MAP kinase phosphatase activity sets the threshold for thymocyte positive selection

Phosphorylation of MAP kinases is important for proper translation of T cell antigen receptor (TCR) signals into thymocyte cell fates, but the role of MAP kinase phosphatase (MKP) activity in thymocyte development has not been characterized. To explore the role of MKP in thymocytes, we constructed a double mutant MKP-3 (DM-MKP3) that acts as a dominant-negative inhibitor of ERK- and JNK-specific MKP. Thymocytes developing in the presence of DM-MKP3 have enhanced frequencies of both CD4 and CD8 mature, single-positive cells and no increase in apoptosis. Expression of DM-MKP3 also results in an increased proportion of thymocytes with high levels of both CD69 and TCRbeta, suggesting that the increased proportion of mature thymocytes is the result of an increased probability that CD4(+)CD8(+) cells will be positively selected. Thus, MKP activity controls thymocyte cell fate by regulating the threshold of TCR signaling that is able to induce positive selection.     

Conditional expression of mitogen-activated protein kinase phosphatase-1, MKP-1, is cytoprotective against UV-induced apoptosis

UV irradiation induces apoptosis in U937 human leukemic cells that is accompanied by the activation of both the stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (MAPK) signal transduction pathways. The MAPK phosphatase, MKP-1, is capable of inactivating both SAPK and p38 MAPK in vivo. To determine whether MKP-1-mediated inhibition of SAPK and/or p38 MAPK activity provided cytoprotection against UV-induced apoptosis, a U937 cell line conditionally expressing MKP-1 from the human metallothionein IIa promoter was established. Conditional expression of MKP-1 was found to abolish UV-induced SAPK and p38 MAPK activity, and inhibit UV-induced apoptosis as judged by both morphological criteria and DNA fragmentation. MKP-1 was also found to inhibit other biochemical events associated with apoptosis, including activation of caspase-3 and the proteolytic cleavage of the caspase-3 substrate, poly(ADP ribose) polymerase. These findings demonstrate that MKP-1 acts at a site upstream of caspase activation within the apoptotic program. The cytoprotective properties of MKP-1 do not appear to be mediated by its ability to inhibit p38 MAPK because the p38 MAPK specific inhibitor SB203580 had no effect on UV-induced apoptosis in U937 cells. Furthermore, by titrating the level of MKP-1 expression it was found that MKP-1 inhibited UV-induced SAPK activity, DNA fragmentation, and caspase-3 activation in a similar dose-dependent manner. The dual-specificity phosphatase, PAC1, which does not inhibit UV-induced activation of SAPK, did not provide a similar cytoprotection against UV-induced apoptosis. These results are consistent with a model whereby MKP-1 provides cytoprotection against UV-induced apoptosis by inhibiting UV-induced SAPK activity.     

Down-regulation of the dual-specificity phosphatase MKP-1 suppresses tumorigenicity of pancreatic cancer cells

BACKGROUND & AIMS: In both pancreatic cancer and chronic pancreatitis, there is enhanced expression of mitogenic growth factors and their tyrosine kinase receptors, which have the capacity to activate mitogen-activated protein kinase (MAPK). In view of the important role of MAPK kinase phosphatase (MKP)-1 in the regulation of MAPK activation, the expression and functional role of MKP-1 was analyzed. METHODS: Pancreatic tissues were analyzed by Northern blotting, Western blotting, and immunohistochemistry. Pancreatic cancer cells were transfected with a full-length MKP-1 antisense construct. Growth characteristics and tumorigenicity in vivo and the effects of mitogenic growth factors on cell growth and MAPK activation were determined in transfected and control cells. RESULTS: MKP-1 messenger RNA (mRNA) levels were increased in pancreatic cancer and chronic pancreatitis (CP) tissues. Moderate to strong MKP-1 immunoreactivity was present in the cancer cells, ductal cells of pancreatic intraepithelial neoplasia, and in tubular complexes in CP. Down-regulation of MKP-1 resulted in decreased anchorage-dependent and -independent growth of pancreatic cancer cells, and decreased tumorigenicity in a nude mouse tumor model. MKP-1 down-regulation led to decreased proliferation and sustained MAPK activation in response to mitogens. CONCLUSIONS: Suppression of MKP-1 expression reduces the tumorigenicity of pancreatic cancer cells in vivo, suggesting that MKP-1 contributes to enhanced mitogenic signaling in pancreatic cancer cells.     

