Involvement of the Receptor-Associated Prorenin System in the Pathogenesis of Human Conjunctival Lymphoma

Abstract

Purpose.: Extranodal marginal zone B-cell lymphoma (EMZL) is the most common subtype of conjunctival lymphoma, though its molecular mechanisms of pathogenesis are largely unknown. We attempted to explore the association of the renin-angiotensin system (RAS) and (pro)renin receptor ([P]RR) in the pathogenesis of conjunctival lymphoma.

Methods.: Surgically removed conjunctiva EMZL samples were used for gene expression, and immunohistochemical and immunofluorescence analyses of (P)RR and RAS components. Human B-lymphoblast IM-9 cells were treated with prorenin or angiotensin II (Ang II), and gene expression levels were analyzed using real-time quantitative PCR (qPCR). In addition, immunofluorescence analysis of EMZL samples was used to evaluate the in vivo expression of those components.

Results.: Gene expression and immunohistochemical analyses revealed the expression of RAS components, including (P)RR and angiotensin II type 1 receptor (AT1R), in EMZL tissues. Double-labeling analyses demonstrated that (P)RR and AT1R were detected in cells positive for CD20, a marker for B-cells, where they colocalized with prorenin and angiotensinogen, respectively. Prorenin stimulation of human B-lymphoblast IM-9 cells increased mRNA expression levels of fibroblast growth factor 2 (FGF2), while angiotensin II treatment upregulated the expression levels of basigin (BSG), matrix metallopeptidase (MMP)2, 9, and 14, which were abolished by (P)RR and AT1R blockades, respectively. Immunofluorescence analyses of clinical samples showed colocalizations of (P)RR and AT1R with the products of these upregulated genes.

Conclusions.: The present study suggests that activation of (P)RR and AT1R is associated with the pathogenesis of conjunctival EMZL by stimulating the production of FGF2 and MMPs.

Introduction

The conjunctiva is an ocular tissue that constitutes the first contact barrier against pathogens from the external environment. This barrier contains an immune system composed of conjunctival lymphoid tissue, which recruits B-cells, T-cells, and dendritic cells when the conjunctiva is exposed to pathogenic agents.¹ Conjunctival lymphoma is one of the common malignancies found in the ocular adnexa. Extranodal marginal zone B-cell lymphoma (EMZL), a malignant tumor involving inflammatory cells, constitutes 7% of non-Hodgkin's B-cell lymphoma cases, and is the most common histological subtype of conjunctival lymphoma.^2–4 Several studies reported that conjunctival EMZL develops as a result of orbital inflammation.^5,6 Moreover, we recently demonstrated that VEGF, a major angiogenic factor, was expressed in human EMZL tissues.⁷ However, little is known about the molecular mechanism of its pathogenesis. 

The renin-angiotensin system (RAS) traditionally has been regarded as a key regulatory mechanism for systemic blood pressure and water balance (circulatory RAS). Recently, components of RAS also were found to be expressed in various tissues independent of the circulatory RAS, hence called tissue RAS. Tissue RAS has diverse roles in the regulation of growth, inflammation and pathological vascular conditions in several organs.^8,9 (Pro)renin receptor ([P]RR) binds with prorenin to exert renin activity through the conformational change of the prorenin molecule (nonproteolytic activation of prorenin causing tissue RAS) instead of the conventional proteolysis of the prorenin prosegment by processing enzymes (proteolytic activation of prorenin causing circulatory RAS). Binding of prorenin to (P)RR triggers dual activation of RAS and RAS-independent signaling pathways, referred to as the receptor-associated prorenin system (RAPS), involved in the molecular pathogenesis of end-organ damage, such as inflammation and angiogenesis, including ocular disorders (e.g., proliferative diabetic retinopathy).^10–12 Blockades of RAS and/or RAPS resulted in beneficial effects on the onset and progression of various diseases.^9–11,13,14 There also is increasing evidence from experimental models and clinical studies that the inhibitors of RAS reduce tumor growth and metastasis,^14–17 suggesting a useful strategy against malignancies independent of their classical cardiovascular actions. In this study, we aimed to analyze the expression of RAS components in EMZL tissues and examined the role of RAS/RAPS in the pathogenesis of EMZL of the conjunctiva. 

