OP0020 HOMEOBOX D TRANSCRIPTION FACTORS SHAPE DIFFERENTIAL JOINT ENVIRONMENT BETWEEN ANTERIOR FINGER JOINTS AND THUMB

Abstract

Background:The expression of embryonic Homeobox (HOX) genes is tightly regulated based on anatomic location in human adult dermal and synovial fibroblasts. Previously, we showed that HOX-D10,-D11 and -D13 are higher expressed in synovial fibroblasts from small distal joints from the hands and feet, in particular in digits II-V and wrists compared to thumb and that this expression pattern is epigenetically imprinted1. The consequences of the tightly restricted expression of these transcription factors are largely unknown.Objectives:To elucidate the function of HOXD10, -D11 and -D13 in synovial fibroblasts.Methods:Synovial tissues were isolated from paws of naïve C57BL/6 mice (n=8), from patients with rheumatoid arthritis (RA), osteoarthritis (OA) and from healthy controls. Synovial fibroblasts were cultured and transfected with GapmeR to silence HOXD10, -D11, and -D13, respectively or with control GapmeR. RNA sequencing was performed on the NovaSeq platform and pathway analysis was done using R packages and web-based tools (GSEA, EnrichR, Cytoscape). HOXD target gene expression was measured by qPCR (n=3-6).Results:To confirm and further analyze the distinctive expression pattern of HOXD genes, we measured their expression in healthy synovial tissues of different joints of human feet and mouse paws. Similar to what we had found in hands, HOXD10, -D11 and -D13 were less abundant in the joints of the first digit of human feet compared to digits II-V (n=3-4 in each joint). Measurements in joints of mouse paws showed lower expression of HoxD10, -D11 and -D13 in distal interphalangeal joints compared to proximal interphalangeal joints and metacarpophalangeal (MCP) joints, respectively. Silencing of HOXD10, -D11 and –D13, affected the expression of 5333, 2217 and 7347 genes, respectively, in cultured RA synovial fibroblasts from human wrists (n=3).There were more transcripts equally regulated by HOXD10 and -D13 (40% of all HOXD10 and 31% of all HOXD13 regulated transcripts), than by HOXD11 and either -D10 or -D13 (18% of all HOXD10 regulated genes and 16% of all HOXD13 regulated genes), suggesting most redundancy between HOXD10 and -D13 (Figure 1). Among genes differentially expressed in SF isolated from MCP II-V versus thumb joints, 19%, 4% and 33% were regulated by HOXD10, -D11 or –D13, respectively, supporting a role for HOXD13 in particular in shaping the joint specific environment. All three HOXD transcription factors regulated genes involved in cell cycle progression, demonstrating dependence of synovial fibroblasts on these HOX genes for cell division. Other enriched pathways were Toll-like receptor and integrin signaling pathways, regulation of unsaturated fatty acid synthesis and autophagy and extra-cellular matrix protein organization. We could confirm several targets of HOXD10, -D11, and –D13 by qPCR, e.g. NR4A1, ROR2, LIF, ATF3.Figure 1.Comparison of the genes which were differentially expressed after HOXD10-11-13 silencingConclusion:The expression of HOXD10, -D11 and –D13 in synovial fibroblasts and tissues strikinglyoverlaps with predilection sites for RA. Silencing experiments suggested that these embryonic HOX transcription factors have a crucial role in regulating fibroblast functions and might shape a joint specific environment that modulates the development and course of RA in specific joints.