ZAP-70 expression is associated with enhanced ability to respond to migratory and survival signals in B-cell chronic lymphocytic leukemia (B-CLL)

Molecular markers like IgV(H) mutational status, chromosomal abnormalities, and CD38 and ZAP-70 expression have prognostic value in B-cell chronic lymphocytic leukemia (B-CLL). These may be pathogenetic because of the coincidental expression of ZAP-70 and increased B-cell receptor (BCR) signaling and the signaling function of CD38 in CLL. This study shows that ZAP-70(+) CLL B cells respond in vitro more readily than ZAP-70(-) CLL and normal B cells to chemokine migratory signals through enhanced surface CCR7 expression (P = .009; P < .001) and increased responsiveness to its ligands CCL19 and CCL21, demonstrated by F-actin polymerization (P < .05) and cellular migration (P < .01). In addition, ZAP-70(+) CLL cells exhibit sustained ERK phosphorylation/activation following stimulation with CXCL12 (SDF1-alpha, a survival factor produced by stromal cells) compared with ZAP-70(-) cells (P = .004). Following coculture with nurse-like cells, the survival of ZAP-70(+) but not ZAP-70(-) CLL cells is significantly enhanced by the addition of CXCL12 (P < .05), an effect that is partially blocked by the MEK inhibitor PD98059. These advantageous migratory and survival responses may promote easier access to and greater proliferation in pseudo-germinal centers and explain in part the more progressive nature of ZAP-70(+) disease.     

Signaling through ZAP-70 is required for CXCL12-mediated T-cell transendothelial migration

Transendothelial migration of activated lymphocytes from the blood into the tissues is an essential step for immune functions. The housekeeping chemokine CXCL12 (or stroma cell-derived factor-1alpha), a highly efficient chemoattractant for T lymphocytes, drives lymphocytes to sites where they are highly likely to encounter antigens. This suggests that cross-talk between the T-cell receptor (TCR) and CXCR4 (the CXCL12 receptor) might occur within these sites. Here we show that the zeta-associated protein 70 (ZAP-70), a key element in TCR signaling, is required for CXCR4 signal transduction. The pharmacologic inhibition of ZAP-70, or the absence of ZAP-70 in Jurkat T cells and in primary CD4(+) T cells obtained from a patient with ZAP deficiency, resulted in an impairment of transendothelial migration that was rescued by the transfection of ZAP-70. Moreover, the overexpression of mutated forms of ZAP-70, whose kinase domain was inactivated, also abrogated the migratory response of Jurkat T cells to CXCL12. In contrast, no involvement of ZAP-70 in T-cell arrest on inflammatory endothelium under flow conditions or in CXCL12-induced actin polymerization was observed. Furthermore, CXCL12 induced time-dependent phosphorylation of ZAP-70, Vav1, and extracellular signal-regulated kinases (ERKs); the latter were reduced in the absence of functional ZAP-70. However, though a dominant-negative Vav1 mutant (Vav1 L213A) blocked CXCL12-induced T-cell migration, pharmacologic inhibition of the ERK pathway did not affect migration, suggesting that ERK activation is dispensable for T-cell chemotaxis. We conclude that cross-talk between the ZAP-70 signaling pathway and the chemokine receptor CXCR4 is required for T-cell migration.     

ZAP-70 enhances migration of malignant B lymphocytes toward CCL21 by inducing CCR7 expression via IgM-ERK1/2 activation

ZAP-70 in chronic lymphocytic leukemia (CLL) has been associated with enhanced B-cell receptor (BCR) signaling, survival, and migration. We investigated whether ZAP-70 can directly govern migration and the underlying mechanisms. In the ZAP-70 stably transfected Ramos cell line, IgM stimulation, but no IgD, enhanced phosphorylation of ERK1/2, Akt and Syk, and delayed IgM and CD79b internalization. In contrast, in the Raji cell line, where ZAP-70 was constitutively phosphorylated, ERK1/2, but not Akt, was phosphorylated, suggesting that MAPK pathway mediates ZAP-70 effects. BCR stimulation modulated the expression of CCR7, CXCR4, CXCR5, CD44, CD49d, and CD62L, which were up-regulated in ZAP-70-positive CLL primary subclones. The most dramatic change after BCR engagement in ZAP-70-transfected cells was CCR7 up-regulation, this being impaired by ERK1/2 inhibition and translating into both increased signaling and migration toward CCL21. Primary CLL subclones with high ZAP-70 expression showed increased migration toward CCL21. In conclusion, ZAP-70 ectopic expression led to enhanced BCR signaling after IgM stimulation and increased the expression of CCR7 predominantly via ERK1/2, increasing the response and migration toward CCL21. In primary CLL samples, cellular subsets with high ZAP-70 expression had increased expression of adhesion molecules and chemokine receptors in addition to an enhanced ability to migrate toward CCL21.     