Methods

Human Surgical Samples

Conjunctival EMZL samples (Ann Arbor classification, Stage 1E or 2E) were surgically removed from five patients and used for gene expression and immunohistochemical analyses. This study was conducted in accordance with the tenets of the Declaration of Helsinki and after receiving approval from the institutional review board of Hokkaido University Hospital. Written informed consent was obtained from all patients after an explanation of the purpose and procedures of the study. 

The presence of small- to medium-sized atypical lymphoid cells was confirmed by hematoxylin and eosin (H&E) staining. Immunohistochemistry with anti-CD3, CD5, CD10, CD20, and cyclin D1 (DAKO, Carpinteria, CA, USA), as well as the detection of κ/λ deviation were analyzed. Immunoglobulin heavy chain gene rearrangement was determined by Southern blot analysis or PCR methods, and flow cytometry was applied to confirm the B-cell monoclonality in the tumor tissue, as described previously.⁷ Systemic involvements were evaluated using positron emission tomography–computed tomography, magnetic resonance imaging, and bone marrow puncture. 

Cell Culture and Chemicals

Human B-lymphoblast IM-9 cells were obtained from American Type Culture Collection (Manassas, VA, USA), and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. 

To cover the handle region of the prorenin molecule, the binding site of (P)RR, decoy peptide NH₂-RIFLKRMPSI-COOH was synthesized as a human (P)RR blocker (PRRB).¹⁰ After serum deprivation, IM-9 cells were pretreated with 1 μM PRRB or 10 μM angiotensin II (Ang II) type 1 receptor (AT1R) blocker valsartan (Sigma-Aldrich Corp., St. Louis, MO, USA) for 1 hour. Prorenin or Ang II then was added at a final concentration of 10 nM or 1 μM, respectively. Cells were incubated for 24 hours and processed for analyses to detect the mRNA expression levels. 

Reverse Transcription-PCR (RT-PCR) and Real-Time Quantitative PCR (qPCR) Analyses

Total RNA was isolated from EMZL tissues and cells using TRIzol (Life Technologies, Carlsbad, CA, USA), according to the manufacturer's protocol. The RT was performed with GoScript Reverse Transcriptase (Promega, Madison, WI, USA) and oligo dT(20) primers, as described previously.^10,18 Real-time qPCR was performed using the GoTaq qPCR Master Mix (Promega) and StepOne plus System (Life Technologies). The quantity of mRNA expression was calculated by normalizing the threshold cycle (Ct) of the target genes to the Ct of hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene in the same sample, according to the comparative ddCt method. The primers are listed in the Table. 

Table

 

Primer Sequences Used in RT-PCR and Real-Time qPCR

Target Gene	Sequence
(P)RR	forward 5′‐AGG CAG TGT CAT TTC GTA CC‐3′
	reverse 5′‐GCC TTC CCT ACC ATA TAC ACT C‐3′
REN	forward 5′‐GTG TCT GTG GGG TCA TCC ACC TTG‐3′
	reverse 5′‐GGA TTC CTG AAA TAC ATA GTC CGT‐3′
AGT	forward 5′‐CTG CAA GGA TCT TAT GAC CTG C‐3′
	reverse 5′‐TAC ACA GCA AAC AGG AAT GGG C‐3′
ACE	forward 5′‐CCG AAA TAC GTG GAA CTC ATC AA‐3′
	reverse 5′‐CAC GAG TCC CCT GCA TCT ACA‐3′
AT1R	forward 5′‐AGG GCA GTA AAG TTT TCG TG‐3′
	reverse 5′‐CGG GCA TTG TTT TGG CAG TG‐3′
AT2R	forward 5′‐GGC CTG TTT GTC CTC ATT GC‐3′
	reverse 5′‐CAC GGG TTA TCC TGT TCT TC‐3′
HPRT1	forward 5′‐ACC CCA CGA AGT GTT GGA TA‐3′
	reverse 5′‐AAG CAG ATG GCC ACA GAA CT‐3′
FGF2	forward 5′‐GCG GCT GTA CTG CAA AAA ACG‐3′
	reverse 5′‐AAG TTG TAG CTT GAT GTG AGG G‐3′
BSG	forward 5′‐CCA TGC TGG TCT GCA AGT CAG‐3′
	reverse 5′‐CCG TTC ATG AGG GCC TTG TC‐3′
MMP2	forward 5′‐TGA TGG TGT CTG CTG GAA AG‐3′
	reverse 5′‐GAC ACG TGA AAA GTG CCT TG‐3′
MMP9	forward 5′‐TTG ACA GCG ACA AGA AGT GG‐3′
	reverse 5′‐GCC ATT CAC GTC GTC CTT AT‐3′
MMP14	forward 5′‐GAA GCC TGG CTA CAG CAA TAT G‐3′
	reverse 5′‐TGC AAG CCG TAA AAC TTC TGC‐3′

