Huntingtin-Interacting Protein 1-Related (HIP1R) Regulates Rheumatoid Arthritis Synovial Fibroblast Invasiveness

Abstract

Huntingtin-interacting protein 1-related (HIP1R) shares some function similarities with HIP1, and HIP1 regulates arthritis and RA fibroblast-like synoviocytes (FLS) invasiveness. Therefore, we hypothesized that HIP1R might be involved in the regulation of FLS phenotypes and molecular processes relevant to RA. siRNA was used to knockdown HIP1R, HIP1 or control in RA FLS, followed by cell studies for invasion in Matrigel, migration, proliferation, and adhesion. RNA was sequenced and analyzed. HIP1R knockdown significantly reduced RA FLS invasiveness and migration (p < 0.05). The DEGs in siRNA HIP1R had an enrichment for GO processes "astrocyte and glial cell projection", "small GTPase signaling", and "PDGFR signaling". The most significantly DEGs had decreased expression in siRNA HIP1R and included AKT1S1, GABBR2, GPR56, and TXNDC12. siRNA HIP1 RA FLS had an enrichment for the "Rap1 signaling pathway" and "Growth factor receptor binding". The most significantly DEGs in HIP1 siRNA included FGF2, PGF, and SLC39A8. HIP1R and HIP1 DEG lists had a greater than expected number of similar genes (p = 0.0015), suggesting that, despite the major differences detected, both have partially overlapping functions in RA FLS. The most significantly DEGs in both HIP1R and HIP1 analyses are involved in cancer cell behaviors and outcomes. HIP1R is a new gene implicated in RA FLS invasiveness and migration, and regulates unique pathways and cell processes relevant to both RA as well as cancer biology. Our study provides new insight into processes implicated in FLS invasiveness, which is relevant for joint damage in RA, and identify new potential gene targets for FLS-specific treatments.

Keywords: HIP1; HIP1R; fibroblast; rheumatoid; signaling; synovial; synovitis.

Figure 1

HIP1R protein is present in RA FLS and knockdown significantly affects cell behavior. (A) Immunofluorescence staining demonstrating that HIP1R (green) protein is expressed in RA FLS, and localized near the cell edge. Phalloidin stain actin filaments (red) and DAPI stains of the nucleus (blue) (three representative cell lines are shown; 600× magnification). (B) Western blots done with RA FLS cell lysates confirming the presence of the HIP1R protein (green) in RA FLS (n = 6). GAPDH (red) was used as loading control. (C) siRNA knockdown of HIP1R in RA FLS significantly reduced cell invasiveness (n = 8, p = 0.0419, paired t-test), (D) and also reduced cell migration in the scratch/wound healing assay (n = 5, p = 0.0413, paired t-test). (E) HIP1R knockdown did not have any significant effect in RA FLS adhesion (n = 4) (F) or on three-day proliferation (n = 5). (RA1 to RA6 refers to individual RA patient cell line; ABS = Absorption; see Table 1 for demographic data). (* statistically significant with p < 0.05).

Figure 2

Biologic Pathways and genes enriched in HIP1R knockdown studies. (A) GO pathways enriched among DEGs in siRNA HIP1R versus control (adj p-value = adjusted p value). (B) Numbers of DEGs in siRNA HIP1R versus siRNA control, and in siRNA HIP1 versus control. (C) Volcano plot showing DEGs (red = up in siRNA HIP1R; green = down in siRNA HIP1R; grey = unchanged). (D) DEGs that remained significant after p-value adjustment, including down-regulated genes TXNDC12, GABBR2, GPR56, AKT1S1, PDGFRB, HIP1R, SLC2A4RG, and EZR, and up-regulated genes CYP26B1, STEAP4, and ADAMTS15.

Figure 3

Biologic pathways and genes enriched in HIP1 knockdown studies. (A) DO terms such as “malignant mesothelioma”, “proliferative diabetic retinopathy”, “liver cirrhosis”, and “osteosarcoma” were enriched in siRNA HIP1 versus siRNA control analyses (adj p-value = adjusted p value). (B) KEGG pathway “Rap1 signaling pathway” was the most significant, followed by GO pathways “growth factor receptor binding”, “heparin binding”, “glycosaminoglycan binding”, and “sulfur compound binding”. (C) Volcano plot showing DEGs (red = up in siRNA HIP1; green = down in siRNA HIP1; grey = unchanged; HIP1 significance and fold change is shown in the upper left corner). (D) DEGs that remained significant after p-value adjustment for multiple tests included down-regulated FGF2, KCNS3, MT-TV, ORC6, PGF, and SLC39A8, and up-regulated CLEC3B, DMKN, PSKH1, and NPTX1.