Poly(ADP-Ribose) May Signal Changing Metabolic Conditions to the Chromatin of Mammalian Cells

Abstract

In mammalian cells, NADI serves a dual role as a respiratory coenzyme and as a substrate for the posttranslational poly(ADP-ribose) modification of chromatin proteins, catalyzed by the nuclear enzyme poly(ADP-ribose) polymerase EC 2.4.2.30]. Biological evidence strongly suggests that poly(ADP-ribosyl)ation modulates chromatin functions, although the precise molecular mechanisms involved have not yet been elucidated. Here we describe conditions for the rapid uptake of exogenously supplied NADI by living hepatocytes in primary monolayer culture. Raising the intracellular NADI concentration by 70% caused a 5-fold increase of chromatin-bound poly(ADP-ribose). We conclude that the constitutive level of posttranslational poly(ADP-ribose) modifications of chromatin proteins in mammalian cells is related to the availability of NAD+, which varies in different physiological and pathological states. We propose that poly-(ADP-ribose) may serve a hitherto unrecognized function by signaling altered metabolic conditions to the chromatin and thus modulate its functions in tune with changing metabolic states.Many lines of biological evidence support the concept that the posttranslational modification of chromatin -roteins by the nuclear enzyme, poly(ADP-ribose) polymei.Xe [NAD+ ADP-ribosyltransferase, EC 2.4.2.30], modulates the phenotypic expression of various chromatin function, in higher eukaryotes (1, 2). The respiratory coenzyme NAD-serves as the specific substrate of this chromatin enzyme. Thi nrompts the question, Why is there such a direct linkage b, w-veen a posttranslational regulatory mechanism peculiar to chromatin and cellular redox reactions? A partial explanation of this is provided by the concept of "suicidal NAD+ depletion" as proposed by Berger (3). This concept emphasizes that carcinogen-inflicted DNA damage in mammalian cells dtamatically stimulates the utilization of NAD+ for poly(ADPribose) biosynthesis and concomitantly depletes the cellular NAD+ pool(s). Thus, the chromatin-associated enzyme poly-(ADP-ribose) polymerase may serve as part of a metabolic "shut-off' mechanism under conditions of excessive DNA damage (3). Here we examined the hypothesis that metabolic conditions affecting the availability of the respiratory coenzyme NAD+ may be signaled to chromatin by poly(ADPribose). Therefore, experimental conditions were set up to determine whether an altered level of posttranslational poly(ADP-ribose) modification could be forced upon the chromatin of mammalian cells by directly manipulating the intracellular NAD+ pool with exogenously supplied NAD+.MATERIALS AND METHODS NAD+ Loading of Hepatocytes. Hepatocytes were isolated from adult rats (male SIVZ rats, 180-240 g, fed ad lib) and cultured in 100-mm Coming tissue culture dishes (seeding density, 1.2 x i07 cells per dish, in 10 ml of medium L-15 per dish) as described (4). Following a 4-hr adaptation to culture conditions, the monolayers were washed and maintained under serum-free conditions with another medium change after 24 hr. The hepatocy...

Evidence for linkage of a candidate chromosome 1 region to human systemic lupus erythematosus.

“…HLX1 (homeo box 1) is expressed in hematopoietic progenitors and activated lymphocytes and may regulate development of CD4ϩ T cells (62). ADPRT (ADP-ribosyltransferase) is induced by DNA damage and plays a role in cellular repair (63). The report that SLE patients and their family members had decreased poly(ADPribose) metabolism makes ADPRT a possible candidate (64).…”

Adaptive changes in NAD+ metabolism in ultraviolet light-irradiated murine lymphoma cells

“…In mammalian cells, the level of chromatin-bound ADP-ribose polymers depends on the availability of NAD + (Loetscher et aL, 1987). Likewise, it has been shown that lactate affects ADP-ribose polymer levels in cultured fibroblasts by decreasing the NAD+/NADH ratios in these cells (Hussain et al, 1989).…”

“…DNA damage drastically stimulates utilization of NAD § for the poly ADPribosylation reaction, catalyzed by the nuclear enzyme poly(ADP-ribose) polymerase [EC 2.4.2.30], and this may cause a substantial depletion of cellular NAD § pools (for review see Althaus and Richter, 1987). On the other hand, the level of proteinbound poly(ADP-ribose) in chromatin depends on the availability of NAD § (Loetscher et al, 1987). At present, the biological significance of this functional relationship is not well understood.…”

Maintenance of connexin 32 and 26 expression in primary cultured rat hepatocytes treated with 3‐acetylpyridine

“…In addition, the cells treated with 3‐AP could maintain highly differentiated functions. It has been reported that intracellular NAD + accelerates the activity of PARP and that, by contrast, NAD + depletion can subsequently inhibit the enzymatic activity 41 . Although nicotinamide is a strong inhibitor of PARP, retained NAD + may stimulate the enzymatic activity and partially suppress the expression related to hepatic differentiation.…”