Original Article Biomol Ther 26(3), 298-305 (2018) Collagen-Induced Arthritis Analysis in Rhbdf2 Knockout Mouse Min­Young Lee1,2, Ju­Seong Kang1, Ryeo­Eun Go2, Yong­Sub Byun1, Young Jin Wi3, Kyung­A Hwang2, Jae­Hoon Choi3, Hyoung­Chin Kim1, Kyung­Chul Choi2,* and Ki­Hoan Nam1,* 1Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, Cheongwon 28116, 2Laboratory of Biochemistry and Immunology, College of Veterinary Medicine, Chungbuk National University, Cheongju 28644, 3Department of Life Science, College of Natureal Sciences, Research Institute of Natural Sciences, Hanyang University, Seoul 04763, Republic of Korea Abstract Rhomboid family member 2 gene (Rhbdf2) is an inactive homologue lacking essential catalytic residues of rhomboid intramem- brane serine proteases. The protein is necessary for maturation of tumor necrosis factor-alpha (TNF-α) converting enzyme, which is the molecule responsible for the release of TNF-α. In this study, Rhbdf2 knockout (KO) mice were produced by CRISPR/CAS9. To see the effects of the failure of TNF-α release induced by Rhbdf2 gene KO, collagen-induced arthritis (CIA), which is the rep- resentative TNF-α related disease, was induced in the Rhbdf2 mutant mouse using chicken collagen type II. The severity of the CIA was measured by traditional clinical scores and histopathological analysis of hind limb joints. A rota-rod test and grip strength test were employed to evaluate the severity of CIA based on losses of physical functions. The results indicated that Rhbdf2 mutant mice showed clear alleviation of the clinical severity of CIA as demonstrated by the significantly lower severity indexes. Moreover, a grip strength test was shown to be useful for the evaluation of physical functional losses by CIA. Overall, the results showed that the Rhbdf2 gene has a significant effect on the induction of CIA, which is related to TNF-α. Key Words: Rhbdf2 knockout mouse, Collagen-induced arthritis, TNF-α INTRODUCTION domains (Ha et al., 2013). Rhomboid proteases have con- served transmembrane segments of their polytopic rhomboid Rhomboids are a family of proteins consisting of intramem- core domain. Catalytic motif is in the fourth transmembrane brane serine proteases and their inactive homologues (Free- domain of Rhomboid proteases, and an Engelman helix di- man, 2014). The common ancestor of all members of the fam- merization motif in the sixth transmembrane domain (Urban ily was probably an active intramembrane protease, although et al., 2001, 2002; Lemberg et al., 2005; Urban and Wolfe, the majority of existing members are not active proteases 2005). A tryptophan-arginine motif in loop 1 present between (Freeman, 2014). Rhomboid protease was initially discovered the first and second transmembrane domains is another in- in Drosophila (Sturtevant et al., 1993; Freeman, 1994), and variant structure observed in rhomboid proteases. Drosophila rhomboid protease cuts epidermal growth factor Although the first mammalian rhomboid protease was receptor (EGFR) ligand Spitz and a homologue for mamma- cloned and named RHBDL1 for rhomboid-like protein1 before lian tumor growth factor (TGF-α), triggering the secretion of Drosophila Rhomboid-1 was recognized as an intramembrane the factors (Rutledge et al., 1992; Schweitzer et al., 1995). protease (Pascall and Brown, 1998), the function of RHBDL2 Homologs of Drosophila rhomboid have been identified in has yet to be elucidated. However, RHBDL2 has been shown most prokaryotic and eukaryotic organisms (Lemberg and to share the catalytic activity of Drosophila Rhomboid-1. The Freeman, 2007). localizations of the five known mammalian rhomboid proteases Rhomboid family members have been shown to have a are diversely scattered being found in the Golgi for RHBDL1, common structure composed of six or seven transmembrane plasma membrane for RHBDL2, endosomes for RHBDL3, Open Access https://doi.org/10.4062/biomolther.2017.103 Received May 7, 2017 Revised Jul 21, 2017 Accepted Jul 24, 2017 Published Online Dec 8, 2017 This is an Open Access article distributed under the terms of the Creative Com- mons Attribution Non-Commercial License (http://creativecommons.org/licens- *Corresponding Authors es/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, E-mail: [email protected] (Choi KC), [email protected] (Nam KH) and reproduction in any medium, provided the original work is properly cited. Tel: +82-43-261-3664 (Choi KC), +82-43-240-6561 (Nam KH) Fax: +82-43-267-3150 (Choi KC), +82-43-240-6569 (Nam KH) Copyright © 2018 The Korean Society of Applied Pharmacology www.biomolther.org 298 Lee et al. Collagen-Induced Arthritis endoplasmic reticulum (ER) for RHBDL4 and mitochondrial types of features. Accordingly, it was predicted that the induc- inner membrane for PARL (Bergbold and Lemberg, 2013), tion of arthritis would be impaired in this mutant mouse be- suggesting that they have distinct and diverse functions. Only cause TNF-α is not released in the Rhbdf2 KO mouse. RHBDL2 can cleave and activate the mammalian proEGF (Adrain et al., 2011), and EGFR signaling is negatively modu- lated