Expression of Ror2 Associated with Fibrosis of the Submandibular Gland

. The submandibular gland (SMG) is one of the major salivary glands that play important roles for variety of physiological functions, such as digestion of foods, prevention of infection, and lubrication of the mouth. Dysfunction of the SMG, often associated with a salivary inflammation, adversely influences a person’s quality of life. However, the mechanism underlying inflammation-driven dysfunction of the SMG is largely unknown. Here, we used a mouse model in which the main excretory duct of the SMG is ligated unilaterally to induce inflammation of the gland and examined the expression of Wnt5a , Ror1 and Ror2 genes, encoding Wnt5a ligand and its cognate receptors, which have been implicated in tissue damage or inflammatory responses in variety of tissues. We show that expression levels of Ror1 , Ror2 , and Wnt5a are increased in the ligated SMG undergoing interstitial fibrosis, which is accompanied by robust expression of fibrosis-associated genes, such as TGF-β1 , TNF-α , IL-1β , and MMP-2 . Increased immunostaining signal of Ror2 was detected in the fibrotic tissues with abundant accumulation of fibroblasts and collagen fibers in the ligated SMG, suggesting that Ror2-mediated signaling might be activated in response to tissue damage and associated with progression of fibrosis in the SMG.

Wnt5a, Ror2 as well as Ror1 have been implicated in tissue-damage and inflammatory responses in adult tissues.For example, expression of Wnt5a and Ror2 is induced in the damaged kidney after unilateral ureteral obstruction (UUO), leading to renal fibrosis in mice (Li et al., 2013).Ror2-mediated signaling seems to elicit expression of MMP-2, thereby disrupting the tubular basement membrane in the kidney (Li et al., 2013).Expression of Ror2 is also induced in reactive astrocytes following brain injury to promote their proliferation in mice, which is important for the formation of glial scars during the tissue repair (Endo et al., 2017).Furthermore, in demyelinating disorders, inflammatory cytokines released from activated microglia induce expression of Ror2 in neurons, contributing to the progression of neurodegeneration (Shimizu et al., 2016).Wnt5a-Ror2 signaling has also been shown to promote dextran sodium sulfate (DSS)-induced colitis by enhancing proinflammatory cytokine production in the colon (Sato et al., 2015).More recently, we have shown that Ror1 is expressed inducibly in satellite cells by inflammatory cyto-kines in the injured skeletal muscles and promotes its proliferation, required for skeletal muscle regeneration (Kamizaki et al., 2017).
The submandibular gland (SMG) is one of the major salivary glands (Pedersen et al., 2002).Saliva has a variety of physiological functions, such as digestion of foods, prevention of infection, and lubrication of the mouth.Salivary inflammation, often associated with Sjögren's syndrome and radiotherapy of head and neck cancers, causes dysfunction of the salivary glands, adversely influencing a person's quality of life (Fox et al., 2000;Henson et al., 2001;Murakami et al., 2015).Current treatments for the salivary dysfunction are limited to the administration of saliva substitutes and sialogogues (Vissink et al., 2010).
Here we used a mouse model in which the main excretory duct of the SMG is ligated unilaterally to induce salivary inflammation.We show that expression levels of Ror1, Ror2, and Wnt5a are increased in the ligated SMG undergoing interstitial fibrosis, which is accompanied by robust expression of fibrosis-associated genes, such as TGF-β1, TNF-α, IL-1β, and MMP-2.Increased immunostaining of Ror2 was detected in the fibrotic tissues with abundant accumulation of fibroblasts and collagen fibers in the ligated SMG, suggesting that Ror2-mediated signaling might be activated in response to tissue damage and associated with progression of fibrosis in the SMG.

Animals
All the experiments using animals in this study were approved by the Institutional Animal Care and Use Committee (Permission number: P160401) and carried out according to the Kobe University Animal Experimentation Regulations.Male C57BL/6J mice (8 weeks of age) obtained from Japan SLC (Shizuoka, Japan) were used in the present study.Mice were anesthetized using isoflurane controlled by small-animal anesthetizer (TK-7, Biomachinery, Chiba, Japan).The main excretory duct of the right SMG was exposed through an incision in the neck and ligated with surgical suture (7-0 braided silk suture, Akiyama MFG, Tokyo, Japan).The main excretory ducts of the sham-operated mice were exposed and were not ligated.The mice were anesthetized and euthanized on day 1, 3, or 7 after the ductal ligation.The ligated, contralateral, and sham-operated glands were excised and processed for quantitative real-time PCR (qRT-PCR) and histological analyses as describe below.

