Notch signaling imposes two distinct blocks in the differentiation of C2C12 myoblasts

Notch signal transduction regulates expression of downstream genes through the activation of the DNA-binding protein Su(H)/CBF1. In Drosophila most of Notch signaling requires Su(H); however, some Notch-dependent processes occur in the absence of Su(H) suggesting that Notch signaling does not always involve activation of this factor. Using constitutively active forms of Notch lacking CBF1-interacting sequences we identified a Notch signaling pathway that inhibits myogenic differentiation of C2C12 myoblasts in the absence of CBF1 activation. Here we show that ligand-induced Notch signaling suppresses myogenesis in C2C12 myoblasts that express a dominant negative form of CBF1, providing additional evidence for CBF1-independent Notch signal transduction. Surprisingly mutant forms of Notch deficient in CBF1 activation are unable to antagonize MyoD activity, despite the fact that they inhibit myogenesis. Moreover, Notch-induced antagonism of MyoD requires CBF1 suggesting that the CBF1-dependent pathway mediates a cell-type-specific block in the myogenic program. However, Notch signaling in the absence of CBF1 activation blocks both myogenesis and osteogenesis, indicative of a general block in cellular differentiation. Taken together our data provide evidence for two distinct Notch signaling pathways that function to block differentiation at separate steps during the process of myogenesis in C2C12 myoblasts.     

Calcineurin enhances MAPK phosphatase-1 expression and p38 MAPK inactivation in cardiac myocytes

Multiple intracellular signaling pathways have been shown to regulate the hypertrophic growth of cardiac myocytes including mitogen-activated protein kinase (MAPK) and calcineurin-nuclear factor of activated T-cells. However, it is uncertain if individual regulatory pathways operate in isolation or if interconnectivity between unrelated pathways is required for the orchestration of the entire hypertrophic response. To this end, we investigated the interconnectivity between calcineurin-mediated cardiac myocyte hypertrophy and p38 MAPK signaling in vitro and in vivo. We show that calcineurin promotes down-regulation of p38 MAPK activity and enhances expression of the dual specificity phosphatase MAPK phosphatase-1 (MKP-1). Transgenic mice expressing activated calcineurin in the heart were characterized by inactivation of p38 and increased MKP-1 expression during early postnatal development, before the onset of cardiac hypertrophy. In vitro, cultured neonatal cardiomyocytes infected with a calcineurin-expressing adenovirus and stimulated with phenylephrine demonstrated reduced p38 phosphorylation and increased MKP-1 protein levels. Activation of endogenous calcineurin with the calcium ionophore decreased p38 phosphorylation and increased MKP-1 protein levels. Inhibition of endogenous calcineurin with cyclosporin A decreased MKP-1 protein levels and increased p38 activation in response to agonist stimulation. To further investigate potential cross-talk between calcineurin and p38 through alteration in MKP-1 expression, the MKP-1 promoter was characterized and determined to be calcineurin-responsive. These data suggest that calcineurin enhances MKP-1 expression in cardiac myocytes, which is associated with p38 inactivation.     

Alterations in subcellular localization of p38 MAPK potentiates endothelin-stimulated COX-2 expression in glomerular mesangial cells

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide with mitogenic actions linked to activation of tyrosine kinase signaling pathways. ET-1 induces cyclooxygenase-2 (COX-2), an enzyme that converts arachidonic acid to pro-inflammatory eicosanoids. Activation of each of the three major mitogen-activated protein kinase (MAPK) pathways, ERK1/2, JNK/SAPK, and p38 MAPK (p38), have been shown to enhance the expression of COX-2. Negative regulation of MAPK may occur via a family of dual specificity phosphatases referred to as mitogen-activated protein kinase phosphatases (MKP). The goal of this work was to test the hypothesis that wild type MKP-1 regulates the expression of ET-1-induced COX-2 expression by inhibiting the activation of p38 in cultured glomerular mesangial cells (GMC). An adenovirus expressing both wild type and a catalytically inactive mutant of MKP-1 (MKP-1/CS) were constructed to study ET-1-regulated MAPK signaling and COX-2 expression in cultured GMC. ET-1 stimulated the phosphorylation of ERK and p38 alpha MAPK and induced the expression of COX-2. Expression of COX-2 was partially blocked by U0126, a MEK inhibitor, and SB 203580, a p38 MAPK inhibitor. Adenoviral expression of MKP-1/CS augmented basal and ET-1-induced phosphorylation of p38 alpha MAPK with less pronounced effects on ERK1/2 phosphorylation. Ectopic expression of wild type MKP-1 blocked the phosphorylation of p38 alpha MAPK by ET-1 but increased the phosphorylation of p38 gamma MAPK. Co-precipitation studies demonstrated association of MKP-1 with p38 alpha MAPK and ERK1/2. Immunofluorescent image analysis demonstrated trapping of phospho-p38 MAPK in the cytoplasm by MKP-1/CS/green fluorescent protein. ET-1-stimulated expression of COX-2 was increased in MKP-1/CS versus LacZ or green fluorescent protein-infected control cells. These results indicate that MKP-1 demonstrates a relative selectivity for p38 alpha MAPK versus p38 gamma MAPK in GMC and is likely to indirectly regulate the expression of COX-2.     

