Total ERK1/2 activity regulates cell proliferation

Regulating ERK activity is essential for normal cell proliferation to occur. In mammals and most vertebrates ERK activity is provided by ERK1 and ERK2 that are highly similar, ubiquitously expressed and share activators and substrates. By combining single and double silencings of ERK1 and ERK2 we recently demonstrated that the apparent dominant role of ERK2 to regulate cell proliferation was due to its markedly higher expression level than ERK1. The contribution of ERK1 was revealed when ERK2 activation was clamped to avoid compensating over-activation of ERK2. We found no evidences in the literature for insulated isoform-specific modules in the Ras/Raf/MEK signaling cascade that could activate specifically ERK1 or ERK2. Obviously in frogs all signal integration and fine modulation provided by three Ras and three Raf isoforms is conducted by only one MEK and one ERK isoform. In mammals, ERK1 and ERK2 display similar specific activities and are activated respectively to their expression levels. After integrating signals from Ras, Raf and MEK isoforms, ERK1 and ERK2 regulate positively cell proliferation according to their expression levels.     

TRPM6 expression and cell proliferation are up-regulated by phosphorylation of ERK1/2 in renal epithelial cells

Transient receptor potential melastatin 6 (TRPM6) is a magnesium channel and expressed in the intestine and renal distal tubules. Little is known about the regulatory mechanism of TRPM6 expression and the role of magnesium influx. EGF increased the phosphorylation of ERK1/2 and TRPM6 expression that were inhibited by U0126 in renal epithelial NRK-52E cells. Furthermore, EGF enhanced the influx of magnesium, whereas U0126 and TRPM6 siRNA inhibited it. EGF increased the proportion of cells in S phase, whereas U0126 and TRPM6 siRNA increased the proportion in G1 phase. The phosphorylation of ERK1/2 may up-regulate TRPM6 expression and magnesium influx, resulting in an increase in cell proliferation with a shift from G1 to S phase.     

Cystathionine gamma-lyase overexpression inhibits cell proliferation via a H2S-dependent modulation of ERK1/2 phosphorylation and p21Cip/WAK-1

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway. CSE uses L-cysteine as a substrate to produce hydrogen sulfide (H2S). The CSE/H2S system has been shown to play an important role in regulating cellular functions in different systems. In the present study, we used CSE stably overexpressed HEK-293 cells to explore the effect of the CSE/H2S system on cell growth and proliferation. The overexpression of CSE resulted in increases in CSE mRNA levels, CSE proteins, and intracellular H2S production rates, as well as the inhibition of cell proliferation and DNA synthesis. These effects were accompanied by a sustained ERK activation and up-regulation of the cyclin-dependent kinase inhibitor p21Cip/WAK-1. Blocking the action of ERK with U0126 inhibited the induction of p21Cip/WAK-1, suggesting that ERK activation functions upstream of p21Cip/WAK-1 activation to initiate the CSE overexpression-induced cell growth inhibition. The antiproliferative effect of CSE is likely mediated by endogenously produced H2S because the H2S scavenger methemoglobin (10 microm) significantly decreased the H2S production rate and reversed the antiproliferative effect afforded by CSE. Exogenous H2S (100 microm) also inhibited cell proliferation. However, the other CSE-catalyzed products, ammonium and pyruvate, failed to inhibit cell proliferation. Methemoglobin also abolished the inhibitory effect of exogenous H2S on cell proliferation. Moreover, exogenous H2S induced a sustained ERK and p21Cip/WAK-1 activation. These findings support the hypothesis that endogenously produced H2S may play a fundamental role in cell proliferation and survival.     

