ICAP-1 loss impairs CD8+ thymocyte development and leads to reduced marginal zone B cells in mice.

ICAP-1 regulates ß1-integrin activation and cell adhesion. Here, we used ICAP-1-null mice to study ICAP-1 potential involvement during immune cell development and function. Integrin α4ß1-dependent adhesion was comparable between ICAP-1-null and control thymocytes, but lack of ICAP-1 caused a defective single-positive (SP) CD8+ cell generation, thus, unveiling an ICAP-1 involvement in SP thymocyte development. ICAP-1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP-1 and reduced levels in SP CD8+ thymocytes of Runx3, a transcription factor required for CD8+ thymocyte generation. In the spleen, ICAP-1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP-1-/- spleen T and B cells displayed upregulation of α4ß1-mediated adhesion, indicating that ICAP-1 negatively controls their attachment. Furthermore, CD3+ - and CD19+ -selected spleen cells from ICAP-1-null mice showed reduced proliferation in response to T- and B-cell stimuli, respectively. Finally, loss of ICAP-1 caused a remarkable decrease in marginal zone B- cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP-1 involvement in the generation of SP CD8+ thymocytes and in the control of marginal zone B-cell numbers.

Matrix metalloproteinase-9 induces a pro-angiogenic profile in chronic lymphocytic leukemia cells.

Increased angiogenesis is commonly observed in chronic lymphocytic leukemia (CLL) tissues in correlation with advanced disease. CLL cells express pro- and anti-angiogenic genes and acquire a pro-angiogenic pattern upon interaction with the microenvironment. Because MMP-9 (a microenvironment component) plays important roles in solid tumor angiogenesis, we have studied whether MMP-9 influenced the angiogenic pattern in CLL cells. Immunofluorescence analyses confirmed the presence of MMP-9 in CLL tissues. MMP-9 interaction with CLL cells increased their MMP-9 expression and secretion into the medium. Accordingly, the conditioned media of MMP-9-primed CLL cells significantly enhanced endothelial cell proliferation, compared to control cells. MMP-9 also increased VEGF and decreased TSP-1 and Ang-2 expression, all at the gene and protein level, inducing a pro-angiogenic pattern in CLL cells. Mechanistic analyses demonstrated that downregulation of the selected gene TSP-1 by MMP-9 involved α4ß1 integrin, Src kinase family activity and the STAT3 transcription factor. Regulation of angiogenic genes is a novel contribution of MMP-9 to CLL pathology.

A catalytically inactive gelatinase B/MMP-9 mutant impairs homing of chronic lymphocytic leukemia cells by altering migration regulatory pathways.

We previously showed that MMP-9 overexpression impairs migration of primary CLL cells and MEC-1 cells transfected with MMP-9. To determine the contribution of non-proteolytic activities to this effect we generated MEC-1 transfectants stably expressing catalytically inactive MMP-9MutE (MMP-9MutE-cells). In xenograft models in mice, MMP-9MutE-cells showed impaired homing to spleen and bone marrow, compared to cells transfected with empty vector (Mock-cells). In vitro transendothelial and random migration of MMP-9MutE-cells were also reduced. Biochemical analyses indicated that RhoAGTPase and p-Akt were not downregulated by MMP-9MutE, at difference with MMP-9. However, MMP-9MutE-cells or primary cells incubated with MMP-9MutE had significantly reduced p-ERK and increased PTEN, accounting for the impaired migration. Our results emphasize the role of non-proteolytic MMP-9 functions contributing to CLL progression.

Evaluation of the potential therapeutic benefits of macrophage reprogramming in multiple myeloma.