Sphingosine 1-phosphate induces MKP-1 expression via p38 MAPK- and CREB-mediated pathways in airway smooth muscle cells

Sphingosine 1-phosphate (S1P), a bioactive sphingolipid elevated in asthmatic airways, is increasingly recognized as playing an important role in respiratory disease. S1P activates receptor-mediated signaling to modulate diverse cellular functions and promote airway inflammation. Although many of the stimulatory pathways activated by S1P have been delineated, especially mitogen-activated protein kinases (MAPK), the question of whether S1P exerts negative feedback control on its own signaling cascade via upregulation of phosphatases remains unexplored. We show that S1P rapidly and robustly upregulates mRNA and protein expression of the MAPK deactivator-MAPK phosphatase 1 (MKP-1). Utilizing the pivotal airway structural cell, airway smooth muscle (ASM), we confirm that S1P activates all members of the MAPK family and, in part, S1P upregulates MKP-1 expression in a p38 MAPK-dependent manner. MKP-1 is a cAMP response element binding (CREB) protein-responsive gene and here, we reveal for the first time that an adenylate cyclase/PKA/CREB-mediated pathway also contributes to S1P-induced MKP-1. Thus, by increasing MKP-1 expression via parallel p38 MAPK- and CREB-mediated pathways, S1P temporally regulates MAPK signaling pathways by upregulating the negative feedback controller MKP-1. This limits the extent and duration of pro-inflammatory MAPK signaling and represses cytokine secretion in ASM cells. Taken together, our results demonstrate that S1P stimulates both kinases and the phosphatase MKP-1 to control inflammation in ASM cells and may provide a greater understanding of the molecular mechanisms responsible for the pro-asthmatic functions induced by the potent bioactive sphingolipid S1P in the lung.     

The functional analysis of transiently upregulated miR-101 suggests a “braking” regulatory mechanism during myogenesis

Skeletal muscle differentiation is a highly coordinated process that involves many cellular signaling pathways and micro RNAs(mi RNAs). A group of muscle-specific mi RNAs has been reported to promote myogenesis by suppressing key signaling pathways for cell growth. However, the functional role and regulatory mechanism of most non-muscle-specific mi RNAs with stage-specific changes during differentiation are largely unclear. Here, we describe the functional characterization of mi R-101 a/b, a pair of non-muscle-specific mi RNAs that show the largest change among a group of transiently upregulated mi RNAs during myogenesis in C2 C12 cells. The overexpression of mi R-101 a/b inhibits myoblast differentiation by suppressing the p38/MAPK,Interferon Gamma, and Wnt pathways and enhancing the C/EBP pathway. Mef2 a, a key protein in the p38/MAPK pathway, was identified as a direct target of mi R-101 a/b. Interestingly, we found that the long non-coding RNA(lnc RNA) Malat1, which promotes muscle differentiation, interacts with mi R-101 a/b, and this interaction competes with Mef2 a m RNA to relieve the inhibition of the p38/MAPK pathway during myogenesis. These results uncovered a "braking" role in differentiation of transiently upregulated mi RNAs and provided new insights into the competing endogenous RNA(ce RNA) regulatory mechanism in myoblast differentiation and myogenesis.     

