Modulation of lipid metabolic defects rescues cleft palate in Tgfbr2 mutant mice

Abstract

Mutations in transforming growth factor beta (TGFβ) receptor type II (TGFBR2) cause Loeys-Dietz syndrome, characterized by craniofacial and cardiovascular abnormalities. Mice with a deletion of Tgfbr2 in cranial neural crest cells (Tgfbr2(fl/fl);Wnt1-Cre mice) develop cleft palate as the result of abnormal TGFβ signaling activation. However, little is known about metabolic processes downstream of TGFβ signaling during palatogenesis. Here, we show that Tgfbr2 mutant palatal mesenchymal cells spontaneously accumulate lipid droplets, resulting from reduced lipolysis activity. Tgfbr2 mutant palatal mesenchymal cells failed to respond to the cell proliferation stimulator sonic hedgehog, derived from the palatal epithelium. Treatment with p38 mitogen-activated protein kinase (MAPK) inhibitor or telmisartan, a modulator of p38 MAPK activation and lipid metabolism, blocked abnormal TGFβ-mediated p38 MAPK activation, restoring lipid metabolism and cell proliferation activity both in vitro and in vivo. Our results highlight the influence of alternative TGFβ signaling on lipid metabolic activities, as well as how lipid metabolic defects can affect cell proliferation and adversely impact palatogenesis. This discovery has broader implications for the understanding of metabolic defects and potential prevention of congenital birth defects.

WNT/β-Catenin Signaling Regulates Multiple Steps of Myogenesis by Regulating Step-Specific Targets

“…To investigate the effect of IWR1-endo on maintenance of myofibers, well-differentiated C2C12 cells were cultured with vehicle or 1 M IWR1-endo in differentiation medium for another 3 to 5 days. The small interfering RNA (siRNA) knockdown for Ctnnb1 (Invitrogen), Ccna2, Cdc25c, and Fermt2 (Sigma-Aldrich) was performed as described previously (17,19). The overexpression of Fermt2 (OriGene Technologies, Inc., Rockville, MD) was also performed as described previously (17).…”

“…For cell proliferation assays, total RNAs isolated from C2C12 cells treated with 80 M IWR1-endo or control vehicle for 36 h (n ϭ 6 per group) were dissected with a QIAshredder and RNeasy mini-extraction kit (Qiagen), as described previously (19,23). Gene expression profiling for cell cycle regulators was performed by PCR arrays (PrimePCR assay; Bio-Rad Laboratories).…”

“…The UCSC genome browser was used to obtain the genomic sequences of the following murine genes, including 5 kbp upstream of the respective transcription start site: (16,19). Multiple alignments for these sequences were obtained using the Clustal Omega tool with default parameters and settings (24).…”

“…Cell extracts were incubated with ␤-catenin antibody (Abcam) overnight at 4°C, followed by precipitation with magnetic beads. Washing and elution of the immune complexes, as well as precipitation of DNA, were performed according to standard procedures, as described previously (16,19). The putative LEF1 target sites of the genes in the immune complexes were detected by PCR using the following primers: Ccna2 gene, 5=-TGGTGTTGCAGATCTAC CGT-3= and 5=-TCTGCTAACAAAATGGCAATGC-3= (Ϫ2364 to Ϫ2154); Cdc25c gene, 5=-TTCATCGGTCTCAGCTTCCC-3= and 5=-AG TCACCACTGAGCCTTGTC-3= (Ϫ3164 to Ϫ3029); and Fermt2 gene, 5=-TAGAGGTTCTAGCGGGGGTT-3= and 5=-TTCAGGCCTTGGCTTTG AGT-3= (Ϫ3138 to Ϫ2949).…”

Integration of comprehensive 3D microCT and signaling analysis reveals differential regulatory mechanisms of craniofacial bone development

“…We have recently reported that non-canonical TGFβ signaling is activated whereas canonical TGFβ signaling is compromised in Tgfbr2 fl/fl ;Wnt1-Cre mice (Iwata et al, 2014; Iwata et al, 2012). In addition, craniofacial deformities in Tgfbr2 mutant mice were largely restored by a haploinsufficiency of Tgfbr1 (Tgfbr2 fl/fl ;Wnt1-Cre;Alk5 fl/+ ) , a key receptor that is involved in ectopic non-canonical TGFβ signaling, indicating that altered canonical TGFβ signaling causes craniofacial deformities (Iwata et al, 2012).…”