Heparan sulfate proteoglycan binding promotes APRIL-induced tumor cell proliferation

APRIL, a proliferation-inducing ligand, is a member of the tumor necrosis factor (TNF) family that is expressed by various types of tumors and influences their growth in vitro and in vivo. Two receptors, transmembrane activator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA), bind APRIL, but neither is essential for the tumor-promoting effects, suggesting that a third receptor exists. Here, we report that APRIL specifically binds to heparan sulfate proteoglycans (HSPG) on the surface of tumor cells. This binding is mediated by the heparin sulfate side chains and can be inhibited by heparin. Importantly, BCMA and HSPG do not compete, but can bind APRIL simultaneously, suggesting that different regions in APRIL are critical for either interaction. In agreement, mutation of three lysines in a putative heparin sulfate-binding motif, which is not part of the TNF fold, destroys interaction with HSPG, while binding to BCMA is unaffected. Finally, whereas interaction of APRIL with HSPG does not influence APRIL-induced proliferation of T cells, it is crucial for its tumor growth-promoting activities. We therefore conclude that either HSPG serve as a receptor for APRIL or that HSPG binding allows APRIL to interact with a receptor that promotes tumor growth.     

The TNF family members BAFF and APRIL: the growing complexity

B cell activating factor belonging to the TNF family (BAFF) and apoptosis-inducing ligand (APRIL) are two related members of the TNF ligand superfamily. Although they share two receptors, TACI and BCMA, transgenic and knockout mice in this system reveal that their functions are not redundant. BAFF is a critical survival/maturation factor for peripheral B cells and this activity is mediated through a BAFF-specific receptor, BAFF-R. Overexpression of BAFF has been linked to autoimmune disease and aspects of B cell neoplasia. APRIL appears to play a role in T-independent type II antigen responses and T cell survival, but can also induce proliferation/survival of non-lymphoid cells. Elevated expression of APRIL has been found in some tumor cell lines and in tumor tissue libraries. Therapies designed to inhibit the BAFF and APRIL pathways holds great promise for the future.     

B-lymphocyte stimulator/a proliferation-inducing ligand heterotrimers are elevated in the sera of patients with autoimmune disease and are neutralized by atacicept and B-cell maturation antigen-immunoglobulin

Introduction

B-lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) are members of the tumor necrosis factor (TNF) family that regulate B-cell maturation, survival, and function. They are overexpressed in a variety of autoimmune diseases and reportedly exist in vivo not only as homotrimers, but also as BLyS/APRIL heterotrimers.

Methods

A proprietary N-terminal trimerization domain was used to produce recombinant BLyS/APRIL heterotrimers. Heterotrimer biologic activity was compared with that of BLyS and APRIL in a 4-hour signaling assay by using transmembrane activator and CAML interactor (TACI)-transfected Jurkat cells and in a 4-day primary human B-cell proliferation assay. A bead-based immunoassay was developed to quantify native heterotrimers in human sera from healthy donors (n = 89) and patients with systemic lupus erythematosus (SLE; n = 89) or rheumatoid arthritis (RA; n = 30). Heterotrimer levels were compared with BLyS and APRIL homotrimer levels in a subset of these samples.

Results

The recombinant heterotrimers consisted mostly of one BLyS and two APRIL molecules. Heterotrimer signaling did not show any significant difference compared with APRIL in the TACI-Jurkat assay. Heterotrimers were less-potent inducers of B-cell proliferation than were homotrimeric BLyS or APRIL (EC₅₀, nMol/L: BLyS, 0.02; APRIL, 0.17; heterotrimers, 4.06). The soluble receptor fusion proteins atacicept and B-cell maturation antigen (BCMA)-immunoglobulin (Ig) neutralized the activity of BLyS, APRIL, and heterotrimers in both cellular assays, whereas B-cell activating factor belonging to the TNF family receptor (BAFF-R)-Ig neutralized only the activity of BLyS. In human sera, significantly more patients with SLE had detectable BLyS (67% versus 18%; P < 0.0001), APRIL (38% versus 3%; P < 0.0002), and heterotrimer (27% versus 8%; P = 0.0013) levels compared with healthy donors. Significantly more patients with RA had detectable APRIL, but not BLyS or heterotrimer, levels compared with healthy donors (83% versus 3%; P < 0.0001). Heterotrimer levels weakly correlated with BLyS, but not APRIL, levels.

