MKK3 and -6-dependent activation of p38alpha MAP kinase is required for cytoskeletal changes in pulmonary microvascular endothelial cells induced by ICAM-1 ligation

Previous studies demonstrated that neutrophil adherence induces ICAM-1-dependent cytoskeletal changes in TNF-alpha-treated pulmonary microvascular endothelial cells that are prevented by a pharmacological inhibitor of p38 MAP kinase. This study determined whether neutrophil adherence induces activation of p38 MAP kinase in endothelial cells, the subcellular localization of phosphorylated p38, which MAP kinase kinases lead to p38 activation, which p38 isoform is activated, and what the downstream targets may be. Confocal microscopy showed that neutrophil adhesion for 2 or 6 min induced an increase in phosphorylated p38 in endothelial cells that was punctate and concentrated in the central region of the endothelial cells. Studies using small interfering RNA (siRNA) to inhibit the protein expression of MAP kinase kinase 3 and 6, either singly or in combination, showed that both MAP kinase kinases were required for p38 phosphorylation. Studies using an antisense oligonucleotide to p38alpha demonstrated that inhibition of the protein expression of p38alpha 1) inhibited activation of p38 MAP kinase without affecting the protein expression of p38beta; 2) prevented phosphorylation of heat shock protein 27, an actin binding protein that may induce actin polymerization upon phosphorylation; 3) attenuated cytoskeletal changes; and 4) attenuated neutrophil migration to the EC borders. Thus MAP kinase kinase3- and 6-dependent activation of the alpha-isoform of p38 MAP kinase is required for the cytoskeletal changes induced by neutrophil adherence and influences subsequent neutrophil migration toward endothelial cell junctions.     

Mitogen-activated protein kinase up-regulation and activation during rat parotid gland atrophy and regeneration: role of epidermal growth factor and beta2-adrenergic receptors

Abstract The rat secretory ductal obstruction model has been widely used to assess salivary gland injury, growth, and differentiation. In this study, a novel ductal obstruction and release procedure was used to explore the signaling pathways leading to salivary gland regeneration. Rats underwent bilateral parotid ductal obstruction in which the duct was occluded against a plastic disk subcutaneously and released by external ligature removal. This ductal obstruction/release procedure was validated to produce glandular atrophy and regeneration with histological analysis and periodic acid-Schiff staining. Immunoblot analysis indicated that during ductal obstruction and the early post-release period (day 7), expression of immunoreactive proliferating cell nuclear antigen and vimentin was increased in the parotid glands compared with sham-operated animals. Immunohistochemical staining and immunoblots revealed up-regulation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated receptor kinase (ERK)1/2, and p38 during the atrophic and regeneration phases of ductal obstruction/release. Similarly, increases in activated, i.e., phosphorylated, ERK1/2 (pERK1/2) and p38 (phospho-p38) were demonstrable in both ductal and recovering acinar cells, with pERKs expressed predominantly in the nuclei and phospho-p38 distributed throughout the cells. Furthermore, levels of epidermal growth factor (EGF) receptor and β2-adrenergic receptor (β2-AR) were elevated in the ligated glands and at day 7 post-release; β1-AR levels did not change over the same time period. These results support the view that cell proliferation is involved in duct ligation-induced atrophy of the rat parotid gland and gland recovery upon ligature removal. Up-regulation of ERKs and p38, and the activation of these MAPKs by up-regulated EGF and β2-ARs, may be important signaling components underlying glandular atrophy and subsequent regeneration.     

Platelet-derived growth factor activates p38 mitogen-activated protein kinase through a Ras-dependent pathway that is important for actin reorganization and cell migration.

