Lactate down‐regulates cellular poly(ADP‐ribose) formation in cultured human skin fibroblasts

Abstract

Polyadenosine diphosphate-ribose (poly(ADP-ribose)) is a nuclear polymer which is derived from nicotinamide adenine dinucleotide (NAD(+)) catalysed by poly(ADP-ribose) polymerase 1 (PARP-1). Aside from the well known role of poly(ADP-ribosyl)ation (pADPR) in DNA repair, pADPR is also involved in other cellular processes such as apoptosis and gene expression. However, the factors that regulate the level of pADPR are not fully elucidated. In view of the fact that healing wounds contain high concentrations of lactate (10-15 mM) and exogenous lactate reduce the NAD(+) pool in cultured fibroblasts, we propose that high lactate lowers the level of nuclear pADPR. Neonatal human dermal fibroblasts (NHDF) were plated to subconfluence and allowed to adhere. Cells were treated with 15 mM l-lactate and pADPR production was assessed by immunofluorescence analysis using 10H antibody. Difference in pADPR production was determined by calculation of positively stained cells compared to total cell numbers. Inhibition of PARP activity was tested by treatment with 100 microM 3-aminobenzamide (3-AB). Specificity of the lactate effect on pADPR synthesis was verified by using the analogue d-lactate. The contents of nicotinamide adenine dinucleotide (NAD(+)) and its reduced form (NADH) in lactated and non-lactated cell cultures were quantified by the enzymatic cyclic assay. We found that exogenous l-lactate (15 mM) can significantly depress pADPR content in cultured fibroblasts. PARP-1 activity was inhibited by 3-AB and analogue d-lactate showed no effect on pADPR synthesis. NAD(+)/NADH ratio was significantly lowered in lactated compared to non-lactated cell culture. Exogenous l-lactate (15 mM) can depress pADPR content in cultured fibroblasts. In view of the fact that healing wounds contain such high concentrations of lactate, we propose that down regulation of pADPR is associated with elevated tissue repair via pADPR dependent gene expression. This observation is important in understanding the stimulation of lactate-mediated protein expression during wound healing.