High glucose and insulin inhibit VSMC MKP-1 expression by blocking iNOS via p38 MAPK activation

Our laboratory has recently demonstrated arole for the phosphatidylinositol 3-kinase-mediatedinducible NO synthase (iNOS) signaling pathway in acute regulation ofinsulin-induced mitogen-activated protein phosphatase-1 (MKP-1)expression in primary cultures of rat aortic vascular smooth musclecells (VSMCs) (N. Begum, L. Ragolia, M. McCarthy, and N. Duddy.J. Biol. Chem. 273: 25164-25170, 1998). We now show that prolonged treatment of VSMCs with 100 nMinsulin and high glucose (25 mM) for 12-24 h, to mimichyperinsulinemia and hyperglycemia, completely blocked MKP-1 mRNA andprotein expression in response to subsequent acute insulin treatment.To understand the mechanism of insulin resistance induced by highglucose and insulin, we studied the regulation of iNOS proteininduction in these cells. Both high glucose and chronic insulintreatment caused a marked impairment of iNOS induction in response toacute insulin. Blocking of signaling via the p38 mitogen-activatedprotein kinase (MAPK) pathway by prior treatment for 1 h withSB-203580, a synthetic p38 MAPK inhibitor, completely prevented theinhibition of iNOS induced by high glucose and insulin and restoredMKP-1 induction to levels observed with acute insulin treatment. Incontrast, PD-98059, a MEK inhibitor, had no effect. Furthermore, highglucose and chronic insulin treatment caused sustained p38 MAPKactivation. We conclude 1) thatchronic insulin and high glucose-induced insulin resistance isaccompanied by marked reductions in both iNOS and MKP-1 inductions dueto p38 MAPK activation that leads to excessive cell growth and2) that p38 MAPK/extracellularsignal-regulated kinase pathways regulate iNOS induction, therebycontrolling MKP-1 expression, which in turn inactivates MAPKs as afeedback mechanism and inhibits cell growth.

     

Casein kinase 2 regulates the mRNA-destabilizing activity of tristetraprolin

Tristetraprolin (TTP) is an AU-rich element-binding protein that regulates mRNA stability. We previously showed that TTP acts as a negative regulator of VEGF gene expression in colon cancer cells. The p38 MAPK pathway is known to suppress the TTP activity. However, until now the signaling pathway to enhance TTP function is not well known. Here, we show that casein kinase 2 (CK2) enhances the TTP function in the regulation of the VEGF expression in colon cancer cells. CK2 increased TTP protein levels and enhanced VEGF mRNA decaying activity of TTP. TTP was not a direct target of CK2. Instead, CK2 increased the phosphorylation of MKP-1, which led to a decrease in the phosphorylation of p38 MAPK. Inhibition of MKP-1 by siRNA attenuated the increase in TTP function and the decrease of p38 phosphorylation induced by CK2α overexpression. TGF-β1 increased the expressions of CK2 and TTP and the TTP function. The siRNA against CK2α or TTP reversed TGF-β1-induced increases in the expression of CK2 and TTP and the TTP function. Our data suggest that CK2 enhances the protein level and activity of TTP via the modulation of the MKP-1-p38 MAPK signaling pathway and that TGF-β1 enhances the activity of CK2.     

CD40-modulated dual-specificity phosphatases MAPK phosphatase (MKP)-1 and MKP-3 reciprocally regulate Leishmania major infection

The macrophage-expressed CD40 regulates immune responses to Leishmania major infection by reciprocal signaling through p38 MAPK and ERK1/2. CD40-induced IL-10 or IL-12 plays crucial roles in the promotion or protection from L. major infection, respectively. Because p38 MAPK and ERK1/2 are dephosphorylated by dual-specificity MAPK phosphatases (MKPs), we tested the role of CD40 in the regulation of MKPs in L. major infection. MKP-1 expression and activity increased whereas MKP-3 expression and activity decreased in virulent L. major-infected macrophages. CD40 differentially regulated the expression and activity of MKP-1 and MKP-3, which, in turn, reciprocally regulated CD40-induced p38 MAPK and ERK1/2 phosphorylation and effector functions in macrophages. Triptolide, an inhibitor of MKP-1 expression, and lentivirally expressed MKP-1 short hairpin RNA enhanced CD40-induced anti-leishmanial functions and significantly protected susceptible BALB/c mice from L. major infection. Similarly, lentivirally overexpressed MKP-3 significantly reduced disease progression and parasite burden in susceptible BALB/c mice. Thus, to our knowledge, our data show for the first time that CD40 reciprocally regulates MKP-1 and MKP-3 expression and activity while the MKPs contribute to the reciprocal CD40 signaling-regulated anti-leishmanial functions. The findings reveal a novel parasite-devised immune evasion strategy and an effective target to redirect CD40-regulated immune responses.     

