Smad proteins are central mediators of the transcriptional effects of transforming growth factor beta (TGF-beta) superfamily that regulate a wide variety of biological processes. Smad7, an inhibitory Smad protein that prevents TGF-beta signaling by interacting with the activated type I TGF-beta receptor, was recently shown to induce sensitization of cells to different forms of cell death. Here we examined the effect of Smad7 on the c-Jun N-terminal kinase (JNK) cascade and investigated the role of this cascade in both the inhibitory and apoptotic functions of Smad7. The transient and stable expression of Smad7 caused a strong and sustained activation of JNK Expression of a dominant-interfering mutant of mitogen-activated protein kinase kinase 4, which completely abolished Smad7-induced activation of JNK, had no effect on Smad7-mediated inhibition of TGF-beta signaling, indicating that the inhibitory function of Smad7 is independent of the JNK cascade. In contrast, expression of the dominant-interfering mutant of mitogen-activated protein kinase kinase 4 impaired the ability of Smad7 to promote cell death. These experiments reveal a novel link between Smad7 and the JNK cascade, which is essential for potentiation of cell death by this inhibitory Smad.

We previously reported that long term treatment with insulin led to sustained inhibition of c-Jun N-terminal kinases (JNKs) in CHO cells overexpressing insulin receptors. Here we investigated the signaling molecules involved in insulin inhibition of JNKs, focusing on phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase phosphatase-1 (MKP-1). In addition, we examined the relevance of JNK inhibition for insulin-mediated proliferation and survival. Insulin inhibition of JNKs was mediated by PI 3-K, as it was blocked by wortmannin and LY294002 and required the de novo synthesis of a phosphatase(s), as it was abolished by orthovanadate and actinomycin D. MKP-1 was a good candidate because 1) insulin stimulation of MKP-1 expression correlated with insulin inhibition of JNKs; 2) insulin stimulation of MKP-1 expression, like insulin inhibition of JNKs, was mediated by PI 3-K; and 3) the transient expression of an antisense MKP-1 RNA reduced the insulin inhibitory effect on JNKs. The overexpression of a dominant negative JNK1 mutant increased insulin stimulation of DNA synthesis and mimicked the protective effect of insulin against serum withdrawal-induced apoptosis. The overexpression of wild-type JNK1 or antisense MKP-1 RNA reduced the proliferative and/or antiapoptotic responses to insulin. Altogether, these results demonstrate that insulin inhibits JNKs through a PI 3-K- and MKP-1-dependent pathway and provide evidence for a key role for JNK inhibition in insulin regulation of proliferation and survival.

We recently showed that the antiapoptotic function of insulin requires nuclear factor kappa B (NF-kappa B) activation (Bertrand, F., Atfi, A, Cadoret, A., L'Allemain, G., Robin, H., Lascols, O., Capeau, J., and Cherqui, G. (1998) J. Biol. Chem. 273, 2931-2938). Here we sought to identify the NF-kappa B-dependent survival genes that are activated by insulin to mediate this function. Insulin increased the expression of tumor necrosis factor receptor-associated factor 2 (TRAF2) mRNA and protein in Chinese hamster ovary cells overexpressing insulin receptors (IRs). This effect required (i) IR activation since it was abrogated by IR mutation at tyrosines 1162 and 1163 and (ii) NF-kappa B activation since it was abolished by overexpression of dominant-negative I kappa B-alpha(A32/36) and mimicked by overexpression of the NF-kappa B c-Rel subunit. TRAF2 contributed to insulin protection against serum withdrawal-induced apoptosis since TRAF2 overexpression mimicked insulin protection, whereas overexpression of dominant-negative TRAF2-(87-501) reduced this process. Along with its protective effect, overexpressed TRAF2 increased basal and insulin-stimulated NF-kappa B activities. All effects were inhibited by I kappa B-alpha(A32/36), suggesting that an amplification loop involving TRAF2 activation of NF-kappa B is implicated in insulin antiapoptotic signaling. We also show that insulin increased manganese-superoxide dismutase (Mn-SOD) mRNA expression through NF-kappa B activation and that Mn-SOD contributed to insulin antiapoptotic signaling since expression of antisense Mn-SOD RNA decreased this process. This study provides the first evidence that insulin activates the NF-kappa B-dependent survival genes encoding TRAF2 and Mn-SOD and thereby clarifies the role of NF-kappa B in the antiapoptotic function of insulin.