CD38 and ZAP-70 are functionally linked and mark CLL cells with high migratory potential

Our interest in chronic lymphocytic leukemia (CLL) derives primarily from the exploitation of human diseases as strategic models for defining the in vivo biological roles of CD38. Using this model, we showed that CD38 triggers robust proliferation/survival signals modulated through the interactions with the CD31 ligand expressed by nurse-like cells and by the stromal/endothelial components. By analyzing a cohort of 56 patients with clinically and molecularly characterized CLL, we show that (1) patients with CD38(+)/ZAP-70(+) are characterized by enhanced migration toward Stromal derived factor-1alpha (SDF-1alpha)/CXCL12; (2) CD38 ligation leads to tyrosine phosphorylation of ZAP-70, showing that these markers are functionally linked; (3) ZAP-70 represents a limiting factor for the CD38 pathway in the CLL context, as shown by studying CD38-mediated signal transduction in 26 molecularly characterized patients; and (4) the CLL subgroup of patients defined on the basis of migratory potential is marked by a specific genetic signature, with a significant number of differentially expressed genes being involved in cell-cell interactions and movement. Altogether, the results of this work provide biological evidence for why the combined analysis of CD38 and ZAP-70 expression as determined in several clinical trials results in more dependable identification of patients with CLL who have aggressive disease.     

ZAP-70 enhances B-cell-receptor signaling despite absent or inefficient tyrosine kinase activation in chronic lymphocytic leukemia and lymphoma B cells.

Expression of ZAP-70 is an important negative prognostic factor in chronic lymphocytic leukemia (CLL). This protein tyrosine kinase is a key mediator of T-cell receptor (TCR) signaling and is structurally homologous to Syk, which plays an analogous role in B-cell receptor (BCR) signaling. Recent studies indicate that ZAP-70 may participate in BCR signaling as well, but the mechanism of action is not completely understood. We have now compared antigen receptor-induced activation of ZAP-70 in B cells and T cells by analyzing phosphorylation of critical regulatory tyrosine residues. We show that BCR-mediated activation of ZAP-70 is very inefficient in CLL and lymphoma B cells and is negligible when compared to activation of Syk. Despite the inefficient catalytic activation, the ability of ZAP-70 to recruit downstream signaling molecules in response to antigen receptor stimulation appeared relatively preserved. Moreover, ectopic expression of ZAP-70 enhanced and prolonged activation of several key mediators of BCR signaling, such as the Syk, ERK, and Akt kinases, and decreased the rate of ligand-mediated BCR internalization. We conclude that the role of ZAP-70 in BCR signaling is quite distinct from its role in TCR signaling and is likely mediated by inhibition of events that terminate the signaling response.     

Expression of ZAP-70 is associated with increased B-cell receptor signaling in chronic lymphocytic leukemia

