Prevention of TNF-alpha-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-alpha/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by alpha-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-alpha/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-alpha/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-alpha/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-alpha/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-alpha/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-alpha/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-alpha/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.     

Spermidine/spermine N1-acetyltransferase overexpression in kidney epithelial cells disrupts polyamine homeostasis, leads to DNA damage, and causes G2 arrest

Expression of spermidine/spermine N¹-acetyltransferase (SSAT) increases in kidneys subjected to ischemia-reperfusion injury (IRI). Increased expression of SSAT in vitro leads to alterations in cellular polyamine content, depletion of cofactors and precursors of polyamine synthesis, and reduced cell proliferation. In our model system, a >28-fold increase in SSAT levels in HEK-293 cells leads to depletion of polyamines and elevation in the enzymatic activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, suggestive of a compensatory reaction to increased polyamine catabolism. Increased expression of SSAT also led to DNA damage and G₂ arrest. The increased DNA damage was primarily due to the depletion of polyamines. Other factors such as increased production of H₂O₂ due to polyamine oxidase activity may play a secondary role in the induction of DNA lesions. In response to DNA damage the ATM/ATR Chk1/2 DNA repair and cell cycle checkpoint pathways were activated, mediating the G₂ arrest in SSAT-expressing cells. In addition, the activation of ERK1 and ERK2, which play integral roles in the G₂/M transition, is impaired in cells expressing SSAT. These results indicate that the disruption of polyamine homeostasis due to enhanced SSAT activity leads to DNA damage and reduced cell proliferation via activation of DNA repair and cell cycle checkpoint and disruption of Raf MEK ERK pathways. We propose that in kidneys subjected to IRI, one mechanism through which increased expression of SSAT may cause cellular injury and organ damage is through induction of DNA damage and the disruption of cell cycle. ischemia-reperfusion injury; polyamine depletion; cell proliferation; DNA repair; cell cycle arrest     

Oxalate inhibits renal proximal tubule cell proliferation via oxidative stress, p38 MAPK/JNK, and cPLA2 signaling pathways

Exposure of renal proximal tubule cells to oxalate may play an important role in cell proliferation, but the signaling pathways involved in this effect have not been elucidated. Thus the present study was performed to examine the effect of oxalate on ³H-labeled thymidine incorporation and its related signal pathway in primary cultured rabbit renal proximal tubule cells (PTCs). The effects of oxalate on [³H]thymidine incorporation, lactate dehydrogenase (LDH) release, Trypan blue exclusion, H₂O₂ release, activation of mitogen-activated protein kinases (MAPKs), and ³H-labeled arachidonic acid (AA) release were examined in primary cultured renal PTCs. Oxalate inhibited [³H]thymidine incorporation in a time- and dose-dependent manner. However, its analogs did not affect [³H]thymidine incorporation. Oxalate (1 mM) significantly increased H₂O₂ release, which was blocked by N-acetyl-L-cysteine (NAC) and catalase (antioxidants). Oxalate significantly increased p38 MAPK and stress-activated protein kinase (SAPK)/c-Jun NH₂-terminal kinase (JNK) activity, not p44/42 MAPK. Oxalate stimulated [³H]AA release and translocation of cytosolic phospholipase A₂ (cPLA₂) from the cytosolic fraction to the membrane fraction. Indeed, oxalate significantly increased prostaglandin E₂ (PGE₂) production compared with control. Oxalate-induced inhibition of [³H]thymidine incorporation and increase of [³H]AA release were prevented by antioxidants (NAC), a p38 MAPK inhibitor (SB-203580), a SAPK/JNK inhibitor (SP-600125), or PLA₂ inhibitors [mepacrine and arachidonyl trifluoromethyl ketone (AACOCF₃)], but not by a p44/42 MAPK inhibitor (PD-98059). These findings suggest that oxalate inhibits renal PTC proliferation via oxidative stress, p38 MAPK/JNK, and cPLA₂ signaling pathways. kidney; mitogen-activated protein kinase; phospholipase A₂     

