Background/Purpose:

Gene expression and functions of synovial fibroblasts (SF) differ profoundly between distinct joints. This might lead to site-specific activation of arthritis-relevant pathways with implications for arthritis pathogenesis and drug targeting ¹. Homeobox (HOX) transcription factors guide the proper morphogenesis of limb structural elements and appear to influence site-specific development of various diseases. Here we analyzed the expression and epigenetic regulation of HOXD genes in SF from different joints of the hand and identified their target genes and functional effects.

Methods:

The expression of HOXD in SF was analyzed by RNA sequencing (n=21), quantitative Real-time PCR (qPCR; n=14) and Western blotting (n=10). The histone marks H3K4me1 (enhancers), H3K4me3 (promoters), H3K27me3 (repressed chromatin) and H3K27ac (active chromatin) were analyzed by Chromatin Immunoprecipitation DNA sequencing (ChIPseq) in SF from one RA (finger II) and one osteoarthritis (OA; thumb) patient. HOXD10, HOXD11 and HOXD13 were silenced using antisense LNA™ GapmeRs in finger II-IV SF (n=4). The expression of potential target genes was analyzed by qPCR in joints of the hand (n=7). Growth of transfected SF (n=3) was analyzed using an impedance-based system (xCELLigence).

Results:

Among the genes in the HOX cluster, HOXD10, HOXD11 and HOXD13 transcripts and proteins were significantly increased in SF in digits II-IV (MCP, PIP) and wrists compared to SF from the thumb (CM I, MP I). This signature was independent of disease and recapitulated the embryonic HOXD expression pattern in the developing hand. ChIPseq showed an increase of H3K27ac and H3K4me3 marks in the genomic region between HOXD9 and HOXD13 in SF from an RA finger II compared to SF from an OA thumb. This was paralleled by a loss of the repressive histone mark H3K27me3 in this region in RA finger II. Similar to the low expression of HOXD10-13 in thumbs, the expression of thrombospondin 2 (THBS2; p<0.05), receptor tyrosine kinase like orphan receptor 2 (ROR2; p<0.05), collagen type XI alpha 1 chain (COL11A1; p<0.01) and ATP binding cassette subfamily C member 9 (ABCC9; p<0.05) was decreased in joints of the thumb compared to joints of digits II-IV as measured by RNAseq and qPCR. Accordingly, silencing of HOXD13 decreased the expression of THBS2 (p<0.001), ROR2 (p<0.001) and COL11A1 (p<0.01), whereas ABCC9 (p<0.01) was decreased by silencing of HOXD10 (p<0.01) and HOXD13 (p<0.01). Silencing of HOXD13 but not HOXD10 and HOXD11 reduced the proliferation of SF by 83,7% (p<0.01).

Conclusion:

HOXD10-13 exhibit an epigenetically regulated, increased expression in fingers II-IV compared to thumb that determines the site-specific signature of downstream target genes and the proliferative capacity of SF. This might influence the differential activation of arthritis-relevant pathways and patterns of OA and RA in the small joints of the fingers II-IV versus the thumb.

References: ¹Frank-Bertoncelj et al., Nat Commun, 2017.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/hoxd-genes-regulate-arthritis-relevant-pathways/