reconsolidation to Extinction through ERK-Mediated Prevention of Reconsolidation.

The retrieval of fear memory induces two opposite memory process, i.e., reconsolidation and extinction. Brief retrieval induces reconsolidation to maintain or enhance fear memory, while prolonged retrieval extinguishes this memory. Although the mechanisms of reconsolidation and extinction have been investigated, it remains unknown how fear memory phases are switched from reconsolidation to extinction during memory retrieval. Here, we show that an extracellular signal-regulated kinase (ERK)-dependent memory transition process after retrieval regulates the switch of memory phases from reconsolidation to extinction by preventing induction of reconsolidation in an inhibitory avoidance (IA) task in male mice. First, the transition memory phase, which cancels the induction of reconsolidation, but is insufficient for the acquisition of extinction, was identified after reconsolidation, but before extinction phases. Second, the reconsolidation, transition, and extinction phases after memory retrieval showed distinct molecular and cellular signatures through cAMP responsive element binding protein (CREB) and ERK phosphorylation in the amygdala, hippocampus, and medial prefrontal cortex (mPFC). The reconsolidation phase showed increased CREB phosphorylation, while the extinction phase displayed several neural populations with various combinations of CREB and/or ERK phosphorylation, in these brain regions. Interestingly, the three memory phases, including the transition phase, showed transient ERK activation immediately after retrieval. Most importantly, the blockade of ERK in the amygdala, hippocampus, or mPFC at the transition memory phase disinhibited reconsolidation-induced enhancement of IA memory. These observations suggest that the ERK-signaling pathway actively regulates the transition of memory phase from reconsolidation to extinction and this process functions as a switch that cancels reconsolidation of fear memory.SIGNIFICANCE STATEMENT Retrieval of fear memory induces two opposite memory process; reconsolidation and extinction. Reconsolidation maintains/enhances fear memory, while extinction weakens fear memory. It remains unknown how memory phases are switched from reconsolidation to extinction during retrieval. Here, we identified an active memory transition process functioning as a switch that inhibits reconsolidation. This memory transition phase showed a transient increase of extracellular signal-regulated kinase (ERK) phosphorylation in the amygdala, hippocampus and medial prefrontal cortex (mPFC). Interestingly, inhibition of ERK in these regions at the transition phase disinhibited the reconsolidation-mediated enhancement of inhibitory avoidance (IA) memory. These findings suggest that the transition memory process actively regulates the switch of fear memory phases of fear memory by preventing induction of reconsolidation through the activation of the ERK-signaling pathway.

RCE‑4, a potential anti‑cervical cancer drug isolated from Reineckia carnea, induces autophagy via the dual blockade of PI3K and ERK pathways in cervical cancer CaSki cells.

