Activation of mammalian target of rapamycin controls the loss of TCRzeta in lupus T cells through HRES-1/Rab4-regulated lysosomal degradation

Abstract

Persistent mitochondrial hyperpolarization (MHP) and enhanced calcium fluxing underlie aberrant T cell activation and death pathway selection in systemic lupus erythematosus. Treatment with rapamycin, which effectively controls disease activity, normalizes CD3/CD28-induced calcium fluxing but fails to influence MHP, suggesting that altered calcium fluxing is downstream or independent of mitochondrial dysfunction. In this article, we show that activity of the mammalian target of rapamycin (mTOR), which is a sensor of the mitochondrial transmembrane potential, is increased in lupus T cells. Activation of mTOR was inducible by NO, a key trigger of MHP, which in turn enhanced the expression of HRES-1/Rab4, a small GTPase that regulates recycling of surface receptors through early endosomes. Expression of HRES-1/Rab4 was increased in CD4(+) lupus T cells, and in accordance with its dominant impact on the endocytic recycling of CD4, it was inversely correlated with diminished CD4 expression. HRES-1/Rab4 overexpression was also inversely correlated with diminished TCRzeta protein levels. Pull-down studies revealed a direct interaction of HRES-1/Rab4 with CD4 and TCRzeta. Importantly, the deficiency of the TCRzeta chain and of Lck and the compensatory up-regulation of FcepsilonRIgamma and Syk, which mediate enhanced calcium fluxing in lupus T cells, were reversed in patients treated with rapamcyin in vivo. Knockdown of HRES-1/Rab4 by small interfering RNA and inhibitors of lysosomal function augmented TCRzeta protein levels in vitro. The results suggest that activation of mTOR causes the loss of TCRzeta in lupus T cells through HRES-1/Rab4-dependent lysosomal degradation.

Fig. 1

Western blot detection of SOD2, VDAC1, FKBP12, TAL, and NOSIP in "untouched" T cells of female patients with SLE and age-matched healthy female controls isolated with the negative T-cell isolation kit (Dynal, Lake Success, NY). SOD2 was detected with rabbit antibody from Santa Cruz (Cat No SC-30080). VDAC1 was detected with goat antibody from Santa Cruz (Cat No SC-8828. FKBP12 was detected with a monoclonal antibody from BD Biosciences (Cat No 554091). Transaldolase was detected with antibody 170 . NOSIP was detected with rabbit antibody . Actin was detected with a monoclonal antibody from Chemicon (Cat No 1501R). The expression of SOD2, VDAC1, FKBP12, TAL, and NOSIP relative to actin was determined in each donor by automated densitometry with Kodak Image Station 440CF. Representative blots are shown. The p values reflect fold changes in protein levels between 14 SLE patients and 10 controls compared with two-tailed t-test.

Fig. 2

Activation of the mammalian target of rapamycin (mTOR) in lupus T cells. A, Western blot analysis of mTOR substrate p70 S6 kinase phosphorylation. Protein lysates of negatively isolated naïve T cells from healthy controls (Controls) and SLE patients treated without (SLE) or with 2mg/day rapamycin (SLE + Rapa) were analyzed by Western blot using antibodies against p70 S6 kinase (S6K) (Santa Cruz, Cat No sc-8418), phospho-S6K (pS6K) (Cell Signaling, Cat#9206) and actin (Chemicon, Cat# MAB1501). Diagram shows fold changes of the phosphorylated S6 kinase signal relative to total S6K. Data represent mean ± SE. B, Western blot analysis of mTOR substrate 4E-BP1 phosphorylation. Phospho-4E-BP1 (Cell Signaling, Cat#2855) and 4E-BP1 were detected rabbit antibodies (Cell Signaling, Cat#9644). C, Effect of rapamycin on SOD2 expression, detected with rabbit antibody from Santa Cruz (Cat No SC-30080), using the samples analyzed in panel A. D, Effect of rapamycin on FKBP12 expression, detected with a monoclonal antibody from BD Biosciences (Cat No 554091), using the samples analyzed in panel A. E, Effect of rapamycin on NOSIP expression, detected with a rabbit antibody , using the samples analyzed in panel A.

Fig. 3

A) Western blot detection of Rab GTPases Rab5A and HRES-1/Rab4 in negatively isolated T cells of female SLE and control subjects. Rab5A was detected with rabbit antibody SC 309 (Santa Cruz). HRES-1/Rab4 was detected as earlier . The expression of HRES-1/Rab4 or CD4 relative to actin was determined in each donor by automated densitometry with Kodak Image Station 440CF. The p values reflect fold changes in protein levels between 14 SLE patients and 10 controls compared with two-tailed t-test. B) Western blot detection of HRES-1/Rab4 and CD4 in negatively isolated CD4+T cells of female Caucasian SLE patients and age-matched female Caucasian controls.

