Mutations in SKI in Shprintzen–Goldberg syndrome lead to attenuated TGF-β responses through SKI stabilization

Abstract

Shprintzen–Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys–Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-β signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional co-repressor SKI, which is a negative regulator of TGF-β signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing, and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-β responses, both in knockin cells expressing an SGS mutation and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-β-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes.

Epigenetic modulation in the pathogenesis and treatment of inherited aortic aneurysm conditions

“…More recently, it was reported by Hill and colleagues that SGS mutations can promote enhanced stability of SKI, and that this was associated with decreased expression of selected TGFβ target genes in either cells transfected with mutant SKI or in SGS fibroblasts (31). Notably, this study only assessed the acute phase-response to administered TGFβ ligand (1 and 8 hours after delivery), and focused on genes that are predominantly expressed in neurons or polarized epithelial cells, but not the aortic wall or dermal fibroblasts.…”

“…SKIL or SMAD7) or factors involved in chronic compensation. Importantly, our prior studies in dermal fibroblasts derived from SGS patients assessed TGFβ target gene expression at steady-state, with no overlap between the repertoire of genes previously assessed and those specifically examined by Hill and colleagues (21, 31). We now unequivocally show that constitutive or VSMC-specific expression of a heterozygous SGS-associated SKI mutation leads to a substantial and sustained increase in H3K27 acetylation in vivo, as predicted by increased CBP/P300 occupancy, in association with high expression of TGFβ target genes relevant to aortic disease and reliably assayed in dermal fibroblasts.…”

“…In comparison, there is no documented example of decreased TGFβ signaling in tissues derived from people or mouse models of these conditions. Yet, we view the evidence for primary functional impairment of the TGFβ signaling response by underlying mutations to be equally compelling, including the evidence by Hill and colleagues that SGS mutations can stabilize SKI and associate with relative impairment of transcriptional responses in selected culture systems (31). The challenge – and, we would argue, the opportunity - lies in embracing all inconvenient truths to arrive at reconciling and testable models that have the true potential to inform disease pathogenesis and therapeutic opportunities.…”

“…The observations that SGS mutations clustered in the R-SMAD binding domain of SKI, and that SGS patient fibroblasts showed high expression of TGFb-responsive genes known to contribute to aneurysm progression suggested that increased TGFb-dependent events may cause the multisystem manifestations of SGS. This might also inform the mechanism for similar manifestations in MFS and LDS.More recently, it was reported by Hill and colleagues that SGS mutations can promote enhanced stability of SKI, and that this was associated with decreased expression of selected TGFb target genes in either cells transfected with mutant SKI or in SGS fibroblasts(31). Notably, this study only assessed the acute phase-response to administered TGFb ligand (1 and 8 hours after delivery), and focused on genes that are predominantly expressed in neurons or polarized epithelial cells, but not the aortic wall or dermal fibroblasts.…”

“…Interestingly, a version of SKI lacking this R-SMAD binding domain retained its ability to regulate SMAD-mediated transcriptional activation in a transient transfection-based reporter system, but failed to dissociate CBP/P300 from the SMAD complex (28, 30), suggesting that CBP/P300 could play a role in SGS. More recently, it was shown that regulated degradation of SKI requires interaction with SMAD2 or SMAD3 and SMAD4; SGS mutations that prevent SMAD2/3 binding resulted in increased stability and hence abundance of mutant SKI, which retained the ability for transcriptional repression of some TGFβ target genes, as evidenced by reduced induction of these transcripts in cultured cells expressing mutant SKI to acute stimulation with exogenous TGFβ (31). Many questions remain regarding the potentially opposing influences of different aspects of altered SKI homeostasis in SGS including increased stability and abundance that is perhaps offset by altered efficiency for recruitment to regulatory elements of target genes.…”