Conclusions

Recombinant BLyS/APRIL heterotrimers have biologic activity and are inhibited by atacicept and BCMA-Ig, but not by BAFF-R-Ig. A novel immunoassay demonstrated that native BLyS/APRIL heterotrimers, as well as BLyS and APRIL homotrimers, are elevated in patients with autoimmune diseases.     

Identification of the sAPRIL Binding Peptide and Its Growth Inhibition Effects in the Colorectal Cancer Cells

Background

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) super family. It binds to its specific receptors and is involved in multiple processes during tumorigenesis and tumor cells proliferation. High levels of APRIL expression are closely correlated to the growth, metastasis, and 5-FU drug resistance of colorectal cancer. The aim of this study was to identify a specific APRIL binding peptide (BP) able to block APRIL activity that could be used as a potential treatment for colorectal cancer.

Methods

A phage display library was used to identify peptides that bound selectively to soluble recombinant human APRIL (sAPRIL). The peptides with the highest binding affinity for sAPRIL were identified using ELISA. The effects of sAPRIL-BP on cell proliferation and cell cycle/apoptosis in vitro were evaluated using the CCK-8 assay and flow cytometry, respectively. An in vivo mouse model of colorectal cancer was used to determine the anti-tumor efficacy of the sAPRIL-BP.

Results

Three candidate peptides were characterized from eight phage clones with high binding affinity for sAPRIL. The peptide with the highest affinity was selected for further characterization. The identified sAPRIL-BP suppressed tumor cell proliferation and cell cycle progression in LOVO cells in a dose-dependent manner. In vivo in a mouse colorectal challenge model, the sAPRIL-BP reduced the growth of tumor xenografts in nude mice by inhibiting proliferation and inducing apoptosis intratumorally. Moreover, in an in vivo metastasis model, sAPRIL-BP reduced liver metastasis of colorectal cancer cells.

Conclusions

sAPRIL-BP significantly suppressed tumor growth in vitro and in vivo and might be a candidate for treating colorectal cancers that express high levels of APRIL.     

Heparan Sulfate Proteoglycans Play a Dual Role in Regulating Fibroblast Growth Factor-2 Mitogenic Activity in Human Breast Cancer Cells

The human breast cancer cell lines MCF-7 and MDA-MB-231 differ in their responsiveness to fibroblast growth factor-2 (FGF-2). This growth factor stimulates proliferation in well-differentiated MCF-7 cells, whereas the less well-differentiated MDA-MB-231 cells are insensitive to this molecule. To investigate the potential regulation of FGF-2 mitogenic activity by heparan sulfate proteoglycans (HSPG), we have treated human breast cancer cells by glycosaminoglycan degrading enzymes or a metabolic inhibitor of proteoglycan sulfation: sodium chlorate. The interaction between FGF-2 and proteoglycans was assayed by examining the binding of¹²⁵I-FGF-2 to breast cancer cell cultures as well as to cationic membranes loaded with HSPG. Using MCF-7 cells, we showed that heparinase treatment inhibited FGF-2 binding to HSPG and completely abolished FGF-2 induced growth; chlorate treatment of MCF-7 cells decreased FGF-2 binding to HSPG and cell responsiveness in a dose-dependent manner. This demonstrates a requirement of adequately sulfated HSPG for FGF-2 growth-promoting activity on MCF-7 cells. In highly invasive MDA-MB-231 cells which produce twice as much HSPG as MCF-7 cells and which are not normally responsive to exogenously added FGF-2, chlorate treatment decreased FGF-2 binding to HSPG and induced FGF-2 mitogenic effect. This chlorate effect was dose dependent and observed at concentrations of 10–30 mM;higher chlorate concentrations completely abolished the FGF-2 effect. This shows that the HSPG level of sulfation can also negatively regulate the biological activity of FGF-2. Taken together, these results demonstrate a crucial role for HSPG in both positive and negative control of FGF-2 mitogenic activity in breast cancer cell proliferation.     