Fig. 4

Enhanced recycling of CD3 and CD4 of lupus T cells. Negatively isolated T cells from of 14 SLE patients, 9 healthy and 6 RA controls were stained on ice with APC-Cy7-conjugated antibody CD3 (BD Cat# 557832 ), Alexa 647-conjugated antibody to CD4 (BD Cat# 557707), and PE-conjugated antibody to CD8 (BD Cat# 555367) for 30 minutes (not shown), then washed 3 times in ice cold RPMI 1640 medium. The cells were resuspended in warm medium and incubated at 37°C for 3 hours, removing samples at 30 minute intervals and placing them on ice. At the conclusion, the cells were restained with the original antibodies on ice for 30 minutes, then washed 3 times with ice cold RPMI and analyzed by flow cytometry. The degree of recycling is given by the increase in mean fluorescence intensity in later time points over the baseline (time 0 sample, kept on ice after initial staining throughout the recycling assay).

Fig. 5

Effect of NO and H₂O₂ on mTOR activity and expression of HRES-1/Rab4 in PBMC. A, Effect of 24 h treatment with NO, released by NOC-18 (100, 150 and 300 µM), and H₂O₂ (25 and 50 µM) on mTOR activity assessed by pS6K/S6K/actin ratio. B, Effect of 24 h treatment with NO, released by NOC-18 (150 and 300 µM), and H₂O₂ (25 and 50 µM), rapamycin (50 nM, dissolved in 0.1% DMSO), DMSO alone (0.1%), GSH (10 mM) and L-NMMA (100 µM) on expression of HRES-1/Rab4 relative to actin. Data represent six independent experiments.

Fig. 6

Expression of Rab5A, HRES-1/Rab4, and CD4 relative to actin in healthy controls (Controls) and SLE patients treated without (SLE) or with 2mg/day rapamycin (SLE + Rapa). Protein lysates of negatively isolated naïve T cells were analyzed by Western blot. P values < 0.05 reflecting fold changes in protein levels are shown.

Fig. 7

Expression of TCR/CD3ζ, Lck, FcεRIγ, and Syk in negatively isolated naïve T cells from healthy controls (Controls) and SLE patients treated without (SLE) or with 2mg/day rapamycin (SLE + Rapa). Protein lysates were analyzed by Western blot. P values < 0.05 reflecting fold changes in protein levels are shown.

Fig. 8

Interaction of HRES-1/Rab4 with T-cell surface and adaptor proteins. A, Direct interaction of the TFR and CD4 with HRES-1/Rab4 in Jurkat cells. 10 Jurkat cells were lysed in 1% NP-40, 10% glycerol, 200 mM NaCl, 5 mM MgCl₂, 50 mM Tris pH 8.0, 1 mM PMSF, 1 mM sodium orthovanadate, 20 mM NaF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin for 30 min at 4°C and the supernatant was obtained by centrifugation at 14,000 × g for 20 min at 4°C. 3 ml of the supernatant was pre-cleared with 1 ml of swollen GSH-agarose beads. 500 µl of pre-cleared supernatant was incubated with 5 µg of HRES-1/Rab4-GST-bound agarose beads or 2.6 µg of GST-bound control beads (~100 pmol of each fusion protein) for 2 h at 4°C with and without 1 mM GTPγS. The beads were pelleted at 500 × g for 5 min at 4°C, washed twice in lysis buffer, resuspended in Laemmli buffer, and analyzed by SDS-PAGE stained with Coomassie brilliant blue (left panel) and by Western blot with the indicated antibodies (right panel). B, Pull-down of CD4, CD2AP, and TCRζ, but not CD8 or Rab5A, from PBL lysates prepared as described for Jurkat cells using agarose beads coupled to HRES-1/Rab4-GST. Antibodies to CD2AP (sc-25272), TCR/CD3ζ (sc-1239), CD8 (sc-53212) and Rab5A (sc-28570) were obtained from Santa Cruz. The results represent four independent experiments.

Fig. 9

Localization of TCRζ, HRES-1/Rab4, and lysosomes in negatively isolated resting T cells from healthy controls and lupus patients using confocal microscopy. TCRζ was visualized with antibody sc-1239 directly conjugated to Alexa-488 (green). HRES-1/Rab4 was detected with antibody sc-312 directly conjugated with Alexa-647 (blue). Lysosomes were detected with Lysotracker Red (LTR). In control T cells, TCRζ and HRES-1/Rab4 were present in the cell membrane or intracellular vesicles but not in lysosomes stained with LTR. In lupus T cells, TCRζ was found predominantly in HRES-1/Rab4-positive intracellular membrane clusters that co-localized with lysosomes. Green color intensities (lower right 8 panels) were enhanced to visualize the localization and compensate for lower expression of TCRζ in lupus T cells.

Fig. 10

Reversal of TCR/CD3ζ depletion in lupus T cells by down-regulation of HRES-1/Rab4 expression and inhibitors of lysosomal function. A, Effect of siRNA-mediated knock-down of HRES-1/Rab4 on TCRζ protein levels in lupus T cells. 10 cells were electroporated with siRNA specifc for HRES1/Rab4 nucleotides 377–399 using the Nucleofector protocol for primary human T cells (Amaxa, Gaithersburg, MD) and assayed 36 h later by western blot. Rab4 and TCRζ were detected with antibodies sc-312 and sc-1239. B, Effect of lysosomal inhibitors bafilomycin A1 and folimycin on TCRζ levels of lupus T-cells. The results represent studies of 5 lupus patients.