Coordinate expression of OPN and associated receptors during monocyte/macrophage differentiation of HL-60 cells

Promyelocytic leukemia HL-60 cells promoted by PMA to differentiate along the monocyte pathway adhere to tissue culture plates. To explore the regulation of adhesion molecules in cells promoted to differentiate, the expression and secretion of osteopontin (OPN) and expression of associated cell surface receptors, CD44 and integrin subunits α_v, β₃, β₁, were examined. Results were as follows: (1) PMA induced OPN mRNA and OPN secretion into media; (2) untreated cells expressed β₁ and CD44 mRNA, and PMA induced α_v and β₃ mRNA and increased β₁ and CD44 mRNA expression; (3) PMA increased levels of α_v, β₃, β₁ and CD44 protein on the cell surface; and (4) retinoic acid, which promotes granulocytic differentiation of HL-60 cells, did not affect OPN, α_v, β₃, β₁, or CD44 mRNA or protein expression. These data suggest that induction of OPN and associated receptors may play a role during monocytic differentiation of HL-60 cells. J. Cell. Physiol. 175:229–237, 1998. © 1998 Wiley-Liss, Inc.     

Differentiation of human promyelocytic leukemia cells is accompanied by an increase in insulin receptors

Changes in insulin receptors accompanying cell differentiation in human promyelocytic leukemia cells (HL-60) were studied. Cell differentiation was induced by 1α,25-dihydroxyvitamin D₃, vitamin A, dimethyl sulfoxide, or phorbol esters. 1α,25-dihydroxy-vitamin D₃ increased the ability of HL-60 cells to bind insulin in a dose-dependent manner. The increase in insulin binding was due to an increase in the number of insulin receptors. Vitamin A, dimethyl sulfoxide and phorbol esters were also effective in increaseing insulin receptors. Thus, the differentiation of HL-60 cells was accompanied by an increase in insulin receptors.     

Extensive Clear Zone and Defective Ruffled Border Formation in Osteoclasts of Osteopetrotic (ia/ia) Rats: Implications for Secretory Function

The cellular distribution of osteoclast integrin subunits α_v and β₃, the tissue distribution, and level of the apparent ligand osteopontin (OPN) as well as of the putative regulatory enzyme tartrate-resistant acid phosphatase (TRAP) were studied along with the intracellular distribution of the activation marker c-src in osteopetrotic ia/ia (incisors-absent) mutant rats and their normal littermates. In ia/ia rats, the osteoclasts are incapable of bone matrix resorption. Ultrastructurally the cells exhibit extended clear zones at the expense of ordinary ruffled borders. A secretory dysfunction in the mutant is strongly suggested by the absence of detectable extracellular TRAP, concomitant with an accumulation of the enzyme in abundant small cytoplasmic vesicles. Moreover, TRAP mRNA, protein content, as well as enzymatic activity were elevated. Furthermore, increased levels of integrin subunits α_v and β₃ were detected at the clear zone of mutant osteoclasts. OPN mRNA levels were elevated in long bones from mutants. In ia/ia rats, immunolabeling for OPN was homogeneously distributed at the surface facing osteoclasts, while in normal littermates it was concentrated at the clear zone area and barely detectable at ruffled borders. The absence of OPN labeling in the abundant, putative intracellular secretory vesicles in mutant osteoclasts suggests that these cells do not produce OPN. The osteoclasts of ia/ia rats appeared to produce and translocate the c-src protein to the cell membrane.In ia/ia a defect ruffled border-formation is observed along with extensive clear zone formation and decreased secretory function. The lesion may be due to a signaling defect, but in that case the defect seems to be located downstream to or not involving the c-src pathway. Our results illustrate the close relationship between secretory function and ruffled border formation in osteoclasts, a relationship that appears to be necessary for proper resorptive function.     