Hypotonic stress upregulates β- and γ-ENaC expression through suppression of ERK by inducing MKP-1

We investigated a physiological role for ERK, a member of the MAPK family, in the hypotonic stimulation of epithelial Na(+) channel (ENaC)-mediated Na(+) reabsorption in renal epithelial A6 cells. We show that hypotonic stress causes a major dephosphorylation of ERK following a rapid transient phosphorylation. PD98059 (a MEK inhibitor) increases dephosphorylated ERK and enhances the hypotonic-stress-stimulated Na(+) reabsorption. ERK dephosphorylation is mediated by MAPK phosphatase (MKP). Hypotonic stress activates p38, which in turn induces MKP-1 and to a lesser extent MKP-3 mRNA expression. Inhibition of p38 suppresses MKP-1 induction, preventing hypotonic stress from dephosphorylating ERK. Inhibition of MKP-1 and -3 by the inhibitor NSC95397 also suppresses the hypotonicity-induced dephosphorylation of ERK. NSC95397 reduces both β- and γ-ENaC mRNA expression and ENaC-mediated Na(+) reabsorption stimulated by hypotonic stress. In contrast, pretreatment with PD98059 significantly enhances mRNA and protein expression of β- and γ-ENaC even under isotonic conditions. However, PD98059 only stimulates Na(+) reabsorption in response to hypotonic stress, suggesting that ERK inactivation by itself (i.e., under isotonic conditions) is not sufficient to stimulate Na(+) reabsorption, even though ERK inactivation enhances β- and γ-ENaC expression. Based on these results, we conclude that hypotonic stress stimulates Na(+) reabsorption through at least two signaling pathways: 1) induction of MKP-1 that suppresses ERK activity and induces β- and γ-ENaC expression, and 2) promotion of translocation of the newly synthesized ENaC to the apical membrane.     

Down-regulation of cytokine-induced interleukin-8 requires inhibition of p38 mitogen-activated protein kinase (MAPK) via MAPK phosphatase 1-dependent and -independent mechanisms

Down-regulation of overabundant interleukin (IL)-8 present in cystic fibrosis (CF) airways could ease excessive neutrophil burden and its deleterious consequences for the lung. IL-8 production in airway epithelial cells, stimulated with e.g. inflammatory cytokines IL-1β and tumor necrosis factor (TNF)-α, is regulated by several signaling pathways including nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK). We previously demonstrated that the anti-inflammatory drugs dexamethasone and ibuprofen suppress NF-κB; however, only dexamethasone down-regulates cytokine-induced IL-8, highlighting the importance of non-NF-κB mechanisms. Here, we tested the hypothesis that down-regulation of cytokine-induced IL-8 requires modulation of the MAPK phosphatase (MKP)-1/p38 MAPK/mRNA stability pathway. The effects of dexamethasone (5 nm) and ibuprofen (480 μm) on this pathway and IL-8 were studied in CF (CFTE29o-, CFBE41o-) and non-CF (1HAEo-) airway epithelial cells. We observed that dexamethasone, but not ibuprofen, destabilizes IL-8 mRNA and up-regulates MKP-1 mRNA. Further, siRNA silencing of MKP-1, via p38 MAPK, leads to IL-8 overproduction and diminishes the anti-IL-8 potential of dexamethasone. However, MKP-1 overexpression does not significantly alter IL-8 production. By contrast, direct inhibition of p38 MAPK (inhibitor SB203580) efficiently suppresses IL-8 with potency comparable with dexamethasone. Similar to dexamethasone, SB203580 decreases IL-8 mRNA stability. Dexamethasone does not affect p38 MAPK activation, which excludes its effects upstream of p38 MAPK. In conclusion, normal levels of MKP-1 are necessary for a full anti-IL-8 potential of pharmacological agents; however, efficient pharmacological down-regulation of cytokine-induced IL-8 also requires direct effects on p38 MAPK and mRNA stability independently of MKP-1.     

