Activation of Cdk2 stimulates proteasome-dependent truncation of tyrosine phosphatase SHP-1 in human proliferating intestinal epithelial cells

SHP-1 is expressed in the nuclei of intestinal epithelial cells (IECs). Increased SHP-1 expression and phosphatase activity coincide with cell cycle arrest and differentiation in these cells. Suspecting the tumor-suppressive properties of SHP-1, a yeast two-hybrid screen of an IEC cDNA library was conducted using the full-length SHP-1 as bait. Characterization of many positive clones revealed sequences identical to a segment of the Cdk2 cDNA sequence. Interaction between SHP-1 and Cdk2 was confirmed by co-immunoprecipitations whereby co-precipitated Cdk2 phosphorylated SHP-1 protein. Inhibition of Cdk2 (roscovitine) or proteasome (MG132) was associated with an enhanced nuclear punctuate distribution of SHP-1. Double labeling localization studies with signature proteins of subnuclear domains revealed a co-localization between the splicing factor SC35 and SHP-1 in bright nucleoplasmic foci. Using Western blot analyses with the anti-SHP-1 antibody recognizing the C terminus, a lower molecular mass species of 45 kDa was observed in addition to the full-length 64-65-kDa SHP-1 protein. Treatment with MG132 led to an increase in expression of the full-length SHP-1 protein while concomitantly leading to a decrease in the levels of the lower mass 45-kDa molecular species. Further Western blots revealed that the 45-kDa protein corresponds to the C-terminal portion of SHP-1 generated from proteasome activity. Mutational analysis of Tyr(208) and Ser(591) (a Cdk2 phosphorylation site) residues on SHP-1 abolished the expression of the amino-truncated 45-kDa SHP-1 protein. In conclusion, our results indicate that Cdk2-associated complexes, by targeting SHP-1 for proteolysis, counteract the ability of SHP-1 to block cell cycle progression of IECs.     

Activation of p53 stimulates proteasome-dependent truncation of eIF4E-binding protein 1 (4E-BP1)

Background information. The translational inhibitor protein 4E‐BP1 [eIF4E (eukaryotic initiation factor 4E)‐binding protein 1] regulates the availability of polypeptide chain initiation factor eIF4E for protein synthesis. Initiation factor eIF4E binds the 5′ cap structure present on all cellular mRNAs. Its ability to associate with initiation factors eIF4G and eIF4A, forming the eIF4F complex, brings the mRNA to the 43S complex during the initiation of translation. Binding of eIF4E to eIF4G is inhibited in a competitive manner by 4E‐BP1. Phosphorylation of 4E‐BP1 decreases the affinity of this protein for eIF4E, thus favouring the binding of eIF4G and enhancing translation. We have previously shown that induction or activation of the tumour suppressor protein p53 rapidly leads to 4E‐BP1 dephosphorylation, resulting in sequestration of eIF4E, decreased formation of the eIF4F complex and inhibition of protein synthesis. Results. We now report that activation of p53 also results in modification of 4E‐BP1 to a truncated form. Unlike full‐length 4E‐BP1, which is reversibly phosphorylated at multiple sites, the truncated protein is almost completely unphosphorylated. Moreover, the latter interacts with eIF4E in preference to full‐length 4E‐BP1. Inhibitor studies indicate that the p53‐induced cleavage of 4E‐BP1 is mediated by the proteasome and is blocked by conditions that inhibit the dephosphorylation of full‐length 4E‐BP1. Measurements of the turnover of 4E‐BP1 indicate that the truncated form is much more stable than the full‐length protein. Conclusions. The results suggest a model in which proteasome activity gives rise to a stable, hypophosphorylated and truncated form of 4E‐BP1, which may exert a long‐term inhibitory effect on the availability of eIF4E, thus contributing to the inhibition of protein synthesis and the growth‐inhibitory and pro‐apoptotic effects of p53.     

Activation of p53 stimulates proteasome-dependent truncation of eIF4E-binding protein 1 (4E-BP1).