The steroidal saponin RCE‑4 (1β, 3β, 5β, 25S)‑spirostan‑1, 3‑diol 1‑[α‑L‑rhamnopyranosyl‑(1→2)‑β‑D‑xylopyranoside], isolated from Reineckia carnea, exerts significant anti‑cervical cancer activity by inducing apoptosis. The potential effect of RCE‑4 on proliferation inhibition and autophagy induction has rarely been studied. Therefore, the focus of the present study was to investigate the effects of RCE‑4 on proliferation, and to elucidate the detailed mechanisms involved in autophagy induction in cervical cancer cells. CaSki cells were treated with RCE‑4 or/and autophagy inhibitors, and the effect of RCE‑4 on cellular proliferation was assessed by MTT assay. The pro‑autophagic properties of RCE‑4 were subsequently confirmed using monomeric red fluorescent protein‑green fluorescent protein‑microtubule‑associated proteins1A/1B light chain3B (LC3) adenoviruses and CYTO‑ID autophagy assays, and by assessing the accumulation of lipid‑modifiedLC3 (LC3II). The mechanisms of RCE‑4‑induced autophagy were investigated by western blot analysis. The results demonstrated that inhibiting autophagy significantly promoted RCE‑4‑induced cell death, indicating that autophagy served a protective role following RCE‑4 treatment. In addition, RCE‑4‑induced autophagy was reflected by increased expression levels of the serine/threonine‑protein kinase ULK1, phosphorylated (p)‑ULK1, p‑Beclin‑1 and LC3II, the formation of autophagosomes and autolysosomes, and sequestosome1 (p62) degradation. Subsequent analysis indicated that RCE‑4 activated the AMP‑activated protein kinase (AMPK) pathway by upregulating AMPK and p‑AMPK, and also inhibited the PI3K and extracellular signal‑regulated kinase (ERK) signaling pathways by downregulating p‑PI3K, p‑Akt, p‑mTOR, Ras, c‑Raf, p‑c‑Raf, dual specificity mitogen‑activated protein kinase kinase (MEK)1/2, p‑MEK1/2 and p‑Erk1/2. Additionally, with increased treatment times RCE‑4 may impair lysosomal cathepsin activity and inhibit autophagy flux by suppressing the expression of AMPK, p‑AMPK, ULK1, p‑ULK1 and p‑Beclin‑1, and upregulating that of p62. These results indicated that the dual RCE‑4‑induced inhibition of the PI3K and ERK pathways may result in a more significant anti‑tumor effect and prevent chemoresistance, compared with the inhibition of either single pathway; furthermore, dual blockade of PI3K and ERK, and the AMPK pathway may be involved in the regulation of autophagy caused by RCE‑4. Taken together, RCE‑4 induced autophagy to protect cancer cells against apoptosis, but AMPK‑mediated autophagy was inhibited in the later stages of RCE‑4 treatment. In addition, autophagy inhibition improved the therapeutic effect of RCE‑4. These data highlight RCE‑4 as a potential candidate for cervical cancer treatment.

Extracellular signal-regulated kinase 2 has duality in function between neuronal and astrocyte expression following neonatal hypoxic-ischaemic cerebral injury.

Key points This study identifies phosphorylated extracellular signal‐regulated kinase (ERK) to be immediately diminished followed by a rapid if transient increase for up to 4 h following hypoxic–ischaemic insult (HI) in the neonatal mouse.Phosphorylated ERK up‐regulation was prevented with systemic injection of the mitogen‐activated protein kinase kinase (MEK) inhibitor SL327. Treatment with SL327 both pre‐ and post‐HI gave a strong reduction in the number of dying cells and microgliosis.By utilising transgenic mouse mutations, we observe that neuronal ERK2 significantly contributes to tissue damage, while ERK1 and astrocytic ERK2 are neuroprotective.Compared to global inactivation, selective cell‐specific interference with ERK activity could result in stronger neuroprotection. Hypoxia–ischaemia (HI) is a major cause of neonatal brain injury resulting in cerebral palsy, epilepsy, cognitive impairment and other neurological disabilities. The role of extracellular signal‐regulated kinase (ERK) isoforms and their mitogen‐activated protein kinase kinase (MEK)‐dependent phosphorylation in HI has previously been explored but remains unresolved at cellular level. This is pertinent given the growing awareness of the role of non‐neuronal cells in neuroprotection. Using a modified Rice–Vannucci model of HI in the neonatal mouse we observed time‐ and cell‐dependent ERK phosphorylation (pERK), with strongly up‐regulated pERK immunoreactivity first in periventricular white matter axons within 15–45 min of HI, followed by forebrain astrocytes and neurons (1–4 h post‐HI), and return to baseline by 16 h. We explored the effects of pharmacological ERK blockade through the MEK inhibitor SL327 on neonatal HI‐brain damage following HI alone (30 or 60 min) or lipopolysaccharide (LPS)‐sensitised HI insult (30 min). Global inhibition of ERK phosphorylation with systemically applied SL327 abolished forebrain pERK immunoreactivity, and significantly reduced cell death and associated microglial activation at 48 h post‐HI. We then explored the effects of cell‐specific ERK2 deletion alone or in combination with global ERK1 knockout under the same conditions of HI insult. Neuronal ERK2 deletion strongly decreased infarct size, neuronal cell death and microglial activation in grey matter following both HI alone or LPS‐sensitised HI. ERK1 deletion attenuated the protective effect of neuronal ERK2 deletion. Removal of astroglial ERK2 produced a reverse response, with a 3‐ to 4‐fold increase in microglial activation and cell death. Our data suggest a cell‐specific and time‐dependent role of ERK in neonatal HI, with a predominant, neurotoxic effect of neuronal ERK2, which is counteracted by neuroprotection by ERK1 and astrocytic ERK2. Overall, global pharmacological inhibition of ERK phosphorylation is strongly neuroprotective.

