TAPI-1 – Z-4-hydroxytamoxifen

GRO-α/CXCR2 System and ADAM17 Correlated Expression in Sjögren’s Syndrome

Abstract—The chemokine GRO-α and its receptor CXCR2 are associated with the chronic inﬂa- mmation in Sjögren’s syndrome (SS). To better understand the molecular mechanisms by which the GRO-α/CXCR2 system is involved in the SS inﬂammatory condition, our studies were designed to clarify the role of ADAM17 activation in the modulation of the GRO-α/CXCR2 chemokine system in epithelial cells (SGEC) from SS salivary glands. The CXCR2 overexpression observed in SS SGEC was dramatically decreased by ADAM17 inhibitor TAPI-1. In addition, comparing the ex- pression levels of ADAM17 in healthy SGEC in presence or not of GRO-α treatment, we observed that GRO-α dose-dependently inﬂuences ADAM17 activation, an effect that was inhibited by bl- ocking the interaction of GRO-α with its CXCR2 receptor. Our data show for the ﬁrst time that ADAM17 has an important role in GRO-α/CXCR2 system activity regulation, suggesting that regulating CXCR2/ADAM17 interaction could be an attractive therapeutic target in SS.

KEY WORDS: ADAM 17; TAPI-1; CXCR2; GRO-α; sjögren´s syndrome.

INTRODUCTION

Sjögren’s syndrome (SS) is a systemic disease in which the deﬁning clinical features, dryness of the eyes and mouth, arise from an autoimmune process affecting the lacrimal and salivary glands. It may occur either alone or in the context of another autoimmune disease, such as rheumatoid arthritis or systemic lupus erythematosus [1]. The Sjögren’s syndrome is one of the most prevalent systemic rheumatic diseases with a unique predilection for post-menopausal women [2]. The disease may affect the nervous system, lungs, and kidneys in addition to the exocrine glands. Late complications may include blindness, dental destruction, oral candidiasis, and non-Hodgkin lymphoma [3]. The pathogenesis of SS is not clearly known.

The matrix metalloproteinases are proteolytic enzymes involved in the degradation of extracellular matrix (ECM) and implicated in the pathogenesis of many autoimmune inﬂammatory diseases including SS [4–9]. Inﬂammatory diseases are characterized by the recruitment of immune cells to the inﬂammation site and the mechanisms leading to immune cell recruitment involve coordinated processes of leuko- cyte recirculation and leukocyte migration, which require complex cell–cell and cell–substratum inter- actions. Furthermore, it is currently believed that there exists a relation between angiogenesis and leukocyte inﬁltration in chronic inﬂammation. The two phenomena appear to be dependent on each other since inﬂammatory mediators released by immune cells promote angiogenesis, which, in turn, contributes to inﬂammation by increasing the recruit- ment of immune cells to inﬂammatory sites [4, 10]. These migration processes require remodelling of ECM through proteolytic degradation [11]. Matrix metalloproteinases are a family of zinc binding, calcium-dependent, enzymes crucial in normal tissue remodelling during embryogenesis, growth, and wound healing. Metalloproteinases, indeed, have been described as involved in processes as diverse as cell fusion, growth factor and cytokine shedding, cell differentiation, cell migration, and cell adhesion [12]. Excessive metalloproteinases activity is associated with inﬂammatory conditions leading to destruction of normal tissue architecture [4–11]; in fact, during the inﬂammatory response, various cell surface proteins undergo ectodomain shedding that leads to the release of a soluble extracellular domain fragment [13].

Among the metalloproteinase, the disintegrin and metalloproteinase-17 (ADAM17), originally referred to as TNF-α converting enzyme (TACE), plays a broad role in mediating ectodomain shedding of several inﬂammatory mediators [14], and researchers have recently demonstrated that ADAM17 has an important regulatory function in the chronic inﬂammation ob- served in SS [4–9].

Growth-related oncogene-alpha (GRO-α), a mem- ber of the CXC chemokine family, and its receptor CXC chemokine receptor 2 (CXCR2) are involved in the inﬂammatory processes. In many models of acute and chronic inﬂammatory diseases, blockade of CXCR2 substantially reduces leukocyte recruitment, tissue dam- age, and mortality [15]. It has been demonstrated previously by our group that an increased expression of GRO-α/CXCR2 complex occurs in SS, depending on the inﬂammatory response observed, indicating the GRO-α/CXCR2 system as a novel therapeutic target for this chronic inﬂammatory disease [16].