BACKGROUND INFORMATION: The translational inhibitor protein 4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] regulates the availability of polypeptide chain initiation factor eIF4E for protein synthesis. Initiation factor eIF4E binds the 5' cap structure present on all cellular mRNAs. Its ability to associate with initiation factors eIF4G and eIF4A, forming the eIF4F complex, brings the mRNA to the 43S complex during the initiation of translation. Binding of eIF4E to eIF4G is inhibited in a competitive manner by 4E-BP1. Phosphorylation of 4E-BP1 decreases the affinity of this protein for eIF4E, thus favouring the binding of eIF4G and enhancing translation. We have previously shown that induction or activation of the tumour suppressor protein p53 rapidly leads to 4E-BP1 dephosphorylation, resulting in sequestration of eIF4E, decreased formation of the eIF4F complex and inhibition of protein synthesis. RESULTS: We now report that activation of p53 also results in modification of 4E-BP1 to a truncated form. Unlike full-length 4E-BP1, which is reversibly phosphorylated at multiple sites, the truncated protein is almost completely unphosphorylated. Moreover, the latter interacts with eIF4E in preference to full-length 4E-BP1. Inhibitor studies indicate that the p53-induced cleavage of 4E-BP1 is mediated by the proteasome and is blocked by conditions that inhibit the dephosphorylation of full-length 4E-BP1. Measurements of the turnover of 4E-BP1 indicate that the truncated form is much more stable than the full-length protein. CONCLUSIONS: The results suggest a model in which proteasome activity gives rise to a stable, hypophosphorylated and truncated form of 4E-BP1, which may exert a long-term inhibitory effect on the availability of eIF4E, thus contributing to the inhibition of protein synthesis and the growth-inhibitory and pro-apoptotic effects of p53.     

Triiodothyronine stimulates CMO1 gene expression in human intestinal Caco-2 BBe cells

Vitamin A is derived from provitamin A carotenoids, mainly beta-carotene, by beta-carotene 15,15'-monooxygenase (CMO1; EC 1.13.11.21). We previously found that enhancement of CMO1 mRNA expression was related to the levels of hormones, such as thyroid hormones, in chick duodenum. We investigated whether CMO1 expression was increased by triiodothyronine (T3), a thyroid hormone, using human intestinal Caco-2 BBe cells. Treatment of 7 days post-confluent Caco-2 BBe cells with T3 significantly enhanced CMO1 mRNA levels in both dose- and time-dependent manners. This T3-inducing effect on CMO1 mRNA level was blocked by actinomycin D. The levels of mRNAs for the thyroid hormone receptors TRalpha1 and TRbeta1 were significantly increased in 7 days post-confluent Caco-2 BBe cells. CMO1 enzyme activity was also significantly increased by T3 treatment in medium supplemented with fetal bovine serum. Furthermore, T3 treatment also increased the level of mRNA for lecithin:retinol acyltransferase (LRAT), but not those for cellular retinol-binding protein, type II (CRBPII) and retinal dehydrogenase 1 (RALDH1), in Caco-2 BBe cells. These results indicate that T3 is an important hormone for the regulation of vitamin A and beta-carotene metabolism-related gene expression in human small intestinal cells.     

Activation of protease-activated receptor (PAR)-1, PAR-2, and PAR-4 stimulates IL-6, IL-8, and prostaglandin E2 release from human respiratory epithelial cells

Epithelia from many tissues express protease-activated receptors (PARs) that play a major role in several different physiological processes. In this study, we examined their capacity to modulate IL-6, IL-8, and PGE(2) production in both the A459 and BEAS-2B cell lines and primary human bronchial epithelial cells (HBECs). All three cell types expressed PAR-1, PAR-2, PAR-3, and PAR-4, as judged by RT-PCR and immunocytochemistry. Agonist peptides corresponding to the nascent N termini of PAR-1, PAR-2, and PAR-4 induced the release of cytokines from A549, BEAS-2B, and HBECs with a rank order of potency of PAR-2 > PAR-4 > PAR-1 at 400 microM. PAR-1, PAR-2, and PAR-4 also caused the release of PGE(2) from A549 and HBECs. The PAR-3 agonist peptide was inactive in all systems tested. PAR-1, PAR-2, or PAR-4, in combination, caused additive IL-6 release, but only the PAR-1 and PAR-2 combination resulted in an additive IL-8 response. PAR peptide-induced responses were accompanied by changes in intracellular calcium ion concentrations. However, Ca(2+) ion shutoff was approximately 2-fold slower with PAR-4 than with PAR-1 or PAR-2, suggesting differential G protein coupling. Combined, these data suggest an important role for PAR in the modulation of inflammation in the lung.     

