The Ufm1 System Is Upregulated by ER Stress During Osteogenic Differentiation

bioRxiv preprint doi: https://doi.org/10.1101/720490; this version posted July 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The Ufm1 system is upregulated by ER stress during osteogenic differentiation Michal Dudek1* and Gillian A. Wallis1 1 Wellcome Centre for Cell Matrix Research, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine & Health, Manchester Academic Health Science Centre, University of Manchester, Oxford Road, Manchester M13 9PT, UK * Corresponding author: Dr Michal Dudek e-mail: [email protected] Abstract Objective: Mutations in the catalytic site of the ubiquitin-fold modifier 1(UFM1)-specific peptidase 2 (UFSP2) gene have been identified to cause autosomal dominant Beukes hip dysplasia in a large multigenerational family and a novel form of autosomal dominant spondyloepimetaphyseal dysplasia in a second family. We investigated the expression of the UFSP2/UFM1 system during mouse joint development the connection to ER stress induced during osteogenic differentiation. Methods: The pattern of expression of Ufsp2 was determined by radioactive RNA in situ hybridisation on mouse tissue sections. qPCR was used to monitor expression during in vitro osteogenic differentiation and chemically induced ER stress. Affinity purification and mass spectrometry was used for isolation and identification of Ufm1 conjugation targets. Luciferase reporter assay was used to investigate the activity of Ufm1 system genes’ promoters. Results: We found that Ufsp2 was predominantly expressed in the bone and secondary ossification centres of 10-day old mice. The Ufm1 system was upregulated during in vitro osteogenic differentiation and in response to chemically induced ER stress. We identified unfolded protein response elements in the upstream sequences of Uba5, Ufl1, Ufm1and Lzap. We identified putative Ufm1 conjugation targets where conjugation was increased in response to ER stress. Conclusion: Higher expression of Ufsp2 in bone and secondary ossification centres as well as upregulation of components of the Ufm1system in response to ER stress suggests that the molecular pathway between the UFSP2 mutations and form of skeletal dysplasia may relate to abnormal ER stress responses during osteoblast differentiation. Keywords: Ufm1, Ufsp2, ER Stress, Osteoarthritis, enzyme cascade the major components of which are: i) the E1- 1. Introduction activating enzyme, Uba5 (ubiquitin like modifier activating enzyme 5); ii) the E2 enzyme, Ufc1 (UFM1 conjugating We have previously identified a unique mutation in the Ufm1- enzyme 1), responsible for conjugation of Ufm1 to target specific peptidase 2 gene, UFSP2, that results in loss of proteins; iii) Ufl1, a Ufm1 E3 ligase, and iv) Ufsp1 and Ufsp2 UFSP2 catalytic activity (in vitro) of in a large (the UFM1 specific peptidases 1 and 2) [3]. Additionally, multigeneration family with Beukes Hip Dysplasia (BHD) [1]. Lzap (Cdk5rap3) is a substrate adaptor for Ufl1 and Ddrgk1 is BHD is an autosomal dominant disorder characterised by one of few known Ufm1 conjugation targets. Loss-of-function bilateral dysmorphism that is limited to the proximal femur mutation in DDRGK1gene was found to cause Shohat-type and delayed ossification of the secondary centre, which results SEMD, a condition characterised by disproportionate short in severe progressive degenerative osteoarthropathy from stature, lordosis, genu varum, and joint hypermobility. childhood. A second novel mutation in the UFSP2 gene, also Radiographically, patients with SEMD have delayed predicted to cause loss of UFSP2 catalytic activity [2] has been epiphyseal ossification, platyspondyly with central notches in identified in affected members of a family with a novel form the vertebral end plates, radiolucency of the femoral of autosomal dominant spondyloepimetaphyseal dysplasia metaphyses, and relative fibular overgrowth. Genetic deletion (SEMD) involving the epiphyses predominantly of the hips, of DDRGK1in zebrafish and in mice led to disruption of but also of knees, ankles, wrists and hands, with variable cartilage formation by decreasing Sox9 protein level [4]. degrees of metaphysis and spine involvement and characterised by short stature, joint pain and genu varum. Components of the Ufm1 system localise to the ER, and functional studies point to specialised roles for ufmylation in UFSP2 is a component of the Ufm1 (ubiquitin-fold modifier regulating ER-related stress [3]. The ER is a major site of 1) system, which like the ubiquitin system, comprises a multi- protein quality control, housing the synthesis machinery for 1 bioRxiv preprint doi: https://doi.org/10.1101/720490; this version posted July 30, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. proteins destined to be secreted or membrane-trafficked of buffer per slide) and heated to 95°C. The hybridisation mix within the cell, as well as chaperones to assist protein