We examined isolated leukemia B cells of patients with chronic lymphocytic leukemia (CLL) for expression of zeta-associated protein 70 (ZAP-70). CLL B cells that have nonmutated immunoglobulin variable region genes (V genes) expressed levels of ZAP-70 protein that were comparable to those expressed by normal blood T cells. In contrast, CLL B cells that had mutated immunoglobulin variable V genes, or that had low-level expression of CD38, generally did not express detectable amounts of ZAP-70 protein. Leukemia cells from identical twins with CLL were found discordant for expression of ZAP-70, suggesting that B-cell expression of ZAP-70 is not genetically predetermined. Ligation of the B-cell receptor (BCR) complex on CLL cells that expressed ZAP-70 induced significantly greater tyrosine phosphorylation of cytosolic proteins, including p72(Syk), than did similar stimulation of CLL cells that did not express ZAP-70. Also, exceptional cases of CLL cells that expressed mutated immunoglobulin V genes and ZAP-70 also experienced higher levels tyrosine phosphorylation of such cytosolic proteins following BCR ligation. Following BCR ligation, ZAP-70 underwent tyrosine phosphorylation and became associated with surface immunoglobulin and CD79b, arguing for the involvement of ZAP-70 in BCR signaling. These data indicate that expression of ZAP-70 is associated with enhanced signal transduction via the BCR complex, which may contribute to the more aggressive clinical course associated with CLL cells that express nonmutated immunoglobulin receptors.     

In-tandem insight from basic science combined with clinical research: CD38 as both marker and key component of the pathogenetic network underlying chronic lymphocytic leukemia

The absence of mutations in the IgV genes, together with the presence of ZAP-70 and CD38, are the most reliable negative prognostic markers for chronic lymphocytic leukemia (CLL) patients. Several lines of evidence indicate that CD38 may be not only a diagnostic marker but also a key element in the pathogenetic network in CLL. First, CD38 is a receptor that induces proliferation and increases survival of CLL cells. Second, CD38 signals start upon interaction with the CD31 ligand expressed by stromal and nurse-like cells. Third, CD38/CD31 contacts up-regulate CD100, a semaphorin involved in sustaining CLL growth. Fourth, evidence that nurselike cells express high levels of CD31 and plexin-B1, the high-affinity ligand for CD100, offers indirect confirmation for this model of receptor cross-talk. Elements of variation in the clinical course of CD38(+) CLL patients include (1) potential intersection with ZAP-70, a kinase involved in the CD38 signaling pathway in T and natural killer (NK) cells, and (2) the effects of genetic polymorphisms of the receptors involved, at least of CD38 and CD31. Consequently, CD38 together with ZAP-70 appear to be the key elements of a coreceptor pathway that may sustain the signals mediated by the B-cell receptor and potentially by chemokines and their receptors. This would result in acquisition of increased survival potential, providing clues to the poorer prognosis of CD38(+) patients.     

ZAP-70 is a novel conditional heat shock protein 90 (Hsp90) client: inhibition of Hsp90 leads to ZAP-70 degradation, apoptosis, and impaired signaling in chronic lymphocytic leukemia

The zeta-associated protein of 70 kDa (ZAP-70) is expressed in patients with aggressive chronic lymphocytic leukemia (CLL). We found that ZAP-70+ CLL cells expressed activated heat-shock protein 90 (Hsp90) with high binding affinity for Hsp90 inhibitors, such as 17-allyl-amino-demethoxy-geldanamycin (17-AAG), whereas normal lymphocytes or ZAP-70- CLL cells expressed nonactivated Hsp90. Activated Hsp90 bound and stabilized ZAP-70, which behaved like an Hsp90 client protein only in CLL cells. Treatment with Hsp90 inhibitors such as 17-AAG and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) induced ZAP-70 degradation and apoptosis in CLL cells but not in T cells, and also impaired B-cell receptor signaling in leukemia cells. Transduction of ZAP-70- CLL cells with an adenovirus encoding ZAP-70 activated Hsp90 and specifically rendered the leukemia cells sensitive to 17-AAG. These data indicate that Hsp90 is necessary for ZAP-70 expression and activity; that ZAP-70 is unique among Hsp90 clients, in that its chaperone-dependency is conditional on the cell type in which it is expressed; and also that ZAP-70 is required for cell survival and signaling in CLL. Additionally, ZAP-70 expression in CLL cells confers markedly heightened sensitivity to 17-AAG or 17-DMAG, suggesting that these or other Hsp90 inhibitors could be valuable therapeutically in patients with aggressive CLL.     

