title: Endoplasmic reticulum stress and apoptosis contribute to the pathogenesis of dominantly inherited isolated GH deficiency due to GH1 gene splice site mutations.
abstract: Dominantly inherited isolated GH deficiency is mainly caused by a heterozygous donor site mutation of intron 3 in the GH1 gene. An exon 3 deletion in GH (del32-71 GH) is produced from a mutant allele, whereas wild-type GH is produced from the other allele. Several studies have demonstrated a dominant negative effect of del32-71 GH on wild-type GH secretion, but the precise molecular mechanisms remain unclear. We hypothesized that unfolded del32-71 GH accumulates in the endoplasmic reticulum (ER) and causes ER stress and apoptosis in somatotrophs, promoting GH deficiency. To evaluate del32-71 GH-mediated ER stress, we established GH4C1 cell lines with doxycycline (dox)-controlled del32-71 GH expression. In 20 of 23 dox-controlled cell lines, the concentration of wild-type GH in the culture medium significantly decreased with del32-71 GH induction, demonstrating the dominant negative effect of this mutant. Cell viability, mRNA abundance of ER stress-response genes, caspase activation, and DNA fragmentation were evaluated in 5 dox-controlled cell lines selected as cellular models. In 4 of the 5 cell lines, del32-71 GH induction decreased cell viability, increased expression of 3 major ER stress response pathways (PRKR-like endoplasmic reticulum kinase [PERK], activating transcription factor-6 [ATF6], and inositol requirement 1 [IRE1]), and induced caspase-3 and caspase-7 activation. In 1 of the 4 cell lines, DNA fragmentation was demonstrated. Finally, overexpression of XBP1(S), a nuclear transcription factor downstream of IRE1, completely reversed the observed caspase activation. These data suggested that del32-71 GH-mediated ER stress and apoptosis contributed to the decrease in wild-type GH secretion observed in GH deficiency due to the GH1 gene slice-site mutations.
authors.affiliation: Department of Endocrinology and Metabolism, Tokyo Metropolitan Children’s Medical Center, Tokyo 183–8561, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Ariyasu
authors.forename: Hiderou
authors.initials: H
authors.lastname: Yoshida
authors.forename: Makoto
authors.initials: M
authors.lastname: Yamada
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Hasegawa
mesh_terms: D000483:Alleles; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002999:Clone Cells; D004318:Doxycycline / Q000494:pharmacology; D004393:Dwarfism, Pituitary / Q000235:genetics* / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D017353:Gene Deletion; D019382:Human Growth Hormone / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000187:drug effects; D022821:RNA Splice Sites; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects; D052683:Somatotrophs / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000900:Anti-Bacterial Agents; D022821:RNA Splice Sites; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D004318:Doxycycline