We report here that antiinsulin receptor (anti-IR) autoantibodies (AIRs) from a newly diagnosed patient with type B syndrome of insulin resistance induced cellular resistance not only to insulin but also to insulin-like growth factor I (IGF-I) for the stimulation of phosphatidylinositol 3-kinase and mitogen-activated protein kinase activities and of glycogen and DNA syntheses. The molecular mechanisms of this dual resistance were investigated. Patient AIRs bound the IR at the insulin-binding site and caused insulin resistance at the IR level by inducing a 50% decrease in cell surface IRs and a severe defect in the tyrosine kinase activity of the residual IRs, manifested by a loss of insulin-stimulated IR autophosphorylation and IR substrate-1 (IRS-1)/IRS-2 phosphorylation. In contrast, cell resistance to IGF-I occurred at a step distal to IGF-I receptors (IGF-IRs), as AIRs altered neither IGF-I binding nor IGF-I-induced IGF-IR autophosphorylation, but inhibited the ability of IGF-IRs to mediate tyrosine phosphorylation of IRS-1 and IRS-2 in response to IGF-I. Coimmunoprecipitation assays showed that in AIR-treated cells, IRs, but not IGF-IRs, were constitutively associated with IRS-1 and IRS-2, strongly suggesting that AIR-desensitized IRs impeded IGF-I action by sequestering IRS-1 and IRS-2. Accordingly, AIRs had no effect on the stimulation of mitogen-activated protein kinase activity or DNA synthesis by vanadyl sulfate, FCS, epidermal growth factor, or platelet-derived growth factor, all of which activate signaling pathways independent of IRS-1/IRS-2. Thus, AIRs induced cell resistance to both insulin and IGF-I through a novel mechanism involving a constitutive and stable association of IRS-1 and IRS-2 with the IR.

We previously reported that insulin activates nuclear factor kappaB (NF-kappaB) in Chinese hamster ovary (CHO)-R cells overexpressing wild-type insulin receptors (IRs) through a pathway requiring IR tyrosine kinase and Raf-1 kinase activities. We now investigated whether the activation of NF-kappaB by insulin could serve an antiapoptotic function. Insulin (10(-9)-10(-7) M) inhibited apoptosis induced by serum withdrawal in CHO-R cells in a concentration-dependent manner. Insulin antiapoptotic signaling: (i) was dependent on IR number and IR tyrosine kinase activity since it was reduced in parental CHO cells and was abolished in CHO-Y2 cells overexpressing IRs mutated at Tyr1162/1163; (ii) was, like insulin activation of NF-kappaB, dependent on Raf-1 but not on mitogen-activated protein kinase activity since both processes were decreased by the dominant-negative Raf-1 mutant Raf-C4 whereas they persisted in mitogen-activated protein kinase-depleted cells; and (iii) required NF-kappaB activation since it was decreased by proteasome inhibitors and the dominant-negative IkappaB-alpha (A32/36) mutant and was mimicked by overexpression of the NF-kappaB c-Rel subunit. We also show that insulin antiapoptotic signaling but not insulin activation of NF-kappaB involved phosphatidylinositol 3-kinase (PI 3-kinase), as supported by the inhibition of the former but not of the latter process by the PI 3-kinase inhibitor LY294002. Inhibition of both NF-kappaB and PI 3-kinase totally abolished insulin antiapoptotic signaling. Thus insulin exerts a specific antiapoptotic function which is dependent on IR tyrosine kinase activity and is mediated by both a Raf-1-dependent pathway that leads to NF-kappaB activation and a PI 3-kinase-dependent pathway.