by RHBDL2-mediated lysosomal degradation of EGFR MATERIALS AND METHODS (Haglund and Dikic, 2012) and (or) EGFR cleavage (Liao and Carpenter, 2012). Conversely, RHBDL4 localizing to the ER Rhbdf2 mutant mouse can induce degradation of various substrates (Bergbold and A Rhbdf2 KO founder mouse with the C57BL/6J back- Lemberg, 2013) as a part of the ER-associated protein degra- ground was produced at the Korea Research Institute of Bio- dation (ERAD) machinery (Fleig et al., 2012). science and Biotechnology (KRIBB) using CRISPR/Cas9, and However, there are other subgroups of rhomboid family the mutation was confirmed by a T7E1 assay. For the mutant members, known as iRhoms, which have high sequence simi- lineage establishment, the founder mouse was crossed with larities. These rhomboid family members, iRhom1 and iRhom2 C57BL/6J mice to maintain the pure C57BL/6J background. (another name of Rhbdf2), are inactive rhomboids that have no the key catalytic motif observed in other rhomboid proteases Mutant allele analysis of Rhbdf2 mutant mouse (Lemberg and Freeman, 2007; Ha et al., 2013). iRhom1 and To identify the exact mutated sequences, the tail DNA from iRhom2 lost their protease activity during their evolution but F1 mutants was extracted and used as a template for PCR am- have retained key non-protease functions, which have been plification with the primer pairs F1 (5’-TTCCATAAACACGAG- implicated in regulation of the epidermal growth factor (EGF) CACCA-3’) and R1 (5’-CTCTGGCTACTCCCATCTGC-3’), signaling pathway (Adrain et al., 2011) and the tumor necrosis which span the target area for the guide RNA for CRISPR/ factor-alpha (TNF-α) signaling pathway (Adrain et al., 2012). Cas9. The PCR amplicon was TA cloned for sequence deter- As more distant rhomboid family members, many other mination using a TA cloning kit TOPO® TA Cloning® Kit (Invitro- genes without the key catalytic motif, such as derins, UBAC2, gen, Carlsbad, CA, USA). The PCR primer pairs for genotyp- RHBDDs and TMEM115, have also been annotated as ing the mutant allele were then designed based on the results rhomboid-like proteins by bioinformatics searches based on of the wild and mutant allele sequences. their sequence similarities (Koonin et al., 2003; Lemberg and To confirm the null mutation of Rhbdf2 gene, Western blot Freeman, 2007; Finn et al., 2010). However, the structural re- analysis for Rhbdf2 was performed with mouse tissues. Brief- lationships among these proteins remain to be investigated ly, lysates from mouse tissues were subjected to 8% SDS- because of their limited overall sequence conservation (Berg- PAGE, and the resolved proteins were transferred to PVDF bold and Lemberg, 2013). membranes (Bio-Rad, Hercules, CA, USA). The membranes Currently, there are 14 rhomboid family members, five were blocked for 1 h at room temperature in 5% skim milk, and rhomboid proteases and nine catalytically inactive homo- then incubated with anti-Rhbdf2 (MyBioSource, San Diego, logues (Bergbold and Lemberg, 2013). Among these rhom- CA, USA). The bands were visualized with enhanced chemi- boids, iRhoms comprise a unique family, not only possessing luminescence reagents (Ab Frontier, Seoul, Korea). Further- the key catalytic motif and highly conserved sequences be- more, to confirm the functional impairment in TNF-α secretion tween species, but also the unique iRhom homology domain in the mutant mouse, TNF-α secretion assay was perfromed and cytosolic N-terminal cytosolic domain, suggesting that with bone marrow derived macrophages (BMDM). Briefly, these proteins have an important biological role, despite their bone marrow cells were collected from femurs and tibias of lack of protease activity (Koonin et al., 2003; Lemberg and mice, and red blood cells (RBCs) were lysed with ACK lysing Freeman, 2007; Freeman, 2014). Rhomboid is a family mem- buffer (CMABREX Bio Science, Walkersville, MD, USA). The ber 2 protein in humans that is encoded by the RHBDF2 gene bone marrow cells were cultured with M-CSF (20 ng/ml) for 6 (Puente et al., 2003). The alternative name iRhom2 has been days to generate BMDM. BMDMs (1x105 cells/well) were cul- proposed to clarify that it is a catalytically inactive member of tured in a 96-well plate with RPMI1640 medium (Gibco Labo- the rhomboid family of intramembrane serine proteases (Lem- ratories, Grand Island, NY, USA) supplemented with 10% fetal berg and Freeman, 2007) . bovine serum, 50 nM 2-mercaptoethanol (Life Technologies, Rheumatoid arthritis (RA) is a chronic inflammatory disor- Gaithersburg, MD, USA), 100 µg/mL streptomycin, 100 U/ der characterized by synovitis and joint damage. The etiology mL penicillin and with or without 1 µg/ml of lipopolysaccha- of RA is multi-factorial, including various genetic and environ- ride (LPS) (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. The mental factors, and its pathogenesis is complex, involving sy- concentrations of TNF-α secreted from BMDM was measured novial tissue proliferation, pannus formation, and cartilage and using commercially available ELISA kits (Quantikine Mouse bone destruction.