Androgen receptor inclusions acquire GRP78/BiP to ameliorate androgen-induced protein misfolding stress in embryonic stem cells

Abstract

Commitment of differentiating embryonic stem cells (ESCs) toward the various lineages is influenced by many factors, including androgens. However, the mechanisms underlying proteotoxic stress conferred by androgen receptor (AR) actions on embryonic cell fate remains unclear. Here we show that mouse ESCs display stress-related cellular phenotypes in response to androgens during early phase of differentiation. Androgen induced a significant increase in the percentage of ESCs and embryoid bodies with the intranuclear and juxtanuclear AR inclusions, which were colocalized with the E3 ubiquitin ligase, C terminus of Hsc70-interacting protein. Caspase-3 activity corresponded with AR expression, was enhanced in cells engaged more differentiation phenotypes. Androgen-mediated accumulation of AR aggregates exacerbated endoplasmic reticulum (ER) stress and rendered ESCs susceptible to apoptosis. Increasing expression levels of the ER chaperones, GRP78/BiP and GRP94, as well as ER stress markers, such as ATF6, phosphorylated PERK, GADD153/CHOP and spliced XBP-1 mRNA, were dramatically elevated in ESCs overexpressing AR. We found that androgen induced GRP78/BiP to dissociate from ATF6, and act as an AR-interacting protein, which was recruited into AR inclusions in ESCs. GRP78/BiP was also colocalized with AR inclusions in the cells of spinal bulbar muscular atrophy transgenic mouse model. Overexpression of GRP78/BiP suppressed ubiquitination of AR aggregates and ameliorated the misfolded AR-mediated cytopathology in ESCs, whereas knockdown of GRP78/BiP increased the accumulation of AR aggregates and significantly higher levels of caspase-3 activity and cell apoptosis. These results generate novel insight into how ESCs respond to stress induced by misfolded AR proteins and identify GRP78/BiP as a novel regulator of the AR protein quality control.

Figure 1

Androgen induces aggregation of AR protein with non-expanded polyQ tracts. (a) Immunofluorescence of ESCs revealed the formation of juxtanuclear and intranuclear inclusions of AR, which distort the nuclear architecture after treatment with DHT for 3 days (left panel). AR inclusions were also formed in EBs treated with DHT or left untreated for 3 days (middle panel). High-magnification images of the regions are indicated by arrows to show nuclear AR with small aggregates (right panel). Quantitation of cells with AR inclusions showed that DHT significantly promotes AR inclusions in ESCs whereas EBs exhibit a higher percentage of AR inclusions than ESCs, regardless of DHT treatment (n=3). Data are presented as means±S.D. (b) The assay for steady-state protein expression showed that 3-day DHT treatment promoted AR aggregation, which was significantly increased in EBs compared with ESCs. Oct3/4 was used as a self-renewal marker and β-tubulin as a loading control. (c) Ubiquitin (red, top left panel) or CHIP (red, top right panel) partially colocalized with AR (green) and a higher colocalization index was observed after DHT treatment of ESCs for 3 days (merge). Scale bars: (ES) 10 μM; (EB) 50 μM. The quantification of the colocalization was performed by using the colocalization finder plugin of ImageJ. The colocalization index was estimated by Pearson's correlation coefficient; *P<0.05, ANOVA

Figure 2

Increased AR expression and apoptosis upon induction of ES cell differentiation. (a) Caspase-3 activity assay (top panel) and WB analysis (bottom panel) showed a correlation between increasing expression of AR protein and induction of caspase-3 activity in undifferentiated ESCs, differentiated ESCs and EBs. Oct3/4 was employed as a self-renewal marker and β-tubulin as the loading control. (b) DHT-treated ESCs were cultured with LIF or left untreated for the indicated times. Both caspase-3 activity and AR expression showed a time-dependent increase from undifferentiated to differentiated ESCs. (c) Clones stably expressing either shAR or control shRNA (shLuc) were established in ESCs and EBs. (d) Caspase-3 activity assay and WB analysis using caspase-3, -12 and PARP antibodies showed a significant increase in activation of caspase-3 and -12 in control ESCs treated with DHT and T, but not ESCs expressing shAR. N suppressed the androgen-mediated effect in ESCs. (e) TUNEL staining of ESCs and EBs expressing shAR or shLuc. EBs contained more apoptotic cells than ESCs, and knockdown of AR led to significant suppression of cell death in both ESCs and EBs (n=3, *P<0.05, ANOVA). Data are presented as means±S.D. The insert represents high magnification projections of apoptotic EB cells. Main image scale bars: (ESCs) 25 μM, (EBs) 100 μM