Tumor-associated macrophages (TAM) are important components of the multiple myeloma (MM) microenvironment that support malignant plasma cell survival and resistance to therapy. It has been proposed that macrophages (MØ) retain the capacity to change in response to stimuli that can restore their antitumor functions. Here, we investigated several approaches to reprogram MØ as a novel therapeutic strategy in MM. First, we found tumor-limiting and tumor-supporting capabilities for monocyte-derived M1-like MØ and M2-like MØ, respectively, when mixed with MM cells, both in vitro and in vivo. Multicolor confocal microscopy revealed that MM-associated MØ displayed a predominant M2-like phenotype in the bone marrow of MM patient samples, and a high expression of the pro-M2 cytokine macrophage migration inhibitory factor (MIF). To reprogram the protumoral M2-like MØ present in MM toward antitumoral M1-like MØ, we tested the pro-M1 cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) plus blockade of the M2 cytokines macrophage colony-stimulating factor or MIF. The combination of GM-CSF plus the MIF inhibitor 4-iodo-6-phenyl-pyrimidine achieved the best reprogramming responses toward an M1 profile, at both gene and protein expression levels, as well as remarkable tumoricidal effects. Furthermore, this combined treatment elicited MØ-dependent therapeutic responses in MM xenograft mouse models, which were linked to upregulation of M1 and reciprocal downregulation of M2 MØ markers. Our results reveal the therapeutic potential of reprogramming MØ in the context of MM.

Inhibition of MMP-9-dependent Degradation of Gelatin, but Not Other MMP-9 Substrates, by the MMP-9 Hemopexin Domain Blades 1 and 4.

Degradation and remodeling of the extracellular matrix by matrix metalloproteinases (MMPs) plays important roles in normal development, inflammation, and cancer. MMP-9 efficiently degrades the extracellular matrix component gelatin, and the hemopexin domain of MMP-9 (PEX9) inhibits this degradation. To study the molecular basis of this inhibition, we generated GST fusion proteins containing PEX9 or truncated forms corresponding to specific structural blades (B1-B4) of PEX9. GST-PEX9 inhibited MMP-9-driven gelatin proteolysis, measured by gelatin zymography, FITC-gelatin conversion, and DQ-gelatin degradation assays. However, GST-PEX9 did not prevent the degradation of other MMP-9 substrates, such as a fluorogenic peptide, αB crystalline, or nonmuscular actin. Therefore, PEX9 may inhibit gelatin degradation by shielding gelatin and specifically preventing its binding to MMP-9. Accordingly, GST-PEX9 also abolished the degradation of gelatin by MMP-2, confirming that PEX9 is not an MMP-9 antagonist. Moreover, GST-B4 and, to a lesser extent, GST-B1 also inhibited gelatin degradation by MMP-9, indicating that these regions are responsible for the inhibitory activity of PEX9. Accordingly, ELISAs demonstrated that GST-B4 and GST-B1 specifically bound to gelatin. Our results establish new functions of PEX9 attributed to blades B4 and B1 and should help in designing specific inhibitors of gelatin degradation.

Circular trimers of gelatinase B/matrix metalloproteinase-9 constitute a distinct population of functional enzyme molecules differentially regulated by tissue inhibitor of metalloproteinases-1.

Gelatinase B/matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) cleaves many substrates and is produced by most cell types as a zymogen, proMMP-9, in complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1). Natural proMMP-9 occurs as monomers, homomultimers and heterocomplexes, but our knowledge about the overall structure of proMMP-9 monomers and multimers is limited. We investigated biochemical, biophysical and functional characteristics of zymogen and activated forms of MMP-9 monomers and multimers. In contrast with a conventional notion of a dimeric nature of MMP-9 homomultimers, we demonstrate that these are reduction-sensitive trimers. Based on the information from electrophoresis, AFM and TEM, we generated a 3D structure model of the proMMP-9 trimer. Remarkably, the proMMP-9 trimers possessed a 50-fold higher affinity for TIMP-1 than the monomers. In vivo, this finding was reflected in a higher extent of TIMP-1 inhibition of angiogenesis induced by trimers compared with monomers. Our results show that proMMP-9 trimers constitute a novel structural and functional entity that is differentially regulated by TIMP-1.

A novel CD44-binding peptide from the pro-matrix metalloproteinase-9 hemopexin domain impairs adhesion and migration of chronic lymphocytic leukemia (CLL) cells.