Vasopressin-mediated mitogenic signaling in intestinal epithelial cells

The role of G protein-coupled receptorsand their ligands in intestinal epithelial cell signaling andproliferation is poorly understood. Here, we demonstrate that argininevasopressin (AVP) induces multiple intracellular signal transductionpathways in rat intestinal epithelial IEC-18 cells via aV_1A receptor. Addition of AVP to these cells induces arapid and transient increase in cytosolic Ca²⁺concentration and promotes protein kinase D (PKD) activation through aprotein kinase C (PKC)-dependent pathway, as revealed by in vitrokinase assays and immunoblotting with an antibody that recognizesautophosphorylated PKD at Ser⁹¹⁶. AVP also stimulates thetyrosine phosphorylation of the nonreceptor tyrosine kinaseproline-rich tyrosine kinase 2 (Pyk2) and promotes Src family kinasephosphorylation at Tyr⁴¹⁸, indicative of Src activation.AVP induces extracellular signal-related kinase (ERK)-1(p44^mapk) and ERK-2 (p42^mapk) activation, aresponse prevented by treatment with mitogen-activated protein kinasekinase (MEK) inhibitors (PD-98059 and U-0126), specific PKC inhibitors(GF-I and Ro-31-8220), depletion of Ca²⁺ (EGTA andthapsigargin), selective epidermal growth factor receptor (EGFR)tyrosine kinase inhibitors (tyrphostin AG-1478, compound 56), or theselective Src family kinase inhibitor PP-2. Furthermore, AVP acts as apotent growth factor for IEC-18 cells, inducing DNA synthesis and cellproliferation through ERK-, Ca²⁺-, PKC-, EGFR tyrosinekinase-, and Src-dependent pathways.

     

Antimitogenic effect of green tea (-)-epigallocatechin gallate on 3T3-L1 preadipocytes depends on the ERK and Cdk2 pathways

Green tea catechins, especially (–)-epigallocatechin gallate (EGCG), have been proposed as a chemopreventative for obesity, diabetes, cancer, and cardiovascular diseases. However, relatively little is known about the mechanism of the action of EGCG on fat cell function. This study was designed to investigate the pathways of EGCG's modulation of the mitogenesis of 3T3-L1 preadipocytes. Preadipocyte proliferation as indicated by an increased number of cells and greater incorporation of bromodeoxyuridine (BrdU) was inhibited by EGCG in dose-, time-, and growth phase-dependent manners. Also, EGCG dose and time dependently decreased levels of phospho-ERK1/2, Cdk2, and cyclin D₁ proteins, reduced Cdk2 activity, and increased levels of G₀/G₁ growth arrest, p21^waf/cip, and p27^kip1, but not p18^ink, proteins and their associations to Cdk2. However, neither MEK1, ERK1/2, p38 MAPK, phospho-p38, JNK, nor phospho-JNK was changed. Increased phospho-ERK1/2 content and Cdk2 activity, respectively, via the transfection of MEK1 and Cdk2 cDNA into preadipocytes prevented EGCG from reducing cell numbers. These data demonstrate the ERK- and Cdk2-dependent antimitogenic effects of EGCG. Moreover, EGCG was more effective than epicatechin, epicatechin gallate, and epigallocatechin in changing the mitogenic signals. The signal of EGCG in reducing growth of 3T3-L1 preadipocytes differed from that of 3T3 fibroblasts. Results of this study may relate to the mechanism by which EGCG modulates body weight. 3T3-L1 preadipocyte; mitogen-activated protein kinase; cyclin-dependent kinase     