Activation of NKCC1 by hyperosmotic stress in human tracheal epithelial cells involves PKC-delta and ERK

Hyperosmotic stress activates Na+-K+-2Cl- cotransport (NKCC1) in secretory epithelia of the airways. NKCC1 activation was studied as uptake of 36Cl or 86Rb in human tracheal epithelial cells (HTEC). Application of hypertonic sucrose or NaCl increased bumetanide-sensitive ion uptake but did not affect Na+/H+ and Cl-/OH-(HCO3-) exchange carriers. Hyperosmolarity decreased intracellular volume (Vi) after 10 min from 7.8 to 5.4 microl/mg protein and increased intracellular Cl- (Cl-i) from 353 to 532 nmol/mg protein. Treatment with an alpha-adrenergic agent rapidly increased Cl-i and Vi in a bumetanide-sensitive manner, indicating uptake of ions by NKCC1 followed by osmotically obligated water. These results indicate that HTEC act as osmometers but lose intracellular water slowly. Hyperosmotic stress also increased the activity of PKC-delta and of the extracellular signal-regulated kinase ERK subgroup of the MAPK family. Activity of stress-activated protein kinase JNK was not affected by hyperosmolarity. PD-98059, an inhibitor of the ERK cascade, reduced ERK activity and bumetanide-sensitive 36Cl uptake. PKC inhibitors blocked activation of ERK indicating that PKC may be a downstream activator of ERK. The results indicate that hyperosmotic stress activates NKCC1 and this activation is regulated by PKC-delta and ERK.     

ERK1/2 mediates lung adenocarcinoma cell proliferation and autophagy induced by apelin-13

The aim of this study was to investigate the role of apelin in the cell proliferation and autophagy of lung adenocarcin- oma. The over-expression of APJ in lung adenocarcinoma was detected by immunohistochemistry, while plasma apelin level in lung cancer patients was measured by enzyme-linked immunosorbent assay. Our findings revealed that apelin-13 significantly increased the phosphorylation of ERK1/2, the expression of cyclin D1, microtubule-associated protein 1 light chain 3A/B （LC3A/B）, and beclinl, and con- fwmed that apelin-13 promoted A549 cell proliferation and induced A549 cell autophagy via ERK1/2 signaling. More- over, there are pores on the surface of human lung adeno- carcinoma cell line A549 and apelin-13 causes cell surface smooth and glossy as observed under atomic force micros- copy. These results suggested that ERK1/2 signaling pathway mediates apelin-13-induced lung adenocarcinoma cell proliferation and autophagy. Under our experimental condition, autophagy associated with 3-methyladenine was not involved in cell proliferation.     

Ferulic acid inhibits endothelial cell proliferation through NO down-regulating ERK1/2 pathway

The aim of this study was to determine the antiproliferative mechanism of ferulic acid (FA) on serum induced ECV304 cell, a human umbilical vein endothelial line. The results suggest that FA significantly suppressed ECV304 cells proliferation and blocked the cell cycle in G0/G1 phase. Treatment of the cells with FA increased nitric oxide (NO) production and inactivated the extracellular signal-regulated kinase (EERK1/2), and the NO donor, sodium nitroprusside, inhibited both ECV304 cells proliferation and phosphorylation of ERK1/2. However, the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester, caused ECV304 cells proliferation. PD 98059, the inhibitor of ERK1/2, had no effect on the NO production. These results indicate that NO suppressed ECV304 cells proliferation through down-regulating ERK1/2 pathway. Moreover, the inhibition of cell cycle progression was associated with the decrement of cyclin D1 expression and phosphorylation of retinoblastoma protein (pRb) by increment of p21 level. The findings not only present the first evidence that FA is a potent inhibitor on ECV304 cells proliferation, but also reveal the potential signaling molecules involved in its action.     