(pro)MMP-9 binds to CLL cells through the PEX9 domain and contributes to CLL progression. To biochemically characterize this interaction and identify potential therapeutic targets, we prepared GST-PEX9 forms containing structural blades B1B2 or B3B4. We recently described a sequence in blade B4 (P3 sequence) that bound α4ß1 integrin and partially impaired cell adhesion and migration. We have now studied the possible contribution of the B1B2 region to cell interaction with PEX9. CLL cells bound to GST-B1B2 and CD44 was the primary receptor. GST-B1B2 inhibited CLL cell migration as effectively as GST-B3B4. Overlapping synthetic peptides spanning the B1B2 region identified the sequence FDAIAEIGNQLYLFKDGKYW, present in B1 and contained in peptide P6, as the most effective site. P6 inhibited cell adhesion to PEX9 in a dose-dependent manner and with an IC50 value of 90 µM. P6 also inhibited cell adhesion to hyaluronan but had no effect on adhesion to VCAM-1 (α4ß1 integrin ligand), confirming its specific interaction with CD44. Spatial localization analyses mapped P6 to the central cavity of PEX9, in close proximity to the previously identified P3 sequence. Both P6 and P3 equally impaired cell adhesion to (pro)MMP-9. Moreover, P6 synergistically cooperated with P3, resulting in complete inhibition of CLL cell binding to PEX9, chemotaxis, and transendothelial migration. Thus, P6 is a novel sequence in PEX9 involved in cell-PEX9/(pro)MMP-9 binding by interacting with CD44. Targeting both sites, P6 and P3, should efficiently prevent (pro)MMP-9 binding to CLL cells and its pathological consequences.

Arsenic trioxide synergistically potentiates the cytotoxic effect of fludarabine in chronic lymphocytic leukemia cells by further inactivating the Akt and ERK signaling pathways.

CLL remains an incurable disease, making it crucial to continue searching for new therapies efficient in all CLL cases. We have studied the effect of combining arsenic trioxide (ATO) with fludarabine, a frontline drug in CLL. We have found a synergistic interaction between 1 µM ATO and 5 µM fludarabine that significantly enhanced the cytotoxic effect of the individual drugs. Importantly, ATO sensitized fludarabine-resistant cells to the action of this drug. The mechanism behind this effect included the downregulation of phospho-Akt, phospho-ERK, and the Mcl-1/Bim and Bcl-2/Bax ratios. The combination of ATO and fludarabine partially overcame the survival effect induced by co-culturing CLL cells with stromal cells. Therefore, low concentrations of ATO combined with fludarabine may be an efficient therapeutic strategy in CLL patients.

Sphingosine-1-phosphate activates chemokine-promoted myeloma cell adhesion and migration involving α4ß1 integrin function.

Myeloma cell adhesion dependent on α4ß1 integrin is crucial for the progression of multiple myeloma (MM). The α4ß1-dependent myeloma cell adhesion is up-regulated by the chemokine CXCL12, and pharmacological blockade of the CXCL12 receptor CXCR4 leads to defective myeloma cell homing to bone marrow (BM). Sphingosine-1-phosphate (S1P) regulates immune cell trafficking upon binding to G-protein-coupled receptors. Here we show that myeloma cells express S1P1, a receptor for S1P. We found that S1P up-regulated the α4ß1-mediated myeloma cell adhesion and transendothelial migration stimulated by CXCL12. S1P promoted generation of high-affinity α4ß1 that efficiently bound the α4ß1 ligand VCAM-1, a finding that was associated with S1P-triggered increase in talin-ß1 integrin association. Furthermore, S1P cooperated with CXCL12 for enhancement of α4ß1-dependent adhesion strengthening and spreading. CXCL12 and S1P activated the DOCK2-Rac1 pathway, which was required for stimulation of myeloma cell adhesion involving α4ß1. Moreover, in vivo analyses indicated that S1P contributes to optimizing the interactions of MM cells with the BM microvasculture and for their lodging inside the bone marrow. The regulation of α4ß1-dependent adhesion and migration of myeloma cells by CXCL12-S1P combined activities might have important consequences for myeloma disease progression.

