Janus Kinase 2 Regulates Transcription Factor EB Expression and Autophagy Completion in Glomerular Podocytes

Abstract

The nonreceptor kinase Janus kinase 2 (JAK2) has garnered attention as a promising therapeutic target for the treatment of CKD. However, being ubiquitously expressed in the adult, JAK2 is also likely to be necessary for normal organ function. Here, we investigated the phenotypic effects of JAK2 deficiency. Mice in which JAK2 had been deleted from podocytes exhibited an elevation in urine albumin excretion that was accompanied by increased podocyte autophagosome fractional volume and p62 aggregation, which are indicative of impaired autophagy completion. In cultured podocytes, knockdown of JAK2 similarly impaired autophagy and led to downregulation in the expression of lysosomal genes and decreased activity of the lysosomal enzyme, cathepsin D. Because transcription factor EB (TFEB) has recently emerged as a master regulator of autophagosome-lysosome function, controlling the expression of several of the genes downregulated by JAK2 knockdown, we questioned whether TFEB is regulated by JAK2. In immortalized mouse podocytes, JAK2 knockdown decreased TFEB promoter activity, expression, and nuclear localization. In silico analysis and chromatin immunoprecipitation assays revealed that the downstream mediator of JAK2 signaling STAT1 binds to the TFEB promoter. Finally, overexpression of TFEB in JAK2-deficient podocytes reversed lysosomal dysfunction and restored albumin permselectivity. Collectively, these observations highlight the homeostatic actions of JAK2 in podocytes and the importance of TFEB to autophagosome-lysosome function in these cells. These results also raise the possibility that therapeutically modulating TFEB activity may improve podocyte health in glomerular disease.

Keywords: JAK2; autophagy; lysosome; podocyte.

Figure 1.

JAK2 deletion impairs podocyte autophagy completion in vivo. (A) Enzymatic X-gal staining of kidney sections from a Podocin-cre⁻ mouse and a Podocin-cre⁺R26R^fl/fl mouse showing glomerular β-galactosidase expression in the Podocin-cre⁺R26R^fl/fl mouse. Original magnification, ×400. (B) Phase-contrast microscopy (original magnification, ×100) and immunoblotting for nephrin in primary cultured mouse podocytes. Lysates from 3T3 cells are provided as a comparator. (C) Immunoblotting for JAK2 in lysates of primary podocytes isolated from JAK2^Ctrl and JAK2^podKO mice. (D) Immunofluorescence dual staining for nephrin and JAK2 in glomerular sections from JAK2^Ctrl and JAK2^podKO mice. The merged image shows colocalization of JAK2 and nephrin (yellow-orange color) in JAK2^Ctrl but not in JAK2^podKO. Blue is 4',6-diamidino-2-phenylindole (DAPI). (E) Urine albumin excretion in JAK2^Ctrl and JAK2^podKO mice ages 10 weeks old and 6 months old. (F) Transmission electron micrographs of podocytes from JAK2^Ctrl and JAK2^podKO mice and autophagosome volume fraction. The transmission electron micrographs illustrate autophagosomes (thick black arrows) and lysosomes (thin black arrows) in the podocyte from the JAK2^podKO mouse. Insets are a higher magnification. Original magnification, ×25,000. (G) Immunoblotting primary cultured podocytes from JAK2^Ctrl and JAK2^podKO mice for LC3. (H) Immunofluorescence dual staining for nephrin and p62 in glomerular sections of JAK2^Ctrl and JAK2^podKO mice. Insets represent zoomed-in images of the dashed areas. The white arrows point to p62 puncta in podocytes (nephrin positive) from the JAK2^podKO mouse. (I) Immunoblotting primary cultured podocytes from JAK2^Ctrl and JAK2^podKO mice for p62. (J) Immunofluorescence staining for nephrin and LAMP2 in glomerular sections of JAK2^Ctrl and JAK2^podKO mice. (K) Immunoblotting primary cultured podocytes from JAK2^Ctrl and JAK2^podKO mice for LAMP2. AU, arbitrary units. *P<0.05; ^†P<0.01.

Figure 2.