ERK1/2 mediates PDGF-BB stimulated vascular smooth muscle cell proliferation and migration on laminin-5

During restenosis following arterial injury, vascular smooth muscle cells (VSMCs) form a neointimal layer in arteries by changing from a differentiated, contractile phenotype to a dedifferentiated, migratory, and proliferative phenotype. Several growth factors, cytokines, and extracellular matrix components released following injury have been implicated in these phenotypic changes. We have recently detected the expression of laminin-5, an ECM protein found predominantly in epithelial tissues, in the arterial vasculature. Here we report that ln-5 expression by VSMC is upregulated by platelet-derived growth factor (PDGF-BB), epidermal growth factor, basic fibroblast growth factor, and transforming growth factor-beta1. Adhesion to ln-5 specifically enhances PDGF-BB-stimulated VSMC proliferation and migration. PD98059, a specific inhibitor of the ERK1/2 members of the Mitogen Activated Protein kinase family, increases both VSMC adhesion to ln-5 and blocks PDGF-BB-stimulated VSMC migration on ln-5. These results suggest that adhesion to ln-5 mediates a PDGF-BB-stimulated VSMC response to vascular injury via an ERK1/2 signaling pathway.     

Unsaturated fatty acids promote proliferation via ERK1/2 and Akt pathway in bovine mammary epithelial cells

GPR40 has recently been identified as a G protein-coupled cell-surface receptor for long-chain fatty acids (LCFAs). The mRNA of the bovine ortholog of GPR40 (bGPR40) was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and in the bovine mammary gland at various stages of lactation. Oleate and linoleate caused an increase in intracellular Ca²⁺ concentrations in these cells, and significantly reduced forskolin-induced cAMP concentrations. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase, which regulates cell proliferation and survival, was rapidly increased by oleate. Incubation with oleate and linoleate for 24 h significantly promoted cell proliferation. Moreover, in serum-free medium, oleate significantly stimulated cell proliferation during a 7-day culture. These results suggest that bGPR40 mediates LCFA signaling in mammary epithelial cells and thereby plays an important role in cell proliferation and survival.     

Heme oxygenase inhibits human airway smooth muscle proliferation via a bilirubin-dependent modulation of ERK1/2 phosphorylation

The aim of this study was to investigate whether the heme oxygenase (HO) pathway could modulate proliferation of airway smooth muscle (ASM) and the mechanism(s) involved in this phenomenon. In cultured human ASM cells, 10% fetal calf serum or 50 ng/ml platelet-derived growth factor AB induced cell proliferation, extracellular and intracellular reactive oxygen species (ROS) production and ERK1/2 phosphorylation. Pharmacological HO-1 induction (by 10 microm hemin or by 20 microm cobalt-protoporphyrin) and HO inhibition (by 25 microm tin-protoporphyrin or by an antisense oligonucleotide), respectively, reduced and enhanced significantly both cell proliferation and ROS production. Neither the carbon monoxide scavenger myoglobin (5-20 microm) nor the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one could reverse ASM proliferation induced by tin-protoporphyrin, making a role of the CO-cGMP pathway in HO-modulated proliferation unlikely. By contrast, bilirubin (1 microm) and the antioxidant N-acetyl-cysteine (1 mm) significantly reduced mitogen-induced cell proliferation, ROS production, and ERK1/2 phosphorylation. Furthermore, both bilirubin and N-acetyl-cysteine and the ERK1/2 inhibitor PD98059 significantly reversed the effects of HO inhibition on ASM proliferation. These results could be relevant to ASM alterations observed in asthma because activation of the HO pathway prevented the increase in bronchial smooth muscle area induced by repeated ovalbumin challenge in immunized guinea pigs, whereas inhibition of HO had the opposite effect. In conclusion, this study provides evidence for an antiproliferative effect of the HO pathway in ASM in vitro and in vivo through a bilirubin-mediated redox modulation of phosphorylation of ERK1/2.     