Low-level laser irradiation modulates brain-derived neurotrophic factor mRNA transcription through calcium-dependent activation of the ERK/CREB pathway.

Low-level laser (LLL) irradiation has been reported to promote neuronal differentiation, but the mechanism remains unclear. Brain-derived neurotrophic factor (BDNF) has been confirmed to be one of the most important neurotrophic factors because it is critical for the differentiation and survival of neurons during development. Thus, this study aimed to investigate the effects of LLL irradiation on Bdnf messenger RNA (mRNA) transcription and the molecular pathway involved in LLL-induced Bdnf mRNA transcription in cultured dorsal root ganglion neurons (DRGNs) using Ca2+ imaging, pharmacological detections, RNA interference, immunocytochemistry assay, Western blot, and qPCR analysis. We show here that LLL induced increases in the [Ca2+] i level, Bdnf mRNA transcription, cAMP-response element-binding protein (CREB) phosphorylation, and extracellular signal-regulated kinase (ERK) phosphorylation, mediated by Ca2+ release via inositol triphosphate receptor (IP3R)-sensitive calcium (Ca2+) stores. Blockade of Ca2+ increase suppressed Bdnf mRNA transcription, CREB phosphorylation, and ERK phosphorylation. Downregulation of phosphorylated (p)-CREB reduced Bdnf mRNA transcription triggered by LLL. Furthermore, blockade of ERK using PD98059 inhibitor reduced p-CREB and Bdnf mRNA transcription induced by LLL. Taken together, these findings establish the Ca2+-ERK-CREB cascade as a potential signaling pathway involved in LLL-induced Bdnf mRNA transcription. To our knowledge, this is the first report of the mechanisms of Ca2+-dependent Bdnf mRNA transcription triggered by LLL. These findings may help further explore the complex molecular signaling networks in LLL-triggered nerve regeneration in vivo and may also provide experimental evidence for the development of LLL for clinical applications.

Trametinib plus 4-Methylumbelliferone Exhibits Antitumor Effects by ERK Blockade and CD44 Downregulation and Affects PD-1 and PD-L1 in Malignant Pleural Mesothelioma

IntroductionMalignant pleural mesothelioma (MPM) is a highly aggressive malignancy in which the mitogen-activated protein kinase pathway plays a critical role in the regulation of tumorigenesis. Hyaluronic acid (HA) is a major component of the extracellular matrix, and elevated HA levels with a concurrent increase in malignant properties are associated with MPM. MethodsWe evaluated the effects of trametinib, a mitogen-activated protein kinase (MEK) inhibitor, and 4-methylumbelliferone (4-MU), an HA synthesis inhibitor, alone and in combination on MPM cells in vitro and in vivo. We studied the effects of trametinib, 4-MU, and their combination on MPM cells by using cell viability assays, Western blot analysis, and a mouse xenograft model. ResultsTrametinib and 4-MU exhibited antiproliferative activity in MPM cells. Trametinib blocked MEK-dependent extracellular signal-regulated kinase (ERK) phosphorylation and decreased CD44 expression in a concentration-dependent manner. Trametinib inhibited the expression of Fra-1 (the activator protein 1 [AP1] component), inhibited ERK phosphorylation, and decreased CD44 expression. 4-MU inhibited ERK phosphorylation but not CD44 expression. In a mouse xenograft model, trametinib and 4-MU alone suppressed tumor growth compared with a control. The combination had a greater inhibitory effect than either monotherapy. Immunohistochemical analysis showed that trametinib treatment alone significantly reduced expression of programmed cell death 1 ligand 1. Furthermore, the combination of trametinib and 4-MU resulted in higher expression of programmed cell death 1 and programmed cell death 1 ligand 1 than did the 4-MU treatment alone. ConclusionsOur results suggest that trametinib and 4-MU are promising therapeutic agents in MPM and that further study of the combination is warranted.

