Maryse Remacle-Bonnet

Protein kinases C-γ and -δ are involved in insulin-like growth factor I–induced migration of colonic epithelial cells

Abstract Background & Aims: The mechanisms by which epithelial cells migrate during the repair of damaged colonic mucosa are poorly understood. This study investigated the insulin-like growth factor I (IGF-I) signaling pathway leading to HT29-D4 human colonic epithelial cell line migration. Methods: IGF-stimulated cell migration was determined using a wound model in the presence or absence of kinase inhibitors. Activation of protein kinase C (PKC) was determined by immunodetection. Results: IGF-I and insulin induce cell migration without affecting cell proliferation through their cognate receptors. Des(1-3)-IGF-I, a truncated analogue of IGF-I, was more potent than IGF-I, suggesting that IGF-binding proteins secreted in the medium modulated IGF-I–induced cell migration. Inhibition of phosphatidylinositol 3-kinase, PKC, and mitogen-activated protein kinases eliminated cell restitution. Long-term exposure of cells to phorbol myristate acetate caused the depletion of PKC-δ and -γ and prevented also IGF-I–induced cell motility. IGF-I also induced activation of PKC-δ and -γ only. Conclusions: IGF-I stimulates colonic restitution through the activation of multiple signaling pathways including activation of phosphatidylinositol 3-kinase, PKC-δ and -γ, and mitogen-activated protein kinases. GASTROENTEROLOGY 1999;116:64-77

Deficient Processing and Activity of Type I Insulin-Like Growth Factor Receptor in the Furin-Deficient LoVo-C5 Cells1

To investigate endoproteolytic processing of the type I insulin-like growth factor receptor (IGF-IR), we have examined its structure and activity in the furin-deficient LoVo-C5 cell line. Immunoprecipitation experiments using the monoclonal anti-IGF-IR antibody (α-IR3) showed that LoVo-C5 cells expressed a major high molecular mass receptor (200 kDa) corresponding to the unprocessed α/β pro-receptor. A small amount of successfully cleaved α/β heterodimers was also produced, indicating a residual endoproteolytic cleavage activity in these cells. In vitro, a soluble form of recombinant furin was able to cleave the pro-IGF-IR (200 kDa) into α-subunit (130 kDa) andβ -subunit (97 kDa). Measurement of IGF binding parameters in LoVo-C5 cells indicated a low number of typical type I IGF-binding sites (binding capacity, 5 × 103 sites/cell; Kd, 1.9 nm for IGF-I and 7.0 nm for IGF-II). These findings in LoVo-C5 contrast with those in HT29-D4 cells, which have active furin, and where IGF-IR (2.8 × 104 sites/cell) was...

Activation of Protein Tyrosine Kinases by Coxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis

ABSTRACT Coxiella burnetii, the agent of Q fever, is an obligate intracellular microorganism that grows in monocytes/macrophages. The internalization of virulent organisms by monocytes is lower than that of avirulent variants and is associated with actin cytoskeleton reorganization. We studied the activation of protein tyrosine kinases (PTKs) by C. burnetii in THP-1 monocytes. Virulent organisms induced early PTK activation and the tyrosine phosphorylation of several endogenous substrates, including Hck and Lyn, two Src-related kinases. PTK activation reflects C. burnetiivirulence since avirulent variants were unable to stimulate PTK. We also investigated the role of PTK activation in C. burnetii-stimulated F-actin reorganization. Tyrosine-phosphorylated proteins were colocalized with F-actin inside cell protrusions induced by C. burnetii, and PTK activity was increased in Triton X-100-insoluble fractions. In addition, lavendustin A, a PTK inhibitor, and PP1, a Src kinase inhibitor, prevented C. burnetii-induced cell protrusions and F-actin reorganization. We finally assessed the role of PTK activation in bacterial phagocytosis. Pretreatment of THP-1 cells with lavendustin A and PP1 upregulated the uptake of virulent C. burnetii but had no effect on the phagocytosis of avirulent organisms. Thus, it is likely that PTK activation by C. burnetii negatively regulates bacterial uptake by interfering with cytoskeleton organization.

Insulin-like growth factor (IGF)–binding protein-3 (IGFBP-3) proteolysis in patients with colorectal cancer: possible association with the metastatic potential of the tumor