Pitfalls and limitations of ZAP-70 detection in chronic lymphocytic leukemia

Zeta-associated protein of 70 kDa (ZAP-70) is a tyrosine kinase that plays a role in signal transduction from the T-cell receptor. ZAP-70 is expressed in normal T-cells and NK-cells. Increased expression of ZAP-70 has been identified in chronic lymphocytic leukemia (CLL). CLL patients with increased ZAP-70 expression have significantly worse prognosis in terms of both progression-free survival and overall survival. There are several methods to quantify ZAP-70: polymerase chain reaction (PCR), immunoblotting, immunohistochemistry, and flow cytometry. Use of flow cytometry for ZAP-70 detection seems to be advantageous as this technique enables us to assess the presence of ZAP-70 separately on CLL clone, T-cells, and NK-cells. On the other hand, detection of ZAP-70 by flow cytometry is substantially influenced by many variables. The principal drawback of flow cytometry is the absence of consensus regarding selection of optimal anti-ZAP-70 antibody, fluorochrome conjugate, the most reliable staining technique, and optimal positivity threshold. This article summarizes pitfalls of flow cytometric analysis of ZAP-70 in CLL.     

ZAP-70 enhances IgM signaling independent of its kinase activity in chronic lymphocytic leukemia

We transduced chronic lymphocytic leukemia (CLL) cells lacking ZAP-70 with vectors encoding ZAP-70 or various mutant forms of ZAP-70 and monitored the response of transduced CLL cells to treatment with F(ab)(2) anti-IgM (anti-mu). CLL cells made to express ZAP-70, a kinase-defective ZAP-70 (ZAP-70-KA(369)), or a ZAP-70 unable to bind c-Cbl (ZAP-YF(292)) experienced greater intracellular calcium flux and had greater increases in the levels of phosphorylated p72(Syk), B-cell linker protein (BLNK), and phospholipase C-gamma, and greater activation of the Ig accessory molecule CD79b in response to treatment with anti-mu than did mock-transfected CLL cells lacking ZAP-70. Transfection of CLL cells with vectors encoding truncated forms of ZAP-70 revealed that the SH2 domain, but not the SH1 domain, was necessary to enhance intracellular calcium flux in response to treatment with anti-mu. We conclude that ZAP-70 most likely acts as an adapter protein that facilitates B-cell receptor (BCR) signaling in CLL cells independent of its tyrosine kinase activity or its ability to interact with c-Cbl.     

Pitfalls and limitations of ZAP-70 detection in chronic lymphocytic leukemia

Abstract

Zeta-associated protein of 70 kDa (ZAP-70) is a tyrosine kinase that plays a role in signal transduction from the T-cell receptor. ZAP-70 is expressed in normal T-cells and NK-cells. Increased expression of ZAP-70 has been identified in chronic lymphocytic leukemia (CLL). CLL patients with increased ZAP-70 expression have significantly worse prognosis in terms of both progression-free survival and overall survival. There are several methods to quantify ZAP-70: polymerase chain reaction (PCR), immunoblotting, immunohistochemistry, and flow cytometry. Use of flow cytometry for ZAP-70 detection seems to be advantageous as this technique enables us to assess the presence of ZAP-70 separately on CLL clone, T-cells, and NK-cells. On the other hand, detection of ZAP-70 by flow cytometry is substantially influenced by many variables. The principal drawback of flow cytometry is the absence of consensus regarding selection of optimal anti-ZAP-70 antibody, fluorochrome conjugate, the most reliable staining technique, and optimal positivity threshold. This article summarizes pitfalls of flow cytometric analysis of ZAP-70 in CLL.     

Roles of the MEK1/2 and AKT pathways in CXCL12/CXCR4 induced cholangiocarcinoma cell invasion