Zinc preconditioning protects against neuronal apoptosis through the mitogen-activated protein kinase-mediated induction of heat shock protein 70

During brain ischemic preconditioning (PC), mild bursts of ischemia render neurons resistant to subsequent strong ischemic injuries. Previously, we reported that zinc plays a key role in PC-induced neuroprotection in vitro and in vivo. Zinc-triggered p75NTR induction transiently activates caspase-3, which cleaves poly(ADP-ribose) polymerase-1 (PARP-1). Subsequently, the PARP-1 over-activation-induced depletion of nicotinamide adenine dinucleotide (NAD+)/adenosine triphosphate (ATP) after exposures to lethal doses of zinc or N-methyl-d-aspartate is significantly attenuated in cortical neuronal cultures. In the present study, zinc-mediated preconditioning (Zn PC) reduced apoptotic neuronal death that was caused by N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), etoposide, or staurosporine in mouse cortical cells. We focused on heat shock protein 70 (HSP70) because NAD+/ATP depletion does not directly cause apoptosis, and HSP70 can inhibit the activation of caspase-9 or caspase-3 by preventing apoptosome formation or cytochrome C release. Zn PC-mediated HSP70 induction was required for neuroprotection against neuronal apoptosis, and geldanamycin-induced HSP70 induction sufficiently blocked neuronal apoptotic cell death. Furthermore, Zn PC-mediated HSP70 induction was blocked by chemical inhibitors of extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein kinase (MAPK) signaling, but not c-Jun N-terminal protein kinase. Similarly, neuroprotection by Zn PC against TPEN-induced apoptosis was almost completely reversed by the blockade of ERK or p38 MAPK signaling. Our findings suggest that the ERK- or p38 MAPK-mediated induction of HSP70 plays a key role in inhibiting caspase-3 activation during Zn PC.

Coordinated activation of AMP‐activated protein kinase, extracellular signal‐regulated kinase, and autophagy regulates phorbol myristate acetate‐induced differentiation of SH‐SY5Y neuroblastoma cells

We explored the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)-induced neuronal differentiation of SH-SY5Y human neuroblastoma cells. PMA-triggered expression of neuronal markers (dopamine transporter, microtubule-associated protein 2, β-tubulin) was associated with an autophagic response, measured by the conversion of microtubule-associated protein light chain 3 (LC3)-I to autophagosome-bound LC3-II, increase in autophagic flux, and expression of autophagy-related (Atg) proteins Atg7 and beclin-1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference-mediated silencing of AMPK suppressed PMA-induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA-induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA-induced differentiation of SH-SY5Y cells. Therefore, PMA-induced neuronal differentiation of SH-SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response. Phorbol myristate acetate (PMA) induces the expression of dopamine transporter, microtubule-associated protein 2, and β-tubulin, and subsequent neuronal differentiation of SH-SY5Y neuroblastoma cells through AMP-activated protein kinase (AMPK)-dependent activation of extracellular signal-regulated kinase (ERK). The activation of AMPK/ERK axis also induces the expression of beclin-1 and Atg7, and increases LC3 conversion, thereby triggering the autophagic response that counteracts differentiation process.

ACE2 activity was increased in atherosclerotic plaque by losartan: Possible relation to anti-atherosclerosis.

Angiotensin-converting enzyme 2 (ACE2) is a new member of the renin-angiotensin system (RAS) and it has been proposed that ACE2 is a potential therapeutic target for the control of cardiovascular disease. The effect of losartan on the ACE2 activity in atherosclerosis was studied. Atherosclerosis was induced in New Zealand white rabbits by high-cholesterol diet for 3 months. An Angiotensin II (Ang II) receptor blocker (losartan, 25 mg/kg/d) was given for 3 months. ACE2 activity was measured by fluorescence assay and the extent of atherosclerosis was evaluated by H&E and Oil Red O staining. In addition, the effect of losartan on ACE2 activity in smooth muscle cells (SMCs) in vitro was also evaluated. Losartan increased ACE2 activity in atherosclerosis in vivo and SMCs in vitro. Losartan inhibited atherosclerotic evolution. Addition of losartan blocked Ang II-induced down-regulation of ACE2 activity, and blockade of extracellular signal-regulated kinase (ERK1/2) with PD98059 prevented Ang II-induced down-regulation of ACE2 activity. The results showed that ACE2 activity was regulated in atherosclerotic plaque by losartan, which may play an important role in treatment of atherosclerosis. The mechanism involves Ang II-AT1R-mediated mitogen-activated protein kinases, MAPKs (MAPKs) signaling pathway.