Adhesion Properties and Integrin Expression of Cultured Human Osteoclast-like Cells

Osteoclast interaction with extracellular matrix drives the sequential events that end with bone resorption. However, the role of matrix proteins is not yet fully understood. We studied this problem on human osteoclast-like cells derived from giant cell tumors of bone (GCT cells). On GCT cells we considered cytoskeletal organization, adhesion properties, and integrin expression upon plating in serum-free medium onto fibronectin (FN), collagen (COL), thrombospondin (TSP), bone sialoprotein (BSPII), and osteopontin (OPN). GCT cells promptly adhered and spread on FN, BSPII, and OPN, while only 50% adhered on COL and none on TSP. The integrin β₁ chain was always associated to focal adhesions, while the α_vβ₃ heterodimer was detected in focal contacts only upon plating on BSPII, OPN, and FN. The focal clustering of β₁ was impaired by monensin treatment, indicating that endogenous FN secretion was required to drive β₁ into focal contacts. Conversely, α_vβ₃ clustering was also not affected by monensin when cells were plated onto plasma FN. Immunoprecipitation of metabolically labeled GCT cell lysates showed that three different heterodimers (α_vβ₃, α₃β₁, and α₅β₁) were assembled. Adhesion to FN was completely inhibited by β₁ antibodies at dilutions up to 1:400, while β₃ antibodies, at similar dilutions, impaired spreading but not adhesion. We conclude that α_vβ₃3 is the main integrin used by GCT cells in bone recognition. We also suggest that selected substrata may induce the release and the organization of endogenous FN that eventually drives the recruitment of a β₁ integrin receptor into focal contacts.     

Use of a β1 Integrin-deficient Human T Cell to Identify β1 Integrin Cytoplasmic Domain Sequences Critical for Integrin Function

T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in β1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the β1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human β1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the β1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the β1 integrin with the activating β1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the β1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of β1 integrin structure and function in human T cells.     

Amiloride inhibits phorbol ester-stimulated Na+/H+ exchange and protein kinase C. An amiloride analog selectively inhibits Na+/H+ exchange

The human leukemic cell line, HL-60, differentiates in response to tumor-promoting phorbol esters. Recently, we have reported that one of the first events evoked by phorbol esters in HL-60 cells is the stimulation of Na+-dependent H+ efflux. In efforts to determine whether stimulation of Na+/H+ exchange by phorbol esters is coupled to induction of cellular differentiation, we found that 1) amiloride, a frequently used inhibitor of Na+/H+ exchange, rapidly inhibits phorbol ester-stimulated protein phosphorylation in vivo and protein kinase C-mediated phosphorylation in vitro, both with potency similar to that with which amiloride inhibits Na+/H+ exchange; 2) an amiloride analog, dimethylamiloride, is a far more potent inhibitor of Na+/H+ exchange than is amiloride, while being no more potent than amiloride in inhibiting phorbol ester/protein kinase C-mediated phosphorylation; and 3) at concentrations sufficient to completely inhibit Na+/H+ exchange, amiloride blocked phorbol ester-induced adhesion of HL-60 cells (adhesion being a property indicative of the differentiated state), but dimethylamiloride (as well as ethylisopropylamiloride, another very potent amiloride analog) did not. Thus, dimethylamiloride represents a potential tool for distinguishing protein kinase C-coupled from Na+/H+ exchange-coupled events in phorbol ester-stimulated cells.     

A Novel Cell Adhesion Region in Tropoelastin Mediates Attachment to Integrin αVβ5

Tropoelastin protein monomers assemble to form elastin. Cellular integrin α_Vβ₃ binds RKRK at the C-terminal tail of tropoelastin. We probed cell interactions with tropoelastin by deleting the RKRK sequence to identify other cell-binding interactions within tropoelastin. We found a novel human dermal fibroblast attachment and spreading site on tropoelastin that is located centrally in the molecule. Inhibition studies demonstrated that this cell adhesion was not mediated by either elastin-binding protein or glycosaminoglycans. Cell interactions were divalent cation-dependent, indicating integrin dependence. Function-blocking monoclonal antibodies revealed that α_V integrin(s) and integrin α_Vβ₅ specifically were critical for cell adhesion to this part of tropoelastin. These data reveal a common α_V integrin-binding theme for tropoelastin: α_Vβ₃ at the C terminus and α_Vβ₅ at the central region of tropoelastin. Each α_V region contributes to fibroblast attachment and spreading, but they differ in their effects on cytoskeletal assembly.     

