Implications of TGFβ on transcriptome and cellular biofunctions of palatal mesenchyme

ORIGINAL RESEARCH ARTICLE published: 10 April 2012 doi: 10.3389/fphys.2012.00085 Implications ofTGFβ on transcriptome and cellular biofunctions of palatal mesenchyme Xiujuan Zhu 1 † , Ferhat Ozturk 1 † , Sanjit Pandey 2 , Chittibabu (Babu) Guda 2 and Ali Nawshad 1 * 1 2 Department of Oral Biology, University of Nebraska Medical Center, Lincoln, NE, USA Center for Bioinformatics and Systems Biology, University of Nebraska Medical Center, Omaha, NE, USA Edited by: Daniel Graf, University of Zurich, Switzerland Reviewed by: Xiu-Ping Wang, Harvard School of Dental Medicine, USA Juhee Jeong, New York University College of Dentistry, USA *Correspondence: Ali Nawshad , Department of Oral Biology, College of Dentistry, University of Nebraska Medical Center, 40th and Holdrege Street, Lincoln, NE 68583, USA. e-mail: † Xiujuan Zhu and Ferhat Ozturk have contributed equally to this work. Development of the palate comprises sequential stages of growth, elevation, and fusion of the palatal shelves. The mesenchymal component of palates plays a major role in early phases of palatogenesis, such as growth and elevation. Failure in these steps may result in cleft palate, the second most common birth defect in the world. These early stages of palatogenesis require precise and chronological orchestration of key physiological processes, such as growth, proliferation, differentiation, migration, and apoptosis.There is compelling evidence for the vital role ofTGFβ-mediated regulation of palate development. We hypothesized that the isoforms of TGFβ regulate different cellular biofunctions of the palatal mesenchyme to various extents. Human embryonic palatal mesenchyme (HEPM) cells were treated with TGFβ1, β2, and β3 for microarray-based gene expression studies in order to identify the roles of TGFβ in the transcriptome of the palatal mesenchyme. Following normalization and modeling of 28,869 human genes, 566 transcripts were detected as differentially expressed in TGFβ-treated HEPM cells. Out of these altered transcripts, 234 of them were clustered in cellular biofunctions, including growth and proliferation, development, morphology, movement, cell cycle, and apoptosis. Biological interpretation and network analysis of the genes active in cellular biofunctions were performed using IPA. Among the differentially expressed genes, 11 of them are known to be crucial for palatogenesis (EDN1, INHBA, LHX8, PDGFC, PIGA, RUNX1, SNAI1, SMAD3, TGF β1, TGF β2, and TGF βR1).These genes were used for a merged interaction network with cellular behaviors. Overall, we have determined that more than 2% of human transcripts were differentially expressed in response to TGFβ treatment in HEPM cells. Our results suggest that both TGFβ1 and TGFβ2 orchestrate major cellular biofunctions within the palatal mesenchyme in vitro by regulating expression of 234 genes. Keywords: TGFβ, microarray, transcriptome, palatogenesis, mesenchyme, HEPM, craniofacial, palate INTRODUCTION Cleft lip and/or palate is one of the most prevalent birth defects worldwide (1 in 800 live births; Schutte and Murray, 1999; Spritz, 2001), and is caused by failures in palate development. The formation of a continuous palate is a complex process composed of multiple steps, including palatal shelf growth, elevation, attachment, and fusion. Palatogenesis in the human spans from approximately gestational day 48 to 59 and the outgrowth of the secondary palate can generally be detected around day 49. During day 54–55, the palatine processes rapidly elevate, assuming a horizontal position which allows them to grow toward each other, attach, and fuse (Wyszynski, 2002). In general, with slight variation among strains, the stages of palatogenesis in mice [12.5–16.5 days post coitum (dpc)] are extremely similar and comparable to that of humans; therefore, mice have been used as a model to study human palate development (Ferguson, 1988). The failure of palatal shelves to Abbreviations: FC, fold change; HEPM, human embryonic palatal mesenchyme; IKB, ingenuity knowledge base; IPA, ingenuity pathway analysis; MEE, medial edge epithelium; TGFβ, transforming growth factor β. www.frontiersin.org grow and adhere after elevation is the most common type of cleft palate defect documented in murine models (Chai and Maxson, 2006). The palatal cellular components originate from the cranial neural crest (CNC)-derived palatal mesenchyme, concealed with a veneer of pharyngeal ectoderm-derived epithelium (Ito et al., 2003; Nakajima et al., 2010). A precise and time-sensitive regulation of various mesenchymal biofunctions, such as cellular movement, cell death (apoptosis), cell morphology, cell cycle progression, development, and growth and proliferation, is fundamental for the proper development of the palate. These cellular functions are coordinated by numerous genes encoding a range of growth factors, signaling mediators, transcriptional factors, cytokines, and extracellular matrix proteins (Richman and Tickle, 1989; Greene and Pisano, 2004, 2005). Therefore it is immensely important to explore the genes and the molecules that regulate the plethora of these biofunctions to understand cellular behavior during palatogenesis. The TGFβ family consists of more than 30 ligand proteins, including activins, BMP, and TGFβ cytokines, regulating a wide variety of biological processes such as cellular development, April 2012 | Volume 3 | Article 85 | 1 Zhu et al. morphology, movement, growth and proliferation, survival, mitotic regulation, apoptosis, and epithelial–mesenchymal transition (EMT). Although the three isoforms of TGFβ (β1, β2, and β3) are highly conserved between species (Rotzer et al., 2001) and share 71–76% sequence identity, these ligands have isoformspecific activities that cannot be compensated by other family members (Iwata et al., 2011). Based on knockout mouse models, TGFβ isoforms have been found to be essential for normal morphogenesis of the palate (Iordanskaia and Nawshad, 2011). TGFβ1 null mice are embryonically lethal and die before 11 dpc (Brunet et al., 1995), so its role in palate development cannot be evaluated. TGFβ2 knockout mice, which are also embryonic lethal at 18 dpc, have defects in their mandible and maxilla, with 23% of cases resulting in cleft palate (Sanford et al., 1997); whereas all TGFβ3 null mice develop cleft palate (Kaartinen et al., 1995; Proetzel et al., 1995) and die within 24 h after birth. Furthermore, TGFβ mutations and expression patterns have been shown to be associated with the occurrence of cleft lip and/or palate (Stoll et al., 2004). Mutations in TGFβ1 and TGFβ3, as well as their variants, are associated with cleft palate in humans (Lidral et al., 1998; Mitchell et al., 2001; Kim et al., 2003; Vieira et al., 2003; Rullo et al., 2006; Guo et al., 2010; Salahshourifar et al., 2011). Moreover, in vitro studies of human tissues showed that TGFβ1 and β3 are differently expressed and correlated with the cleft lip and/or (...truncated)