Akt down-regulates ERK1/2 nuclear localization and angiotensin II-induced cell proliferation through PEA-15

Angiotensin II (AngII) type 1 receptors (AT1) regulate cell growth through the extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol 3-kinase (PI3K) pathways. ERK1/2 and Akt/protein kinase B, downstream of PI3K, are independently activated but both required for mediating AngII-induced proliferation when expressed at endogenous levels. We investigate the effect of an increase in the expression of wild-type Akt1 by using Chinese hamster ovary (CHO)-AT1 cells. Unexpectedly, Akt overexpression inhibits the AT1-mediated proliferation. This effect could be generated by a cross-talk between the PI3K and ERK1/2 pathways. A functional partner is the phosphoprotein enriched in astrocytes of 15 kDa (PEA-15), an Akt substrate known to bind ERK1/2 and to regulate their nuclear translocation. We report that Akt binds to PEA-15 and that Akt activation leads to PEA-15 stabilization, independently of PEA-15 interaction with ERK1/2. Akt cross-talk with PEA-15 does not affect ERK1/2 activation but decreases their nuclear activity as a result of the blockade of ERK1/2 nuclear accumulation. In response to AngII, PEA-15 overexpression displays the same functional consequences on ERK1/2 signaling as Akt overactivation. Thus, Akt overactivation prevents the nuclear translocation of ERK1/2 and the AngII-induced proliferation through interaction with and stabilization of endogenous PEA-15.     

Abl regulates smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 activation

Abl is a nonreceptor tyrosine kinase that has a role in regulating migration and adhesion of nonmuscle cells as well as smooth muscle contraction. The role of Abl in smooth muscle cell proliferation has not been investigated. In this study, treatment with endothelin-1 (ET-1) and platelet-derived growth factor (PDGF) increased Abl phosphorylation at Tyr(412) (an indication of Abl activation) in vascular smooth muscle cells. To assess the role of Abl in smooth muscle cell proliferation, we generated stable Abl knockdown cells by using lentivirus-mediated RNA interference. ET-1- and PDGF-induced cell proliferation was attenuated in Abl knockdown cells compared with cells expressing control shRNA and uninfected cells. Abl silencing also arrested cell cycle progression from G(0)/G(1) to S phase. Furthermore, activation of smooth muscle cells with ET-1 and PDGF induced phosphorylation of ERK1/2 and Akt. Abl knockdown attenuated ERK1/2 phosphorylation in smooth muscle cells stimulated with ET-1 and PDGF. However, Akt phosphorylation upon stimulation with ET-1 and PDGF was not reduced. Because Abl is known to regulate actin polymerization in smooth muscle, we also evaluated the effects of inhibition of actin polymerization on phosphorylation of ERK1/2. Pretreatment with the actin polymerization inhibitor latrunculin-A also blocked ERK1/2 phosphorylation during activation with ET-1 and PDGF. The results suggest that Abl may regulate smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 phosphorylation during mitogenic activation.     

Urotensin II mediates ERK1/2 phosphorylation and proliferation in GPR14-transfected cell lines

Urotensin-II (U-II), a vasoactive cyclic neuropeptide, was recently identified as the natural ligand for the G-protein coupled receptor GPR14. The expression pattern of U-II and GPR14 are consistent with a role as a neurohormonal regulatory system in cardiovascular homeostasis. Urotensin-II induces a rapid and short-lasting rise in intracellular calcium in recombinant GPR14 expressing cells. In the present study we show that U-II induces signal transduction pathways leading to the long-lasting activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in chinese hamster ovary cells expressing human GPR14 (CHO-GPR14). Furthermore, we observed a growth-stimulating and PD98059 sensitive activity of U-II in CHO-GPR14 cells, but not CHO-K1 cells. The investigation of the GPR14 induced signal transduction pathways leading to ERKI/2 phosphorylation revealed a previously unsuspected role for G(i/o)-protein coupling and showed an involvement of phospatidylinositol-3-kinase, phospholipase C and calcium channel mediated mechanisms. Our results suggest that U-II and its receptor GPR14 may be involved in long-lasting physiological effects such as cardiovascular remodeling.     