Structures of APRIL-receptor complexes: like BCMA, TACI employs only a single cysteine-rich domain for high affinity ligand binding

TACI is a member of the tumor necrosis factor receptor superfamily and serves as a key regulator of B cell function. TACI binds two ligands, APRIL and BAFF, with high affinity and contains two cysteine-rich domains (CRDs) in its extracellular region; in contrast, BCMA and BR3, the other known high affinity receptors for APRIL and BAFF, respectively, contain only a single or partial CRD. However, another form of TACI exists wherein the N-terminal CRD is removed by alternative splicing. We find that this shorter form is capable of ligand-induced cell signaling and that the second CRD alone (TACI_d2) contains full affinity for both ligands. Furthermore, we report the solution structure and alanine-scanning mutagenesis of TACI_d2 along with co-crystal structures of APRIL.TACI_d2 and APRIL.BCMA complexes that together reveal the mechanism by which TACI engages high affinity ligand binding through a single CRD, and we highlight sources of ligand-receptor specificity within the APRIL/BAFF system.     

The TNF family member APRIL promotes colorectal tumorigenesis

The tumor necrosis factor (TNF) family member APRIL (A proliferation inducing ligand) is a disease promoter in B-cell malignancies. APRIL has also been associated with a wide range of solid malignancies, including colorectal cancer (CRC). As evidence for a supportive role of APRIL in solid tumor formation was still lacking, we studied the involvement of APRIL in CRC. We observed that ectopic APRIL expression exacerbates the number and size of adenomas in Apc^Min mice and in a mouse model for colitis-associated colon carcinogenesis. Furthermore, knockdown of APRIL in primary spheroid cultures of colon cancer cells and both mouse and human CRC cell lines reduced tumor clonogenicity and in vivo outgrowth. Taken together, our data therefore indicate that both tumor-derived APRIL and APRIL produced by non-tumor cells is supportive in colorectal tumorigenesis.     

Interferon-Gamma-Induced Nitric Oxide Inhibits the Proliferation of Murine Renal Cell Carcinoma Cells

Renal cell carcinoma (RCC) remains one of the most resistant tumors to systemic chemotherapy, radiotherapy, and immunotherapy. Despite great progress in understanding the basic biology of RCC, the rate of responses in animal models and clinical trials using interferons (IFNs) has not improved significantly. It is likely that the lack of responses can be due to the tumor''s ability to develop tumor escape strategies. Currently, the use of targeted therapies has improved the clinical outcomes of patients with RCC and is associated with an increase of Th1-cytokine responses (IFNγ), indicating the importance of IFNγ in inhibiting tumor proliferation. Thus, the present study was designed to investigate a new mechanism by which IFNγ mediates direct anti-proliferative effects against murine renal cell carcinoma cell lines. When cultured RCC cell lines were exposed to murine recombinant IFNγ, a dose dependent growth inhibition in CL-2 and CL-19 cells was observed; this effect was not observed in Renca cells. Growth inhibition in CL-2 and CL-19 cell lines was associated with the intracellular induction of nitric oxide synthase (iNOS) protein, resulting in a sustained elevation of nitric oxide (NO) and citrulline, and a decrease in arginase activity. The inhibition of cell proliferation appears to be due to an arrest in the cell cycle. The results indicate that in certain RCC cell lines, IFNγ modulates L-arginine metabolism by shifting from arginase to iNOS activity, thereby developing a potent inhibitory mechanism to encumber tumor cell proliferation and survival. Elucidating the cellular events triggered by IFNγ in murine RCC cell lines will permit anti-tumor effects to be exploited in the development of new combination therapies that interfere with L-arginine metabolism to effectively combat RCC in patients.     