The glycosylated IgII Extracellular domain of EMMPRIN is implicated in the induction of MMP-2

Insulin-like growth factor (IGF)-I and IGF-II play major roles in the regulation of skeletal muscle growth and differentiation, and both are locally expressed in muscle cells. Recent studies have demonstrated that IGF-II up-regulates its own gene expression during myogenesis and this auto-regulatory loop is critical for muscle differentiation. How local IGF-I is regulated in this process is unclear. Here, we report that while IGF-II up-regulated its own gene expression, it suppressed IGF-I gene expression during myogenesis. These opposite effects of IGF-II on IGF-I and IGF-II genes expression were time dependent and dose dependent. It has been shown that IGFs activate the PI3K-Akt-mTOR, p38 MAPK, and Erk1/2 MAPK pathways. In myoblasts, we examined their role(s) in mediating the opposite effects of IGF-II. Our results showed that both the PI3K-Akt-mTOR and p38 MAPK pathways played critical roles in increasing IGF-II mRNA expression. In contrast, mTOR was required for down-regulating the IGF-I gene expression by IGF-II. In addition, Akt, Erk1/2 MAPK, and p38 MAPK pathways were also involved in the regulation of basal levels of IGF-I and IGF-II genes during myogenesis. These findings reveal a previously unrecognized negative feedback mechanism and extend our knowledge of IGF-I and IGF-II gene expression and regulation during myogenesis.     

p38 MAPK-induced nuclear factor-kappaB activity is required for skeletal muscle differentiation: role of interleukin-6

p38 MAPK and nuclear factor-kappaB (NF-kappaB) signaling pathways have been implicated in the control of skeletal myogenesis. However, although p38 is recognized as a potent activator of myoblast differentiation, the role of NF-kappaB remains controversial. Here, we show that p38 is activated only in differentiating myocytes, whereas NF-kappaB activity is present both in proliferation and differentiation stages. NF-kappaB activation was found to be dependent on p38 activity during differentiation, being NF-kappaB an effector of p38, thus providing a novel mechanism for the promyogenic effect of p38. Activation of p38 in C2C12 cells induced the activity of NF-kappaB, in a dual way: first, by reducing IkappaBalpha levels and inducing NF-kappaB-DNA binding activity and, second, by potentiating the transactivating activity of p65-NF-kappaB. Finally, we show that interleukin (IL)-6 expression is induced in C2C12 differentiating myoblasts, in a p38- and NF-kappaB-dependent manner. Interference of IL-6 mRNA reduced, whereas its overexpression increased, the extent of myogenic differentiation; moreover, addition of IL-6 was able to rescue significantly the negative effect of NF-kappaB inhibition on this process. This study provides the first evidence of a crosstalk between p38 MAPK and NF-kappaB signaling pathways during myogenesis, with IL-6 being one of the effectors of this promyogenic mechanism.     

Resveratrol inhibits EMMPRIN expression via P38 and ERK1/2 pathways in PMA-induced THP-1 cells

The extracellular matrix metalloproteinase inducer (EMMPRIN) is significant in the regulation of matrix metalloproteinase (MMP) synthesis in atherosclerosis-related cells, and is possibly involved in the progression of atherosclerotic plaque. EMMPRIN expression is also up-regulated in PMA-induced THP-1 cells and is inhibited by resveratrol. However, it remains unclear how resveratrol inhibits EMMPRIN expression. We thus investigated the role of the MAPK signaling pathway in resveratrol inhibiting the up-regulation of EMMPRIN in PMA-induced THP-1 cells. We found that the ERK1/2 and p38 pathways, but not the JNK, are activated during the up-regulation of EMMPRIN expression. We also observed that while resveratrol suppresses the up-regulation of EMMPRIN, it also suppresses both the ERK1/2 and p38 pathways in a dose-dependent manner. Taken together, we established that it is through both the ERK1/2 and p38 MAPK pathways that resveratrol inhibits the expression of EMMPRIN in PMA-induced THP-1 cells.     

N-cadherin-dependent cell-cell contact regulates Rho GTPases and beta-catenin localization in mouse C2C12 myoblasts

N-cadherin, a member of the Ca(2+)-dependent cell-cell adhesion molecule family, plays an essential role in skeletal muscle cell differentiation. We show that inhibition of N-cadherin-dependent adhesion impairs the upregulation of the two cyclin-dependent kinase inhibitors p21 and p27, the expression of the muscle-specific genes myogenin and troponin T, and C2C12 myoblast fusion. To determine the nature of N-cadherin-mediated signals involved in myogenesis, we investigated whether N-cadherin-dependent adhesion regulates the activity of Rac1, Cdc42Hs, and RhoA. N-cadherin-dependent adhesion decreases Rac1 and Cdc42Hs activity, and as a consequence, c-jun NH2-terminal kinase (JNK) MAPK activity but not that of the p38 MAPK pathway. On the other hand, N-cadherin-mediated adhesion increases RhoA activity and activates three skeletal muscle-specific promoters. Furthermore, RhoA activity is required for beta-catenin accumulation at cell-cell contact sites. We propose that cell-cell contacts formed via N-cadherin trigger signaling events that promote the commitment to myogenesis through the positive regulation of RhoA and negative regulation of Rac1, Cdc42Hs, and JNK activities.     