Activation of αVβ3 on Vascular Cells Controls Recognition of Prothrombin

Regulation of vascular homeostasis depends upon collaboration between cells of the vessel wall and blood coagulation system. A direct interaction between integrin α_Vβ₃ on endothelial cells and smooth muscle cells and prothrombin, the pivotal proenzyme of the blood coagulation system, is demonstrated and activation of the integrin is required for receptor engagement. Evidence that prothrombin is a ligand for α_Vβ₃ on these cells include: (a) prothrombin binds to purified α_Vβ₃ via a RGD recognition specificity; (b) prothrombin supports α_Vβ₃-mediated adhesion of stimulated endothelial cells and smooth muscle cells; and (c) endothelial cells, either in suspension and in a monolayer, recognize soluble prothrombin via α_Vβ₃. α_Vβ₃-mediated cell adhesion to prothrombin, but not to fibrinogen, required activation of the receptor. Thus, the functionality of the α_Vβ₃ receptor is ligand defined, and prothrombin and fibrinogen represent activation- dependent and activation-independent ligands. Activation of α_Vβ₃ could be induced not only by model agonists, PMA and Mn²⁺, but also by a physiologically relevant agonist, ADP. Inhibition of protein kinase C and calpain prevented activation of α_Vβ₃ on vascular cells, suggesting that these molecules are involved in the inside-out signaling events that activate the integrin. The capacity of α_Vβ₃ to interact with prothrombin may play a significant role in the maintenance of hemostasis; and, at a general level, ligand selection by α_Vβ₃ may be controlled by the activation state of this integrin.     

Osteopontin-Stimulated Vascular Smooth Muscle Cell Migration Is Mediated by β3 Integrin

Osteopontin (OPN), a 41-kDa phosphorylated glycoprotein, has been detected in rat aorta and carotid arteries, and expression of its mRNA in blood vessels is strongly increased in response to vascular injury. To investigate the potential role of OPN in vascular pathophysiology, we studied the effect of rat OPN on aortic smooth muscle cell migration and proliferation in vitro. OPN enhanced the migration of rat smooth muscle cells in a time- and concentration-dependent manner with an EC₅₀ value of 46 ± 11 nmol/liter (n = 5). The maximal increase in cell migration by OPN was 29-fold over basal levels. OPN-induced smooth muscle cell migration was inhibited in a concentration-dependent manner by the monoclonal antibody F11, which recognizes the rat integrin subunit β₃. In contrast, polyclonal antiserum recognizing the rat integrin β₁ subunit did not inhibit smooth muscle cell migration in response to OPN, but did block fibronectin-promoted migration. Moreover, OPN-induced smooth muscle cell migration was dependent on the presence of extracellular divalent cations and was significantly inhibited by anti-OPN antibodies. OPN did not stimulate [³H]thymidine incorporation into cultured smooth muscle cells, indicating that it selectively enhanced migration. In view of the pathological significance of arterial smooth muscle cell migration in the formation of intimal thickening, our results suggest that smooth muscle cell recognition of OPN, probably through the vitronectin receptor, α_vβ₃, could play a role in the cells' response to vascular injury and especially neointima formation.     

A prognostic gene signature in advanced ovarian cancer reveals a microfibril-associated protein (MAGP2) as a promoter of tumor cell survival and angiogenesis