p53 independent G(1) arrest induced by DL-alpha-difluoromethylornithine

Ornithine decarboxylase (ODC), which catalyzes polyamine biosynthesis, plays an essential role in cell growth. DL-alpha-Difluoromethylornithine (DFMO), a synthetic inhibitor of ODC, inhibits cell growth. However, the exact mechanism by which polyamine depletion by DFMO results in growth inhibition remains to be elucidated. We clarified the mechanisms by which DFMO inhibits human gastric cancer cell (MKN45) growth. DFMO induced MKN45 cell G(1) phase arrest after 48 h, and the percentage of G(1) arrest cells continued to increase until 72 h. Expression of p21 and phosphorylation of Stat1 were significantly induced by DFMO at 24 h. Luciferase assay and gel shift assay showed specific binding of Stat1 to the p21 promoter, and promoter activity was activated at 24 h. In dominant negative p53 expressing cells, DFMO significantly induced p21 expression, arrested cells at G(1) phase, and suppressed cell growth effectively. These results suggest that DFMO induced MKN45 cell arrest at G(1) phase in a p53 independent manner, and Stat1 is, at least in part, involved in G(1) arrest.     

Role of K(+) channel expression in polyamine-dependent intestinal epithelial cell migration

Polyamines are essential for cell migrationduring early mucosal restitution after wounding in the gastrointestinaltract. Activity of voltage-gated K⁺ channels (Kv) controlsmembrane potential (E_m) that regulates cytoplasmicfree Ca²⁺ concentration([Ca²⁺]_cyt) by governing thedriving force for Ca²⁺ influx. This study determinedwhether polyamines are required for the stimulation of cell migrationby altering K⁺ channel gene expression,E_m, and[Ca²⁺]_cyt in intestinal epithelialcells (IEC-6). The specific inhibitor of polyamine synthesis,-difluoromethylornithine (DFMO, 5 mM), depleted cellularpolyamines (putrescine, spermidine, and spermine), selectivelyinhibited Kv1.1 channel (a delayed-rectifier Kv channel) expression,and resulted in membrane depolarization. Because IEC-6 cells did notexpress voltage-gated Ca²⁺ channels, the depolarizedE_m in DFMO-treated cells decreased [Ca²⁺]_cyt as a result of reduceddriving force for Ca²⁺ influx through capacitativeCa²⁺ entry. Migration was reduced by 80% in thepolyamine-deficient cells. Exogenous spermidine not only reversed theeffects of DFMO on Kv1.1 channel expression, E_m,and [Ca²⁺]_cyt but also restoredcell migration to normal. Removal of extracellular Ca²⁺ orblockade of Kv channels (by 4-aminopyridine, 1-5 mM) significantly inhibited normal cell migration and prevented the restoration of cellmigration by exogenous spermidine in polyamine-deficient cells. Theseresults suggest that polyamine-dependent intestinal epithelial cellmigration may be due partially to an increase of Kv1.1 channelexpression. The subsequent membrane hyperpolarization raises[Ca²⁺]_cyt by increasing the drivingforce (the electrochemical gradient) for Ca²⁺ influx andthus stimulates cell migration.

     

RhoA stimulates IEC-6 cell proliferation by increasing polyamine-dependent Cdk2 activity

Although RhoA plays an important role in cell proliferation and in Ras transformation in fibroblasts and mammary epithelial cells, its role in intestinal epithelial cells (IEC) is unknown. In a previous study (Ray RM, Zimmerman BJ, McCormack SA, Patel TB, and Johnson LR. Am J Physiol Cell Physiol 276: C684-C691, 1999), we showed that polyamine depletion [dl-alpha-difluoromethylornithine (DFMO) treatment] strongly inhibits the proliferation of IEC. In this report, we examined the effect of RhoA on IEC-6 cell proliferation and whether polyamine depletion inhibits cell proliferation in the presence of constitutively active RhoA. Constitutively active RhoA and vector-transfected IEC-6 cell lines were grown in the presence or absence of DFMO, which causes polyamine depletion by inhibiting ornithine decarboxylase, the first rate-limiting step in polyamine synthesis. Constitutively active RhoA significantly increased the rate of cell proliferation. These cells also lost contact inhibition and formed conspicuous foci when they were fully confluent. Decreased p21Waf1/Cip1 expression and increased cyclin-dependent kinase (Cdk2) mRNA levels and activity accompanied the increased proliferation. The inhibition of p21Waf1/Cip1 was independent of p53. There was no activation of the Ras-Raf-MEK-ERK pathway in the RhoA-transfected cell line. Polyamine depletion totally prevented the effect of activated RhoA on IEC-6 cell proliferation, focus formation, and Cdk2 expression. The stability of mRNA and protein for Cdk2 and p21Waf1/Cip1 in V14-RhoA cells was not significantly different from that of vector-transfected cells. In conclusion, RhoA activation decreased p21Waf1/Cip1 expression and increased basal and serum-induced ornithine decarboxylase activity, Cdk2 expression, Cdk2 protein, and Cdk2 activity, leading to the stimulation of IEC proliferation and transformation. Polyamine depletion totally prevented RhoA's effect on proliferation by decreasing Cdk2 expression and activity.     

