The Phosphoinositide Kinase Pikfyve Regulates Lysosomal Biogenesis in Macrophages By Modulating the Transcription Factor EB (TFEB)

Macrophages are professional phagocytes that are essential for host defense and tissue homeostasis. Phagocytic functions critically depend on the proper membrane trafficking and degradative functions of the endolysosomal system. Membrane trafficking is modulated by several signaling molecules including phosphoinositides. Particularly, the endolysosomal trafficking involves the endosomal phosphoinositide phosphatidylinostol 3,5 bisphosphate [PI(3,5)P2], which is synthesized by the kinase PIKfyve. Studies using PIKfyve inhibitors or genetically modified mice demonstrate that PIKfyve is critical for lysosomal homeostasis. In this study, we analyzed the role of PIKfyve in macrophages as well as the potential mechanism by which PIKfyve regulates lysosomal homeostasis.

Mice were genetically engineered to lack PIKfyve in their macrophages (PIKfyve^fl/fl LysM-Cre). These mice develop diffuse tissue infiltration of foamy macrophages, hepatosplenomegaly, and a systemic inflammation response that is reminiscent of lysosomal storage disorders. Their macrophages were isolated from the spleen or bone marrow using F4/80 antibody and the biogenesis and function of their endolysosomes was analyzed. PIKfyve-null macrophages displayed an enlarged LAMP1-positive compartment by immunofluorescence microscopy. Moreover, the enlarged endolysosomes showed reduced proteolysis of endocytosed fluorogenic substrates demonstrating these cells have decreased lysosomal degradative activity. It is notable that the endolysosomal compartment was normally acidified in the PIKfyve-null macrophages. In addition, RT-PCR showed increased mRNA of lysosomal genes such as Cathepsin D and LAMP1 in the PIKfyve-null macrophages compared to WT macrophages. Immunoblotting confirmed increased expression of these lysosomal proteins in PIKfyve-null macrophages. Together, these findings indicate that PIKfyve is a critical regulator of the endolysosomal morphology, function, and biogenesis in macrophages.

TFEB is the master transcription factor for lysosomal gene network. Activation and nuclear translocation of TFEB is inhibited by mTORC1-dependent and mTORC1-independent phosphorylation of TFEB. We hypothesized that the upregulation of lysosomal genes in the PIKfyve-null macrophages may be driven by activation of TFEB. Surprisingly, we found that both mRNA expression and protein expression of full-length TFEB were reduced in PIKfyve-null macrophages compared to WT macrophages. Interestingly, mTORC1 activity was reduced, and as expected, the phosphorylation of full-length TFEB was decreased in PIKfyve-null macrophages. However, significantly elevated protein expression of shorter variants of TFEB was also detected in PIKfyve-null macrophages. Given that these shorter forms of TFEB were not previously reported, we analyzed them by mass spectrometry. The shorter forms of TFEB were identified to be truncated at the N-terminus of TFEB upstream of Aa 71 or Aa 197. Since the canonical phosphorylation and inactivation sites of TFEB include serine 142 and serine 211, we postulate that truncated variants of TFEB function as active forms of TFEB that translocate into the nucleus and mediate transcription of the lysosomal gene network. Together, our data show that PIKfyve deficiency may upregulate the expression of lysosomal genes by reducing mTORC1 activity and generating truncated variants of TFEB.

In conclusion, our study demonstrates that PIKfyve is a critical regulator of lysosome biogenesis and function in macrophages, and suggests a novel mechanism for the regulation of TFEB-mediated lysosome biogenesis.