Abstract
Frizzled 6 (FZD6) encodes an integral membrane protein that functions in multiple signal transduction pathways, for example, as a receptor in Wnt/planar cell polarity (PCP) signaling pathway for polarized cell migration and organ morphogenesis. Mutations in FZD6 have been identified in a variety of tumors. In this study, the full-length cDNA of Sus scrofa FZD6 (Sfz6) was cloned and characterized. Nucleotide sequence analysis demonstrates that the Sfz6 gene encodes the 712 amino-acid (aa) protein with seven transmembrane domain. Tissue distribution analysis showed that Sfz6 mRNA is ubiquitously expressed in various tissues, being highest in kidney, moderate in jejunum, ileum, colon, liver, and spleen. However, FZD6 protein is highly expressed in the heart and there was no significant difference in other tissues. The relative abundance and localization of FZD6 protein in jejunum along the crypt-villus axis was determined by Western blot and immunohistochemical localization. The results show that in the jejunum, FZD6 protein is highly expressed in the villus and less in the crypt cells. Cellular proliferation and viability assays indicate that knockdown of FZD6 with small interfering RNAs (siRNA) significantly reduced the cell viability of the intestinal porcine enterocyte cells (IPEC-J2). Furthermore, qPCR and Western blot analysis revealed that expressions of ras-related C3 botulinum toxin substrate 1 (Rac1); ras homolog gene family member A (RhoA) and c-Jun N-terminal kinase 1 (JNK1), some components of PCP signaling pathway were upregulated (P < 0.05) by knockdown of FZD6 in IPEC-J2 cells. In conclusion, these results showed that FZD6 abundance in the villus was higher than that in crypt cells and knockdown of FZD6 induces PCP signal pathway components expression in IPEC-J2 cells. Our findings provide the foundation for further investigation into porcine FZD6 gene.
Highlights
Wnts, a family of evolutionarily conserved, cysteine-rich, secreted glycoproteins, functions as central mediators of vertebrate and invertebrate development, influencing cell proliferation, differentiation, and migration [1, 2]
Translation of the nucleotide sequence was carried out using the open reading frame (ORF) Finder and an ORF of scrofa FZD6 (Sfz6) cDNA was identified constituting 2139 bp in length and encoding a predicted 712 amino acid SFz6 protein, which is similar in size to that of other mammalian counterparts: 706 aa, 709 aa, and 714 aa
The results show that the pig Frizzled 6 (FZD6) gene was highly expressed in the kidney (P
Summary
A family of evolutionarily conserved, cysteine-rich, secreted glycoproteins, functions as central mediators of vertebrate and invertebrate development, influencing cell proliferation, differentiation, and migration [1, 2]. The Wnt gene family consists of at least 19 members encoding secreted glycoproteins functioning as ligands for receptors [3, 4]. Wnts bind cell surface Frizzled receptors and activate at least one of the three distinct signaling pathways, which include β-catenin-dependent Wnt/β-catenin pathway, β-catenin-independent Wnt/planar cell polarity (PCP) and Wnt/calcium pathway[5]. Frizzled (FZD), a family of seven transmembrane domain proteins, have extracellular cysteine-rich domain (CRD) at the amino terminus that have been implicated as the Wnt binding domain [5]. In addition to FZD, the Wnt/β-catenin pathway requires the low-density lipoprotein receptor related proteins 5 and 6 (Lrp5/6) as co-receptor [8]
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