Evidence for ERK1/2 phosphorylation controlling contact inhibition of proliferation in Madin-Darby canine kidney epithelial cells

Increasing cell density arrests epithelial cell proliferation by a process termed contact inhibition. We investigated mechanisms of contact inhibition using a model of contact-inhibited epithelial cells. Hepatocyte growth factor (HGF) treatment of contact-inhibited Madin-Darby canine kidney (MDCK) cells stimulated cell proliferation and increased levels of phosphorylated ERK1/2 (phospho-ERK1/2) and cyclin D1. MEK inhibitors PD-98059 and U0126 inhibited these HGF-dependent changes, indicating the dependence on phosphorylation of ERK1/2 during HGF-induced loss of contact inhibition. In relation to contact-inhibited high-density cells, low-density MDCK cells proliferated and had higher levels of phospho-ERK1/2 and cyclin D1. PD-98059 and U0126 inhibited low-density MDCK cell proliferation. Trypsinization of high-density MDCK cells immediately increased phospho-ERK1/2 and was followed by a transient increase in cyclin D1 levels. Reformation of cell junctions after trypsinization led to decreases in phospho-ERK1/2 and cyclin D1 levels. High-density MDCK cells express low levels of both cyclin D1 and phospho-ERK1/2, and treatment of these cells with fresh medium containing HGF but not fresh medium alone for 6 h increased phospho-ERK1/2 and cyclin D1 levels compared with cells without medium change. These data provide evidence that HGF abrogates MDCK cell contact inhibition by increasing ERK1/2 phosphorylation and levels of cyclin D1. These results suggest that in MDCK cells, contact inhibition of cell proliferation in the presence of serum occurs by cell density-dependent regulation of ERK1/2 phosphorylation. cell density; cyclin D1; hepatocyte growth factor; cell cycle; extracellular signal-regulated kinases     

Glutamate accelerates RPE cell proliferation through ERK1/2 activation via distinct receptor-specific mechanisms

The proliferation and migration of Retinal Pigment Epithelium cells resulting from an epithelial-mesenchymal transition plays a key role in proliferative vitreoretinopathy, which leads to retinal detachment and the loss of vision. In neurons, glutamate has been shown to activate the Ras/Raf/MEK/ERK cascade, which participates in the regulation of proliferation, differentiation, and survival processes. Although glutamate-stimulation and the activation of ERK1/2 by different stimuli have been shown to promote RPE cell proliferation, the signaling pathway(s) linking these effects has not been established. We analyzed the molecular mechanisms leading to glutamate-induced proliferation by determining ERK1/2 and CREB phoshporylation in chick RPE cells in primary culture and the human-derived RPE cell line ARPE-19. This study shows for the first time, that glutamate promotes RPE cell proliferation by activating two distinct signaling pathways linked to selective glutamate receptor subtypes. Results demonstrate that glutamate stimulates RPE cell proliferation as well as ERK and CREB phosphorylation. These effects were mimicked by the mGluR agonist ACPD and by NMDA, and were prevented by the respective receptor inhibitors MCPG and MK-801, indicating a cause-effect relationship between these processes. Whereas mGluR promoted proliferation by activating the MEK/ERK/CREB cascade, NMDA stimulated proliferation through the MEK-independent activation of Ca(2+)/calmodulin-dependent kinases. The blockage of both signaling pathways to proliferation by KN-62 suggests the involvement of CaMKs in the control of glutamate-induced proliferation at a common step, downstream of CREB, possibly the regulation of cell cycle progression. Based on these findings, the participation of glutamate in the development of PVR can be considered.     

FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signalling.