Activation of cyclic AMP-dependent protein kinases by vasoactive intestinal peptide (VIP) in isolated intestinal epithelial cells from rat.

VIP stimulates protein kinase activity in intestinal epithelial cells isolated from rat jejuno-ileum. The stimulation is time-dependent and is potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The response occurs in the 0.1–10 nM range of VIP concentrations, half-maximal stimulation being observed with 0.7 nM VIP. The VIP-induced protien kinase activation is thus observed at concentrations similar to those promoting the accumulation of cyclic AMP (11). Secretin also stimulates protien kinase activity but with a 100-times lower potency than VIP, in agreement with the fact that secretin is a VIP agonist of 100-times lower potency with respect to cyclic AMP increase. Prostaglandins E₁ and E₂ (10^?5 M), are also found to increase protein kinase activity.     

The novel role of the C-terminal region of SHP-2. Involvement of Gab1 and SHP-2 phosphatase activity in Elk-1 activation

SHP-2, a nontransmembrane-type protein-tyrosine phosphatase that contains two Src homology 2 (SH2) domains, is thought to participate in growth factor signal transduction pathways via SH2 domain interactions. To determine the role of each region of SHP-2 in platelet-derived growth factor signaling assayed by Elk-1 activation, we generated six deletion mutants of SHP-2. The large SH2 domain deletion SHP-2 mutant composed of amino acids 198-593 (SHP-2-(198-593)), but not the smaller SHP-2-(399-593), showed significantly higher SHP-2 phosphatase activity in vitro. In contrast, SHP-2-(198-593) mutant inhibited wild type SHP-2 phosphatase activity, whereas SHP-2-(399-593) mutant increased activity. To understand these functional changes, we focused on the docking protein Gab1 that assembles signaling complexes. Pull-down experiments with Gab1 suggested that the C-terminal region of SHP-2 as well as the SH2 domains (N-terminal region) associated with Gab1, but the SHP-2-(198-593) mutant did not associate with Gab1. SHP-2-(1-202) or SHP-2-(198-593) inhibited platelet-derived growth factorinduced Elk-1 activation, but SHP-2-(399-593) increased Elk-1 activation. Co-expression of SHP-2-(1-202) with SHP-2-(399-593) inhibited SHP-2-(399-593)/Gab1 interaction, and the SHP-2-(399-593) mutant induced SHP-2 phosphatase and Elk-1 activation, supporting the autoinhibitory effect of SH2 domains on the C-terminal region of SHP-2. These data suggest that both SHP-2/Gab1 interaction in the C-terminal region of SHP-2 and increased SHP-2 phosphatase activity are important for Elk-1 activation. Furthermore, we identified a novel sequence for SHP-2/Gab1 interactions in the C-terminal region of SHP-2.     

SHP-1 as a critical regulator of Mycoplasma pneumoniae-induced inflammation in human asthmatic airway epithelial cells

Asthma is a chronic inflammatory disease in which airway epithelial cells are the first line of defense against exposure of the airway to infectious agents. Src homology protein (SHP)-1, a protein tyrosine phosphatase, is a negative regulator of signaling pathways that are critical to the development of asthma and host defense. We hypothesize that SHP-1 function is defective in asthma, contributing to the increased inflammatory response induced by Mycoplasma pneumoniae, a pathogen known to exacerbate asthma. M. pneumoniae significantly activated SHP-1 in airway epithelial cells collected from nonasthmatic subjects by bronchoscopy with airway brushing but not in cells from asthmatic subjects. In asthmatic airway epithelial cells, M. pneumoniae induced significant PI3K/Akt phosphorylation, NF-κB activation, and IL-8 production compared with nonasthmatic cells, which were reversed by SHP-1 overexpression. Conversely, SHP-1 knockdown significantly increased IL-8 production and PI3K/Akt and NF-κB activation in the setting of M. pneumoniae infection in nonasthmatic cells, but it did not exacerbate these three parameters already activated in asthmatic cells. Thus, SHP-1 plays a critical role in abrogating M. pneumoniae-induced IL-8 production in nonasthmatic airway epithelial cells through inhibition of PI3K/Akt and NF-κB activity, but it is defective in asthma, resulting in an enhanced inflammatory response to infection.     

