Enhanced SMAD1 Signaling Contributes to Impairments of Early Development in CFC-iPSCs

Abstract

Cardio-facio-cutaneous (CFC) syndrome is a developmental disorder caused by constitutively active ERK signaling manifesting mainly from BRAF mutations. Little is known about the role of elevated ERK signaling in CFC syndrome during early development. Here, we show that both SMAD1 and ERK signaling pathways may contribute to the developmental defects in CFC syndrome. Induced pluripotent stem cells (iPSCs) derived from dermal fibroblasts of a CFC syndrome patient (CFC-iPSCs) revealed early developmental defects in embryoid body (EB) development, b-catenin localization, and neuronal differentiation. Both SMAD1 and ERK signalings were significantly activated in CFC-iPSCs during EB formation. Most of the b-catenin was dissociated from the membrane and preferentially localized into the nucleus in CFC-EBs. Furthermore, activation of SMAD1 signaling recapitulated early developmental defects in wildtype iPSCs. Intriguingly, inhibition of SMAD1 signaling in CFC-iPSCs rescued aberrant EB morphology, impaired neuronal differentiation, and altered b-catenin localization. These results suggest that SMAD1 signaling may be a key pathway contributing the pathogenesis of CFC syndrome during early development.

SHP2 mutations induce precocious gliogenesis of Noonan syndrome-derived iPSCs during neural development in vitro

“…1c). Similarly, CFC syndrome-iPSCs with the BRAF mutation show elevated SMAD1 signaling, which leads to failures of EB development and NR formation [40]. In the present study, the activities of p-SHP2, p-ERK, p-SMAD1, and p-SMAD2 were enhanced in the NS-EBs compared with the WT-EBs (Fig.…”

“…Other neurological manifestations of NS patients include motor or cognitive developmental delays, learning disabilities, socialization problems, attention-deficit hyperactivity disorder (ADHD), and autism aspects [29][30][31][32][33][34][35][36]. Recently, modeling other rasopathies such as Costello syndrome (CS) and cardio-facio cutaneous (CFC) syndrome using human induced pluripotent stem cells (iPSCs) demonstrates neurodevelopmental abnormalities in vitro [37][38][39][40]. However, modeling of the neurological dysfunctions of NS patients has not been reported.…”

Impaired Osteogenesis of Disease-Specific Induced Pluripotent Stem Cells Derived from a CFC Syndrome Patient

“…In contrast to a downregulated SMAD1 pathway during osteogenesis of CFC-MSCs ( Figure 4 a), increment in the SMAD1 pathway was detected in CFC-iPSCs during early neuronal development in our previous study [ 20 ]. Although ERK signaling was activated in both CFC-EBs and CFC-MSCs due to the gain-of-function mutation of BRAF , the activity of the SMAD1 pathway was different between CFC-EBs and CFC-MSCs.…”

“…Even though the patient has been treated with human growth hormone, defects of his skeletal developmental marks such as height and bone mineral density have not been alleviated ( Table S1 ). To study the pathological mechanisms, CFC-iPSCs were generated from dermal fibroblasts of the CFC syndrome patient with BRAF Q257R (c. 770A > G), which is the most frequent mutation found in CFC syndrome [ 20 ]. The two lines of CFC-iPSCs (CFC2 and CFC7) employed in this study were characterized.…”

“…One WT-iPSC line and two CFC-iPSC lines (CFC2 and CFC7) used in this study were characterized as previously reported [ 20 , 47 ]. For immunostaining, cells were fixed with 4% formaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 15 min.…”