folding. was then applied on the slides and the slides were covered with Chondrocytes and osteoblasts actively secrete the proteins that glass cover slips. The slides were then placed in hybrydisation form the vast extracellular matrix of cartilage and bone. Forms chamber containing 50% formamide and incubated overnight of skeletal dysplasia caused by mutations in matrix genes have at 55°C. On the next day cover slips were removed and slides been identified where ER stress is a common feature of their were incubated in following wash solutions and buffers: Wash molecular pathology [5, 6]. solution twice at 55°C for 20 min and once at 65°C for 20 minutes; RNase buffer twice at 37°C for 15 minutes; RNase A In this study, following identifying that Ufsp2 is expressed by at 37°C for 30 minutes; RNase buffer at 37°C for 15 minutes; osteoblasts during mouse joint development, we investigated Wash solution twice at 65°C for 20 min; SSC/DTT buffer at whether the Ufm1 system is differentially regulated during 5°C for 20 min and then 5 minutes at room temperature; osteogenic differentiation and in response to ER stress. ammonium acetate / ethanol for 2 minutes at room temperature. The slides were then dehydrated through the 2. Methods graded ethanol series, air dried and exposed to 2.1 Radioactive in situ hybridisation autoradiography film overnight to confirm the hybridisation worked before covering the slides with photoreactive Probe synthesis: DNA template for the synthesis of RNA emulsion. probe was prepared by linearisation of plasmids containing probe sequence with a restriction enzyme cutting at the 5’ end of RNA polymerase transcription site generating antisense Developing: After confirming the hybridisation with RNA product. The template DNA was then separated on autoradiography film the slides were covered with K5 nuclear agarose gel and extracted using QIAquick Gel Extraction Kit. emulsion (Ilford, UK) prepared as 50:50 solution with 2% The radio-labelled probe was synthesised replacing UTP with glycerol, dried and stored in dark for 2 weeks before 35 S UTP using Riboprobe® System (Promega) for 40 min at developing. The slides were developed in D19 developer 37°C. After this time 1μl of RNA polymerase was added and (Kodak) and fixed using Unifix (Kodak). After developing the reaction was incubated at 37°C for an additional 1 hour. slides were H&E stained using Thermo Shandon Linistain Following transcription the DNA template was digested with GLX stainer. DNase at 37°C for 10 minutes. The RNA probe was then precipitated. The pellet was washed with 50 μl of 10 mM DTT 2.2 Protein extraction diluted in 100% EtOH. After drying the RNA pellet was resuspended in 50 μl of 50 mM DTT diluted in DEPC H2O. Cartilage tissues were pulverized using a liquid-nitrogen- cooled tissue grinder and proteins extracted as previously Preparation of tissue sections: Mouse tissues were fixed described [5]. Briefly, cartilage samples were reconstituted in overnight in 4% PFA in PBS, processed in Shandon Citadel 100 μL of 100 mM Tris acetate buffer pH 8.0 containing 10 2000 tissue processor and paraffin embedded. Tissues from mM EDTA and protease/phosphatase inhibitors and mice older than newborn were decalcified for three days in deglycosylated by treatment with 0.1 units of chondroitinase decalcification solution (20% EDTA, 4% PFA in PBS pH 7.4) ABC for 6 h at 37 °C. Proteins were sequentially extracted in at 4°C before embedding. Slides were prepared by sectioning a chaotropic buffer containing guanidine hydrochloride (4M paraffin blocks on Microm HM 355S microtome with feed set GuHCl, 65 mM DTT, 10 mM EDTA in 50 mM sodium to 5 and drying them overnight in 50°C. The sections were acetate, pH 5.8). Protein samples were precipitated with nine then de-waxed in xylene and rehydrated by passing through a volumes of ethanol, washed once in 70% ethanol, then graded ethanol series. The slides were incubated in 2% HCl resuspended in 120 μL of solubilisation buffer (7 M urea, 2 M for 20 minutes, washed in 2x SSC and incubated in proteinase thiourea, and 30 mM Tris, pH 8.0) and the volume was K buffer for 10 minutes at 37°C. The proteinase was then adjusted to achieve a concentration of ∼1 mg/mL, as estimated inactivated by dipping the slides in 2mg/ml glycine for 2 using the EZQ protein quantitation protocol (Thermo Fisher). minutes at room temperature. Slides were washed twice in Samples were then stored at −80 °C until required. Protein PBS, incubated for 20 minutes in 4% PFA-PBS and washed in samples were analysed by SDS-PAGE and detected by silver PBS again. The slides were then incubated in triethanolamine staining as previously described. (triethanolamine 4.65 ml, 875 μl acetic anhydride and 345ml H2O) for 10 minutes at room temperature and washed in PBS 2.3 Osteogenic differentiation followed by wash in H2O and dehydrating through the graded Subconfluent 2T3 cells were trypsinized and plated on ethanol series.

Load more