A 17-residue sequence from the matrix metalloproteinase-9 (MMP-9) hemopexin domain binds α4ß1 integrin and inhibits MMP-9-induced functions in chronic lymphocytic leukemia B cells.

We previously showed that pro-matrix metalloproteinase-9 (proMMP-9) binds to B chronic lymphocytic leukemia (B-CLL) cells and contributes to B-CLL progression by regulating cell migration and survival. Induction of cell survival involves a non-proteolytic mechanism and the proMMP-9 hemopexin domain (PEX9). To help design specific inhibitors of proMMP-9-cell binding, we have now characterized B-CLL cell interaction with the isolated PEX9. B-CLL cells bound soluble and immobilized GST-PEX9, but not GST, and binding was mediated by α4ß1 integrin. The ability to recognize PEX9 was observed in all 20 primary samples studied irrespective of their clinical stage or prognostic marker phenotype. By preparing truncated forms of GST-PEX9 containing structural blades B1B2 or B3B4, we have identified B3B4 as the primary α4ß1 integrin-interacting region within PEX9. Overlapping synthetic peptides spanning B3B4 were then tested in functional assays. Peptide P3 (FPGVPLDTHDVFQYREKAYFC), a sequence present in B4 or smaller versions of this sequence (peptides P3a/P3b), inhibited B-CLL cell adhesion to GST-PEX9 or proMMP-9, with IC(50) values of 138 and 279 µm, respectively. Mutating the two aspartate residues to alanine rendered the peptides inactive. An anti-P3 antibody also inhibited adhesion to GST-PEX9 and proMMP-9. GST-PEX9, GST-B3B4, and P3/P3a/P3b peptides inhibited B-CLL cell transendothelial migration, whereas the mutated peptide did not. B-CLL cell incubation with GST-PEX9 induced intracellular survival signals, namely Lyn phosphorylation and Mcl-1 up-regulation, and this was also prevented by the P3 peptides. The P3 sequence may, therefore, constitute an excellent target to prevent proMMP-9 contribution to B-CLL pathogenesis.

Molecular and functional characterization of mouse S5D-SRCRB: a new group B member of the scavenger receptor cysteine-rich superfamily.

The scavenger receptor cysteine-rich superfamily (SRCR-SF) members are transmembrane and/or secreted receptors exhibiting one or several repeats of a cysteine-rich protein module of ∼100 aa, named scavenger receptor cysteine-rich (SRCR). Two types of SRCR domains (A or B) have been reported, which differ in the number of coding exons and intradomain cysteines. Although no unifying function has been reported for SRCR-SF members, recognition of pathogen-associated molecular patterns (PAMPs) was recently shown for some of them. In this article, we report the structural and functional characterization of mouse S5D-SRCRB, a new group B member of the SRCR-SF. The s5d-srcrb gene maps at mouse chromosome 7 and encompasses 14 exons extending over 15 kb. The longest cDNA sequence found is 4286 bp in length and encodes a mature protein of 1371 aa, with a predicted M(r) of 144.6 kDa. Using an episomal mammalian-expression system, a glycosylated soluble recombinant form >200 kDa was obtained and used as immunogen for the generation of specific rat mAbs. Subsequent immunohistochemical and real-time PCR analysis showed significant S5D-SRCRB expression in murine genitourinary and digestive tracts. S5D-SRCRB was shown to bind endogenous extracellular matrix proteins (laminin and galectin-1), as well as PAMPs present on Gram-positive and Gram-negative bacteria and fungi. PAMP binding by S5D-SRCRB induced microbial aggregation and subsequent inhibition of PAMP-induced cytokine release. These abilities suggest that S5D-SRCRB might play a role in the innate defense and homeostasis of certain specialized epithelial surfaces.