Members of the fibroblast growth factor (FGF) family induce lens epithelial cells to undergo cell division and differentiate into fibres; a low dose of FGF can stimulate cell proliferation (but not fibre differentiation), whereas higher doses of FGF are required to induce fibre differentiation. To determine if these cellular events are regulated by the same signalling pathways, we examined the role of mitogen-activated protein kinase (MAPK) signalling in FGF-induced lens cell proliferation and differentiation. We show that FGF induced a dose-dependent activation of extracellular regulated kinase 1/2 (ERK1/2) as early as 15 minutes in culture, with a high (differentiating) dose of FGF stimulating a greater level of ERK phosphorylation than a lower (proliferating) dose. Subsequent blocking experiments using UO126 (a specific inhibitor of ERK activation) showed that activation of ERK is required for FGF-induced lens cell proliferation and fibre differentiation. Interestingly, inhibition of ERK signalling can block the morphological changes associated with FGF-induced lens fibre differentiation; however, it cannot block the synthesis of some of the molecular differentiation markers, namely, beta-crystallin. These findings are consistent with the in vivo distribution of the phosphorylated (active) forms of ERK1/2 in the lens. Taken together, our data indicate that different levels of ERK signalling may be important for the regulation of lens cell proliferation and early morphological events associated with fibre differentiation; however, multiple signalling pathways are likely to be required for the process of lens fibre differentiation and maturation.     

p38 and ERK1/2 coordinate cellular migration and proliferation in epithelial wound healing: evidence of cross-talk activation between MAP kinase cascades

One important action of growth factors is their participation in tissue repair; however, the signaling pathways involved are poorly understood. In a model of corneal wound healing, we found that two paracrine growth factors, hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF), induced rapid and marked activation and prompt nuclear accumulation of phospho-p38 (p-p38) and -ERK1/2 (p-ERK1/2), but not of JNK (p-JNK1/2), in corneal epithelial cells. Interruption of p38 and ERK1/2 signaling pathways by pretreatment with inhibitors SB203580 and PD98059 and subsequent stimulation with HGF or KGF abolished the activation and nuclear localization. Inhibition of either one of these mitogen-activated protein kinases, p38 or ERK1/2, induced a robust cross-activation of the other. In immunofluorescence studies of wounded cornea, p-p38, unlike p-ERK1/2, was immediately detectable in epithelium after injury. Inhibition of p38 by SB203580 blocked migration of epithelial cells almost completely. In contrast, PD98059 seemed to slightly increase the migration, through concomitant activation of p38. Unlike ERK1/2, p38 did not significantly contribute to proliferation of epithelial cells. Inhibition of either the ERK1/2 or p38 pathway resulted in delayed corneal epithelial wound healing. Interruption of both signaling cascades additively inhibited the wound-healing process. These findings demonstrate that both p38 and ERK1/2 coordinate the dynamics of wound healing: while growth factor-stimulated p38 induces epithelial migration, ERK1/2 activation induces proliferation. The cross-talk between these two signal cascades and the selective action of p38 in migration appear to be important to corneal wound healing, and possibly wound healing in general, and may offer novel drug targets for tissue repair.     

ANG II-induced cell proliferation is dually mediated by c-Src/Yes/Fyn-regulated ERK1/2 activation in the cytoplasm and PKCzeta-controlled ERK1/2 activity within the nucleus

High-affinity binding of angiotensin II (ANG II) to the ANG II type 1 receptor (AT₁R) results in the activation of ERK1/2 mitogen-activated protein kinases (MAPK). However, the precise mechanism of ANG II-induced ERK1/2 activation has not been fully characterized. Here, we investigated the signaling events leading to ANG II-induced ERK1/2 activation using a c-Src/Yes/Fyn tyrosine kinase-deficient mouse embryonic fibroblast (MEF) cell line stably transfected with the AT₁R (SYF/AT₁). ERK1/2 activation was reduced by 50% within these cells compared with wild-type controls (WT/AT₁). The remaining 50% of intracellular ERK1/2 activation was dependent upon heterotrimeric G protein and protein kinase C zeta (PKC) activation. Therefore, ANG II-induced ERK1/2 activation occurs via two independent mechanisms. We next investigated whether a loss of either c-Src/Yes/Fyn or PKC signaling affected ERK1/2 nuclear translocation and cell proliferation in response to ANG II. ANG II-induced cell proliferation was markedly reduced in SYF/AT₁ cells compared with WT/AT₁ cells (P < 0.01), but interestingly, ERK2 nuclear translocation was normal. ANG II-induced nuclear translocation of ERK2 was blocked via pretreatment of WT/AT₁ cells with a PKC pseudosubstrate. ANG II-induced cell proliferation was significantly reduced in PKC pseudosubstrate-treated WT/AT₁ cells (P < 0.01) and was completely blocked in SYF/AT₁ cells treated with this same compound. Thus ANG II-induced cell proliferation appears to be regulated by both ERK1/2-driven nuclear and cytoplasmic events. In response to ANG II, the ability of ERK1/2 to remain within the cytoplasm or translocate into the nucleus is controlled by c-Src/Yes/Fyn or heterotrimeric G protein/PKC signaling, respectively. Src family tyrosine kinases; angiotensin II     