Immunohistochemistry

The EMZL tissue samples were fixed in 4% paraformaldehyde in the operating room soon after excision. Following fixation, the samples were preserved as paraffin-embedded blocks. The slides were dewaxed, rehydrated, and rinsed in PBS. As a pretreatment, microwave-based antigen retrieval was performed in 10 mM citrate buffer (pH 6.0). The slides were incubated with normal goat serum, followed by peroxidase block solution. Sections were incubated with rabbit polyclonal antibodies against human (P)RR (Sigma-Aldrich) and AT1R (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Visualization was performed using the Envision HRP kit (DAKO). Normal rabbit IgG was used as a negative antibody control. Sections were examined using a BIOREVO microscope (Keyence, Osaka, Japan). 

Immunofluorescence Microscopy

Immunofluorescence analyses were performed as described previously.^10,18 Sections were incubated with the following primary antibodies: rabbit anti-(P)RR, rabbit anti-AT1R, mouse anti-prorenin (Abcam, Cambridge, MA, USA ), goat anti-angiotensinogen (AGT; Santa Cruz Biotechnology), mouse anti-CD20 (DAKO), mouse anti-CD31 (DAKO), mouse anti-fibroblast growth factor 2 (FGF2; Millipore, Temecula, CA, USA), mouse anti-basigin (BSG; Millipore), goat anti-matrix metallopeptidase 2 (MMP2; Santa Cruz Biotechnology), goat anti-MMP9 (Santa Cruz Biotechnology), and mouse anti-MMP14 (Millipore) antibodies. Secondary antibodies for fluorescent detection were AlexaFluor 488 and 546 (Life Technologies). Sections were visualized under a BIOREVO microscope (Keyence). 

Statistical Analysis

All results were expressed as mean ± SD with n-numbers as indicated. Two-tailed Student's t-test was used for determining significant differences between the groups. Differences between the means were considered statistically significant when P < 0.05. 

Results

Expression and Localization of RAS Components in EMZL Tissues and IM-9 Cells

In previous reports, we showed that (P)RR and other RAS components were expressed in surgically excised fibrovascular tissues obtained from patients with proliferative diabetic retinopathy, human retinal cell lines, and the mouse retina.^8,10,18 To understand the pathological role of RAS/RAPS in conjunctival EMZL, we investigated the gene expression of RAS components in EMZL tissues and human B lymphoma cell line IM-9. Transcripts of RAS components were detected in all clinical EMZL samples and IM-9 cells (Fig. 1A). To validate gene expression results, we performed immunohistochemical analyses on conjunctival EMZL tissues. Signals of (P)RR and AT1R were observed widely in conjunctival EMZL tissues, including atypical lymphoid cells and vascular endothelial cells (Figs. 1B, 1C). 