There are evidences that the CXCR2 expression can also be regulated, by enzymatic cleavage via metalloproteinases [17]. Actually, the ADAM17 inhib- itors are under development for the treatment of a variety of inﬂammatory autoimmune disorders [5, 18]. In addition, the ADAM17 activation was recently associat- ed with the chronic inﬂammatory condition observed in SS [4–9], the ADAM17 inhibitor TAPI-1 was demon- strated to attenuate the release of pro-inﬂammatory cytokines by SGEC from SS patients [5], and the CXCR2 expression is regulated by an inﬂammatory microenvironment that mimics the inﬂammatory process observed in SS [16]. Then, we reasoned that it could be possible that the ADAM17-dependent pathway could be involved in the modulation of CXCR2 in SS SGEC, and this report described a study designed to clarify the role of ADAM17 activation in the modulation of the GRO-α/ CXCR2 chemokine system in epithelial cells (SGEC) from SS salivary glands.

MATERIALS AND METHODS

Patient Selection

Twenty SS patients were recruited for this study. The patients all had deﬁnite disease according to the revised 2002 American–European criteria [19]. All patients had the clinical symptoms of dry eyes and mouth, a positive Schirmer’s test (less than 5 mm wetting of a strip of ﬁlter paper per 5 min), and Rose Bengal staining (increased uptake of Rose Bengal dye in devitalised areas in the conjunctiva and cornea) along with the presence of at least one of the following autoantibodies: anti-Ro/SSA, anti-La/SSB, antinuclear antibodies, rheumatoid factor. All SS patients reported here had a focus score values of greater than 1. The patients had all given their written consent, the study was approved by the local Ethical Review Committee, and the experiments were conducted according to the tenets of the Declaration of Helsinki. After obtaining informed consent, LSG biopsy samples were taken from SS patients. Labial minor glands were harvested from the lower lip under local anaesthesia through normal mucosa, according to the explant outgrowth technique [20].

Microdissection and primary explant cultures of human salivary gland epithelial cells

Following surgery, SS samples were immediately processed to obtain primary cultures of human SGEC or to extract RNA and proteins. For the cultures, the cells were isolated from the healthy and SS labial glands by microdissection and collagenase (Worthington Diagnos- tic Division, Millipore, freehold, NJ, USA) digestion in physiological saline containing 1 mm Ca2+. Following dispersal, cells were re-suspended in McCoy’s 5a modiﬁed medium supplemented with 10 % fetal bovine serum, 1 % antibiotic solution, 2 mM L-Glutamine,
20 ng/ml epidermal growth factor (EGF, Promega, Madison, WI), 0.5 μg/ml insulin (Novo, Bagsvaerd, Denmark) and incubated at 37 °C, 5 % CO2 in air. Contaminating ﬁbroblasts were selectively removed by treatment of the cultures with 0.02 % EDTA. The epithelial origin of cultured cells was routinely conﬁrmed by staining with monoclonal antibodies against epithelial-speciﬁc markers, including the various cytokeratins and epithelial membrane antigens, and the absence of myoepithelial ﬁbroblastoid and lymphoid markers, using immunocytochemistry as previously described [21].

Pharmacological Treatments to Inhibit TACE

Human SS SGEC were pretreated with 10 μM of the metalloprotease inhibitor TAPI-1 (Calbiochem, San Diego, CA, USA) or with the DMSO vehicle used to dissolve the TAPI-1 for 24 h. This substance was not cytotoxic at the concentration used and had no effect with the ability of SGEC to execute normal acinar and ductal morphogenesis in the presence of soluble EGF (data not shown).

GRO-α Treatment and CXCR2 Block

Human healthy SGEC were treated with growing concentration of human recombinant GRO-α (eBio- science, San Diego, CA, USA; from 10 to 100 ng/ml) for 24 h in presence or not of the CXCR2 blocking antibody [anti-human CXCR2 monoclonal Ab (mAb), R&D system, Minneapolis, MN USA] (5 μg/ml).

