Clock gene activities, metalloproteinase and TGFβ1 mRNA levels in human dental pulp fibroblasts treated with cyclosporin A

Introduction. Circadian rhythm of clock gene expression is linked to basic cellular functions and the dysregulation of the endogenous cell circadian time structure is associated with pathological conditions, including extracellular matrix (ECM) disorders. Circadian rhythmicity has been also demonstrated in transcripts that encode for cytokines and enzymes. Cyclosporin A (CsA), an effective immunosuppressive drug, affects ECM components and remodelling, also modifying the metalloproteinases (MMPs) and cytokines such as TGFβ1. Since both ECM and clock genes are important for maintaining normal cell and organ functions, we examined the clock gene expression, the MMPs and TGFβ1 mRNA levels in human dental pulp fibroblast cultures after CsA treatment. Materials and methods. Cells obtained from the dental pulp of three healthy third molars, extracted in 18-year-old orthodontic patients, were grown in culture flasks with 199 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics, at 37°C and 5% CO2. At the 4th passage, fully confluent cultures were treated with 50% FBS for 2 hours and then maintained in 199 medium containing 10% FBS alone or 800 ng/ml CsA for 24 hours. After this time we determined MMP-1, MMP-2, and TGFβ1 mRNA levels by RT-PCR analysis, while Bmal1, Clock, Per1 and Cry1 gene expression by microarray methods. Results and conclusions. The treated dental pulp fibroblasts showed a significant decrease of TGFβ1, and MMP-2 mRNA levels; microarrays showed the inactivation of Bmal1 and the stimulation of Clock gene expression. Since MMP-2 and TGFβ1 are involved in ECM remodelling, and clock genes regulate cell activities and tissue-specific processes, these modifications could affect the normal functions of tissues and organs. Considering that oncologic patients treated by chronomodulated therapy have good responses, the role of changes in circadian cell time structure and ECM turnover in multiple human diseases is an intriguing area for future studies.