A Knockout of the Tsg101 Gene Leads to Decreased Expression of ErbB Receptor Tyrosine Kinases and Induction of Autophagy Prior to Cell Death

The Tumor Susceptibility Gene 101 (Tsg101) encodes a multi-domain protein that mediates a variety of molecular and biological processes including the trafficking and lysosomal degradation of cell surface receptors. Conventional and conditional knockout models have demonstrated an essential requirement of this gene for cell cycle progression and cell viability, but the consequences of a complete ablation of Tsg101 on intracellular processes have not been examined to date. In this study, we employed mouse embryonic fibroblasts that carry two Tsg101 conditional knockout alleles to investigate the expression of ErbB receptor tyrosine kinases as well as stress-induced intracellular processes that are known to be associated with a defect in growth and cell survival. The conditional deletion of the Tsg101 gene in this well-controlled experimental model resulted in a significant reduction in the steady-state levels of the EGFR and ErbB2 but a stress-induced elevation in the phosphorylation of mitogen activated protein (MAP) kinases independent of growth factor stimulation. As part of an integrated stress response, Tsg101-deficient cells exhibited extensive remodeling of actin filaments and greatly enlarged lysosomes that were enriched with the autophagy-related protein LC3. The increase in the transcriptional activation and expression of LC3 and its association with Lamp1-positive lysosomes in a PI3K-dependent manner suggest that Tsg101 knockout cells utilize autophagy as a survival mechanism prior to their ultimate death. Collectively, this study shows that a knockout of the Tsg101 gene causes complex intracellular changes associated with stress response and cell death. These multifaceted alterations need to be recognized as they have an impact on defining particular functions for Tsg101 in processes such as signal transduction and lysosomal/endosomal trafficking

Remodeling of actin filaments and enlargement of lysosomes in response to the conditional deletion of the <i>Tsg101</i> gene.

<p><b>A</b>. Confocal microscopic images of a phalloidin-based fluorescent staining of F-actin in Tsg101 conditional knockout cells (<i>Tsg101^−/−</i>) and their controls (<i>Tsg101^fl/fl</i>). <b>B</b>., <b>C</b>. Co-staining of Tsg101-deficient cells and their wildtype controls with antibodies against Lamp1 and Cathepsin D; panel B illustrates the quantitative analysis of the colocalization of both signals (<i>P</i> value, <i>t</i> test). Note that Cathepsin D, which was more abundant in Tsg101-deficient cells, was clearly present within the lumen of the enlarged lysosomes (panel C, arrows). Bar in panel C represents 10 µm.</p

Tsg101-deficient cells initiate autophagy to prolong their survival prior to cell death.

<p><b>A</b>. Confocal images of wildtype cells and Tsg101 conditional knockout cells that were grown in the presence or absence of 3-Methyladenine (3MA) to inhibit the fusion and maturation of LC3 containing autophagosomes with lysosomes. Tsg101-deficient cells were maintained in the presence of the inhibitor following one day after deletion of <i>Tsg101</i> using an adenovirus-based delivery of Cre recombinase; bar represents 10 µm. <b>B</b>. Quantitative analysis of the colocalization of LC3 and Lamp1 following 2 and 3 days of treatment with 3MA (<i>P</i> value, <i>t</i> test). <b>C</b>. Counts of Tsg101-deficient cells and their controls following treatment with 1.5 mM 3MA. The inhibitor was administered 24 hrs after infection with AdCre and deletion of <i>Tsg101</i>.</p

A conditional knockout of Tsg101 results in reduced expression of EGFR and ErbB2 but a stress-induced activation of Erk1/2 in a growth factor independent manner.

<p><b>A</b>., <b>B</b>. Western blot analyses to assess the steady-state levels of endogenous EGFR and ErbB2 (A) as well as exogenous human EGFR in Tsg101 (B) conditional knockout fibroblasts (−/−) and their isogenic controls expressing Tsg101 (fl/fl). <b>C</b>. Semi-quantitative RT-PCR to examine the transcriptional activation of the endogenous <i>EGFR</i> gene in response to Tsg101 ablation. Genomic DNA (gDNA) was used as a control for the PCR with exon-specific primers that span intronic sequences of the mouse <i>EGFR</i>. <b>D</b>. EGFR and Erk1/2 expression and activation in response to EGF stimulation (0, 30, and 60 min) in cells lacking Tsg101 and their isogenic controls. <b>E</b>. Western blot analysis to assess immediate and long-term changes in EGFR expression and activation of Erk1/2 between one and four days following the conditional deletion of <i>Tsg101</i>. <b>F</b>. Analysis of Erk1/2 phosphorylation in the presence and absence of growth factors (GF) in response to Tsg101 deficiency.</p

Permanent rescue of Tsg101 knockout cells from the stress-induced induction of autophagy through expression of exogenous, HA-tagged Tsg101 from a retroviral vector.

<p><b>A</b>. Experimental design. <b>B</b>. Western blot analysis to verify the reduction in the levels of LC3 and Cathepsin D (CtsD) upon re-expression of Tsg101. <b>C</b>., <b>D</b>. Analysis of confocal images of Tsg101 conditional knockout cells with and without exogenous HA-tagged Tsg101 and their wildtype controls that were co-stained with antibodies against endogenous LC3 and Lamp1. Panel C illustrates the quantitative analysis of the colocalization of LC3 and Lamp1 (<i>P</i> value, <i>t</i> test). Bar in panel D represents 10 µm.</p

Tsg101 deficiency leads to induction of macroautophagy.

<p><b>A</b>. Quantitative real-time RT-PCR to assess the transcriptional activation of the <i>Microtubule-associated protein 1/light chain 3</i> (<i>Map1lc3</i>) gene in Tsg101 conditional knockout cells and their wildtype controls. <b>B</b>. Western blot analysis to verify the increase in expression of LC3 in response to Tsg101 deficiency. Wildtype cells maintained in the absence of growth factors (GF) were used as a positive control. <b>C</b>., <b>D</b>. Analysis of confocal images of Tsg101 conditional knockout cells and their wildtype controls that were maintained in the presence or absence of growth factors (GF) prior to fixation and co-staining with antibodies against endogenous LC3 and Lamp1. Panel C illustrates the quantitative analysis of the colocalization of LC3 and Lamp1 (<i>P</i> value, <i>t</i> test). The bar in panel D represents 10 µm and arrows indicate areas of intensive co-localization of LC3 and Lamp1.</p