Strain-specific probiotic (Lactobacillus helveticus) inhibition of Campylobacter jejuni invasion of human intestinal epithelial cells

Campylobacter jejuni is the most common bacterial cause of enterocolitis in humans, leading to diarrhoea and chronic extraintestinal diseases. Although probiotics are effective in preventing other enteric infections, beneficial microorganisms have not been extensively studied with C. jejuni . The aim of this study was to delineate the ability of selected probiotic Lactobacillus strains to reduce epithelial cell invasion by C. jejuni . Human colon T84 and embryonic intestine 407 epithelial cells were pretreated with Lactobacillus strains and then infected with two prototypic C. jejuni pathogens. Lactobacillus helveticus , strain R0052 reduced C. jejuni invasion into T84 cells by 35–41%, whereas Lactobacillus rhamnosus R0011 did not reduce pathogen invasion. Lactobacillus helveticus R0052 also decreased invasion of one C. jejuni isolate (strain 11168) into intestine 407 cells by 55%. Lactobacillus helveticus R0052 adhered to both epithelial cell types, which suggest that competitive exclusion could contribute to protection by probiotics. Taken together, these findings indicate that the ability of selected probiotics to prevent C. jejuni -mediated disease pathogenesis depends on the pathogen strain, probiotic strain and the epithelial cell type selected. The data support the concept of probiotic strain selectivity, which is dependent on the setting in which it is being evaluated and tested.     

BCL-2 modifying factor (BMF) is a central regulator of anoikis in human intestinal epithelial cells

BCL-2 modifying factor (BMF) is a sentinel considered to register damage at the cytoskeleton and to convey a death signal to B-cell lymphoma 2. B-cell lymphoma 2 is neutralized by BMF and thereby facilitates cytochrome C release from mitochondria. We investigated the role of BMF for intestinal epithelial cell (IEC) homeostasis. Acute colitis was induced in Bmf-deficient mice (Bmf(-/-)) with dextran sulfate sodium. Colonic crypt length in Bmf(-/-) mice was significantly increased as compared with WT mice. Dextran sulfate sodium induced less signs of colitis in Bmf(-/-) mice, as weight loss was reduced compared with the WT. Primary human IEC exhibited increased BMF in the extrusion zone. Quantitative PCR showed a significant up-regulation of BMF expression after initiation of anoikis in primary human IEC. BMF was found on mitochondria during anoikis, as demonstrated by Western blot analysis. RNAi mediated knockdown of BMF reduced the number of apoptotic cells and led to reduced caspase 3 activity. A significant increase in phospho-AKT was determined after RNAi treatment. BMF knockdown supports survival of IEC. BMF is induced in human IEC by the loss of cell attachment and is likely to play an important role in the regulation of IEC survival.     

Relationship between structure and permeability of tryptophan derivatives across human intestinal epithelial (Caco-2) cells

L-Trp and its derivatives were used as model compounds to clarify structural factors which influence the intestinal epithelial permeation and metabolism of amino-acid derivatives. Permeability of model compounds through Caco-2 cells was used as an in vitro absorption model for human intestinal epithelial cells. The influence of compound concentration, the effects of various transporter substrates on permeability coefficients, and pH dependency of permeability coefficients were investigated. The transcellular permeability of Trp and Trp-NH2 in the direction from the apical side to the basolateral side, in which nutrients and drugs were ordinarily absorbed, declined with increasing concentration and saturated at more than 1 and 0.4 mM, respectively. The permeability coefficients for N-terminal protected Trp derivatives and Ac-Trp-NH2 showed similar and constant values in both from the apical-to-basolateral and basolateral-to-apical directions. In addition, significant inhibition of the apical-to-basolateral permeation of Trp by Leu and Phe was observed. The permeability coefficient ratio at pH 6.3 to that at pH 7.3 was explained by the ratio of the ionic form to the neutral form of the compounds. Based upon these results and the partition coefficients in the 1-octanol/water system, possible absorption mechanism of Trp and its derivatives across Caco-2 cells was proposed.     