Cellular iron depletion stimulates the JNK and p38 MAPK signaling transduction pathways, dissociation of ASK1-thioredoxin, and activation of ASK1

The role of signaling pathways in the regulation of cellular iron metabolism is becoming increasingly recognized. Iron chelation is used for the treatment of iron overload but also as a potential strategy for cancer therapy, because iron depletion results in cell cycle arrest and apoptosis. This study examined potential signaling pathways affected by iron depletion induced by desferrioxamine (DFO) or di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). Both chelators affected multiple molecules in the mitogen-activated protein kinase (MAPK) pathway, including a number of dual specificity phosphatases that directly de-phosphorylate MAPKs. Examination of the phosphorylation of major MAPKs revealed that DFO and Dp44mT markedly increased phosphorylation of stress-activated protein kinases, JNK and p38, without significantly affecting the extracellular signal-regulated kinase (ERK). Redox-inactive DFO-iron complexes did not affect phosphorylation of JNK or p38, whereas the redox-active Dp44mT-iron complex significantly increased the phosphorylation of these kinases similarly to Dp44mT alone. Iron or N-acetylcysteine supplementation reversed Dp44mT-induced up-regulation of phospho-JNK, but only iron was able to reverse the effect of DFO on JNK. Both iron chelators significantly reduced ASK1-thioredoxin complex formation, resulting in the increased phosphorylation of ASK1, which activates the JNK and p38 pathways. Thus, dissociation of ASK1 could serve as an important signal for the phosphorylation of JNK and p38 activation observed after iron chelation. Phosphorylation of JNK and p38 likely play an important role in mediating the cell cycle arrest and apoptosis induced by iron depletion.     

Activation of K(+) channels and increased migration of differentiated intestinal epithelial cells after wounding

Earlymucosal restitution occurs by epithelial cell migration to resealsuperficial wounds after injury. Differentiated intestinal epithelialcells induced by forced expression of the Cdx2 gene migrateover the wounded edge much faster than undifferentiated parental cellsin an in vitro model. This study determined whether thesedifferentiated intestinal epithelial cells exhibit increased migrationby altering voltage-gated K⁺ (Kv) channel expression andcytosolic free Ca²⁺ concentration([Ca²⁺]_cyt). StableCdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 andKv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1cells nor parental IEC-6 cells expressed voltage-dependent Ca²⁺ channels. The increased expression of Kv channels indifferentiated IEC-Cdx2L1 cells was associated with an increase inwhole cell K⁺ currents, membrane hyperpolarization, and arise in [Ca²⁺]_cyt. The migration rates indifferentiated IEC-Cdx2L1 cells were about four times those of parentalIEC-6 cells. Inhibition of Kv channel expression by polyamine depletiondecreased [Ca²⁺]_cyt, reduced myosin stressfibers, and inhibited cell migration. Elevation of[Ca²⁺]_cyt by ionomycin promoted myosin IIstress fiber formation and increased cell migration. These resultssuggest that increased migration of differentiated intestinalepithelial cells is mediated, at least partially, by increasing Kvchannel activity and Ca²⁺ influx during restitution.