Figure 3

UPR signaling is activated by androgen/AR. (a) Expression and activation of UPR transducers (ATF-6α, p-PERK and XBP-1^s) and chaperones (GRP78 and GRP94) were increasingly detected in ESCs and EBs treated with DHT on WBs. Oct3/4 was used as a differentiation marker and β-tubulin as the loading control. General ES medium (GM) was used for basal level treatment and TM for ER stress induction. (b) ES cell clones stably expressing either AR (ESCs-FLAG-AR) or empty vector (ESCs-FLAG) were treated with DHT, or left untreated. WB analysis revealed increased expression and activation of UPR sensors and chaperones in ESCs-FLAG-AR in the presence of DHT. (c) In RT-PCR experiments, ESCs-FLAG-AR contained higher levels of spliced Xbp-1 mRNA than control ESCs-FLAG in a DHT- and time-dependent manner. Unspliced (u) and spliced (s) Xbp-1 mRNA products are indicated. The asterisk (*) represents the position of a hybrid amplicon. β-tubulin was used as the loading control and TM treatment as ER stress-induction control. (d) Real-time PCR data showed higher DHT-dependent Chop mRNA expression in ESCs-FLAG-AR than control ESCs-FLAG. *P<0.05 was defined as significant

Figure 4

Androgens promote AR and GRP78/BiP interactions and colocalization with intranuclear AR inclusions in ESCs, EB and cells of a mouse model of SBMA. (a) The endogenous AR-GRP78/BiP protein complex was immunoprecipitated with anti-GRP78/BiP antibodies, but not anti-IgG control antibodies. (b) Overexpression of AR enhanced the DHT-induced interactions between AR and GRP78/BiP. The AR-GRP78/BiP complex was detected with an immunoprecipitation assay using anti-AR antibodies, followed by immunoblotting with anti-GRP78/BiP antibodies. (c) Decreasing association of GRP78/BiP with ATF6 in DHT-treated AR-expressing cells was detected via immunoprecipitation with anti- GRP78/BiP or anti-ATF6 antibodies and immunoblotting with anti- GRP78/BiP or anti-ATF6, respectively. (d) Nuclear AR inclusions colocalized with GRP78/BiP in DHT-treated ESCs and EBs. Scale bars: (ES) 10 μM; (EB) 50 μM. Immunofluorescence in muscle (e and f) sections from AR-24Q and AR-97Q mice revealed a marked increase in nuclear inclusions number of the AR-97Q mice compared with the AR-24Q mice. The boxed area shown at high-magnification images of the regions is a group of cells in an adjacent section, immunolabeled for AR and GRP78 (inset). Intense staining of PolyQ with small aggregates was frequently seen in the AR-97Q mice, whereas staining was infrequently in the AR-24Q mice. (g) Colocalization of AR inclusions and GRP78 in muscle cells of the AR-24Q and AR-97Q mice by double immunofluorescence staining. The extent of colocalization of GRP78 and polyQ AR was higher in the cells of muscle from AR-97Q than AR-24Q mice. The quantification of the colocalization was performed by using the colocalization finder plugin of ImageJ. The index of colocalization corresponds to the mean of the Pearson's coefficient. (h) The histograms show the density of polyQ+/GRP78+ cells is significantly higher than polyQ-/GRP78+, polyQ+/GRP78− cells from both AR-24Q mice and AR-97Q mice, and a higher density in polyQ+/GRP78+ cells in muscle of the AR-97Q mice compared with the AR-24Q mice. Scale bars: (low-magnification image) 100 μM; (high-magnification image) 10 μM. (n=4, mean±S.D., *P<0.05, ANOVA)

Figure 5

GRP78 is required for suppressing AR aggregation and androgen/AR-mediated ESC death. (a) Ubiquitination assay showed both poly-and mono-ubiquitination of AR were decreased in GRP78/BiP-overexpressing ESCs. (b) Knockdown of GRP78/BiP increases AR inclusions formation in ESCs with DHT treatment. (c) Flag-AR ESCs with GRP78 overexpression or vector control were treated with DHT for 72 h. The high molecular weight AR aggregates were trapped in the CA membrane and the monomer AR was retained in the NC membrane. The expression of β-tubulin trapped in the NC membrane is shown as a loading control. (d) Immunofluorescence of AR aggregates in parental ESCs control and ESCs-FLAG-AR. GRP78/BiP expression regulate the AR aggregates accumulation. The percentage of cell with AR aggregates is shown. Data are presented as means±S.D., n=3. *P<0.05 was defined as significant

Figure 6

GRP78/BiP mitigates the misfolded AR-induced cytopathology. ESCs-FLAG-AR and ESCs-FLAG were treated with DHT 10⁻⁶ M or vehicle control. (a) More ESCs-FLAG-AR were positive for TUNEL staining than control ESCs-FLAG in response to DHT treatment. The number of TUNEL-positive cells was significantly reduced upon overexpression of GRP78/BiP, but increased upon GRP78/BiP knockdown. Scale bars: 25 μM. (b) Each value of caspase-3 activity was determined in triplicate. Both ESCs-FLAG-AR and ESCs-FLAG overexpressing GRP78/BiP exhibited a marked decrease in caspase-3 activity and cell death. Conversely, knockdown of GRP78/BiP expression promoted apoptosis. *P<0.05 was defined as significant. (c) Model for GRP78-dependent processing of misfolded AR proteins and aggregates formation. Numbers indicate various steps during misfolded AR proteins and aggregates biogenesis, as described in the discussion