KIF5B transports BNIP-2 to regulate p38 mitogen-activated protein kinase activation and myoblast differentiation

The Cdo-p38MAPK (p38 mitogen-activated protein kinase) signaling pathway plays important roles in regulating skeletal myogenesis. During myogenic differentiation, the cell surface receptor Cdo bridges scaffold proteins BNIP-2 and JLP and activates p38MAPK, but the spatial-temporal regulation of this process is largely unknown. We here report that KIF5B, the heavy chain of kinesin-1 motor, is a novel interacting partner of BNIP-2. Coimmunoprecipitation and far-Western study revealed that BNIP-2 directly interacted with the motor and tail domains of KIF5B via its BCH domain. By using a range of organelle markers and live microscopy, we determined the endosomal localization of BNIP-2 and revealed the microtubule-dependent anterograde transport of BNIP-2 in C2C12 cells. The anterograde transport of BNIP-2 was disrupted by a dominant-negative mutant of KIF5B. In addition, knockdown of KIF5B causes aberrant aggregation of BNIP-2, confirming that KIF5B is critical for the anterograde transport of BNIP-2 in cells. Gain- and loss-of-function experiments further showed that KIF5B modulates p38MAPK activity and in turn promotes myogenic differentiation. Of importance, the KIF5B-dependent anterograde transport of BNIP-2 is critical for its promyogenic effects. Our data reveal a novel role of KIF5B in the spatial regulation of Cdo–BNIP-2–p38MAPK signaling and disclose a previously unappreciated linkage between the intracellular transporting system and myogenesis regulation.     

Akt2, a novel functional link between p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in myogenesis

Activation of either the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt or the p38 mitogen-activated protein kinase (MAPK) signaling pathways accelerates myogenesis but only when the reciprocal pathway is functional. We therefore examined the hypothesis that cross-activation between these signaling cascades occurs to orchestrate myogenesis. We reveal a novel and reciprocal cross-talk and activation between the PI 3-kinase/Akt and p38 MAPK pathways that is essential for efficient myoblast differentiation. During myoblast differentiation, Akt kinase activity correlated with S473 but not T308 phosphorylation and occurred 24 h after p38 activation. Inhibition or activation of p38 with SB203580, dominant-negative p38, or MKK6EE regulated Akt kinase activity. Analysis of Akt isoforms revealed a specific increase in Akt2 protein levels that coincided with AktS473 phosphorylation during myogenesis and an enrichment of S473-phosphorylated Akt2. Akt2 promoter activity and protein levels were regulated by p38 activation, thus providing a mechanism for communication. Subsequent Akt activation by S473 phosphorylation was PI 3-kinase dependent and specific for Akt2 rather than Akt1. Complementary to p38-mediated transactivation of Akt, activation or inhibition of PI 3-kinase regulated p38 activity upstream of MKK6, demonstrating reciprocal communication and positive feedback characteristic of myogenic regulation. Our findings have identified novel communication between p38 MAPK and PI 3-kinase/Akt via Akt2.     

Mitogen-activated protein kinase phosphatase-1 (MKP-1) preferentially dephosphorylates p42/44MAPK but not p38MAPK in rat pinealocytes

We recently reported a diurnal and norepinephrine (NE) -induced expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the rat pineal gland and postulated that this MKP-1 expression might impact adrenergic-regulated arylalkylamine- N -acetyltransferase (AA-NAT) activity via modulation of MAPKs. In this study, we investigated the effect of depletion of MKP-1 expression by using doxorubicin, a topoisomerase inhibitor that suppresses the expression of MKP-1 in other cell types and small interfering RNA targeted against Mkp1 in NE-stimulated pinealocytes. We found that both treatments were effective in inhibiting NE induction of MKP-1 expression. Moreover, both treatments also resulted in a prolonged activation of p42/44MAPK and an increase in AA-NAT induction by NE. In contrast, treatment of pinealocytes with PD98059, an inhibitor of MAPK kinase, reduced NE-stimulated AA-NAT activity. Interestingly, suppressing MKP-1 expression had no effect on the time profile of NE-stimulated p38MAPK activation. These results indicate that MKP-1 modulates the profile of AA-NAT activity by selectively shaping the activation profile of p42/44MAPK but not that of p38MAPK.