Role of the different mitogen-activated protein kinase subfamilies in the stimulation of dog and human thyroid epithelial cell proliferation by cyclic adenosine 5'-monophosphate and growth factors

We have investigated the role of the different classes of MAPKs, i.e. ERKs, c-Jun N-terminal kinases (JNKs), and p38 MAPK in the proliferation of dog and human thyroid epithelial cells (thyrocytes) in primary cultures. In these cells, TSH, acting through cAMP, epidermal growth factor, hepatocyte growth factor (HGF), and phorbol 12-myristate 13-acetate induce DNA synthesis. With the exception of HGF, all of these factors require the presence of insulin for mitogenic effects to be expressed. We found that TSH and forskolin are without effect on the phosphorylation and activity of the different classes of MAPKs. In contrast, all the cAMP-independent growth factors, whereas without effect on the phosphorylation and activity of JNKs and p38 MAPK, stimulated the ERKs. This effect was strong and sustained in response to HGF, epidermal growth factor and 12-myristate 13-acetate but weak and transient in response to insulin. Moreover, whereas in stimulated cells DNA synthesis was inhibited by PD 098059, an inhibitor of MAPK kinase 1 and consequently of ERKs, it was not modified by SB 203580, an inhibitor of p38 MAPK. Taken together, these data 1) exclude a role of JNKs and p38 MAPK in the proliferation of dog and human thyrocytes; 2) suggest that the mitogenic action of the cAMP-independent agents requires a strong and sustained activation of both ERKs and phosphatidylinositol 3-kinase/protein kinase B as realized by HGF alone or by the other agents together with insulin; and 3) show that TSH and cAMP do not activate ERKs but that the weak activation of ERKs by insulin is nevertheless necessary for DNA synthesis to occur.     

The p38 mitogen-activated protein kinase cascade is not required for the stimulation of insulin secretion from rat islets of Langerhans

The expression of the p38 subfamily of mitogen-activated protein kinases (MAPKs) was examined in rat islets of Langerhans and pancreatic beta-cell lines, and its involvement in the regulation of insulin secretion was investigated. Rat islets and several rodent beta-cell lines were shown to express p38 MAPK by Western blotting. The cellular stress agents sodium arsenite and hyperosmotic sorbitol significantly stimulated p38 MAPK activity, as did the tyrosine phosphatase inhibitor sodium pervanadate and the serine/threonine phosphatase inhibitor okadaic acid. Increases in p38 MAPK activity were not consistently correlated with increases in insulin secretion, and the dissociation between p38 MAPK activity and the regulation of insulin secretion was further demonstrated in studies using the specific p38 MAPK inhibitor SB203580, which was without significant effect on the stimulation of insulin secretion by glucose, 4beta phorbol myristate acetate and forskolin. These studies indicate that although p38 MAPK is expressed in pancreatic beta-cells and can be activated pharmacologically, its activity can be dissociated from the exocytotic release of insulin from rat islets of Langerhans.     

Regulation of FRTL-5 thyroid cell growth by phosphatidylinositol (OH) 3 kinase-dependent Akt-mediated signaling.

Thyrotropin (TSH)-initiated cell cycle progression from G1 to S phase in FRTL-5 thyroid cells requires serum, insulin, or insulin-like growth factor 1 (IGF-1) and involves activation of 3-hydroxy-3-methylglutaryl-CoA reductase, geranylgeranylation of RhoA, p27Kip1 degradation, and activation of cyclin-dependent kinase (cdk) 2. In the present report, we show that the serine-threonine kinase Akt is an important mediator of insulin/IGF-1/serum effects on cell cycle progression in FRTL-5 thyroid cells. The phosphoinositol (OH) 3 kinase inhibitors, Wortmannin (WM) and Ly294002 (LY), block the ability of insulin/IGF-1 to reduce p27 expression, to induce expression of cyclins E, D1, and A as well as cdk 2 and 4, and to phosphorylate retinoblastoma protein. They also inhibit insulin/IGF-1-increased DNA synthesis and cell cycle entrance (S+G2/M). Insulin/IGF-1 rapidly induced activation of Aktl in a PI3 kinase-dependent manner, and increased Aktl RNA levels. Most importantly, FRTL-5 cells transfected with a constitutively active form of Aktl have higher basal rates of DNA synthesis and no longer require exogenous insulin/IGF-1 or serum for TSH-induced growth. In sum, Aktl appears to have an important role in insulin/IGF-1 regulation of FRTL-5 thyroid cell growth and cell cycle progression.     

