Dexamethasone Downregulates Autophagy through Accelerated Turn-Over of the Ulk-1 Complex in a Trabecular Meshwork Cells Strain: Insights on Steroid-Induced Glaucoma Pathogenesis

Abstract

Steroid-induced glaucoma is a severe pathological condition, sustained by a rapidly progressive increase in intraocular pressure (IOP), which is diagnosed in a subset of subjects who adhere to a glucocorticoid (GC)-based therapy. Molecular and clinical studies suggest that either natural or synthetic GCs induce a severe metabolic dysregulation of Trabecular Meshwork Cells (TMCs), an endothelial-derived histotype with phagocytic and secretive functions which lay at the iridocorneal angle in the anterior segment of the eye. Since TMCs physiologically regulate the composition and architecture of trabecular meshwork (TM), which is the main outflow pathway of aqueous humor, a fluid which shapes the eye globe and nourishes the lining cell types, GCs are supposed to trigger a pathological remodeling of the TM, inducing an IOP increase and retina mechanical compression. The metabolic dysregulation of TMCs induced by GCs exposure has never been characterized at the molecular detail. Herein, we report that, upon dexamethasone exposure, a TMCs strain develops a marked inhibition of the autophagosome biogenesis pathway through an enhanced turnover of two members of the Ulk-1 complex, the main platform for autophagy induction, through the Ubiquitin Proteasome System (UPS).

Keywords: autophagy; glaucoma; glucocorticoids; intraocular pressure; trabecular meshwork; ubiquitin proteasome system.

Figure 1

Reduced growth and apoptosis in TMCs repeatedly stimulated with Dexa. (A) TMCs were seeded at the same dilution (3 wells for each experimental condition) and challenged with vehicle or Dexa following the scheme and dosage indicated. At selected timepoints, cell proliferation and viability were assessed by MTT assay. A nominal value of 1 was assigned to the O.D. of the first vehicle well. A representative experiment of two independent observations is reported (n = 3). Comparisons have been run between Dexa and correspondent vehicle cells at each timepoint. * p < 0.005, unpaired τ Student’s test. (B) Immunodetection of the p25 fragment of PARP in whole cell lysates by Wb. Although β-actin pattern is shown, normalization was performed on total proteins (Ponceau S stain). A representative experiment of three independent observations is reported (n = 3) * p < 0.04, ** p < 0.0008. (C) Immunodetection of the p21, p53, phospho-p53(Ser46) and determination of the phospho-p53(ser46)/p53 ratio in whole cell lysates by Wb. Normalization was performed on β-actin. A representative experiment of three independent observations is reported (n = 3) * p < 0.0001; ** p < 0.008; one-way ANOVA followed by Tukey’s post hoc significance test.

Figure 2

Proteasome activity is not significantly altered by Dexa treatment. (A) Proteasome assemblies of crude cell extracts of TMCs were assayed for the kinetics of LLVY-amc cleavage. The release of fluorescence (λ_exc.: 380 nm; λ_em.: 460 nm) was monitored until linearity was observed (typically 2 h). Individual slopes were calculated by subtracting the fluorescence release in the presence of 500 nM epoxomicin (null). Data are presented as fold variation of the slope between Dexa and correspondent vehicle-treated cells. A nominal value of 1 was assigned to the slope calculated for the first (1 out of 3 wells) vehicle cells. Comparisons have been run between Dexa and correspondent vehicle cells at each timepoint. (B) Immunodetection of α7 and Rpn10 subunits and (C) ubiquitinated proteins by denaturing and reducing Wb in whole cell lysates. β-actin was used as internal control. Notably, (C) has been manipulated to clear out one lane loaded with an additional vehicle cell lysate. * p < 0.04; ** p < 0.005; *** p < 0.0001. A representative experiment of three independent observations is reported (n = 3); one-way ANOVA followed by Tukey’s post hoc significance test.

Figure 3

Impaired autophagy flux in Dexa-treated TMCs. Immunoblotting of LC3B in the presence/absence of 20 µM CQ (A) and of Beclin-1 and p62/SQSTM1 (B) in whole cell lysates of TMCs * p < 0.0001. (C) p62/SQSTM1 accumulation in vehicle-treated cells and Dexa-6d* cells in the presence of 20 µM CQ. Normalization in both figures was performed on β-actin. * p < 0.005. A representative experiment of three independent observations is reported (n = 3); one-way ANOVA followed by Tukey’s post hoc significance test.

Figure 4

Decreased autophagosomes count in Dexa-treated TMCs. (A) Immunofluorescence microscopy analysis of LC3B⁺ autophagosomes (e.g., red spheres) in vehicle and Dexa-6d* cells. Nuclei were stained by Hoechst. Images were acquired at 40X and 100X magnification. Autophagosome count was analyzed by means of either percentage of cells showing at least 10 LC3B⁺ dots and average number of dots per cell. * p < 0.0001; ** p < 0.004. A representative experiment of two independent observations is reported (n = 10); unpaired Students’ τ test.

Figure 5

Downregulation of ULK1 members in Dexa-treated TMCs. Immunoblotting of Ulk-1, pUlk1(Ser555), pUlk1(Ser757), Fip200, Atg13, and Atg101 in whole cell lysates of TMCs. Normalization was performed on total proteins (Ponceau S stain). * p < 0.0001, ** p < 0.009, *** p < 0.001. A representative experiment of three independent observations is reported (n = 3); one-way ANOVA followed by Tukey’s post hoc significance test.

Figure 6

Autophagy gene expression analysis of Dexa-treated TMCs. RT-PCR results of the expression of ULK-1, ATG101, ATG13, and Beclin-1. Gapdh was used as internal control, as indicated in the Section 4. Data are reported as fold-increase with respect to control group. The 2^−ΔΔCt formula was used to calculate the fold-increase versus vehicle cells.

Figure 7

Enhanced turnover of Ulk-1 and Atg101 in Dexa-treated TMCs. (A) Immunoblotting of Ulk-1, Atg101 and Atg13 in whole cell lysates of TMCs. Normalization was performed on total proteins (Ponceau S stain). * p < 0.02; ** p < 0.0004; *** p < 0.0001; **** p < 0.001. A representative experiment of three independent observations is reported (n = 3); one-way ANOVA followed by Tukey’s post hoc significance test.

Figure 8

Turnover of poly-Ub protein and IkBα in Dexa-treated TMCs. Immunoblotting of Ub and IkBα in whole cell lysates of TMCs. β-actin was used as internal control. A representative experiment of three independent observations is reported (n = 3); * p < 0.0001; one-way ANOVA followed by Tukey’s post hoc significance test.