Identification of cell type–specific correlations between ERK activity and cell viability upon treatment with ERK1/2 inhibitors

Increased MAPK signaling is a hallmark of various cancers and is a central regulator of cell survival. Direct ERK1/2 inhibition is considered a promising approach to avoid ERK1/2 reactivation caused by upstream kinases BRAF, MEK1/2, and KRAS, as well as by receptor tyrosine kinase inhibitors, but the dynamics and selectivity of ERK1/2 inhibitors are much less studied compared with BRAF or MEK inhibitors. Using ERK1/2 and downstream kinase ELK1 reporter cell lines of lung cancer (H1299; NRAS^Q61K), colon cancer (HCT-116; KRAS^G13D), neuroblastoma (SH-SY5Y), and leukemia (U937), we examined the relationship between ERK inhibition and drug-induced toxicity for five ERK inhibitors: SCH772984, ravoxertinib, LY3214996, ulixertinib, and VX-11e, as well as one MEK inhibitor, PD0325901. Comparing cell viability and ERK inhibition revealed different ERK dependencies for these cell lines. We identify several drugs, such as SCH772984 and VX-11e, which induce excessive toxicity not directly related to ERK1/2 inhibition in specific cell lines. We also show that PD0325901, LY3214996, and ulixertinib are prone to ERK1/2 reactivation over time. We distinguished two types of ERK1/2 reactivation: the first could be reversed by adding a fresh dose of inhibitors, while the second persists even after additional treatments. We also showed that cells that became resistant to the MEK1/2 inhibitor PD0325901 due to ERK1/2 reactivation remained sensitive to ERK1/2 inhibitor ulixertinib. Our data indicate that correlation of ERK inhibition with drug-induced toxicity in multiple cell lines may help to find more selective and effective ERK1/2 inhibitors.     

Monocyte chemoattractant protein-1 mediates angiotensin II-induced vascular smooth muscle cell proliferation via SAPK/JNK and ERK1/2

Abnormal vascular smooth muscle cells proliferation is the pathophysiological basis of cardiovascular diseases, such as hypertension, atherosclerosis, and restenosis after angioplasty. Angiotensin II can induce abnormal proliferation of vascular smooth muscle cells, but the molecular mechanisms of this process remain unclear. Here, we explored the role and molecular mechanism of monocyte chemotactic protein-1, which mediated angiotensin II-induced proliferation of rat aortic smooth muscle cells. 1,000 nM angiotensin II could stimulate rat aortic smooth muscle cells' proliferation by angiotensin II type 1 receptor (AT(1)R). Simultaneously, angiotensin II increased monocyte chemotactic protein-1 expression and secretion in a dose-and time-dependent manner through activation of its receptor AT(1)R. Then, monocyte chemotactic protein-1 contributed to angiotensin II-induced cells proliferation by CCR2. Furthermore, we found that intracellular ERK and JNK signaling molecules were implicated in angiotensin II-stimulated monocyte chemotactic protein-1 expression and proliferation mediated by monocyte chemotactic protein-1. These results contribute to a better understanding effect on angiotensin II-induced proliferation of rat smooth muscle cells.