RT-PCR

For the reverse transcriptase polymerase chain reaction (RT-PCR), total RNA was isolated from TAPI-1-treated and untreated SS SGEC following the manufacturer’s protocol. RNA quality was checked by gel electrophoresis to conﬁrm the integrity of the RNA preparations. To determine CXCR2 mRNA expression, we treated 2 μg of RNA with DNase I (GIBCO, Life Technologies, Carlsbad, CA, USA) prior to reverse transcription with Moloney murine leukemia virus reverse transcriptase (GIBCO) in the presence of RNaseOUT (GIBCO) and used 1/10 of the cDNA preparation for each PCR. PCR was performed in a 50-μl reaction mixture composed of 2 μM of each sense and antisense primer, 1×PCR buffer, 2.4 mM MgCl2, 0.2 mM each dNTP, 10 μl of transcribed cDNA, and 0.04 U/μl Taq DNA polymerase. After initial denatur- ation at 94 °C for 5 min, 35 cycles were performed (denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min) followed by 10 min at 72 °C. Equal amounts of PCR products were run on a 1.5 % agarose gel containing ethidium bromide. The expected size of the PCR products is 465 bp for GADPH and 300 bp for CXCR2. PCR primers were designed according to published sequen- ces (ref. source: www.ncbi.nlm.nih.gov) and were as follows: Forward 5′-CTCCAATAACAGCAGGTCAC-3′ and reverse 5′-GGCTCAGCAGGAATACCA-3′; GADPH was used as internal control, and the primers used to amplify it were: GADPH forward 5′-CAACG- GATTTGGTCGTATT-3′ and GADPH reverse 5′- GATGGCAACAATATCCACTT-3′. Densitometric anal- ysis, performed by gel image software (Bio-Proﬁl Bio-1D; ltf Labortechnik GmbH, Wasserburg, Ger- many), was done to quantify mRNA expression levels by determining the intensity values for each GADPH band (internal control for lane loading). Results were averaged from four sets of independent experiments and expressed as arbitrary units. The identity of each PCR product was conﬁrmed by the size and direct sequencing of the ampliﬁed cDNA eluted from the gel.

Real-Time PCR

For the real-time quantitative PCR, total RNA was isolated from SS SGEC following the manufacturer’s protocol. RNA quality was checked by gel electrophoresis to conﬁrm the integrity of the RNA preparations. Two micrograms of RNA was treated with DNase I (GIBCO, Life Technologies, Carlsbad, CA, USA) prior to reverse transcription with Moloney murine leukemia virus reverse transcriptase (GIBCO) in the presence of RNaseOUT (GIBCO), and 1/10 of the cDNA preparation for each PCR was used. Real-time PCR was performed in 96-well microtiter plates with TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA), following the manufacturer’s recommendation. Forward and reverse primers for human CXCR2 and the internal control gene β-2 microglobulin (part n°4326319E; β2M) were purchased from Applied Biosystems (Assays-On- Demand, Applied Biosystems). Taqman qPCR ampliﬁca- tions were performed as duplex reactions with assays for the test and control in the same well. The result of the relative increase in reporter ﬂuorescent dye emission was analyzed by an ABI PRISM 7700 sequence detector (Applied Biosystems).The threshold was determined as ten times the SD of the baseline ﬂuorescence signal. The cycle number at the threshold was used as the threshold cycle (Ct). The different expression of mRNA was deducted from 2−ΔΔCt.

Western Blot Analysis

Protein lysates, obtained from variously treated human cultured SGEC from SS patients, were subjected to sodium dodecyl sulfate-polyacrylamide gel electro- phoresis according to standard electophoresis and trans- fer techniques. Membranes were incubated for 90 min with mouse anti-human CXCR2 mAb (R&D System) and with the relative secondary antibodies-HRP con- jugates (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Proteins recognized by the antibodies were revealed using the chemoluminescence luminal reagent (Santa Cruz Biotechnology) according to the protocol, and β-actin was used as protein loading control.

Flow Cytometry

SS SGEC, treated or not with TAPI-1, were incubated with anti-human CXCR2-phycoerythrin mAb (R&D Systems). Healthy SGEC treated with growing concentration of GRO-α in presence or not of the CXCR2 blocking antibody were incubated with mono- clonal anti-human TACE-phycoerythrin Ab (R&D Sys- tems). The protein expression was analyzed by a Becton Dickinson FACSCanto™ II ﬂow cytometer and BD FACS Diva software (BD, Becton Dickinson, Franklin Lakes, NJ USA). Values are given as mean ﬂuorescence intensity (M.F.I.).