     

Caco-2 intestinal cell differentiation is associated with G1 arrest and suppression of CDK2 and CDK4

The cellular mechanisms regulating intestinal proliferation anddifferentiation remain largely undefined. Previously, we showed anearly induction of the cyclin-dependent kinase (CDK) inhibitor p21^Waf1/Cip1 in Caco-2 cells, ahuman colon cancer line that spontaneously differentiates into a smallbowel phenotype. The purpose of our present study was to assess thetiming of cell cycle arrest in relation to differentiation in Caco-2cells and to examine the mechanisms responsible for CDK inactivation.Caco-2 cells undergo a relativeG₁/S block and cease toproliferate at day3 postconfluency; an increase in theactivity of terminally differentiated brush-border enzymes (sucrase andalkaline phosphatase) was noted at day6 postconfluency. Cell cycle block wasassociated with suppression of both CDK2 and CDK4 activities, which areimportant for G₁/S progression.Treatment of the CDK immune complexes with the detergent deoxycholate(DOC) resulted in restoration of CDK2, but not CDK4, activity atday 3 postconfluency, suggesting the presence of inhibitory protein(s)binding to the cyclin/CDK2 complex at this time point. An increasedbinding of p21^Waf1/Cip1 to CDK2complexes at day3 postconfluency was noted, suggesting a potential role for p21^Waf1/Cip1in CDK2 inactivation; however, immunodepletion ofp21^Waf1/Cip1 from Caco-2 proteinextracts demonstrated thatp21^Waf1/Cip1 is only partiallyresponsible for CDK2 suppression atday 3 postconfluency. A decrease in the cyclin E/CDK2 complex appears tocontribute to the CDK2 inactivation noted atdays6 and12 postconfluency. Taken together, ourresults suggest that multiple mechanisms contribute to CDK suppressionduring Caco-2 cell differentiation. Inhibition of CDK2 and CDK4 leadsto G₁ arrest and inhibition ofproliferation that precede Caco-2 cell differentiation.

     

Polyamine depletion partially reduces the radiation-induced cell death via cell cycle delay mediated by thioredoxin

In previous studies, polyamine depletion by DFMO (α-difluoromethylornithine)-treatment reduced H₂O₂-induced apoptotic cell death by reduction of ferric ion uptake. In the present study, we analyzed the reduction of radiation-induced cell death by polyamine depletion. Exposure of HT29 cells to radiation induced severe cell death, but when cells were pretreated with DFMO, a specific inhibitor of polyamine biosynthesis, radiation-induced cell death was reduced to 50–60% of control. Cell cycle analysis showed that, in these cells, the time to reach the G₂/M phase arrest was delayed for 20–24 h compared to the control cells, at which stage the fate of cells exposed to ionizing radiation is determined. DFMO-treated cells also showed a low level of thioredoxin, which is a high-level determinant of the cellular fate. To investigate the relationship between the G₂/M phase arrest and the reduction of thioredoxin caused by polyamine depletion, we also analyzed thioredoxin-antisensed (asTRX) HT29 cells as for DFMO-treated cells. In asTRX-transfected cells, the γ-irradiation-induced G₂/M phase arrest was also significantly delayed and radiation-induced cell death was profoundly reduced, as in the DFMO-treated cells. Both sets of cells showed a decrease of cyclin D1 and an increment of HSP25, which are involved in radiation-induced cell cycle progress. Overall, these results suggest that polyamines are essential for normal cell death of HT29 cells triggered by γ-radiation and that this is partially mediated by the regulation of thioredoxin expression.     

Growth regulatory effects of cyclic AMP and polyamine depletion are dissociable in cultured mouse lymphoma cells

Treatment of mouse lymphoma S49 cells with D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, depleted cellular polyamine levels and stopped cell growth. The cells were arrested predominantly in G1. Thus, polyamine depletion may lead to a regulatory growth arrest in S49 cells. We tested two hypotheses regarding the relationship of growth arrest mediated by polyamine limitation to that mediated by cyclic AMP (cAMP). The hypothesis that cAMP-induced arrest results from polyamine depletion is not tenable, because the arrest could not be reversed by addition of exogenous polyamines, and because cellular polyamine levels do not drop in dibuturyl cyclic AMP (Bt2cAMP)-arrested cells. The hypothesis that polyamine-mediated growth arrest is effected via modulation of cAMP levels or cAMP-dependent protein kinase activity was also shown to be incorrect, because a S49 variant deficient in cAMP-dependent protein kinase was arrested by DFMO. The activities of the polyamine-synthesizing enzymes ornithine decarboxylase (ODC) and S-adenosyl methionine decarboxylase (SAMD) are both reduced in Bt2cAMP-treated cells to about 10% of that in control populations, as shown previously. DFMO diminishes ODC activity and augments SAMD activity in both untreated and Bt2cAMP-treated cells, leading to polyamine depletion in both cases.     

