Gilbert Pommier

Integrins and E-cadherin cooperate with IGF-I to induce migration of epithelial colonic cells.

Although the detailed mechanisms of cell migration remain largely unknown, it is now clear that growth factors and cell adhesion molecules are crucial for this process. We have shown that type I insulin‐like growth factor (IGF‐I) promotes migration of human colonic tumour cells. Since morphological analysis suggested an involvement of adhesion molecules, we have now examined the role of integrins (cell‐matrix adhesion molecules) and E‐cadherin/catenins complex (cell‐cell adhesion molecules) in the IGF‐I‐induced migration. Using a monolayer wounding assay, we have determined that, except for α2β1, all of the integrins expressed in HT29‐D4 cells are involved in the induced cell migration. Immunofluorescence studies revealed that upon IGF‐I stimulation the integrins reorganized at the leading edge of migrating cells. We also demonstrate that E‐cadherin is involved in cell migration. A rapid tyrosine phosphorylation of E‐cadherin and β‐catenin was detected upon IGF‐I stimulation. Tyrosine phosphorylation was associated with reduced membranous expression of E‐cadherin and promotion of cell motility, suggesting a regulation of the E‐cadherin/catenins complex. This effect can be reversed by incubating cells with tyrosine kinase inhibitors. Taken together, our results suggest that IGF‐I promotes colonic cell migration through reorganization of integrin receptors and through modulation of E‐cadherin/catenins complex function. Int. J. Cancer 83: 497–505, 1999.

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

Insulin-like growth factor-I receptor, E-cadherin and αv integrin form a dynamic complex under the control of α-catenin

Dynamic crosstalk between cell adhesion molecules, extracellular matrix and soluble informative factors is essential for cancer cell migration and invasion. Here, we investigated the mechanisms by which the E‐cadherin/catenin complex and αv integrin can modulate insulin‐like growth factor‐I (IGF‐I)‐induced cell migration. Human colon mucosa, human colon cancer cell lines, HT29‐D4 and HCT‐8 derivatives that differ in their expression of α‐catenin, were used as models. Interactions between E‐cadherin, αv integrin and IGF‐I receptor (IGF‐IR) were analyzed by coimmunoprecipitation and immunolocalization experiments. The impact of these interactions on cell mobility was determined by haptotaxis assays. We report that αv integrin, E‐cadherin and IGF‐IR form a ternary complex in both cultured cancer cells and human normal colonic mucosa. α‐Catenin regulates the scaffolding of this complex. IGF‐IR ligation by IGF‐I induces the disruption of the complex and the relocalization of αv integrin from cell–cell contacts to focal contact sites. This perturbation is correlated with the observed increase in cell migration. These results suggest that regulation of the αv integrin/E‐cadherin/IGF‐IR scaffolding is essential for the modulation of cell mobility. Its alteration could be of major importance to sustain alterations in cell adhesion that occur during cancer cell invasion and metastasis.

Gα(q/11)-coupled P2Y2 nucleotide receptor inhibits human keratinocyte spreading and migration

Reepithelialization is a critical step in wound healing. It is initiated by keratinocyte migration at the wound edges. After wounding, extracellular nucle‐otides are released by keratinocytes and other skin cells. Here, we report that activation of P2Y2 nucleotide receptor by ATP/UTP inhibits keratinocyte cell spreading and induces lamellipodium withdrawal. Kymogra‐phy analysis demonstrates that these effects correlate with a durable decrease of lamellipodium dynamics. P2Y2 receptor activation also induces a dramatic dismantling of the actin network, the loss of a3 integrin expression at the cell periphery, and the dissolution of focal contacts as indicated by the alteration of av integrins and focal contact protein distribution. In addition, activation of P2Y2R prevents growth factor‐induced phosphorylation of Erk1,2 and Akt/PkB. The use of a specific pharmacological inhibitor (YM‐254890), the depletion of Ga(q/11) by siRNA, or the expression of a constitutively active Ga(q/11) mutant (Q209L) show that activation of Ga(q/11) is responsible for these ATP/ UTP‐induced effects. Finally, we report that ATP delays growth factor‐induced wound healing of keratino‐cyte monolayers. Collectively, these findings provide evidence for a unique and important role for extracellular nucleotides as efficient autocrine/paracrine regulators of keratinocyte shape and migration during wound healing.— Taboubi, S., Milanini, J., Delamarre, E., Parat, F., Garrouste, F., Pommier, G., Takasaki, J., Hubaud, J‐C., Kovacic, H., Lehmann, M. Ga(q/11)‐coupled P2Y2 Nucleotide Receptor Inhibits Human Keratinocyte Spreading and Migration. FASEB J. 21, 4047–4058 (2007)

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.

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 ...

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.