In the present study, we compared the ability of insulin to regulate SAPKs/JNKs and ERKs in CHO cells overexpressing human insulin receptors. We show that acute insulin treatment induced a time-dependent increase both in SAPK/JNK and ERK activity but with distinct kinetics. PI-3-kinase inhibition by wortmannin completely blocked insulin activation of SAPKs/JNKs, whereas it partially decreased ERK activation. Prolonged exposure to insulin caused a marked inhibition of SAPK/JNK activity while it induced a sustained activation of ERKs. Insulin inhibition of SAPKs/JNKs was partly due to decreased tyrosine phosphorylation of JNK2. These data indicate that insulin differentially regulates SAPKs/JNKs and ERKs. Moreover, they provide the first evidence that insulin exerts opposite effects on SAPK/JNK activity according to the time of cell treatment.

BACKGROUND/AIMS:In the liver, intrahepatic biliary cells are the sole site of expression of the cystic fibrosis transmembrane conductance regulator, the product of the cystic fibrosis gene. We examined the regulation of cystic fibrosis transmembrane conductance regulator gene expression by protein kinase C in the recently characterized human liver epithelial BC1 cell line which expresses, at early confluence, both biliary (cystic fibrosis transmembrane conductance regulator, cytokeratin 19) and hepatocytic (albumin) specific markers.METHODS:Expression of the cystic fibrosis transmembrane conductance regulator was examined at the mRNA level by Northern blot, reverse transcription-polymerase chain reaction and nuclear run-on assays and at the protein level by Western blotting. The functionality of this protein was tested by measurement of chloride efflux. Protein kinase C isotype expression and cytosol-to-membrane translocation were analysed by Western blotting.RESULTS:1) Phorbol ester down-regulated cystic fibrosis transmembrane conductance regulator mRNA expression in a time- and dose-dependent manner through a post-transcriptional mechanism with concomitant inhibition of stimulated chloride efflux. 2) Phorbol ester also activated protein kinase C as indicated by the cytosol-to-membrane translocation of both protein kinase C alpha and epsilon the two major protein kinase C isotypes expressed by BC1 cells. 3) Further, maximal down-regulation of the cystic fibrosis transmembrane conductance regulator mRNA by the phorbol ester was inhibited by H7 and by GF 109203X, two known protein kinase C inhibitors.CONCLUSIONS:These findings provide the first evidence for phorbol ester-induced down-regulation of cystic fibrosis transmembrane conductance regulator mRNA expression in a human liver epithelial cell line and point to a role for the classical protein kinase C alpha and the novel protein kinase C epsilon in this process.

We examined the effect of insulin on protein kinase C alpha (PKCalpha) expression and the implication of the mitogen-activated protein kinase kinase 1 mitogen-activated protein kinase (MAPK) pathway in this effect. PKCalpha expression was measured by quantitative RT-PCR and Western blotting using Chinese hamster ovary (CHO) cells overexpressing human insulin receptors of the wild type (CHO-R) or insulin receptors mutated at Tyr1162/1163 autophosphorylation sites (CHO-Y2). In CHO-R cells, insulin caused a time- and concentration-dependent increase in PKCalpha messenger RNA, with a maximum at 6 h and 10-(8)M insulin. This increase involved a transcriptional mechanism, as it was not due to stabilization of PKCalpha messenger RNA and was associated with a similar increase in the immunoreactive PKCalpha level. Insulin induction of PKCalpha expression involved the MEK1MAPK pathway, as it was 1) almost completely suppressed by the potent MEK1 inhibitor PD98059, 2) mimicked by the dominant-active MEK1 (S218D/S222D) mutant, and 3) associated with sustained MAPK activation. In CHO-Y2 cells in which the early phase of MAPK activation by insulin was lost and only the late and sustained phase of activation was observed, insulin signaling of PKCalpha expression was preserved and again involved the MEK1-MAPK pathway. Moreover, we show that in both CHO-R and CHO-Y2 cells, insulin stimulation of PKCalpha gene expression was associated with prolonged activation of nuclear p44MAPK. These results indicate that induction of PKCalpha gene expression by insulin is independent of Tyr1162/1163 autophosphorylation sites and correlates with sustained activation of p44MAPK at the nuclear level.