Immunochemistry
The sections were permeabilized with 0.1% (v/v) Triton X-100 for 20 min, washed in PBS, and then immersed in 0.1% (v/v) hydrogen peroxide for 60 min.After washing with PBS and blocking with Blocking serum (VECTASTAIN ABC KIT; Vector Laboratories, Burlingame, CA, USA) for 60 min, the specimens were incu-bated with anti-Ror2 rabbit polyclonal antibody (Kani et al., 2004) diluted 1:100 in Can Get Signal immunostain Solution A (Toyobo, Osaka, Japan) overnight at 4°C, followed by incubation with biotinylated anti-rabbit IgG (Vector Laboratories) for 120 min at room temperature.The sections were incubated with VECTASTAIN ABC reagent (Vector Laboratories) for 60 min at room temperature, developed with DAKO ENVISION kit/HRP (DAB) (DAKO, Carpinteria, CA, USA), and counterstained with hematoxylin.The slides were evaluated under the light microscope (BZ-X710).

Immunofluorescence staining
The sections were permeabilized with 0.1% (v/v) Triton X-100 for 20 min and blocked with 5% (w/v) BSA for 60 min.The specimens were incubated with anti-vimentin antibody (D21H3; Cell Signaling Technology, Danvers, MA, USA) diluted 1:100 in Can Get Signal immunostain Solution B (Toyobo) overnight at 4°C and then with Alexa Fluor 546-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) diluted 1:500 in Can Get Signal immunostain Solution B. The specimens were counterstained with DAPI and mounted with the Fluoro-KEEPER Antifade Reagent, Non-Hardening Type (Nacalai tesque, Kyoto, Japan).Fluorescent images were obtained using BZ-X710 and processed using Image J (National Institutes of Health, Bethesda, MD, USA).

Statistical methods
Data collection and statistical analyses were carried out with Excel 2013 (Microsoft Corporation, Redmond, WA, USA).The associations of each variable were tested by Student's t-test.A value of p<0.05 was considered statistically significant.

Ductal ligation induces fibrosis within the SMG
We used a mouse model in which the main excretory duct of the SMG was ligated unilaterally to induce inflammation in the gland.The contralateral SMG and sham-operated SMG were used as controls.The SMGs of mice are composed primarily of acinar cells, intercalated ducts, granular convoluted tubules, striated ducts, and excretory ducts.One day after ligation, most of the ducts in the ligated SMGs were dilated, while acinar cells appeared to be unaffected by the ductal ligation (Fig. 1A).On day 4 and 7 after ligation, in the ligated SMGs epithelial cells of the dilated ducts as well as acinar cells were atrophied, and interstitial spaces between and within the lobules contained large number of fibroblast-like cells (Fig. 1D, G), which were not observed on day 1 (Fig. 1A).On the other hand, the contralateral and sham-operated SMGs failed to show any apparent alterations at any time points examined (Fig. 1B,  C, E, F, H, I).
We next examined whether inflammation within the SMGs, induced by the ductal ligation, would bring about fibrotic lesions.Immunofluorescence staining with antivimentin antibodies revealed that the fibroblast-like cells, characterized by vimentin expression, were accumulated markedly between and within the lobules on day 7 of the ligated SMG, while such vimentin-positive cells were detected marginally in the contralateral SMG (Fig. 2A, B), indicating that the ductal ligation induces a robust increase in fibroblasts or fibroblast-like cells in interstitial tissues of the SMGs.Since increased fibroblasts are associated with fibrotic lesions within damaged tissues, we examined the ligated SMGs by azan staining that identifies collagen fibers.In agreement with the distribution of fibroblasts, marked depositions of collagen fibers were detected between and within the lobules of the SMG on day 4 and 7, but not day 1, after the ductal ligation (Fig. 2C-K), indicating that the ductal ligation might induce fibrotic lesions or fibrosis within the SMGs.