Down-regulation of the P-glycoprotein relevant for multidrug resistance by intracellular acidification through the crosstalk of MAPK signaling pathways

In our previous study, we have found that the tumor multidrug resistance mediated by P-glycoprotein could be reversed by sustained intracellular acidification through down-regulating the multidrug resistance gene 1 mRNA and P-glycoprotein expression. However, the molecular events linking the intracellular acidification and the regulation of P-glycoprotein remain unclear. In the present study, the molecular pathways involved in the regulation of P-glycoprotein expression by the intracellular acidification were investigated. We found that the P-glycoprotein expression was down-regulated by the intracellular acidification through inhibition of p38 mitogen-activated protein kinase (MAPK) and the activation of c-Jun N-terminal kinase (JNK) in the resisitant K562/DOX cells. In the sensitive K562 and HL60 cell lines, the changes of the p38 MAPK expression after the acidification are not as obvious as that of K562/DOX cells, but the activation of extracellular signal-regulated kinase (ERK) is also observed, which indicates that the down-regulation of p38 MAPK by the intracellular acidification might be the resistant cell line specific. Blockade of ERK and JNK signaling by the inhibitors or RNA interference increased p38MAPK activities suggesting that cross-talk within MAPKs is also important for this response. Our study provides the first direct evidence that the reversal of P-glycoprotein-mediated multidrug resistance by intracellular acidification is mediated by the crosstalk of MAPK signaling pathways.

Icariin promotes self-renewal of neural stem cells: An involvement of extracellular regulated kinase signaling pathway

To investigate the effects and underlying molecular mechanisms of icariin (ICA) on self-renewal and differentiation of neural stem cells (NSCs). NSCs were derived from forebrains of mice embryos by mechanical dissociation into single cell suspension. The self-renewal of NSCs was measured by neurosphere formation assay. The proliferation of NSCs was detected by water-soluble tetrazolium (WST) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Protein expression of neuron-specific marker tubulin-βIII(TuJ1) and astrocyte-specific marker glial fibrillary acidic protein (GFAP) were measured by immunofluorescence and Western blotting. Using microarray, the differentially expressed genes (DEGs) were screened between NSCs with or without ICA treatment. The signaling pathways enriched by these DEGs and their role in mediating effects of ICA were analyzed. ICA significantly promoted neurosphere formation of NSCs cultured in growth protocol in a dose-dependent manner and achieved the maximum effects at 100 nmol/L. ICA also increased optical absorbance value and EdU incorporation into nuclei of NSCs. ICA had no significant effects on the percentage of TuJ1 or GFAP-positive cells, and TuJ1 or GFAP protein expression in NSCs cultured in differentiation protocol. A total of 478 genes were found to be differentially regulated. Among signaling pathways significantly enriched by DEGs, mitogen activated protein kinase (MAPK) pathway was of interest. Blockade of extracellular signal-regulated kinase (ERK)/MAPK, other than p38/MAPK subfamily pathway partially abolished effects of ICA on neurosphere formation and EdU incorporation of NSCs. ICA can promote the selfrenewal of NSCs at least partially through ERK/MAPK signaling pathway.

Regulation of Renal Fibrosis by Smad3 Thr388 Phosphorylation

Transforming growth factor-β (TGF-β) promotes tissue fibrosis via receptor-mediated phosphorylation of the receptor-activated Smad2/3, together with Smad4. Of these, Smad3 plays a major profibrotic role in mouse models of tissue fibrosis. Transcriptional activity of the Smad3 protein is regulated by phosphorylation of residues in the C-terminal domain and the linker region. Herein, we examined the role of a novel phosphorylation site within the MH2 domain (T388) in the regulation of Smad3 activity. Confocal microscopy using an Smad3 phosphorylated T388-specific antibody identified phosphorylation of Smad3 T388 in myofibroblasts and tubular epithelial cells in human focal and segmental glomerulosclerosis and mouse models of unilateral ureteric obstruction and diabetic nephropathy, whereas phosphorylated T388 was largely absent in normal kidney. In vitro, TGF-β1 induced phosphorylation of Smad3 T388 in a biphasic pattern. A point mutation of T388/V in an Smad3 construct demonstrated that phosphorylation of T388 promotes Smad3 binding to Smad4 and CDK8, but was not necessary for nuclear translocation. Furthermore, T388 phosphorylation was required for TGF-β-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts. In conclusion, our study identifies phosphorylation of T388 in the Smad3 MH2 domain as an important mechanism that regulates the profibrotic TGF-β/Smad3 signaling pathway, which has direct relevance to human and experimental fibrotic kidney disease.