AIM:To evaluate the expression of C-X-C motif chemokine receptor 4(CXCR4)and its signaling cascades,which were previously identified as a key factor for cancer cell progression and metastasis,in cholangiocarcinoma cell lines.METHODS:The expression of CXCR4 and its signaling cascades were determined in the cholangiocarcinoma cell lines(RMCCA1 and KKU100)by Western blotting.The invasion assays and the detection of actin polymerization were tested in these cholangiocarcinoma cells treated with CXC chemokine ligand-12(CXCL12).RESULTS:Expression of CXCR4 was detected in both cholangiocarcinoma cell lines and activation of CXCR4 with CXCL12 triggered the signaling via the extracellular signal-regulated kinase-1/2(ERK1/2)and phosphoinositide 3-kinase(PI3K)and induction of cholangiocarcinoma cell invasion,and displayed high levels of actin polymerization.Addition of CXCR4 inhibitor(AMD3100)abrogated CXCL12-induced phosphorylation of MEK1/2 and Akt in these cells.Moreover,treatment with MEK1/2 inhibitor(U0126)or PI3K inhibitor(LY294 002)also attenuated the effect of CXCL12-induced cholangiocarcinoma cell invasion.CONCLUSION:These results indicated that the activation of CXCR4 and its signaling pathways(MEK1/2 and Akt)are essential for CXCL12-induced cholangiocarcinoma cell invasion.This rises Implications on a potential role for the inhibition of CXCR4 or its signal cascades in the treatment of cholangiocarcinoma.     

Mechanisms and clinical significance of BIM phosphorylation in chronic lymphocytic leukemia

B-cell receptor and microenvironment-derived signals promote accumulation of chronic lymphocytic leukemia (CLL) cells through increased proliferation and/or decreased apoptosis. In this study, we investigated the regulation of BIM, a proapoptotic BCL2-related protein, which is tightly regulated by phosphorylation. Surface IgM stimulation increased phosphorylation of 2 BIM isoforms, BIM(EL) and BIM(L), in a subset of CLL samples. In contrast, in normal B cells, anti-IgM triggered selective phosphorylation of BIM(EL) only. In CLL, anti-IgM-induced BIM phosphorylation correlated with unmutated IGHV gene status and with progressive disease. Strikingly, it was also associated with progressive disease within the mutated IGHV gene subset. BIM phosphorylation was dependent on MEK1/2 kinase activity, and we identified BIM(EL) serine 69, previously linked to pro-survival responses, as the major site of phosphorylation in CLL and in Ramos cells. BIM(EL)/BIM(L) phosphorylation was associated with release of the pro-survival protein MCL1. Coculture of CLL cells with HK cells, a model of the CLL microenvironment, promoted CLL cell survival and was associated with MEK1/2 activation and BIM(EL) phosphorylation. Hence, BIM phosphorylation appears to play a key role in apoptosis regulation in CLL cells, potentially coordinating antigen and microenvironment-derived survival signals. Antigen-mediated effects on BIM may be an important determinant of clinical behavior.     

Overexpression of the CXCR5 chemokine receptor, and its ligand, CXCL13 in B-cell chronic lymphocytic leukemia

CXCL13 is a homeostatic chemokine for lymphocyte homing and positioning within follicles of secondary lymphoid tissues, acting through its cognate receptor, CXCR5. Moreover, the CXCR5-CXCL13 axis plays a unique role in trafficking and homing of B1 cells. Here, we report that chronic lymphocytic leukemia (CLL) B cells express high levels of functional CXCR5. CXCR5 expression levels were similar on CLL B cells and normal CD5+ B cells, and higher compared with normal CD5- B cells, follicular B-helper T cells (T(FH) cells), or neoplastic B cells from other B-cell neoplasias. Stimulation of CLL cells with CXCL13 induces actin polymerization, CXCR5 endocytosis, chemotaxis, and prolonged activation of p44/42 mitogen-activated protein kinases. Anti-CXCR5 antibodies, pertussis toxin, and wortmannin inhibited chemotaxis to CXCL13, demonstrating the importance of Gi proteins and PI3 kinases for CXCR5 signaling. Moreover, CLL patients had significantly higher CXCL13 serum levels than volunteers, and CXCL13 levels correlated with beta2 microglobulin. We detected CXCL13 mRNA expression by nurselike cells, and high levels of CXCL13 protein in supernatants of CLL nurselike cell cultures. By immunohistochemistry, we detected CXCL13+ expression by CD68+ macrophages in situ within CLL lymph nodes. These data suggest that CXCR5 plays a role in CLL cell positioning and cognate interactions between CLL and CXCL13-secreting CD68+ accessory cells in lymphoid tissues.     