Stimulation of macrophages with endotoxin and/or cytokines is responsible for the expression of the inducible isoform of nitric oxide synthase (iNOS). Because macrophages are exposed to low pH within the microenvironment of inflammatory lesions, the potential role of acidic pH as an additional regulator of iNOS was investigated. Substitution of the culture medium of rat peritoneal macrophages at pH 7.4 with medium at pH 7.0 up-regulated iNOS activity, as reflected by a 2.5-fold increase in nitrite accumulation. The increase in iNOS activity was associated with a similar increase in iNOS mRNA expression that reflected an increase in iNOS mRNA synthesis rather than stability. Low environmental pH-induced iNOS gene transcription involved the activation of nuclear factor-κB (NF-κB) transcription factor since exposure of macrophages to low environmental pH both increased NF-κB binding activity in the nucleus and enhanced NF-κB-driven reporter gene expression. In addition, treatment of macrophages with pyrrolidine dithiocarbamate orn-acetyl-leucinyl-leucinyl-norleucinal, two drugs preventing NF-κB translocation to the nucleus, canceled low pH-induced nitrite accumulation. The overall mechanism required the synthesis of tumor necrosis factor α (TNFα). Indeed, 1) elevated TNFα bioactivity was observed in the medium of macrophages exposed to pH 7.0, and 2) incubation of macrophages with a neutralizing anti-TNFα antibody impaired both NF-κB activation and nitrite accumulation in response to acid challenge. In summary, exposure of macrophages to acidic microenvironment in inflammatory lesions leads to the up-regulation of iNOS activity through the activation of NF-κB.

We previously reported that the enterocytic differentiation of human colonic Caco-2 cells correlated with down-regulation of fibronectin (FN) and laminin (LN), two extracellular matrix components interacting with cell surface integrin receptors. We now investigated whether Caco-2 cell differentiation was associated with alterations in integrin signaling with special interest in the expression and activity of focal adhesion kinase (FAK) and mitogen-activated protein (MAP) kinase. The differentiation of Caco-2 cells was associated with: 1) down-regulation of beta 1 integrin expression at the mRNA and protein levels, 2) increased FAK expression together with decreased FAK autophosphorylation; 3) decreased FAK's ability to associate with PI3-kinase and pp60(c-src); and 4) increased MAP kinase expression along with decreased MAP activity. In addition, we show that FAK and MAP kinase belong to distinct integrin signaling pathways and that both pathways remain functional during Caco-2 cell differentiation since the coating of differentiating cells on FN and LN but not on polylysine increased the tyrosine phosphorylation of FAK and of its endogenous substrate paxillin, and stimulated MAP kinase activity. In conclusion, our results provide evidence that FAK and MAP kinase, two signaling molecules activated independently by beta 1 integrins in Caco-2 cells, undergo alterations of both expression and activity during the enterocytic differentiation of this cell line. J Cell Physiol 177:618-627, 1998. (C) 1998 Wiley-Liss, Inc.

We previously reported that ras and polyoma middle T (PyMT), a constitutive activator of the src proto-oncogene product, up-regulated Caco-2 cell proliferation along with protein kinase C (PKC) alpha expression and PKC activity. We aimed to investigate whether oncogene-induced up-regulation of Caco-2 cell proliferation involved stimulation of the autocrine IGF-II/IGF-I receptor (IGF1R) loop described in these cells and if so, to analyse the role of overexpressed and activated PKC. Compared with control vector transfected Caco-2 cells, ras- and PyMT-transfected cells exhibited increased expression of the 6.0 and 4.8 kb IGF-II transcripts. This was due to increased activity of the P3 and P4 promoters of the IGF-II gene which correlated with increased expression and DNA-binding activity of Sp1, a transcription factor interacting with several specific sites in P3 and P4 promoters. Oncogene-transfected cells displayed enhanced autocrine IGF-II production, which was fully responsible for the oncogene-induced increase in their proliferation since this increase was blunted by anti-human IGF-II and IGF1R (αIR3) antibodies. PKC mediated oncogene activation of the IGF-II gene presumably through action on Sp1 since (i) PKC activation by phorbol 12-myristate 13-acetate increased Sp1 expression, P3 and P4 activity and IGF-II mRNA in control but not in oncogene-transfected cells; and (ii) PKC inhibition by the PKC inhibitor Gö6976 reduced Sp1, P3 and P4 activity and IGF-II mRNA in all three cell lines. This is the first evidence that ras- and PyMT/src oncogenes up-regulate Caco-2 cell proliferation through a PKC-mediated pathway which stimulates IGF-II gene transcription and thereby increases autocrine IGF-II production. The mechanisms underlying IGF-II gene activation by PKC most probably involve action on Sp1.

