Axenfeld-Rieger syndrome is an autosomal-dominant disorder caused by mutations in the PITX2 homeodomain protein. We have studied the mechanism underlying the dominant negative K88E mutation, which occurs at position 50 of the homeodomain. By using yeast two-hybrid and in vitro pulldown assays, we have documented that PITX2a can form homodimers in the absence of DNA. Moreover, the K88E mutant had even stronger dimerization ability, primarily due to interactions involving the C-terminal region. Dimerization allowed cooperative binding of wild-type (WT) PITX2a to DNA containing tandem bicoid sites in a head-to-tail orientation (Hill coefficient, 1.73). In contrast, the WT-K88E heterodimer bound the tandem sites with greatly reduced cooperativity and decreased transactivation activity. To further explore the role of position 50 in PITX2a dimerization, we introduced a charge-conservative mutation of lysine to arginine (K88R). The K88R protein had greatly reduced binding to a TAATCC element and did not specifically bind any other TAATNN motif. Like K88E, K88R formed relatively stronger dimers with WT. As predicted by our model, the K88R protein acted in a dominant negative manner to suppress WT PITX2a activity. These results suggest that the position 50 residue in the PITX2 homeodomain plays an important role in both DNA binding and dimerization activities.The PITX2 gene was cloned based on its linkage in Axenfeld-Rieger syndrome (ARS) (30). ARS is an autosomal-dominant human disorder characterized by ocular anterior chamber anomalies causing glaucoma in more than 50% of affected individuals as well as dental hypoplasia, mild craniofacial dysmorphism, and umbilical stump abnormalities. Other features associated with this syndrome include abnormal cardiac, limb, and pituitary development (28,30). Three isoforms of PITX2 (designated a, b, and c) that differ only in their amino termini have been identified (9, 15). PITX2a and the related PITX1 protein have been shown to interact with Pit-1, a transcription factor that regulates pituitary cell differentiation, including the expression of the thyroid-stimulating hormone, growth hormone, and prolactin genes (4, 16, 31). We have used the prolactin promoter as a model target for studying PITX2a activities. Transient transfection assays with the prolactin promoter, which contains both Pit-1 and PITX2a binding sites, have shown a strong synergistic effect of PITX2a and Pit-1 on transactivation (4). Consistent with these data, knockout mice lacking Pitx2 show arrested pituitary gland development (10).PITX2a is a 33-kDa homeodomain protein that is a functional member of the bicoid homeodomain subfamily, as defined by a lysine at residue 50 of the homeodomain (4). It has been demonstrated that this residue in the bicoid protein interacts with base pairs 5 and 6 of the hexanucleotide consensus 5Ј-TAATCC-3Ј (12, 32). PITX2a has been shown to specifically bind to this consensus sequence and transactivate corresponding reporter genes (4). We have previously reported an ARS mutation th...

Purpose. The cornea is protected by apical hydrophilic transmembrane mucins and tears. In pathologic states the mucin barrier is disrupted, creating potential for meibomian lipids to adhere more strongly. Undisplaced lipids create an unwettable surface. The hypothesis that pathologic ocular surfaces alter lipid binding and the ability of tear proteins to remove lipids was tested. Methods. Corneas with pathologic surfaces were studied for lipid adhesion and removal by tears. Capture of fluorescence-labeled phospholipids by human tears was assessed by steady state fluorometry. Tear proteins were separated by gel filtration chromatography and analyzed for bound lipids. Results. Contact angle measurements revealed strong lipid adherence to corneas submerged in buffer. Lower contact angles are observed for lipids on completely de-epithelialized corneas compared with intact corneas (P = 0.04). Lipid removal from these surfaces is greater with whole tears than with tears depleted of tear lipocalin (P < 0.0005). Significantly fewer lipids are captured by tears from Bowman's layer than from epithelial-bearing surfaces (P < 0.025). The only tear component to bind the fluorescence-tagged lipid is tear lipocalin. The histology of a rare case of dry eye disease demonstrates the dominant features of contemporaneous bullous keratopathy. Lipid sequestration from this cornea by tear lipocalin was robust. Conclusions. Lipid is captured by tear lipocalin from corneas with bullous keratopathy and dry eye. Lipid removal is slightly abrogated by greater lipid adhesion to Bowman's layer. Reduced secretion of tear lipocalin documented in dry eye disease could hamper lipid removal and exacerbate ocular surface pathology.

The consistency of DRS measurements suggests that the posterior surface of the cornea does not change appreciably after keratorefractive surgery and is imaged more accurately using DRS compared with SSB. The DRS system affords confidence in interpreting data that are useful for discerning morphologic abnormalities of the cornea, both before and after keratorefractive surgery.

Background: Protein inhibitors of activated STAT (Pias) proteins can modulate the activity of transcription factors. Results: Pias1 and Piasy enhance or repress pituitary homeobox 2 (PITX2) transcriptional activity. Conclusion: Pias interactions regulate PITX2 transcriptional activity. Significance: Pias proteins can differentially regulate PITX2 activity.

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