Regulation of cell survival by sphingosine-1-phosphate receptor S1P1 via reciprocal ERK-dependent suppression of Bim and PI-3-kinase/protein kinase C-mediated upregulation of Mcl-1

Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P1 is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P1 renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P1-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P1-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P1 regulation of Bim. However, S1P1 suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P1 in disease, analysis of tissue microarrays from ER+ breast cancer patients revealed a significant correlation between S1P1 expression and tumour cell survival. In these tumours, S1P1 expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P1 is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context.

N-methyl-D-aspartic acid receptor activation downregulates expression of δ subunit-containing GABAA receptors in cultured hippocampal neurons.

Neurosteroids are endogenous allosteric modulators of GABAA receptors (GABARs), and they enhance GABAR-mediated inhibition. However, GABARs expressed on hippocampal dentate granule neurons of epileptic animals are modified such that their neurosteroid sensitivity is reduced and δ subunit expression is diminished. We explored the molecular mechanisms triggering this GABAR plasticity. In the cultured hippocampal neurons, treatment with N-methyl-D-aspartic acid (NMDA) (10 μM) for 48 hours reduced the surface expression of δ and α4 subunits but did not increase the expression of γ2 subunits. The tonic current recorded from neurons in NMDA-treated cultures was reduced, and its neurosteroid modulation was also diminished. In contrast, synaptic inhibition and its modulation by neurosteroids were preserved in these neurons. The time course of NMDA's effects on surface and total δ subunit expression was distinct; shorter (6 hours) treatment decreased surface expression, whereas longer treatment reduced both surface and total expression. Dl-2-amino-5-phosphonopentanoic acid (APV) blocked NMDA's effects on δ subunit expression. Chelation of calcium ions by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) or blockade of extracellular signal-regulated kinase (ERK) 1/2 activation by UO126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene) also prevented the effects of NMDA. Thus, prolonged activation of NMDA receptors in hippocampal neurons reduced GABAR δ subunit expression through Ca(2+) entry and at least in part by ERK1/2 activation.

P38 mitogen‐activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture

Articular cartilage is an avascular tissue with poor regenerative capacity following injury, a contributing factor to joint degenerative disease. Cell-based therapies for cartilage tissue regeneration have rapidly advanced; however, expansion of autologous chondrocytes in vitro using standard methods causes ‘dedifferentiation’ into fibroblastic cells. Mitogen-activated protein kinase (MAPK) signalling is crucial for chondrocyte metabolism and matrix production, and changes in MAPK signals can affect the phenotype of cultured cells. We investigated the effects of inhibition of MAPK signalling on chondrocyte dedifferentiation during monolayer culture. Blockade of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signalling caused a significant increase in cartilage gene expression, however, also caused up-regulation of fibrotic gene expression. Inhibition of p38 MAPK (p38) caused a significant up-regulation of collagen type II while suppressing collagen type I expression. P38 inhibition also resulted in consistently more organized secretion of collagen type II protein deposits on cell culture surfaces. Follow-on pellet culture of treated cells revealed that MAPK inhibition reduced cell migration from the pellet. ERK and JNK inhibition caused more collagen type I accumulation in pellets versus controls while p38 inhibition strongly promoted collagen type II accumulation with no effect on collagen type I. Blockade of all three MAPKs caused increased GAG content in pellets. These results indicate a role for MAPK signalling in chondrocyte phenotype loss during monolayer culture, with a strong contribution from p38 signalling. Thus, blockade of p38 enhances chondrocyte phenotype in monolayer culture and may promote more efficient cartilage tissue regeneration for cell-based therapies.