Members of the mitogen activated protein (MAP) kinase family, extracellular signal-regulated kinase, stress-activated protein kinase-1/c-Jun NH2-terminal kinase, and p38, are central elements that transduce the signal generated by growth factors, cytokines, and stressing agents. It is well known that the platelet-derived growth factor (PDGF) activates extracellular signal-regulated kinase, which leads to cellular mitogenic response. On the other hand, the role of the other MAP kinases in mediating the cellular function of PDGF remains unclear. In the present study, we have investigated the functional role of the other MAP kinases in PDGF-mediated cellular responses. We show that ligand stimulation of PDGF receptors leads to the activation of p38 but not stress-activated protein kinase-1/c-Jun NH2-terminal kinase. Experiments using a specific inhibitor of p38, SB203580, show that the activation of p38 is required for PDGF-induced cell motility responses such as cell migration and actin reorganization but not required for PDGF-stimulated DNA synthesis. Analyses of tyrosine residue-mutated PDGF receptors show that Src homology 2 domain-containing proteins including Src family kinases, phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, the Src homology 2 domain-containing phosphatase SHP-2, phospholipase C-gamma, and Crk do not play a major role in mediating the PDGF-induced activation of p38. Finally, the expression of dominant-negative Ras but not dominant-negative Rac inhibited p38 activation by PDGF, suggesting that Ras is a potent mediator in the p38 activation pathway downstream of PDGF receptors. Taken together, our present study proposes the existence of a Ras-dependent pathway for the activation of p38, which is important for cell motility responses elicited by PDGF stimulation.     

Modulation of PKCdelta tyrosine phosphorylation and activity in salivary and PC-12 cells by Src kinases

Protein kinase C (PKC) delta becomes tyrosine phosphorylated in rat parotid acinar cells exposed to muscarinic and substance P receptor agonists, which initiate fluid secretion in this salivary cell. Here we examine the signaling components of PKCdelta tyrosine phosphorylation and effects of phosphorylation on PKCdelta activity. Carbachol- and substance P-promoted increases in PKCdelta tyrosine phosphorylation were blocked by inhibiting phospholipase C (PLC) but not by blocking intracellular Ca2+ concentration elevation, suggesting that diacylglycerol, rather than D-myo-inositol 1,4,5-trisphosphate production, positively modulated this phosphorylation. Stimuli-dependent increases in PKCdelta activity in parotid and PC-12 cells were blocked in vivo by inhibitors of Src tyrosine kinases. Dephosphorylation of tyrosine residues by PTP1B, a protein tyrosine phosphatase, reduced the enhanced PKCdelta activity. Lipid cofactors modified the tyrosine phosphorylation-dependent PKCdelta activation. Two PKCdelta regulatory sites (Thr-505 and Ser-662) were constitutively phosphorylated in unstimulated parotid cells, and these phosphorylations were not altered by stimuli that increased PKCdelta tyrosine phosphorylation. These results demonstrate that PKCdelta activity is positively modulated by tyrosine phosphorylation in parotid and PC-12 cells and suggest that PLC-dependent effects of secretagogues on salivary cells involve Src-related kinases.     

Redistribution of carbonic anhydrase VI expression from ducts to acini during development of ovine parotid and submandibular glands

 Carbonic anhydrase VI (CA VI) is a secreted enzyme produced predominantly by serous acinar cells of submandibular and parotid glands. We have investigated the developmental pattern of CA VI production by these glands in the sheep, from fetal life to adulthood, using immunohistochemistry. Also, a specific radioimmunoassay for CA VI was used to measure changes in enzyme expression in the parotid gland postnatally. CA VI is detectable by immunohistochemistry in parotid excretory ducts from 106 days gestation (term is 145 days), in striated ducts from 138 days and in acinar cells from 1 day postnatal. The duct cell content of CA VI declined as the acinar cell population increased, a feature also of CA VI immunoreactivity in the submandibular gland. Production of CA VI by submandibular duct cells was detectable initially at 125 days gestation, and acinar production was not seen before 29 days post-natal. Apart from the differing ontogeny of CA VI production in ducts and acini of parotid and submandibular glands, there was a parallel pattern of CA VI expression during the development of these major salivary glands.With the development of the acinar tissues in the postnatal lamb, there was a dramatic increase (about 600-fold) in the level of expression of CA VI in the parotid gland between days 7 and 59 as measured by radioimmunoassay. Accepted: 19 December 1996     

