Fetal bovine serum contains biologically available ATP.

ATP is a ubiquitous extracellular messenger released in a wide number of pathophysiological conditions. ATP is known to be present in minute amounts in the extracellular space in healthy tissues and in the blood, and to modulate a multiplicity of cell responses. Cell culture systems are widely used to explore purinergic signaling. We show here that currently used fetal bovine sera contain ATP in the 300-1300 pmol/L range. Serum ATP is associated with albumin as well as with microparticle/microvesicle fraction. Serum microparticles/microvesicles affect in vitro cell responses due to their content of miRNAs, growth factors, and other bioactive molecules. ATP is likely to be one of these bioactive factors found in a variable amount in sera of different commercial sources. ATP in serum supports ATP-dependent biochemical reactions such as the hexokinase-dependent phosphorylation of glucose to glucose 6-phosphate, and affects purinergic signaling. These findings show that cells growing in vitro in serum-supplemented media are exposed to varying levels of extracellular ATP, and thus to varying degrees of purinergic stimulation.

Reciprocal Regulation of TRPS1 and miR-221 in Intervertebral Disc Cells.

Intervertebral disc (IVD), a moderately moving joint located between the vertebrae, has a limited capacity for self-repair, and treating injured intervertebral discs remains a major challenge. The development of innovative therapies to reverse IVD degeneration relies primarily on the discovery of key molecules that, occupying critical points of regulatory mechanisms, can be proposed as potential intradiscal injectable biological agents. This study aimed to elucidate the underlying mechanism of the reciprocal regulation of two genes differently involved in IVD homeostasis, the miR-221 microRNA and the TRPS1 transcription factor. Human lumbar IVD tissue samples and IVD primary cells were used to specifically evaluate gene expression and perform functional analysis including the luciferase gene reporter assay, chromatin immunoprecipitation, cell transfection with hTRPS1 overexpression vector and antagomiR-221. A high-level expression of TRPS1 was significantly associated with a lower pathological stage, and TRPS1 overexpression strongly decreased miR-221 expression, while increasing the chondrogenic phenotype and markers of antioxidant defense and stemness. Additionally, TRPS1 was able to repress miR-221 expression by associating with its promoter and miR-221 negatively control TRPS1 expression by targeting the TRPS1-3'UTR gene. As a whole, these results suggest that, in IVD cells, a double-negative feedback loop between a potent chondrogenic differentiation suppressor (miR-221) and a regulator of axial skeleton development (TRPS1) exists. Our hypothesis is that the hostile degenerated IVD microenvironment may be counteracted by regenerative/reparative strategies aimed at maintaining or stimulating high levels of TRPS1 expression through inhibition of one of its negative regulators such as miR-221.