Inhibitory Activity of Bevacizumab to Differentiation of Retinoblastoma Cells

Vascular endothelial growth factor (VEGF) is a major regulator in retinal and choroidal angiogenesis, which are common causes of blindness in all age groups. Recently anti-VEGF treatment using anti-VEGF antibody has revolutionarily improved the visual outcome in patients with vaso-proliferative retinopathies. Herein, we demonstrated that bevacizumab as an anti-VEGF antibody could inhibit differentiation of retinoblastoma cells without affection to cellular viability, which would be mediated via blockade of extracellular signal-regulated kinase (ERK) 1/2 activation. The retinoblastoma cells expressed VEGFR-2 as well as TrkA which is a neurotrophin receptor associated with differentiation of retinoblastoma cells. TrkA in retinoblastoma cells was activated with VEGF treatment. Interestingly even in the concentration of no cellular death, bevascizumab significantly attenuated the neurite formation of differentiated retinoblastoma cells, which was accompanied by inhibition of neurofilament and shank2 expression. Furthermore, bevacizumab inhibited differentiation of retinoblastoma cells by blockade of ERK 1/2 activation. Therefore, based on that the differentiated retinoblastoma cells are mostly photoreceptors, our results suggest that anti-VEGF therapies would affect to the maintenance or function of photoreceptors in mature retina.

Synergistic Action of a RAF Inhibitor and a Dual PI3K/mTOR Inhibitor in Thyroid Cancer

In thyroid cancer clinical trials, agents targeting VEGF receptors (VEGFR) and RET, among other kinases, have led to partial responses but few complete or durable responses. The RAF-MEK-ERK and PI3K-AKT-mTOR signaling pathways are frequently activated in differentiated and medullary thyroid cancer (DTC and MTC) and may provide therapeutic targets for these diseases. We tested a novel drug combination targeting RAF, phosphoinositide 3-kinase (PI3K), and mTOR, plus VEGFR2 and RET, in thyroid cancer preclinical models with defined genetic backgrounds. RAF265, an ATP-competitive pan-RAF inhibitor active against VEGFR2, and BEZ-235, a PI3K inhibitor also active against Torc1 and Torc2, were tested alone and in combination in a panel of thyroid cancer lines. We tested RAF265 and BEZ-235 for kinase inhibition, growth inhibition and cell-cycle alterations, and inhibition of signaling targets and tumor growth in xenograft models. Both drugs potently inhibited their kinase targets in the extracellular signal-regulated kinase (ERK) and PI3K pathways. In addition, RAF265 had significant RET inhibitory activity (IC₅₀ = 25-50 nmol/L for RET(C634W)). The combination strongly inhibited proliferation of DTC and MTC cell lines with mutations in RAS, BRAF, PTEN, and RET. Synergy was shown for B-CPAP (BRAF(V600E)) and TT cells (RET(C634W)). The combination of both drugs significantly inhibited growth of CAL62 (KRAS(G12R/G12R)) and TT xenografts, thoroughly inhibiting ERK and PI3K pathway signaling. Combined blockade of ERK and PI3K signaling potently inhibits growth in preclinical models representing the key genotypes seen in refractory thyroid cancer. These targets and therapies are promising for further development in both differentiated and medullary thyroid cancers.

Angiotensin II–Induced MMP-2 Activity and MMP-14 and Basigin Protein Expression Are Mediated via the Angiotensin II Receptor Type 1–Mitogen-Activated Protein Kinase 1 Pathway in Retinal Pigment Epithelium: Implications for Age-Related Macular Degeneration