The dual-specificity phosphatase MKP-1 limits the cardiac hypertrophic response in vitro and in vivo

Mitogen-activated protein kinase (MAPK) signaling pathways are important regulators of cell growth, proliferation, and stress responsiveness. A family of dual-specificity MAP kinase phosphatases (MKPs) act as critical counteracting factors that directly regulate the magnitude and duration of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) activation. Here we show that constitutive expression of MKP-1 in cultured primary cardiomyocytes using adenovirus-mediated gene transfer blocked the activation of p38, JNK1/2, and ERK1/2 and prevented agonist-induced hypertrophy. Transgenic mice expressing physiological levels of MKP-1 in the heart showed (1) no activation of p38, JNK1/2, or ERK1/2; (2) diminished developmental myocardial growth; and (3) attenuated hypertrophy in response to aortic banding and catecholamine infusion. These results provide further evidence implicating MAPK signaling factors as obligate regulators of cardiac growth and hypertrophy and demonstrate the importance of dual-specificity phosphatases as counterbalancing regulatory factors in the heart.     

Interleukin-1alpha stimulates steroidogenic acute regulatory protein expression via p38 MAP kinase in immature rat Leydig cells

We have investigated the involvement of the steroidogenic acute regulatory (StAR) protein in interleukin-1alpha (IL-1alpha)-induced steroidogenesis in immature (40-day-old) and adult Leydig cells in vitro. Further, IL-1alpha-mediated signaling pathway(s) controlling StAR expression in immature Leydig cells were also studied. IL-1alpha stimulated both androgen production and StAR protein expression in a dose- and time-dependent manner in immature but not adult Leydig cells. These effects of IL-1alpha were prevented by pretreatment of the cells with the specific inhibitors of the p38 MAP kinase, SB203580 and PD169316, suggesting that this kinase is an important part of IL-1alpha signaling in the immature Leydig cell. The present results suggest that IL-1alpha, which is constitutively produced by the rat testis from postnatal day 25, is an important paracrine regulator of postnatal Leydig cell maturation. Regulation of StAR protein expression is one of the possible mechanisms by which IL-1alpha contributes to the differentiation of immature Leydig cells into adult cells.     

Brain death-induced expression of ICAM-1 and VCAM-1 on rat hepatocytes

BACKGROUND/AIMS: Brain death is a significant risk factor for donor organs used for transplantation. Adhesion molecules, ICAM-1 and VCAM-1 are induced in the liver of brain-dead patients, which have important roles in allograft rejection. However, the role of these molecules on hepatocytes has not been investigated. To investigate morphological changes and expression of ICAM-1 and VCAM-1 on hepatocytes, we employed a standardized rat model of brain death. METHODOLOGY: Isolated hepatocytes were examined by histology and immunohistology. RESULTS: The hepatocytes of brain-dead rats were less stained with eosin indicating the decreased permeability and impaired integrity of the cell membranes. Immunohistologically, expression of ICAM-1 was weak on hepatocytes of normal rats, and clearly enhanced and after brain death. Similarly, expression of VCAM-1 was not identified in the normal, but strongly induced after brain death. CONCLUSIONS: These findings indicate the increased immunogenicity of donor hepatocytes after brain death. Immunomodulation before engraftment of cellular grafts to diminish or decrease the immunogenicity may result in better outcome of hepatocytes from brain-dead donor.     