The protein encoded by ras and src protooncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study, we investigated the effect of oncogenic p21ras and Py-MT/pp60c-src on the synthesis of syndecan-1, a membrane anchored proteoglycan playing a role in cell-matrix interaction and neoplastic growth control. To this end, we used Caco-2 cells transfected with an activated (Val-12) human Ha-ras gene or the polyoma middle T (Py-MT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. As compared to control vector-transfected Caco-2 cells, both oncogene-transfected cells exhibited: (1) a decrease in syndecan-1 specific activity; (2) a decrease in size and sulfation of syndecan-1 ectodomain glycosaminoglycan side chains; and (3) an active heparanase specifically degrading the heparan sulfate chains. In conclusion, the tumorigenic progression induced by oncogenic p21ras or Py-MT/pp60c-src is associated with marked alterations of syndecan-1 at the post-translational level.

Initially described as a unique entity, protein kinase C (PKC) is now represented by a family of 11 isoforms which differ in their structural and biochemical properties as well as in their tissular distribution, subcellular localization and substrate specificity. So far a lot of studies have attempted to approach the role of each of these PKC isoforms in the deregulation of growth signaling that leads to carcinogenesis. Among the various strategies developed, the surexpression of specific isoforms in different cellular models is the strategy which led to major advances as concern the PKC-cancer relationship. This review reports the main results obtained in this field especially in that of colorectal cancer.

The protein encoded by ras and src protooncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study, we investigated the effect of oncogenic p21(ras) and Py-MT/pp60(c-src) on the synthesis of syndecan-1, a membrane anchored proteoglycan playing a role in cell-matrix interaction and neoplastic growth control To this end, we used Caco-2 cells transfected with an activated (Val-12) human Ha-ras gene or the polyoma middle T (Py-MT) oncogene, a constitutive activator of pp60(c-src) tyrosine kinase activity. Re compared to control vector-transfected Caco-2 cells, both oncogene-transfected cells exhibited (1) a decrease in syndecan-1 specific activity; (2) a decrease in size and sulfation of syndecan-1 ectodomain glycosaminoglycan side chains; and (3) an active heparanase specifically degrading the heparan sulfate chains. In conclusion, the tumorigenic progression induced by oncogenic p21(ras) or Py-MT/pp60(c-src) is associated with marked alterations of syndecan-1 at the post-translational level.

The products of ras and src proto-oncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study we attempted to establish whether the tumorigenic progression induced by oncogenic activation of p21ras and pp60c-src in human colonic Caco-2 cells is associated with specific alterations of syndecan-1, a membrane-anchored proteoglycan playing a role in cell-matrix interaction and neoplastic growth control. To this end, we used Caco-2 cells made highly tumorigenic by transfection with an activated (Val 12) human Ha-ras gene or with the polyoma middle T (Py-MT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. Compared with control vector-transfected Caco-2 cells, both oncogene-transfected cell lines (1) contained smaller amounts of membrane-anchored PGs; (2) exhibited decreased syndecan-1 expression at the protein but not the mRNA level; (3) synthesized 35S-labelled syndecan-1 with decreased specific activity; (4) produced a syndecan-1 ectodomain with a lower molecular mass and reduced GAG chain size and sulphation; and (5) expressed heparanase degradative activity. These results show that the dramatic activation of the tumorigenic potential induced by oncogenic p21ras or Py-MT/pp60c-src in Caco-2 cells is associated with marked alterations of syndecan-1 expression at the translational and post-translational levels.

