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.

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.

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.

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.

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.

Bone marrow stroma-induced resistance of chronic lymphocytic leukemia cells to arsenic trioxide involves Mcl-1 upregulation and is overcome by inhibiting the PI3Kδ or PKCß signaling pathways.

CLL remains an incurable disease in spite of the many new compounds being studied. Arsenic trioxide (ATO) induces apoptosis in all CLL cell types and could constitute an efficient therapy. To further explore this, we have studied the influence of stromal cells, key components of the CLL microenvironment, on the response of CLL cells to ATO. Bone marrow stromal cells induced CLL cell resistance to 2 µM ATO and led to activation of Lyn, ERK, PI3K and PKC, as well as NF-κB and STAT3. Mcl-1, Bcl-xL, and Bfl-1 were also upregulated after the co-culture. Inhibition experiments indicated that PI3K and PKC were involved in the resistance to ATO induced by stroma. Moreover, idelalisib and sotrastaurin, specific inhibitors for PI3Kδ and PKCß, respectively, inhibited Akt phosphorylation, NF-κB/STAT3 activation and Mcl-1 upregulation, and rendered cells sensitive to ATO. Mcl-1 was central to the mechanism of resistance to ATO, since: 1) Mcl-1 levels correlated with the CLL cell response to ATO, and 2) blocking Mcl-1 expression or function with specific siRNAs or inhibitors overcame the protecting effect of stroma. We have therefore identified the mechanism involved in the CLL cell resistance to ATO induced by bone marrow stroma and show that idelalisib or sotrastaurin block this mechanism and restore sensibility to ATO. Combination of ATO with these inhibitors may thus constitute an efficient treatment for CLL.

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.

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.

Overexpression of progelatinase B/proMMP-9 affects migration regulatory pathways and impairs chronic lymphocytic leukemia cell homing to bone marrow and spleen.

This study addresses the role of (pro)MMP-9 overexpression in CLL cell migration. We have used primary CLL cells and CLL-derived MEC-1 cells transfected with empty (mock cells) or proMMP-9-encoding (MMP-9 cells) lentiviral vectors. The constitutive (pro)MMP-9 expression in mock cells and primary CLL cells was similar, whereas in MMP-9 cells, expression resembled that of CLL cells incubated with proMMP-9. In xenograft models, in NOD/SCID mice, MMP-9-MEC-1 transfectants showed significantly reduced homing to bone marrow and spleen compared with mock cells. Likewise, incubation of primary CLL cells with proMMP-9, before injection into mice, inhibited their homing to these organs. This inhibition was specific, dose-dependent, and observed in all CLL tested, independently of prognostic markers or disease stage. Additionally, the MMP-9 catalytic activity was only partially involved, as the inactive mutant proMMP-9MutE had a partial effect. MMP-9 cells also showed impaired migration in vitro, which was reverted by reducing (pro)MMP-9 expression with siRNAs. CLL migration thus requires optimal (pro)MMP-9 expression levels, below or above which migration is hampered. Biochemical analysis of the (pro)MMP-9 effect indicated that MMP-9 cells or primary CLL cells incubated with proMMP-9 had reduced activation of migration regulatory molecules, including RhoAGTPase, Akt, ERK, and FAK. In contrast, p190RhoGAP (RhoA inhibitor) and PTEN (Akt/ERK/FAK inhibitor) were up-regulated in MMP-9 cells. Reduction of (pro)MMP-9 expression by siRNAs restored RhoA activity and diminished PTEN levels. Our results reveal a novel function for (pro)MMP-9 in modulating signaling pathways leading to CLL cell arrest. Therefore, local high (pro)MMP-9 expression may contribute to malignant cell retention in lymphoid organs and disease progression.

Matrix metalloproteinase-9 is involved in chronic lymphocytic leukemia cell response to fludarabine and arsenic trioxide.