VEGF/VEGFR2 interaction down-regulates matrix metalloproteinase-9 via STAT1 activation and inhibits B chronic lymphocytic leukemia cell migration.

B-cell chronic lymphocytic leukemia (B-CLL) migration involves several molecules, including matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF). We have studied whether VEGF regulates MMP-9. VEGF significantly reduced MMP-9 protein expression in a dose-dependent manner, measured by gelatin zymography. Blocking the VEGFR2 receptor restored MMP-9 levels, implicating this receptor in the observed effect. Down-regulation of MMP-9 by VEGF resulted in significant inhibition of B-CLL cell migration through Matrigel or human umbilical vein endothelial cells, confirming the crucial role of MMP-9 in these processes. Reverse-transcription polymerase chain reaction analyses revealed that VEGF regulated MMP-9 at the transcriptional level. Indeed, VEGF induced STAT1 tyrosine phosphorylation, and this was blocked by inhibiting VEGFR2. STAT1 was responsible for MMP-9 down-regulation, as STAT1 gene silencing restored MMP-9 production and B-CLL cell migration in the presence of VEGF. Thus, the levels of VEGF and MMP-9 influence B-CLL cell expansion and both molecules could constitute therapeutic targets for this disease.

Alpha4beta1 integrin and 190-kDa CD44v constitute a cell surface docking complex for gelatinase B/MMP-9 in chronic leukemic but not in normal B cells.

As B-cell chronic lymphocytic leukemia (B-CLL) progresses, malignant cells extravasate and infiltrate lymphoid tissues. Several molecules, including gelatinase B/MMP-9, contribute to these processes. Although mainly a secreted protease, some MMP-9 is present at the B-CLL cell surface and the function, mode of anchoring, and interactions of this MMP-9 are unknown. Here we show that anti-MMP-9 antibodies immunoprecipitated a 190-kDa CD44v isoform and alpha4beta1 integrin from B-CLL cells, but not from normal B cells. Function-blocking antibodies to alpha4beta1 or CD44, or transfection with specific siRNAs, decreased cell-associated proMMP-9 and increased the secreted form. B-CLL cells attached to and bound proMMP-9 and active MMP-9, and this was inhibited by blocking the expression or function of alpha4beta1 or CD44. The MMP-9 hemopexin domain was critical in these interactions. alpha4beta1 and 190-kDa CD44v (but not CD44H) formed a complex at the cell surface, since they both coimmunoprecipitated with anti-alpha4, anti-beta1, or anti-CD44 antibodies. Immunofluorescence analyses confirmed that alpha4beta1 and CD44v colocalized with MMP-9. Binding of proMMP-9 inhibited B-CLL cell migration, and this required MMP-9 proteolytic activity. Thus, we have identified alpha4beta1 and CD44v as a novel proMMP-9 cell surface docking complex and show that cell-associated MMP-9 may regulate B-CLL cell migration and arrest.

The CS1 segment of fibronectin is involved in human OSCC pathogenesis by mediating OSCC cell spreading, migration, and invasion.