Ductal ligation induces expression of Ror2 in fibrotic tissues
To study a possible involvement of Wnt5a-Ror signaling in damage responses within the SMG, expression levels of Ror1, Ror2 and Wnt5a within the SMGs after the ductal ligation were examined by qRT-PCR.We found that expression levels of all of these genes are increased significantly on day 4 and 7, but not day 1, after the ductal ligation, compared to the respective contralateral SMGs (Fig. 3).There were not any apparent differences in these expression levels in the contralateral and sham-operated SMGs throughout the time course examined (Fig. 3).We next examined expression pattern of Ror2 in the ligated SMG by immunohistochemical analysis.Immunostining signals of Ror2 were detected in most of the ductal cells, but not acinar cells, of both ligated and control (contralateral and sham-operated) SMGs, irrespective of the periods of ligation (Fig. 4).Obviously, on day 4 and 7, but not day 1, Ror2 expression was increased markedly at the interstitial spaces between and within the lobules, and the Ror2positive fibroblast-like cells were juxtaposed to the atrophied acinar cells (Fig. 4).Such increase in Ror2expressing cells was not observed in the respective contralateral or sham-operated SMGs (Fig. 4).Thus, increased Ror2 expression seems to be correlated with the progression of fibrosis within the ligated SMG.

Effect of the ductal ligation on expression of fibrosis-related genes in the SMG
To explore the molecular nature of the SMG fibrosis, gene expression profiling of the ligated SMGs (day 7) was performed using a fibrosis PCR array.Genes whose expression was increased or decreased at least 2-fold in the ligated SMGs, compared to the respective contralateral SMGs, are shown in Table I.As expected from their key roles in regulating fibrosis of various organs (Liu, 2006;Mallat and Lotersztajn, 2013;Tatler and Jenkins, 2012;Thenappan et al., 2010), expression of Transforming growth factor-β (TGF-β) ligands, TGF-β1 and TGF-β3, were upregulated in the ligated SMGs (Table I).We also detected increased expression of the components and target genes of TGF-β signaling, such as TGF-β receptor II, TGFB-induced factor homeobox 1, Smad7, Serpine1, connective tissue growth factor (Ctgf), and Snail1 (Table I), suggesting that TGF-β  I).
We also examined the expression of TGF-β1, TNF-α, IL-1β, and MMP-2 on day 1, day 4, and day 7 after the ductal ligation by qRT-PCR.Expression of TGF-β1 and TNF-α was upregulated significantly in the ligated SMGs on day 1, and their levels were further increased on day 4 and day 7, compared to the respective contralateral SMGs (Fig. 5A,  B).Significant increases in expression levels of IL-1β and MMP-2 were observed in the ligated SMGs on day 4 and day 7 (Fig. 5C, D).