Statistical Analysis

Experimental values were expressed as mean ± SE, and the differences in means for paired observations were analyzed by Student’s t test; values of p <0.05 were considered statistically signiﬁcant. RESULTS Inhibition of CXCR2 Expression in Human SS SGEC in Response to Treatment by ADAM17 Inhibitor TAPI-1 We sought to investigate the possibility that TAPI- 1, blocking ADAM17 activation, regulates CXCR2 expression in SS. To directly investigate the effect of ADAM17 inhibition on the expression of CXCR2, we used healthy and SS SGEC treated or not with the TAPI- 1. To validate this hypothesis, RT-PCR was performed on RNA samples from independent experiments. As shown in Fig. 1a, b, using speciﬁc primers, designed to evaluate CXCR2 mRNA expression in TAPI-1-treated or untreated SS SGEC, we demonstrated that CXCR2 expression was downregulated at a transcriptional level in SS SGEC following TAPI-1-inhibition of ADAM17. Real-time PCR (panel C), performed using total RNA isolated from treated and untreated SS SGEC, conﬁrmed that CXCR2 was indeed underexpressed (by 2.7-fold; P<0.01) in SS SGEC by TAPI-1 treatment, and the decreased expression of CXCR2 gene resulted more consistent when compared to that observed in TAPI-1- treated SGEC from healthy donors. To determine whether this change in mRNA expression also resulted in differential regulation of protein expression by TAPI-1, Western blot (Fig. 2a) was performed. Total protein extracts of untreated and TAPI-1-treated healthy and SS SGEC were treated with a mAb speciﬁc to human CXCR2, and a protein band was speciﬁcally recognized in cell lysates of SS and healthy SGEC. As suggested by the results of the densitometric analysis (Fig. 2b), CXCR2 receptor expression was abundantly decreased both in healthy and in the SS SGEC treated with TAPI-1. Flow cytometric analysis conﬁrmed these results. Evaluation of the M.F.I. (Fig. 2c) revealed that the TAPI-1 treatment determined a reduction of CXCR2 expression in SS SGEC (M.F.I 012,567± 189 for untreated SS SGEC versus 5,584 ± 154 for TAPI-1-treated SS SGEC; P<0.01) as well as in the SGEC from healthy donors (M.F.I06,607±198 for untreated healthy SGEC versus 2,178±123 for TAPI-1-treated healthy SGEC; P<0.01). The Effect of CXCR2 Ligand GRO-α on ADAM17 Activation in SGEC We have previously reported increased expression of CXCR2 and its ligand GRO-α at the gene and protein levels in cultured SS SGEC in comparison with healthy donors [16]. Our ﬁndings so far demonstrated increased expression of GRO-α and its receptor CXCR2 both in acinar and ductal cells in SS biopsies [16]. To map any potential pathogenic consequences of the raised GRO-α levels in SS disease, we, in addition, examined the effect of GRO-α treatment on the activation of ADAM17 in healthy SGEC. Figure 3a shows that GRO-α treatment boosted the ADAM17 activation in a dose-dependent manner. This enhancing effect was attenuated by adding an anti-human CXCR2 to block GRO-α/CXCR2 bind- ing, suggesting that this GRO-α-mediated effect may involve the interaction with CXCR2 (Fig. 3b). A schematic representation of the mechanism hypothesized was reported in Fig. 4. Fig. 1. Inhibition of CXCR2 expression in SS SGEC by TAPI-1. a, b Reduced CXCR2 gene expression revealed by RT-PCR in SGEC from healthy (H) and Sjögren’s syndrome (SS) biopsies after TAPI-1 treatment. M marker. GAPDH served as an internal control for ampliﬁcation performed. Results presented are from one experiment and are representative of three independent experiments. c Real-time PCR analysis for CXCR2 mRNA expression in TAPI-1-treated H and SS SGEC. mRNA expression levels of CXCR2 were analyzed in triplicate real-time PCR assays. Data are presented as fold induction of CXCR2 mRNA levels after normalizing to β-2-mycroglobulin expression. DISCUSSION The chemokine system has been associated with the regulation of leukocyte trafﬁcking to normal and inﬂamed tissues [22]. However, in addition to leuko- cytes, other nonhematopoietic cell types, including endothelial cells, smooth muscle cells, neurons, epithe- lial cells, ﬁbroblasts, and several tumor lineage cells, also produce chemokines and express chemokine recep- tors [23]. This unrestricted cell type expression, added to the fact that chemokines can couple to distinct signalling pathways, suggests that the functions of chemokine receptors are not limited to cell trafﬁc regulation. In this regard, gene transcription, cell proliferation, and neo-vascolarization among others are described as effects mediated by the chemokine system [16, 23]. However, although the chemokine system has been shown to be involved in many functions in several cell types, the actual contribution to the mechanisms involved in the chronic inﬂammation observed in SS remains to be established. Based