Antagonistic Regulation of <I>Dlx2</I> Expression by PITX2 and Msx2: Implications for Tooth Development

Abstract

The transcriptional mechanisms underlying tooth development are only beginning to be understood. Pitx2, a bicoid-like homeodomain transcription factor, is the first transcriptional marker observed during tooth development. Because Pitx2, Msx2, and Dlx2 are expressed in the dental epithelium, we examined the transcriptional activity of PITX2 in concert with Msx2 and the Dlx2 promoter. PITX2 activated while Msx2 unexpectedly repressed transcription of a TK-Bicoid luciferase reporter in a tooth epithelial cell line (LS-8) and CHO cell line. Surprisingly, Msx2 binds to the bicoid element (5'-TAATCC-3') with a high specificity and competes with PITX2 for binding to this element. PITX2 binds to bicoid and bicoid-like elements in the Dlx2 promoter and activates this promoter 45-fold in CHO cells. However, it is only modestly activated in the LS-8 tooth epithelial cell line that endogenously expresses Msx2 and Pitx2. RT-PCR and Western blot assays reveal that two Pitx2 isoforms are expressed in the LS-8 cells. We further demonstrate that PITX2 dimerization can occur through the C-terminus of PITX2. Msx2 represses the Dlx2 promoter in CHO cells and coexpression of both PITX2 and Msx2 resulted in transcriptional antagonism of the Dlx2 promoter. Electrophoretic mobility shift assays demonstrate that factors in the LS-8 cell line specifically interact with PITX2. Thus, Dlx2 gene transcription is regulated by antagonistic effects between PITX2, Msx2, and factors expressed in the tooth epithelia.

Cell-specific Activation of the Atrial Natriuretic Factor Promoter by PITX2 and MEF2A

“…Because Pitx2 is expressed in the oral epithelium and MEF2 proteins also play roles in non-muscle lineages, we decided to test PITX2a and MEF2A activation of the ANF promoter in the LS8 oral epithelial cell line. The LS8 cells express Pitx2, and we have shown previously that exogenous PITX2a can activate target promoters in these cells (24,30).…”

“…The LS8 oral epithelial cells have been reported to express the endogenous Pitx2 gene (30). For comparison with heterologous cells that do not express Pitx2, we used the CHO cell line (30). MEF2 immunohistochemical staining was detected in the nuclei of C2C12 and LS8 cells but not in those of CHO cells (Fig.…”

“…The C2C12 myoblastlike cells are known to express both the endogenous Pitx2 and MEF2 genes (5,28,29). The LS8 oral epithelial cells have been reported to express the endogenous Pitx2 gene (30). For comparison with heterologous cells that do not express Pitx2, we used the CHO cell line (30).…”

PITX2 Isoform-specific Regulation of Atrial Natriuretic Factor Expression

“…Electrophoretic Mobility Shift Assay (EMSA)-Complementary oligonucleotides containing the PLOD1 promoter sequence (Ϫ1120 to Ϫ1092) with flanking partial BamHI ends were annealed and filled with Klenow polymerase to generate 32 P-labeled probes for EMSAs, as described (33). Standard binding assays were performed as previously described (34).…”

“…Approximately, 200 ng of purified PITX2 proteins and 20 g of cell lysates were analyzed by Western blots. Following SDS gel electrophoresis, the proteins were transferred to polyvinylidene difluoride filters (Millipore), immunoblotted, and detected using PITX2 antibody P2R10 (25,33) and ECL reagents from Amersham Biosciences.…”