Figure 1

 

Gene expression and localization of (P)RR and AT1R in human conjunctival EMZL tissues and IM-9 cells. (A) Gene expression of RAS components in EMZL tissues of human conjunctiva and IM-9 cell line. Semiquantitative reverse transcription PCR analysis was performed to check the expression of RAS components (REN, AGT, ACE, AT1R, and AT2R) in five conjunctival EMZL tissues (EMZL #1–5) and IM-9 cells. (B–D) Immunohistochemical staining of (P)RR and AT1R in human conjunctival EMZL tissues. Arrowhead, endothelial cells; open arrowhead, B-cells. Scale bar: 100 μm.

Localization of Prorenin and (P)RR in Endothelial and Lymphoid Cells of EMZL Tissues

To further study the localization of (P)RR in conjunctival EMZL tissues, we performed immunofluorescence analysis. Double-staining experiments demonstrated colocalization of (P)RR signal with CD20, a B-cell marker (Figs. 2A–C), and with CD31, a vascular endothelial cell marker (Figs. 2D–F). Moreover, expression of (P)RR in B-cells was abundantly colocalized with prorenin (Figs. 2G–I). 

Figure 2

 

Immunofluorescent analyses of prorenin and (P)RR in human conjunctival EMZL tissues. (A–C) Double-labeling of CD20 (green), (P)RR (red), and DAPI (blue). (D–F) Double-labeling of CD31 (green), (P)RR (red), and DAPI (blue). (G–I) Double-labeling of prorenin (green), (P)RR (red), and DAPI (blue). Scale bar: 20 μm.

Localization of AGT and AT1R in Endothelial and Lymphoid Cells of EMZL Tissues

To further verify our immunohistochemical analysis results, we performed double-staining of AT1R with CD20 or CD31. The AT1R colocalized with CD20 and CD31, which indicates that AT1R is expressed in the B-lymphoid cells (Figs. 3A–C) and vascular endothelial cells of EMZL tissues (Figs. 3D–F). In addition, AT1R immunoreactivity also colocalized with AGT on lymphoid cells of EMZL (Figs. 3G–I). 

Figure 3

 

Immunofluorescent analyses of AGT and AT1R in human conjunctival EMZL tissues. (A–C) Double-labeling of CD20 (green), AT1R (red), and DAPI (blue). (D–F) Double-labeling of CD31 (green), AT1R (red), and DAPI (blue). (G–I) Double-labeling of AGT (green), AT1R (red), and DAPI (blue). Scale bar: 20 μm.

Upregulation of FGF2 Expression via Prorenin-(P)RR Interaction

Bindings of prorenin to (P)RR and of Ang II to AT1R have been shown to upregulate various gene expressions in vivo and in vitro, related to the pathogenesis of numerous diseases.^{10,11,19–23} In retinal vascular endothelial cells, we and other groups have shown that prorenin and Ang II stimulations significantly upregulated mRNA expressions, such as MMP2.^10,19,21 However, there are no reports on B-cells. In the present study, to investigate the effect of prorenin-(P)RR interaction on B-lymphoid cells, we examined whether stimulation of prorenin affects mRNA expression levels in human B-lymphoma cell line IM-9 by real-time qPCR analysis. We found that FGF2 expression levels significantly increased (fold change = 2.24, P < 0.01) in IM-9 cells stimulated with prorenin compared to control, but not with Ang II (fold change = 0.87, P > 0.05). Importantly, increased FGF2 expression was inhibited by pretreatment with PRRB (fold change = 1.41, P < 0.05, Fig. 4A). 

Figure 4

 

(P)RR-mediated upregulation of FGF2 in human B-lymphoma cells. (A) Relative mRNA expression level of FGF2 in Ang II or prorenin-stimulated IM-9 cells with or without PRRB compared with control. n = 4. *P < 0.05, **P < 0.01. N.S., nonsignificant. (B–D) Double-labeling of FGF2 (green), (P)RR (red), and DAPI (blue) in human conjunctival EMZL tissues. Scale bar: 20 μm.

Based on these results, we performed immunofluorescence experiments to investigate the expression of FGF2 with (P)RR in conjunctival lymphoma samples. Immunofluorescence analysis revealed the colocalization of FGF2 with (P)RR in conjunctival lymphoma (Figs. 4B–D). These data suggested that activation of RAPS, through the interaction between prorenin and (P)RR, induces an increase in FGF2 expression levels in B-lymphocytes, and possibly leads to angiogenesis in EMZL of the conjunctiva. 