Activation of p38 mitogen-activated protein kinase mediates thyroid hormone-stimulated osteocalcin synthesis in osteoblasts

It is well known that thyroid hormone modulates osteoblast cell function. We have previously shown that triiodothyronine (T(3)) activates p44/p42 mitogen-activated protein (MAP) kinase, which limits T(3)-induced alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether p44/p42 MAP kinase or p38 MAP kinase is involved in the thyroid hormone-stimulated osteocalcin synthesis in these cells. T(3) markedly induced the phosphorylation of p38 MAP kinase in addition to p44/p42 MAP kinase. PD98059 and U0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, had little effect on the T(3)-induced synthesis of osteocalcin. On the contrary, the T(3)-induced osteocalcin synthesis was significantly reduced by SB203580 and PD169316, inhibitors of p38 MAP kinase. SB203580, PD169316 or PD98059 suppressed the T(3)-phosphorylation of myelin basic protein. T(3)-induced osteocalcin synthesis was significantly reduced by SB203580 or PD169316 also in primary cultured mouse osteoblasts. These results strongly suggest that p38 MAP kinase but not p44/p42 MAP kinase takes part in the thyroid hormone-stimulated osteocalcin synthesis in osteoblasts.     

p38 mitogen-activated protein (MAP) kinase but not p44/p42 MAP kinase is involved in prostaglandin E1-induced vascular endothelial growth factor synthesis in osteoblasts

We investigated the mechanism underlying vascular endothelial growth factor (VEGF) synthesis stimulated by prostaglandin E1 (PGE1) in osteoblast-like MC3T3-E1 cells. PGE1 induced the phosphorylation of both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. SB203580, a specific inhibitor of p38 MAP kinase, inhibited the PGE1-stimulated VEGF synthesis as well as PGE1-induced phosphorylation of p38 MAP kinase. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, which reduced the PGE1-induced phosphorylation of p44/p42 MAP kinase, had little effect on the VEGF synthesis stimulated by PGE1. AH-6809, an antagonist of the subtypes of the PGE receptor, EP1 and EP2, or SC-19220, an antagonist of EP1 receptor, did not inhibit the PGE1-induced VEGF synthesis. H-89, an inhibitor of cAMP-dependent protein kinase, and SQ22536, an inhibitor of adenylate cyclase, reduced the VEGF synthesis induced by PGE1. Cholera toxin, an activator of G(s), and forskolin, an activator of adenylate cyclase, induced VEGF synthesis. SB203580 and PD169316, another specific inhibitor of p38 MAP kinase, reduced the cholera toxin-, forskolin- or 8bromo-cAMP-stimulated VEGF synthesis. However, PD98059 failed to affect the VEGF synthesis stimulated by cholera toxin, forskolin or 8-bromoadenosine-3',5'-cyclic monophosphate (8bromo-cAMP). SB203580 reduced the phosphorylation of p38 MAP kinase induced by forskolin or 8bromo-cAMP. These results strongly suggest that p44/p42 MAP kinase activation is not involved in the PGE1-stimulated VEGF synthesis in osteoblasts but that p38 MAP kinase activation is involved.     

An efficient proteomics method to identify the cellular targets of protein kinase inhibitors

Small molecule inhibitors of protein kinases are widely used in signal transduction research and are emerging as a major class of drugs. Although interpretation of biological results obtained with these reagents critically depends on their selectivity, efficient methods for proteome-wide assessment of kinase inhibitor selectivity have not yet been reported. Here, we address this important issue and describe a method for identifying targets of the widely used p38 kinase inhibitor SB 203580. Immobilization of a suitable SB 203580 analogue and thoroughly optimized biochemical conditions for affinity chromatography permitted the dramatic enrichment and identification of several previously unknown protein kinase targets of SB 203580. In vitro kinase assays showed that cyclin G-associated kinase (GAK) and CK1 were almost as potently inhibited as p38alpha whereas RICK [Rip-like interacting caspase-like apoptosis-regulatory protein (CLARP) kinase/Rip2/CARDIAK] was even more sensitive to inhibition by SB 203580. The cellular kinase activity of RICK, a known signal transducer of inflammatory responses, was already inhibited by submicromolar concentrations of SB 203580 in intact cells. Therefore, our results warrant a reevaluation of the vast amount of data obtained with SB 203580 and might have significant implications on the development of p38 inhibitors as antiinflammatory drugs. Based on the procedures described here, efficient affinity purification techniques can be developed for other protein kinase inhibitors, providing crucial information about their cellular modes of action.     