BACKGROUND: The alternatively spliced V region or type III connecting segment III (IIICS) of fibronectin is important in early development, wound healing, and tumorigenesis, however, its role in oral cancer has not been fully investigated. Thus, we investigated the role of CS-1, a key site within the CSIII region of fibronectin, in human oral squamous cell carcinoma (OSCC). METHODS: To determine the expression of CS-1 in human normal and oral SCC tissue specimens immunohistochemical analyses were performed. The expression of CS1 was then associated with clinicopathological factors. To investigate the role of CS-1 in regulating OSCC cell spreading, migration and invasion, OSCC cells were assayed for spreading and migration in the presence of a CS-1 peptide or a CS-1 blocking peptide, and for invasion using Matrigel supplemented with these peptides. In addition, integrin alpha4siRNA or a focal adhesion kinase (FAK) anti-sense oligonucleotide was transfected into OSCC cells to examine the mechanistic role of integrin alpha4 or FAK in CS1-mediated cell spreading and migration, respectively. RESULTS: CS-1 expression levels were significantly higher in OSCC tissues compared to normal tissues (p < 0.05). Also, although, high levels of CS-1 expression were present in all OSCC tissue samples, low-grade tumors stained more intensely than high grade tumors. OSCC cell lines also expressed higher levels of CS-1 protein compared to normal human primary oral keratinocytes. There was no significant difference in total fibronectin expression between normal and OSCC tissues and cells. Inclusion of CS-1 in the in vitro assays enhanced OSCC cell spreading, migration and invasion, whereas the CS1 blocking peptide inhibited these processes. Suppression of integrin alpha4 significantly inhibited the CS1-mediated cell spreading. Furthermore, this migration was mediated by focal adhesion kinase (FAK), since FAK suppression significantly blocked the CS1-induced cell migration. CONCLUSION: These data indicate that the CS-1 site of fibronectin is involved in oral cancer pathogenesis and in regulating OSCC cell spreading, migration and invasion.

Molecular Players in Hematologic Tumor Cell Trafficking.

The trafficking of neoplastic cells represents a key process that contributes to progression of hematologic malignancies. Diapedesis of neoplastic cells across endothelium and perivascular cells is facilitated by adhesion molecules and chemokines, which act in concert to tightly regulate directional motility. Intravital microscopy provides spatio-temporal views of neoplastic cell trafficking, and is crucial for testing and developing therapies against hematologic cancers. Multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia (ALL) are hematologic malignancies characterized by continuous neoplastic cell trafficking during disease progression. A common feature of these neoplasias is the homing and infiltration of blood cancer cells into the bone marrow (BM), which favors growth and survival of the malignant cells. MM cells traffic between different BM niches and egress from BM at late disease stages. Besides the BM, CLL cells commonly home to lymph nodes (LNs) and spleen. Likewise, ALL cells also infiltrate extramedullary organs, such as the central nervous system, spleen, liver, and testicles. The α4ß1 integrin and the chemokine receptor CXCR4 are key molecules for MM, ALL, and CLL cell trafficking into and out of the BM. In addition, the chemokine receptor CCR7 controls CLL cell homing to LNs, and CXCR4, CCR7, and CXCR3 contribute to ALL cell migration across endothelia and the blood brain barrier. Some of these receptors are used as diagnostic markers for relapse and survival in ALL patients, and their level of expression allows clinicians to choose the appropriate treatments. In CLL, elevated α4ß1 expression is an established adverse prognostic marker, reinforcing its role in the disease expansion. Combining current chemotherapies with inhibitors of malignant cell trafficking could represent a useful therapy against these neoplasias. Moreover, immunotherapy using humanized antibodies, CAR-T cells, or immune check-point inhibitors together with agents targeting the migration of tumor cells could also restrict their survival. In this review, we provide a view of the molecular players that regulate the trafficking of neoplastic cells during development and progression of MM, CLL, and ALL, together with current therapies that target the malignant cells.

MMP-9 affects gene expression in chronic lymphocytic leukemia revealing CD99 as an MMP-9 target and a novel partner in malignant cell migration/arrest.