Upregulation of MMP Family Members Via Ang II–AT1R Interaction

To study the effects of Ang II–AT1R binding in B-lymphoma cells, we administered Ang II to IM-9 cells and examined the changes in gene expression levels using real-time qPCR analysis. Ang II stimulation to cells significantly increased expression levels of BSG (fold change = 2.25, P < 0.05), MMP2 (fold change = 2.56, P < 0.05), MMP9 (fold change = 3.64, P < 0.05), and MMP14 (fold change = 1.83, P < 0.05) compared to those of controls, while pretreatment of valsartan suppressed Ang II-induced BSG, MMP2, MMP9, and MMP14 expressions (BSG, fold change = 0.90, P < 0.01; MMP2, fold change = 1.75, P < 0.05; MMP9, fold change = 0.91, P < 0.05; MMP14, fold change = 1.03, P < 0.05, Figs. 5A–D). 

Figure 5

 

AT1R-mediated upregulation of BSG, MMP2, MMP9, and MMP14 in human B-lymphoma cells. (A–D) Relative mRNA expression levels of BSG, MMP2, MMP9, and MMP14 in Ang II-stimulated IM-9 cells with or without valsartan (Val) compared with control. n = 4. *P < 0.05, **P < 0.01. Double-labeling of BSG (E–G), MMP2 (H–J), MMP9 (K–M), and MMP14 ([N–P]; green), AT1R (red), and DAPI (blue) in human conjunctival EMZL tissues. Scale bar: 20 μm.

Following these in vitro experiment results, we carried out immunofluorescence analysis to examine the colocalization of BSG, MMP2, MMP9, and MMP14 with AT1R in EMZL samples. Immunoreactivity for BSG, MMP2, MMP9, and MMP14 was widely distributed (Figs. 5E, 5H, 5K, and 5N) and colocated with AT1R (Figs. 5F, 5G, 5I, 5J, 5L, 5M, 5O, and 5P) in the EMZL sections. The results suggest that the binding of Ang II to AT1R causing tissue RAS stimulation increases expression levels of MMP family genes and leads to the following sequence of events at the molecular level to trigger angiogenesis and invasion in conjunctival EMZL. 

Discussion

In this study, we provide several unique mechanistic insights into the roles of RAPS and RAS in the pathogenesis of conjunctival EMZL. First, expression of RAS component genes was confirmed in surgically excised EMZL tissues as well as human B-lymphoma cell lines (Fig. 1). The (P)RR and AT1R proteins were immunopositive in B-lymphocytes in the tissues, and colocalized with prorenin and AGT, respectively (Figs. 11552–3). Stimulation of prorenin to B-lymphoma cell culture triggered the upregulation of FGF2 through (P)RR interaction (Fig. 4), and (P)RR colocalized with FGF2 in conjunctiva EMZL tissues. Activation of AT1R with Ang II increased BSG and MMPs gene expression levels, all of which were suppressed by pretreatment with valsartan, and immunofluorescence analyses showed colocalization of AT1R with these MMP-related molecules (Fig. 5). 

The pro-angiogenic molecule FGF2 is widely expressed in various tumor tissues and its elevated levels are associated with inflammation leading to tumor growth, progression, and metastasis.²⁴ Expression of FGF2 was found to be significantly elevated in sera or tissue specimens of Hodgkin's and non-Hodgkin's lymphomas.^25,26 In addition, through its interaction with FGF receptor 1, FGF2 is demonstrated to promote angiogenesis in the tumor microenvironment leading to angiogenesis and lymphatic metastasis.²⁷ Excessive FGF2 expression levels in diabetes and hypertension were suppressed by RAS inhibitors.^28,29 Here, we found that colocalization of (P)RR and FGF2 in EMZL tissues and prorenin stimulation to B-lymphocyte cell line significantly increased the expression level of FGF2, which was suppressed by PRRB. Taken together, our results suggested that prorenin-(P)RR interaction (i.e., activation of RAPS) is responsible for the increase in the expression of FGF2, induction of angiogenesis and inflammation in B-cells, and eventually the pathogenesis of conjunctival lymphoma. 

