Transforming Growth Factor-β Regulates Basal Transcriptional Regulatory Machinery to Control Cell Proliferation and Differentiation in Cranial Neural Crest-derived Osteoprogenitor Cells

Abstract

Transforming growth factor-␤ (Tgf-␤) signaling is crucial for regulating craniofacial development. Loss of Tgf-␤ signaling results in defects in cranial neural crest cells (CNCC), but the mechanism by which Tgf-␤ signaling regulates bone formation in CNCC-derived osteogenic cells remains largely unknown. In this study, we discovered that Tgf-␤ regulates the basal transcriptional regulatory machinery to control intramembranous bone development. Specifically, basal transcription factor Taf4b is down-regulated in the CNCC-derived intramembranous bone in Craniofacial skeletal elements are mainly formed by intramembranous ossification through a mechanism that remains relatively uncharacterized. The majority of osteoblasts and chondrocytes in the craniofacial region are derived from cranial neural crest cells (CNCC), 2 which produce the facial skeleton (1, 2). Tgf-␤ signaling plays a crucial role in craniofacial development, and loss of Tgf-␤ signaling in CNCC results in craniofacial skeletal malformations (3, 4). Tgf-␤ transmits signals through a membrane receptor serine/threonine kinase complex that phosphorylates Smad2 and Smad3, and activated Smads form transcriptional complexes with Smad4 and translocate into the nucleus (5). These Tgf-␤ signaling complexes contain other transcription factors and target a variety of genes in an embryonic stage-dependent and cell type-specific manner, but the factors involved in this transcriptional regulatory machinery have yet to be identified. During development, the expression of many genes is associated with changes accompanied by dynamic restructuring of chromatin (6, 7). Recent studies demonstrate that basal transcriptional factors have cell-and promoter-specific functions during embryogenesis (8 -12).RNA polymerase II requires the assembly of a multiprotein complex around the transcriptional start site (13). The general transcriptional factor IID (TFIID) is a large multiprotein transcriptional factor, consisting of the TATA-binding protein and a set of 13-14 TATA-binding protein-associated factors (TAFs), that is responsible for specific binding to the TATA element found in many polymerase II promoters and also demonstrates a coactivator function during transcriptional initiation (14). TAFs are able to regulate gene transcription at multiple steps, with functions in promoter recognition, selective binding to core promoter elements, as well as direct interactions with transcriptional activators (15-17). Mutation and loss of TAFs in yeast and mammalian cells lead to cell cycle arrest and gene-specific transcriptional effects (16). The function of TAFs in gene regulation during embryogenesis has yet to be determined. Here, we show that the interaction between Tgf-␤ signaling and TAFs has a crucial role in regulating CNCC-derived osteogenesis during craniofacial morphogenesis. EXPERIMENTAL PROCEDURES Animals-Mating Tgfbr2fl/ϩ ;Wnt1-Cre with Tgfbr2 fl/fl mice generated Tgfbr2 fl/fl ;Wnt1-Cre conditional null alleles that were genotyped using PCR primers as described previously ...

Smad4-Irf6 genetic interaction and TGFβ-mediated IRF6 signaling cascade are crucial for palatal fusion in mice

“…Hematoxylin and Eosin (H&E), immunohistochemical and 5-bromo-2Ј-deoxyuridine (BrdU) staining were performed as described previously (Iwata et al, 2012;Iwata et al, 2010). Antibodies used for immunohistochemistry were rabbit polyclonal antibodies against p21 (Santa Cruz Biotechnology), IRF6 (Aviva Systems Biology), phosphorylated histone H3 (Millipore) and Lex/SSEA1 (FUT4 -Mouse Genome Informatics) (Cell Signaling Technology), and mouse monoclonal antibody against p63 (Santa Cruz Biotechnology).…”

“…Immunoblots were performed as described previously (Iwata et al, 2006;Iwata et al, 2010). Antibodies used for immunoblotting were rabbit polyclonal antibodies against IRF6 (Aviva Systems Biology) and p21 (Abcam), and mouse monoclonal antibodies against p63 (Santa Cruz Biotechnology) and GAPDH (Chemicon).…”

“…Total RNA was isolated from dissected mouse MEE at E14.5 with the QIAshredder and RNeasy Micro Extraction Kit (QIAGEN), as described previously (Iwata et al, 2010). The following PCR primers were used: Irf6, 5Ј-AGGGCTCTGTCATTAATCCAG-3Ј and 5Ј-TGATTCGGGGCT -GCAGTTTC-3Ј; p21 (Cdkn1a), 5Ј-AGCCTGAAGACTGTGATGGG-3Ј and 5Ј-AAAGTTCCACCGTTCTCGG-3Ј; ΔNp63, 5Ј-CAAAACCC -TGGAAGCAGAAA-3Ј and 5Ј-GAGGAGCCGTTCTGAATCTG-3Ј; and Gapdh, 5Ј-AACTTTGGCATTGTGGAAGG-3Ј and 5Ј-ACACATTG -GGGGTAGGAACA-3Ј.…”

“…Tgfbr2 and Irf6 siRNA duplexes were purchased from Santa Cruz Biotechnology. siRNA mixture in transfection medium was incubated with cells for 7 hours at 37°C in a CO 2 incubator, as described previously (Iwata et al, 2010).…”

TGFβ regulates epithelial-mesenchymal interactions through WNT signaling activity to control muscle development in the soft palate

“…Hematoxylin and Eosin (H&E), BrdU and immunohistochemical staining were performed as described previously (Iwata et al, 2010;Iwata et al, 2012;Iwata et al, 2013a). Antibodies used for immunohistochemistry were mouse monoclonal antibody against myosin heavy chain (MyHC; SigmaAldrich) and DKK1 (Santa Cruz Biotechnology).…”