Ovarian cancer is the most lethal gynecologic cancer, and this is largely related to its late diagnosis. High grade serous cancers often initially respond to chemotherapy, resulting in a better survival rate, compared to other ovarian carcinoma subtypes. We review recent work identifying a survival-associated gene expression profile for advanced serous ovarian cancer. Within this signature, the authors identified MAGP2, also known as microfibrillar associated protein 5 (MFAP5), as a highly significant indicator of survival and chemosensitivity. MAGP2 is a multifunctional secreted protein—important for elastic microfibril assembly and modulating endothelial cell behavior—with a newly identified role in cell survival. Through α_Vβ₃ integrin-mediated signaling, MAGP2 promotes tumor and endothelial cell survival and endothelial cell motility, providing a potential mechanistic link between MAGP2 and angiogenesis as well as patient survival.Key words: ovarian cancer, cell survival, MAGP2, α_Vβ₃, endothelial cellAmong reproductive cancers, ovarian cancer has the highest associated mortality rate-the majority of patients present with late—stage disease that has already spread beyond the ovaries.¹ Surgery and chemotherapy are used to eliminate localized and metastatic cancer cells, and the size of the residual legion after surgery is the main prognostic factor for survival. Biomarkers or gene expression profiles associated with prognosis or chemosensitivity in different ovarian cancer subtypes could aid treatment decisions—perhaps improving patient mortality rates by identifying patients that will not respond to traditional surgical or chemotherapeutic approaches. While the underlying mechanisms for ovarian cancer are incompletely understood, cellular alterations important for cancer initiation and progression have been described.² Distinguishing features of cancer cells include the ability to survive and proliferate under adverse conditions, induce and maintain angiogenesis, and invade into the surrounding tissues. The contribution of secreted proteins to these processes is of particular interest, as these proteins can modulate cell signaling in both tumor and normal cells. This review summarizes recent work by Mok et al.³ describing the identification of a gene expression signature correlating with serous ovarian cancer survival and chemosensitivity. They validate the secreted protein, Microfibril-Associated Glycoprotein 2 (MAGP2), gene name, MFAP5 (also known as microfibrillar associated protein 5), as an independent prognostic biomarker of survival and chemosensitivity and report a role for MAGP2 in cell survival.To identify a gene expression signature predicting survival in late stage, high-grade papillary serous ovarian tumors, the authors used laser capture microdissection to isolate epithelial cancer cells from paraffin-embedded tissue and analyzed gene expression changes using microarrays. The authors were able to identify and validate a gene expression signature that correlated with survival in this patient cohort. The median hazard ratio was used as a threshold to group high- and low-risk patients and the representative Kaplan Meier curves were compared using the log-rank test, revealing a significant association between the high-risk group and shorter survival times. Of all the genes examined, microfibril-associated glycoprotein 2 (MAGP2) had the highest correlation with poor patient prognosis. MAGP2 expression was further explored as an independent prognostic marker, at both the mRNA and protein levels. Mok and colleagues confirmed increased MAGP2 expresssion by both real time PCR and immunohistochemical staining. Both high MAGP2 mRNA and protein levels stratified the low and high risk patient groups, with positive MAGP2 predicting a poor prognosis.To explore the signaling pathways affecting ovarian cancer patient survival, the authors compared gene expression profiles from the advanced stage ovarian cancer samples to those from normal ovarian surface epithelial cells and compared them with the patient survival gene signature. The α_Vβ₃ integrin receptor was found to be a central signaling pathway in this network. MAGP2 binds and activates α_Vβ₃ integrin, and the downstream effectors, focal adhesion kinase (FAK), paxillin (PXN), growth factor receptor-bound protein 2 (GRB2) and son of sevenless homolog 1 (SOS 1) were overexpressed. These may contribute toward cell cycle progression and increased cell survival. Changes in a subset of these genes, including FAK and MAGP2, were verified using quantitative Real Time-PCR, indicating that the α_Vβ₃ signaling pathway is over-activated in ovarian cancer. Given that α_Vβ₃ integrin signaling correlated with chemotherapy resistance in ovarian cancer cell lines,⁴ the authors also examined the utility of MAGP2 as an indicator of chemosensitivity. By stratifying patients based on response to chemotherapy, the authors determined that high MAGP2 levels correlate with chemoresistance—suggesting that these patients might require alternative treatment strategies to improve patient outcome.To characterize the role of secreted MAGP2 in modulating tumor and endothelial cells via α_Vβ₃ integrin, an important mediator of cell adhesion and survival, the authors initially employed an in vitro approach. Ovarian cancer cell lines were screened for MAGP2 and α_Vβ₃ integrin expression and were evaluated for their ability to adhere to synthetic MAGP2 protein. Cell lines with (A224, OVCA429 and SKOV3) and without (UCI107) α_Vβ₃ integrin were used. A224 cell adhesion to MAGP2 was diminished in the presence of a α_Vβ₃ integrin blocking antibody, whereas no change in UCI107 cell adhesion was observed, demonstrating a role for MAGP2 in α_Vβ₃ integrin-mediated adhesion. Improved OVCA429 cell survival in serum-free media was also observed in the presence of recombinant MAGP2, suggesting that MAGP2 is important for both α_Vβ₃ integrin-mediated tumor cell survival and adhesion (Fig. 1).Open in a separate windowFigure 1Ovarian serous cancer cells secrete MAGP2, which acts to increase tumor cell survival and endothelial cell motility and survival through the α_Vβ₃ integrin.Previous reports have demonstrated a role for MAGP2 in elastic fiber assembly⁵ and endothelial cell behavior.^6,7 The ability of MAGP2 to modulate endothelial cell behavior via α_Vβ₃ integrin was next examined. The authors observed an increase in human umbilical vein endothelial cell (HUVEC) adhesion to recombinant MAGP2, which was inhibited by pre-incubation with a α_Vβ₃ integrin antibody. HUVEC survival in the absence of serum, and cell motility and invasion were also increased by MAGP2 (Fig. 1). The MAGP2 protein sequence includes the classical integrin-binding RGD domain. To determine whether this domain was important for MAGP2-mediated alterations in endothelial cell behavior, the authors generated recombinant proteins with amino acid substitutions in the RGD motif. Enhanced MAGP2-mediated cell motility was diminished in the presence of α_Vβ₃ integrin blocking antibody or mutated recombinant MAGP2, but not with α₅ or β₁ integrin blocking antibodies, demonstrating the MAGP2 RGD motif was important for α_Vβ₃ integrin signaling in endothelial cells. To further explore MAGP2-mediated changes in endothelial cells, microarray analysis was used to compare gene expression profiles in MAGP2-treated and untreated HUVEC cells. Increased expression of genes involved in cell adhesion such as FAK (focal adhesion kinase) and ITGAV (integrin alpha v) were observed, together with increased cell motility genes, such as CDC42 and APC, while an increase in phosphorylated FAK was observed via western blotting. These data provide mechanistic insight into MAGP2-modulation of cellular activity.To understand MAGP2 function during tumorigenesis, the authors generated SKOV3 ovarian cancer clonal cell lines stably expressing either an empty vector or shRNA against MAGP2. The authors evaluated the ability of subcutaneously injected cells to form tumors in nude mice, showing that cells with reduced MAGP2 expression formed smaller tumors than controls. This effect was confirmed using additional MAGP2-targeting shRNAs in both SKOV3 and OVCAR3 cells. Finally, as MAGP2 affects endothelial cell behavior, the effects on the angiogenic microenvironment was evaluated. Immunohistochemical analysis of MAGP2-knockdown tumor tissue in mice revealed decreased microvessel density (MVD) as visualized by CD34⁺ staining, while MVD and MAGP2 expression significantly correlated in 30 human serous ovarian cancer tissue specimens. Together these data suggest an in vivo role for MAGP2 in promoting angiogenesis.     