Heparanase degrades syndecan-1 and perlecan heparan sulfate: functional implications for tumor cell invasion

Heparanase (HPSE-1) is involved in the degradation of both cell-surface and extracellular matrix (ECM) heparan sulfate (HS) in normal and neoplastic tissues. Degradation of heparan sulfate proteoglycans (HSPG) in mammalian cells is dependent upon the enzymatic activity of HPSE-1, an endo-beta-d-glucuronidase, which cleaves HS using a specific endoglycosidic hydrolysis rather than an eliminase type of action. Elevated HPSE-1 levels are associated with metastatic cancers, directly implicating HPSE-1 in tumor progression. The mechanism of HPSE-1 action to promote tumor progression may involve multiple substrates because HS is present on both cell-surface and ECM proteoglycans. However, the specific targets of HPSE-1 action are not known. Of particular interest is the relationship between HPSE-1 and HSPG, known for their involvement in tumor progression. Syndecan-1, an HSPG, is ubiquitously expressed at the cell surface, and its role in cancer progression may depend upon its degradation. Conversely, another HSPG, perlecan, is an important component of basement membranes and ECM, which can promote invasive behavior. Down-regulation of perlecan expression suppresses the invasive behavior of neoplastic cells in vitro and inhibits tumor growth and angiogenesis in vivo. In this work we demonstrate the following. 1) HPSE-1 cleaves HS present on the cell surface of metastatic melanoma cells. 2) HPSE-1 specifically degrades HS chains of purified syndecan-1 or perlecan HS. 3) Syndecan-1 does not directly inhibit HPSE-1 enzymatic activity. 4) The presence of exogenous syndecan-1 inhibits HPSE-1-mediated invasive behavior of melanoma cells by in vitro chemoinvasion assays. 5) Inhibition of HPSE-1-induced invasion requires syndecan-1 HS chains. These results demonstrate that cell-surface syndecan-1 and ECM perlecan are degradative targets of HPSE-1, and syndecan-1 regulates HPSE-1 biological activity. This suggest that expression of syndecan-1 on the melanoma cell surface and its degradation by HPSE-1 are important determinants in the control of tumor cell invasion and metastasis.     

To target or not to target APRIL in systemic lupus erythematosus: that is the question!

Among the cytokines that regulate B-cell homeostasis are the TNF-like ligands B-lymphocyte stimulator (BLyS; also B-cell activation factor) and a proliferation-inducing ligand (APRIL). BLyS and APRIL share two receptors; that is, B-cell maturation antigen and transmembrane activator and CAML interactor. Therapeutic approaches using biologics are limited for treatment of lupus patients. One previously approved drug is belimumab, which antagonizes the B-cell stimulator BLyS. Atacicept, another biologic inhibiting BLyS and APRIL, was terminated for serious adverse events - raising the question of whether APRIL should be neutralized in autoimmune diseases.Treamtrakanpon and coworkers analyzed B-lymphocyte stimulator (BLyS; also B-cell activation factor) and a proliferation-inducing ligand (APRIL) expression in patients with lupus nephritis and observed a correlation with renal disease activity and APRIL serum levels [1]. In addition, the authors describe that, upon treatment with immunosuppressors, nonresponding patients had higher APRIL serum levels. They thus concluded that APRIL could be a potential biomarker for predicting difficult-to-treat cases of lupus nephritis, and propose the use of APRIL antagonists such as atacicept for treatment of lupus nephritis patients with high APRIL serum levels.These conclusions might be premature, as Treamtrakanpon and coworkers have not found a correlation between the level of APRIL in kidney tissue and renal disease activity. Another hypothesis could be that APRIL has a protective effect in autoimmune diseases. Indeed, the crucial role of BLyS in B-cell maintenance became evident by the analysis of BLyS-deficient mice displaying lower numbers of mature B cells and of BLyS transgenic mice developing severe B-cell hyperplasia. Although APRIL can trigger different B-cell responses in vitro, including proliferation and survival of human and murine B cells, it is less critical than BLyS in B-cell maintenance as APRIL knockout and transgenic mice reveal no gross abnormalities in lymphoid homeostasis [2]. In fact, APRIL was found to modulate specific B-cell responses such as IgA isotype switching, increased IgM secretion and B1 cell activity.Meanwhile, BLyS is an established promoter of B-cell-triggered autoimmmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, whereas the role of APRIL in these pathologies is rather controversial. Neutralizing BLyS with the mAb belimumab displayed a modest, although statistically significant, therapeutic effect in systemic lupus erythematosus [3,4]. But blocking both BLyS and APRIL with atacicept (TACI-Fc) was associated with a pronounced reduction of immunoglobulins, and occurrence of serious infections led to a premature termination of a phase II/III trial in lupus nephritis [5]. The combination of mycofenolate mofetil with atacicept may have contributed to the decrease of immunoglobulins. However, at acicept combined with another drug such as methotrexate in patients with rheumatoid arthritis was also associated with a significant reduction of immunoglobulins (especially IgM). In this autoimmune disease, atacicept failed to demonstrate efficacy on American College of Rheumatology 20 criteria [6]. In contrast, administration of belimumab showed a modest but significant efficacy using the same evaluation criteria in rheumatoid arthritis [7].These findings suggest distinct roles for BLyS and APRIL in lupus and other B-cell-mediated autoimmune diseases. Elevated serum levels are found for both cytokines in lupus patients, and for BLyS there is a consensus in the literature that this reflects its disease-promoting activity. Elevated APRIL serum levels, however, have been - depending on the respective study - either positively or negatively correlated with disease features [8]. One possible explanation for this discrepancy could be differences in the patient cohorts analyzed. A recent study by Jacob and colleagues analyzed a murine lupus model in APRIL-deficient mice and observed elevated numbers of splenocytes, increased autoantibody production and a tendency towards increased IgG production [9]. Notably, ectopic APRIL expression does not result - in contrast to BLyS transgenic mice - in lupus-like symptoms. In fact, we found that APRIL does dampen collagen-induced arthritis, the most common mouse model for human arthritis [10].Experimental mouse models for autoimmune diseases obviously cannot entirely mimic human diseases. Nevertheless, in vivo data are accumulating that do not support a disease-supporting role for APRIL in B-cell-mediated autoimmunity. The study by Treamtrakanpon and colleagues is putting forward the need to better elucidate the role of APRIL in B-cell-driven diseases before concluding a therapeutic approach.     