TRAF6 and Src kinase activity regulates Cot activation by IL-1

Cot is one of the MAP kinase kinase kinases that regulates the ERK1/ERK2 pathway under physiological conditions. Cot is activated by LPS, by inducing its dissociation from the inactive p105 NFkappaB-Cot complex in macrophages. Here, we show that IL-1 promotes a 10-fold increase in endogenous Cot activity and that Cot is the only MAP kinase kinase kinase that activates ERK1/ERK2 in response to this cytokine. Moreover, in cells where the expression of Cot is blocked, IL-1 fails to induce an increase in IL-8 and MIP-1betamRNA levels. The activation of Cot-MKK1-ERK1/ERK2 signalling pathway by IL-1 is dependent on the activity of the transducer protein TRAF6. Most important, IL-1-induced ERK1/ERK2 activation is inhibited by PP1, a known inhibitor of Src tyrosine kinases, but this tyrosine kinase activity is not required for IL-1 to activate other MAP kinases such as p38 and JNK. This Src kinases inhibitor does not block the dissociation and subsequently degradation of Cot in response to IL-1, indicating that other events besides Cot dissociation are required to activate Cot. All these data highlight the specific requirements for activation of the Cot-MKK1-ERK1/ERK2 pathway and provide evidence that Cot controls the functions of IL-1 that are mediated by ERK1/ERK2.     

High glucose-mediated effects on endothelial cell proliferation occur via p38 MAP kinase

The mitogen-activated protein (MAP) kinases contribute to altered cell growth and function in a variety of disease states. However, their role in the endothelial complications of diabetes mellitus remains unclear. Human endothelial cells were exposed for 72 h to 5 mM (control) or 25 mM (high) glucose or 5 mM glucose plus 20 mM mannitol (osmotic control). The roles of p38 and p42/44 MAP kinases in the high glucose-induced growth effects were determined by assessment of phosphorylated MAP kinases and their downstream activators by Western blot and by pharmacological inhibition of these MAP kinases. Results were expressed as a percentage (means +/- SE) of control. High glucose increased the activity of total and phosphorylated p38 MAP kinase (P < 0.001) and p42/44 MAP kinase (P < 0.001). Coexposure of p38 MAP kinase blocker with high glucose reversed the antiproliferative but not the hypertrophic effects associated with high-glucose conditions. Transforming growth factor (TGF)-beta1 increased the levels of phosphorylated p38 MAP kinase, and p38 MAP kinase blockade reversed the antiproliferative effects of this cytokine. The high glucose-induced increase in phosphorylated p38 MAP kinase was reversed in the presence of TGF-beta1 neutralizing antibody. Although hyperosmolarity also induced antiproliferation (P < 0.0001) and cell hypertrophy (P < 0.05), there was no change in p38 activity, and therefore inhibition of p38 MAP kinase had no influence on these growth responses. Blockade of p42/44 MAP kinase had no effect on the changes in endothelial cell growth induced by either high glucose or hyperosmolarity. High glucose increased p42/44 and p38 MAP kinase activity in human endothelial cells, but only p38 MAP kinase mediated the antiproliferative growth response through the effects of autocrine TGF-beta1. High glucose-induced endothelial cell hypertrophy was independent of activation of the MAP kinases studied. In addition, these effects were independent of any increase in osmolarity associated with high-glucose exposure.     

Differential activation of mitogen-activated protein kinases in AGS gastric epithelial cells by cag+ and cag- Helicobacter pylori.

The aim of this study was to determine whether Helicobacter pylori activates mitogen-activated protein (MAP) kinases in gastric epithelial cells. Infection of AGS cells with an H. pylori cag+ strain rapidly (5 min) induced a dose-dependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases, as determined by Western blot analysis and in vitro kinase assay. Compared with cag+ strains, cag- clinical isolates were less potent in inducing MAP kinase, particularly JNK and p38, activation. Isogenic inactivation of the picB region of the cag pathogenicity island resulted in a similar loss of JNK and p38 MAP kinase activation. The specific MAP kinase inhibitors, PD98059 (25 microM; MAP kinase kinase (MEK-1) inhibitor) and SB203580 (10 microM; p38 inhibitor), reduced H. pylori-induced IL-8 production in AGS cells by 78 and 82%, respectively (p < 0.01 for each). Both inhibitors together completely blocked IL-8 production (p < 0.001). However, the MAP kinase inhibitors did not prevent H. pylori-induced IkappaBalpha degradation or NF-kappaB activation. Thus, H. pylori rapidly activates ERK, p38, and JNK MAP kinases in gastric epithelial cells; cag+ isolates are more potent than cag- strains in inducing MAP kinase phosphorylation and gene products of the cag pathogenicity island are required for maximal MAP kinase activation. p38 and MEK-1 activity are required for H. pylori-induced IL-8 production, but do not appear to be essential for H. pylori-induced NF-kappaB activation. Since MAP kinases regulate cell proliferation, differentiation, programmed death, stress, and inflammatory responses, activation of gastric epithelial cell MAP kinases by H. pylori cag+ strains may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia.     