We previously showed that MMP-9 contributes to CLL pathology by regulating cell survival and migration and that, when present at high levels, MMP-9 induces cell arrest. To further explore the latter function, we studied whether MMP-9 influences the gene-expression profile in CLL. Microarray analyses rendered 131 differentially expressed genes in MEC-1 cells stably transfected with MMP-9 (MMP-9-cells) versus cells transfected with empty vector (Mock-cells). Ten out of twelve selected genes were also differentially expressed in MEC-1 cells expressing the catalytically inactive MMP-9MutE mutant (MMP-9MutE-cells). Incubation of primary CLL cells with MMP-9 or MMP-9MutE also regulated gene and protein expression, including CD99, CD226, CD52, and CD274. Because CD99 is involved in leukocyte transendothelial migration, we selected CD99 for functional and mechanistic studies. The link between MMP-9 and CD99 was reinforced with MMP-9 gene silencing studies, which resulted in CD99 upregulation. CD99 gene silencing significantly reduced CLL cell adhesion, chemotaxis and transendothelial migration, while CD99 overexpression increased cell migration. Mechanistic analyses indicated that MMP-9 downregulated CD99 via binding to α4ß1 integrin and subsequent inactivation of the Sp1 transcription factor. This MMP-9-induced mechanism is active in CLL lymphoid tissues, since CD99 expression and Sp1 phosphorylation was lower in bone marrow-derived CLL cells than in their peripheral blood counterparts. Our study establishes a new gene regulatory function for MMP-9 in CLL. It also identifies CD99 as an MMP-9 target and a novel contributor to CLL cell adhesion, migration and arrest. CD99 thus constitutes a new therapeutic target in CLL, complementary to MMP-9.

The heparin III-binding domain of fibronectin (III4-5 repeats) binds to fibronectin and inhibits fibronectin matrix assembly.

Fibronectin matrix assembly involves interactions among various regions of the molecule, which contribute to elongation and stabilization of the fibrils. In this study, we examined the possible role of the heparin III domain of fibronectin (repeats III4-5) in fibronectin fibrillogenesis. We show that a recombinant fragment comprising these repeats (FNIII4-5 fragment) blocked fibronectin fibril formation and the incorporation of 125I-fibronectin into cell layers. Binding assays using a biosensor revealed that FNIII4-5 bound fibronectin and the amino-terminal 70 kDa and 29 kDa fragments. It also bound to itself, indicating a previously unidentified self-association site in repeats III4-5. These interactions were specific since FNIII4-5 did not bind to the FNIII7-10 fragment, representing a central region in fibronectin. The fibronectin-binding property of the III4-5 domain, but not its matrix assembly inhibitory function, was apparently cryptic in larger fragments. By mutating the arginine residues in the WTPPRAQITGYRLTVGLTRR proteoglycan-binding sequence (HBP/III5 site) of FNIII4-5 [Moyano, J.V., Carnemolla, B., Albar, J.P., Leprini, A., Gaggero, B., Zardi, L., Garcia-Pardo, A., 1999. Cooperative role for activated alpha4beta1 integrin and chondroitin sulfate proteoglycans in cell adhesion to the heparin III domain of fibronectin. Identification of a novel heparin and cell binding sequence in repeat III5. J. Biol. Chem. 274, 135-142.], we found that the first two arginine residues in HBP/III5 were involved in the fibronectin-binding property of FNIII4-5, while the last two arginine residues in HBP/III5 were required for inhibition of matrix assembly and the binding of 125I-fibronectin to cell layers. Both properties appear to function independently from each other, depending on the conformation of the fibronectin dimer.

Alpha4beta1 integrin/ligand interaction inhibits alpha5beta1-induced stress fibers and focal adhesions via down-regulation of RhoA and induces melanoma cell migration.

We have studied the function of the Hep III fibronectin domain in the cytoskeletal response initiated by alpha5beta1 integrin-mediated adhesion. Melanoma cells formed stress fibers and focal adhesions on the RGD-containing FNIII7-10 fragment. Coimmobilization of FNIII4-5, a fragment spanning Hep III and containing the alpha4beta1 ligand H2 with FNIII7-10, or addition of soluble FNIII4-5 to cells preattached to FNIII7-10, inhibited stress fibers and induced cytoplasmic protrusions. This effect involved alpha4beta1 since: 1) mutations in H2 reverted the inhibition; 2) other alpha4beta1 ligands (CS-1, VCAM-1), an anti-alpha4 mAb, or alpha4 expression in HeLa cells inhibited stress fibers. This activity was apparently cryptic in fibronectin or large fibronectin fragments, but exposed upon proteolytic degradation. Indeed purified peptic fragments containing H2, inhibited stress fibers when mixed with FNIII7-10 or fibronectin. RhoA activation with LPA or transfection with V14RhoA reverted the inhibitory effect and induced stress fibers on FNIII7-10+FNIII4-5. Furthermore, addition of alpha4beta1 ligands to FNIII7-10, down-regulated RhoA and activated p190RhoGAP, which localized to cytoplasmic protrusions. alpha4beta1/ligand interaction induced cell migration, monitored by video microscopy and wound healing assays. These data indicate that alpha4beta1 provides an antagonistic signal to alpha5beta1 by interfering with the RhoA activation pathway and this leads to melanoma cell migration.

