Purpose: In a pilot study, selected gene expression in tissue samples from patients with OA and osteoporosis (OP) were compared. The gene, SLC9A3R1 which encodes for a member of the sodium/hydrogen regulatory factor protein family (NHERF1) was found to be differentially expressed in osteoblasts. NHERF1 plays a role in tumour suppression, via PTEN, and intracellular pH (pHi) regulation by acting on the sodium/hydrogen exchanger (NHE). We are investigating whether a decrease in NHERF1 causes a change in the pHi of chondrocytes and leads to chondrocyte proliferation. The aim of the project is to test the hypothesis that dysregulated expression of NHERF1 is a key part of the abnormal behaviour of articular chondrocytes. Methods: Full depth cartilage explants or shavings were removed from osteoarthritic femoral heads or knee joints, and from healthy controls (amputees or osteoporotic femoral heads). Explants from 17 patients (4 OA and 4 non-OA femoral heads, 4 OA knees and 5 amputees) were decalcified and fixed in formalin for paraffin embedding and sectioning, whilst shavings were placed in Dulbecco’s Modified Eagles Medium (DMEM) and washed prior to enzymatic chondrocyte isolation. Sections were deparaffinised and either stained with Fast-Green-FCF and Safranin ‘O’, or treated with Target Retrieval Solution and hyaluronidase prior to incubation with glycine, blocking with non-immune serum and overnight incubation with either rabbit anti-human NHE3 (pAb, C-20), mouse anti-human NHERF1 (mAb, IgG2b), or goat anti-human PTEN (pAb, N19) diluted in Dako Antibody Diluent (1:50: PTEN 1:100). Bound NHE3, NHERF1 or PTEN antibodies were detected with goat anti-rabbit (488 nm), goat anti-mouse (555 nm) or donkey anti-goat (488 nm) secondary antibodies (1:100, PTEN 1:200) respectively. Sections were mounted with Prolong Gold anti-fade mounting media containing DAPI and visualized with a Zeiss Imager M2 fluorescent or LSM 700 Zeiss Imager M2 confocal laser scanning microscope (CLSM). Cell counts and co-localisation were analysed using Image J software. Isolated articular chondrocytes (n=3) were incubated with BCECF-AM (5 μM, 15 mins, 37 oC), washed in DMEM without phenol red, aliquoted ( 14 x 104) and placed in a sterile Ibidi μ-Slide I (0.4) Flow Chamber (Thistle Scientific, UK) for 20-30 min (37 oC) to attach. They were washed into the appropriate saline,After saline washing, and BCECF was then alternately excited at 488 nm and 458 nm, and emission collected at >505 nm using a CLSM (Zeiss LSM 710) from individual cells. The background from each wavelength was subtracted and the 488:458 nm ratio (R) converted to pHi using a calibration curve constructed by permeabilizing the chondrocytes to H+ in high-potassium (K+) solutions of different pH containing nigericin (2 μM). Results: NHE3, NHERF1 and PTEN were expressed in all zones of OA and non-OA articular cartilage. Image J analysis showed that NHERF1 co-localised with both proteins in the cytoplasm and perinuclear region, but little was found near the cell membrane. It was also expressed in the nucleus in 50% of chondrocytes in OA and non-OA samples. The median diameter of the nuclei of OA chondrocytes was also found to be significantly larger (7.47 μm) than in non-OA chondrocytes (6.78 μm, P=0.001???). The pHi of normal, healthy chondrocytes was 6.9 + 0.2 (n=3) but OA chondrocytes appeared to be more acidic. The pHi of these chondrocytes was too low to be recorded successfully and reliably using BCECF (pKa 6.98) which is used for measuring near neutral pH, so this necessitated exploring the use of another fluorescein derivative, DCFDA (pKa 4.8) for pH measurements from pH 4-5 Conclusions: This is the first study to identify NHE3, NHERF1 and PTEN expression and co-localisation in human articular cartilage, and to measure pHi in single isolated chondrocytes using confocal microscopy and the fluorescent dyes, BCECF-AM and DCFDA. NHERF1 was expected at the cell membrane but, surprisingly, location was predominantly cytoplasmic. Detection of NHERF1 expression and function in articular cartilage may open new approaches for OA therapy in the future.
|Journal||Osteoarthritis and Cartilage|
|Issue number||Supplement 2|
|Publication status||Published - Apr 2015|
|Event||World Congress of the Osteoarthritis-Research-Society-International (OARSI) on Osteoarthritis 2015 - Seattle, Seattle, United States|
Duration: 30 Apr 2015 → 3 May 2015