The limited proteolysis of insulin‐like growth factor (IGF)–binding protein (IGFBP)‐3 is a key event in the regulation of endocrine bioavailability of IGFs. Here, we investigated IGFBP‐3 and IGFBP‐3 proteolysis in serum from patients with colorectal cancer both before and at different times following surgery. In vivo IGFBP‐3 proteolysis, estimated by immunoblot analysis of IGFBP‐3 fragments in serum, andin vitro IGFBP‐3 protease activity of serum, estimated by a 125I‐IGFBP‐3 degradation assay, allowed us to identify 2 groups of patients (IGF‐M vs. IGF‐NM) with respect to their status for mobilizing the IGF system. In IGF‐M patients, in vivo and in vitro IGFBP‐3 proteolysis were significantly elevated (156% and 181% of the age‐matched control pool, respectively) and accompanied by a decrease in intact IGFBP‐3 (38% of the control pool). The IGFBP‐3 proteolytic processing was further increased in response to surgical ablation of the tumor (mean increase 45–55%), then gradually returned to levels comparable with controls. In contrast, IGF‐NM patients exhibited a minimal alteration of in vitro IGFBP‐3 protease activity and even an inhibition of in vivo IGFBP‐3 proteolysis, whereas intact IGFBP‐3 was unaltered when compared with controls. Moreover, this pattern was not further significantly altered in response to the surgical stress. None (0/6) of the IGF‐M patients vs. 70% (5/7) of the IGF‐NM patients developed a metastatic disease (median duration of follow‐up 26 months). Neither elevated amounts of pro‐IGF‐II nor presence of detectable IGFBP‐3 protease inhibitors in the circulation could explain the observed suppression of IGFBP‐3 proteolytic processing in IGF‐NM patients. These results indicate that inhibition of IGFBP‐3 proteolysis and invasive properties of cancer cells are related in colorectal cancer patients. Int. J. Cancer (Pred. Oncol.) 79:460–467, 1998.© 1998 Wiley‐Liss, Inc.

Immunosuppressive properties of human placenta: study of supernatants from short-term syncytiotrophoblast cultures

Using collagenase and mechanical treatment to attempt to eliminate cellular contamination such as macrophages and decidual cells, trophoblast enriched cell suspensions were isolated from the human placenta. With a view to assessing the role of trophoblast in impairing maternal rejection of the fetus, supernatants (SPl4) were prepared from these placental cells after short-term culture (4 h). The immunosuppressive activity of these supernatants was studied following application to mitogen-stimulated human lymphocyte cultures and mixed lymphocyte cultures. In both cases a reproducible inhibition was observed. The ability of these substances to induce a non-specific inhibitory effect was ascertained by observing mouse lymphocyte responses to mitogens or alloantigens. To gain further insight into in vivo fetal protection against anti-paternal cells, we also examined the effects of SPl4 on CTL generation. It was found not only that CTL generation was markedly depressed but also that SPl4 drastically impaired cell-mediated lympholysis at the effector level. To characterize the factors involved in our observations, SPl4 was subjected to dialysis and to chromatography. In the first case, it was found that these factors were not amenable to dialysis. In the second case, we obtained on an Ultrogel AcA 44 column two fractions with immunosuppressive activity. Following our previous work on human syncytiotrophoblast, we analyzed only the low molecular weight inhibitory fraction, which was chromatographed again on Ultrogel AcA 202. The molecular weight of the immunosuppressive factor(s) was estimated to be around 3.5 kDa. We postulate that human trophoblast releases soluble factors around the fetus which may act to protect it against maternal immunological rejection.

Surface-bound plasmin induces selective proteolysis of insulin-like-growth-factor (IGF)-binding protein-4 (IGFBP-4) and promotes autocrine IGF-II bio-availability in human colon-carcinoma cells.

Limited proteolysis of insulin‐like‐growth‐factor (IGF)‐binding proteins (IGFBPs) represents a key process to modulate IGF bio‐availability at the cellular level. In human colon carcinomas, urokinase‐type plasminogen activator (u‐PA) produced by stroma cells can bind to cancer‐cell‐associated u‐PA receptor (u‐PAR), and then catalyze the conversion of plasminogen (Pg) into plasmin (Pm). We therefore investigated the interplay between the IGF and Pm systems in the HT29‐D4 human colon‐carcinoma‐cell model. HT29‐D4 cells secreted IGF‐II totally complexed to IGFBP‐2, IGFBP‐4 and IGFBP‐6. Approximately 15% of IGFBP‐4 was associated with the extracellular matrix. HT29‐D4 cells produced neither u‐PA‐ nor IGFBP‐specific proteases. However, activation of Pm at the HT29‐D4 cell surface obtained by the sequential addition of exogenous u‐PA and Pg to mimic the stromal complementation induced selective proteolysis targeted to IGFBP‐4 only (>95%). IGFBP‐2 and IGFBP‐6, though sensitive to proteolysis by soluble Pm, were not altered by cell‐bound Pm. IGFBP‐4 proteolysis yielded 18‐ and 14‐kDa immunoreactive fragments which were not detectable by Western ligand blotting, indicating that they bound IGF‐II with poor affinity. Release of IGF‐II from IGF‐II‐IGFBP complexes after IGFBP‐4 proteolysis by cell‐bound Pm was indicated by the observation that approximately 20% of the 125I‐IGF‐II initially associated with endogenous IGFBP in reconstituted complexes was transferred to HT29‐D4 cell‐surface IGF‐I receptors. These results suggest that IGFBP‐4 proteolysis by cell‐bound Pm can promote autocrine/paracrine IGF‐II bio‐availability in colon‐cancer cells. This may have important consequences on the behavior of cancer cells at the interface between stroma and malignant cells in carcinomas of the colon in vivo. Int. J. Cancer 72:835–843, 1997.

