Abstract

CD38, originally described as a differentiation marker, has emerged as an important multifunctional transmembrane protein. Its most intriguing and well-characterized function is its ability to catalyze the synthesis of cyclic ADP-ribose (cADPR) from NAD. Of particular interest is its presence on the inner membrane of the nucleus, suggesting that CD38/cADPR may play a direct role in mediating nuclear activation and gene expression. Our studies on ex vivo expansion of Hematopoietic Stem Cells (HSCs) have led us to test whether alteration of CD38 function carries the potential of affecting cell fate decisions of HSCs. Inhibition of CD38 enzymatic activity was achieved by treating CD34+ cell cultures with nicotinamide (NA), a well-known base-exchange inhibitor demonstrated to inhibit the synthesis of cADPR from NAD.

We report here that exogenously added nicotinamide (5–10 mM) to CD34+ cell cultures supplemented with cytokines (SCF, TPO, IL-6, FLt3, +/− IL-3) resulted in significant enrichment of CD34+CD38− (79±9.3%, n=9) and CD34+CD38−Lin− (19±3%, n=8) cells, as compared with control cultures treated only with cytokines (6.3±1.8%, n=9, and 0.7±0.06%, n=8, respectively, p<0.01). The functionality of these early progenitor subsets was demonstrated using the extended LTC-CFC assay, performed in the absence of NA. These results raised the intriguing possibility that cADPR production may have a pivotal role in regulation of CD34+ cell fate. However, inhibition of cADPR downstream signal transduction pathways by its specific antagonist, 8-amino-cADPR did not yield any effect on CD34+ cell cultures, excluding the possibility that nicotinamide modulates CD34+ cell fate solely by inhibition of cADPR synthesis.

Nicotinamide is also a well-known potent inhibitor of SIRT2, a unique NAD(+)-dependent type III histone deacetylase (HDAC) with mono-ADP-ribosyltransferase activity involved in gene silencing, metabolism, apoptosis and aging. NA blocks NAD(+) hydrolysis by binding to an adjacent conserved pocket, and is therefore suggested as the physiologically relevant regulator of SIRT2 enzymes. This additional function of nicotinamide raises the intriguing possibility that HSC enrichment achieved by nicotinamide treatment may be related to specific inhibition of SIRT2 deacetylase activity and modulation of chromatin architecture leading to re-activation of previously silenced genes.

In line with this hypothesis, Milhem et all. recently reported that addition of trichostatin A, a specific HDAC (type I and II) inhibitor, along with a DNA hypomethylating agent, modulated HSC fate ex vivo resulting in the retention of stem cell phenotype, number, and function (

Blood
,
2004
;
103
;
4102
).

Ongoing work is aimed at elucidating whether inhibition of SIRT2 is specifically involved in NA mechanism of activity leading to modulation of hematopoietic stem cell fate in ex vivo conditions.

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