NF-kappaB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells

The maintenance of intestinal mucosal integrity depends on a balance between cell renewal and cell death, including apoptosis. The natural polyamines, putrescine, spermidine, and spermine, are essential for mucosal growth, and decreasing polyamine levels cause G(1) phase growth arrest in intestinal epithelial (IEC-6) cells. The present study was done to determine changes in susceptibility of IEC-6 cells to apoptosis after depletion of cellular polyamines and to further elucidate the role of nuclear factor-kappaB (NF-kappaB) in this process. Although depletion of polyamines by alpha-difluoromethylornithine (DFMO) did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced apoptosis increased significantly as measured by changes in morphological features and internucleosomal DNA fragmentation. In contrast, polyamine depletion by DFMO promoted resistance to apoptotic cell death induced by the combination of tumor necrosis factor-alpha (TNF-alpha) and cycloheximide. Depletion of cellular polyamines also increased the basal level of NF-kappaB proteins, induced NF-kappaB nuclear translocation, and activated the sequence-specific DNA binding activity. Inhibition of NF-kappaB binding activity by sulfasalazine or MG-132 not only prevented the increased susceptibility to STS-induced apoptosis but also blocked the resistance to cell death induced by TNF-alpha in combination with cycloheximide in polyamine-deficient cells. These results indicate that 1) polyamine depletion sensitizes intestinal epithelial cells to STS-induced apoptosis but promotes the resistance to TNF-alpha-induced cell death, 2) polyamine depletion induces NF-kappaB activation, and 3) disruption of NF-kappaB function is associated with altered susceptibility to apoptosis induced by STS or TNF-alpha. These findings suggest that increased NF-kappaB activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the death stimulus.     

Differential role of p38 and c-Jun N-terminal kinase 1 mitogen-activated protein kinases in NK cell cytotoxicity

The serine-threonine mitogen-activated protein kinase (MAPK) family includes extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 kinases. In NK cells, spontaneous or Ab-mediated recognition of target cells leads to activation of an ERK-2 MAPK-dependent biochemical pathway(s) involved in the regulation of NK cell effector functions. Here we assessed the roles of p38 and JNK MAPK in NK cell-mediated cytotoxicity. Our data indicate that p38 is activated in primary human NK cells upon stimulation with immune complexes and interaction with NK-sensitive target cells. FcgammaRIIIA-induced granule exocytosis and both spontaneous and Ab-dependent cytotoxicity were reduced in a dose-dependent manner in cells pretreated with either of two specific inhibitors of this kinase. Target cell-induced IFN-gamma and FcgammaRIIIA-induced TNF-alpha mRNA accumulation was similarly affected under the same conditions. Lack of inhibition of NK cell cytotoxicity in cells overexpressing an inactive form of JNK1 indicates that this kinase, activated only upon FcgammaRIIIA ligation, does not play a significant role in cytotoxicity. These data underscore the involvement of p38, but not JNK1, in the molecular mechanisms regulating NK cell cytotoxicity.     