BACKGROUND: Matrix metalloproteinase-9 (MMP-9) contributes to chronic lymphocytic leukemia (CLL) pathology by regulating cell migration and preventing spontaneous apoptosis. It is not known if MMP-9 is involved in CLL cell response to chemotherapy and we address this in the present study, using arsenic trioxide (ATO) and fludarabine as examples of cytotoxic drugs. METHODS: We used primary cells from the peripheral blood of CLL patients and MEC-1 cells stably transfected with an empty vector or a vector containing MMP-9. The effect of ATO and fludarabine was determined by flow cytometry and by the MTT assay. Expression of mRNA was measured by RT-PCR and qPCR. Secreted and cell-bound MMP-9 was analyzed by gelatin zymography and flow cytometry, respectively. Protein expression was analyzed by Western blotting and immunoprecipitation. Statistical analyses were performed using the two-tailed Student's t-test. RESULTS: In response to ATO or fludarabine, CLL cells transcriptionally upregulated MMP-9, preceding the onset of apoptosis. Upregulated MMP-9 primarily localized to the membrane of early apoptotic cells and blocking apoptosis with Z-VAD prevented MMP-9 upregulation, thus linking MMP-9 to the apoptotic process. Culturing CLL cells on MMP-9 or stromal cells induced drug resistance, which was overcome by anti-MMP-9 antibodies. Accordingly, MMP-9-MEC-1 transfectants showed higher viability upon drug treatment than Mock-MEC-1 cells, and this effect was blocked by silencing MMP-9 with specific siRNAs. Following drug exposure, expression of anti-apoptotic proteins (Mcl-1, Bcl-xL, Bcl-2) and the Mcl-1/Bim, Mcl-1/Noxa, Bcl-2/Bax ratios were higher in MMP-9-cells than in Mock-cells. Similar results were obtained upon culturing primary CLL cells on MMP-9. CONCLUSIONS: Our study describes for the first time that MMP-9 induces drug resistance by modulating proteins of the Bcl-2 family and upregulating the corresponding anti-apoptotic/pro-apoptotic ratios. This is a novel role for MMP-9 contributing to CLL progression. Targeting MMP-9 in combined therapies may thus improve CLL response to treatment.

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.

Overexpression of progelatinase B/proMMP-9 affects migration regulatory pathways and impairs chronic lymphocytic leukemia cell homing to bone marrow and spleen.

This study addresses the role of (pro)MMP-9 overexpression in CLL cell migration. We have used primary CLL cells and CLL-derived MEC-1 cells transfected with empty (mock cells) or proMMP-9-encoding (MMP-9 cells) lentiviral vectors. The constitutive (pro)MMP-9 expression in mock cells and primary CLL cells was similar, whereas in MMP-9 cells, expression resembled that of CLL cells incubated with proMMP-9. In xenograft models, in NOD/SCID mice, MMP-9-MEC-1 transfectants showed significantly reduced homing to bone marrow and spleen compared with mock cells. Likewise, incubation of primary CLL cells with proMMP-9, before injection into mice, inhibited their homing to these organs. This inhibition was specific, dose-dependent, and observed in all CLL tested, independently of prognostic markers or disease stage. Additionally, the MMP-9 catalytic activity was only partially involved, as the inactive mutant proMMP-9MutE had a partial effect. MMP-9 cells also showed impaired migration in vitro, which was reverted by reducing (pro)MMP-9 expression with siRNAs. CLL migration thus requires optimal (pro)MMP-9 expression levels, below or above which migration is hampered. Biochemical analysis of the (pro)MMP-9 effect indicated that MMP-9 cells or primary CLL cells incubated with proMMP-9 had reduced activation of migration regulatory molecules, including RhoAGTPase, Akt, ERK, and FAK. In contrast, p190RhoGAP (RhoA inhibitor) and PTEN (Akt/ERK/FAK inhibitor) were up-regulated in MMP-9 cells. Reduction of (pro)MMP-9 expression by siRNAs restored RhoA activity and diminished PTEN levels. Our results reveal a novel function for (pro)MMP-9 in modulating signaling pathways leading to CLL cell arrest. Therefore, local high (pro)MMP-9 expression may contribute to malignant cell retention in lymphoid organs and disease progression.

The dioxin receptor controls ß1 integrin activation in fibroblasts through a Cbp-Csk-Src pathway.

Recent studies have suggested a regulatory role for the dioxin receptor (AhR) in cell adhesion and migration. Following our previous work, we report here that the C-terminal Src kinase-binding protein (Cbp) signaling pathway controls ß1 integrin activation and that this mechanism is AhR dependent. T-FGM AhR-/- fibroblasts displayed higher integrin ß1 activation, revealed by the increased binding of the activation reporter 9EG7 anti-ß1 mAb and of a soluble fibronectin fragment, as well as by enhanced talin-ß1 association. AhR-/- fibroblasts also showed increased fibronectin secretion and impaired directional migration. Notably, interfering Cbp expression in AhR-/- fibroblasts reduced ß1 integrin activation, improved cell migration and rescued wild-type cell morphology. Cbp over-expression in T-FGM AhR-/- cells enhanced the formation of inhibitory Csk-Cbp complexes which in turn reduced c-Src p-Tyr(416) activation and focal adhesion kinase (FAK) phosphorylation at the c-Src-responsive residues p-Tyr(576) and p-Tyr(577). The c-Src target and migration-related protein Cav1 was also hypophosphorylated at p-Tyr(14) in AhR-/- cells, and such effect was rescued by down-modulating Cbp levels. Thus, AhR regulates fibroblast migration by modulating ß1 integrin activation via Cbp-dependent, Src-mediated signaling.

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.