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.
Highlights
From sex differentiation during embryogenesis to the onset and the maintenance of reproductive capacity in sexual maturity, androgen signaling is required for proper development and function
Previous studies showed that GRP78 À / À embryos did not hatch from the zona pellucida in vitro, failed to grow in culture, and exhibited proliferation defects and a massive increase in apoptosis in the inner cell mass (ICM),[12] suggesting GRP78/BiP is required for cell proliferation and protecting the ICM from apoptosis
We identified GRP78/BiP is colocalized with androgen receptor (AR) inclusions in both embryonic stem cells (ESCs) and a mouse model of spinal bulbar muscular atrophy (SBMA)
Summary
From sex differentiation during embryogenesis to the onset and the maintenance of reproductive capacity in sexual maturity, androgen signaling is required for proper development and function. In rats[3] and mice,[4] excess of testosterone (T) or other androgens can cause infanticide, reduced litter size or low pup viability.[5] In regard to human, enhanced ovarian androgen secretion is associated with embryo degeneration.[6] Functional AR requires interaction with a wide variety of cellular proteins that facilitate AR conformation, nuclear localization, DNA binding and interaction with the basal transcriptional machinery to induce expression of androgen-responsive AR-target genes.[7,8,9]. We present a novel insight in which AR proteins in ESCs form nuclear aggregated inclusions in response to androgen treatment, leading to exacerbated ER stress and cell death with apoptotic features during ESCs differentiation. GRP78/BiP acts as an AR-associated protein to suppress ubiquitination of AR aggregates, and required for amelioration of androgenmediated cytopathology of ESCs
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