Interleukin-1beta stimulates macrophage inflammatory protein-1alpha and -1beta expression in human neuronal cells (NT2-N)

Chemokines are important mediators in immune responses and inflammatory processes of neuroimmunologic and infectious diseases. Although chemokines are expressed predominantly by cells of the immune system, neurons also express chemokines and chemokine receptors. We report herein that human neuronal cells (NT2-N) produce macrophage inflammatory protein-1alpha and -1beta (MIP-1alpha and MIP-1beta), which could be enhanced by interleukin (IL)-1beta at both mRNA and protein levels. The addition of supernatants from human peripheral blood monocyte-derived macrophage (MDM) cultures induced MIP-1beta mRNA expression in NT2-N cells. Anti-IL-1beta antibody removed most, but not all, of the MDM culture supernatant-induced MIP-1beta mRNA expression in NT2-N cells, suggesting that IL-1beta in the MDM culture supernatants is a major factor in the induction of MIP-1beta expression. Investigation of the mechanism(s) responsible for IL-1beta-induced MIP-1alpha and -1beta expression demonstrated that IL-1beta activated nuclear factor kappa B (NF-kappaB) promoter-directed luciferase activity in NT2-N cells. Caffeic acid phenethyl ester, a potent and specific inhibitor of activation of NF-kappaB, not only blocked IL-1beta-induced activation of the NF-kappaB promoter but also decreased IL-1beta-induced MIP-1alpha and -1beta expression in NT2-N cells. These data suggest that NF-kappaB is at least partially involved in the IL-1beta-mediated action on MIP-1alpha and -1beta in NT2-N cells. IL-1beta-mediated up-regulation of beta-chemokine expression may have important implications in the immunopathogenesis of inflammatory diseases in the CNS.     

Gene expression profiles of normal proliferating and differentiating human intestinal epithelial cells: a comparison with the Caco-2 cell model

cDNA microarray technology enables detailed analysis of gene expression throughout complex processes such as differentiation. The aim of this study was to analyze the gene expression profile of normal human intestinal epithelial cells using cell models that recapitulate the crypt-villus axis of intestinal differentiation in comparison with the widely used Caco-2 cell model. cDNA microarrays (19,200 human genes) and a clustering algorithm were used to identify patterns of gene expression in the crypt-like proliferative HIEC and tsFHI cells, and villus epithelial cells as well as Caco-2/15 cells at two distinct stages of differentiation. Unsupervised hierarchical clustering analysis of global gene expression among the cell lines identified two branches: one for the HIEC cells versus a second comprised of two sub-groups: (a) the proliferative Caco-2 cells and (b) the differentiated Caco-2 cells and closely related villus epithelial cells. At the gene level, supervised hierarchical clustering with 272 differentially expressed genes revealed distinct expression patterns specific to each cell phenotype. We identified several upregulated genes that could lead to the identification of new regulatory pathways involved in cell differentiation and carcinogenesis. The combined use of microarray analysis and human intestinal cell models thus provides a powerful tool for establishing detailed gene expression profiles of proliferative to terminally differentiated intestinal cells. Furthermore, the molecular differences between the normal human intestinal cell models and Caco-2 cells clearly point out the strengths and limitations of this widely used experimental model for studying intestinal cell proliferation and differentiation.     

Activation of the proteolytic activity of ADAMTS4 (aggrecanase-1) by C-terminal truncation.

Proteolysis of the hyalectans (aggrecan, versican, brevican) in vivo appears to result from the activity of ADAMTS4 (aggrecanase-1, herein referred to as an hyalectanase). To examine the mode of activation of ADAMTS4, a human chondrosarcoma cell line, JJ012, has been stably transfected with the full-length c-DNA for human ADAMTS4. The cells synthesized a high molecular weight form of the enzyme (p100), which in serum-free culture was processed to three truncated forms, p75, p60, and p50. Treatment of the p100 form with recombinant furin indicated that the p75 form is generated by the removal of the prodomain by a furin-like activity. Analysis with domain-specific antisera showed that the p60 and p50 forms are generated by C-terminal truncation of the p75 form. The appearance of the p60 and p50 forms in culture medium was prevented by inclusion of a furin inhibitor, inhibitors of glycosylphosphatidylinositol synthesis, glucosamine, a hydroxamate-based matrix metalloproteinase (MMP) inhibitor, and TIMP-1, but not by AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride) or E64. Only medium samples containing the p60/p50 forms exhibited aggrecanase activity, and isolation of the p75, p60, and p50 forms by preparative SDS-PAGE showed that only p60 and p50 were active in aggrecanase and versicanase assays. Pig synovium and human cartilages also contained ADAMTS4 in the p75, p60, and p50 forms. We suggest that in vivo production of proteolytically active ADAMTS4 requires not only removal of the prodomain by a furin-like activity but also MMP-mediated removal of a portion of the C-terminal spacer domain.