Activation of Integrin αVβ3 Regulates Cell Adhesion and Migration to Bone Sialoprotein

α_Vβ₃, a broadly distributed member of the integrin family of adhesion receptors, has been implicated in a variety of physiological and pathophysiological events, including control of bone density, angiogenesis, apoptosis, tumor growth, and metastasis. Recently, it has been shown that activation of α_Vβ₃, its transition from a low- to a high-affinity/avidity state, influences its recognition of certain ligands. Bone sialoprotein (BSP) is recognized as an important ligand for α_Vβ₃ in processes ranging from bone formation to the homing of metastatic tumor cells. Here, the influence of α_Vβ₃ activation on the adhesion and migration of relevant cells to BSP has been examined. Stimulation of lymphoblastoid, osteoblastoid, and human umbilical vein endothelial cells (HUVEC) with PMA or Mn²⁺ markedly enhanced α_Vβ₃-dependent adhesion to BSP. α_Vβ₃-mediated migration of HUVEC or osteoblastic cells to BSP was substantially enhanced by stimulation, demonstrating that α_Vβ₃ activation enhances both adhesive and migratory responses. However, adhesion and/or migration of certain tumor cell lines, including M21 melanoma and MDA MB435 and SKBR3 breast carcinoma cell lines, to BSP was constitutively high and was not augmented by α_Vβ₃-activating stimuli. Inhibitors of the intracellular signaling molecules, phosphatidylinositol 3-kinase with wortmannin, hsp90-dependent kinases with geldanamycin, and calpain with calpeptin, but not MAPKK with PD98059, reduced the high spontaneous adhesion and migration of the M21 cells to BSP, consistent with the constitutive activation of the receptor on these tumor cells. These results indicate that the activation state of α_Vβ₃ can regulate cell migration and adhesion to BSP and, by extension, to other ligands of this receptor. The constitutive activation of α_Vβ₃ on neoplastic cells may contribute to tumor growth and metastatic potential.     

