Expression Analysis of FGF/FGFR and FOX Family Proteins in Mucosal Tissue Obtained from Orofacial Cleft-Affected Children

Abstract

Orofacial clefts affect hundreds of thousands of children worldwide annually and are usually corrected by a series of surgeries extending to childhood. The underlying mechanisms that lead to clefts are still unknown, mainly because of the multifactorial etiology and the myriad of interactions between genes and environmental factors. In the present study, we investigated the role and expression of candidate genes belonging to the FGF/FGFR signaling pathway and FOX family in tissue material obtained from 12 pediatric patients undergoing cleft correction surgery. The expression was investigated using immunohistochemistry (IHC) and chromogenic in-situ hybridization (CISH) in three cell/tissue types—epithelial cells, connective tissue, and endothelial cells. We found elevated expression of FGFR1 in epithelial cells while no expression was observed in endothelial cells. Further, our results elucidate the potential pathogenetic role of FGFR1 in cellular proliferation, local site inflammation, and fibrosis in cleft patients. Along with bFGF (also called FGF2), FGFR1 could play a pro-inflammatory role in clefts. Over-amplification of FGFR2 in some patients, along with bFGF, could potentially suggest roles for these genes in angiogenesis. Additionally, increased expression of FOXE1 (also called TTF2) contributes to local site inflammation. Finally, zero to low amplification of FOXO1 could suggest its potential role in inducing oxidative stress in the endothelium along with reduced epithelial apoptosis.

Evaluating the Expression of Candidate Homeobox Genes and Their Role in Local-Site Inflammation in Mucosal Tissue Obtained from Children with Non-Syndromic Cleft Lip and Palate

“…It is known that PTX3 possess FGF2-binding and inhibitory capacity (basic fibroblast growth factor/bFGF) which confers it anti-angiogenic properties [38,39]. In our last study (again, with the same cohort as the present study) we showed that FGF2 was over-expressed in cleft mucosal tissue and hypothesized on the possible interaction and role of PTX3 in cleft tissue [40].…”

“…The relevance and importance of the studies like the present one has been discussed and reported previously [40]. However, the results obtained in the present study are constrained by certain limitations.…”

“…DLX4 has been shown to directly stimulate expression of both the low and high molecular weight isoforms of FGF2 (Figure 7) which in turn stimulate VEGF (vascular endothelial growth factor) and promote an angiogenetic environment [53]. We also reported an increase in FGF2 expression in cleft tissue [40] which can lead to accelerated wound healing (FGF2 can further promote EMT and wound closure). Another possible pathway for angiogenetic promotion has been postulated by the means of DLX4 induced expression of iNOS (inducible nitric oxide synthetase) [54].…”

“…Bone formation and maintenance is a dynamic process that is under the regulatory control of various factors. While we noted previously an increase in FGF/FGFR pathways [40] which promote osteoblast differentiation, a chronic inflammatory state coupled with decrease in BMPs and TGF-β1/3 expression [26,74] usually results in greater bone resorption in cleft patients. This process is further compounded by an increase in the NF-κB expression due to activation of the classical pathway by TNF-α, IL-1β, IL-6, and IL-17 produced by T cells and other cells, all of which have been reported to be elevated in cleft tissue [26].…”

“…First, the limited number of control and cleft patients in the present study limits our ability to generalize our findings. This is rather understandable due to genuine concerns of the parents, given the tender age at which corrective surgery is undertaken [26,40]. Secondly, the complex etio-pathogenesis of cleft lip and palate makes it difficult to predict or state whether neonatal gene expression resembles embryonic gene expression, especially during the events just prior and during the palatal closure.…”