Extracellular matrix heparan sulfate proteoglycans modulate the mitogenic capacity of acidic fibroblast growth factor

Confluent cultures of human endothelial cells deposit into extracellular matrix (ECM) distinct heparan sulfate proteoglycans (HSPG) which modulate acidic fibroblast growth factor's (aFGF) ability to stimulate human endothelial cell mitogenic capacity. Extracellular matrix 35S-HSPG were isolated from cultures metabolically labelled with Na235SO4 by DEAE-Sepharose, Sepharose CL-4B, and aFGF-Affi-Gel 15 column chromatography and identified by resistance to chondroitinase ABC and sensitivity to nitrous acid. Fifty to sixty percent of the 35S-HSPG deposited into ECM do not bind aFGF. The bound 35S-HSGP (40-50% of the total counts applied) eluted from the aFGF-Affi-Gel column after the addition of buffer containing 2 M NaCl. aFGF-binding and aFGF-nonbinding 35S-HSPG were individually pooled and further purified by Sepharose CL-4B column chromatography. 35S-HSPG which bind aFGF, designated HSPGP, were 100-fold superior to heparin in augmenting the mitogenic efficacy of aFGF in sparse proliferating cultures. In contrast, however, 35S-HSPG, which did not bind aFGF, designated HSPG1, inhibited aFGF-stimulated proliferation in both sparse and subconfluent endothelial cell cultures. The majority of the biological activity of both aFGF-potentiating HSPGP and aFGF-inhibitory HSPG1 was contained in the glycosaminoglycan chains released by alkaline borohydride treatment of intact HSPGP or HSPG1, respectively. 3H-Core protein derived from HSPGP or HSPG1 contained only minor biological activity. The ability of heparitinase or heparinase (Flavobacterium heparinum) to abolish biological activity differed, depending upon the HSPG tested, also suggested that these are two distinct HSPGs.     

