Identification of Smad-dependent and -independent signaling with transforming growth factor-β type 1/2 receptor inhibition in palatogenesis

Yoshimi Suzuki; Akira Nakajima; Takayuki Kawato; Koichi Iwata; Mitsuru Motoyoshi; Charles F Shuler

doi:10.1016/j.jobcr.2020.01.002

Identification of Smad-dependent and -independent signaling with transforming growth factor-β type 1/2 receptor inhibition in palatogenesis

J Oral Biol Craniofac Res. 2020 Apr-Jun;10(2):43-48. doi: 10.1016/j.jobcr.2020.01.002. Epub 2020 Jan 16.

Authors

Yoshimi Suzuki¹, Akira Nakajima^{1

2}, Takayuki Kawato^{2

3}, Koichi Iwata^{2

4}, Mitsuru Motoyoshi^{1

2}, Charles F Shuler⁵

Affiliations

¹ Department of Orthodontics, Nihon University School of Dentistry, 1-8-13 Kanda Surugadai, Chiyoda-ku, Tokyo, 1018310, Japan.
² Dental Research Center, Nihon University School of Dentistry, 1-8-13 Kanda Surugadai, Chiyoda-ku, Tokyo, 1018310, Japan.
³ Department of Oral Health Science, Nihon University School of Dentistry, 1-8-13 Kanda Surugadai, Chiyoda-ku, Tokyo, 1018310, Japan.
⁴ Department of Physiology, Nihon University School of Dentistry, 1-8-13 Kanda Surugadai, Chiyoda-ku, Tokyo, 1018310, Japan.
⁵ Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, 2194 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.

Abstract

TGF-β signaling is one of important function during palatal fusion. Three types of TGF-β receptor (TβR1, TβR2, and TβR3) have been identified, and play essential roles in mechanisms leading to palatal fusion. However, the balance between Smad-dependent/-independent signaling during palatal fusion with inhibited TβR1/2 functions is not fully understood. The objective of this study was to investigate palatal fusion via TGF-β signaling when TβR1 and TβR2, but not TβR3, were inhibited. In addition, the present study examined the functional balance between Smad-dependent/-independent signaling and related gene expression. Palatal organ cultures were treated with TβR1/2 inhibitor in vitro. Control palates were cultured without inhibitor. We observed histological phenotype of palatal fusion, and evaluation of expression pattern by Western blot or real time RT-PCR. Palatal organ cultures treated with the inhibitor did not fuse and the medial edge epithelium remained at embryonic 13 day +72 h in culture. The inhibitor decreased TβR1 and TβR2 expression by approximately 90%, but did not affect TβR3 expression. The expression of p-Smad2 and p-Smad3 was significantly decreased in treated palates compared with controls. The expression of p-Smad4 was slightly decreased in treated palates compared with controls. Smad-independent signaling was also affected by the inhibitor; p-ERK, p-JNK, and p-p38 expressions was significantly reduced in treated palates compared with controls. The expression of transcription factors (Runx1 and Msx1) and extracellular matrix proteins (MMP2/13) was also significantly decreased by inhibitor exposure. Treatment with TβR1/2 inhibitor altered the patterns of the Smad-dependent and -independent signaling pathways during palatal fusion.

Keywords: Cleft palate; Medial edge epithelial cell; Palatal fusion; Smad-dependent/-independent; TGF-β signaling.