Integrin α5β1 Expression Is Required for Inhibition of Keratinocyte Migration by Ganglioside GT1b

Polysialoganglioside GT1b, a keratinocyte membrane glycosphingolipid, inhibits normal keratinocyte adhesion and migration on a fibronectin matrix. The specificity of the inhibition for cells plated on a fibronectin matrix and competition of GT1b inhibition with peptide RGDS suggest that GT1b abrogates the α₅β₁/fibronectin interaction. We examined the effects of GT1b on the adhesion and migration of keratinocyte-derived cell lines and correlated GT1b responsiveness and α₅β₁integrin expression. GT1b (5 nM) significantly inhibited migration of normal human keratinocytes, immortalized keratinocytes, and squamous cell carcinoma SCC12F2 cells on fibronectin, but not on collagen I. Concentrations as high as 5 μM had no effect on SCC13 or HaCaT cells. Likewise, GT1b inhibited fibronectin-dependent cell adhesion of normal human keratinocytes, immortalized keratinocytes, and SCC12F2 cells, but had no effect on SCC13 or HaCaT cells. Flow cytometric and Western immunoblot analysis of integrin expression showed significantly decreased α₅and β₁integrin expression in SCC13 and HaCaT cells compared to normal keratinocytes, immortalized keratinocytes, and SCC12F2 cells. Incubation with TGF-β1 increased α₅β₁integrin expression and induced responsiveness to GT1b in HaCaT cells. These data imply that GT1b “response” requires sufficient expression of α₅β₁and further suggest that the mechanism of the inhibitory effect of GT1b involves GT1b/α₅β₁interaction.     

Osteopontin-Enhanced Hepatic Metastasis of Colorectal Cancer Cells

Liver metastasis is a major cause of mortality from colorectal cancer (CRC). However, mechanisms underlying this process are largely unknown. Osteopontin (OPN) is a secreted phosphorylated glycoprotein that is involved in tumor migration and metastasis. The role of OPN in cancer is currently unclear. In this study, OPN mRNA was examined in tissues from CRC, adjacent normal mucosa, and liver metastatic lesions using quantitative real-time PCR analysis. The protein expression of OPN and its receptors (integrin αv and CD44 v6) was detected by using an immunohistochemical (IHC) method. The role of OPN in liver metastasis was studied in established colon cancer Colo-205 and SW-480 cell lines transfected with sense- or antisense-OPN eukaryotic expression plasmids by flow cytometry and cell adhesion assay. Florescence redistribution after photobleaching (FRAP) was used to study gap functional intercellular communication (GJIC) among OPN-transfected cells. It was found that OPN was highly expressed in metastatic hepatic lesions from CRC compared to primary CRC tissue and adjacent normal mucosa. The expression of OPN mRNA in tumor tissues was significantly related with the CRC stages. OPN expression was also detected in normal hepatocytes surrounding CRC metastatic lesions. Two known receptors of OPN, integrin αv and CD44v6 proteins, were strongly expressed in hepatocytes from normal liver. CRC cells with forced OPN expression exhibited increased heterotypic adhesion with endothelial cells and weakened intercellular communication. OPN plays a significant role in CRC metastasis to liver through interaction with its receptors in hepatocytes, decreased homotypic adhesion, and enhanced heterotypic adhesion.     

Lipid Raft Is Required for PSGL-1 Ligation Induced HL-60 Cell Adhesion on ICAM-1

P-selectin glycoprotein ligand-1 (PSGL-1) and integrins are adhesion molecules that play critical roles in host defense and innate immunity. PSGL-1 mediates leukocyte rolling and primes leukocytes for integrin-mediated adhesion. However, the mechanism that PSGL-1 as a rolling receptor in regulating integrin activation has not been well characterized. Here, we investigate the function of lipid raft in regulating PSGL-1 induced β2 integrin-mediated HL-60 cells adhesion. PSGL-1 ligation with antibody enhances the β2 integrin activation and β2 integrin-dependent adhesion to ICAM-1. Importantly, with the treatment of methyl-β-cyclodextrin (MβCD), we confirm the role of lipid raft in regulating the activation of β2 integrin. Furthermore, we find that the protein level of PSGL-1 decreased in raft fractions in MβCD treated cells. PSGL-1 ligation induces the recruitment of spleen tyrosine kinase (Syk), a tyrosine kinase and Vav1 (the pivotal downstream effector of Syk signaling pathway involved in cytoskeleton regulation) to lipid raft. Inhibition of Syk activity with pharmacologic inhibitor strongly reduces HL-60 cells adhesion, implicating Syk is crucial for PSGL-1 mediated β2 integrin activation. Taken together, we report that ligation of PSGL-1 on HL-60 cells activates β2 integrin, for which lipid raft integrity and Syk activation are responsible. These findings have shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion.     