Role of the von Hippel-Lindau tumor suppressor gene in the formation of beta1-integrin fibrillar adhesions.

The von Hippel-Lindau tumor suppressor gene (VHL) is absent or inactivated in the VHLcancer syndrome and in most sporadic renal cancers. VHL is requiredfor the assembly of a proper extracellular fibronectin matrix, although the exact mechanism remains unknown. In this report, we demonstrate that 786-O renal cancer cells are unable to organize an adequate matrix even in the presence of an excess of exogenous fibronectin. Because the formation of integrin fibrillar adhesions plays a pivotal role in the organization of extracellular fibronectin, we next examined the expression and subcellular distribution of integrins in VHL- cells and their wild-type VHL stably transfected counterparts. The levels of beta1 and alphav integrins were increased in VHL- cells when compared with VHL+ transfectants. Early after plating, both VHL+ and VHL- cells were capable of assembling classic "patch-like" alphav focal contacts. As the culture advanced and cells became confluent, alphav integrins partly relocated to the intercellular junctions in VHL+ transfectants, which then developed large beta1 fibrillar-type adhesions and anchored firmly to the substrate. In contrast, confluent VHL- cells were unable to assemble beta1 fibrillar adhesions, and alphav focal contacts remained unchanged at all stages of the culture. Exogenous activation of beta1 integrins with either divalent cations or activating antibodies partly restored the capability of VHL- cells to assemble beta1 fibrillar adhesions and fibronectin fibers. Finally, pulse-chase studies of metabolically labeled 786-O cells revealed that the maturation of the common beta1-integrin chain was delayed in VHL- cells when compared with VHL+ cells. Our results show that VHL is an important regulator of integrins and is essential for the formation of beta1 fibrillar adhesions. These findings help to explain the abnormal extracellular matrix organization and increased motility of VHL- renal cancer cells.

Gene expression profile induced by arsenic trioxide in chronic lymphocytic leukemia cells reveals a central role for heme oxygenase-1 in apoptosis and regulation of matrix metalloproteinase-9.

CLL remains an incurable disease in spite of the many new compounds being tested. Arsenic trioxide (ATO) induces apoptosis in all CLL cell types and could constitute an efficient therapy. To further explore this, we have studied the gene expression profile induced by ATO in CLL cells. ATO modulated many genes, largely involved in oxidative stress, being HMOX1 the most upregulated gene, also induced at the protein level. ATO also increased MMP-9, as we previously observed, both at the mRNA and protein level. Using specific inhibitors, qPCR analyses, and gene silencing approaches we demonstrate that upregulation of MMP-9 by ATO involved activation of the p38 MAPK/AP-1 signaling pathway. Moreover, gene silencing HMOX1 or inhibiting HMOX1 activity enhanced p38 MAPK phosphorylation and c-jun expression/activation, resulting in transcriptional upregulation of MMP-9. Overexpression of HMOX1 or enhancement of its activity, had the opposite effect. Cell viability analyses upon modulation of HMOX1 expression or activity demonstrated that HMOX1 had a pro-apoptotic role and enhanced the cytotoxic effect of ATO in CLL cells. We have therefore identified a new mechanism in which HMOX1 plays a central role in the response of CLL cells to ATO and in the regulation of the anti-apoptotic protein MMP-9. Thus, HMOX1 arises as a new therapeutic target in CLL and the combination of HMOX1 modulators with ATO may constitute an efficient therapeutic strategy in CLL.