Up-regulation of insulin/insulin-like growth factor-I hybrid receptors during differentiation of HT29-D4 human colonic carcinoma cells.

To assess the autocrine function of insulin-like growth factor II (IGF-II) in the balance of proliferation and differentiation in HT29-D4 human colonic cancer cells, we studied the expression of IGF-I receptors (IGF-IR) and insulin receptors (IR) in relation to the state of cell differentiation. IGF-IR and IR were expressed in both undifferentiated and enterocyte-like differentiated HT29-D4 cells. IGF-IR had two isoforms with a 97-kDa and a 102-kDa β-subunit. In addition, HT29-D4 cells expressed hybrid receptors (HR) formed by the association of two αβ heterodimers from both IR and IGF-IR. HR were evidenced through 1) inhibition of IGF-I binding by the B6 anti-IR antibody and 2) immunoprecipitation with the α-IR3 anti-IGF-IR antibody, which revealed an additional 95-kDa IR β-subunit that disappeared when the heterotetrameric receptor was dissociated by disulfide reduction into αβ heterodimers before immunoprecipitation. Like IGF-IR, HR had a high affinity for IGF-I (Kd, ∼1.5 nm), but did not bind insulin ...

Colorectum cell-derived growth factor (CRDGF) is homologous to amphiregulin, a member of the epidermal growth factor family.

We have previously shown that an autocrine factor (CRDGF) of molecular weight 25,000 is produced by the HT29 human colon cancer cell line. Although CRDGF was shown to inhibit the binding of epidermal growth factor (EGF) to its receptor, several lines of evidence suggested that it was distinct from EGF or transforming growth factor-alpha (TGF-alpha). In order to check the possibility that CRDGF represents a new member of the EGF family, a four-step purification protocol involving acid gel filtration, cation-exchange high-performance liquid chromatography (HPLC), C18 reversed-phase HPLC and gel permeation HPLC was used to purify this protein to homogeneity. The purified material exhibited a 22 kDa molecular mass on SDS-PAGE. Partial N-terminal amino acid sequence of CRDGF showed identity to amphiregulin (AR), an EGF-related protein. Western blotting experiments using AR-specific antiserum confirmed that CRDGF and AR are identical proteins. In addition, we showed that AR, like EGF or TGF-alpha stimulated the phosphorylation of the epidermal growth factor receptor (EGF-R) on tyrosine residues. This indicates that the AR intracellular signalling pathway involves the activation of EGF-R kinase.

Cell polarity of the insulin-like growth factor system in human intestinal epithelial cells. Unique apical sorting of insulin-like growth factor binding protein-6 in differentiated human colon cancer cells.

In this study, we have used enterocyte-like differentiated HT29-D4 human colonic carcinoma cells cultured in a glucose-free medium (HT29-D4-GAL cells) on semi-permeable supports in order to investigate the polarity of the insulin-like growth factor (IGF) system. We report that these cells secrete endogenous IGF-II predominantly (66%) from the basolateral cell surface where type I IGF receptors are almost all (> 96%) localized. HT29-D4-GAL cells also secrete IGF-binding protein (IGFBP) -2, -4, and -6 as evidenced by Western ligand and immunoblot analyses of conditioned medium. IGFBP-2 and IGFBP-4 are secreted primarily into the basolateral side (71 and 87%, respectively), whereas IGFBP-6 is targeted to the apical surface (76%) as a possible consequence of an active sorting. Finally, HT29-D4-GAL cells are found to display responses to IGF-II added to the basolateral but not the apical membrane side in terms of intracellular tyrosine phosphorylation and long-term stimulation of amino acid uptake. This study indicates (a) that IGF-II is potentially capable of autocrine regulation on the basolateral side of HT29-D4-GAL cell, and (b) that IGFBP-6 has a unique pattern of secretory polarity. It supports the concept that a differential sorting of the various forms of IGFBPs might play a modulatory role in the maintenance of a functional polarity in the differentiated HT29-D4-GAL cells.

Non-specific immunoregulatory factors in the cytosol fraction of human trophoblast

Cytosol extracts from syncytiotrophoblast of human placenta (HP) have been shown to contain substances capable of suppressing the proliferation of normal human lymphocytes stimulated by mitogens. This suppressive effect has also been observed on lymphocytes stimulated by mitogens and by allogenic cells in various species. The extracts did not, however, inhibit spontaneous (i.e. unstimulated) lymphoproliferation. In addition, HP in some cases exercised an immunostimulatory effect solely on stimulated fractionated lymphocytes. In preliminary experiments the suppressive activity was shown to depend on at least two factors: the first had the ability to bind the mitogen while the second acted irreversibly after 8 h of contact on the lymphocyte itself and was thermostable.