USF inhibits cell proliferation through delay in G2/M phase in FRTL-5 cells

Upstream stimulatory factor (USF) has a negative effect on the cell proliferation in some cell types. However, its effect on thyrocytes is not clear. Therefore, we investigated the effects of USF on the proliferation and function of thyroid follicular cells. Complementary DNAs of the USF-1 and USF-2 were synthesized using RT-PCR from FRTL-5 cells, and each was transfected to FRTL-5 cells and papillary thyroid carcinoma cell lines. Cyclic AMP (cAMP) production and [methyl-3H] thymidine uptake after thyroid stimulating hormone (TSH) treatment were measured in FRTL-5 cells. In the carcinoma cell lines, 5-bromo-2'-deoxyuridine (BrdU) uptake was assayed to evaluate cell proliferation. Apoptosis was tested by Hoechst staining and cell cycle analysis was done using a fluorescence activated cell sorting. Expression of cell cycle regulating genes was evaluated by Northern and Western blotting. Overexpression of USF-1 and USF-2 significantly suppressed TSH-stimulated [methyl-3H] thymidine uptake (p<0.05), while it maintained TSH-stimulated cAMP production in FRTL-5 cells. Overexpression of USF significantly suppressed BrdU uptake in each carcinoma cell line, NPA and TPC-1 cells (p<0.05). It induced delay of cell cycle at the G2/M phase, but did not increase apoptosis in FRTL-5 cells. It was accompanied by a decrease of cyclin B1 and cyclin-dependent kinase (CDK)-1, and an increase of p27 expression. USF-1 and USF-2 suppressed cell proliferation of normal thyrocytes and thyroid carcinoma cells. However, they retained the ability to produce cAMP after TSH stimulation. Their inhibitory effect on cell proliferation might be caused partly by the delay in G2/M phase.     

Diallyl disulfide-induced G2／M arrest of human gastric cancer MGC803 cells involves activation of p38 MAP kinase pathways

AIM: To determine the role of p38 MAP kinase signal transduction pathways in diallyl disulfide (DADS)-induced G2/M arrest in human gastric cancer MGC803 cells. METHODS: MGC803 cell growth inhibition was measured by MTT assay. Phase distribution of cell cycle was analyzed by flow cytometry. Expression of Cdc25C, p38, phosphorylation of p38 (pp38) were determined by Western blotting. RESULTS: MTT assay showed that SB203580, a specific p38 MAPK inhibitor blocked DADS-induced growth inhibition. Flow cytometry analysis revealed that treatment of MGC803 cells with 30 mg/L DADS increased the percentage of cells in the G2/M phase from 9.3% to 39.4% (P<0.05), whereas inhibition of p38 activity by SB203580 abolished induction of G2/M arrest by DADS. Western blotting showed that phosphorylation of p38 was increased 3.52-fold following treatment of MGC803 cells with 30 mg/L DADS for 20 min (P<0.05), whereas Cdc25C was decreased 68% following treatment of MGC803 cells with 30 mg/L DADS for 24 h (P<0.05). Decreased Cdc25C protein expression by DADS was attenuated by SB203580 (P<0.05). CONCLUSION: DADS-induced G2/M arrest of MGC803 cells involves activation of p38 MAP kinase pathways. Decreased Cdc25C protein expression by p38 MAPK played a crucial role in G2/M arrest after treatment with DADS.     

Novel nonclassical antifolate, 2-[N-(2´-Hydroxyethyl)amino]methyl-3H-quinazolin-4-one, with a potent antineoplastic activity toward leukemia cells

This study was aimed to investigate the therapeutic potential of novel nonclassical antifolate, 2-[N-(2′-Hydroxyethyl)ami-no]methyl-3H-quinazolin-4-one (HEAMQ), toward human promonocytic U937 and murine lymphoblastic L1210 cell lines. The antiproliferative activity of HEAMQ was determined by MTT assay and its effects on cell cycle progression and apoptosis were studied by flow cytometry, and by immunoblots, respectively. In addition, combination chemotherapy of HEAMQ with cisplatin and temozolomide under in vitro and in vivo conditions was tested. HEAMQ showed concentration- and time-dependent cytotoxicity toward U937 and L1210 cells. It induced G2/M arrest that in U937 cells was associated with a marked decrease in the protein expressions of cyclin A, cyclin B, and cyclin-dependent kinase Cdk1. HEAMQ-induced apoptosis was accompanied with up-regulation of the protein expression of Bax and down-regulation of the protein expression of Bcl-2, Mcl-1, XIAP, and survivin, resulting in cytochrome c release and activation of caspases. Inhibitors of JNK (SP600125) and p38 MAPK (SB203580) suppressed HEAMQ-induced apoptosis and G2/M phase arrest, attenuated the activation of Bax, and blocked down-regulation of Bcl-2, XIAP and survivin in HEAMQ-treated U937 cells. In addition, combinations of HEAMQ with cisplatin and temozolomide resulted in synergistic inhibition of cell growth, producing long-term survivors of L1210 tumor-bearing mice. In conclusion, these results indicate that HEAMQ antineoplastic activity toward leukemia cells is associated with cell cycle arrest and apoptosis. The in vivo studies further confirmed the antitumor activity of HEAMQ and highlighted that this agent could be used to further increase therapeutic efficacies of traditional chemotherapeutic agents. Keywords: nonclassical antifolate, quinazolinone, antineoplastic, apoptosis, cell cycle arrest, synergism.     

