Altered regulation of platelet-derived growth factor receptor-alpha gene-transcription in vitro by spina bifida-associated mutant Pax1 proteins

Abstract

Mouse models show that congenital neural tube defects (NTDs) can occur as a result of mutations in the platelet-derived growth factor receptor-alpha gene (PDGFRalpha). Mice heterozygous for the PDGFRalpha-mutation Patch, and at the same time homozygous for the undulated mutation in the Pax1 gene, exhibit a high incidence of lumbar spina bifida occulta, suggesting a functional relation between PDGFRalpha and Pax1. Using the human PDGFRalpha promoter linked to a luciferase reporter, we show in the present paper that Pax1 acts as a transcriptional activator of the PDGFRalpha gene in differentiated Tera-2 human embryonal carcinoma cells. Two mutant Pax1 proteins carrying either the undulated-mutation or the Gln --> His mutation previously identified by us in the PAX1 gene of a patient with spina bifida, were not or less effective, respectively. Surprisingly, Pax1 mutant proteins appear to have opposing transcriptional activities in undifferentiated Tera-2 cells as well as in the U-2 OS osteosarcoma cell line. In these cells, the mutant Pax1 proteins enhance PDGFRalpha-promoter activity whereas the wild-type protein does not. The apparent up-regulation of PDGFRalpha expression in these cells clearly demonstrates a gain-of-function phenomenon associated with mutations in Pax genes. The altered transcriptional activation properties correlate with altered protein-DNA interaction in band-shift assays. Our data provide additional evidence that mutations in Pax1 can act as a risk factor for NTDs and suggest that the PDGFRalpha gene is a direct target of Pax1. In addition, the results support the hypothesis that deregulated PDGFRalpha expression may be causally related to NTDs.

Figure 1

In vitro transcription and translation of Pax1 and derived mutants. The in vitro transcription/translation products of wt-Pax1 (wt), sb-derived-Pax1 (sb), and un-Pax1 (un) were detected on Western blot by a specific anti-Pax1 antibody. As a positive (pc) and negative control (nc) total protein extract from the developing vertebral column of 13.5-day-old wt and a homozygous undulated shorttail (Un^S) embryos were used, respectively. In Un^S mutant mice, the Pax1 gene has been deleted completely. The antibody detects a predicted band of ≈40 kDa, which is not present in the negative control. No Pax1 was detected in the transcription/translation lysate (not shown).

Figure 2

Electrophoretic mobility shift assays with Pax1 and derived mutants. In vitro transcription/translation products of wt-Pax1, sb-derived Pax1, and un-Pax1 were assayed for binding activity on the PRS4 probe derived from the e5 site in the Drosophila even-skipped promoter. The lanes Ly, un, wt, sb, SC, and 0, correspond to incubation of the probe with reticulocyte lysate, un-Pax1, wt-Pax1, sb-Pax1, and wt-Pax1 + unlabeled PRS4 probe in a 500-fold excess, and H₂O, respectively. The Pax1 bands are indicated with arrows. Nonspecific bands are indicated with asterisks.

Figure 3

Regulation of transcriptional activity of the PDGFRα promoter by Pax-1 and derived mutants. The activity of the −944/+118 promoter luciferase construct (P1) is regulated differently by wt-Pax1, sb-Pax1, and un-Pax1 in RA-differentiated Tera-2 RA cells (A, hatched bars), undifferentiated Tera-2 EC cells (B, open bars), and U-2 OS (C, black bars). Values are presented as mean promoter activity relative to the activity of the −944/+118 clone cotransfected with the expression construct without a Pax1 insert, which was set to 1. Error bars indicate the sample SD after four luciferase/β-galactosidase measurements.