Introduction
Differentiation of macrophages from monocytes involves adoption of a glycolytic metabolism. As emerging evidences are linking autophagy and metabolic reprogramming in monocyte differentiation, we investigated the molecular mechanisms connecting these two events upon monocyte differentiation.
Methods
Using the phorbol myristate acetate (PMA)-induced cell differentiation model of primary and human monocytic cell line THP-1, the relationship between autophagy and metabolism in monocyte differentiation was investigated using Western blot analysis, co-immuno-precipitation, siRNA mediated knock down, and FACS. Cytokine bead array and Real time PCR was used to dissect the effect of PMA on expression of inflammatory mediators, and acquisition of macrophage phenotype.
Results
An increase in PPARg (Peroxisome proliferator-activated receptor) HK2 (Hexokinase 2) and SIRT6 (Sirtuin) expression was observed upon PMA-induced monocyte differentiation. While PPARg positively regulated HK2 and SIRT6 expression, the latter served as a negative regulator of HK2. Changes in expression of these metabolic modelers were accompanied by decreased glucose uptake and increase in Chibby. Knockdown of Chibby abrogated PMA induced differentiation. Inhibition of HK2 by Lonidamine or siRNA mediated knock-down further elevated PMA induced Chibby, mitochondrial ROS (Reactive Oxygen Species), TIGAR (TP53-Induced Glycolysis and Apoptosis Regulator) and LC3-II levels. Inhibition of autophagy increased HK2 and abrogated Chibby levels. Importantly, siRNA mediated knock-down of SIRT6 exhibited opposite effect as compared to HK2. The acquisition of specific M1 vs M2 macrophage phenotypes and the release of pro-inflammatory mediators from differentiated macrophages was HK2 dependent.
Conclusions
Our findings highlight a previously unknown role of SIRT6-HK2 axis in affecting autophagy dependent monocyte differentiation. By providing mechanistic insights into metabolism driven macrophage differentiation, our findings raise the possibility of pharmacologically manipulating the SIRT6-HK2 cross-talk to affect the differentiation potential of monocytes.