on our previous data, showing that the chemokine system is considered to be implicated in SS pathogenesis [16], in the present study, we investi- gated the mechanism of regulation of the chemokine receptor CXCR2 in SS SGEC by the metalloproteinase ADAM17. Inﬂammatory conditions are, in fact, almost always characterized by deregulated, often increased metalloproteinases activities [24], and the central question is whether the metalloproteinases can inﬂuence the outcome of inﬂammation and if so, how they do so. Fig. 2. Effect of ADAM17 inhibitor TAPI-1 on CXCR2 protein expression in SS SGEC. a, b All protein isolations and Western blots were repeated a minimum of three times. H and SS SGEC were treated for 24 h with ADAM17 inhibitor TAPI-1. Western blot analysis was done, and blots were probed with anti-human CXCR2. Western blot and densitometric analysis show the downmodulation of CXCR2 expression in SS SGEC following TAPI-1 treatment. c Levels of CXCR2 expression on TAPI-1-treated H and SS SGEC were measured by ﬂow cytometry and expressed as mean ﬂuorescence intensity (M.F.I). We demonstrated that the treatment with TAPI-1 markedly reduced CXCR2 expression level in SS SGEC, suggesting that the upregulation of CXCR2 expression observed in SS SGEC requires ADAM17 activation. These data provide novel insight into the mechanism of chemokine receptor regulation in SS. Recently, high-level expression of CXCR2 ligand GRO-α by SS SGEC has been detected in salivary gland biopsies from SS patients [16]. This discovery is in agreement with published data supporting the ﬁnding that the CXCR2 expression is correlated to GRO-α production in inﬂammatory pathological conditions. A correlated expression of both GRO-α and CXCR2 was found in activated multiple sclerosis lesions [25], high level of expression of CXCR2 and GRO-α was detected in atherosclerotic lesions, potentially contributing to inﬂammation, matrix degradation, and lipid accumulation within the atherosclerotic plaques [26]. Further- more, chemokine and chemokine receptor expression is tightly interrelated to composition of inﬂammatory cells in experimental autoimmune encephalomyelitis [27]. In accordance with these published data, our laboratory recently extends these ﬁndings by identifying GRO-α as one of the most strongly induced cytokines in SGEC from SS patients when compared to healthy individuals. Upregulation of GRO-α was also modulated by the pro-inﬂammatory cytokines, as demonstrated both at the protein and mRNA levels [16]. However, at our knowledge, none of published studies has combined investigations to assess whether GRO-α expression inﬂuences ADAM17 activation and the CXCR2 expression on SGEC from SS patients. In this study, we examined the effect of GRO-α on the activation of ADAM17 in healthy SGEC. The expres- sion levels of active ADAM17 were measured in healthy SGEC treated with growing concentrations of GRO-α,showing that GRO-α increased the active ADAM17 levels in a dose-dependent manner. GRO-α-treated healthy SGEC exhibited signiﬁcantly higher AD- AM17 activity than did the untreated healthy control cells. Furthermore, depletion of GRO-α/CXCR2 interaction, through the use of an anti-human CXCR2, markedly suppressed ADAM17 activation,suggesting that GRO-α plays a role in the CXCR2- mediated ADAM17 activation. Fig. 3. Changes in ADAM17 activation induced by GRO-α ligand treatment in H SGEC. a H SGEC were treated for 24 h with growing concentrations of GRO-α (10–100 ng/ml), and active ADAM17 content was measured by ﬂow cytometry, as described in “Materials and Methods” section, and expressed as M.F.I. b H SGEC were treated for 24 h with GRO-α (100 ng/ml) in presence or absence of anti-human CXCR2 antibody to block GRO-α/CXCR2 interaction, and ADAM17 activation was assessed by ﬂow cytometry. M.F.I. values (mean ± S.E. of four independent experiments) are reported in the graph. Fig. 4. Schematic diagram of our proposed model showing the effect of ADAM17 inhibitor TAPI-1 on the chemokine GRO-α/CXCR2 sy- stem expression in SS SGEC. In vitro efﬁcacy for the TAPI-1 inhibition of ADAM17 on GRO-α/CXCR2 system. Activation of ADAM17 by GRO-α ligand markedly increases CXCR2 expression leading to per- sistent inﬂammation observed in SS. This process was efﬁciently inh- ibited by ADAM17 inhibitor TAPI-1.

In summary, the ﬁndings of the current study suggest a broad involvement of the chemokine GRO-α/CXCR2 system in SS pathogenesis that is mediated by the involvement of ADAM17. These results demonstrate that metalloproteinases play a role in modulating inﬂammatory reactions linked to autoimmune conditions and provide support for anti-chemokine therapies in SS to alleviate inﬂammation or for proceeding with the development of metalloproteinases into effective drug targets.