The MMP2 and MMP9 have important roles in the process of tumor neovascularization, and the extent of neovascularization correlates with MMPs expression during the progression such as in multiple myeloma and skin T-cell lymphoma.³⁰ Our current study showed that Ang II induced an AT1R-mediated increase in gene expression levels of BSG, MMP2, MMP9, and MMP14 in cultured human B-lymphocytes. Supporting in vitro data, BSG, MMP2, MMP9, and MMP14 colocalized with AT1R in EMZL tissues. Alternatively called “extracellular MMP inducer” or CD147, BSG is a member of the immunoglobulin superfamily and abundantly expressed on the surface of tumor cells.³¹ The BSG-positive tumor cells and their supernatants increase expression levels of MMPs, including MMP2 and MMP9.^32,33 The MMP2 and MMP9 also are regarded as tumor biomarkers in monitoring response to cancer treatment.³⁴ Degradation of type IV collagen by MMP2 is a significant hallmark of metastasis and invasion in carcinoma.³⁵ The MMPs also can activate growth factor signaling by increasing the bioavailability of factors, such as FGF2, and initiate tumor progression through stimulation of angiogenesis.³⁶ The release of angiogenic growth factors, cytokines, and proteases, like FGF2, MMP2, MMP9, and MMP14, into the surrounding extracellular matrix initiates tumor angiogenesis.^37–39 Studies on MMPs in clinical samples reported that BSG and MMP9 expression levels are elevated in non-Hodgkin's and Hodgkin's lymphomas, which are associated with clinical stages.^40,41 Activation of AT1R by Ang II has been reported to trigger upregulation of BSG and MMPs, including MMP2, MMP9, and MMP14, in various cells.^21,22,42 Moreover, activity of serum angiotensin-converting enzyme (ACE) increased in non-Hodgkin's and other types of lymphoma patients. In the bone marrow, ACE cleaves N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), an inhibitor of hematopoietic stem cell proliferation, and renders it inactive.⁴³ Abnormal increased ACE activity may lead to the acceleration of AcSDKP degradation, resulting in hematopoietic cell proliferation. In accordance with these findings, our data suggested that binding of Ang II-AT1R (i.e., stimulation of tissue RAS) has roles in the extracellular matrix turnover and remodeling in B-lymphomas, induces the subsequent sequence of molecular events in the microenvironment, and leads to formation and development of conjunctival lymphoma. 

The treatments of conjunctival EMZL are performed mainly with surgical excision and/or irradiation due to their low-grade and radiosensitive malignancy. However, still there are cases that do not achieve complete remission even after these treatments. Therefore, development of additional therapeutic options are required. Our data from clinical human EMZL samples and B-lymphoid cells show the evidence that activations of RAPS and tissue RAS via (P)RR and AT1R are associated with the pathogenesis of conjunctival lymphoma. Interactions of prorenin-(P)RR and AngII-AT1R have been reported to activate mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) 1/2 and nuclear factor-κB (NF-κB) pathways, and induce proliferation and differentiation in various cells.^10,11,44 Several reports propose the NF-κB signaling pathway as an attractive therapeutic target in T- and B-cell malignancies including EMZL.⁴⁵ Blockades of (P)RR and AT1R may be promising to prevent the cascade of events essential in the pathogenesis of EMZL of the conjunctiva and serve as clinical tools in the treatment of conjunctival lymphoma. 

Acknowledgments

The authors thank Ikuyo Hirose and Shiho Yoshida (Hokkaido University) for their skillful technical assistance. 

Supported in part by the Takeda Science Foundation, Institute of Science of Blood Pressure and Hormone, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program, and a grant-in-aid from the Ministry of Education, Science and Culture of Japan (#24791823; AK). The authors alone are responsible for the content and writing of the paper. 

Disclosure: E.T. Ishizuka, None; A. Kanda, None; S. Kase, None; K. Noda, None; S. Ishida, None