Involvement of p21(WAF1/Cip1) and p27(Kip1) in intestinal epithelial cell differentiation

Using theconditionally immortalized human cell line tsFHI, we have investigatedthe role of cyclin-dependent kinase inhibitors (CKIs) in intestinalepithelial cell differentiation. Expression of cyclins,cyclin-dependent kinases (Cdk), and CKIs was examined under conditionspromoting growth, growth arrest, or expression of differentiatedtraits. Formation of complexes among cell cycle regulatory proteins andtheir kinase activities were also investigated. The tsFHI cells expressthree CKIs: p16, p21, and p27. With differentiation, p21 and p27 werestrongly induced, but with different kinetics: the p21 increase wasrapid but transient and the p27 increase was delayed but sustained. Ourresults suggest that the function of p16 is primarily to inhibit cyclinD-associated kinases, making tsFHI cells dependent on cyclin E-Cdk2 forpRb phosphorylation and G₁/Sprogression. Furthermore, they indicate that p21 is the main CKIinvolved in irreversible growth arrest during the early stages of celldifferentiation in association with D-type cyclins, cyclin E, and Cdk2,whereas p27 may induce or stabilize expression of differentiated traitsacting independently of cyclin-Cdk function.

     

Cholinergic agonists transactivate EGFR and stimulate MAPK to induce goblet cell secretion

Conjunctival goblet cells are the primarysource of mucins in the mucous layer, the innermost layer of the tearfilm. Conjunctival goblet cell mucin secretion is under neural controlbecause exogenous addition of parasympathetic agonists stimulatesgoblet cell secretion. To elucidate the intracellular signal pathwaysused by cholinergic agonists to stimulate goblet cell mucin secretion,we determined whether p42/p44 mitogen-activated protein kinase (MAPK)is activated during cholinergic agonist-stimulated mucin secretion. Ratconjunctiva was removed, preincubated with or without antagonists, andstimulated with the cholinergic agonist carbachol (10⁴M). Carbachol statistically significantly stimulated thephosphorylation of MAPK in a time- and concentration-dependent manner.U-0126, an inhibitor of MAPK activation, completely inhibited both the activation of MAPK and goblet cell secretion stimulated by carbachol. The M₁ muscarinic antagonist pirenzepine, theM₂ muscarinic antagonist gallamine, and theM₁/M₃ muscarinic receptor antagonistN-(3-chloropropyl)-4-piperidinyl diphenylacetate (4-DAMP)also inhibited carbachol-stimulated MAPK activation. Increasing theintracellular Ca²⁺ concentration with a Ca²⁺ionophore increased MAPK activation, and chelation of extracellular Ca²⁺ inhibited carbachol-stimulated activation. Carbacholalso increased tyrosine phosphorylation of Pyk2, p60Src, and theepidermal growth factor receptor (EGFR). The Src inhibitor PP1 and theEGFR inhibitor AG-1478 completely inhibited carbachol-stimulated MAPKactivation. AG-1478 also inhibited goblet cell secretion. We concludethat carbachol transactivates the EGFR to activate MAPK, leading to conjunctival goblet cell secretion. In addition, carbachol also activates Pyk2 and p60Src that could play a role in the transactivation of the EGFR.

     

Gas1 cooperates with Cdo and promotes myogenic differentiation via activation of p38MAPK

Skeletal myogenesis is a multistep process that involves cell cycle exit, expression of muscle-specific genes and formation of multinucleated myotubes. Growth arrest specific gene 1 (Gas1) is a GPI-linked membrane protein and originally identified as a growth arrest-linked gene in fibroblasts. Promyogenic cell surface protein, Cdo functions as a component of multiprotein complexes that include other cell adhesion molecules, like Cadherins to mediate cell contact signaling. Here we report that Gas1 and Cdo are coexpressed in muscle cells and form a complex in differentiating myoblasts. Interestingly, Cdo^−/− myoblasts display defects in Gas1 induction during differentiation. Overexpression or depletion of Gas1 enhances or decreases myogenic differentiation, respectively. During myoblast differentiation, Gas1 depletion causes defects in downregulation of Cdk2 and Cyclin D1 and up-regulation of miR-322, a negative regulator of Cdk2 activities. Furthermore overexpression or knockdown of Gas1 either enhances or decreases activation of p38MAPK that functions downstream of Cdo. Additionally, Gas1 overexpression in Cdo-depleted C2C12 cells restores p38MAPK activities and differentiation abilities. These data suggest that Gas1 promotes myogenic differentiation through regulation of cell cycle arrest and is critical to activate p38MAPK, most likely via association with Cdo/Cadherin multiprotein complexes.