Activation of MAP kinases in growth responsive pancreatic cancer cells.

The implication of MAP kinases in the proliferation control of pancreatic cancer cells is still unknown. This study was undertaken to examine the contribution of the p44/p42 and p38 MAP kinases in the mitogenic response to epidermal growth factor (EGF) and bombesin in human pancreatic cancer cells, MIA PaCa-2 and PANC-1. Data indicate that EGF and bombesin stimulated growth of both cell lines. In MIA PaCa-2 cells, EGF and bombesin stimulated the in gel activation of p38 while p44/p42 kinases exhibited high basal activity and no response to stimuli. Growth and p38 activation were inhibited by genistein, wortmannin, PD98059 and SB203580, specific inhibitors of tyrosine kinase, phosphatidylinositol 3-kinase, MEK-1 and p38 kinases, respectively. In PANC-1 cells, EGF and bombesin stimulated p42 in gel activation; p44 remained highly activated and unresponsive to stimuli and p38 did not respond. Stimulated growth and p42 activation were inhibited by genistein, wortmannin and PD98059. Estimation of MAPK activities with a specific anti-active MAP kinase antibody indicated, however, that EGF increased the intensity of the bands corresponding to p42 and p44 MAP kinases in both cell lines, indicating that the mitogenic factor can regulate MAP kinase activity. Data also pointed out that ATP is sufficient to increase MAP kinase activity within the in gel assay technique and may thus explain the discrepancies existing between the in gel assay data and those obtained with the anti-active MAP kinase antibody.     

Protein kinase C delta is essential for etoposide-induced apoptosis in salivary gland acinar cells.

We have previously shown that parotid C5 salivary acinar cells undergo apoptosis in response to etoposide treatment as indicated by alterations in cell morphology, caspase-3 activation, DNA fragmentation, sustained activation of c-Jun N-terminal kinase, and inactivation of extracellular regulated kinases 1 and 2. Here we report that apoptosis results in the caspase-dependent cleavage of protein kinase C-delta (PKCdelta) to a 40-kDa fragment, the appearance of which correlates with a 9-fold increase in PKCdelta activity. To understand the function of activated PKCdelta in apoptosis, we have used the PKCdelta-specific inhibitor, rottlerin. Pretreatment of parotid C5 cells with rottlerin prior to the addition of etoposide blocks the appearance of the apoptotic morphology, the sustained activation of c-Jun N-terminal kinase, and inactivation of extracellular regulated kinases 1 and 2. Inhibition of PKCdelta also partially inhibits caspase-3 activation and DNA fragmentation. Immunoblot analysis shows that the PKCdelta cleavage product does not accumulate in parotid C5 cells treated with rottlerin and etoposide together, suggesting that the catalytic activity of PKCdelta may be required for cleavage. PKCalpha and PKCbeta1 activities also increase during etoposide-induced apoptosis. Inhibition of these two isoforms with G?6976 slightly suppresses the apoptotic morphology, caspase-3 activation, and DNA fragmentation, but has no effect on the sustained activation of c-Jun N-terminal kinase or inactivation of extracellular regulated kinase 1 and 2. These data demonstrate that activation of PKCdelta is an integral and essential part of the apoptotic program in parotid C5 cells and that specific activated isoforms of PKC may have distinct functions in cell death.     