The role of BAFF and APRIL in rheumatoid arthritis

Development and activation of B cells quickly became clear after identifying new ligands and receptors in the tumor necrosis factor superfamily. B cell–activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are the members of membrane proteins Type 2 family released by proteolytic cleavage of furin to form active, soluble homotrimers. Except for B cells, ligands are expressed by all such immune cells like T cells, dendritic cells, monocytes, and macrophages. BAFF and APRIL have two common receptors, namely TNFR homolog transmembrane activator and Ca2+ modulator and CAML interactor (TACI) and B cell–maturation antigen. BAFF alone can also be coupled with a third receptor called BAFFR (also called BR3 or BLyS Receptor). These receptors are often expressed by immune cells in the B-cell lineage. The binding of BAFF or APRIL to their receptors supports B cells differentiation and proliferation, immunoglobulin production and the upregulation of B cell–effector molecules expression. It is possible that the overexpression of BAFF and APRIL contributes to the pathogenesis of autoimmune diseases. In BAFF transgenic mice, there is a pseudo-autoimmune manifestation, which is associated with an increase in B-lymphocytes, hyperglobulinemia, anti-single stranded DNA, and anti-double-stranded DNA antibodies, and immune complexes in their peripheral blood. Furthermore, overexpressing BAFF augments the number of peripheral B220+ B cells with a normal proliferation rate, high levels of Bcl2, and prolonged survival and hyperactivity. Therefore, in this review article, we studied BAFF and APRIL as important mediators in B-cell and discussed their role in rheumatoid arthritis.     

Alterations in both Heparan Sulfate Proteoglycans and Mitogenic Activity of Fibroblast Growth Factor-2 Are Triggered by Inhibitors of Proliferation in Normal and Breast Cancer Epithelial Cells

Heparan sulfate proteoglycans (HSPG) are involved in the regulation of cellular proliferation, differentiation, and migration. We have studied the effect of three inhibitors of proliferation on³⁵S incorporation into HSPG of the breast cancer cell lines MCF-7 and MDA-MB-231 and the normal breast epithelial cells (NBEC). Transforming growth factor β-1 (TGFβ-1), which inhibits the proliferation of NBEC, but not of MCF-7 and MDA-MB-231, cells induced an increase in³⁵S incorporation of HSPG in NBEC, but had no effect on cancer cells. Sodium butyrate (NaB), which inhibits NBEC as well as cancer cell proliferation, induced an increase in³⁵S incorporation into HSPG in all cell types studied. In contrast, retinoic acid had no effect on HSPG of breast epithelial cells. Modification of HSPG induced by TGFβ-1 or NaB treatments in normal and breast cancer epithelial cells resulted in an increase in¹²⁵I-fibroblast growth factor-2 (FGF-2) binding on HSPG. More importantly, NaB pretreatment resulted in an inhibition of the MCF-7 cell responsiveness to FGF-2, even though these cells remained sensitive to growth stimulation induced by serum or epidermal growth factor. These results indicate that changes in HSPG production are a key process involved in the mechanism of breast epithelial cell growth regulation.     

Characterization and high‐yield production of non‐N‐glycosylated recombinant human BCMA‐Fc in Pichia pastoris

B‐cell maturation antigen (BCMA) fused at the C‐terminus to the Fc portion of human IgG1 (BCMA‐Fc) blocks B‐cell activating factor (BAFF) and proliferation‐inducing ligand (APRIL)‐mediated B‐cell activation, leading to immune disorders. The fusion protein has been cloned and produced by several engineering cell lines. To reduce cost and enhance production, we attempted to express recombinant human BCMA‐Fc (rhBCMA‐Fc) in Pichia pastoris under the control of the AOX1 methanol‐inducible promoter. To produce the target protein with uniform molecular weight and reduced immunogenicity, we mutated two predicted N‐linked glycosylation sites. The secretory yield was improved by codon optimization of the target gene sequence. After fed‐batch fermentation under optimized conditions, the highest yield (207 mg/L) of rhBCMA‐Fc was obtained with high productivity (3.45 mg/L/h). The purified functional rhBCMA‐Fc possessed high‐binding affinity to APRIL and dose‐dependent inhibition of APRIL‐induced proliferative activity in vitro through three‐step purification. Thus, this yeast‐derived expression method could be a low‐cost and effective alternative to the production of rhBCMA‐Fc in mammalian cell lines.     