Prognostic significance of combined analysis of ZAP-70 and CD38 in chronic lymphocytic leukemia

The clinical heterogeneity that characterizes chronic lymphocytic leukemia (CLL) poses critical questions concerning the identification of high risk patients. Unmutated IgV(H) genes, CD38 and ZAP-70 expression have emerged as the most useful tools in identifying aggressive CLL. The simultaneous expression of ZAP-70 and CD38 in 157 patients with CLL has been evaluated. Fifty-seven patients (36%) were positive for ZAP-70 and 46 patients (29%) were positive for CD38. Both molecules were highly correlated and predictive of the clinical course of the disease. According to the simultaneous evaluation of ZAP-70 and CD38, patients were divided into three groups. In 81 patients (52%), there was a negative concordance of both molecules (ZAP-70(-)/CD38(-)); in 27 patients (17%) there was a positive concordance (ZAP-70(+)/CD38(+)); in 49 patients (31%) there was a discordant expression (ZAP-70(+)/CD38(-) and ZAP-70(-)/CD38(+)). A comparison of the clinical and laboratory data showed in ZAP-70(+)/CD38(+) patients a significantly higher bone marrow and peripheral blood lymphocytosis, lower hemoglobin levels, more advanced clinical stage, and higher number of unmutated IgV(H) status with respect to the other two groups. Furthermore, ZAP-70(+)/CD38(+) patients displayed a much shorter treatment-free interval (median 12 months vs 42 months in discordant patients and not reached in ZAP-70(-)CD38(-) patients). These results prove that the concomitant evaluation of ZAP-70 and CD38 expression allows the separation of CLL patients in prognostic subgroups and suggest that their simultaneous assessment should become an integral component of the CLL diagnostic grid.     

Flt3 ligand and the Flt3 receptor regulate hematopoietic cell migration by modulating the SDF-1alpha(CXCL12)/CXCR4 axis

Flt3 ligand (FL) enhances hematopoietic cell proliferation and facilitates hematopoietic stem cell mobilization in vivo, while the stromal-derived factor 1alpha (SDF-1alpha, CXC ligand 12 [CXCL12])/CXC receptor 4 (CXCR4) axis is critical for their homing and trafficking. We investigated if FL and its receptor, Flt3, functionally interact with CXCL12/CXCR4 to regulate hematopoietic cell migration. FL stimulated chemokinetic activity when used alone, but synergistically enhanced short-term migration of CD34+ cells, Ba/F3 cells expressing human Flt3 (Ba/F3-Flt3), and human RS4;11 acute leukemia cells, induced by CXCL12. Moreover, overexpression of constitutively activated internal tandem duplication (ITD)-Flt3 mutants in Ba/F3 cells dramatically enhanced migration toward CXCL12. In Ba/F3-Flt3 cells, synergistic cell migration to FL plus CXCL12 was associated with enhanced phosphorylation of mitogen-activated protein kinase p42/p44 (MAPK(p42/p44)), cyclic adenosine monophosphate response element binding protein (CREB), and Akt, and was partially inhibited by pretreatment of cells with selective inhibitors for MAPK(p42/p44), protein kinase A (PKA), or phosphatidylinositol 3-kinase (PI3-kinase), implicating these pathways in migration to FL plus CXCL12. In contrast, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulated CXCR4 expression, inhibited CXCL12-mediated phosphorylation of MAPK(p42/p44), CREB, and Akt, and impaired migration toward CXCL12. These findings suggest that FL/Flt3 may facilitate hematopoietic cell migration/homing and mobilization by enhancing or inhibiting CXCL12/CXCR4 signaling pathways and that the FL/Flt3 axis participates in trafficking of normal and transformed hematopoietic cells.     

CXCL4L1 and CXCL4 signaling in human lymphatic and microvascular endothelial cells and activated lymphocytes: involvement of mitogen-activated protein (MAP) kinases,Src and p70S6 kinase