Tumor necrosis factor-alpha induces insulin resistance in endothelial cells via a p38 mitogen-activated protein kinase-dependent pathway

Chronic inflammation contributes to vascular insulin resistance and endothelial dysfunction. Systemic infusion of TNF-alpha abrogates insulin's action to enhance skeletal muscle microvascular perfusion. In skeletal muscle TNF-alpha induces insulin resistance via the p38 MAPK pathway. To examine whether p38 MAPK also regulates TNF-alpha-induced vascular insulin resistance, bovine aortic endothelial cells (bAECs) were incubated+/-TNF-alpha (5 ng/ml) for 6 h in the presence or absence of SB203580 (p38 MAPK specific inhibitor, 10 microM) after serum starvation for 10 h. For the last 30 min, cells were treated+/-1 nM insulin, and insulin receptor substrate (IRS)-1, Akt, endothelial nitric oxide synthase (eNOS), p38 MAPK, ERK1/2, c-Jun N-terminal kinase, and AMP-activated protein kinase (AMPK) phosphorylation, and eNOS activity were measured. TNF-alpha increased p38 MAPK phosphorylation, potently stimulated IRS-1 serine phosphorylation, and blunted insulin-stimulated IRS-1 tyrosine and Akt phosphorylation and eNOS activity. TNF-alpha also potently stimulated the phosphorylation of ERK1/2 and AMPK. Treatment with SB203580 decreased p38 MAPK phosphorylation back to the baseline and restored insulin sensitivity of IRS-1 tyrosine and Akt phosphorylation and eNOS activity in TNF-alpha-treated bAECs without affecting TNF-alpha-induced ERK1/2 and AMPK phosphorylation. We conclude that in cultured bAECs, TNF-alpha induces insulin resistance in the phosphatidylinositol 3-kinase/Akt/eNOS pathway via a p38 MAPK-dependent mechanism and enhances ERK1/2 and AMPK phosphorylation independent of the p38 MAPK pathway. This differential modulation of TNF-alpha's actions by p38 MAPK suggests that p38 MAPK plays a key role in TNF-alpha-mediated vascular insulin resistance and may contribute to the generalized endothelial dysfunction seen in type 2 diabetes mellitus and the cardiometabolic syndrome.     

Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase mediate macrophage proliferation induced by oxidized low-density lipoprotein

We previously reported that oxidized low-density lipoprotein (Ox-LDL)-induced expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) via PKC, leading to activation of phosphatidylinositol-3 kinase (PI-3K), was important for macrophage proliferation [J Biol Chem 275 (2000) 5810]. The aim of the present study was to elucidate the role of extracellular-signal regulated kinase 1/2 (ERK1/2) and of p38 MAPK in Ox-LDL-induced macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages assessed by [3H]thymidine incorporation and cell counting assays was significantly inhibited by MEK1/2 inhibitors, PD98059 or U0126, and p38 MAPK inhibitors, SB203580 or SB202190, respectively. Ox-LDL-induced GM-CSF production was inhibited by MEK1/2 inhibitors but not by p38 MAPK inhibitors in mRNA and protein levels, whereas recombinant GM-CSF-induced macrophage proliferation was inhibited by p38 MAPK inhibitors but enhanced by MEK1/2 inhibitors. Recombinant GM-CSF-induced PI-3K activation and Akt phosphorylation were significantly inhibited by SB203580 but enhanced by PD98059. Our results suggest that ERK1/2 is involved in Ox-LDL-induced macrophage proliferation in the signaling pathway before GM-CSF production, whereas p38 MAPK is involved after GM-CSF release. Thus, the importance of MAPKs in Ox-LDL-induced macrophage proliferation was confirmed and the control of MAPK cascade could be targeted as a potential treatment of atherosclerosis.     