Discussion
Here, we found that expression of Ror1, Ror2, and Wnt5a was upregulated at mRNA levels during fibrosis of the SMG induced by the ligation of the main excretory ducts.Immunohistochemical analyses also revealed that expression of Ror2 was upregulated primarily at the interstitial spaces between and within the lobules, where fibroblastic cells, fibroblasts and collagen fibers were accumulated, following the ductal ligation, indicating that these fibroblasts express Ror2.Tissue fibrosis is a common cause of dysfunctions and diseases in various organs, including the SMG (Shimizu et al., 2013).TGF-β has been shown to play key roles in the regulation of fibrosis in various organs (Liu, 2006;Mallat and Lotersztajn, 2013;Tatler and Jenkins, 2012;Thenappan et al., 2010), and in fact, our PCR array data indicated an upregulation of TGF-β1 and TGF-β3 as well as their target genes in the ligated SMGs, consistent with the previous report (Woods et al., 2015).We also found that increased expression of TGF-β1 could be detected as early as day 1 after the ductal ligation, the time point when apparent increases in the expression of Ror1, Ror2, and Wnt5a were not detectable, suggesting that TGF-β1 is involved primarily in early steps during tissue damage responses.In agreement with this notion, expression of TGF-β1 is induced at earlier steps than expression of Ror2 and Wnt5a during renal fibrosis induced by UUO (Li et al., 2013).
Previous report has shown that duct ligation of the SMG induces expression of TGF-β receptor I and activation of TGF-β/Smad signaling in acinar cells, indicating that acinar cells would be primary targets of TGF-β1 and TGF-β3 in the ligated SMG, even though the source of TGF-β production has not been identified (Woods et al., 2015).Thus, it can be envisaged that acinar cells express TGF-β-target genes, such as Serpine1, a most highly upregulated gene in the ligated SMGs (Table I).Serpine1, also known as plasminogen activator inhibitor-1, contributes to tissue fibrosis of various organs by regulating epithelial-to-mesenchymal and/or endothelial-to-mesenchymal transition, or migration of inflammatory cells, depending on the cell types (Ghosh and Vaughan, 2012).Ctgf is another TGF-β-target gene that was also upregulated highly in the ligated SMGs (Table I).(Lipson et al., 2012).It also activates the myofibroblasts to stimulate ECM deposition and tissue remodeling (Lipson et al., 2012).Future studies will be necessary to clarify the roles of these and other upregulated genes in fibrosis of the SMGs.Inflammation plays a fundamental role in tissue fibrosis (Wynn, 2007).In fact, previously reported evidence demonstrates that inflammatory cytokines, including TNF-α and IL-1β, play an essential role in the fibrosis of various tissues (Kamari et al., 2011;Zhang et al., 1997).Our PCR array and qRT-PCR analyses have revealed that expression of TNF-α and IL-1β is highly induced in the ligated SMGs.Importantly, we have recently shown that TNF-α and IL-1β induce expression of Ror1 and Ror2 in injured skeletal muscles in mice (Kamizaki et al., 2017).Furthermore, IL1β has been shown to induce expression of Wnt5a and Ror2 in human mesenchymal stem cells (Sonomoto et al., 2012), suggesting that these inflammatory cytokines might be involved in expression of Ror1, Ror2, and Wnt5a in the ligated SMGs undergoing fibrosis.It is currently unknown which cells within the ligated SMGs are major source of these inflammatory cytokines.
The ductal ligation induces expression of MMP-2, a ubiquitous metalloproteinase that is involved in various biological processes, such as inflammation, wound healing, and tumor invasion (Lovett et al., 2013;Yang et al., 2017;Zhang et al., 2016).We have previously shown that Ror2 is required for induced expression of MMP-2 during renal fibrosis induced by UUO (Li et al., 2013), suggesting that Ror2-mediated signaling might induce expression of MMP-2 in the ligated SMGs to regulate fibrosis.Despite the fact that Ror1 plays essential roles in tissue-and organogenesis during embryonic development and cancer progression (Endo et al., 2015;Minami et al., 2010), its roles in tissue-damage responses have not been reported, except for our recent finding about the critical role of Ror1 during regeneration of the injured skeletal muscles (Kamizaki et al., 2017).Since the SMGs have capacity to regenerate after long-term obstruction (Watanabe et al., 2017), it would be of interest to examine a possible functional implication of Ror1 in regeneration of the damaged SMGs.
In summary, we found that the ductal ligation induces expression of Ror1, Ror2, and Wnt5a in the SMGs undergoing fibrosis, which is accompanied by robust expression of fibrosis-associated genes, such as TGF-β1, TNF-α, IL-1β, and MMP-2.Increased immunostaining signals of Ror2 were detected in the fibrotic lesions with abundant accumulation of fibroblastic cells, fibroblasts and collagen fibers in the ligated SMGs, suggesting that Ror2-mediated signaling might be activated in response to tissue damage and involved in the progression of fibrosis within the SMGs.Further studies will be required to understand the exact functional roles of Ror1, Ror2, and Wnt5a during fibrosis of the SMGs.

Fig. 4 .
Fig. 4. Immunohistochemical analysis of Ror2 in the ligated (A, D, G), contralateral (B, E, H), and sham-operated SMGs (C, F, I) on day 1 (A-C), day 4 (D-F), and day 7 (G-I) after the ductal ligation.Immunostaining signals of Ror2 are markedly increased at the interstitial spaces between and within the lobules on day 4 and day 7 in the ligated SMGs (arrowheads), but not in the contralateral and sham-operated SMGs.Insets show magnified images of boxed regions.Scale bar, 100 μm.Ac, acinar cells.

Table I .
Genes differentially expressed in the ligated SMGs Profiler PCR Array was used to examine the expression of 84 genes closely associated with fibrosis in the ligated and contralateral SMGs (day 7).Positive and negative values represent increased and decreased fold expression of the genes of interest in the ligated SMGs compared to the contralateral SMGs, respectively.Data for genes whose expression was changed at least 2 fold were summarized.Data presented are average values from two independent experiments.CTGF is a central mediator of tissue fibrosis in various organs, acting to generate myofibroblasts by modulating differentiation of epithelial cells, stellate cells, resident fibroblasts, or fibrocytes