Differential role of I kappa B kinase 1 and 2 in primary rat hepatocytes

Nuclear factor-kappa B (NF-kappa B) protects hepatocytes from undergoing apoptosis during embryonic development and during liver regeneration. Activation of NF-kappa B is mediated through phosphorylation of its inhibitor, I kappa B, by a kinase complex that contains 2 I kappa B kinases. We analyzed the differential role of I kappa B kinase 1 (IKK1) and I kappa B kinase 2 (IKK2) in tumor necrosis factor alpha (TNF-alpha)- and interleukin-1 beta (IL-1 beta)-mediated NF-kappa B activation in primary rat hepatocytes. Maximal induction of IKK activity was observed 5 minutes after TNF-alpha and 15 minutes after IL-1 beta treatment, and activated IKK was able to phosphorylate GST-I kappa B (1-54) and GST-p65 (354-551), but not a GST-p65 (354-551) substrate with a serine-to-alanine substitution at position 536. Infection with an adenovirus containing catalytically inactive IKK2K44M (Ad5IKK2dn) completely blocked both TNF-alpha- and IL-1 beta-induced GST-I kappa B and GST-p65 phosphorylation, I kappa B degradation, and NF-kappa B DNA binding. Adenovirally transduced, catalytically inactive IKK1K44M (Ad5IKK1dn) reduced IKK activity and NF-kappa B DNA binding only slightly. Accordingly, Ad5IKK2dn induced apoptosis in 75% (+/-6%) of hepatocytes after 12 hours of TNF-alpha, which was accompanied by activation of caspases 3 and 8, nuclear fragmentation, and DNA laddering. In contrast, Ad5IKK1dn led to 21% (+/-2%) apoptosis in TNF-alpha-treated hepatocytes after 12 hours and comparatively low activity of caspases 3 and 8. Furthermore, Ad5IKK2dn completely blocked the induction of inducible nitric oxide synthase (iNOS), whereas Ad5IKK1dn had no influence on the expression of iNOS. Thus, IKK2 is the main mediator for cytokine-induced NF-kappa B activation in primary hepatocytes and protects against TNF-alpha-induced apoptosis, whereas IKK1 kinase activity is not required for NF-kappa B activation.     

Structural basis of docking interactions between ERK2 and MAP kinase phosphatase 3

Mitogen-activated protein (MAP) kinases are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. Signaling efficiency and specificity are modulated in large part by docking interactions between individual MAP kinase and the kinase interaction motif (KIM), (R/K)(2-3)-X(1-6)-Phi(A)-X-Phi(B), in its cognate kinases, phosphatases, scaffolding proteins, and substrates. We have determined the crystal structure of extracellular signal-regulated protein kinase 2 bound to the KIM peptide from MAP kinase phosphatase 3, an extracellular signal-regulated protein kinase 2-specific phosphatase. The structure reveals that the KIM docking site, situated in a noncatalytic region opposite of the kinase catalytic pocket, is comprised of a highly acidic patch and a hydrophobic groove, which engage the basic and Phi(A)-X-Phi(B) residues, respectively, in the KIM sequence. The specific docking interactions observed in the structure consolidate all known biochemical data. In addition, structural comparison indicates that the KIM docking site is conserved in all MAP kinases. The results establish a structural model for understanding how MAP kinases interact with their regulators and substrates and provide new insights into how MAP kinase docking specificity can be achieved.     

Glucocorticoids activate TGF-β induced PAI-1 and CTGF expression in rat hepatocytes

Background  

In addition to the activation of hepatic stellate cells TGF-β govern apoptosis and growth control of hepatocytes in liver injury. In non-parenchymal cells, TGF-β induces plasminogen activator inhibitor 1 (PAI-1) and connective tissue growth factor (CTGF) expression, which are involved in extra cellular matrix formation. Both genes were also regulated by glucocorticoids, which in certain cases showed antagonistic effects to the TGF-β-Smad 3 pathway. The purpose of our work was to investigate the influence of TGF-β and dexamethasone on PAI-1 and CTGF expression and secretion in primary hepatocytes.     

Dynamic regulation of pro- and anti-inflammatory cytokines by MAPK phosphatase 1 (MKP-1) in innate immune responses

Engagement of Toll-like receptors (TLRs) on macrophages leads to activation of the mitogen-activated protein kinases (MAPKs), which contribute to innate immune responses. MAPK activity is regulated negatively by MAPK phosphatases (MKPs). MKP-1, the founding member of this family of dual-specificity phosphatases, has been implicated in regulating lipopolysaccharide (LPS) responses, but its role in TLR-mediated immune responses in vivo has not been defined. Here, we show that mice deficient in MKP-1 were highly susceptible to endotoxic shock in vivo, associated with enhanced production of proinflammatory cytokines TNF-alpha and IL-6 and an anti-inflammatory cytokine, IL-10. We further examined the regulation and function of MKP-1 in macrophages, a major cell type involved in endotoxic shock. MKP-1 was transiently induced by TLR stimulation through pathways mediated by both myeloid differentiation factor 88 (MyD88) and TIR domain-containing adaptor inducing IFN-beta (TRIF). MKP-1 deficiency led to sustained activation of p38 MAPK and c-Jun N-terminal kinase (JNK) in LPS-treated macrophages. In response to TLR signals, MKP-1-deficient macrophages produced 5- to 10-fold higher IL-10, which could be blocked by a p38 MAPK inhibitor. Thus, p38 MAPK plays a critical role in mediating IL-10 synthesis in TLR signaling. TNF-alpha was found to be more abundant in MKP-1-deficient macrophages within 2 hours of TLR stimulation, but its production was rapidly down-regulated by IL-10. Our studies demonstrate that MKP-1 attenuates the activities of p38 MAPK and JNK to regulate both pro- and anti-inflammatory cytokines in TLR signaling. These results highlight the complex mechanisms by which the MAPKs regulate innate immunity.     

