Niacin restriction upregulates NADPH oxidase and reactive oxygen species (ROS) in human keratinocytes

Abstract

NAD(+) is a substrate for many enzymes, including poly(ADP-ribose) polymerases and sirtuins, which are involved in fundamental cellular processes including DNA repair, stress responses, signaling, transcription, apoptosis, metabolism, differentiation, chromatin structure, and life span. Because these molecular processes are important early in cancer development, we developed a model to identify critical NAD-dependent pathways potentially important in early skin carcinogenesis. Removal of niacin from the cell culture medium allowed control of intracellular NAD. Unlike many nonimmortalized human cells, HaCaT keratinocytes, which are immortalized and have a mutant p53 and aberrant NF-kB activity, become severely NAD depleted but divide indefinitely under these conditions. Niacin-deficient HaCaTs develop a decreased growth rate due to an increase in apoptotic cells and an arrest in the G(2)/M phase of the cell cycle. Long-term survival mechanisms in niacin-deficient HaCats involve accumulation of reactive oxygen species and increased DNA damage. These alterations result, at least in part, from increased expression and activity of NADPH oxidase, whose downstream effects can be reversed by nicotinamide or NADPH oxidase inhibitors. Our data support the hypothesis that glutamine is a likely alternative energy source during niacin deficiency and we suggest a model for NADPH generation important in ROS production.

Fig. 1

Effects of nicotinamide restriction on HaCaT keratinocytes. (A) Growth rates of HaCaT cells grown under control (33 μM Nam, squares), or restricted (0 μM Nam, triangles) conditions. Inset shows details of growth for days 0 to 21. Values correspond to duplicate samples (Mean ± SEM). (B) Cell viability was assessed for 33 μM Nam, 0 μM Nam and repleted cells using Annexin V/PI flow cytometry. Values represent percentage of viable cells from triplicate samples (Mean ± SEM). (C) Cell cycle analysis was assessed for 33 μM Nam (black) and 0 μM Nam (white). Bars represent the mean of triplicate analyses (± SEM) by PI fluorescence and flow cytometry. (D) DNA damage was analyzed by alkaline comet assay. Dots represent single cells (n=50 for day 7; n=100 for day 14); mean tail moment is shown by the lines. * p < 0.01, ** p < 0.001, *** p < 0.0001 versus control (Student's t-test).

Fig. 2

ROS accumulation in NAD-depleted HaCaT keratinocytes. (A) Determination of intracellular ROS (A) after 7 and 14 days. (B) reduced GSH (left), GSSG (middle), and GSH/GSSG ratio quantification 14 days of growth. (C) ROS after treatment with 0.3 mM apocynin. (D) Cell viability after treatment with 0.3 mM apocynin. 33 μM Nam (black), 0 μM Nam (white), or repleted (gray). All values represent the mean of triplicate samples (± SEM).* p < 0.01, ** p < 0.001, *** p < 0.0001 versus control (Student's t-test).

Fig. 3

Effect of GDH inhibition on niacin deficient HaCaT keratinocytes. Growth rates of HaCaT cells grown under control (33 μM Nam, squares), or restricted (0 μM Nam, triangles) conditions in the presence (dotted line) or absence (continuous line) of DON. Values represent triplicate samples (Mean ± SEM).

Fig. 4

Model of NOX activation in nicotinamide-restricted HaCaT keratinocytes. Under NAD-depleted conditions, the ability of the cell to use glycolysis as a source of energy is greatly reduced. Cells can utilize glutamate, synthesized from glutamine in the culture medium as an energy source. Glutamate dehydrogenase (GDH) coverts glutamate to α-ketoglutarate, with reduction of NADP⁺ to NADPH. The activity of GDH may be regulated by SIRT4, which is dependent on NAD⁺; therefore, under NAD-depleted conditions, GDH activity may be upregulated providing α-ketoglutarate as a carbon source for TCA cycle intermediates and energy production. Additional NADPH may be generated by IDH2. NADPH accumulation from these reactions could support NOX dependent ROS formation and recycling of NADP⁺. In pre-cancerous cells, this ROS dependent signaling may lead to progression in carcinogenesis.