CXC chemokines influence a variety of biological processes, such as angiogenesis, both in a physiological and pathological context. Platelet factor-4 (PF-4)/CXCL4 and its variant PF-4var/CXCL4L1 are known to favor angiostasis by inhibiting endothelial cell proliferation and chemotaxis. CXCL4L1 in particular is a potent inhibitor of angiogenesis with anti-tumoral characteristics, both through regulation of neovascularization and through attraction of activated lymphocytes. However, its underlying signaling pathways remain to be elucidated. Here, we have identified various intracellular pathways activated by CXCL4L1 in comparison with other CXCR3 ligands, including CXCL4 and interferon-γ-induced protein 10/CXCL10. Signaling experiments show involvement of the mitogen-activated protein kinase (MAPK) family in CXCR3A-transfected cells, activated lymphocytes and human microvascular endothelial cells (HMVEC). In CXCR3A transfectants, CXCL4 and CXCL4L1 activated p38 MAPK, as well as Src kinase within 30 and 5 min, respectively. Extracellular signal-regulated kinase (ERK) phosphorylation occured in activated lymphocytes, yet was inhibited in microvascular and lymphatic endothelial cells. CXCL4L1 and CXCL4 counterbalanced the angiogenic chemokine stromal cell-derived factor-1/CXCL12 in both endothelial cell types. Notably, inhibition of ERK signaling by CXCL4L1 and CXCL4 in lymphatic endothelial cells implies that these chemokines might also regulate lymphangiogenesis. Furthermore, CXCL4, CXCL4L1 and CXCL10 slightly enhanced forskolin-stimulated cAMP production in HMVEC. Finally, CXCL4, but not CXCL4L1, induced activation of p70S6 kinase within 5 min in HMVEC. Our findings confirm that the angiostatic chemokines CXCL4L1 and CXCL4 activate both CXCR3A and CXCR3B and bring new insights into the complexity of their signaling cascades.     

Functional expression of CXCR4 in somatotrophs: CXCL12 activates GH gene, GH production and secretion, and cellular proliferation

The interaction of chemokine (C-X-C motif) ligand 12 (CXCL12) and its receptor CXCR4 may play an important role in the regulation of anterior pituitary function. In this study, we investigated the expression of CXCL12 and CXCR4 and their role in normal rat pituitary and GH-producing GH3 tumor cell line. RT-PCR analysis and immunohistochemistry revealed that CXCR4 was expressed in normal rat anterior pituitary and GH3 tumor cells. Double immunofluorescent staining showed the complete colocalization of CXCR4 with GH in rat pituitary, indicating that CXCR4 is specifically expressed in rat somatotrophs. Using rat primary pituitary cell cultures and GH releasing hormone receptor expressing stable GH3 cells (GH3-GHRHR), we evaluated the function of CXCL12 compared with GHRH. CXCL12 stimulated GH gene activation in both primary rat anterior pituitary cells and GH3-GHRHR cells. CXCL12 also stimulated GH secretion from primary rat pituitary cells in a dose-dependant manner. BrdU incorporation was increased in response to CXCL12 addition in GH3 cell culture, indicating CXCL12-induced cell proliferation. CXCL12-dependent phosphorylation of ERK1/2 was also confirmed by western blot analysis, supporting the evidence that MAPK is an intracellular mediator of CXCL12/CXCR4 interaction in GH3 cell proliferation. In conclusion, these results indicate that CXCL12/CXCR4 interaction plays an important role in GH production, secretion, and the proliferation of somatotrophs.     

ZAP-70 directly enhances IgM signaling in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) B cells that express unmutated immunoglobulin heavy-chain variable region genes (IgV(H)) generally express ZAP-70, in contrast to normal B cells or most CLL cases with mutated IgV(H). Following IgM ligation, ZAP-70+ CLL cells had significantly higher levels of phosphorylated p72(Syk), BLNK, and phospholipase-Cgamma (PLCgamma) and had greater[Ca2+]i flux than did ZAP-70-negative CLL cases, including unusual ZAP-70-negative cases with unmutated IgV(H). IgM ligation of ZAP-70-negative CLL B cells infected with an adenovirus vector encoding ZAP-70 induced significantly greater levels of phosphorylated p72(Syk), BLNK, and PLCgamma and had greater[Ca2+]i flux than did similarly stimulated, noninfected CLL cells or CLL cells infected with a control adenovirus vector. We conclude that expression of ZAP-70 in CLL allows for more effective IgM signaling in CLL B cells, a feature that could contribute to the relatively aggressive clinical behavior generally associated with CLL cells that express unmutated IgV(H).