Site-specific Phosphorylation of Kindlin-3 Protein Regulates Its Capacity to Control Cellular Responses Mediated by Integrin αIIbβ3

The contributions of integrins to cellular responses depend upon their activation, which is regulated by binding of proteins to their cytoplasmic tails. Kindlins are integrin cytoplasmic tail binding partners and are essential for optimal integrin activation, and kindlin-3 fulfills this role in hematopoietic cells. Here, we used human platelets and human erythroleukemia (HEL) cells, which express integrin α_IIbβ₃, to investigate whether phosphorylation of kindlin-3 regulates integrin activation. When HEL cells were stimulated with thrombopoietin or phorbol 12-myristate 13-acetate (PMA), α_IIbβ₃ became activated as evidenced by binding of an activation-specific monoclonal antibody and soluble fibrinogen, adherence and spreading on fibrinogen, colocalization of β₃ integrin and kindlin-3 in focal adhesions, and enhanced β₃ integrin-kindlin-3 association in immunoprecipitates. Kindlin-3 knockdown impaired adhesion and spreading on fibrinogen. Stimulation of HEL cells with agonists significantly increased kindlin-3 phosphorylation as detected by mass spectrometric sequencing. Thr⁴⁸² or Ser⁴⁸⁴ was identified as a phosphorylation site, which resides in a sequence not conserved in kindlin-1 or kindlin-2. These same residues were phosphorylated in kindlin-3 when platelets were stimulated with thrombin. When expressed in HEL cells, T482A/S484A kindlin-3 decreased soluble ligand binding and cell spreading on fibrinogen compared with wild-type kindlin-3. A membrane-permeable peptide containing residues 476–485 of kindlin-3 was introduced into HEL cells and platelets; adhesion and spreading of both cell types were blunted compared with a scrambled control peptide. These data identify a role of kindlin-3 phosphorylation in integrin β₃ activation and provide a basis for functional differences between kindlin-3 and the two other kindlin paralogs.     

Secreted Heat Shock Protein 90α Induces Colorectal Cancer Cell Invasion through CD91/LRP-1 and NF-κB-mediated Integrin αV Expression

HCT-8 colon cancer cells secreted heat shock protein 90α (HSP90α) and had increased invasiveness upon serum starvation. The concentrated conditioned medium of serum-starved HCT-8 cells was able to stimulate the migration and invasion of non-serum-starved cells, which could be prevented by treatment with an anti-HSP90α antibody. Recombinant HSP90α (rHSP90α) also enhanced HCT-8 cell migration and invasion, suggesting a stimulatory role of secreted HSP90α in cancer malignancy. HSP90α binding to CD91α and Neu was evidenced by the proximity ligation assay, and rHSP90α-induced HCT-8 cell invasion could be suppressed by the addition of anti-CD91α or anti-Neu antibodies. Via CD91α and Neu, rHSP90α selectively induced integrin α_V expression, and knockdown of integrin α_V efficiently blocked rHSP90α-induced HCT-8 cell invasion. rHSP90α induced the activities of ERK, PI3K/Akt, and NF-κB p65, but only NF-κB activation was involved in HSP90α-induced integrin α_V expression. Additionally, we investigated the serum levels of HSP90α and the expression status of tumor integrin α_V mRNA in colorectal cancer patients. Serum HSP90α levels of colorectal cancer patients were significantly higher than those of normal volunteers (p < 0.001). Patients with higher serum HSP90α levels significantly exhibited elevated levels of integrin α_V mRNA in tumor tissues as compared with adjacent non-tumor tissues (p < 0.001). Furthermore, tumor integrin α_V overexpression was significantly correlated with TNM (Tumor, Node, Metastasis) staging (p = 0.001).