Endoplasmic Reticulum Stress and Apoptosis Contribute to the Pathogenesis of Dominantly Inherited Isolated GH Deficiency Due to GH1 Gene Splice Site Mutations

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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.

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