Les effets métaboliques majeurs de l'insuline sur le métabolisme glucidique, lipidique et protéique sont connus depuis longtemps. Plus récemment, un rôle dans la prolifération et la différenciation cellulaire a été affirmé. La connaissance des mécanismes moléculaires et cellulaires par lesquels le message hormonal est transmis a largement progressé ces dernières années. Le récepteur de l'insuline (RI) y joue un rôleclé. Activé par la liaison de l'hormone, il va, à son tour, activer séquentiellement des protéines substrats cytosoliques en les phosphorylant sur des tyrosines. Celles-ci vont amplifier le signal et le faire diverger vers différentes voies intracellulaires conduisant, en particulier, à l'augmentation du transport du glucose, de la synthèse de glycogène et de la lipogenèse, de la synthèse protéique et de la prolifération cellulaire. La complexité des voies de signalisation est renforcée par le fait que les mêmes substrats sont également activés par d'autres récepteurs de facteurs de croissance ou de cytokines indiquant la nécessité de mécanismes permettant l'intégration des signaux et la génération de réponses spécifiques à l'insuline.

We investigated whether the oncogenic activation of p21ras or pp60c-src, which is frequently observed in colorectal cancers, induced alterations of sugar uptake in human colonic cells. We therefore examined hexose transporter expression and/or activity in Caco-2 cells transfected either with an activated human (Val-12) Ha-ras gene or with the polyoma middle T (PyMT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. Experiments were performed at day 20 of culture, when Caco-2 cells express enterocyte-specific GLUT-2, GLUT-5, and SGLT-1 transporters in addition to GLUT-1 and GLUT-3. Along with increased glucose consumption rates, both oncogene-transfected cells exhibited increased levels of GLUT-1 and GLUT-3 mRNAs and/or immunoreactive proteins compared with control vector Caco-2 cells. In contrast, oncogene-transfected cells lost GLUT-2, GLUT-5, and SGLT-1 expression as determined by Northern and/or Western blot analyses and/or specific transport assays. The oncogene-induced repressive effect on these enterocyte-specific hexose transporters extended to brush-border hydrolases and villin but not to tight junctional protein ZO-1. In conclusion, oncogenic p21ras and PyMT/pp60c-src induce severe deregulation of hexose transporter expression in Caco-2 cells, which is manifested by 1) increased GLUT-1 and GLUT-3 expression and 2) repression of GLUT-2, GLUT-5, and SGLT-1, which parallels repression of some markers of the enterocyte-like differentiated phenotype of Caco-2 cells.

We evaluated the role of the activatedRasandSrc/PyMT(Polyoma Middle T) signaling pathways on the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in human colonic Caco-2 cell lines. Control vector-transfected Caco-2 cell monolayer preparations (Caco-2-H) responded to forskolin with an increase in short circuit current (ISC) mediated by CFTR. Furthermore, Caco-2-H cells responded to ATP, a reported stimulator of intracellular Ca2+(Ca2+i), and a potential source of adenosine-mediated elevation of cAMP. In contrast, Caco-2 cells transfected with PyMT (Caco-2-MT), expressing high levels of PKC, showed no sustained ISCresponse to forskolin or ATP. Pretreatment of Caco-2-MT cells with 2.5 μM phorbol 12-myristate 13-acetate (PMA) for 24 hr. effectively down-regulated PKC activity and restored expression of CFTR mRNA but failed to re-establish functional CFTR. These data suggest that, stable up-regulation of PKCα, consequent to activation of theRasorSrc/PyMTpathways, leads to an absence of CFTR expression and Cl−secretion mediated by either cAMP or Ca2+i. Moreover, Cl−secretion in the colonic Caco-2 epithelial cell line is mediated primarily by CFTR and an alternate Ca2+i-activated Cl−channel is not functional in these cells.