Nonreceptor tyrosine phosphatase Shp2 promotes adipogenesis through inhibition of p38 MAP kinase

The molecular mechanism underlying adipogenesis and the physiological functions of adipose tissue are not fully understood. We describe here a unique mouse model of severe lipodystrophy. Ablation of Ptpn11/Shp2 in adipocytes, mediated by aP2-Cre, led to premature death, lack of white fat, low blood pressure, compensatory erythrocytosis, and hepatic steatosis in Shp2^fat−/− mice. Fat transplantation partially rescued the lifespan and blood pressure in Shp2^fat−/− mice, and administration of leptin also restored partially the blood pressure of mutant animals with endogenous leptin deficiency. Consistently, homozygous deletion of Shp2 inhibited adipocyte differentiation from embryonic stem (ES) cells. Biochemical analyses suggest a Shp2-TAO2-p38-p300-PPARγ pathway in adipogenesis, in which Shp2 suppresses p38 activation, leading to stabilization of p300 and enhanced PPARγ expression. Inhibition of p38 restored adipocyte differentiation from Shp2^−/− ES cells, and p38 signaling is also suppressed in obese patients and obese animals. These results illustrate an essential role of adipose tissue in mammalian survival and physiology and also suggest a common signaling mechanism involved in adipogenesis and obesity development.     

Alterations in the functional expression of receptors on cirrhotic rat hepatocytes

Reduced hepatic uptake and clearance of macromolecules in liver cirrhosis is due to two major factors: increased diffusional barriers, resulting primarily from the deposition of excessive connective tissue in the space of Disse, and hepatocellular dysfunction, manifested by receptor and/or postreceptor defects. To probe the mechanisms underlying hepatocellular dysfunction in liver cirrhosis, we have investigated receptor-ligand interactions for asialoorosomucoid, insulin and epidermal growth factor in hepatocytes isolated from the livers of rats chronically exposed to phenobarbital and carbon tetrachloride for up to 12 weeks. Viable cells were allowed to attach at 37 degrees C and the high-affinity cell surface binding sites for each ligand were assessed at 4 degrees C in the presence of [125I]-ligand. In parallel incubations, digitonin (0.055%) was added to the binding medium to assess total cellular binding sites. Results demonstrated that chronic treatment of rats with phenobarbital increased hepatocyte asialoorosomucoid surface receptor affinity (p less than 0.05) but had no affect on the number of asialoglycoprotein binding sites. Treatment with CCl4 and phenobarbital significantly reduced the number of surface binding sites for asialoorosomucoid (p less than 0.05) and epidermal growth factor (p less than 0.02), although this treatment had no effect on either the binding affinity or the number of binding sites for insulin. The decrease in cell surface binding sites for asialoorosomucoid and epidermal growth factor was not due to a redistribution of the surface sites to intracellular locations, since the total number of cellular binding sites also was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin regulation of GnRH gene expression through MAP kinase signaling pathways

In mammals, reproduction is acutely regulated by metabolic status. Insulin is an important nutritional signal from the periphery that may regulate the reproductive axis. To determine whether insulin acts directly on the GnRH neuron, we performed studies in mouse-derived GnRH-expressing cell lines. Both insulin receptor protein and mRNA were detected in these cells. A saturation radioligand binding assay revealed high affinity, low capacity binding sites for insulin in GnRH neurons. Insulin also stimulated GnRH promoter activity in GnRH neurons. This effect was blocked by pretreatment with the MEK inhibitor, PD98059, indicating a role for MAP kinase signaling. In transient transfection studies, insulin treatment stimulated expression of a 1250 bp mouse GnRH gene promoter fragment four-fold when compared to promoter activity in untreated cells. In contrast, insulin did not stimulate activity of a 587 bp fragment of the mGnRH gene promoter, indicating that the promoter elements mediating insulin stimulation of the GnRH promoter are located between -1250 and -587 bp. Our studies suggest that insulin may regulate reproductive function by direct effects on the GnRH neurons and specifically by stimulating GnRH gene expression.