Reduction of insulin-stimulated glucose uptake in L6 myotubes by the protein kinase inhibitor SB203580 is independent of p38MAPK activity

Insulin increases glucose uptake through translocation of the glucose transporter GLUT4 to the plasma membrane. We previously showed that insulin activates p38MAPK, and inhibitors of p38MAPKalpha and p38MAPKbeta (e.g. SB203580) reduce insulin-stimulated glucose uptake without affecting GLUT4 translocation. This observation suggested that insulin may increase GLUT4 activity via p38alpha and/or p38beta. Here we further explore the possible participation of p38MAPK through a combination of molecular strategies. SB203580 reduced insulin stimulation of glucose uptake in L6 myotubes overexpressing an SB203580-resistant p38alpha (drug-resistant p38alpha) but barely affected phosphorylation of the p38 substrate MAPK-activated protein kinase-2. Expression of dominant-negative p38alpha or p38beta reduced p38MAPK phosphorylation by 70% but had no effect on insulin-stimulated glucose uptake. Gene silencing via isoform-specific small interfering RNAs reduced expression of p38alpha or p38beta by 60-70% without diminishing insulin-stimulated glucose uptake. SB203580 reduced photoaffinity labeling of GLUT4 by bio-LC-ATB-BMPA only in the insulin-stimulated state. Unless low levels of p38MAPK suffice to regulate glucose uptake, these results suggest that the inhibition of insulin-stimulated glucose transport by SB203580 is likely not mediated by p38MAPK. Instead, changes experienced by insulin-stimulated GLUT4 make it susceptible to inhibition by SB203580.     

Involvement of p38MAPK in the regulation of proteolysis by liver cell hydration

BACKGROUND & AIMS: Liver cell hydration is a major determinant of proteolysis control; however, the underlying mechanisms are unknown. METHODS: The role of mitogen-activated protein kinases for proteolysis control was studied in perfused rat liver. RESULTS: Hyposmolarity led to a rapid activation of Erk-2 and p38(MAPK), but not of c-Jun-N-terminal kinase 1. Likewise, isosmotic cell swelling induced by insulin, ethanol, or glutamine/glycine activated p38(MAPK). Inhibition of hyposmotic Erk activation by pertussis or cholera toxin, erbstatin, or genistein had no effect on the swelling-induced inhibition of proteolysis. Likewise, wortmannin, rapamycin, and okadaic acid were ineffective, but proteolysis recovery from hyposmotic inhibition was okadaic acid sensitive. SB203580, an inhibitor of p38(MAPK), abolished both the antiproteolytic effect of hyposmotic cell swelling and the hyposmolarity-induced inhibition of autophagic vacuole formation. Also, the antiproteolytic effect of isotonic cell swelling induced by ethanol, glutamine/glycine, or insulin was abolished by SB203580, but not the swelling potency of these agents. SB203580 had no effect on the cell hydration-independent control of proteolysis exerted by NH4Cl, asparagine, or phenylalanine. CONCLUSIONS: The data suggest an important role of p38(MAPK) in the regulation of autophagic proteolysis by cell volume in liver.     

Angiotensin II stimulates protein synthesis and inhibits proliferation in primary cultures of rat adrenal glomerulosa cells

Angiotensin II (Ang II) is one of the most important stimuli of rat adrenal glomerulosa cells. The aim of the present study was to investigate whether Ang II can stimulate cell proliferation and/or hypertrophy and investigate pathways and intracellular targets. A 3-d treatment with Ang II (5-100 nm), through the Ang II type 1 receptor subtype, abolished cell proliferation observed in control cells but increased protein synthesis. Preincubation with PD98059 (a MAPK kinase inhibitor) abolished basal proliferation and had no effect on basal protein synthesis but did reverse the effect of Ang II on protein synthesis. The p38 MAPK inhibitor SB203580 reversed the inhibitory effect on cell proliferation and abolished the increase in protein synthesis, whereas the c-Jun N-terminal kinase inhibitor SP600125 had no effect. Time-course studies revealed that Ang II stimulated phosphorylation of both p42/p44mapk and p38 MAPK but did not activate c-Jun N-terminal kinase. Ang II had no effect on the level of cyclin E expression but increased the expression of the cyclin-dependent kinase, p27Kip1, an effect abolished in cells preincubated with SB203580 and PD98059. In conclusion, in cultured rat glomerulosa cells, a 3-d treatment with Ang II increases protein synthesis, with a concomitant decrease in proliferation. These effects are mediated by both the p42/p44mapk and p38 MAPK pathways, which increase expression of the steroidogenic enzymes, steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase and p27Kip1, a protein known to block the cell cycle in G1 phase. Together these results support the key role of Ang II as a stimulus of steroid synthesis rather than a proliferating factor.