Human PRKC Apoptosis WT1 Regulator Is a Novel PITX2-interacting Protein That Regulates PITX2 Transcriptional Activity in Ocular Cells

Abstract

Mutations in the homeobox transcription factor PITX2 result in Axenfeld-Rieger syndrome (ARS), which is associated with anterior segment dysgenesis and an increased risk of glaucoma. To understand the pathogenesis of the defects resulting from PITX2 mutations, it is essential to know the normal functions of PITX2 and its interaction with the network of proteins in the eye. Yeast two-hybrid screening was performed using a cDNA library from a human trabecular meshwork primary cell line to detect novel PITX2-interacting proteins and study their role in ARS pathogenesis. After screening of ϳ1 ؋ 10 6 clones, one putative interacting protein was identified named PRKC apoptosis WT1 regulator (PAWR). This interaction was further confirmed by retransformation assay in yeast cells as well as co-immunoprecipitation in ocular cells and nickel pulldown assay in vitro. PAWR is reportedly a proapoptotic protein capable of selectively inducing apoptosis primarily in cancer cells. Our analysis indicates that the homeodomain and the adjacent inhibitory domain in PITX2 interact with the C-terminal leucine zipper domain of PAWR. Endogenous PAWR and PITX2 were found to be located in the nucleus of ocular cells and to co-localize in the mesenchyme of the iridocorneal angle of the developing mouse eye, consistent with a role in the development of the anterior segment of the eye. PAWR was also found to inhibit PITX2 transcriptional activity in ocular cells. These data suggest PAWR is a novel PITX2-interacting protein that regulates PITX2 activity in ocular cells. This information sheds new light in understanding ARS and associated glaucoma pathogenesis. Pituitary homeobox transcription factor 2 (PITX2)2 is a member of the paired-bicoid family of homeodomain (HD) transcription factors. Members of pituitary homeobox proteins are actively involved in a wide range of developmental processes including formation of pituitary gland and hind limb and of anterior segment of the eye as well as brain morphogenesis (1-3). Expression of PITX2 is found during ocular development (4, 5). The PITX2 gene is represented by several different splicing and transcriptional isoforms. The four best characterized are PITX2A, -B, -C (5), and -D (6) These isoforms of PITX2 vary in their N termini but share common HD and C-terminal sequences. The PITX2D isoform, however, has a truncated and non-functional HD (7). These alternate transcripts (A, B, and C) encode 271, 317, and 324 amino acids, respectively (4, 8).Numerous pathologic PITX2 mutations including missense variations, splice site alterations, and insertions/deletions have been described, producing a continuum of clinical phenotypes including Axenfeld-Rieger syndrome (ARS), iridogoniodysgenesis, and iris hypoplasia (5, 9, 10), as well as rarer cases of a Peters-like anomaly (11). Of these, ϳ82% of PITX2 mutations were observed in ARS, ϳ7% were observed both in iridogoniodysgenesis and in iris hypoplasia, and less than 3% were observed in Peters-like anomaly (Human Gene Mutation Database (HGMD)). ARS itself...

Neural crest derivatives in ocular development: Discerning the eye of the storm

“…Thus, PITX2 is a functional downstream target of RA in the POM, which mediates RA regulation of neural crest derivatives in the eye disrupted neural crest-derived corneal endothelial and iris stromal formation. Clinical mutations in PITX2 in individuals with ARS illustrate the essential role of the neural crest in ocular development (Acharya et al, 2009; Weisschuh et al, 2006). Animal studies demonstrate that Pitx2 along with RA signaling in the neural crest-derived POM is a critical regulator of anterior segment development and understanding these developmental mechanisms yield insight into the pathogenesis of this and other congenital eye diseases.…”

A novel PITX2c loss-of-function mutation underlies lone atrial fibrillation

“…A schematic diagram of PITX2c showing the structural domains and the location of the identified mutation is exhibited in Fig. 3 [modified from previous studies (55,56)]. This missense mutation was neither observed in the control population nor reported in the EVS and NCBI SNP databases.…”

PITX2c Loss-of-Function Mutations Responsible for Congenital Atrial Septal Defects

“…The sequence electropherograms showing the identified heterozygous PITX2c variations in contrast to corresponding control sequences are illustrated in Figure 1. A schematic diagram of PITX2c showing the structural domains and the locations of the detected mutations is presented in Figure 2 35,36. The variations were neither observed in 400 control chromosomes nor reported in the EVS's and NCBI's SNP databases, which were consulted again on May 31, 2013.…”