Equilibrative Nucleoside Transporter 3 Deficiency Perturbs Lysosome Function and Macrophage Homeostasis

Abstract

Lysosomal storage diseases (LSDs) are a group of heterogeneous disorders caused by defects in lysosomal enzymes or transporters, resulting in accumulation of undegraded macromolecules or metabolites. Macrophage numbers are expanded in several LSDs, leading to histiocytosis of unknown pathophysiology. Here, we found that mice lacking the equilibrative nucleoside transporter 3 (ENT3) developed a spontaneous and progressive macrophage-dominated histiocytosis. In the absence of ENT3, defective apoptotic cell clearance led to lysosomal nucleoside buildup, elevated intralysosomal pH, and altered macrophage function. The macrophage accumulation was partly due to increased macrophage colony-stimulating factor and receptor expression and signaling secondary to the lysosomal defects. These studies suggest a cellular and molecular basis for the development of histiocytosis in several human syndromes associated with ENT3 mutations and potentially other LSDs.

Inhibition of Transient Receptor Potential Channel Mucolipin-1 (TRPML1) by Lysosomal Adenosine Involved in Severe Combined Immunodeficiency Diseases

“…To test this, HEK293T cells were transfected with surface-expressed mutant TRPML1 channels (TRPML1-L15A/L16A/L577A/L578A; abbreviated as TRPML1-4A), which serve as a surrogate of lysosomal TRPML1 (27)(28)(29). Because lysosomal pH is elevated to around 5.5 by adenosine accumulation (7), to mimic the environments of lysosomes with adenosine accumulation, extracellular solutions (analogous to the intralysosomal luminal side) were adjusted to pH 5.5. Consistent with our previous study, TRPML1 currents were activated by mucolipin synthetic agonist 1 (ML-SA1; a TRPML1 agonist; 5 M) (29,30) in cells transfected with GFP-TRPML1-4A but not in untransfected cells or cells transfected with GFP alone (data not shown).…”

“…In contrast, ENT3 (6 -8) and ENT4 (9) are predominantly expressed in intracellular membranes (10). Uniquely, ENT3 is pH-sensitive and optimally active under acidic conditions (optimal pH value of 5.5), probably reflecting the subcellular location of the transporters in acidic compartments, like lysosomes (6,7). In addition, adenosine can be broken down by adenosine deaminase (ADA).…”

“…However, the molecular mechanisms underlying the ADA Ϫ/Ϫ phenotypes remain unclear. Interestingly, loss of ENT3 in mice results in lysosomal adenosine overload (7), subsequently causing enlarged and impaired lysosomes and defects in the clearance of apoptotic cells (7). These phenocopy mice with deficiency in transient receptor potential channel mucolipin-1 (TRPML1), a lysosome Ca 2ϩ -permeable channel regulating lysosomal membrane trafficking, lysosome function, and apoptotic cell clearance (16 -22).…”

A Case of SLC29A3 Spectrum Disorder—Unresponsive to Multiple Immunomodulatory Therapies

“…SLC29A3 mutations typically cause altered stability of the ENT protein (8). SLC29A3-null mice develop lysosomal accumulation of nucleotides and altered macrophage function (9), which provides a molecular basis for some clinical aspects of the SLC29A3 spectrum disorders, however it does not explain the entire phenotype.…”

Regulation of lysosome biogenesis and functions in osteoclasts

“…Interestingly, in macrophages, ENT3 is required for the proper clearance of the nucleosides from the lysosomes, and in Ent3 −/− macrophages intralysosomal pH is elevated and phagocytic function altered. 41 Whether nucleoside transport may also be important for lysosomal acidification in osteoclasts remains to be tested.…”