RNASeq-based identification of extracellular matrix components impacted by FasL deficiency in osteoblasts

Osteoblasts and osteoclasts are two critical cell populations in bone remodelling. FasL is known from osteoblast-induced osteoclast apoptosis. Notably, FasL deficient (gld) mice display increased bone mass pointing to non-apoptotic functions of FasL. Some key extracellular matrix associated molecules, such as osteoprotegerin, osteocalcin, osteopontin and alkaline phosphatase were reported to be affected by FasL deficiency in osteoblasts but a complex insight has been missing. To fill this gap and provide a robust dataset, gld calva-derived cells were differentiated along with controls and subjected to RNASequencing. The analysis revealed statistically significant changes in expression of 1,710 genes. 15 % (263) out of them were associated with the extracellular matrix. This applied for the collagen components, but also non-collagenous proteoglycans including versican and aggrecan. This report provides enrichment analysis of statistically significant expression changes in calva-derived osteoblastic cells with focus on extracellular matrix components. The dataset further provides a solid background for further functional analyses revealing the exact mechanisms behind and consequences of such dysregulations in pathophysiology. Overall design: Mice homozygous for the FasL gld mutation (B6Smn.C3-Faslgld/J) were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 were used as a wild-type control. Mice were kept at the Department of Biomedical Research of the Medical University of Vienna. Calvaria-derived cells (gld and wt) were isolated and expanded as described in Kratochvilova et al. (2022). Differentiation was performed in osteogenic media consisting of MEM alpha (Gibco, Waltham, MA, USA), 10% fetal bovine serum (Sigma-Aldrich, Burlington, MA, USA), 1% penicillin/streptomycin (Sigma-Aldrich), 50 mg/ml ascorbate and 4 mM ß-glycerophosphate in 37°C and 5% CO2. For the stimulatory experiment of gld cells, the osteogenic medium was supplemented with 150 ng/ml recombinant human sFas ligand (Peprotech, 310-03H) at day 14 of differentiation. The cells were then cultured for 72 h, the medium/medium+FasL was changed once during this time, wt cells (wt), gld cells (gld) and gld cells treated by FasL (gld+FasL) were cultured in parallel. Samples for RNA isolation were lysed in 350 µl of RLT lysis buffer (Qiagen) with ß-ME (Sigma-Aldrich), total RNA was extracted using RNeasy Mini Kit (Qiagen, Germany) and cDNA was synthesized using the gb Elite Reverse Transcription Kit (Generi Biotech, Czech Republic). Total RNA samples were sent for QuantSeq analysis to Lexogen GmbH, Campus Vienna Biocenter 5, Vienna, where the samples were processed and sequenced according to the company's standard operating procedure. In brief, the library for sequencing was prepared using the QuantSeq 3'mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen) and the Lexogen i5 6 nt Unique Dual Indexing Add-on Kit (Lexogen). Single-read RNA-Seq (1 × 75) was performed using an Illumina NextSeq 500 System (Illumina). Comprehensive analysis of the RNA-Seq data, including demultiplexing, read quality assessment, trimming and filtering, read mapping, and differential expression analysis, was carefully performed by Lexogen GmbH. This analysis was performed according to their standardized pipeline for QuantSeq FWD data. In brief, read quality was assessed using the FastQC , RSeQC and MultiQC packages. Samples were trimmed using the Cutadapt tool and aligned to the reference genome using the STAR aligner.