Src family kinase activity is required for Kit-mediated mitogen-activated protein (MAP) kinase activation, however loss of functional retinoblastoma protein makes MAP kinase activation unnecessary for growth of small cell lung cancer cells.

Small cell lung cancer (SCLC) is characterized by multiple genetic alterations that include inactivation of the retinoblastoma protein (Rb), the establishment of several autocrine loops including that induced by coexpression of stem cell factor (SCF) and Kit, and the ectopic expression and activation of Src family kinases. Previous studies have shown that Lck associates with, and becomes activated by, Kit after SCF stimulation of SCLC cells. In the present study, we have demonstrated that PP1, a pharmacological inhibitor of Src kinases, blocked SCF-mediated activation of mitogen-activated protein (MAP) kinase, but it also inhibited Kit activation. However, MAP kinase activation was more sensitive than Kit activation to the effects of PP1. Overexpression of Lck reduced the sensitivity of MAP kinase activation to PP1 without altering the sensitivity of Kit activation, which suggested a role for Lck in SCF-mediated MAP kinase activation. Inducible expression of a dominant negative Lck inhibited MAP kinase activation in a dose-dependent manner, which confirmed that Src family kinase activity is required for SCF-induced MAP kinase activation. The growth of cells that expressed dominant negative Lck was unaffected, however, despite the inhibition of MAP kinase. Growth was also unaffected by the inhibition of the MAP kinase pathway using PD 98059, but sensitivity to the MAP/extracellular signal-regulated kinase kinase inhibitor could be partially restored by expression of wild-type Rb. Therefore, MAP kinase activation seems to be dispensable for the growth of SCLC only in the absence of Rb expression. These data suggest that the SCF/Kit autocrine loop, through activation of Lck and subsequently MAP kinase, and the mutational inactivation of Rb contribute to the loss of G1-S phase checkpoint regulation during the pathogenesis of SCLC. Furthermore, the data demonstrate that, in established SCLC cell lines, proliferative signal transduction initiated by Kit is mediated by pathways other than the classic MAP kinase pathway.     

Characterization of rat parotid and submandibular acinar cell apoptosis in primary culture

Summary Apoptosis is a highly organized cellular process that is critical for maintaining glandular homeostasis. We have used primary rat salivary acinar cells from the parotid and submandibular glands to investigate the critical regulatory events involved in apoptosis. Caspase-3 activity, cleavage of caspase substrates, and deoxyribonucleic acid (DNA) fragmentation were assayed in cells treated with etoposide, a DNA-damaging agent, or brefeldin A (BFA), a Golgi toxin. Dose-response studies showed that the sensitivity of both cell types to etoposide and BFA was similar, with 150 μM etoposide or 1.5 μM BFA inducing maximal caspace activation. However, BFA induced a more robust activation of caspase and DNA fragmentation in both cell types. Similar results were observed when the caspase cleavage of poly(adenosine 5′-diphosphate ribose) polymerase and protein kinase C delta were analyzed by Western blot. Analysis of the kinetics of apoptosis showed that caspace-3 activation was maximal at 8 h of etoposide or BFA treatment in the parotid cells and at 8–18 h in the submandibular cells. A similar time course was observed when DNA fragmentation was assayed, although maximal DNA fragmentation in BFA-treated cells was two-to threefold higher than that observed in etoposide-treated cells. Despite slight kinetic differences, it would appear that the apoptotic cascade is very similar in both primary parotid and submandibular acinar cells. Although limited in their long-term stability in culture, the use of primary, nonimmortalized salivary acinar cultures will also permit the use of specific transgenic animals to further characterize the molecular events involved in the regulation of salivary gland acinar cell apoptosis.     

Coordination of murine parotid secretory protein and salivary amylase expression.

PSP, parotid secretory protein, and salivary amylase are the major secretory proteins of mouse parotid gland where they appear in a constant ratio. Here we describe the isolation of the PSP gene and show through expression analysis on this and the salivary amylase gene that the two genes are transcribed in a coordinate fashion in adult animals, whereas the activation profiles are different during postnatal development. An explanation is put forward that involves activation of the genes at different stages of the acinar cell differentiation, leading in adults to the maximal and thus proportionate expression.