JAK2 knockdown with siRNA causes autophagosome and lysosome accumulation in cultured immortalized mouse podocytes. (A) JAK2 knockdown with siRNA. (B) Immunoblotting for LC3 and p62. (C) Immunoblotting for LC3 in JAK2 siRNA-transfected podocytes (or scramble-transfected cells) incubated in EBSS for 19 hours (5 hours post-transfection), with bafilomycin A1 (100 nM) added for the final 4 hours. (D) Transmission electron micrographs of mouse podocytes transfected with scramble or JAK2 siRNA and autophagosome and lysosome volume fraction. Insets are higher magnification (original magnification, ×25,000). The thick arrow labels an autophagosome, and the thin arrow labels a lysosome. (E) Immunoblotting for LAMP2. (F) Immunofluorescence staining for LAMP2 (red) and 4',6-diamidino-2-phenylindole (DAPI) (blue). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; AU, arbitrary units. *P<0.05 versus scramble; ^†P<0.01 versus scramble.

Figure 3.

JAK2 knockdown or knockout impairs lysosome function and decreases TFEB expression in mouse podocytes. (A) Cathepsin D activity in immortalized podocytes transfected with scramble or JAK2 siRNA for 24 hours. (B) Relative mRNA levels of TFEB targets in primary podocytes from JAK2^Ctrl and JAK2^podKO mice. BECN1, beclin 1; CTSD, cathepsin D; CTNS, cystinosin; MCOLN1, mucopilin-1; RRGAC, Ras-related GTP binding C; STK4, serine/threonine kinase 4. (C–G) Regulation of TFEB expression by JAK2 in immortalized podocytes transfected with scramble or JAK2 siRNA for 24 hours. (C) TFEB promoter activity. (D) TFEB mRNA levels. (E) TFEB protein levels. (F) TFEB nuclear levels. (G) Chromatin immunoprecipitation of the TFEB promoter after STAT1 enrichment. (H) TFEB protein levels in primary podocytes from JAK2^Ctrl and JAK2^podKO mice. AU, arbitrary units. *P<0.05 versus scramble; ^†P<0.05 versus JAK2^Ctrl; ^‡P<0.01 versus scramble; ^§P<0.01 versus IgG.

Figure 4.

TFEB overexpression restores lysosome function and albumin permselectivity in JAK2-deficient mouse podocytes. (A) Immunoblotting for GFP in control mouse podocytes or podocytes transfected with EGFP-tagged TFEB. (B) Cathepsin D mRNA levels under control conditions (scramble) or transfected with JAK2 siRNA, EGFP-tagged TFEB, or JAK2 siRNA and EGFP-tagged TFEB. (C) Cathepsin D activity in podocytes under control conditions (scramble) or transfected with JAK2 siRNA, EGFP-tagged TFEB, or JAK2 siRNA and EGFP-tagged TFEB. (D) Immunoblotting for LC3 in podocytes transfected with EGFP-tagged TFEB in the presence or absence of 100 nM bafilomycin A1 for 4 hours or JAK2 siRNA for 24 hours. (E) Albumin permeability in podocytes under control conditions (scramble) or transfected with JAK2 siRNA, EGFP-tagged TFEB, or JAK2 siRNA and EGFP-tagged TFEB. EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; RPLPO, large ribosomal protein; AU, arbitrary units. *P<0.05 versus control; ^†P<0.05 versus all other groups; ^‡P<0.001 versus all other groups.

Figure 5.

JAK2 regulates autophagy completion in podocytes. (A) Under normal conditions (wild type), signaling through JAK2 induces translocation of STAT1 to the nucleus, where STAT1 binds to the promoter region of the gene encoding the transcription factor TFEB. TFEB, in turn, facilitates the transcription of genes involved in lysosome and autophagosome function, including cathepsin D. Autophagosomes are recognized by the presence of LC3-II and contain proteins bound to p62 and targeted for degradation. Autophagy completion involves the fusion of double-membrane–bound autophagosomes with lysosomes (recognized by the presence of LAMP2) and subsequent degradation of the contents of the resultant autolysosome. (B) When JAK2 is absent (JAK2^podKO), TFEB expression is diminished, leading to decreased expression of lysosomal genes (including cathepsin D) and lysosomal dysfunction, impairing autophagy completion, and leading to podocyte dysfunction, diminished podocyte permselectivity, and consequent albuminuria.