Accumulation of various lipid-rich extracellular matrix (ECM) deposits under the retinal pigment epithelium (RPE) has been observed in eyes with age-related macular degeneration (AMD). RPE-derived matrix metalloproteinase (MMP)-2, MMP-14, and basigin (BSG) are major enzymes involved in the maintenance of ECM turnover. Hypertension (HTN) is a systemic risk factor for AMD. It has previously been reported that angiotensin II (Ang II), one of the most important hormones associated with HTN, increases MMP-2 activity and its key regulator, MMP-14, in RPE, inducing breakdown of the RPE basement membrane, which may lead to progression of sub-RPE deposits. Ang II exerts most of its actions by activating the mitogen-activated protein kinase (MAPK) signaling pathway. Herein is explored the MAPK signaling pathway as a potential key intracellular modulator of Ang II-induced increase in MMP-2 activity and MMP-14 and BSG protein expression. It was observed that Ang II stimulates phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK in RPE cells and ERK/p38 and Jun N-terminal kinase (JNK) in mice. These effects were mediated by Ang II type 1 receptors. Blockade of ERK or p38 MAPK abrogated the increase in MMP-2 activity and MMP-14 and BSG proteins in ARPE-19 cells. A better understanding of the molecular events by which Ang II induces ECM dysregulation is of critical importance to further define its contribution to the progression of sub-RPE deposits in AMD patients with HTN.

Neuropeptide Y Y5 Receptor Promotes Cell Growth through Extracellular Signal-Regulated Kinase Signaling and Cyclic AMP Inhibition in a Human Breast Cancer Cell Line

Overexpression of neuropeptide Y (NPY) and its receptor system has been reported in various types of cancers. NPY Y5 receptor (Y5R) has been implicated in cell growth and angiogenesis. However, the role of Y5R in breast cancer is unknown. To identify the role of Y5R in breast cancer, we screened several breast cancer cell lines to examine the expression of Y5R and its function in breast cancer. All screened cell lines express both Y1 receptor and Y5R except BT-549, which expresses mainly Y5R. Binding studies showed that NPY, Y5R-selective agonist peptide, and Y5R-selective antagonist (CGP71683A) displaced (125)I-PYY binding in BT-549 cell membranes in a dose-dependent manner. The displacement studies revealed the presence of two binding sites in Y5R with IC(50) values of 29 pmol/L and 531 nmol/L. NPY inhibited forskolin-stimulated cyclic AMP accumulation with an IC(50) value of 52 pmol/L. NPY treatment of BT-549 cells induced extracellular signal-regulated kinase phosphorylation but did not alter intracellular calcium. Y5R activation stimulates BT-549 cell growth, which is inhibited by CGP71683A, pertussis toxin, and extracellular signal-regulated kinase blockade. CGP71683A alone induced cell death in a time- and dose-dependent manner in Y5R-expressing cells. The stimulation of MDA MB-231 cell migration by NPY is inhibited by CGP71683A. Together, our results suggest that Y5R plays an important role in cancer cell growth and migration and could be a novel therapeutic target for breast cancer.

ERK activation and cell growth require CaM kinases in MCF-7 breast cancer cells

Previous studies on MCF-7 breast cancer cells have shown that the G-protein coupled receptor (GPCR) agonist carbachol increases intracellular calcium levels and the activation of extracellular signal-regulated kinase (ERK). Calcium and calmodulin regulate the calcium/calmodulin-dependent kinase (CaM kinase) family of proteins that have been proposed to regulate ERK and gene transcription. Our results suggest that both estrogen (E2) and carbachol treatment of MCF-7 breast cancer cells trigger phosphorylation of ERK1/2 and the transcription factor Elk-1. Carbachol and estrogen triggered nearly a four- to sixfold increase in MCF-7 cell proliferation by 96 h, respectively. Carbachol-stimulated ERK activation and cell growth was completely blocked by the Muscarinic M(3)-subtype GPCR inhibitor, 4-DAMP, and siRNA against the M(3)-subtype GPCR. Interestingly, blockade of CaM KK with the selective inhibitor STO-609 prevented carbachol activation CaM KI, ERK, Elk-1, and cell growth. Consistent with these observations, knockdown of CaM KKalpha and CaM KIgamma with shRNA-containing plasmids blocked ERK activation by carbachol. In addition, Elk-1 phosphorylation and luciferase activity in response to carbachol treatment was also dependent upon CaM kinases and was inhibited by U0126, STO-609, and siRNA knockdown of CaM kinases and ERK2. Finally, blockade of either CaM KK (with STO-609) or ERK (with U0126) activities resulted in the inhibition of carbachol- and estrogen-mediated cyclin D1 expression and MCF-7 cell growth. Taken together, our results suggest that carbachol treatment of MCF-7 cells activates CaM KI, ERK, the transcription factor Elk-1, cyclin D1, and cell growth through CaM KK.