Thymus-independent class switch recombination is affected by APRIL

The tumour necrosis factor (TNF) family member a proliferation-inducing ligand (APRIL) is implicated in various B-cell processes, such as class switch recombination, plasma cell differentiation and plasma cell survival. This was suggested from initial studies analysing B-cell responses in APRIL-deficient and transgenic mice, and mice deficient for the TNF receptors of APRIL, transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen. Here, we present additional evidence for the importance of APRIL in thymus-independent (TI) B-cell responses, using APRIL-deficient and transgenic mice. APRIL-deficient mice show an impaired immunoglobulin A (IgA) response towards TI B-cell antigens, whereas APRIL transgenic mice show exaggerated TI B-cell responses. Moreover, antibody titres to TI antigens were sustained in APRIL transgenic mice for a long time and even increased up to 75 days in the case of IgA against 4-hydroxy-nitrophenacetyl-lipopolysaccharide.     

"APRIL hath put a spring of youth in everything": Relevance of APRIL for survival

A proliferation inducing ligand (APRIL or TALL-2 and TRDL-1) was first discovered as a cytokine over-expressed in many transformed cells and with the capacity to stimulate proliferation. APRIL was shown to bind two different receptors of the TNF receptor superfamily: B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), as well as heparan sulphate proteoglycans (HSPGs). APRIL has since been shown to play a physiological role in B cell biology, in particular the survival of plasma B cells in a specialized APRIL-rich niche. However, aberrant expression of APRIL and the subsequent activation of pro-survival pathways, is potentially the driving force for the survival of several B cell malignancies. APRIL has therefore become an important therapeutic target, but many questions regarding its mechanism of action still remain. It is for instance unclear what the exact physiological implications of the APRIL-HSPG interaction could be. Neither do we know the precise signals elicited by APRIL in normal or in malignant cells, and whether blocking these effects could provide real therapeutic gain in cancer patients. In this review we discuss the specific relevance of APRIL for cell survival, in terms of both its physiological role and its role in tumor biology, and highlight some of the key questions that will undoubtedly form the basis of future research in this field.     

Modified high-density lipoprotein modulates aldosterone release through scavenger receptors via extra cellular signal-regulated kinase and Janus kinase-dependent pathways

A proliferation-inducing ligand (APRIL) is overexpressed in most tumor cells and tissues, especially in tumors of the alimentary system, such as colorectal cancer (CRC), gastric cancer, and liver cancer. RNA interference (RNAi) has been proved to be a powerful tool for gene knockdown and holds great promise for the treatment of cancer. In this study, the efficacy of RNAi targeting APRIL was analyzed via relevant experiments on human CRC xenografted in BALB/c nude mice. Both the mRNA and protein levels of APRIL were examined after intratumoral injection of APRIL small interfering RNA (siRNA). Meanwhile, pathological tools were utilized to observe the alterations on the aspects of proliferation, metastasis, apoptosis and cellular necrosis by means of detecting proliferating cell nuclear antigen, Ki-67, MMP-2, MMP-9, TIMP-3, TIMP-4, Bcl-2, Bax and Bcl-xL of CRC. In addition, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) and hematoxylin and eosin staining were also conducted to examine cell apoptosis and necrosis. It was found that grafted human colorectal tumor growth and metastasis were obviously inhibited while tumor cell apoptosis and necrosis were induced after in vivo APRIL siRNA injection into nude mice. The data indicated that silencing of the APRIL gene using RNAi may serve as a novel therapeutic strategy for treatment of CRC.     

Molecular Characterization of Equine APRIL and its Expression Analysis During the Adipogenic Differentiation of Equine Adipose-Derived Stem Cell In Vitro

A proliferation inducing ligand (APRIL) is a member of the TNF superfamily. It shares two receptors with B-cell activating factor (BAFF), B-cell maturation antigen (BCMA), and transmembrane activator and CAML interactor (TACI). Herein, the equine APRIL was identified from equine adipose-derived stem cell (ASC), and the protein expression of APRIL and its related molecules were detected during the adipogenic differentiation of equine ASC in vitro. The equine APRIL gene was located on chromosome 11, spans 1852 base pairs (bp). Its open reading frame covers 753 bp, encoding a 250-amino acid protein with the typical TNF structure domain. During the two weeks’ adipogenic differentiation of equine ASC, although the protein expression of APRIL and TACI had an insignificant change, that of BCMA increased significantly. Moreover, with the addition of recombinant protein His₆-sAPRIL, a reduced differentiation of equine ASC toward adipocyte was detected. These results may provide the basis for investigating the role of APRIL in ASC adipogenic differentiation.