Abstract
IntroductionCircadian rhythms play an important role in the body and in single cells. Rhythms of molecular clocks have not been investigated in synovial fibroblasts (SF) of patients with osteoarthritis (OA) and rheumatoid arthritis (RA). The study was initiated to fill this gap and to study effects of interleukin (IL)-1β/tumor necrosis factor (TNF) on rhythmicity in synovial fibroblasts of RA and OA patients.MethodsThe presence of BMAL-1, CLOCK, Period 1 and Period 2 proteins in synovial tissue was investigated by immunofluorescence. The presence of mRNA of molecular clocks was studied during 72 h by qPCR. Characteristics of rhythms were studied with time series analysis.ResultsBMAL-1, CLOCK, Period 1 and Period 2 proteins were abundantly present in synovial tissue of OA, RA and controls. Receiving synovial tissue at different operation time points during the day (8:00 am to 4:00 pm) did not reveal a rhythm of BMAL-1 or Period 1 protein. In OASF and RASF, no typical rhythm curve of molecular clock mRNA was observed. Time series analysis identified a first peak between 2 and 18 hours after synchronization but a period was not detectable due to loss of rhythm. TNF inhibited mRNA of CLOCK, Period 1 and Period 2 in OASF, while IL-1β and TNF increased these factors in RASF. This was supported by dose-dependently increased levels in MH7A RA fibroblasts. In RASF, IL-1β and TNF shifted the first peak of BMAL-1 mRNA to later time points (8 h to 14 h).ConclusionRhythmicity is not present in primary OASF and RASF, which is unexpected because fibroblasts usually demonstrate perfect rhythms during several days. This might lead to uncoupling of important cellular pathways.
Highlights
Circadian rhythms play an important role in the body and in single cells
Clock proteins in synovial tissue The presence of molecular clocks is a prerequisite for endogenous cellular rhythms
While synovial density of positive cells for CLOCK and Period 2 were similar between disease groups, density of BMAL1-positive cells was higher in rheumatoid arthritis (RA) compared to OA (Figure 2A)
Summary
Circadian rhythms play an important role in the body and in single cells. Rhythms of molecular clocks have not been investigated in synovial fibroblasts (SF) of patients with osteoarthritis (OA) and rheumatoid arthritis (RA). The study was initiated to fill this gap and to study effects of interleukin (IL)-1b/tumor necrosis factor (TNF) on rhythmicity in synovial fibroblasts of RA and OA patients Symptoms, such as swelling, pain, stiffness, and functional ability, follow a circadian rhythm in patients with rheumatoid arthritis (RA) [1]. The circadian rhythm is generated in the superordinate hypothalamic nucleus suprachiasmaticus [7], and this rhythm can be transferred to peripheral cells of the body by hormonal and neuronal signals [8,9,10,11,12]. After synchronization with serum shock in vitro, rhythms of molecular clocks are often self-sustained under constant culture conditions [12,14,15] This has been demonstrated for neurons of the nucleus suprachiasmaticus [14], T cells [12] and fibroblasts [15]. It is thought that these rhythms serve an overall coupling of important bodily functions which is, for example, reflected in coupling of feeding behavior and cardiomyocyte responsiveness to ingested fatty acids [8]
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